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Characterization of Inositol Transporters as a Method for Drug Delivery to the Central Nervous System
by
Daniela Fenili
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Department of Laboratory Medicine and Pathobiology University of Toronto
© Copyright by Daniela Fenili 2010
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Characterization of Inositol Transporters as a Method for Drug Delivery
to the Central Nervous System
Daniela Fenili
Doctor of Philosophy
Department of Laboratory Medicine and Pathobiology University of Toronto
2010 ABSTRACT
A challenge in the treatment of central nervous system (CNS) diseases is the transport of drug
candidates into the brain. Inositol stereoisomers have show promise as therapeutic agents for
CNS disorders. scyllo-Inositol wasan effective prophylactic and therapeutic for Alzheimer’s
disease (AD) inTgCRND8 mice, a model of AD. Thissuggests inositol stereoisomers have
excellent CNS bioavailability. They enter the brain through inositol transporters, of which
thereare three: one hydrogen myo-inositol transporter (HMIT) and two sodium myo-inositol
transporters (SMIT1, SMIT2). HYPOTHESIS: Given the high CNS bioavailability of inositol
stereoisomers, it may be possible to use inositol transporters to shuttle other compounds into the
CNS. OBJECTIVES: 1. To confirm the CNS bioavailability of the two main inositol
stereoisomers, myo- and scyllo-inositol, in both TgCRND8 and wild-type mice. 2. To examine
inositol transporter expression in the brains, as a function of time and disease pathology, inboth
groups. 3. To evaluate the flexibility of the inositol transporters for transporting compounds
by determining the substrate structural features required for active transport. RESULTS:
myo-Inositol and scyllo-inositol accumulated in the brain following oral administration.
Disease pathology did not alter baseline inositol levels or uptake. Brain subregional transporter
expression was unaltered as a function of age or disease pathology. In vitro cell culture
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experiments found HMIT inactive and therefore not a contender for drug transport. In contrast
SMIT1 and SMIT2 were both active and competitive transportassays, revealed distinct criteria
for active transport through each system. However, both were stringent in the substitutions to
the structure of myo-inositol possible to maintain active transport. CONCLUSION: Active
transport through the inositol transporters is very sensitive to changes in the structure of myo-
inositol and only conservative changes arepossible. Therefore, these transporters would not
make effective shuttling systems for drug transport into the brain.
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ACKNOWLEDGEMENTS
I would like to thank JoAnne for her help and guidance throughout my PhD, both towards
designing and interpreting my experiments and during the preparation of my thesis.
I would like to thank my PhD committees, both past and present for their helpful suggestions
and questions.
I’d like to acknowledge my fellow lab members, both past and present for their help with
interpreting and understanding my experiments, as well as for creating a great working
environment full of fun, laughter and inappropriate music (you know who you are).
Finally, I would like to thank my family and friends for their support and understanding
whenever my experiment and thesis took away from our quality time.
Without all of your support, this thesis would not have been possible.
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TABLE OF CONTENTS
ABSTRACT ..................................................................................................................................iiLIST OF TABLES .....................................................................................................................viiLIST OF FIGURES ...................................................................................................................viiLIST OF ABBREVIATIONS.....................................................................................................ixCHAPTER 1 .................................................................................................................................1
Introduction1.1 Brain Barriers ......................................................................................................................2
1.1.1 The blood-brain barrier ................................................................................................21.1.2 The Blood-CSF Barrier ................................................................................................5
1.2 Strategies for transport of drugs across brain barriers ........................................................61.2.1 Barrier Circumvention .................................................................................................71.2.2 Barrier Navigation........................................................................................................9
1.3 Inositol Transporters as a Therapeutic Strategy................................................................131.3.1 Inositol in Health and Disease....................................................................................131.3.2 scyllo-Inositol as a Therapeutic for Alzheimer’s Disease..........................................161.3.3 The Inositol Stereoisomers.........................................................................................231.3.4 Inositol in Nature........................................................................................................251.3.5 Inositol Synthesis and Degradation Pathways ...........................................................251.3.6 The Inositol Transporters ...........................................................................................281.3.7 Inositol Efflux ............................................................................................................341.3.8 Inositol Pools..............................................................................................................35
CHAPTER 2 ...............................................................................................................................36Rationale, Hypothesis and Objectives2.1 Rationale ...........................................................................................................................372.2 Hypothesis.........................................................................................................................372.3 Objectives..........................................................................................................................38
CHAPTER 3 ...............................................................................................................................39Materials and Methods.............................................................................................................39
CHAPTER 4 ...............................................................................................................................53myo- and scyllo-Inositol Levels and Equilibrium in the BrainAbstract ....................................................................................................................................54Introduction ..............................................................................................................................55Results ......................................................................................................................................58Discussion ................................................................................................................................77
CHAPTER 5 ...............................................................................................................................81Quantification of Inositol Transporter Expression LevelsAbstract ....................................................................................................................................82Introduction ..............................................................................................................................83Results ......................................................................................................................................86Discussion ..............................................................................................................................100
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CHAPTER 6 .............................................................................................................................104Substrate Structural Requirements for Inositol TransportAbstract ..................................................................................................................................105Introduction ............................................................................................................................106Results ....................................................................................................................................110Discussion ..............................................................................................................................141
CHAPTER 7 .............................................................................................................................145Discussion, Conclusions and Future DirectionsDiscussion ..............................................................................................................................146Conclusions ............................................................................................................................162Future Directions....................................................................................................................163
REFERENCES.........................................................................................................................168
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LIST OF TABLES
Table 3.1 Mouse QPCR primers. ...............................................................................................45
Table 3.2 Human QPCR primers................................................................................................45
Table 6.1 Comparison of the protein homology of the three inositol transporters to each other in humans, mice and rats. ...........................................................................................................117
Table 6.2 Analysis of the SMIT1 transport model based on the structure of the scyllo-inositol derivatizes that were transported or not. ....................................................................................139
LIST OF FIGURES
Figure 1.1 The inositol stereoisomers. .......................................................................................24
Figure 1.2 Endogenous sources of scyllo-inositol......................................................................27
Figure 4.1 Derivatization............................................................................................................59
Figure 4.2 Gas chromatography method development. .............................................................61
Figure 4.3 Selected ion recording. .............................................................................................63
Figure 4.4 Selected ion recording for plasma samples...............................................................64
Figure 4.5 Examples of myo-inositol and scyllo-inositol concentration curves.........................66
Figure 4.6 Baseline brain myo- and scyllo-inositol levels..........................................................67
Figure 4.7 The effects of myo-inositol treatment on brain myo- and scyllo-inositol. ................69
Figure 4.8 Brain and CSF myo- and scyllo-inositol levels following scyllo-inositol treatment.71
Figure 4.9 Plasma myo- and scyllo-inositol concentrations following scyllo-inositol treatment.......................................................................................................................................................74
Figure 4.10 scyllo-Inositol incorporation into phosphatidylinositol. .........................................76
Figure 5.1 Concentration and dissociation curves for the inositol transporters primers............87
Figure 5.2 Microarray, concentration and dissociation curves for each of the control genes....89
Figure 5.3 HMIT expression as a function of age in TgCRND8 mice and their wild-type littermates. ....................................................................................................................................91
Figure 5.4 SMIT1 expression as a function of age in TgCRND8 mice and their wild-type littermates. ....................................................................................................................................92
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Figure 5.5 SMIT2 expression as a function of age in TgCRND8 mice and their wild-type littermates. ....................................................................................................................................93
Figure 5.6 Relative mRNA expression of the three inositol transporters in the brain. ..............95
Figure 5.7 A comparison of regional expression for each of the inositol transporters. .............97
Figure 5.8 Kidney inositol transporter expression. ....................................................................99
Figure 6.1 Structures of the inositol stereoisomers, derivatives and related compounds. .......109
Figure 6.2 Interspecies protein alignment for HMIT. ..............................................................111
Figure 6.3 Interspecies protein alignment for SMIT1..............................................................113
Figure 6.4 Interspecies protein alignment for SMIT2..............................................................115
Figure 6.5 HMIT transport activation. .....................................................................................119
Figure 6.6 Measurement of HMIT myo-inositol transport in primary cells.............................121
Figure 6.7 QPCR quantification of inositol transporter expression levels in HEK293 cells. ..122
Figure 6.8 myo-Inositol-(2-3H) and scyllo-inositol-(2-3H) transport in HEK 293 cells. ..........123
Figure 6.9 Basic structural model for SMIT1 transport. ..........................................................125
Figure 6.10 Basic structural model for SMIT2 transport. ........................................................126
Figure 6.11 Substrates not transported by SMIT1. ..................................................................128
Figure 6.12 Substrates not transported by SMIT2. ..................................................................129
Figure 6.13 myo-Inositol and scyllo-inositol transport kinetics in HEK293 cells. ..................131
Figure 6.14 A comparison of D- and L-chiro-inositol transport in HEK293 cells. .................134
Figure 6.15 L-fucose-(5,6-3H) transport. .................................................................................136
Figure 6.16 Transport of scyllo-inositol derivatives via SMIT1. .............................................138
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LIST OF ABBREVIATIONS
AD Alzheimer’s disease
BBB blood-brain barrier
CSF cerebral spinal fluid
CNS central nervous system
Gapdh glyceraldehyde-3-phosphate dehydrogenase
GC gas chromatography
GC/MS gas chromatography/mass spectrometry
HEK293 human endothelial kidney cell-line
HMIT hydrogen myo-inositol transporter
MRS magnetic resonance spectroscopy
PBS phosphate buffered saline
PCR polymerase chain reaction
QPCR quantitative polymerase chain reaction
SMIT1 sodium myo-inositol transporter 1
SMIT2 sodium myo-inositol transporter 2
Tbp TATA box binding protein
1
CHAPTER 1
Introduction
Inositol Transporters and Drug Development
Portions of this section have been previously published in:
Fenili D, Ma K, McLaurin J (2010) scyllo-Inositol, a Potential Therapeutic for Alzheimer’s Disease. Emerging Drugs & Targets for AD. Royal Society of Chemistry. Cambridge, UK
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Characterization of inositol transporters as a method for drug delivery to the CNS
For diseases of the central nervous system (CNS), the primary challenge for drug design is
frequently not creating drugs to target and treat the disease, but the successful transportation of
drug candidates into the brain. While certain drugs can be administered via intracerebral
implantation of pumps or through the direct injection of drugs into the brain or the cerebral
spinal fluid (CSF), these methods of administration are very invasive and concerns arise over
how often the drugs need to be administered, how well they perfuse to areas of disease
pathology, whether they reach therapeutically relevant concentrations in those areas and whether
the method of administration will cause significant inflammation and/or cell death. Whenever
possible, the preferred method of drug delivery to the CNS is oral, especially if treatment of the
disease is projected to be long-term. One reason why drug delivery to the CNS is so challenging
is because of the presence of the blood-brain and blood-CSF barriers.
1.1 Brain Barriers
1.1.1 The blood-brain barrier
The blood-brain barrier (BBB) is a physiological barrier made up of endothelial cells and
support cells that divide the brain from the rest of the body, in terms of drug transport. The
presence of the BBB was first observed in the work of the 19th century bacteriologist Paul
Ehrlich, who noted that an intravenous injection of the dye coerulean-S into rats resulted in the
staining of all the rat’s tissues, except the brain and spinal cord (Ehrlich, 1885). Thirty years
later his student, Edwin Goldmann, performed the reverse experiment and found staining only in
the spinal cord and brain and correctly concluded that there must be a barrier dividing the two
regions (Goldmann, 1913). This and other experiments resulted in the term blood-brain barrier
first being coined by Lina Stern at a meeting of the Medical Society of Geneva on April 21st,
1921 (reviewed in Vein, 2008). From her additional work using dyes, Stern concluded that the
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BBB had three main purposes: 1. to protect the brain from any harmful substances present in the
blood stream, 2. to allow the transport of substances needed for its function and 3. to maintain
brain homeostasis (Stern, 1921). She proposed that the BBB was the rate-limiting factor
determining the permeation of therapeutic drugs into the brain (Stern, 1921).
In the 1960s, with the advent of the electron microscope and the use of horseradish peroxidase
to enzymatically amplify visual markers, it was determined that the key component of the BBB
is endothelial cells, joined together in a monolayer by tight junctions (Reese and Karnovsky,
1967). Surrounding these endothelial cells are astrocytic endfeet, which induce the endothelial
cells to form those tight junctions (Janzer and Raff, 1987). While the endothelial cells form
tight junctions, astrocytic endfeet form gap junctions (Brightman and Reese, 1969). Therefore
while astrocytic endfeet do surround the vasculature, they do not impose a physical barrier to
drug transport (Brightman and Reese, 1969). There is a ~20 nm gap between the astrocytic
endfeet that allows any compounds transported across endothelial cells to reach the brain and
vice versa (Brightman and Reese, 1969). Pericytes also interact with and stabilize endothelial
cells, both by surrounding the endothelial cells with processes to contribute to its mechanical
stability and by forming specialized junctions with the endothelial cells, through which they
influence their differentiation or quiescence (reviewed in von Tell et al, 2006). In addition,
pericytes also appear to influence tight junction formation (Hori et al, 2004). Pericytes express
angiopoetin, which induces endothelial cells to express occludin, a major component of tight
junctions (Hori et al, 2004). The extracellular matrix of the basal lamina, alternatively known as
the basement membrane, also acts to stabilize vascular structure, via the interaction of matrix
proteins such as laminin with integrin receptors on endothelial cells (reviewed in Hynes, 1992).
In addition, the expression of matrix proteins such as type IV collagen, fibronectin and laminin,
in the basal lamina also appears to influence tight junction expression (Tilling et al, 1998).
4
Completing this neurovascular unit, composed of endothelial cells, astrocytic endfeet, pericytes
and the basal lamina are neurons that, via neuronal processes that innervate vascular endothelial
cells and astrocytes, regulate cerebral blood flow and BBB permeability (reviewed in Hawkins
and Davis, 2005).
Since the endothelial cells are responsible for providing a physical barrier to drug transport, they
are of particular interest in drug design. They are polarized cells that appear to express certain
transporters on either the luminal (blood) or abluminal (brain) side of their plasma membrane.
This was first noted by Betz and Goldstein (1978), who examined the transport of 14C-labelled
α–(methylamino)isobutyric acid and L-leucine in cerebral cortex capillaries isolated from adult
rats. Using these two amino acids, which are transported by two different neutral amino acid
transporter systems, they observed that the sodium-independent L system transporter was
present on the luminal side, while the sodium-dependent A system transporter was present on
the abluminal side of brain capillary endothelial cells (Betz and Goldstein, 1978). Therefore,
large neutral amino acids, such as phenylalanine, leucine, tryptophan and methionine can be
transported into the brain via the L system transporter, while small neutral amino acids, such as
glycine, alanine, serine, proline and α–(methylamino)isobutyric acid can be transported out of
the brain by the A system transporter. Glucose transporter 1, the only glucose transporter
expressed at the BBB, is polarized in a 4:1 expression ratio between the abluminal and luminal
membranes of endothelial cells (Farrell and Pardridge, 1991). Drug efflux transporters, such as
P-glycoprotein (Thiebaut et al, 1989), are also polar; they are present on the luminal side of
endothelial cells and are responsible for preventing the transport of toxic substances from the
blood into the brain. This transport across concentration gradients requires high levels of
5
energy, as evidenced by the fact that endothelial cells at the BBB contain five to six times as
many mitochondria compared to endothelial cells elsewhere (Oldendorf et al, 1977). In order
for a drug to be transported into the brain from the blood, it must navigate this highly selective
brain barrier.
1.1.2 The Blood-CSF Barrier
In addition to the BBB, a second brain barrier exists, the blood-CSF barrier, through which
drugs in the blood can be transported into the CSF. Lina Stern, along with a colleague of hers,
Constantin von Monakow, both concluded that the brain was supplied with nutrients by both the
blood and the CSF and that the choroid plexus was responsible for functioning as a barrier,
separating the blood from the CSF (Stern, 1921; Monakow, 1921). There are four choroid
plexuses in humans, located in each of the ventricles: one in each of the lateral ventricles, one in
the third and one in the fourth ventricle. The choroid plexus is composed of a network of
capillaries, surrounded by interstitial fluid, separating those capillaries from a layer of epithelial
cells, which act as a barrier to regulate entry into the CSF (reviewed in Johanson et al, 2005). In
contrast to the structure of the BBB, the choroidal capillaries contain gap junctions (Dermietzel
and Schunke, 1975), which allow the unimpeded movement of nutrients from the blood into the
neighbouring interstitial fluid space (Johanson et al, 2005). Like the endothelial cells of the
BBB, the choroid plexus epithelial cells are also connected together by tight junctions, thus
regulating the transport of compounds from the blood into the CSF (Brightman and Reese,
1969). Also like the BBB endothelial cells, the choroid plexus epithelial cells are polarized with
different transporter expression between their apical and basolateral membranes. For example,
the sodium myo-inositol transporter, SMIT1, is located only on the basolateral side of these cells
(Hakvoort et al, 1998).
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Originally, the choroid plexus was thought to be less important for drug transport than the BBB
because of the apparent size differential of the two structures (Pardridge et al, 1981). Now it has
become increasingly apparent that these two barriers actually offer similar opportunities for drug
transport, because of a number of anatomical features found in the choroid plexus. Research has
shown that vascular perfusion into the choroid plexus is five to ten times higher than cerebral
blood flow because of an increased number of capillaries in this region, thus resulting in an
enhanced opportunity for nutrients to enter the brain via this barrier system (Johanson et al,
2005). In addition, unlike brain microvessels, which contain tight junctions to prevent nutrient
transport, the choroidal capillary endothelium contains gap junctions (Dermietzel and Schunke,
1975), which allows for the ready movement of compounds into the interstitial fluid space
(Johanson et al, 2005). Furthermore, the epithelial cells that form the physical barrier between
the blood and the CSF contain interdigitations on their basolateral membrane and a carpet of
microvilli on their apical surface (Keep and Jones, 1990). Therefore, there is an increased
opportunity for compounds to cross into the epithelial cells and an increased surface area on the
apical membrane through which they can exit into the CSF (Keep and Jones, 1990). In the adult
rat, the apical membrane surface area is 75 cm2, compared to 155 cm2 for cerebral capillaries
(Keep and Jones, 1990). The basolateral membrane surface area of the choroidal epithelial cells
is 25 cm2, indicating the degree of extension offered by the microvilli, in comparison to the
interdigitations (Keep and Jones, 1990). All these structural features make both the blood-brain
and blood-CSF barriers, potentially important barriers for drug transport design.
1.2 Strategies for transport of drugs across brain barriers
In animal research, one common way to avoid the brain barriers when delivering drug
candidates to the brain is through intracerebroventricular injection. This is an appealing strategy
because the ependymal cells that line the ventricles contain gap junctions, which are permeable
7
to even macromolecules (discussed in Johanson et al, 2005). However, while this is an effective
short-term solution, this is not an ideal method for drug delivery in humans. Therefore
researchers have been searching for alternative solutions for the transport of drugs into the brain.
Some methods that have been/are being examined target circumvention of the barrier through
the use of techniques, such as intranasal delivery, osmotic and biochemical disruption as well as
barrier navigation strategies, such as designing drugs that will diffuse across the barriers or by
aiding transport through the use of nanoparticle technology and Trojan horses.
1.2.1 Barrier Circumvention
1.2.1.1 Intranasal delivery
Intranasal delivery of drugs has been suggested as one method of circumventing the brain
barriers (reviewed in Hanson and Frey, 2008). First developed by Frey in 1989 for the
administration of neurotrophic factors, such as nerve growth factor (27.5 kDa), to the CNS, it is
now being tested in other drug delivery applications (Hanson and Frey, 2008). Using this
technique, drugs are aerosolized in the nasal cavity and reach the CNS by flowing along the
olfactory and trigeminal neural pathways to reach the brain within minutes. By bypassing the
periphery and the brain barriers, a higher concentration of the drug is able to reach the brain and
the risk of unrelated, systemic effects is reduced (Hanson and Frey, 2008). For antiepileptic
medications, used to stop seizures, intranasal delivery of drugs offers an attractive alternative to
intravenous drug delivery, allowing for faster, more convenient administration outside of a
hospital setting, thereby reducing the duration of the seizures and the risk of brain damage
(Wermeling, 2009).
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1.2.1.2 Osmotic disruption
The ability of substances, such as urea, to alter osmolality and disrupt BBB integrity was first
noted by Rapoport (1970) in cats injected with sodium bicarbonate with or without one of four
solutions: sodium azide, sodium chloride, urea and ethanol. The rate of sodium bicarbonate
transport correlated with each substances ability to alter osmolality. He hypothesized that these
changes in osmolality might cause vascular endothelial cells to shrink and result in the
formation of gaps between the cells, allowing for the passage of the sodium bicarbonate. The
reversibility of this procedure was confirmed in a later experiment in rabbits, injected with an
Evans blue-albumin dye complex, and non-lipid soluble substances were concluded to be the
most effective at reversibly opening the barrier (Rapoport et al, 1971). Next, the ability of
animals to survive a temporary osmotic disruption in their BBB was studied in monkeys
administered 2 M of urea through their left common carotid artery, followed by an intravenous
injection of Evans blue dye (Rapoport et al, 1972). Monkeys were able to survive the procedure
(Rapoport et al, 1972) and following adjustments in their urea administration procedure to avoid
compromising blood supply to the brain, no gross neurological deficits were observed (Rapoport
and Thompson, 1973). Cresyl violet staining of the monkey brains, sacrificed 5 to 10 days
following the procedure, showed no evidence of brain necrosis (Rapoport and Thompson,
1973). Electroencephalogram recordings of the brain, showed a temporary decrease in
amplitude one day following the procedure, which disappeared by the time the animals were
sacrificed (Rapoport and Thompson, 1973). Therefore, following additional adjustments, this
procedure could be used to safely administer drugs to the CNS.
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1.2.1.3 Biochemical disruption
Another method for opening brain barriers is through biochemical disruption. One strategy is to
use the kallikrein-kinin system, activated in ischemic stroke, to cause BBB disruption and brain
edema, to reversibly open brain barriers (Wahl et al, 1983). For example, a low dose of the
bradykinin analog, RMP-7 (0.1 µgram/kg/min) injected into the carotid artery in rats, selectively
increased dextran (40 kD) transport 10-fold into RG2 glial tumors, when compared to the
vehicle control group (Inamura et al, 1994). In contrast, transport into normal brain capillaries
was unaltered by this low dose, which is important to limit the exposure of normal brain tissue
to antitumor drugs (Inamura et al, 1994). In rats, RMP-7 pretreatment increased the uptake of
the antitumor drug, carboplatin (371.3 daltons) into brain tumors (Elliot et al, 1996).
Barrier circumvention strategies offer viable options for drug entry into the brain. However,
strategies such as osmotic and biochemical disruption open the brain barriers indiscriminately,
not only to entry of the desired drugs but also to potential toxins or bacteria and viruses,
therefore, more selective methods of transferring drugs into the brain might be more
advantageous.
1.2.2 Barrier Navigation
Barrier navigation strategies aim to transport drugs through the brain barriers, rather than by
disrupting those barriers. There is an estimated 600 meters of capillaries in the brain, which are
6-10 µm in diameter, creating a large surface area through which transport can occur (Begley
and Brightman, 2003). If the brain barriers are disrupted, this creates a large surface area
through which transport of undesired compounds can occur. By using barrier navigation
strategies, such as diffusion, nanoparticle or Trojan horse delivery methods, this risk can be
removed.
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1.2.2.1 Diffusion
Certain properties will influence whether a compound can diffuse across the BBB, including the
oil/water partition ratio (Mayer et al, 1959), dissociation constant (Rall et al, 1959) and weight
of the compound (discussed in Banks, 2009). When Mayer and colleagues (1959) examined the
ability of different drugs to diffuse into the rabbit brain after injection through the femoral vein,
they found diffusion into the brain was influenced by the oil/water distribution ratio of the
compound at pH 7.4. Compounds such as thiopental and aniline showed the fastest diffusion
rates, reaching equilibrium between CSF and plasma within 5 minutes, while N-acetyl-4-
aminoantipyrine and salicylic acid were the slowest, failing to reach equilibrium within the 3
hour experiment window, but theoretically calculated to reach equilibrium within 16 and 32
hours, respectively (Mayer et al, 1959). The dissociation constant of a compound (pKa),
described as the pH at which 50% of the compound is ionized, also influences whether a
compound will cross the BBB. Compounds are more likely to cross into the brain if they are
predominantly in unionized forms at a physiological pH (Rall et al, 1959). However, when
Brodie and colleagues (1960) compared the diffusion of 17 compounds, differing in their lipid
solubility and their degree of ionization at physiological pH, they found that the oil/water
partition coefficient of a compound, rather than the dissociation constant, was a stronger
determinant of whether a compound would diffuse across the BBB. The dissociation constant
was important for determining what level of the compound would be in an unionized form in the
plasma, but lipid solubility ultimately determined whether the compound would cross into the
brain in any form (Brodie et al, 1960). Perhaps the most important factor determining whether a
compound will cross into the brain is weight. Generally, compounds must be less then 400-600
Daltons to be transported into the brain, however this is not a set rule (Banks, 2009). The
largest compound reported to cross the BBB via transmembrane diffusion was cytokine-induced
11
neutrophil chemoattractant-1 (CINC-1), weighing 7,800 Daltons (Pan and Kastin, 2001; Banks,
2009). Designing drugs to conform to these three variables to transmembrane diffusion is one
way to induce drug transport into the brain. However, this is not always a feasible, therefore
other methods need to be considered.
1.2.2.2 Nanoparticle Technology
Nanoparticle delivery is another mechanism under development for drug transport into the
brain. Nanoparticles are macromolecule assemblies that range in size from 1 to 1000 nm.
Drugs are made to interact with these assemblies in one of three ways: they can either be 1.
entrapped inside the particles, 2. associated with the exterior surface of the particles or 3.
homogeneously dispersed throughout (reviewed in Lockman et al, 2002). There are several
potential advantages of using nanoparticle technology. Through the use of surface ligands it is
theoretically possible to directly deliver drugs to a desired tissue, removing the concern of
systemic drug effects and decreasing the efficacy dose by minimizing non-specific binding
(reviewed in Provenzale and Silva, 2009). In addition, the temporal release of nanoparticles can
theoretically be controlled through the use of light- or heat-labile liposomes (reviewed in
Gazeau et al, 2008; Huang et al, 2008). These two strategies are very appealing for diseases
such as brain tumors, where antitumor drugs are very toxic and directed delivery of the drug and
timing its release has the potential to greatly reduce side effects. The nanoparticle,
polybutylcyanoacrylate, coated with polysorbate-80 to prolong its circulation by inhibiting
scavenging by cells of the reticuloendothelial system, is an example of a nanoparticle that has
shown promise as a drug delivery system to the brain. An intraperitoneal injection of nerve
growth factor, normally unable to cross into the brain, absorbed onto the surface of polysorbate-
12
80 coated polybutylcyanoacrylate nanopoarticles was able to reduce parkinsonian symptoms in
mice, in which a parkinsonian syndrome was chemically induced through an injection of 1-
methyl-4-phenyl-1,2,3,6-tetrahydrophyrindine (Kurakhmaeva et al, 2008).
1.2.2.3 Trojan Horse
A third option for barrier navigation is to conjugate the drug onto another compound that has
access to the brain. Boado and colleagues (2009) describe molecular Trojan horses as
endogenous peptides or peptidomimetic monoclonal antibodies that target endogenous brain
barrier receptor-mediated transport systems. This technique was first developed in vivo by
Trowbridge and Domingo (1981) for the direct delivery of antitumor drugs to human melanoma
cells in nude mice, in an effort to reduce undesired systemic effects. In this study, diphtheria
fragment A from diphtheria toxin was conjugated to monoclonal antibodies against the
transferrin receptor. Tranferrin receptors are preferentially expressed by actively proliferating
tissues, such as tumors, which require higher amounts of iron (Gatter et al, 1983), thus allowing
for directed drug delivery. Transferrin monoclonal antibodies were recently used to transfer
therapeutic single chain Fv antibodies, normally unable to cross the BBB, into the mouse brain
in a proof-of-principle experiment (Boado et al, 2009). By adapting a transport system found
naturally at the brain barriers, into a drug shuttling system, the authors made it possible to
convert a large molecule drug, normally unable to cross into the brain, into a viable CNS drug.
Therefore, barrier navigation strategies, such as diffusion, nanoparticle and Trojan horse drug
delivery are all potentially effective methods for delivering compounds to the brain. In contrast
to barrier circumvention strategies, these techniques try to work with the natural properties of
the BBB to deliver drugs from the blood into the brain.
13
1.3 Inositol Transporters as a Therapeutic Strategy
Receptors, such as the transferrin receptor, appear to be an effective shuttling system for the
delivery of molecules to the CNS (Boado et al, 2009). This is an example of a receptor-
mediated transport system. Another potential option for drug transport is through a carrier-
mediated transport system. An example of carrier-mediate transport is the inositol transporters
(Hager et al, 1995; Uldry et al, 2001; Coady et al, 2002; Bourgeois et al, 2005). Inositol
stereoisomers, such as myo- and scyllo-inositol are transported from the blood into the brain
using these transporters (Spector, 1988; Wiese et al, 1996; Hakvoort et al, 1998; Berry et al,
2003). The inositol stereoisomers have shown promise as therapeutic drugs in a number of CNS
diseases, indicating the activity of this transport system (Benjamin et al, 1995; Levine et al,
1995; Fux et al, 1996; Einat et al, 1998; Bersudsky et al, 1999; Chengappa et al, 2000; Gelber et
al, 2001; Palatnik et al, 2001; Eden et al, 2006; McLaurin et al, 2006). It might be possible to
conjugate other compounds to the inositols to facilitate their transport into the brain or to alter
the inositol stereoisomers into novel drug therapeutics.
1.3.1 Inositol in Health and Disease
Over the past 20+ years there has been interest in the ability of inositol stereoisomers to act as
therapeutic drugs in the treatment of psychiatric disorders, such as depression (Levine et al,
1995), bipolar/affective disorder (Chengappa et al, 2000; Eden et al, 2006), obsessive-
compulsive disorder (Fux et al, 1996), eating disorders (Gelber et al, 2001), panic disorder
(Benjamin et al, 1995; Palatnik et al, 2001) and anxiety (Einat et al, 1998; Bersudsky et al,
1999), as well as for the treatment of respiratory distress syndrome in premature infants
(Hallman et al, 1986; 1992), for preventing neural tube defects (Cogram et al, 2002) and to
increase insulin sensitivity in polycystic ovary syndrome (Nestler et al, 1999; Gerli et al, 2003).
In 1978, Barkai and colleagues first noted a reduction in the levels of myo-inositol in the CSF of
14
people with affective disorder. This led to research into the levels of myo-inositol in psychiatric
and other disorders and to the examination of inositol stereoisomers as potential therapeutic
agents. A double-blind study, comparing 12 g/day of myo-inositol to a placebo, showed a
significant improvement in Hamilton Depression Rating Scale scores in myo-inositol-treated
patients, compared to placebo-treated patients, by the end of the four week study (Levine et al,
1995). When patients with bipolar depression were administered either 12 g/day of myo-inositol
or a placebo for six weeks, 50% of the inositol-treated group showed a significant decrease in
depressive symptoms, as measured using the Hamilton Depression Rating Scale and a Clinical
Global Improvement scale, compared to 30% of placebo controls (Chengappa et al, 2000).
Using the Montgomery-Asberg Depression Rating Scale as an outcome measure, 67% of
inositol-treated patients showed a significant decrease in depressive symptoms, compared to
33% of placebo controls (Chengappa et al, 2000). In patients with obsessive-compulsive
disorder, six weeks of treatment with 18 g/day of myo-inositol resulted in a significant decrease
in obsessive-compulsive behaviour compared to the placebo treatment group in a double-blind,
controlled crossover study (Fux et al, 1996). Similarly, in patients with bulimia nervosa and
binge eating, six weeks of 18 g/day myo-inositol treatment resulted in significant improvements
on the Global Clinical Impression Scale, the Visual Analogue Scale and the Eating Disorders
Inventory, when compared to a placebo treatment group in a double-blind crossover trial
(Gelber et al, 2001). For panic disorder, 12 g/ day myo-inositol treatment was significantly
more effective than placebo at reducing the frequency and severity of panic attacks following
four weeks of treatment (Benjamin et al, 1995). In a double-blind, controlled, crossover study
comparing one month of 18 g/day myo-inositol to 150 mg/day fluvoxamine treatment, which is
an accepted drug for the treatment of panic disorder, myo-inositol was significantly more
effective at reducing the frequency of panic attacks, and patients reported less nausea or
tiredness, when compared to the fluvoxamine-treated group (Palatnik et al, 2001).
15
In addition to acting on psychiatric disorders, myo-inositol supplementation has been proven to
significantly decrease the rates of bronchopulmonary dysplasia and retinopathy of prematurity,
in premature infants suffering from respiratory distress syndrome (Hallman et al, 1986; 1992).
Based on this research it has been determined that infants not receiving breast milk, who receive
parenteral nutrition instead, which is inositol-free, require myo-inositol supplementation to
significantly increase their chances of survival (Hallman et al, 1992).
A second inositol stereoisomer, epi-inositol, has shown promise in the treatment of anxiety
disorder (Einat et al, 1998; Bersudsky et al, 1999). When anxiety in rats was compared
following eleven daily intraperitoneal injectons of epi-inositol, myo-inositol or placebo on an
elevated plus-maze model of anxiety, both epi- and myo-inositol were significantly more
effective than placebo at reducing anxiety in the rats (Einat et al, 1998; Bersudsky et al, 1999).
epi-Inositol treatment resulted in a stronger reduction in anxiety levels than myo-inositol, a
finding that the authors suggested might result because epi-inositol metabolism may be slower,
have a different mechanism of action, and because it is not a substrate of phosphatidylinositol
synthase (Einat et al, 1998).
D-chiro-inositol has also been examined for disease treatment and it has shown promise at
preventing neural tube defects in folate-resistant mice (Cogram et al, 2002) and as an insulin-
sensitizing agent in women with polycystic ovary syndrome (Nestler et al, 1999; Gerli et al,
2003). Thirty percent of women who give birth to babies with neural tube defects are
insensitive to folic acid treatment in early pregnancy as a preventative measure. myo-Inositol
has shown some promise at reversing neural tube defects in the curly tail mouse model of folate-
resistant neural tube defects (Greene and Copp, 1997). However, D-chiro-inositol appears to be
more effective and in this same mouse model, D-chiro-inositol treatment resulted in a 73-86%
16
reduction in spina bifida rates, compared to a 53-56% reduction observed following myo-
inositol treatment (Cogram et al, 2002). D-chiro-inositol also appears to improve insulin
sensitivity, which is a concern in people with diabetes and those with polycystic ovary
syndrome (Nestler et al, 1999). A 1200 mg per day dose of D-chiro-inositol for six to eight
weeks, resulted in a significant improvement in insulin sensitivity, ovulatory function, blood
pressure, androgen levels, and plasma triglycerides compared to the placebo group (Nestler et al,
1999). A randomized, double-blind study, comparing patients receiving 100 mg of D-chiro-
inositol twice daily to those receiving a placebo, still resulted in a significant improvement in
ovarian function and a significant increase in weight loss, in the D-chiro-inositol treatment
group, despite the reduction in D-chiro-inositol concentration, highlighting the sensitivity of this
treatment and the low concern for side effects (Gerli et al, 2003).
1.3.2 scyllo-Inositol as a Therapeutic for Alzheimer’s Disease
Alzheimer’s disease (AD) is the most common form of dementia and affects 12 million people
worldwide (Citron, 2001). Clinically, the patient will exhibit memory impairments and can also
display a loss of language function, sensory perception, exectutive function, as well as agitation,
aggression, delusions, hallucinations and repetitive vocalizations (Sink et al, 2005; Chertkow,
2008).
Neuropathologically, AD is characterized by the appearance of extracellular Aβ deposits, as
either diffuse or neuritic plaques, as well as intraneuronal neurofibrillary tangles of the
hyperphosphorylated tau protein (Selkoe, 2001). Associated with Aβ neuritic plaques are
distrophic neurites, activated microglia and reactive astrocytes, therefore plaques were initially
found to be the more toxic form of aggregated Aβ (Selkoe, 2001). However, soluble Aβ has
17
now been shown to correlate better with AD severity (McLean et al, 1999) and soluble Aβ
oligomers have been proven to be toxic to neurons in culture (Selkoe, 2002). Therefore, the
majority of AD drug design has been focused on targeting either Aβ formation, aggregation,
deposition and/or clearance, based on the hypothesis that limiting the levels of Aβ oligomers
and/or fibrils will slow down disease progression and reduce cognitive and behavioural deficits
in patients.
One potential disease therapeutic for AD is scyllo-inositol, which appears to bind and stabilize
Aβ (McLaurin et al, 1998), thereby inhibiting Aβ oligomerization and fibrillogenesis. The
discovery of scyllo-inositol as a possible treatment for AD started with the investigation of Aβ-
lipid interactions, as a mechanism for the promotion of fibril formation and as a possible
mechanism for Aβ-mediated toxicity (McLaurin and Chakrabartty, 1996; 1997; Choo-Smith and
Surewicz, 1997; McLaurin et al, 1998; Kremer et al, 2000; Yip et al, 2001; Curtain et al, 2003).
Multiple lines of evidence suggest that one of the central events in the pathogenesis of AD is the
accumulation of neurotoxic oligomeric/protofibrillar aggregates of Aβ (McLean et al, 1999;
Sinha, 2002). Acidic phospholipids were shown to induce a structural transition in Aβ40 and
Aβ42 to their more toxic β-form, with a concomitant disruption of the bilayer (McLaurin and
Chakrabartty, 1996; 1997). The most potent phospholipid at causing this transition, was
phosphatidylinositol, therefore components of phosphatidylinositol, i.e. the headgroup,
phosphorylation state and fatty acyl chains were examined to determine the crucial element(s)
for β-structure induction (McLaurin et al, 1998). myo-Inositol, the headgroup of
phosphatidylinositol, mediated a transition to β-structure for Aβ42, while the addition of a single
phosphate group abolished this transition (McLaurin et al, 1998). This suggested that myo-
inositol was responsible for inducing Aβ42 β-structural transition. The effect of myo-inositol
18
on this conformational change within Aβ42 was an immediate, not time-dependent, effect.
Interestingly, even though myo-inositol induced a β-structure formation, it did not induce
fibrillization but maintained Aβ42 in a soluble form (McLaurin et al, 1998). Negative stain
electron microscopy showed Aβ42 in buffer alone formed short thin fibrils whereas, no fibrils
were observed in the presence of myo-inositol (McLaurin et al, 1998). Aβ42 - myo-inositol
interactions suggested that even though β-structure is required for fibril formation, Aβ is also
able to form stable non-fibrillar β-structures (McLaurin et al, 1998).
Since myo-inositol induced stable micelles of Aβ42, the effect of other inositol stereoisomers on
Aβ42 aggregation was similarly investigated (McLaurin et al, 2000). epi-Inositol, scyllo-
inositol and chiro-inositol were examined as potential inhibitors of fibrillogenesis. These
stereoisomers differ in the position of their hydroxyl groups; myo-inositol has five equatorial
hydroxyl groups and one axial hydroxyl group. Compared to myo-inositol, epi-inositol and D-
chiro-inositol have one or two extra hydroxyls in the axial position, respectively. scyllo-
Inositol, on the other hand, has all its hydroxyl groups in the equatorial position. epi-Inositol
and scyllo-inositol but not chiro-inositol induced a random to β-structure transition in Aβ42,
without formation of fibrils (McLaurin et al, 2000). Aβ42 incubated with chiro-inositol formed
fibrils indistinguishable from Aβ42 in buffer alone, displaying an inactive isomer (McLaurin et
al, 2000).
Analysis of Aβ42-inositol conformers in culture, found both PC-12 cells and primary neuronal
cultures protected from any Aβ-mediated neuronal toxicity and death (McLaurin et al, 2000).
Normally, Aβ42 accumulates on the cell surface of cells. Aβ42 in the presence of myo-, scyllo-,
and epi-inositol resulted in decreased cell surface Aβ accumulation, while incubation with
chiro-inositol resulted in no change in the amount of Aβ accumulation (McLaurin et al, 2000).
19
Therefore, the ability of myo-, scyllo-, and epi-inositol to decrease Aβ accumulation on neuronal
membranes offers a possible mechanism for the attenuation of Aβ-induced neurotoxicity.
In vitro studies showed that inositol stereoisomers stabilize Aβ conformers, inhibit Aβ fibril
assembly, accelerate disassembly of preformed fibrils, and protect primary cultured neurons
from Aβ-induced toxicity (McLaurin et al, 1998; 2000). The Aβ conformers stabilized by
inositol were small β-structured spherical micelles that were non-toxic in vitro. These
compounds exhibited stereoisomer-specific differences in their ability to inhibit Aβ aggregation
and cytotoxicity. Aβ aggregation and toxicity were more efficiently inhibited by scyllo-inositol
than by myo-inositol (McLaurin et al, 2000).
To determine the importance of this structure-function relationship, a series of scyllo-inositol
derivatives were synthesized in which one or two hydroxyl groups were replaced with fluoro,
chloro, methoxy or hydrogen substituents. This approach has been previously demonstrated to
be an effective method to garner information about hydrogen bonding requirements of a given
hydroxyl group in the carbohydrate binding sites of lectins or antibodies (Glaudemans, 1991;
Auzanneau et al, 1993; Swaminathan et al, 1997). The hydroxyl groups at positions C-1 and C-
4 were modified in light of evidence that vicinal diols at positions 1, 3, 4 and 6 are recognized
by the epimerases that interconvert inositol stereoisomers (Hipps et al, 1977; Pak et al, 1992).
Therefore, replacement of epimerase-targeted hydroxyl groups at positions 1 and 4 with stable
substituents was hypothesized to increase in vivo compound stability. These derivatives showed
maintenance of Aβ activity with some substitutions and enhanced in vivo stability with respect
to epimerization. The data on the effects of these compounds on Aβ-aggregation suggest that
20
only the most conservative single hydroxyl substitutions are tolerated, thus 1-deoxy-1-fluoro-
scyllo-inositol behaved similarly to, but not as well as, the parent compound (Sun et al, 2008).
The potency of various inositol stereoisomers in vivo was investigated in a transgenic model of
AD, the TgCRND8 mouse (McLaurin et al, 2006). TgCRND8 mice express a human amyloid
precursor protein transgene (APP695) bearing two missense mutations that cause AD in humans
(KM670/671NL and V717F). At about three months of age, the TgCRND8 mice display
progressive spatial learning deficits that are accompanied both by rising cerebral Aβ levels and
by increasing numbers of cerebral amyloid plaques (Chishti et al, 2001). By six months of age,
the levels of Aβ and the morphology, density and distribution of the amyloid plaques in the
brain of TgCRND8 mice are similar to those seen in the brains of humans with well-established
AD (Wang et al, 1999; Näslund et al, 2000; Li et al, 2004). As observed in patients with AD,
the biochemical, behavioural and neuropathological features of this mouse are accompanied by
accelerated mortality (Wang et al, 1999; Näslund et al, 2000; Li et al, 2004).
myo-Inositol had no beneficial effects, while epi-inositol had early effects that were not
sustained with disease progression in this mouse model (McLaurin et al, 2006). In contrast,
scyllo-inositol treatment of TgCRND8 mice increased the survival of treated mice from 42% to
72% at 6 months of age (p=0.02). This increase in survival was accompanied by the rescue of
cognitive deficits observed using the Morris Water Maze test for spatial memory (McLaurin et
al, 2006). scyllo-Inositol treatment decreased total Aβ40 (p<0.001) and Aβ42 (p<0.05) levels
and a 25% reduction in Aβ42 concentrations was maintained over 6 months (p<0.05). Plaque
load was uniformly decreased by 35% across the entire brain indicating that inositol action was
not region specific (p<0.05). A similar reduction was seen in the percent brain area covered by
21
vascular amyloid and in the size of cerebrovascular Aβ deposits. This decrease in deposited Aβ
was due to scyllo-inositol-induced alterations of Aβ species in treated transgenic mice.
Prophylactic treatment reduced soluble Aβ oligomers of mass greater than 40kDa by 40% at
both 4 and 6 months of age. scyllo-Inositol treated 4-month-old TgCRND8 mice showed a
significant decrease in high-molecular weight Aβ oligomers and an increase in trimeric and
monomeric Aβ species.
This cognitive benefit was reflected in scyllo-inositol’s reduction of synaptic toxicity in
transgenic animals, as evidenced by a 148% increase in synatophysin immunoreactive boutons
and cell bodies by 6 months of age (McLaurin et al, 2006). Improvements in
neuroinflammatory status were marked by a reduction in astrogliosis and microgliosis
(McLaurin et al, 2006). Both synaptic and inflammatory changes likely resulted from scyllo-
inositol blockage of Aβ oligomer-induced toxicity.
Aβ oligomer-induced inhibition of long-term potentiation (LTP) was studied using mouse
hippocampal slices and rescue of this phenotype was shown by pre-incubating these naturally
occurring oligomers with scyllo-inositol prior to perfusing brain slices (Townsend et al, 2006).
Application of scyllo-inositol after Aβ perfusion did not confer LTP protection. This effect is
due to the direct binding of scyllo-inositol to Aβ trimers and neutralization of toxicity. Taken
together, binding and neutralizing Aβ trimers could explain the increased amount of trimeric Aβ
species observed in scyllo-inositol treated TgCRND8 animals; stabilizing Aβ trimers would also
cause the decrease in high-molecular weight Aβ oligomers observed in the same animals. Co-
application of scyllo-inositol and Aβ oligomers also prevented oligomer-induced decreases in
dendritic spine density (Shankar et al, 2007). The protective effect of scyllo-inositol on synaptic
22
dysfunction may be partially the result of preventing oligomer-induced reductions in
phosphatidylinositol-4,5-bisphosphate levels (Berman et al, 2008).
These results demonstrate that selected inositols can significantly inhibit the development of
AD-like phenotype in TgCRND8 mice, when given prior to the onset of disease (McLaurin et
al, 2006). However, most AD patients will seek treatment only after their disease state is
significantly advanced - i.e. at a time when Aβ oligomerization, deposition, toxicity and plaque
formation are already well advanced. To assess whether scyllo-inositol could abrogate an
established disease state, treatment in TgCRND8 mice was delayed until five months of age
(McLaurin et al, 2006). At this age, TgCRND8 mice have significant behavioural deficits,
accompanied by significant Aβ peptide and plaque burdens (Chishti et al, 2001). A 28-day
course of scyllo-inositol treatment reduced brain levels of Aβ40 and Aβ42 (e.g. insoluble Aβ40
p<0.05; insoluble Aβ42 p<0.05) and significantly reduced plaque burden (p<0.05). The decrease
in plaque burden was accompanied by a decrease in soluble, high molecular weight oligomers.
These results were comparable in effect size to those observed in the prophylactic studies.
Spatial learning in these mice was significantly improved when compared to untreated
TgCRND8 mice (p<0.02). The cognitive performance of these scyllo-inositol-treated animals
was not significantly different from their non-transgenic littermates (p=0.11). This beneficial
effect of inositol treatment was not due to non-specific effects because scyllo-inositol had no
effect on the cognitive performance of non-transgenic mice (p=0.39).
In vivo studies in rats further confirmed the efficacy of scyllo-inositol in rescuing cognitive
deficits caused by Aβ. Pre-incubation of scyllo-inositol with Aβ oligomers, prior to
intracerebroventricular injection into rats improved delayed alternation and complex reference
23
memory, measured by the Lever Cyclic Ratio assay as switching and perseveration errors
(Townsend et al, 2006). Oral administration of scyllo-inositol via drinking water at least 3 days
prior to intracerebroventricular Aβ injection in rats also restored switching and perseveration
errors to baseline levels (Townsend et al, 2006). These combined results suggest that scyllo-
inositol is effective in multiple model systems of AD.
Overall studies of inositols in health and disease, suggest that inositol stereoisomers, especially
myo- and scyllo-inositol, demonstrate good CNS bioavailability. This suggests that if other
compounds are designed for transport via the inositol transporters, they might show similar
degrees of CNS bioavailability. In order to accomplish this, the inositol transporters and their
system of transport need to be further studied.
1.3.3 The Inositol Stereoisomers
myo-Inositol was first isolated in the mid-19th century from muscle extracts and was accordingly
named inosit, from the Greek root word inos for muscle (Scherer, 1850). Eight years later, a
second, related compound was isolated from the shark Scyllium canicula, the skates Raja batis
and Raja clavataI, and was given the name Scyllit (Staedeler and Frerichs, 1858). These
compounds were later renamed myo- and scyllo-inositol and identified as two of the nine
possible stereoisomers of inositol (Bouveault, 1894).
The inositol isomers belong to the class of compounds known as cyclitols, 5-7 carbon ring
compounds, containing at least 3 carbons with an attached hydroxyl group. Inositols are
cyclohexanehexols and members of the polyol family. The inositol isomers are 6 carbon ring
molecules, with a hydroxyl group attached to each carbon of the ring. The structure of scyllo-
inositol was first determined by Posternak in 1941 (Posternak, 1941; 1942), followed by the
structure of myo-inositol a year later (Dangschat, 1942). The 9 stereoisomers differ from each
24
other based on the spatial orientation of their hydroxyl groups along axial or equatorial planes
(Figure 1.1). Of the nine, seven are optically inactive, whereas D- and L-chiro-inositol are
enantiomers. While nine stereoisomers are possible, only six are found in nature (myo-, scyllo-,
D-chiro, epi-, muco-, and neo-inositol).
Figure 1.1 The inositol stereoisomers.
There are 9 possible stereoisomers for inositol, which differ from each other based on the spatial
orientation of their hydroxyl groups along axial or equatorial planes.
25
1.3.4 Inositol in Nature
The biosynthesis of inositol is an evolutionarily conserved pathway in nature, found across
phylogenic kingdoms, including plants, animals, parasites, bacteria and archaea (Majumder et
al, 2003). The majority of inositol present in humans is in the form of myo-inositol (Fisher et al,
2002). The next most abundant isomer, scyllo-inositol, is present at 8-20% the concentration of
myo-inositol (Michaelis et al, 1993; Seaquist and Gruetter, 1998). scyllo-Inositol has been
documented in a number of other organisms, although dietary sources of scyllo-inositol are
limited (Sherman et al, 1978; Sanz et al, 2004; Soria et al, 2009). In the skate (Raja erinacea),
scyllo-inositol was recorded at higher concentrations than myo-inositol, with the highest levels
reported in the kidney (Sherman et al, 1978). In fruit, scyllo-inositol has been found in trace
amounts in grapes and citrus fruits, with the highest levels reported in grapefruit (Sanz et al,
2004). scyllo-Inositol has also been reported in the Apiaceae family of vegetables, which
includes carrots, fennel, parsley and coriander (Soria et al, 2009). However, the parts per
million levels of scyllo-inositol in dietary sources preclude the use of dietary intervention alone
to significantly alter scyllo-inositol levels within the CNS. In humans, it is unclear what
function scyllo-inositol serves, however, the presence of enzymes for the interconversion
between scyllo-and myo-inositol would suggest that scyllo-inositol does have a function within
the cell, if only as a reserve pool for myo-inositol.
1.3.5 Inositol Synthesis and Degradation Pathways
Inositol levels within the human body are maintained through a combination of diet, synthesis
from D-glucose-6-phosphate, recycling from inositol phosphates and through interconversion
between inositol derivatives (Hipps et al, 1976). The average human ingests 1 g/day of inositol
in the diet, predominantly as myo-inositol and phytic acid (Holub, 1986) and can synthesize up
to 4 g/day in the kidneys (Clements and Diethelm, 1979). myo-Inositol is synthesized from D-
26
glucose-6-phosphate, through the activity of two enzymes, myo-inositol-3-phosphate synthase
and inositol monophosphatase. In the brain, myo-inositol-3-phosphate synthase is only
expressed in the vasculature (Wong et al, 1987), therefore outside of this region inositol must be
actively transported into the cells.
myo-Inositol can be recycled from inositol phosphates through the action of inositol
polyphosphate phosphatases to generate inositol monophosphate, followed by the action of
inositol monophosphatase to generate myo-inositol (for an extensive list of human inositol
phosphatases see Caldwell et al, 2006). In the rat, Northern blot analysis showed strong inositol
monophosphatase expression in all brain regions tested: cortex, hippocampus, striatum and
cerebellum (McAllister et al, 1992), suggesting that unlike the synthesis from glucose-6-
phosphate, which appears to only occur in the vasculature, the creation of myo-inositol from
inositol phosphates can occur throughout the brain.
Enzymes that interconvert the inositol stereoisomers and related derivatives have also been
identified within the CNS (Figure 1.2; Hipps et al, 1976; 1977). In bovine brain, two enzymes
have been isolated that convert myo-inosose-2 into myo- and scyllo-inositol (Hipps et al, 1976).
The first, myo-inositol oxidoreductase, converts myo-inosose-2 to myo- or scyllo-inositol at a
ratio of 10:1 with NADH as a cofactor and 1:1 with NADPH (Figure 1.2A; Hipps et al, 1976).
The second, scyllo-inositol oxidoreductase converts myo-inosose-2 to myo- or scyllo-inositol at a
ratio of 1:3 with NADPH and showed no activity with NADH (Hipps et al, 1976). In addition, a
NADP+ dependent inositol epimerase, which was isolated from bovine brain, was also found to
convert myo-inosose-2 to myo- or scyllo-inositol, at a ratio of 2:1, in the presence of NADP+
27
(Hipps et al, 1977). scyllo-Inositol and myo-inositol are also interconverted by the NADP+
dependent inositol epimerase (Hipps et al, 1977). This epimerase converts myo-inositol to either
scyllo- or neo-inositol, at a ratio of 1:10, and converts scyllo-inositol back to myo-inositol
(Figure 1.2B).
Figure 1.2 Endogenous sources of scyllo-inositol.
A) A number of enzymes have been isolated from bovine brain that convert myo-inositol to
scyllo-inositol, via myo-inosose-2.22,28 The first, myo-inositol oxidoreductase, converts myo-
inosose-2 to myo- or scyllo-inositol, using either NADH or NADPH as a cofactor. In contrast,
scyllo-inositol oxidoreductase and inositol epimerase only function in the presence of NADPH
to convert myo-inosose-2 to scyllo-inositol. B) In addition, another NADPH dependent
epimerase has been isolated that converts myo-inositol to neo-inositol and scyllo-inositol and
can convert scyllo-inositol back into myo-inositol.
28
Removal of inositol from the body occurs by direct excretion in the kidney or by degradation
predominantly in the kidney and the liver. myo-Inositol oxygenase, which is the enzyme
required for the first step in myo-inositol catabolism, is mainly expressed in the proximal tubular
epithelial cells of the kidney cortex (Arner et al, 2006). Western blot analysis of mouse tissue,
showed no detectable levels of myo-inositol oxygenase in the brain, heart, lung, liver, spleen,
intestines or muscles (Arner et al, 2006). Trace amounts of the mRNA were detected in the
sciatic nerve, liver and heart (Arner et al, 2006). This enzyme cleaves the 6-carbon ring of
inositol to form D-glucuronate, which can be metabolized in the liver. These combined studies
suggest that inositol is actively transported throughout the body for both function and
degradation. Three inositol transporters have been identified and characterized in mammals,
one proton-myo-inositol transporter (HMIT) and two sodium-myo-inositol transporters (SMIT1,
SMIT2).
1.3.6 The Inositol Transporters
1.3.6.1 H(+)-myo-inositol Transporter
HMIT, alternatively known as solute carrier family 2 (facilitated glucose transporter), member
13, is a member of the major facilitator superfamily, a group of secondary transporters that
includes uniporters, symporters and antiporters. These transporters are characterized by 12
transmembrane domains and N- and C-terminal tails located on the cytoplasmic side of the
cellular membrane (Mueckler et al, 1985; reviewed by Zhao and Keating, 2007). HMIT is
predominantly expressed in the brain, with limited expression also found in the adipose tissue
and in the kidney (Uldry et al, 2001). In the brain, mRNA expression was observed in both
neurons and glia with high expression observed in all brain regions examined: cerebral cortex,
hippocampus, hypothalamus, cerebellum and brainstem (Uldry et al, 2001). Rat HMIT was
shown to transport myo-inositol preferentially followed by scyllo-inositol > chiro-inositol >
29
muco-inositol at a 1:1 ratio with H+ (Uldry et al, 2001). allo-Inositol was not transported by
HMIT and no other inositols were examined. Despite being labeled a facilitated glucose
transporter, rat HMIT did not transport D- or L-glucose or other related hexoses (galactose,
fructose, mannose, 2-deoxy-glucose, glucosamine or maltose). Homology between rat and
human HMIT is 90%, which suggests that similar transport activities are present.
HMIT is typically internalized in the cell and cell surface translocation and regulation of
expression have been suggested to be stimulated by membrane depolarization, changes in
protein kinase C, internal calcium concentrations and acidification of the extracellular
environment (Uldry et al, 2001; 2004), all of which occur with synaptic activity (Chesler and
Kaila, 1992). In light of this, the primary role of HMIT may be to adjust the intracellular
inositol pools involved in cellular signaling pathways or phosphatidylinositol synthesis, which
are required in areas with high rates of signaling and endo/exocytosis (Uldry et al, 2004).
1.3.6.2 Na(+)-myo-inositol Transporter 1
SMIT1, also known as solute carrier family 5 (sodium/glucose cotransporter), member 3, is a
member of the sodium/solute symporter family (Wright and Turk, 2004), which are
characterized by 13-14 transmembrane domains, with an N-terminal domain located in the
extracellular space and a C-terminal domain, either located in the cytoplasm in members with 13
transmembrane domains, or encased in the membrane as the 14th transmembrane domain, as is
the case for SMIT1 (Wright and Turk, 2004). The human, mouse, rat, canine and bovine
SMIT1 amino acid sequences are more than 92% homologous (Kwon et al, 1992; Berry et al,
1995; Lubrich et al, 2000; McVeigh et al, 2000). In humans, SMIT1 mRNA expression was
found in the kidney, brain, placenta, pancreas, heart, skeletal muscle and the lung, but not in the
liver (Berry et al, 1995), although its expression has been reported in the HepG2 human liver
30
cell line (Ostlund et al, 1996). In brain, SMIT1 mRNA expression was highest in the choroid
plexus (Inoue et al, 1996). High SMIT1 mRNA expression was also observed in the
hippocampus, the locus coeruleus, the suprachiasmatic nucleus, the olfactory bulb and the
Purkinje and granule cell layers of the cerebellum (Inoue et al, 1996). Across the brain, SMIT1
was expressed in almost all neurons and small glia-like cells (Inoue et al, 1996). In the
hippocampus, SMIT1 mRNA was localized to pyramidal cells in areas CA1 to CA3 and to
granule cells in the dentate gyrus (Inoue et al, 1996). In the choroid plexus, SMIT1 has been
specifically localized to the basolateral side of cells, indicating that it is responsible for
transporting inositol from the blood into the epithelial cells (Hakvoort et al, 1998).
SMIT1 appears to be the main transporter responsible for inositol transport into cells, SMIT1-/-
mice, show a 92% reduction in inositol levels in the brain and an 84% reduction in inositol
levels in the periphery (Berry et al, 2003). Heterozygous mice show a 15% reduction in inositol
levels in the frontal cortex and a 25% decrease in the hippocampus (Shaldubina et al, 2007),
which would suggest a dependence on SMIT1 transport in these areas or that the demand for
inositol is higher than elsewhere. When rat astrocytes from the cortex, hippocampus,
cerebellum, diencephalon and tegmentum were cultured and compared, the cortical and
hippocampal cultures had a lower Km and a higher Vmax for myo-inositol than those for the other
three regions, supporting the view that these two regions require more SMIT1 transporters
(Lubrich et al, 2000). In addition, SMIT1 mRNA levels were 2.5-fold higher in the cortex than
in the other regions (Lubrich et al, 2000).
SMIT1 is unique among the inositol transporters because it shows an equal affinity for myo- and
scyllo-inositol, as demonstrated in Xenopus oocytes transfected with the canine SMIT1 gene
(Hager et al, 1995). Overall, the sugar selectivity of the transporter was myo-inositol = scyllo-
31
inositol > L-fucose > L-xylose > L-glucose = D-glucose = α-methyl-D-glucopyranoside > D-
galactose = D-fucose = 3-O-methyl-D-glucose = 2-deoxy-D-glucose > D-xylose (Hager et al,
1995). The ability of scyllo-inositol to strongly compete out myo-inositol transport was
confirmed in murine neuroblastoma, murine cerebral microvessel endothelial and bovine aortic
endothelial cell-lines (Wiese et al, 1996).
Inositol transport through SMIT1 is dependent on the cotransport of sodium with a stochiometry
ratio of two Na+ ions per molecule of myo-inositol (Hager et al, 1995). Not unexpectedly, this
dependence on Na+ resulted in SMIT1 transport inhibition in the presence of the sodium
dependent transport inhibitor, phlorizin (Hager et al, 1995). The two Na+ ions bind to the
transporter first, followed by myo-inositol, this sequential binding may explain the sodium leak
currents observed in the absence of substrate (Hager et al, 1995).
Cells react to changes in osmolality by adjusting the levels of compatible organic osmolytes in
the cell. In cultured rat cortical astrocytes, myo-inositol accounts for 56-100% of the
osmolyte(s) utilized for adaptation to hypertonicity (Strange et al, 1994). Its been shown that
the accumulation of myo-inositol in the cell, as a response to hypertonicity, requires the presence
of myo-inositol in the extracellular space, indicating that this accumulation is not due to
synthesis from glucose-6-phosphate or conversion from other inositol derivatives or phosphates,
but from the transportation of inositol into the cell (Nakanishi et al, 1989). SMIT1 activity is
upregulated by hypertonicity (Nakanishi et al, 1989; Kwon et al, 1991; Paredes et al, 1992;
Cammarata et al, 1994a; 1994b; Miyai et al, 1995; Wiese et al, 1996; Mallee et al, 1997;
Denkert et al, 1998; Matskevitch et al, 1998; Yorek et al, 1998a; 1998b; Matsuoka et al, 1999;
Yorek et al, 1999; Lubrich et al, 2000) and downregulated by hypotonicity (Wiese et al, 1996).
32
SMIT1 mRNA levels and activity can also be regulated by TNF-α (Yorek et al, 1998a; 1998b),
possibly via NFκB, protein kinase C and ceramide activation (Yorek et al, 1998b). Treatment of
bovine aorta, pulmonary endothelial or cerebral microvessel endothelial cells with TNF-α
caused a significant decrease in SMIT1 mRNA levels and activity (Yorek et al, 1998b). The
effects of TNF-α on SMIT1 expression and activity required RNA synthesis and were inhibited
by actinomycin D (Yorek et al, 1998a). TNF-α decreased SMIT1 Vmax without altering the Km,
suggesting a reduction in SMIT1 transporters at the cell surface. In contrast, IGF-1, platelet-
derived growth factor, TGF-β, IL-1α, IL-1β, IL-2 or IL-6 did not affect SMIT1 activity (Yorek
et al, 1998a).
As shown for HMIT, SMIT1 activity is also regulated by changes in pH (Matskevitch et al,
1998; Eladari et al, 2002). However, unlike HMIT, SMIT1 is more active at physiological pH
and inhibited when pH is reduced. This is the opposite pattern to that observed for HMIT
(Uldry et al, 2001) and is thought to be due to a reduction in the transporter’s affinity for sodium
(Eladari et al, 2002). SMIT1 activity was also reduced by depolarizing-concentrations of
potassium (Wiesinger, 1991), again in direct opposition to HMIT regulation (Uldry et al, 2004).
Therefore, it would appear that the body has adapted multiple methods for regulating inositol
concentrations at the cellular level.
1.3.6.3 Na(+)-myo-inositol Transporter 2
The last known member of the sodium/solute symporter family that transports inositol is
SMIT2, also known as solute carrier family 5 (sodium/glucose cotransporter), member 11.
When comparing the human amino acid sequences, SMIT2 is 43% homologous to SMIT1
(Coady et al, 2002) and like SMIT1, the SMIT2 amino acid sequence encodes for 14
transmembrane domains (Roll et al, 2002). In humans, SMIT2 expression is highest in the
33
kidney, liver, heart, skeletal muscles and placenta, with much lower expression observed in the
brain and minimal levels detected in the spleen, small intestine, lungs and lymphocytes (Roll et
al, 2002). Interestingly, in polarized cells, such as Madin-Darby canine kidney cells, SMIT1 is
preferentially expressed at the basolateral membrane and SMIT2 is preferentially expressed at
the apical membrane (Bissonnette et al, 2004; 2008). These studies suggest that SMIT1/2 might
work in concert to regulate inositol within tissues.
In transport studies, SMIT2 displays an equal affinity for myo-inositol and its stereoisomer, D-
chiro-inositol (Coady et al, 2002; Aouameur et al, 2007; Lin et al, 2009). SMIT2 in HepG2
human liver cells showed a transport preference for D-chiro-inositol and D-glucose over their L-
stereoisomers (Ostlund et al, 1996) but did not transport α-methylglucose and L-fucose (Coady
et al, 2002; Lahjouji et al, 2007). This is in contrast to SMIT1, which shows an equal preference
for both D- and L-glucose stereoisomers63 and transports α–methylglucose and L-fucose (Coady
et al, 2002; Lahjouji et al, 2007). The remaining inositol stereoisomers have not been examined
for transport by SMIT2.
As observed for SMIT1, inositol transport by SMIT2 is dependent on the cotransport of two
sodium ions per substrate molecule (Coady et al, 2002; Bourgeois et al, 2005). As was observed
with the other members of the sodium/substrate symporter family, SMIT2 displays a sodium
leak current in the absence of substrate (Coady et al, 2002), and SMIT2 activity is upregulated
by hyperosmotic conditions (Bissonnette et al, 2004; 2008). This upregulation is the result of an
increase in SMIT2 transcription and translation, since upregulation was inhibited by
actinomycin B and cycloheximide treatment (Bissonnette et al, 2008). SMIT2 activity can also
be regulated by the mitogen-activated protein kinases p38 and c-Jun amino-terminal kinase, but
not the extracellular signal-regulated kinase (ERK; Bissonnette et al, 2008). Inhibiting p38 and
34
c-Jun amino-terminal kinase resulted in a 40% and 32% reduction in SMIT2 activity,
respectively (Bissonnette et al, 2008). In addition, SMIT2 activity can be upregulated by insulin
(Lin et al, 2009). Insulin treatment for 24 h caused an 18-fold increase in D-chiro-inositol-3H
transport in rat L6 skeletal muscle cells transiently transfected with human SMIT2 (Lin et al,
2009) and insulin treatment in diabetes has been linked to increased D-chiro-inositol levels
(reviewed in Larner, 2002). In contrast to HMIT and SMIT1, SMIT2 does not appear to be
sensitive to changes in pH (Eladari et al, 2002).
The expression and activity of the inositol transporters both in the periphery and CNS place
them in juxtiposition for the uptake of inositol as a therapeutic agent. In regards to AD, the high
expression and activity of the transporters in the cortex and hippocampus further suggest that
compounds utilizing these transporters could be preferentially targeted to these regions of the
brain.
1.3.7 Inositol Efflux
Inositol efflux from cells is not a well understood process, although fast and slow efflux currents
have been detected. Efflux of inositol may involve both volume-sensitive and volume-
insensitive components (Cammarata et al, 1997; Karihaloo et al, 1997; Isaacks et al, 1999),
which are both active following hypertonicity-induced cell swelling, though perhaps not under
basal conditions. The fast component appears to be mediated by volume-sensitive organic
osmolyte-anion channels (Jackson and Strange, 1993; Karihaloo et al, 1997; Isaacks et al, 1999;
Novak et al, 2000; Loveday et al, 2003). Both fast and slow myo-inositol efflux currents were
observed in bovine lenses exposed to hypertonic solution (Cammarata et al, 1997). The fast
efflux current was the result of the activation of a common anionic (chloride) channel. These
two efflux currents were observed in primary astrocyte cultures, but only the fast component
35
was inhibitable by anion membrane transport inhibitors (Isaacks et al, 1999). In human
teratocarcinoma-derived Ntera2/D1 neuron-like cells, inositol efflux was blocked by a Cl-
channel blocker, supporting the hypothesis that the volume-sensitive organic osmolyte-anion
channel is a common chloride channel (Novak et al, 2000).
1.3.8 Inositol Pools
Experimental data suggest that inositol in the cell exists as two or three separate pools (Diringer
and Rott, 1977; Yorek et al, 1991). This was first suggested by Diringer and Rott (1977), who
examined the effects of inositol metabolism in chick embryo cells and discovered differentially
regulated inositol pools, one smaller pool and one or more larger pools. Only the smaller pool
appeared to regulate the synthesis of phosphatidylinositol (Diringer and Rott, 1977). Behavioral
evidence has also suggested the presence of inositol compartmentalization (Bersudsky et al,
1994). Lithium-pilocarpine administration to rats leads to seizures, through the combined
actions of lithium, which prevents inositol resynthesis and pilocarpine, which increases the use
of inositol for second messenger systems by stimulating cholinergic activity (Kofman et al,
1993). These effects could be prevented by intracerebroventricular administration of myo-
inositol (Kofman et al, 1993) but not by osmotically-induced increases in inositol levels
(Bersudsky et al, 1994), suggesting that the inositol used to sustain phosphatidylinositol levels
must be different from that used to maintain osmotic function, strengthening the argument of
different inositol pools (Bersudsky et al, 1994).
Separate inositol pools have also been suggested by inositol uptake and efflux kinetics (Sigal et
al, 1993; Wolfson et al, 2000). Uptake of myo-inositol-3H into the soluble fraction of cells
showed no evidence of saturation, however incorporation into the lipid fraction had a Km of 0.28
mmol/L and was inhibited by phlorizin, a SMIT1/2 inhibitor. It was proposed that intracellular
36
inositol exists as one larger, metabolically inert, pool and one smaller pool that equilibrates with
external inositol levels and is utilized for the synthesis of phosphoinositides (Sigal et al, 1993).
In astrocytes exposed to myo-inositol-3H, three inositol pools were identified; the largest showed
slow efflux kinetics while the two smaller pools had faster efflux kinetics (Wolfson et al, 2000).
Unlike the findings in hepatocytes, the largest pool was membrane-associated and influenced
the phosphatidylinositide second messenger system, while the two smaller pools were located in
the cytosol and were thought to be involved in osmotic regulation (Wolfson et al, 2000). These
results suggest that it may be possible to alter certain pools of inositol without altering signal
transduction pathways.
36
CHAPTER 2
Rationale, Hypothesis and Objectives
372.1 Rationale
Transport of drug candidates into the brain is one of the key challenges for designing drug
treatments for CNS diseases. Oral administration of drug candidates is the preferred method of
drug delivery wherever possible because of the ease and accessibility of this procedure for the
patient and their caregiver. However this method of drug delivery requires, in the case of CNS
disorders, that drug candidates be able to cross the blood-brain and blood-CSF barriers. Many
compounds fail in clinical trials because, while they are effective in vitro and in animal testing,
they are unable to cross the brain barriers in humans to access their drug targets in the CNS.
Therefore, one goal in CNS drug design research is to generate non-invasive methods for
delivering these otherwise promising compounds to the brain. One possible barrier navigation
strategy is to try and use a compound that enters the brain for other purposes, not only as a drug
candidate but as a drug carrier. For example, inositol stereoisomers, such as myo- and scyllo-
inositol, have shown promise as therapeutic agents for the treatment of certain CNS disorders
(Hallman et al, 1986; Hallman et al, 1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al,
1996; Chengappa et al, 2000; Gelber et al, 2001; Palatnik et al, 2001; Eden et al, 2006;
McLaurin et al, 2006). This is likely due to the presence of transporters for these inositols in the
CNS, more specifically the transporters HMIT, SMIT1 and SMIT2 (Berry et al, 1995; Inoue et
al, 1996; Hakvoort et al, 1998; Uldry et al, 2001). The accessibility of inositol stereoisomers to
the CNS and the presence of transporters for inositol in the brain, suggest that it may be possible
to use these transporters as a shuttling system for the delivery of other compounds to the CNS.
2.2 Hypothesis
Inositol transporters represent an effective shuttling system for drug delivery into the CNS.
382.3 Objectives
1. Inositol levels: Determine the CNS bioavailability of the two main inositol stereoisomers,
myo- and scyllo-inositol, by comparing their baseline levels to their concentrations
following oral administration. These studies were conducted in TgCRND8 mice and their
wild-type littermates, to determine whether disease pathology would affect these outcome
measures.
2. Inositol transporter expression: Inositol transport was examined by quantification of
transporter expression levels in four regions of the brain: cortex, hippocampus, septum,
cerebellum, as well as the kidney as a function of age and disease pathology.
3. Inositol transport kinetics: The flexibility of the inositol transporters for the transportation
of compounds other than myo-inositol was evaluated by determining the structural features
required for active transport through each of the inositol transporters.
39
CHAPTER 3
Materials and Methods
Portions of this section have been previously published in:
Fenili D, Brown M, Rappaport R, McLaurin J. Properties of scyllo-inositol as a therapeutic treatment of AD-like pathology. J Mol Med. 2007 Jun;85(6):603-11.
40Mice
TgCRND8 mice were maintained on an outbred C3H/C57Bl6 background. These mice over
express the human APP gene containing both the Swedish (KM670/671NL) and Indiana
(V717F) mutations under control of the Syrian hamster prion gene promoter (Chishti et al,
2001). Mice were kept on a 12-hour light/dark cycle and given water and standard rodent chow
ad libitum. All experiments were performed according to the Canadian Council on Animal Care
guidelines.
For gas chromatography/mass spectrometry studies, one group of mice was treated with 10
mg/ml myo- or scyllo–inositol ad libitum through their drinking water for one week and changes
in inositol levels were quantified. Each mouse typically consumed between 2.5–3 ml per day,
which is equivalent to a 25–30 mg dose of inositol per animal. A second group of animals were
treated with a once-daily gavage dose of, either 10, 30 or 100 mg/kg/day of scyllo-inositol, for
one month and sacrificed 8 hours following the final gavage treatment.
Materials
All reagents were purchased from Sigma (St. Louis, MO, USA) unless otherwise noted. epi-
Inositol, allo-inositol and cold scyllo–inositol was acquired from Transition Therapeutics
(Toronto, Ontario, Canada). Viburnitol (1-D-3-deoxy-myo-inositol; Cat #: FC-041), D-Ononitol
(1-D-4-O-methyl-myo-inositol; Cat #: FC-040), Sequoyitol (5-O-methyl-myo-inositol; Cat #:
FC-047) and D-Pinitol (3-O-methyl-D-chiro-inositol; Cat #: FC-026) were all
41purchased from Industrial Research Ltd. (Lower Hutt, New Zealand). myo-Inositol-(2-3H) (Cat
#: ART 0116), scyllo-inositol-(2-3H) (Cat #: ART 0264) and L-fucose-(5,6-3H) (Cat #: ART
0106A) were all purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO, USA).
Quantification of myo– and scyllo–inositol
myo–Inositol and scyllo-inositol concentrations in the brain, CSF, and plasma, as well as
synthetic stock solutions of both inositols were quantified using gas chromatography/mass
spectrometry (GC/MS; Fenili et al, 2007). To increase the volatility and thermal stability of
these compounds and to allow for peak separation, these samples were first derivatized. The
derivatization protocol was adapted from Shetty et al. (1995). D-chiro-Inositol was added to all
samples as an internal standard, at a concentration of 50 ng/µl for brain and plasma samples and
1 ng/µl for CSF and synthetic samples. Synthetic myo- and scyllo-inositol were used for initial
method development and for the generation of concentration curves for sample analysis. For
brain samples, one hemisphere was weighed and the tissue, along with D-chiro-inositol, was
homogenized in 2 x 2 ml of methanol, and the resulting suspension was centrifuged for 5 min at
5,000 x g. A volume of supernatant equivalent to 30 mg of brain tissue (based on the weight of
the tissue before homogenization) was analyzed. Similarly, for plasma, CSF and synthetic
inositols, either 100 µl of plasma, 5 µl of CSF or different concentrations of synthetic inositols
were mixed with 1 ml of methanol and D-chiro-inositol; the solution was allowed to stand at
room temperature for 5 min, then centrifuged (5000 x g, 5 min) and the supernatant removed.
These samples were evaporated to dryness (Speedvac; 60°C); 100 µl of pyridine reagent (1
mg/ml 4 dimethylaminopyridine solution in pyridine) and 100 µl of acetic anhydride were
added, and the tubes were flushed dry with nitrogen and heated (80°C, 30 min). After
derivatization, the unreacted acetylating reagent was evaporated under a steady stream of
42nitrogen. The derivatized products were re-dissolved (4 ml; hexane-ethyl acetate (80:20, v/v))
and washed with 1 ml of 5% sodium carbonate. After vortexing (5 min) and centrifugation (3
min, 1000 x g), the organic layer was transferred to a new tube and evaporated (Speedvac;
40°C). The residue was reconstituted (100 µl; ethyl acetate), and 1 µL of this was injected into
the GC/MS system.
Initial GC method development was performed on a Perkin Elmer Autosystem XL, with a
programmable split-splitless injector and a flame ionization detector. GC/MS was performed
using a Perkin Elmer TurboMass Autosystem XL with a quadrupole mass spectrometer and
electron ionization. For both machines, GC was performed using a 30 m x 0.25 mm x 0.25 mm
ZB 5 column (5% diphenyl/95% dimethylpolysiloxane, Phenomenex, Macclesfield, UK) and
helium as the carrier gas (1 ml/min). Samples were injected with the split set to 50 at 1 min and
0 at 5 min; the injector temperature was set at 300°C. The initial optimized GC protocol
consisted of a starting temperature of 190°C. This was increased by 2° increments to 210°C,
held at this temperature for 4 minutes, then increased by 35° increments to 290°C and held at
this temperature for 4 minutes. The final optimized GC/MS protocol, consisted of a GC protocol
where, the initial oven temperature was set to 80°C. After a hold of 1 min, the temperature was
increased by 25° increments to 190°C, then by 3° increments to 220°C and finally by 35°
increments to 290°C, which was held for 1.5 minutes. Sample peaks were analyzed using
selected-ion monitoring at m/z 168 for brain and CSF samples and m/z 373 for plasma samples.
Three selected-ion recording time points, corresponding to chiro-, myo- and scyllo-inositol
43elution from the GC column, were programmed into the MS protocol for increased sampling
accurracy: 11.9-12.3 minutes, 12.4-12.85 minutes and 13.25-13.52 minutes, respectively. The
sample peak areas were compared to the standard concentration curves generated using
synthetic inositols, which were analyzed using selected-ion monitoring at m/z 168.
Lipid extraction and hydrolysis
The method for lipid isolation and analysis was adapted from Kersting et al. (2003). One brain
hemisphere was homogenized in 2 ml of dH2O, and 500 µl was used for lipid isolation. This was
placed in a glass screw-cap tube containing 3.75 ml of chloroform/methanol/HCl (10:20:0.1,
v/v) and vortexed. Chloroform (1.25 ml) and 0.1 M HCl (1.25 ml) were added and the solution
revortexed. The samples were centrifuged (200 x g, 5 min) to separate the phases. The organic
phase containing the lipids was dried under nitrogen gas and resuspended in 200 µl of
chloroform/methanol (6:1, v/v) before streaking onto a silica gel 60 F254 plate (EM Industries,
Merck, Darmstadt, Germany). The plate was placed in hexane/ethyl ether/acetic acid (70:30:1,
v/v). Once the solvent had migrated within 1 cm from the top of the plate, the plate was
removed from the thin-layer chromatography tank and air dried. The origin containing
phosphatidylinositol lipids was collected, and the lipids eluted using four 1 ml washes of
chloroform/methanol/concentrated HCl (2:1:0.1, v/v). The lipids were dried under nitrogen,
redissolved in 1 ml of 6 N HCl, and then acid hydrolyzed (110°C, 56 h), to release the inositols
from inositol-containing phospholipids. The hydrolysate was dried under nitrogen and
derivatized (as above) for GC/MS analysis. The sample peaks were analyzed using selected-ion
monitoring, m/z 168.
44Quantitation of inositol transporter expression
Control gene selection for the tissue studies:
Genes such as, tyrosine kinase receptor (Abl1), a RNA-dependent ATPase (Bat1),
glyceraldehyde-3-phosphate dehydrogenase (Gapdh), ribosomal protein (L32), nitric oxide
synthase 2 (Nos2), proteosome 26S subunit ATPase 4 (PSMC4), mitochondrial ribosomal
protein (S12), succinate dehydrogenase (SDHA), TATA box binding protein (Tbp) and the
transferrin receptor (TFRC) were analyzed as potential control genes for this study. Potential
control gene for this study were chosen based on listed commonly used control genes in QPCR
control gene selection kits, such as, the Mouse Endogenous Control Gene Panel (Tataa
Biocenter, Göteborg, Sweden) and Validated qPCR Control Kits (Eurogentec, San Diego,
USA). Once potential control genes were selected, the National Center for Biotechnology
Information (NCBI), Gene Expression Omnibus website was used to scan mouse microarray
data for each of these genes. Control genes were selected that demonstrated similar levels of
expression in the kidney and in all four of the brain regions examined in this study. The control
genes Gapdh and Tbp each had the most equivalent expression across these tissue regions,
therefore they were selected for this study.
Primer design:
Potential primers for each of the five genes examined in this study were selected using the
Beacon Designer 7.5 software program for Mac OS X. Sequence amplification studies were
conducted using a kidney RNA sample from a 2 month old, wild-type mouse. A concentration
curve of RNA was used to test the amplification efficiency (slope) and accuracy (R2 value) of
each primer pair. In addition, dissociation curve analysis was performed to determine whether
one, or multiple, amplification products were generated by the primer pair under evaluation (see
45Figures 5.1 and 5.2). Primers were selected if their slope was in the range 2.9-3.2, their R2 value
was between 0.98-1 and if they produced one main amplification product with minimal, or
preferably no, secondary amplification products (see Figures 5.1 and 5.2). The final primers
chosen for mouse and human gene amplification are listed in Tables 3.1 and 3.2. β-actin was
chosen as the control gene for human cell-line inositol transporter tests because primers for this
gene had previously been chosen, courtesy of Mary Brown.
Table 3.1 Mouse QPCR primers. Protein Name Gene Accession # Primers TATA-box Binding Protein Tbp NM_013684 Forward: GCC TTC CAC CTT ATG CTC
AG Reverse: GAG TAA GTC CTG TGC CGT AAG
Glyceraldehyde 3-phosphate dehydrogenase
Gapdh XM_001473623 Forward: AAG AAG GTG GTG AAG CAG GCA TC Reverse: CGA AGG TGG AAG AGT GGG AGT TG
Hydrogen/myo-inositol transporter Slc2a13 NM_001033633 Forward: GTC ACC ATC AAC ACC CTC TTC Rerverse: ACC TCC ATC CAT CCT TCT GC
Sodium/myo-inositol transporter 1 Slc5a3 NM_017391 Forward: CTG TGG TGC TGT GGG ATG ATG Reverse: CCT GCT GGG TCT GAA CTT TGC
Sodium/myo-inositol transporter 2 Slc5a11 NM_146198 Forward: CAA GGT GGT GAG GGC TAT CC Reverse: CTA TGA CAG GTT CCG CTT TGC
Table 3.2 Human QPCR primers. Protein Name Gene Accession # Primers β-actin ACTB M10277 Forward: GCC GAG GAC TTT GAT TGC
Reverse: GGA CTT GGG AGA GGA CTG G Hydrogen/myo-inositol transporter SLC2A13 NM_052885 Forward: TTG AAT CAC TCT TTG ACA AC
Rerverse: CCT TTA CCC GAA TAT ATT CA Sodium/myo-inositol transporter 1 SLC5A3 NM_006933 Forward: AGC CTT CTC ACA CCA CCT C
Reverse: CAG TTC TCC TTC ACC ACC AG Sodium/myo-inositol transporter 2 SLC5A11 NM_052944 Forward: TCC TGT GGC TCT GTG GAA TA
Reverse: CGC AGA AAA TGA GGT TGA CG
46
Tissue RNA extraction:
Kidney, cortical, hippocampal, septal and cerebellar tissues were each placed in a homogenizer
with 2 ml of Solution D (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5%
sarcosyl and 0.1 M 2-mercaptoethanol) and the tissue homogenized. The resulting suspension
was transferred to a 50 ml falcon tube and 200 µl of 2 M sodium acetate (pH 4.0), 2 ml of water-
equilibrated phenol and 400 µl of chloroform were added and the solution mixed vigorously for
10 seconds. The tubes were incubated on ice for 20 minutes, parafilmed and centrifuged (8000
rpm, 30 min, 4°C, Beckman Coulter Avanti J-20XP). The top, aqueous interface was removed
and placed in a new Eppendorf tube with 12.5 µl of 1 N acetic acid and 250 µl of 100% ethanol
added for each 0.5 ml of aqueous interface and the resulting solution was mixed thoroughly.
The solution was incubated (-20°C, 1 h), centrifuged (10,000 x g, 20 min, 4°C) and the
supernatant discarded. The RNA pellet was dissolved in 0.3 mL of Solution D, after which 5 µl
of 1 N acetic acid and 100 µl of 100% ethanol were added, the solution incubated (-20°C, 1 h)
and then centrifuged (13,000 x g, 20 min, 4°C). The supernatant was discarded and the pellet
washed three times in 0.5 mL of 70% ethanol (in DEPC-treated water) and centrifuged
following each wash (13,000 x g, 20 min, 4°C). The supernatant was removed and the RNA
pellet resuspended in a small volume of DEPC-treated water. The RNA concentration of the
resulting solution was determined using a NanoDrop™ Spectrometer (Thermo Scientific,
Wilmington, USA).
Cell culture RNA extraction:
Trypsin was added to the cell culture dish and the cells incubated (3-5 min, 37°C) to harvest
from the cell culture dish. Once the cells were lifted, culture medium, at twice the volume of
trypsin, was added to inactivate the trypsin and the resulting suspension was transferred to a 15
47
mL falcon tube for centrifugation (1000 rpm, 5 min). The supernatant was removed and the
cells washed once in ice cold phosphate buffered saline (PBS). The PBS was centrifuged (1000
rpm, 5 min), removed and replaced with 0.5 mL of Solution D (4 M guanidine thiocyanate, 25
mM sodium citrate, pH 7.0, 0.5% sarcosyl and 0.1 M 2-mercaptoethanol). The cells were re-
suspended, transferred to an Eppendorf tube and homogenized using a 1 inch, 20 g needle
attached to a 1 mL syringe. To this tube, 50 µl of 2M sodium acetate (pH 4.0), 500 µl of water-
equilibrated phenol and 100 µl of chloroform were added. The final suspension was shaken
vigorously for 10 seconds, incubated on ice for 20 minutes and centrifuged (10,000 x g, 20 min,
4°C). The top, aqueous interface was removed and the same procedure as tissue RNA
extraction was followed from this point onward. The RNA concentration was determined using
a NanoDrop™ Spectrometer.
DNase I Treatment:
RNA was treated with DNase I (Fermentas, Cat #: EN0521) to remove genomic DNA. RNase
inhibitor (Fermentas, Cat #: EO0381) was added to the reaction mixture to prevent RNA
degradation during DNase I treatment. To a RNase-free tube 500 ng of RNA was added plus
enough DEPC-H2O to equal 1.75 µl and 3.25 µl of the following master mix: 4-parts 10X
reaction buffer with MgCl2, 4-parts Deoxyribonuclease I (DNase I), RNase-free, 1-part
RiboLock™ RNase Inhibitor and 17-parts DEPC-treated water. Samples were incubated (37°C,
30 min), following which 25mM EDTA (0.5 µl) was added and the sample further incubated
(65°C, 10 min). Following DNase I treatment, concentrations were determined again using the
NanoDrop™ Spectrometer and DEPC-treated water was added to the samples to normalize their
concentrations.
48
Reverse transcription:
For each sample, 3 cDNA reactions were performed, each one at a volume of 10 µl using a
SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen, Cat #: 11752).
For this reaction, 5 µl of the kit provided 2X RT Reaction Mix, 1 µl of the RT Enzyme Mix and
60 ng of DNase-treated RNA were added to each tube on ice. Three cDNA tubes were created
per sample. The tube contents were gently mixed and placed in a PCR machine and incubated
at 25ºC for 10 min, 50ºC for 30 min and 85ºC for 5 min. Following this E. coli RNase H (2 U, 1
µl) was added and the mixture incubated (37ºC, 20 min) to remove any residual RNA. Next,
autoclaved distilled water (43.6 µl) was added to each tube to dilute the samples for the
quantitative polymerase chain reaction (QPCR).
Quantitative polymerase chain reaction:
For the QPCR step a SYBR® GreenER™ qPCR SuperMix Universal kit (Invitrogen, Cat #:
11762) was used. To each well of a MicroAmp™ Optical 96-Well Reaction Plate (Applied
Biosystems, Cat #: 4306737), 9.1 ml of diluted cDNA and 10.9 ml of the master mix consisting
of: 10 µl of SYBR® GreenER™ qPCR SuperMix Universal, 10 µM of the forward primer (0.4
µl), 10 µM of the reverse primer (0.4 µl) and 0.1 µl of the ROX Reference Dye were added. A 2
month, non-transgenic, kidney mRNA sample, with ideal 260/280 and 260/230 ratios, was used
as a between plate control. For analysis, relative quantification values for the three inositol
transporters were adjusted to the two control genes, inter-plate calibrators and variations in
primer efficiencies, then adjusted to an average expression level of 1 using the MultiD, GenEx
software program (genex.gene-quantification.info/).
49
Protein homology analysis
Human, mouse and rat HMIT, SMIT1 and SMIT2 sequences were retrieved from the NCBI
database (www.ncbi.nlm.nih.gov/pubmed/). Sequences were aligned using DNAMAN software
(www.lynnon.com/). Alignment was performed using the identity matrix, fast alignment
protocol, with the following non-homology penalty parameters: gap penalty = 4, K-tuple = 2,
gap open = 10 and gap extension = 0.1.
Substrate selectivity analysis
Cell culture:
Embryonic day 18 primary rat cortical neurons (Neuromics, PC35102) - The shipping medium
was removed from the tube containing the tissue, saved and replaced with an enzymatic solution
consisting of Hibernate-Ca medium (2 ml, supplied with kit), containing papain (2mgs/ml) and
the tissue incubated (30°C, 30 min). The enzymatic solution was removed and 1 ml of the
initial shipping medium was added back. The tissue was titurated using a blue plastic tip until
the cells in the tissue were mostly dispersed. Undispersed tissue was allowed to settle by
gravity for 1 min and the supernatant transferred to a 15 ml tube containing an additional 1ml of
the initial shipping medium. The cells were gently mixed by swirling and centrifuged (1100
rpm, 1 min). The supernatant was discarded and the tube flicked a few times to loosen the cell
pellet. Culture medium (NbActiv4 medium, Neuromics, M36107) was added and the cells
resuspended by gentle pipetting. Viable cells were counted using a hemocytometer to a
concentration of 32 x 103 cells/2 cm2 of substrate in 0.4 ml/2 cm2 substrate and plated. Half the
medium was changed with fresh culture medium every 3-4 days and the cells used for
competitive transport studies after 3 weeks in culture.
50
Cell-lines, human epithelial kidney (HEK293) cells and 1321 N1 astrocytoma cells were
cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin. Cells were plated onto 24-well plates and allowed to grow to 90%
confluency before the competitive transport assays were conducted.
Competitive transport assay:
Once 90% confluent, cells were washed in PBS, then incubated in PBS (pH 7.0) containing:
myo-inositol-(2-3H) (3 µCi/mL, 100 µM), BSA (0.1%, used to reduce background radioactivity),
with or without a substrate competitor. The substrate competitors consisted of 10 mM of either
inositol stereoisomers (myo-, scyllo-, epi-, allo-, muco-, neo-, L-chiro-, or D-chiro-inositol)
inositol derivatives (viburnitol, D-ononitol, sequoyitol, L-quebinchitol or D-pinitol) or
structurally similar sugar substrates (D-glucose, L-glucose, D-galactose, D-mannose, D-fucose,
L-fucose, L-rhamnose, D-ribose, L-ribose or D-fructose). One well was dedicated to testing
each competitor and triplicate plates were used for each experiment. Following incubation (3 h,
37°C), cells were washed twice with PBS containing 1 mM cold myo-inositol, to stop
transporter activity, following which the cells were dissolved using 2% SDS and transferred to
vials containing 5 mL scintillation fluid. Their radioactivity was then measured using a
scintillation counter.
For competitor testing against scyllo-inositol transport, myo-inositol-(2-3H) was substituted with
scyllo-inositol-(2-3H) at the same concentration and the same protocol as above was followed.
For competitor testing against L-fucose transport, myo-inositol-(2-3H) was substituted with L-
fucose-(5,6-3H) at the same concentration. L-fucose transport was tested against the following
51
competitors: 1. 10 mM myo-inositol and 10, 50, 100, 150, 200 and 400 mM cold L-fucose. 2.
10 mM of cold L-fucose, D-fucose, myo-, scyllo-, allo-inositol, L-glucose and D-glucose. 3. 0.1
mM D-fucose and 10, 25, 50, 100 and 250 mM cold L-fucose.
For D- and L-chiro-inositol competitor testing, myo-inositol-(2-3H) transport was tested in the
absence or presence of 10 mM cold myo-inositol, 1, 10, 20, 40, 80 or 160 mM of D- or L-chiro-
inositol.
For scyllo-inositol derivative testing, myo-inositol-(2-3H) transport was tested in the absence or
presence of 2 mM of the scyllo-inositol derivatives MN-001, MN-002, MN-003, MN-005, MN-
007, MN-010 or MN-012 (see Figure 6.16a for structures).
For the testing of cold myo- and scyllo-inositol concentration curves on myo-inositol-(2-3H)
transport, transport was tested against a range of concentrations between 62.5 mM and 0.95 µM
for myo-inositol and between 100 mM and 0.024 mM for scyllo-inositol. For the myo-inositol
concentration curve experiments, incubation was limited to 15 minutes to better reflect the
concentration versus transport ratio. In contrast, the scyllo-inositol concentration curve
experiments were conducted with an incubation time of 3 hours, because of a slower uptake rate
for scyllo-inositol.
For HMIT activation, four adjustments to the above competitive transport assay were made in
different combinations to try and activate HMIT.
1. Cell surface depolarization, through an increase in external potassium levels – potassium
levels in the PBS solution, used for cell washes and the radioactivity incubations, was
52
increased from 2 g/L to 5.964 g/L (80 mM). This concentration was chosen based on the
concentration used by Uldry et al. (2004).
2. Acidification of the extracellular environment – the pH of the PBS solution, used to wash
cells and for the incubation solution, was lowered from 7.0 to 6.0.
3. Phloridzin induced SMIT1/2 inhibition – multiple mM concentrations of phloridzin were
tested and 2 mM chosen for SMIT1/2 transport inhibition. For these silencing experiments,
cells were washed in PBS and then incubated in 2 mM of phloridzin (37°C, 30 min) before
the radioactivity step. For the radioactivity step, 2 mM of phloridzin was added to the
solution to maintain the SMIT1/2 silencing.
4. Na-free PBS – The PBS solution, used for cell washes and the incubation solution, was
switched to a Na-free version, which consisted of (per L): 58 g LiCl, 2 g KCl, 2.4 g
KH2PO4 and 17.42 g K2HPO4, adjusted to pH 7.0.
Statistical analysis
Statistical analysis for the GC/MS and competition assay studies were performed using the
Statistical Package for the Social Sciences (SPSS) for Mac OS X. QPCR statistical analysis was
conducted using the Statistical Analysis System (SAS) program (courtesy of Lillian Weng,
Sunnybrook Hospital). Groups were compared using a one-way ANOVA. If a significant F
score was observed (p < 0.05), a Bonferroni post hoc test was used to compare the groups with
the statistical significance set at p < 0.05.
53
CHAPTER 4
myo- and scyllo-Inositol Levels and Equilibrium in the Brain
Portions of this section have been previously published in:
Fenili D, Brown M, Rappaport R, McLaurin J. Properties of scyllo-inositol as a therapeutic treatment of AD-like pathology. J Mol Med. 2007 Jun;85(6):603-11.
54Abstract
Inositol stereoisomers have been examined as potential therapeutic agents with favorable results.
But while both myo- and scyllo-inositol are able to inhibit Aβ aggregation in vitro, only scyllo–
inositol is effective at ameliorating disease pathology in vivo in the TgCRND8 mouse model of
AD. This disparity between in vitro and in vivo results is interesting and unexpected based on
what is known about the transport of both inositols into the brain. Therefore, this project’s
objectives were: 1. To design a gas chromatography/mass spectrometry (GC/MS) protocol to
quantify myo- and scyllo-inositol. 2. To compare brain inositol levels between TgCRND8 mice
and their wild-type littermates. 3. To examine inositol transport and equilibrium dynamics in
these two groups by examining the effects of myo- and scyllo-inositol treatment on brain inositol
levels. and 4. To determine whether an increase in scyllo-inositol levels would result in its
incorporation into phosphatidylinositol. We conclude that brain inositol levels, and the effects
of inositol administration on those levels, was not significantly altered by disease pathology. Ad
libitum scyllo-inositol resulted in a significantly greater increase in its corresponding levels in
the brain, than ad libitum myo-inositol, suggesting that myo-inositol is tightly regulated. Once-
daily gavage treatment did not result in a sustained increase in CNS scyllo-inositol levels and
increases in brain scyllo-inositol levels, did not result in incorporation into phosphatidylinositol
lipids.
55Introduction
Inositol is a simple polyol with nine stereoisomers, of which, the most commonly found in
nature are myo-, D-chiro-, epi- and scyllo-inositol (Fisher et al, 2002). Inositol stereoisomers as
a drug therapy have been extensively studied for more than 20 years. myo–Inositol has been
successfully used in human studies to treat psychiatric disorders such as depression (Levine et
al, 1995), bipolar/affective disorder (Chengappa et al, 2000; Eden et al, 2006), obsessive-
compulsive disorder (Fux et al, 1996), eating disorders (Gelber et al, 2001) and panic disorder
(Benjamin et al, 1995; Palatnik et al, 2001), as well as for the treatment of respiratory distress
syndrome in premature infants (Hallman et al, 1986; 1992). epi–Inositol has been successfully
used in mice to treat anxiety (Einat et al, 1998; Bersudsky et al, 1999). D-chiro-inositol has
been successfully used to prevent neural tube defects in folate-resistant mice (Cogram et al,
2002) and as an insulin-sensitizing agent in women with polycystic ovary syndrome (Nestler et
al, 1999; Gerli et al, 2003). While these inositol stereoisomers have been extensively studied as
potential disease therapeutics, our laboratory was the first to study scyllo–inositol as a
therapeutic agent (McLaurin et al, 2006).
Both myo– and scyllo–inositol were tested as potential disease therapeutics in the TgCRND8
mouse model of AD (McLaurin et al, 2006). This mouse model of AD expresses a mutated
form of the human amyloid precursor protein, which results in an increase in cerebral Aβ levels,
Aβ plaque formation and cognitive deficits by 3 months of age, and a pathology corresponding
to humans in the advanced stages of AD by 6 months of age (Chishti et al, 2001). In vitro, myo–
inositol successfully inhibited Aβ aggregation, but in vivo myo-inositol did not significantly
increase survival or cognitive performance in the TgCRND8 animals (McLaurin et al, 2000;
2006). In contrast, scyllo–inositol successfully inhibited Aβ aggregation both in vitro and in
56vivo (McLaurin et al, 2000; 2006). In vivo scyllo-inositol treatment significantly improved
cognitive function, synaptic function and survival rates in the TgCRND8 mice (McLaurin et al,
2006). In addition, a significant decrease in Aβ40 and Aβ42 levels, vascular amyloid levels,
plaque size and area were also observed (McLaurin et al, 2006). These positive effects occurred
both in animals given scyllo–inositol prophylactically, before the visible onset of symptoms, and
therapeutically at 5 months of age, once symptoms have fully developed. Therefore, scyllo–
inositol is an effective treatment for decreasing disease pathology and improving behavioral
correlates in the TgCRND8 model of AD (McLaurin et al, 2006).
However, it was unclear why myo-inositol would only be effective in vitro, while scyllo-inositol
is effective both in vitro and in vivo. myo–Inositol is the most abundant isomer found in nature
and is a ubiquitous component of all eukaryotic cells. It is a constituent of phosphatidylinositol,
which is an important phospholipid in membranes and second messenger systems. scyllo-
Inositol is the next most abundant isomer and is found in humans at 8-20% of myo-inositol
levels (Michaelis et al, 1993; Seaquist and Gruetter, 1998). The human brain has the highest
concentration of inositol in the body, containing approximately 5 mM of myo–inositol and 0.5
mM of scyllo–inositol (Michaelis et al, 1993); values that are almost 100-fold greater than
circulating myo- and scyllo-inositol levels (Palmano et al, 1977). Inositol content in the brain is
either synthesized in situ from glucose or transported across the blood-brain and blood-CSF
barriers (Spector, 1978; 1988). Endothelial cells, located at the BBB, are the only cells capable
of producing inositol from glucose within the CNS; therefore, utilization of inositol by most
cells in the CNS requires an active transport system. Inositol may enter the brain either by
passive diffusion or through stereospecific saturable transport systems at the brain barriers
(Spector, 1988; Wiesinger, 1991; Rubin and Hale, 1993; Uldry et al, 2001; Coady et al, 2002).
57
myo–Inositol is critical for maintaining osmolarity and signal transduction pathways within the
CNS, and although the physiological concentrations of inositol in the brain and some
mechanisms of its regulation have been reported, this understanding is still very general (Fisher
et al, 2002). Overall, this study sought to better understand myo- and scyllo-inositol regulation
within the brain and whether regulation changes with disease pathology in TgCRND8 mice. To
address this, five objectives were set:
1. To develop a GC/MS method to quantify the levels of myo- and scyllo-inositol in the brain,
CSF and plasma of experimental animals.
2. To compare the brain levels of myo- and scyllo-inositol between TgCRND8 mice and their
wild-type littermates, to examine whether they change as a result of disease pathology.
3. To compare those findings to inositol levels following ad libitum myo-inositol treatment, as
a way to examine the effects of disease pathology on CNS inositol transport and the
regulation of inositol levels in the brain.
4. To examine the effects of scyllo-inositol dosing on CNS inositol levels, by treating animals
with one of four doses of scyllo-inositol and comparing brain inositol levels to an untreated
control group. CSF inositol levels were also examined to see if the same effects observed in
the brain would be reflected in the CSF.
5. Finally, incorporation of scyllo-inositol into phosphatidylinositol, following ad libitum
administration to animals, was examined.
58Results
Gas chromatography/mass spectrometry method development
A GC/MS technique was developed to track inositol levels present in the brain, CSF and plasma
of experimental animals. In order to increase the volatility of the inositol stereoisomers within
the GC and to better chromatographically separate the different inositol stereoisomers, the
compounds were first derivatized (Figure 4.1a). Derivatization consisted of replacing the
hydroxyl groups of inositol with acetyl groups. Samples were derivatized in the presence of
pyridine reagent and acetic anhydride at 80°C for 30 minutes, following which non-derivatized
compounds and lipids were removed using organic extraction. The resulting derivatized
compounds were dried and resolublized in ethyl acetate.
To confirm the effectiveness of the derivatization protocol, samples of synthetic inositol were
derivatized and submitted for analysis using a matrix-assisted laser desorption time-of-flight
mass spectrometer (Proteomic and Mass Spectrometry Centre, Medical Sciences Building). The
resulting chomatograms, showing the peaks generated, were analyzed for the presence of the
derivatization products (Figure 4.1b). The peak at mass 180 identifies the amount of
unacetylated inositol present in the sample. While the peaks at mass 373 and 433 correspond to
derivatized inositol, either in its more predominant form, where there are acetyl groups in place
of 5 of the 6 hydroxyl groups and the final hydroxyl group has been replaced by a hydrogen (ion
373) or in a fully derivatized state, where all six carbons have an attached acetyl group (ion
433). While a small proportion of inositol was present in the sample at different degrees of
acetylation, the majority of the inositol was found with either 5 or 6 acetyl groups attached.
These results confirmed that the derivatization protocol was effective.
59
Figure 4.1 Derivatization.
In order to increase the volatility of the inositol stereoisomers so that they would vapourize
when injected into the GC and to better separate when individual inositol stereoisomers would
leave the GC column, the compounds were first derivatized. Derivatization consisted of
replacing the hydroxyl groups of inositol with acetyl groups (A). Samples were derivatized in
the presence of pyridine reagent and acetic anhydride, non-derivatized compounds and lipids
were removed from the mixture using organic extraction and the remainder was dried and
resolubilized in ethyl acetate. The derivatization protocol was tested by mass spectrometry
analysis (B). The ions corresponding to inositol at each stage of acetylation are highlighted
using red boxes. Most of the inositol was found in a form where the hydroxyl groups had been
replaced by 5 or 6 acetyl groups.
60Once the effectiveness of the derivatization protocol was confirmed, derivatized synthetic
inositols were used for initial GC method development. For the GC method, the mobile gas
phase used was helium and the stationary, liquid phase coating the column was 5%
diphenyl/95% dimethylpolysiloxane. D-chiro-inositol was added to all samples, prior to
derivatization, as an internal standard. By changing the temperature, to influence the
partitioning behaviour of the inositol stereoisomers between the mobile and liquid phases, a
method was developed to separate the stereoisomers into distinct peaks on the chromatogram
(Figure 4.2a). This method consisted of a starting temperature of 190°C, increased first by 2°
increments to 210°C, held there for 4 minutes, then increased by 35° increments to 290°C, at
which point the temperature was held for 4 minutes. This method resulted in excellent peak
separation using synthetic compounds, but as expected, when organic samples were tested,
background noise elevated thereby masking peak detection and preventing quantification.
Therefore, method development was switched to GC/MS instrumentation, which is superior at
reducing background noise when analyzing organic samples. Once again helium and 5%
diphenyl/95% dimethylpolysiloxane were used as the mobile and stationary phases,
respectively. Attached to the GC was a quadrupole mass spectrometer, which allows the
identification of compounds as they exit the GC column based on their ion fragmentation
spectra. Changes in the GC method were made to better separate inositol peaks as a function of
time and each of these peaks was scanned to ensure that it only contained the compound of
interest. One of the most important adjustments made to the GC protocol was to change the
starting temperature from 190°C to 80°C, which is just above the vaporization temperature of
the solvent used, ethyl acetate. Starting at this temperature, rather than at a higher temperature,
61
Figure 4.2 Gas chromatography method development.
Initial method development to separate myo-, scyllo- and D-chiro-inositol into distinct peaks on
a chromatograph was initially conducted using a GC machine and synthetic inositols (A). Once
peak separation was achieved, method development for organic samples was conducted using a
GC/MS machine (B) and the method was further adjusted to reduce background noise (C).
62resulted in the vaporization of only ethyl acetate and well derivatized compounds. After one
minute the remaining, non-vaporized sample was flushed from the system. This resulted in a
focusing of the sample onto the head of the column and a reduction in background noise (Figure
4.2b,c).
Once peak separation was optimized, a further change was made to the GC/MS protocol, from a
total ion chromatography approach to sample analysis by selected ion recording. The distinct
fragmentation pattern that exists for each compound (Figure 4.3a,b) was used to select certain
ions for mass spectrometer detection. Using a selected ion recording protocol results in the
mass spectrometer focusing its ion detection system away from the recording of every ion, to the
quantification of selected ion(s). This increases the accuracy and detection limits of the
machine. Total ion chromatography was used for the initial method development, to determine
when compounds exit the column to be detected as peaks on the chromatogram (Figure 4.3c).
At this stage, mass spectrometry was used to identify which peaks contained the inositol
stereoisomers of interest. Once these peaks were identified, each was carefully scanned to
confirm the presence of only the one compound. Whenever a fusion of peaks was observed,
changes were made to the GC method to improve peak separation. Once the GC protocol was
optimized, the mass spectrometry protocol was modified to track only the selected ion, rather
than the whole ion spectrum (Figure 4.3d). In addition, the mass spectrometry method was
further adjusted to only track the chosen ion at the three elution times for the inositol
compounds, thus further enhancing ion detection (Figure 4.3e). For brain and CSF samples, ion
168 was chosen, because it is present in the highest quantities in the mass spectra of the inositol
63
Figure 4.3 Selected ion recording.
Samples derivatized and run on the GC/MS using total ion chromatography, resulted in a
separation of myo-, scyllo- and D-chiro-inositol over time on the chromatograph. However, in
organic samples these peaks were masked by background noise. By examining the mass
spectrums for myo- (A), scyllo- (B) and D-chiro-inositol, selected ions specific to the inositol
compounds, could be selected to focus mass spectrometry analysis, to enhance peak detection.
This is evidenced when comparing inositol peaks (outlined by red box) in the total ion
chromatograph (C) to those recorded using selected ion recording (D). The mass spectrometer
method was further adjusted to quantify the selected ion (ion 168 for brain and CSF samples) at
the three time points when the inositol compounds were recorded as exiting the GC column.
64compounds. However, for plasma samples, because of a greater degree of background noise
(Figure 4.4a), masking the inositol peaks even following the selection of ion 168 (Figure 4.4b),
further adjustments were made to the mass spectrometry methodology. Analysis of this region
of the spectrum showed that the noise was being caused by large quantities of derivatized
glucose and galactofuranose in plasma (Figure 4.4c,d). Ions 373 and 432 were examined as
alternatives because they are components of the derivatized inositol mass spectra, but not those
of derivatized glucose or galactofuranose. Ion 373 corresponds to the molecular weight of the
predominate species of derivatized inositol with five acetyl groups, one of which
_____________________________________________________________________________
Figure 4.4 Selected ion recording for plasma samples.
Plasma samples derivatized and run on the GC/MS using total ion chromatography, also showed
myo-, scyllo- and D-chiro-inositol peaks masking by unrelated compounds (A). Selected ion
recording for ion 168 still resulted in peak masking, in these samples, in the region of interest
(B). Therefore, two other ions, 373 (C) and 432 (D), present in the mass spectra of the inositol
compounds, were tested as alternatives. Based on this analysis, ion 373 was chosen for the
selected ion recording of plasma samples. The red box highlights the region of the
chromatograph corresponding to where the three inositol compounds exited the column.
65is shared between two carbons, and ion 432, which corresponds to the molecular weight of fully
derivatized inositol. Ion 432 was not present at quantifiable levels in our samples, whereas ion
373 was present and, effectively eliminated noise allowing for the accurate quantification of
inositol levels in plasma samples.
Once method development was completed, inositol levels present in the brain, CSF and plasma
samples were quantified by comparing the selected ion peaks generated to those resulting from
the injection of derivatized synthetic inositols. Concentration curves, generated using these
synthetic inositols, were used to quantify myo- and scyllo-inositol levels (Figure 4.5).
Baseline brain inositol levels in TgCRND8 mice and their wild-type littermates
Baseline brain levels of myo- and scyllo-inositol were quantified and compared between
TgCRND8 mice and their wild-type littermates. The brain levels of myo– and scyllo–inositol in
the mice were comparable to those previously reported in the literature (Seaquist and Gruetter,
1998; Fisher et al, 2002). Brain myo-inositol concentrations have been reported in the range of
2 to 15 mM (Fisher et al, 2002), which is comparable to the 3 mM observed in this study.
scyllo–Inositol concentrations between 8 and 20% of myo–inositol levels have been reported
previously (Seaquist and Gruetter, 1998), which at an average of 14%, equals a concentration
range for scyllo-inositol of 0.28 mM and 2.1 mM. In the present study, an average of 0.9 mM of
scyllo-inositol was recorded in brain samples from untreated TgCRND8 mice and their wild-
type littermates.
66
Figure 4.5 Examples of myo-inositol and scyllo-inositol concentration curves.
Concentration curves were generated for myo- (A) and scyllo-inositol (B) using synthetic stock
solutions, derivatized and analyzed using GC/MS. Curves such as these were generated for each
experiment and used to calculate the concentrations of myo- and scyllo-inositol present in the
organic samples under analysis.
_____________________________________________________________________________
A comparison of brain myo- and scyllo-inositol concentrations between TgCRND8 mice and
their wild-type littermates, found no significant differences between the two groups (Figure 4.6;
myo-inositol: F1,15 = 2.277, p = 0.152; scyllo-inositol: F1,15 = 0.980, p = 0.338). This suggests
that basal myo- and scyllo-inositol levels present in the brain are not altered by disease
pathology, such as that found in this mouse model of AD.
67
Figure 4.6 Baseline brain myo- and scyllo-inositol levels.
myo-Inositol and scyllo-inositol levels were measured in brain homogenates from TgCRND8
mice and their wild-type littermates using GC/MS. A. No significant differences were observed
in brain myo- and scyllo-inositol levels between the two groups. (p > 0.05). B. An analysis of
the distribution of brain myo- and scyllo-inositol concentrations in both groups shows the wide
range of baseline inositol concentrations present, from 2.3 to 3.5 mM for myo-inositol and from
0.3 to 1.5 mM for scyllo-inositol. (n = 17).
68Brain inositol levels in TgCRND8 mice and their wild-type littermates following ad libitum
myo-inositol treatment
To compare the effects of inositol administration on myo– and scyllo–inositol concentrations
within the brain of TgCRND8 mice and their wild-type littermates, mice were administered
myo–inositol ad libitum in their drinking water at a concentration of 10 mg/ml. The purpose of
this test was to determine whether disease pathology would alter inositol transport into the brain
or its equilibrium/regulation within the brain. myo-Inositol administration was chosen for this
study because it is reported to be the primary substrate transported by all three inositol
transporters. Therefore analysis of this inositol would give an accurate estimate of the effects of
disease pathology, caused by cerebrovascular amyloid, on myo- and scyllo-inositol brain levels.
Ad libitum myo-inositol treatment caused a significant increase in brain myo-inositol levels and a
significant decrease in scyllo-inositol levels in both groups, irrespective of disease phenotype
(Figure 4.7; myo-inositol: F3,28 = 6.647, p = 0.002; scyllo-inositol: F3,28 = 13.710, p < 0.001).
Both TgCRND8 mice and their wild-type littermates showed an approximate 17% increase in
myo-inositol levels following myo-inositol treatment (p = 0.015 in wild-type animals versus p =
0.001 in TgCRND8 mice). scyllo-Inositol levels significantly decreased 5-fold in both groups
following myo-inositol administration (p < 0.001 in wild-type animals versus p = 0.001 in
TgCRND8 mice). Therefore, disease pathology did not change inositol transport or its
regulation within the brain. The decrease in scyllo–inositol levels, observed in both groups
following myo–inositol administration, might reflect a shift in inositol equilibrium towards
degradation pathways, in an effort to reestablish/maintain brain homeostasis.
69
Figure 4.7 The effects of myo-inositol treatment on brain myo- and scyllo-inositol.
Both TgCRND8 mice and their wild-type littermates were administered 10 mg/ml of myo-
inositol ad libitum in their drinking water for one week and brain myo- and scyllo-inositol
concentrations were measured using GC/MS. myo-Inositol levels (A,B) were significantly
increased following myo-inositol ad libitum treatment. In contrast, scyllo-Inositol levels (C,D)
were significantly decreased. (*: p < 0.05; control group: n = 17, myo-inositol group: n = 15).
70The effects of single dose versus continuous scyllo-inositol treatment on CNS inositol levels
Baseline brain myo- and scyllo-inositol levels were quantified and compared to their levels
following either a once-daily gavage dose of 10, 30 or 100 mg/kg/day scyllo-inositol for one
month, or an ad libitum dose of 10 mg/ml scyllo-inositol in their drinking water for one week.
These scyllo-inositol doses were chosen, based on previous research on the effects of scyllo-
inositol treatment in TgCRND8 mice (McLaurin et al, 2006). Gavage doses of 0.3 to 30
mg/kg/day of scyllo-inositol, as well as an ad libitum dose of 10 mg/ml scyllo-inositol, showed a
dose-dependent improvement in spatial memory and a corresponding dose-dependent decrease
in brain Aβ oligomers and plaques (McLaurin et al, 2006). Plaque levels were significantly
reduced following the administration of 1 mg/kg/day or higher doses of scyllo-inositol, soluble
Aβ oligomers were significantly reduced following the administration of 3.3 mg/kg/day or
higher doses of scyllo-inositol and soluble Aβ42 levels were significantly reduced following the
administration of 10 mg/kg/day or higher doses of scyllo-inositol (McLaurin et al, 2006). Ad
libitum scyllo-inositol administration also significantly decreased soluble and insoluble Aβ40
and Aβ42 levels, plaque accumulation and improved spatial memory (McLaurin et al, 2006;
Fenili et al, 2007).
Overall, a significant difference in brain myo- and scyllo-inositol levels was observed between
groups (Figure 4.8a,b; myo-inositol: F4,29 = 6.477, p = 0.001; scyllo-inositol: F4,29 = 87.370, p <
0.001). However, none of the gavaged doses of scyllo-inositol resulted in a significant change
in either myo- or scyllo-inositol levels. Only ad libitum scyllo-inositol treatment resulted in a
71
Figure 4.8 Brain and CSF myo- and scyllo-inositol levels following scyllo-inositol treatment.
scyllo-Inositol treatment consisted of either a once-daily gavage dose of either 10 mg/kg, 30
mg/Kg or 100 mg/kg scyllo-inositol for one month or ad libitum administration of 10 mg/ml
scyllo-inositol in the animals drinking water for one week. Gavaged mice were sacrificed 8
hours following their last treatment, when brain scyllo-inositol levels are maximal (see Figure
4.9B). Gavage treatment did not significantly change either myo- or scyllo-inositol levels in the
brain (A,B) or CSF (C,D). However, ad libitum treatment resulted in a significant decrease in
brain myo-inositol levels (A) and a significant increase in scyllo-inositol levels (B). An
examination of CSF inositol levels following ad libitum treatment showed no significant
changes in myo-inositol levels (C) but a significant increase in scyllo-inositol levels (D). (*: p <
0.05; untreated group: n = 17 for brain and 10 for CSF, ad libitum group: n = 5 for both brain
and CSF, once-daily groups: n = 5 per group for both brain and CSF).
72significant 0.3-fold decrease in brain myo-inositol levels in comparison to the untreated control
group (p < 0.001) and a significant 7-fold increase in brain scyllo-inositol levels in comparison
to all other groups (p < 0.001).
Given the lack of change in brain scyllo-inositol levels following once-daily gavage treatments,
yet a marked increase following ad libitum treatment, the corresponding changes in CSF levels
were examined, to correlate with these findings. Once again, a significant difference was
observed in scyllo-inositol levels but not in myo-inositol levels between groups (Figure 4.8c,d;
myo-inositol: F4,28 = 2.603, p = 0.057; scyllo-inositol: F4,27 = 31.926, p < 0.001). As was found
in the brain, none of the gavaged doses resulted in a significant increase in scyllo-inositol
concentrations but ad libitum scyllo-inositol treatment raised CSF scyllo-inositol levels up to 10-
fold in comparison to the other groups (p < 0.001). The 7.5- and 10-fold increases in brain and
CSF scyllo-inositol levels, respectively, observed following ad libitum treatment, indicates a
shift in the inositol influx/efflux equilibrium pathways at the brain barriers in response to an
increase in peripheral scyllo-inositol levels. This finding suggests that a sustained increase in
scyllo-inositol levels in the periphery will be reflected in a subsequent sustained increase in
brain scyllo-inositol levels. This effect was more pronounced following scyllo–inositol
treatment than myo-inositol treatment, which may reflect a tighter control imposed to maintain
myo-inositol equilibrium. These results further suggest that a once-daily dose of scyllo-inositol
is insufficient to elevate scyllo-inositol levels in the CNS, but that more frequent administration
would result in a sustained increase. These results are in agreement with cognitive testing of
TgCRND8 mice treated once- or twice-daily by gavage with scyllo-inositol, in which only
twice-daily administration had beneficial effects (unpublished data).
73Plasma inositol levels as a function of time, post-scyllo-inositol gavage treatment
Preliminary research (courtesy of Mary Brown, Figure 4.9a,b), found a peak increase in scyllo-
inositol-(2-3H) levels in plasma by 2 hours post-gavage treatment and a peak increase in brain
scyllo-inositol-(2-3H) levels by 8 hours post-treatment. GC/MS analysis of brain samples at 8
hours post-gavage treatment, found scyllo-inositol levels were not significantly altered from
control levels. Therefore, myo- and scyllo-inositol levels were tracked in plasma at 1.5, 2, 4 and
8 hours following scyllo-inositol gavage treatment, to track scyllo-inositol export from the
plasma using GC/MS. A significant difference in both plasma myo- and scyllo-inositol levels
was observed as a function of time post-gavage (Figure 9c,d; myo-inositol: F3,9 = 6.612, p =
0.012; scyllo-inositol: F3,9 = 5.674, p = 0.018). myo-Inositol was significantly decreased by 4
hours post-gavage (p = 0.030) and further decreased by 8 hours (p = 0.019). scyllo-Inositol
levels were not significantly decreased by 4 hours post-gavage (p = 0.064) but were
significantly decreased by 8 hours (p = 0.048). The significant decrease in scyllo-inositol levels
observed in plasma by 8 hours post-gavage, supports the initial findings made using scyllo-
inositol-(2-3H), that brain scyllo-inositol levels would be maximal by 8 hours post-gavage
treatment. In addition, these results, along with the partial but not significant increase in brain
scyllo-inositol levels observed at 8 hours post-gavage treatment, support our earlier suggestion
that once-daily gavage treatments of scyllo-inositol are insufficient to significantly increase
scyllo-inositol levels in the CNS. Multiple gavage treatments per day would be required to
cause a sustained increase in brain scyllo-inositol levels.
74
Figure 4.9 Plasma myo- and scyllo-inositol concentrations following scyllo-inositol treatment.
Bioavailability of scyllo-inositol in plasma (A) and brain (B), determined using orally
administered scyllo-inositol-(2-3H) uptake studies, showed that scyllo-inositol levels increased
rapidly, peaking at 2 hours post-administration in plasma and 8 hours post-administration in
brain (experiment courtesy of Mary Brown). Based on these findings, mice were gavaged with
100 mg/kg scyllo-inositol and plasma myo- and scyllo-inositol levels were quantified at 1.5, 2, 4
and 8 hours post-gavage. There was a significant decrease in plasma myo-inositol levels (C) by
4 hours post-gavage and a significant decrease in scyllo-inositol levels (D) by 8 hours post-
gavage. (§: p = 0.064. *: p < 0.05; 1h30, 4h and 8h groups: n = 3, 2h group: n = 4).
75scyllo-Inositol Incorporation into Phosphatidylinositol
The phosphatidylinositol family of lipids are located at the cellular membrane and are important
regulators of second messenger cell signaling pathways. The head group of phosphatidylinositol
is usually myo-inositol, but scyllo-inositol incorporation into phosphatidylinositol has been
reported to occur in lower organisms, such as mycobacteria and in barley seeds (Kinnard et al,
1995; Ryals et al, 1999; Salman et al, 1999; Riggs et al, 2007). Therefore, we examined
whether scyllo-inositol incorporation into phosphatidylinositol lipids occurred in untreated and
treated mice as a result of fluctuations in brain scyllo-inositol levels. For this purpose, brain
phospholipids from both untreated and ad libitum scyllo-inositol treated animals were isolated
using thin layer chromatography and their head group extracted for derivatization and analysis
using GC/MS. scyllo–Inositol could not be detected in phosphatidylinositol lipids isolated from
the brains of either control or scyllo–inositol-treated mice (Figure 4.10). scyllo–Inositol had an
elution time of 18.2 min on the chromatograph, and a point by point examination of the signal
between 17 and 19 minutes failed to detect scyllo–inositol. The lower limit of detection of the
assay was determined to be approximately 0.25 ng/µl, therefore, although we cannot rule out
minor concentrations of scyllo–inositol being present in brain phosphatidylinositol lipids, if this
occurs, it occurs at negligible levels. Overall, these results suggest that scyllo–inositol does not
become incorporated into phosphatidylinositol when present at elevated concentrations within
the CNS. This suggests that increasing scyllo-inositol levels in the brain should not alter
phosphatidylinositol levels and therefore should not alter signal transduction pathways.
76
Figure 4.10 scyllo-Inositol incorporation into phosphatidylinositol.
GC/MS profiles, determining the levels of myo- and scyllo-inositol isolated from
phosphatidylinositol in control mice versus those given scyllo-inositol. The
phosphatidylinositol head groups were isolated through lipid isolation and hydrolysis. D-chiro-
Inositol was added as an internal standard, the inositol compounds were derivatized and single
mass ion 168 was used to track inositol levels. myo-Inositol was readily detected but scyllo-
inositol could not be detected in any of the samples. This suggests that scyllo-inositol does not
substitute myo-inositol as the head group for phosphatidylinositol. The insets show
amplification of the regions of the chromatograph where scyllo-inositol should occur, a small
arrow in the inset points out where the scyllo-inositol peak would be expected (n = 5 per group).
77Discussion
It has previously been shown that the treatment of TgCRND8 mice with scyllo–inositol ad
libitum resulted in improvements in cognition and a decrease in toxic soluble Aβ species within
the CNS, while myo-inositol administration did not significantly improve these same disease
outcome measures (McLaurin et al, 2006). Using GC/MS, the present study found that both
myo- and scyllo-inositol administration resulted in a corresponding significant increase in their
brain levels. The increases observed following myo-inositol treatment were more moderate and
might explain why myo–inositol was not effective for the treatment of AD pathology in
TgCRND8 mice despite the observed in vitro efficacy (McLaurin et al, 2000; 2006). myo-
Inositol might be more tightly regulated than scyllo–inositol within the CNS, because it is
important for osmolarity control and signal transduction pathways.
Since scyllo-inositol has no known functions within the brain, or the body, its levels appear to be
less tightly regulated than those for myo-inositol. This allows for high CNS bioavailability, an
important concern when designing drug therapeutics. Using various administration paradigms
for scyllo-inositol it was found that ad libitum, but not single-dose scyllo–inositol
administration, resulted in a significant elevation in brain scyllo–inositol levels. Drug efficacy
is dependent on a sustained elevation of the drug in the brain for therapeutic benefit. The
present study would suggest that a multiple dosing regiment might be more effective than a
once-daily dose. In support of this suggestion, previous research examining the effects of
different doses of scyllo-inositol in TgCRND8 mice on disease outcome measures found twice-
daily oral gavage administration of scyllo–inositol more effective than once-daily treatment at
reversing cognitive deficits in the animals (Unpublished results: McLaurin et al, 2006).
78The inter-regulation of myo- and scyllo-inositol in the brain is of interest when examining
inositol as a disease therapeutic. Rat, rabbit, and bovine brains are known to contain an
epimerase that converts myo– to scyllo–inositol and vice versa (Sherman et al, 1968a; b; Hipps
et al, 1977). myo–Inositol administration in women has demonstrated a 1% conversion to
scyllo–inositol within the plasma (Groenen et al, 2003), and myo–inositol treatment in rats
showed a 0.06% conversion to scyllo–inositol (Pak et al, 1992). An increase in inositol levels,
with maintenance of the correlation between scyllo- and myo-inositol, has been reported to occur
as a function of age within the white matter of healthy human subjects (Kaiser et al, 2005). A
simultaneous fluctuation of scyllo- with myo-inositol has also been reported in patients with
brain pathology (Griffith et al, 2007). In AD patients, increases in scyllo-inositol/creatine ratios
were positively correlated with increases in myo-inositol/creatine ratios (Griffith et al, 2007).
Our study also found the levels of these two inositols to be inter-regulated. A significant (p <
0.05) decrease in brain scyllo-inositol levels was recorded following ad libitum myo-inositol
administration and vice versa. The severe drop in scyllo-inositol levels that occurred following
myo-inositol administration, compared to the smaller drop in myo-inositol levels that occurred
following scyllo-inositol administration, supports our earlier statement that myo-inositol levels
are more tightly regulated. The apparent inverse correlation between the two inositols following
ad libitum administration, in comparison to the positive correlation observed in aging humans
and as a result of disease pathology, likely results from early shifts in the levels of these
inositols in an effort to re-establish equilibrium.
While scyllo-inositol has no known function in the body, we wanted to confirm that scyllo-
inositol elevation in the brain would not result in competition with myo-inositol for
incorporation into phosphatidylinositol, and thus alter cell signaling systems. In lower
79organisms such as mycobacteria, tetrahymena cells and barley seeds, scyllo-inositol containing
phosphatidylinositols, phosphatidylinositol-linked glycans and polyphosphoinositols have been
observed (Kinnard et al, 1995; Ryals et al, 1999; Salman et al, 1999; Riggs et al, 2007).
However, scyllo-inositol incorporation into phosphatidylinositols has not been observed in
higher organisms (Takenawa and Egawa, 1977). This may be due to the reduced binding
affinity of phosphatidylinositol synthesizing enzymes for scyllo-inositol, even when levels are
elevated (Takenawa and Egawa, 1977; Salman et al, 1999). The final step in the de novo
synthesis of phosphatidylinositol is catalyzed by CDP-diglyceride:inositol transferase, which
has been found to bind myo-inositol with a Km of 2.5 mM, a reaction for which scyllo-inositol
does not appear to compete (Takenawa and Egawa, 1977). When phosphatidylinositol:inositol
phosphatidyl transferase activity was examined in rat liver microsomes, scyllo-inositol
inhibition of myo-inositol-3H incorporation into phosphatidylinositol was two orders of
magnitude lower than that of myo-inositol and had no inhibitory effect if given at the
micromolar concentrations found in most tissues (Irvine, 1998). These findings are supported
by the present study, which found a lack of scyllo-inositol incorporation into
phosphatidylinositols in mice, both under basal conditions and when scyllo-inositol levels were
elevated in the brain. These combined results suggest that scyllo-inositol does not directly affect
phosphatidylinositol pathways via replacement of myo-inositol and that an increase should not
have deleterious effects. In fact, high levels of scyllo–inositol have been observed in a healthy
subject with no apparent neurological problems, despite a myo– to scyllo–inositol ratio of 5:1
rather than the more typical 12:1 ratio (Seaquist and Gruetter, 1998).
80Therefore, baseline myo- and scyllo-inositol levels are comparable between TgCRND8 mice and
their wild-type littermate. Ad libitum myo-inositol administration resulted in a significant
increase in brain myo-inositol levels in both groups, with no significant difference in uptake
observed as a result of disease pathology. Similarly, ad libitum scyllo-inositol administration
resulted in a significant increase in brain scyllo-inositol levels. This increase was significantly
higher than the increase observed following myo-inositol administration. Both results indicate
that myo- and scyllo-inositol, particularly scyllo-inositol, have excellent CNS bioavailability.
This suggests that compounds bound to scyllo-inositol or derivatives of scyllo-inositol that
utilize the same transport systems might have similar CNS bioavailability, thus supporting my
hypothesis.
81
CHAPTER 5
Quantification of Inositol Transporter Expression Levels
82Abstract
GC/MS dose studies found both myo- and scyllo-inositol levels elevated in the brain following
their oral administration, a finding that is particularly pronounced following scyllo-inositol
administration. This elevation in inositol levels occurs across a concentration gradient as a
result of active transport from the periphery. Three inositol transporters have been reported in
the literature: one hydrogen/myo-inositol transporter (HMIT) and two sodium/myo-inositol
transporters (SMIT1, SMIT2). Expression of all three transporters in the brain have been
previously reported. However, limited information is available on their subregional expression,
how their levels compare to each other and the effects of disease pathology and aging on their
expression. Therefore, transporter mRNA levels were examined in four brain regions: the
cortex, hippocampus, septum and cerebellum, all known to be affected by disease pathology in
AD, in TgCRND8 mice and control animals at 2, 4 and 6 months of age. The objectives of this
study were to: 1. Develop a QPCR method to quantify the mRNA levels of HMIT, SMIT1 and
SMIT2, along with control genes in mouse tissues. 2. Examine the effects of age on transporter
expression. 3. Examine the effects of disease pathology on expression, by comparing
expression between TgCRND8 mice and their wild-type littermates. 4. Compare transporter
expression levels to each other, to determine which transporter is present at the highest levels in
each brain region. 5. Compare the expression of each transporter between brain regions. Aging
and disease pathology did not alter inositol transporter levels. Overall, brain inositol transporter
levels were found in the order HMIT > SMIT1 > SMIT2, however, regional differences in the
expression profiles of the three transporters was observed.
83Introduction
Inositol stereoisomers have shown promise as therapeutic agents in a number of diseases
(Hallman et al, 1986; Hallman et al, 1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al,
1996; Einat et al, 1998; Bersudsky et al, 1999; Nestler et al, 1999; Chengappa et al, 2000;
Gelber et al, 2001; Palatnik et al, 2001; Cogram et al, 2002; Gerli et al, 2003; Eden et al, 2006;
McLaurin et al, 2006). In particular myo- and scyllo-inositol have shown promise as disease
therapeutics in psychiatric disorders, respiratory distress syndrome in premature infants and in
the TgCRND8 mouse model of AD (Hallman et al, 1986; Hallman et al, 1992; Benjamin et al,
1995; Levine et al, 1995; Fux et al, 1996; Chengappa et al, 2000; Gelber et al, 2001; Palatnik et
al, 2001; Eden et al, 2006; McLaurin et al, 2006; Fenili et al. 2007). TgCRND8 mice show
many of the hallmark features of AD, including an increase in cerebral Aβ levels, Aβ
aggregation and plaque deposition, followed by cognitive deficits as the disease advances
(Chishti et al, 2001). While both myo- and scyllo-inositol were affective at preventing Aβ
aggregation in vitro, only scyllo-inositol was found to significantly prevent aggregation in vivo
(McLaurin et al, 2000; 2006). This observed discrepancy between in vitro and in vivo data led
to an examination of the effects of myo- and scyllo-inositol administration on their accumulation
in the brains of both TgCRND8 mice and control animals (Chapter 4). GC/MS analysis found
that scyllo-inositol administration caused a more pronounced increase in scyllo-inositol levels
within the brain, when compared to the levels observed following administration of a similar
dose of myo-inositol (Chapter 4). The discrepancy in effects of myo- and scyllo-inositol
administration on the resulting brain concentrations, points to a potential tight regulation of
myo-inositol equilibrium in the brain. This may result because myo-inositol is a key compound
for the regulation of osmolarity in the brain and is a constituent of phosphatidylinositol; an
84important membrane phospholipid and component of second messenger systems, while scyllo-
inositol has no known function in the brain or in the periphery (Fisher et al, 2002).
scyllo-Inositol was a more effective treatment for AD in transgenic mice and levels were
significantly elevated following its administration, however, both myo- and scyllo-inositol have
shown promise as CNS disease therapeutic agents. Physiologically, these two stereoisomers are
the most abundant inositol stereoisomers found in the body (Michaelis et al, 1993; Seaquist and
Gruetter, 1998). In the brain, 5 mM of myo–inositol and 0.5 mM of scyllo–inositol have been
reported (Michaelis et al, 1993), values 100-fold greater than those found in the periphery
(Palmano et al, 1977). This elevation of inositol levels within the brain compared to the
periphery, suggests the presence of active transport systems, in addition to simple diffusion, for
the regulation of brain inositol levels. Therefore, the effectiveness of myo-inositol in the
treatment of psychiatric ailments and respiratory distress syndrome (Hallman et al, 1986;
Hallman et al, 1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al, 1996; Chengappa et al,
2000; Gelber et al, 2001; Palatnik et al, 2001; Eden et al, 2006) and scyllo-inositol in the
treatment of AD in transgenic mice (McLaurin et al, 2006; Fenili et al., 2007) is probably due to
ready access to the brain through these transporters.
Active transport of inositol stereoisomers, in particular myo- and scyllo-inositol, occurs via
inositol transporters. Three inositol transporters have been reported in the literature, HMIT,
SMIT1 and SMIT2 (Kwon et al, 1992; Uldry et al, 2001; Roll et al, 2002). These inositol
transporters exchange one hydrogen atom or two sodium atoms along their concentration
gradients, to generate enough energy to actively transport myo-inositol across its concentration
gradient (Hager et al, 1995; Uldry et al, 2001; Coady et al, 2002; Bourgeois et al, 2005). While
85all three transporters are expressed in the brain (Berry et al, 1995; Inoue et al, 1996; Uldry et al,
2001; Roll et al, 2002), there is limited information on the subregional expression of the
transporters and no information on how expression changes with disease pathology, such as that
which occurs in the TgCRND8 mouse model of AD. In addition, how age affects their
expression and how the levels of the three transporters compare to each other has not been
previously examined. Therefore, to address these questions, several objectives were set:
1. To develop a QPCR method to quantify the mRNA levels of HMIT, SMIT1 and SMIT2,
standardized against control genes, in mouse tissues.
2. To examine the effects of age on inositol transporter mRNA expression levels, by
examining their expression at 2, 4 and 6 months of age in both TgCRND8 mice and their
wild-type littermates. Time points that correspond to different, key stages in AD pathology
in these animals.
3. To compare inositol transporter mRNA expression levels between TgCRND8 mice and
their wild-type littermates, in order to examine the effects of disease pathology on
expression.
4. To compare the levels of HMIT, SMIT1 and SMIT2 to each other, by quantifying their
expression in four brain regions: the cortex, hippocampus, septum and cerebellum, tissues
that are affected to varying degrees in AD and by Aβ aggregation in TgCRND8 mice.
5. To compare the expression of each inositol transporter between the four brain regions
examined.
86Results
Inositol transporter primer design
Primers were designed, for each of the three inositol transporters, to quantify their mRNA levels
using QPCR. To confirm the effectiveness of primers pairs for amplifying transporter cDNA,
concentration curves of cDNA were used to test the amplification efficiency (slope) and
accuracy (R2 value) of each primer pair (Figure 5.1a, c, e). In addition, dissociation curve
analysis was performed to determine whether one, or multiple, amplification products were
generated by each primer pair (Figure 5.1b, d, f). Primers were selected if their slope was
between 2.9-3.2, their R2 value was between 0.98-1 and if they produced one main amplification
product with minimal, or preferably no, secondary amplification products. The selected SMIT1
and SMIT2 primer pairs showed an excellent level of amplification efficiency and accuracy and
only amplified one product. For HMIT, over 30 primer pairs were tested and the primer pair
selected showed acceptable amplification efficiency and accuracy with only minor non-specific
amplification.
Control gene selection and primer design
Several genes were examined as potential control genes for the QPCR study, based on a
literature search of commonly used control genes, including tyrosine kinase receptor (Abl1), a
RNA-dependent ATPase (Bat1), glyceraldehyde-3-phosphate dehydrogenase (Gapdh),
ribosomal protein (L32), nitric oxide synthase 2 (Nos2), proteosome 26S subunit ATPase 4
(PSMC4), mitochondrial ribosomal protein (S12), succinate dehydrogenase (SDHA), TATA
box binding protein (Tbp) and the transferrin receptor (TFRC). The NCBI, Gene Expression
Omnibus website was used to examine mouse microarray data for each of the potential control
genes. Expression of these genes was examined, with the goal of selecting control genes with
87
Figure 5.1 Concentration and dissociation curves for the inositol transporters primers.
Concentrations curves (A,C,E) and dissociation curves (B,D,F) were analyzed for HMIT (A,B),
SMIT1 (C,D) and SMIT2 (E,F). The concentration curves provided information on the
amplification efficiency (slope) and accuracy (R2 value) of each primer set and the dissociation
curves were used to check for the amplification of one, main product.
88similar expression levels in all the brain regions in this study. This is important because inositol
transporter gene amplification values are normalized using the control gene values, therefore,
uneven control gene amplification between tissues can skew the relative expression values.
Based on an examination of the microarray data, two control genes, Gapdh and Tbp, were
chosen (Figure 5.2a, d). Concentration curves were generated, with their corresponding slope
and R2 values, for both of these control genes (Figure 5.2b, e). Dissociation curves were also
examined to determine if only the product of interested was being generated (Figure 5.2c, f).
The Gapdh primers selected showed an excellent accuracy and efficiency of sequence
amplification and the dissociation curve showed no nonspecific amplification. For Tbp primer
design, 20 primer pairs were examined and the primer pair selected showed an acceptable level
of sequence amplification accuracy and efficiency, with only minor nonspecific amplification.
As expected based on the microarray data, expression levels of the two control genes selected
were similar across all the samples analyzed, indicating that they were appropriate control genes
for this study. Therefore, transporter gene expression levels were normalized to both of the
control genes, as well as within plate and between plate control samples. The resulting relative
expression levels were normalized to an average expression of 1 and statistically analyzed.
QPCR was chosen, rather than PCR, for gene expression analysis because it is a more accurate
method for quantifying mRNA expression levels. This is because QPCR, through the use of a
dye that fluoresces when bound to double-stranded DNA and a machine to quantify
fluorescence following each round of DNA amplification, allows the researcher to track DNA
amplification and to quantify and compare gene expression when DNA amplification is in an
89
Figure 5.2 Microarray, concentration and dissociation curves for each of the control genes.
Microarray data (A,D) from the NCBI, Gene Expression Omnibus website was used to select
control genes with comparable expression levels in the regions of interest in the present study
(highlighted using blue boxes). Concentration curves (B,E) and dissociation curves (C,F) were
used to confirm the accuracy and efficiency of the primers. Based on these studies, Gapdh (A-
C) and Tbp (D-F) were selected.
90exponential growth phase for each gene under analysis. In contrast, PCR amplification only
allows the researcher to try and quantify DNA levels after amplification is completed, when
some gene amplification will have reached a plateau, while others will still be in an exponential
amplification phase. This can result in the over/under estimation of DNA expression.
Inositol transporter expression as a function of age
The mRNA expression of the three inositol transporters was examined as a function of age, by
examining their expression at 2, 4 and 6 months of age. No significant differences in the mRNA
expression of any of the three inositol transporters was observed as a function of age, either in
the TgCRND8 mice or in their wild-type littermates (Figure 5.3-5.5; p > 0.05). Therefore,
mRNA expression of all three inositol transporters, remained stable across the time period
examined.
Inositol transporter expression as a function of disease
The mRNA expression of the three inositol transporters was compared between TgCRND8 mice
and their wild-type littermates at 2, 4 and 6 months of age, to examine the effects of disease
pathology on inositol transporter expression. These time points were selected because they
correspond to pre-plaque deposition, mid-stage AD and advanced stage AD in TgCRND8 mice
(Chishti et al, 2001). No significant differences were observed between TgCRND8 mice and
their wild-type littermates, for any of the transporters, in any of the brain regions examined
(Figure 5.3-5.5; p > 0.05). These findings agree with GC/MS results, which found no
differences in myo-inositol uptake between TgCRND8 mice and their wild-type littermates
(Figure 4.7).
91
Figure 5.3 HMIT expression as a function of age in TgCRND8 mice and their wild-type
littermates.
HMIT expression was examined in wild-type (A-D) and TgCRND8 mice (E-H) in the cortex
(A,E), hippocampus (B,F), septum (C,G) and cerebellum (D,H) at 2, 4 and 6 months of age. No
significant change in HMIT expression was observed as a function of time in either group. A
comparison of HMIT expression between TgCRND8 mice and their wild-type littermates also
found no significant differences. (p > 0.05; n = 5 animals per group, 3 wells per sample).
92
Figure 5.4 SMIT1 expression as a function of age in TgCRND8 mice and their wild-type
littermates.
SMIT1 expression was examined in wild-type (A-D) and TgCRND8 mice (E-H) in the cortex
(A,E), hippocampus (B,F), septum (C,G) and cerebellum (D,H) at 2, 4 and 6 months of age. No
significant change in SMIT1 expression was observed as a function of time in either group. A
comparison of SMIT1 expression between TgCRND8 mice and their wild-type littermates also
found no significant differences. (p > 0.05; n = 5 animals per group, 3 wells per sample).
93
Figure 5.5 SMIT2 expression as a function of age in TgCRND8 mice and their wild-type
littermates.
SMIT2 expression was examined in wild-type (A-D) and TgCRND8 mice (E-H) in the cortex
(A,E), hippocampus (B,F), septum (C,G) and cerebellum (D,H) at 2, 4 and 6 months of age. No
significant change in SMIT2 expression was observed as a function of time in either group. A
comparison of SMIT2 expression between TgCRND8 mice and their wild-type littermates also
found no significant differences. (p > 0.05; n = 5 animals per group, 3 wells per sample).
94A comparison of brain inositol transporter gene expression
An examination of the mRNA expression levels of the three inositol transporters were compared
in four regions of the brain: the cortex, hippocampus, septum and cerebellum. These four brain
regions were examined because they are affected to varying degrees by Aβ plaque deposition in
AD (Thal et al, 2002). The earliest plaque deposition is observed in the cortex and
hippocampus, followed by the septum and finally the cerebellum (Thal et al, 2002). Deposition
in the cerebellum is only observed in the very advanced stages of AD and the patient often
succumbs to the disease prior to deposition being observed (Thal et al, 2002). The brain mRNA
expression levels of the three inositol transporters, in comparison to each other, was found to be
significantly different, irrespective of the brain region examined (Figure 5.6; F2,117 = 468.1, p <
0.001). Overall, transporter mRNA expression levels in the brain were in the order HMIT >
SMIT1 > SMIT2. When analyzed in greater detail, HMIT mRNA expression was significantly
higher than SMIT1 and SMIT2 expression in all regions of the brain tested (p < 0.001), except
for the cerebellum, where HMIT levels were significantly higher than SMIT2 (p < 0.05), but not
SMIT1. SMIT1 mRNA expression was significantly higher than SMIT2 levels in the
hippocampus and septum (p < 0.001), but expression of the two transporters was not
significantly different in the cortex or the cerebellum.
Regional brain inositol transporter expression levels
An examination of each inositol transporter’s mRNA expression levels between the four brain
regions examined was found to be significantly different (F3,86 = 54.87, p < 0.001). In addition,
a significant correlation between brain region and the expression levels of each gene were
observed (F6,354 = 39.48, p < 0.001). HMIT showed the highest mRNA expression in the cortex
and septum and expression of the gene was not significantly different between the two
95
Figure 5.6 Relative mRNA expression of the three inositol transporters in the brain.
mRNA expression of the three inositol transporters: HMIT, SMIT1 and SMIT2 were determined
in the: A. cortex, B. hippocampus, C. septum and D. cerebellum. In all regions of the brain
tested, except the cerebellum, expression occurred in the order HMIT > SMIT1 > SMIT2. (n =
30 animals; * = p < 0.05).
96regions (Figure 5.7a). The next highest HMIT expression was observed in the hippocampus,
which had a significantly lower expression of HMIT than the other two regions (p < 0.001).
Finally, HMIT mRNA expression in the cerebellum was significantly lower than the other three
brain regions examined (p < 0.05). Therefore, the order of HMIT expression observed was
cortex = septum > hippocampus > cerebellum (Figure 5.7a).
For SMIT1 mRNA, the highest levels of expression were in the septum, followed by the
cerebellum, hippocampus and cortex (Figure 5.7b). SMIT1 cerebellular mRNA expression was
significantly lower than septal SMIT1 expression (p < 0.05). Cortical and hippocampal SMIT1
mRNA expression were significantly lower than septal expression (p < 0.001) but not cerebellar
SMIT1 expression. Cortical and hippocampal SMIT1 expression were not significantly
different from each other. Overall, SMIT1 expression occurred in the order septum >
cerebellum ≥ cortex = hippocampus (Figure 5.7b).
In contrast, brain SMIT2 mRNA expression was highest in the cerebellum (Figure 5.7c). The
next highest expression was in the septum, followed by the cortex and finally the hippocampus.
Septal and cortical SMIT2 mRNA expression levels were not significantly lower than cerebellar
levels. Hippocampal SMIT2 levels were significantly lower than cerebellar and septal levels (p
< 0.005), but not cortical levels. Overall, SMIT2 expression occurred in the order cerebellum ≥
septum = cortex ≥ hippocampus (Figure 5.7c).
97
Figure 5.7 A comparison of regional expression for each of the inositol transporters.
A comparison of the regional expression patterns of the three inositol transporters, HMIT (A),
SMIT1 (B) and SMIT2 (C) in the brain showed a distinct pattern of expression for each
transporter in the brain. While HMIT levels were highest in both the cortex and the septum,
SMIT1 levels were highest in the septum alone and SMIT2 levels were highest in the
cerebellum. (n = 30 animals; * = p < 0.05).
98Kidney inositol transporter expression
In addition to examining transporter expression levels in the brain, expression was examined in
the kidney as a positive control tissue for the QPCR technique. Expression of all three
transporters has been previously reported in the kidney (Berry et al, 1995; Uldry et al, 2001;
Roll et al, 2002). Similar to what was observed in the brain, disease pathology and age did not
significantly alter mRNA expression for any of the three inositol transporters. However, in
contrast to the brain, expression in the kidney occurred in the order SMIT2 > SMIT1 > HMIT
(Figure 5.8). SMIT2 levels were extremely high in the kidney, significantly higher than the
expression of the other two transporters (p < 0.001). While HMIT levels were the lowest,
significantly lower than the other two transporters (p < 0.001) and could almost be considered
negligible.
99
Figure 5.8 Kidney inositol transporter expression.
Examination of inositol transporter expression in the kidney showed expression in the order
SMIT2 > SMIT1 > HMIT. Relative expression of SMIT2 (A) in the kidney was significantly
higher than that of SMIT1 and HMIT (B). HMIT levels were extremely low in this tissue. (red
= SMIT1, green = HMIT, n = 30 animals).
100Discussion
Treatment of psychiatric and neurodegenerative disorders by inositol stereoisomers, requires
their transport across the blood-brain and blood-CSF barriers to areas of the brain where they
are needed. Since the concentrations of both myo- and scyllo-inositol are 100-fold higher in the
brain than their concentrations in the periphery (Palmano et al, 1977), active transport of inositol
into the brain is required. There are three inositol transporters that have been reported in the
literature: HMIT, SMIT1 and SMIT2 (Kwon et al, 1992; Uldry et al, 2001; Roll et al, 2002), all
of which are expressed in the brain (Berry et al, 1995; Inoue et al, 1996; Uldry et al, 2001; Roll
et al, 2002). Therefore, active transport of inositol into the brain and between regions of the
brain, would be expected to occur through any of these three transporters.
Oral administration of ad libitum myo- or scyllo-inositol to mice increased their corresponding
levels in the brain (Chapter 4). Both TgCRND8 mice and their wild-type littermates showed a
significant increase in myo-inositol levels in the brain following ad libitum administration
(Chapter 4). Baseline and inositol levels following treatment were comparable between the two
groups (Chapter 4). These findings would suggest that TgCRND8 mice and wild-type mice
express all three inositol transporters in the brain and that disease pathology does not alter
inositol transporter expression levels. This hypothesis is supported by the observations made in
the present study, which found matching HMIT, SMIT1 and SMIT2 expression profiles between
TgCRND8 mice and their wild-type littermates at all three of the time points tested. Since these
time points correspond to pre-plaque formation, established disease pathology and advanced
disease pathology in TgCRND8 mice (Chishti et al, 2001), this indicates that expression of all
101three inositol transporters is not altered even when AD pathology is in its advanced stages. A
stable expression pattern, irrespective of disease pathology, is an important factor for successful
CNS drug design, because it removes one potential confounding variable to shuttling drugs into
the brain using these transporters.
In addition to examining the effects of disease pathology on transporter expression, the effect of
age was also examined. A comparison of the transporter expression levels, at 2, 4 and 6 months
was performed in both TgCRND8 mice and their wild-type littermates, to determine whether
drug access to the brain, via these transport systems, would be altered as a function of age.
Fluctuations in inositol transporters with age would complicate drug testing and dose studies by
adding another variable to consider. This study indicates that, at least within the age range
tested, expression of the three inositol transporters is stable and unaffected by age.
Other variables to consider are the relative expression of the three transporters to each other and
their subregional expression in the brain. Based on the literature it is known that all three
inositol transporters are expressed in the brain (Berry et al, 1995; Inoue et al, 1996; Uldry et al,
2001; Roll et al, 2002), however, a more detailed examination of their expression profiles,
allows for a better understanding of active inositol transport within the brain and between
subregions. This study found differences in the relative expression levels of the three
transporters. Overall, expression in the brain occurred in the order HMIT > SMIT1 > SMIT2.
Based on this information, compounds designed for transport via HMIT would be expected to
reach the brain, and subregions of the brain, in higher quantities than compounds designed for
transport through SMIT2. However, each transporter appears to have a distinct regional
expression profile as well, which complicates this basic analysis. In the brain regions examined,
102HMIT expression occurred in the order cortex = septum > hippocampus > cerebellum, while
SMIT1 expression occurred in the order septum > cerebellum ≥ cortex = hippocampus and
SMIT2 expression occurred in the order cerebellum ≥ septum = cortex ≥ hippocampus.
Therefore, if drug targeting to the cerebellum is required, than SMIT2 is the superior
transporter, because brain expression levels were highest in the cerebellum, in comparison to the
other three brain regions. In contrast, if targeted transport to the septum is required, then SMIT1
might be the better choice. In theory, depending on the transporter chosen, drug accumulation
in selected subregions of the brain could be favored, an interesting prospect if increasing drug
bioavailability to subregions of the brain were desired.
One caveate to using mRNA quantification to monitor transporter expression profiles is the
herteogeneity of mRNA transcript half-lives that exist in the cell. Therefore, a combination of
mRNA and protein quantification is usually preferred to add confidence to any conclusions
reached regarding gene expression, transcription and translation. For this reason, the original
project sought, in addition to monitoring mRNA levels of the transporters, to monitor protein
concentrations, using both Western blots and immunohistochemistry. However, repeated efforts
were made to create polyclonal antibodies for each of the transporters, without success. In
addition, commercially available antibodies for each of the transporters were tested. In both
instances, significant background and nonspecific labeling was observed and therefore, this
aspect of the project was abandoned.
An additional variable that can be considered for CNS drug design is the rate at which a drug
will be excreted from the body. In this study, kidney inositol transporter levels were examined
as a positive control, because all three of the inositol transporters are expressed in the kidney
103(Berry et al, 1995; Uldry et al, 2001; Roll et al, 2002). Since the kidney is the main avenue for
inositol removal from the body (Arner et al, 2006), this additional information gives insights
into which transporters are important regulators of inositol excretion. As was observed in the
brain, inositol transporter expression levels were not altered with either disease pathology or as
a function of age in the kidney, therefore suggesting that inositol excretion is unaltered by either
variable. A comparison of the expression of the three inositol transporters, relative to each
other, found expression in the order SMIT2 > SMIT1 > HMIT. This is the opposite of what was
observed in the brain. QPCR analysis found a very high expression of SMIT2 in the kidney, 80-
fold higher than the levels observed in the brain. A higher level of SMIT2 expression in the
kidney, relative to the brain, has been previously reported in the literature (Roll et al, 2002). In
contrast, HMIT levels were much lower in the kidney, relative to the brain, and its levels in the
kidney were barely quantifiable. Uldry and colleagues (2001) also reported predominant HMIT
expression in the brain, with limited expression in the kidney. This would suggest that targeting
drugs to HMIT and SMIT1 would result in higher CNS bioavailability, than targeting drugs to
SMIT2.
In conclusion, inositol transporter expression in both the brain and the kidney is unaltered by
disease pathology, as determined by comparing their expression profiles in TgCRND8 mice and
their wild-type littermates, or as a function of age. Overall, inositol transporters in the brain
were expressed in the order of HMIT > SMIT1 > SMIT2, while kidney inositol transporter
expression occurred in the order SMIT2 > SMIT1 > HMIT. Both these findings would suggest
that drug transport via HMIT or SMIT1 would have a greater likelihood of entering the brain in
quantifiable amounts, than transport via SMIT2. Expression of all three transporters was found
in all the brain regions examined, therefore, once a drug is transported into the brain using any
104of these systems, the drug should be able to travel to any region of the brain. However, the
regional expression profile of each transporter was unique. This feature might be exploitable to
favorably target drug transport to particular subregions of the brain. An examination of the
features of inositol required for transport would give a better indication of the degree to which
each transporter system can be manipulated for targeted drug transport.
104
CHAPTER 6
Substrate Structural Requirements for Inositol Transport
105Abstract
Inositol stereoisomers, such as myo- and scyllo-inositol, are known to cross into the brain and
their ad libitum administration has been shown to result in an increase in their brain levels. This
indicates that inositol stereoisomers, especially scyllo-inositol, have excellent CNS
bioavailability. The main transport mechanism via which inositols enters the brain is the
inositol transporters. Given the excellent CNS bioavailability that is possible through these
transporters, it may be possible to use them to shuttle other compounds into the brain. To
determine whether and to what extent substrates could be adapted and still transported into the
brain using this system, the structural features of myo-inositol required for active transport were
determined. A competitive transport assay was developed, to quantify myo- and scyllo-inositol
transport, in the presence or absence of 23 potential competitive substrates. The substrates
tested consisted of inositol stereoisomers, inositol derivatives and other related compounds. The
results of these transport assays were then used to draw models of the basic structural features
required for active transport. Based on these results, SMIT1 and SMIT2 appear to have
different substrate structural requirements for active transport. Active transport through either
appears to be very sensitive to changes in the structure of myo-inositol and it is concluded that
only conservative changes are possible to maintain active transport.
106Introduction
One of the main challenges in the treatment of CNS diseases is transport of drugs across the
brain barriers to regions of bioactivity. There are several strategies in which this can be
accomplished. One possibility is to design drugs irrespective of this concern and try and use
barrier circumvention strategies, such as intranasal delivery or osmotic disruption, to avoid the
brain barriers entirely or temporarily open them to allow the drugs to get through. Alternatively,
barrier navigation strategies, such as nanoparticle delivery or a Trojan horse delivery system can
be used to convert a compound that cannot enter the brain into one that can. A final strategy is
to try and use a compound, which enters the brain for other purposes, as a drug candidate and
potentially as a drug carrier.
scyllo-Inositol is an example of a compound that is present in the body, crosses into the brain
naturally and is an effective drug for the treatment of AD in TgCRND8 mice (Fisher et al, 2002;
McLaurin et al, 2006). In a previous chapter of this thesis, I showed that ad libitum
administration resulted in a sustained increase in scyllo-inositol levels within the brain (Chapter
4). In addition, twice-daily scyllo-inositol treatment was an effective treatment of disease
pathology in TgCRND8 mice (McLaurin et al, 2006). Both these results indicate that scyllo-
inositol has excellent CNS bioavailability and suggests that derivatives of scyllo-inositol or
compounds attached to scyllo-inositol might show the same rates of CNS bioavailability. In
order for these compounds to be successfully transported into the brain, they need to cross the
brain barriers. Three transporters have been reported in the literature as being inositol
transporters: HMIT, SMIT1 and SMIT2, (Hager et al, 1995; Ostlund et al, 1996; Wiese et al,
1996; Lubrich et al, 2000; Uldry et al, 2001; Coady et al, 2002; Berry et al, 2003; Bissonnette et
107al, 2004; Uldry et al, 2004; Bourgeois et al, 2005; Aouameur et al, 2007; Lahjouji et al, 2007;
Shaldubina et al, 2007; Bissonnette et al, 2008; Lin et al, 2009).
HMIT is mainly expressed in the brain, with limited expression in the kidney and adipose tissue
(Uldry et al, 2001). In the brain, mRNA expression was observed in both neurons and glia, with
high levels found in all the brain regions examined: cerebral cortex, hippocampus,
hypothalamus, cerebellum and brainstem (Uldry et al, 2001). In a previous chapter of this
thesis, I recorded high brain HMIT mRNA expression levels in all four of the brain regions
examined: cerebral cortex, hippocampus, septum and cerebellum (Chapter 5). Again, in
agreement with previous reports, I found kidney HMIT mRNA expression levels to be
extremely low (Chapter 5). In cells, HMIT is internalized because of three internalization
sequences in the protein code of HMIT, but cell surface translocation has been reported to occur
following membrane depolarization or a decrease in extracellular pH; conditions that are typical
byproducts of synaptic activity (Uldry et al, 2001; 2004).
In contrast to HMIT, SMIT1 has a much wider mRNA expression pattern, with expression
reported in the kidney, brain, placenta, pancreas, heart, skeletal muscle and in the lung (Berry et
al, 1995). In the brain, SMIT1 mRNA expression is highest in the choroid plexus (Inoue et al,
1996). In addition, expression has been reported in the hippocampus, locus coeruleus, the
suprachiasmatic nucleus, the olfactory bulb and in certain parts of the cerebellum (Inoue et al,
1996). In Chapter 5, I showed, using QPCR, expression of SMIT1 in all four of the brain
regions studied: cerebral cortex, hippocampus, septum and cerebellum (Chapter 5). Based on
SMIT1 knockout studies, SMIT1 appears to be the main transporter responsible for maintaining
myo-inositol levels both in the brain and in the periphery (Berry et al, 2003). Homozygous
108knockout mice showed a 92% reduction in brain myo-inositol levels and an 84% reduction in
myo-inositol levels in the periphery (Berry et al, 2003). Heterozygous mice showed a 15%
reduction in frontal cortex myo-inositol levels and a 25% reduction in hippocampal myo-inositol
levels, suggesting these regions are particularly dependent on SMIT1 activity (Shaldubina et al,
2007). This high dependence of myo-inositol levels on SMIT1 transport suggests that designing
drugs to target this transporter might be a novel strategy for maximizing drug transport into the
brain, particularly to the frontal cortex and hippocampus.
The final transporter, SMIT2, is part of the same sodium/solute symporter family as SMIT1.
Like SMIT1, SMIT2 shows a wide level of expression in the periphery, with the highest levels
of expression reported in the kidney, liver, heart, skeletal muscles and placenta (Roll et al,
2002). However, unlike what is observed for SMIT1, SMIT2 expression in the brain is much
lower (Roll et al, 2002). Using QPCR, I showed very high levels of SMIT2 mRNA expression
in the mouse kidney and minimal expression in the brain (Chapter 5).
Based on the literature and my work presented in previous chapters of this thesis, it was
concluded that all three of the inositol transporters are expressed in the brain and all three are
potential portals for drug delivery to the CNS. Therefore, the objective of this study was to
characterize the structural features of inositol required for active transport through HMIT,
SMIT1 and SMIT2. A competitive transport assay was developed, to quantify myo- and scyllo-
inositol transport, in the presence or absence of potential competitive substrates, consisting of
inositol stereoisomers, derivatives and related compounds (Figure 6.1). This technique allowed
for an estimation of the structural features of inositol required for active transport by HMIT,
SMIT1 and SMIT2, with the caveate that transport channel blockers would also appear to be
109
Figure 6.1 Structures of the inositol stereoisomers, derivatives and related compounds.
The structures of the inositol stereoisomers, derivatives and related compounds used for the
initial competitive transport assays.
110transported by this system. To alleviate the possibility that channel blockers would be
misinterpreted as transported substrates, a model was drawn of the structural features required
for active transport and a comparison was made between this model and both compounds
apparently transported and not transported by this system. Using this technique, I was able to
identify one SMIT transport channel blocker, L-fucose, and additional experiments were
conducted to support this conclusion.
Results
Interspecies and intertransporter protein homology
Before examining the transport substrate specificity of each of the three transporters,
interspecies protein alignment studies were conducted between the human, mouse and rat
transporter sequences to see the degree of evolutionarily conservation. A large degree of
variability in the protein sequences between species would complicate substrate transport
studies, by requiring species-specific substrate transport models be designed. However,
alignment of the protein sequences showed all three transporter sequences are very similar
between the species examined (Figure 6.2-6.4). Overall, homology of the human, mouse and rat
sequences was 90% for HMIT, 94% for SMIT1 and 89% for SMIT2. The HMIT protein was
87% homologous between rats and humans, 88% homologous between mice and humans and
95% homologous between rats and mice (Figure 6.2). The SMIT1 sequence was 92%
homologous between rats and humans, 94% homologous between mice and humans and 96%
homologous between rats and mice (Figure 6.3). Finally, the SMIT2 protein was 86%
homologous between the human and both the rat and mouse sequences and 94% homologous
between the rat and mouse sequences (Figure 6.4).
111Figure 6.2 Interspecies protein alignment for HMIT.
An alignment of the human, mouse and rat HMIT proteins, to compare their amino acid
sequences. Dots and dotted lines indicate gaps in the sequence between species, added for the
purposes of an improved alignment. The consensus line provides information on where the
three sequences match (wherever an amino acid is listed) and where a difference exists (blank
spaces). Positive amino acids are shown in red, negative amino acids in bold, while the rest are
neutral. The human sequence is 88% and 87% homologous with the mouse and rat sequences,
respectively, while the mouse and rat sequences are 95% homologous with each other. The
protein sequences were acquired using the NCBI database. Accession Numbers: human =
NM_052885, mouse = NM_001033633 and rat = NM_133611.
Human MSRKASENVEYTLRSLSSLMGERRRKQPEPDAASAAGECS 40 Mouse MSRKASEdVEYTLRSLSSLMGERRRrQPEPgApg..GErS 38 Rat ...................MGERRRrQPEPgApg..GErS 19 Consensus mgerrr qpep a ge s Human LLAAAESSTSLQSAGAGGGGVGDLERAARRQFQQDETPAF 80 Mouse LL.AAESaaSLQgAe........LERAARRQFQrDETPAF 69 Rat LL.AAESaaSLQgAe........LERAARRQFQrDETPAF 50 Consensus ll aaes slq a leraarrqfq detpaf Human VYVVAVFSALGGFLFGYDTGVVSGAMLLLKRQLSLDALWQ 120 Mouse VYaaAaFSALGGFLFGYDTGVVSGAMLLLrRQmrLgAmWQ 109 Rat VYaaAaFSALGGFLFGYDTGVVSGAMLLLrRQmrLgAmWQ 90 Consensus vy a fsalggflfgydtgvvsgamlll rq l a wq Human ELLVSSTVGAAAVSALAGGALNGVFGRRAAILLASALFTA 160 Mouse ELLVSgaVGAAAVaALAGGALNGalGRRsAILLASALcTv 149 Rat ELLVSgaVGAAAVaALAGGALNGalGRRsAILLASALcTv 130 Consensus ellvs vgaaav alaggalng grr aillasal t Human GSAVLAAANNKETLLAGRLVVGLGIGIASMTVPVYIAEVS 200 Mouse GSAVLAAAaNKETLLAGRLVVGLGIGIASMTVPVYIAEVS 189 Rat GSAVLAAAaNKETLLAGRLVVGLGIGIASMTVPVYIAEVS 170 Consensus gsavlaaa nketllagrlvvglgigiasmtvpvyiaevs Human PPNLRGRLVTINTLFITGGQFFASVVDGAFSYLQKDGWRY 240 Mouse PPNLRGRLVTINTLFITGGQFFASVVDGAFSYLQKDGWRY 229 Rat PPNLRGRLVTINTLFITGGQFFASVVDGAFSYLQKDGWRY 210 Consensus ppnlrgrlvtintlfitggqffasvvdgafsylqkdgwry Human MLGLAAVPAVIQFFGFLFLPESPRWLIQKGQTQKARRILS 280 Mouse MLGLAAiPAVIQFlGFLFLPESPRWLIQKGQTQKARRILS 269 Rat MLGLAAiPAVIQFlGFLFLPESPRWLIQKGQTQKARRILS 250 Consensus mlglaa paviqf gflflpesprwliqkgqtqkarrils Human QMRGNQTIDEEYDSIKNNIEEEEKEVGSAGPVICRMLSYP 320 Mouse QMRGNQTIDEEYDSIrNsIEEEEKEataAGPiICRMLSYP 309 Rat QMRGNQTIDEEYDSIrNsIEEEEKEasaAGPiICRMLSYP 290
112Consensus qmrgnqtideeydsi n ieeeeke agp icrmlsyp Human PTRRALIVGCGLQMFQQLSGINTIMYYSATILQMSGVEDD 360 Mouse PTRRALvVGCGLQMFQQLSGINTIMYYSATILQMSGVEDD 349 Rat PTRRALaVGCGLQMFQQLSGINTIMYYSATILQMSGVEDD 330 Consensus ptrral vgcglqmfqqlsgintimyysatilqmsgvedd Human RLAIWLASVTAFTNFIFTLVGVWLVEKVGRRKLTFGSLAG 400 Mouse RLAIWLASiTAFTNFIFTLVGVWLVEKVGRRKLTFGSLAG 389 Rat RLAIWLASiTAFTNFIFTLVGVWLVEKVGRRKLTFGSLAG 370 Consensus rlaiwlas taftnfiftlvgvwlvekvgrrkltfgslag Human TTVALIILALGFVLSAQVSPRITFKPIAPSGQNATCTRYS 440 Mouse TTVALIILALGFlLSAQVSPRvTFrPttPSdQNtTCTgYS 429 Rat TTVALtILALGFlLSAQVSPRvTFrPtAPSGQNATCTeYS 410 Consensus ttval ilalgf lsaqvspr tf p ps qn tct ys Human YCNECMLDPDCGFCYKMNKSTVIDSSCVPVNKASTNEAAW 480 Mouse YCNECMLDPDCGFCYKiNgSaVIDSSCVPVNKASTtEAAW 469 Rat YCNECMLDPDCGFCYKiNsSaVIDSSCVPVNKASTNEAAW 450 Consensus ycnecmldpdcgfcyk n s vidsscvpvnkast eaaw Human GRCENETKFKTEDIFWAYNFCPTPYSWTALLGLILYLVFF 520 Mouse GRCdNETKFKaEgahWAYsFCPTPYSWTALvGLvLYLVFF 509 Rat GRCENETKFKaEDvhWAYsFCPTPYSWTALvGLvLYLVFF 490 Consensus grc netkfk e way fcptpyswtal gl lylvff Human APGMGPMPWTVNSEIYPLWARSTGNACSSGINWIFNVLVS 560 Mouse APGMGPMPWTVNSEIYPLWARSTGNACSaGINWIFNVLVS 549 Rat APGMGPMPWTVNSEIYPLWARSTGNACSaGINWIFNVLVS 530 Consensus apgmgpmpwtvnseiyplwarstgnacs ginwifnvlvs Human LTFLHTAEYLTYYGAFFLYAGFAAVGLLFIYGCLPETKGK 600 Mouse LTFLHTAEYLTYYGAFFLYAGFAAVGLLFvYGCLPETKGK 589 Rat LTFLHTAEYLTYYGAFFLYAGFAAVGLLFvYGCLPETKGK 570 Consensus ltflhtaeyltyygafflyagfaavgllf ygclpetkgk Human KLEEIESLFDNRLCTCGTSDSDEGRYIEYIRVKGSNYHLS 640 Mouse KLEEIESLFDhRLCsCGaaDSDEGRYIEYIRVKGSNYHLS 629 Rat KLEEIESLFDhRLCTCGTaDSDEGRYIEYIRVKGSNYHLS 610 Consensus kleeieslfd rlc cg dsdegryieyirvkgsnyhls Human DNDASDVE 648 Mouse DNDASDVE 637 Rat DNDASDVE 618 Consensus dndasdve
113Figure 6.3 Interspecies protein alignment for SMIT1.
An alignment of the human, mouse and rat SMIT1 proteins, to compare their amino acid
sequences. Dots and dotted lines indicate gaps in the sequence between species, added for the
purposes of an improved alignment. The consensus line provides information on where the
three sequences match (wherever an amino acid is listed) and where a difference exists (blank
spaces). Positive amino acids are shown in red, negative amino acids in bold, while the rest are
neutral. The human sequence is 94% and 92% homologous with the mouse and rat sequences,
respectively, while the mouse and rat sequences are 96% homologous with each other. The
protein sequences were acquired using the NCBI database. Accession Numbers: human =
NM_006933, mouse = NM_017391 and rat = NM_053715.
Human MRAVLDTADIAIVALYFILVMCIGFFAMWKSNRSTVSGYF 40 Mouse MRAVLeaADIAvVALYFILVMCIGFFAMWKSNRSTVSGYF 40 Rat MRAVLeTADIAIVALYFvLVMCIGFFAMWKSNRSTVSGYF 40 Consensus mravl adia valyf lvmcigffamwksnrstvsgyf Human LAGRSMTWVTIGASLFVSNIGSEHFIGLAGSGAASGFAVG 80 Mouse LAGRSMTWVaIGASLFVSNIGSEHFIGLAGSGAASGFAVG 80 Rat LAGRSMTWVaIGASLFVSNIGSEHFIGLAGSGAASGFAVG 80 Consensus lagrsmtwv igaslfvsnigsehfiglagsgaasgfavg Human AWEFNALLLLQLLGWVFIPIYIRSGVYTMPEYLSKRFGGH 120 Mouse AWEFNALLLLQLLGWVFIPIYIRSGVYTMPEYLSKRFGGH 120 Rat AWEFNALLLLQLLGWVFIPIYIRSGVYTMPEYLSKRFGGH 120 Consensus awefnallllqllgwvfipiyirsgvytmpeylskrfggh Human RIQVYFAALSLILYIFTKLSVDLYSGALFIQESLGWNLYV 160 Mouse RIQVYFAALSLlLYIFTKLSVDLYSGALFIQESLGWNLYV 160 Rat RIQVYFAALSLlLYIFTKLSVDLYSGALFIQESLGWNLYV 160 Consensus riqvyfaalsl lyiftklsvdlysgalfiqeslgwnlyv Human SVILLIGMTALLTVTGGLVAVIYTDTLQALLMIIGALTLM 200 Mouse SVILLIGMTALLTVTGGLVAVIYTDTLQALLMIIGALTLM 200 Rat SVILLIGMTALLTVTGGLVAVIYTDTLQALLMIIGALTLM 200 Consensus svilligmtalltvtgglvaviytdtlqallmiigaltlm Human IISIMEIGGFEEVKRRYMLASPDVTSILLTYNLSNTNSCN 240 Mouse vISmvkIGGFEEVKRRYMLASPDVaSILLkYNLSNTNaCm 240 Rat vISmMkvGGFEEVKRRYMLASPnVaSILLThNLSNTNSCm 240 Consensus is ggfeevkrrymlasp v sill nlsntn c Human VSPKKEALKMLRNPTDEDVPWPGFILGQTPASVWYWCADQ 280 Mouse VhPKanALKMLRdPTDEDVPWPGFILGQTPASVWYWCADQ 280 Rat VhPKadALKMLRdPTDEDVPWPGFILGQTPASVWYWCADQ 280 Consensus v pk alkmlr ptdedvpwpgfilgqtpasvwywcadq Human VIVQRVLAAKNIAHAKGSTLMAGFLKLLPMFIIVVPGMIS 320 Mouse VIVQRVLAAKNIAHAKGSTLMAGFLKLLPMFIIVVPGMIS 320 Rat VIVQRVLAAKNIAHAKGSTLMAGFLKLLPMFIIVVPGMIS 320
114Consensus vivqrvlaakniahakgstlmagflkllpmfiivvpgmis Human RILFTDDIACINPEHCMLVCGSRAGCSNIAYPRLVMKLVP 360 Mouse RIvFaDeIACINPEHCMqVCGSRAGCSNIAYPRLVMtLVP 360 Rat RILFvDDIACINPEHCMqVCGSRAGCSNIAYPRLVMeLVP 360 Consensus ri f d iacinpehcm vcgsragcsniayprlvm lvp Human VGLRGLMMAVMIAALMSDLDSIFNSASTIFTLDVYKLIRK 400 Mouse VGLRGLMMAVMIAALMSDLDSIFNSASTIFTLDVYKLIRK 400 Rat VGLRGLMMAVMIAALMSDLDSIFNSASTIFTLDVYKLlRn 400 Consensus vglrglmmavmiaalmsdldsifnsastiftldvykl r Human SASSRELMIVGRIFVAFMVVISIAWVPIIVEMQGGQMYLY 440 Mouse SASSRELMIVGRIFVAFMVVISIAWVPIIVEMQGGQMYLY 440 Rat nASSRELMIVGRIFVAFMVVISIAWVPIIVEMQGGQMYLY 440 Consensus assrelmivgrifvafmvvisiawvpiivemqggqmyly Human IQEVADYLTPPVAALFLLAIFWKRCNEQGAFYGGMAGFVL 480 Mouse IQEVADYLTPPVAALFLLAIFWKRCNEQGAFYGGMAGFVL 480 Rat IQEVADYLTPPVAALFLLAIFWKRCNEQGAFYGGMAGFVL 480 Consensus iqevadyltppvaalfllaifwkrcneqgafyggmagfvl Human GAVRLILAFAYRAPECDQPDNRPGFIKDIHYMYVATGLFW 520 Mouse GAVRLILAFtYRAPECDQPDNRPGFIKDIHYMYVATaLFW 520 Rat GAiRLILAFtYRAPECDQPDNRPsFIKDIHYMYVATaLFW 520 Consensus ga rlilaf yrapecdqpdnrp fikdihymyvat lfw Human VTGLITVIVSLLTPPPTKEQIRTTTFWSKKNLVVKENCSP 560 Mouse iTGLITVIVSLLTPPPTKdQIRTTTFWSKKtLVtKEsCSq 560 Rat iTGLITVIVSLLTPPPTKdQIRTTTFWSKKtvVtKEsCSq 560 Consensus tglitvivslltppptk qirtttfwskk v ke cs Human KEEPYQMQEKSILRCSENNETINHIIPNGKSEDSIKGLQP 600 Mouse KdEPYkMQEKSILqCSENsEvIsHtIPNGKSEDSIKGLQP 600 Rat KdEPYkMQEKSILRCSENsEvvsHtIPNGKSEDSIKGLQP 600 Consensus k epy mqeksil csen e h ipngksedsikglqp Human EDVNLLVTCREEGNPVASLGHSEAETPVDAYSNGQAALMG 640 Mouse EDVNLLVTCREEGNPVASmGHSEAETPVDAYSNGQAALMG 640 Rat EDVNLLVTCREEGNPVASmGHSEAETPVDAYSNGQAALMG 640 Consensus edvnllvtcreegnpvas ghseaetpvdaysngqaalmg Human EKERKKETDDGGRYWKFIDWFCGFKSKSLSKRSLRDLMEE 680 Mouse ErEReKETenrsRYWKFIDWFCGFKSKSLSKRSLRDLMdE 680 Rat EraReKETesrsRYWKFIDWFCGFKSrSLSKRSLRDsMdE 680 Consensus e r ket rywkfidwfcgfks slskrslrd m e Human EAVCLQMLEETRQVKVILNIGLFAVCSLGIFMFVYFSL 718 Mouse EAVCLQMLEETpQVKVILNIGLFAVCSLGIFMFVYFSL 718 Rat EAVCLQMLEETprVrVILNIGLFAVCSLGIFMFVYFSL 718 Consensus eavclqmleet v vilniglfavcslgifmfvyfsl
115Figure 6.4 Interspecies protein alignment for SMIT2.
An alignment of the human, mouse and rat SMIT2 proteins, to compare their amino acid
sequences. Dots and dotted lines indicate gaps in the sequence between species, added for the
purposes of an improved alignment. The consensus line provides information on where the
three sequences match (wherever an amino acid is listed) and where a difference exists (blank
spaces). Positive amino acids are shown in red, negative amino acids in bold, while the rest are
neutral. The human sequence is 86% homologous with both the mouse and rat sequences, while
the mouse and rat sequences are 94% homologous with each other. The protein sequences were
acquired using the NCBI database. Accession Numbers: human = NM_052944, mouse =
NM_146198 and rat = NM_00100482.
Human MESGTSSPQPPQLDPLDAFPQKGLEPGDIAVLVLYFLFVL 40 Mouse MESaTiSPQPPQsDsLeAFPQKsmEPaDIAVLVLYFLFVL 40 Rat MEStTSSPQPPpsDaLeAFPQKsmEPaDIvVLVLYFLFVL 40 Consensus mes t spqpp d l afpqk ep di vlvlyflfvl Human AVGLWSTVKTKRDTVKGYFLAGGDMVWWPVGASLFASNVG 80 Mouse AVGLWSTVrTKRDTVKGYFLAGGDMVWWPVGASLFASNVG 80 Rat AVGLWSTVrTKRDTVKGYFLAGGDMVWWPVGASLFASNVG 80 Consensus avglwstv tkrdtvkgyflaggdmvwwpvgaslfasnvg Human SGHFIGLAGSGAATGISVSAYELNGLFSVLMLAWIFLPIY 120 Mouse SGHFIGLAGSGAAvGISVaAYELNGLFSVLMLAWvFLPIY 120 Rat SGHFIGLAGSGAAvGISVaAYELNGLFSVLMLAWIFLPIY 120 Consensus sghfiglagsgaa gisv ayelnglfsvlmlaw flpiy Human IAGQVTTMPEYLRKRFGGIRIPIILAVLYLFIYIFTKISV 160 Mouse IAGQVTTMPEYLRrRFGGnRIsItLAVLYLFIYIFTKISV 160 Rat IAGQVTTMPEYLkrRFGGsRIPItLAVLYLFIYIFpilqV 160 Consensus iagqvttmpeyl rfgg ri i lavlylfiyif v Human DMYAGAIFIQQSLHLDLYLAIVGLLAITAVYTVAGGLAAV 200 Mouse DMYAGAIFIQQSLHLDLYLAIVGLLAITAlYTVAGGLAAV 200 Rat DMYAGAIFIQQSLHLDLYLAIVGLLAvTAlYTVAGGLAAV 200 Consensus dmyagaifiqqslhldlylaivglla ta ytvagglaav Human IYTDALQTLIMLIGALTLMGYSFAAVGGMEGLKEKYFLAL 240 Mouse IYTDALQTvIMLIGAfiLMGYSFAAVGGMEGLKdqYFLAL 240 Rat IYTDALQTvIMLIGAfiLMGYSFAAVGGMEGLKdqYFLAL 240 Consensus iytdalqt imliga lmgysfaavggmeglk yflal Human ASNRSENSSCGLPREDAFHIFRDPLTSDLPWPGVLFGMSI 280 Mouse ASNRSENSSCGLPREDAFHIFRDPLTSDLPWPGiLFGMSI 280 Rat ASNRSENSSCGLPREDAFHIFRDPLTSDLPWPGiLFGMSI 280 Consensus asnrsensscglpredafhifrdpltsdlpwpg lfgmsi Human PSLWYWCTDQVIVQRTLAAKNLSHAKGGALMAAYLKVLPL 320 Mouse PSLWYWCTDQVIVQRsLAAKNLSHAKGGsLMAAYLKVLPL 320 Rat PSLWYWCTDQVIVQRsLAAKNLSHAKGGsLMAAYLKVLPL 320
116Consensus pslwywctdqvivqr laaknlshakgg lmaaylkvlpl Human FIMVFPGMVSRILFPDQVACADPEICQKICSNPSGCSDIA 360 Mouse FlMVFPGMVSRvLFPDQVACAhPdICQrvCSNPSGCSDIA 360 Rat FlMVFPGMVSRILFPDQVACAhPdICQrvCSNPSGCSDIA 360 Consensus f mvfpgmvsr lfpdqvaca p icq csnpsgcsdia Human YPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIF 400 Mouse YPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIF 400 Rat YPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIF 400 Consensus ypklvlellptglrglmmavmvaalmssltsifnsastif Human TMDLWNHLRPRASEKELMIVGRVFVLLLVLVSILWIPVVQ 440 Mouse TMDLWNHiRPRASErELMIVGRiFVfaLVLVSILWIPiVQ 440 Rat TMDLWhHiRPRASErELMIVGRVFVLaLVLVSILWIPVVQ 440 Consensus tmdlw h rprase elmivgr fv lvlvsilwip vq Human ASQGGQLFIYIQSISSYLQPPVAVVFIMGCFWKRTNEKGA 480 Mouse ASQGGQLFIYIQSISSYLQPPVAmVFIMGCFWKRTNEKGA 480 Rat ASQGGQLFIYIQSISSYLQPPVAVVFIMGCFWKRTNEKGA 480 Consensus asqggqlfiyiqsissylqppva vfimgcfwkrtnekga Human FWGLISGLLLGLVRLVLDFIYVQPRCDQPDERPVLVKSIH 520 Mouse FsGLIlGLLLGLVRLiLDFvYaQPRCDQPDdRPavVKdvH 520 Rat FsGLIlGLLLGLVRLiLDFvYVQPRCDQPDdRPavVKdvH 520 Consensus f gli glllglvrl ldf y qprcdqpd rp vk h Human YLYFSMILSTVTLITVSTVSWFTEPPSKEMVSHLTWFTRH 560 Mouse YLYFSMILSftTLITVvTVSWFTEtPSKEMVSrLTWFTRH 560 Rat YLYFSMILSstTLITVfTVSWFTEtPSKEMVSrLTWFTRH 560 Consensus ylyfsmils tlitv tvswfte pskemvs ltwftrh Human DPVVQKEQAPPAAPLSLTLSQNGMPEASSSSSVQFEMVQE 600 Mouse ePVaQKdsAPPetPLSLTLSQNGttEApgtSiqlet.VQE 599 Rat ePVaQKdsvPPenPLSLTiSQNGttEAtgiSiqles.VQE 599 Consensus pv qk pp plslt sqng ea s vqe Human NTSKTHSCDMTPKQSKVVKAILWLCGIQEKGKEELPARAE 640 Mouse sTtKacgdgvsPrhSKVVrAILWLCGm.EKnKEEpPskAE 638 Rat aTtKaHSdgvsPKQSKVlKAILWLCGm.EKdKEEpPskvE 638 Consensus t k p skv ailwlcg ek kee p e Human AIIVSLEENPLVKTLLDVNLIFCVSCAIFIWGYFA 675 Mouse pvIVSLEENPLVKTLLDVNcIvCiSCAIFlWGYFA 673 Rat pvIVSLEENPLVKTLLDVNcIvCiSCAIFlWGYFA 673 Consensus ivsleenplvktlldvn i c scaif wgyfa
117In addition to examining transporter homology between species, intertransporter homology was
examined for each of the three species (Table 6.1). HMIT, SMIT1 and SMTI2 are all part of
glucose transporter superfamilies; HMIT is a member of the solute carrier family 2, facilitated
glucose transporter superfamily, while both SMIT1 and SMIT2 are part of the solute carrier
family 5, sodium/glucose cotransporter superfamily. Therefore, we were curious to see how
closely the three proteins would be homologous. Across the three species examined, HMIT was
6-11% homologous to SMIT1/2, while SMIT1 and SMIT2 were 40-43% homologous to each
other. This indicates that, while the transporter protein sequences are very similar between
species, the three inositol transporters are very different from each other and therefore would be
predicted to have significantly different substrate structural requirements.
Table 6.1 Comparison of the protein homology of the three inositol transporters to each other
in humans, mice and rats.
118To analyze the substrate structural requirements for active transport by each inositol transporter,
competitive transport assays were developed. For these assays, cultures of cell-lines or primary
cells were grown to 90% confluency, washed with PBS, and incubated in medium containing
myo- or scyllo-inositol-(2-3H), without or with potential substrate competitors. Once the
incubation period was completed, cells were washed with PBS to stop radioactive uptake and
myo- and scyllo-inositol-(2-3H) levels were quantified using a liquid scintillation counter.
HMIT activation
The literature reports that HMIT is internalized in the cell and translocates to the cell surface in
response to cellular changes such as depolarization of the cell membrane or acidification of the
extracellular environment, both of which occur following synaptic activity (Uldry et al, 2001).
Based on these findings, four conditions were used to induce HMIT translocation to the cell
surface, with the goal of studying its transporter kinetics. Two conditions were modeled on the
paper by Uldry and colleagues (2001) and involved either cell surface depolarization, through an
elevation in external potassium levels, and/or acidification of the extracellular environment.
The second two conditions try to induce HMIT activity through the inhibition of the sodium
myo-inositol transporters. Since both SMIT1 and SMIT2 cotransport myo-inositol with two
sodium ions, they are inhibited by phlorizin, a sodium-dependent transport inhibitor (Hager et
al, 1995; Coady et al, 2002). Therefore, the effect of phlorizin-induced SMIT1/2 inhibition on
HMIT activity was examined (Figure 6.5a). Alternatively, SMIT1/2 silencing was induced,
through a combination of prewashing cells in Na+-free medium, and using Na+-free medium
during the competition transport assay (Figure 6.5b). While both of these techniques were
effective at silencing SMIT1/2 activity, they did not result in HMIT activation (Figure 6.5c). In
119
Figure 6.5 HMIT transport activation.
HMIT is internalized in cells and activation of the transporter requires translocation to the cell
surface. This has been reported to occur following cell surface depolarization and/or
acidification of the extracellular environment (Uldry et al, 2001). Based on these observations,
multipe conditions to activate HMIT in HEK293 cells were examined, including silencing
SMIT1/2 using phloridzin (A,C) and conducting the transport assay in sodium-free PBS (B).
Based on an analysis of myo-inositol-(2-3H) transport in the presence of increasing
concentrations of phloridzin (A), myo-inositol-(2-3H) competitive transport was tested in the
presence of 2 mM phloridzin to test for activation (C). myo-Inositol-(2-3H) transport was
eliminated by both phloridzin treatment and testing in sodium-free PBS, with only background
radioactivity recorded in both conditions. (n = 3 wells per variable).
120order to isolate HMIT activity during depolarization and acidification tests, these tests were
conducted in the presence of phlorizin and/or Na+-free medium to silence SMIT1/2 activity.
Again, while both phlorizin and Na+-free medium were successful at silencing SMIT1/2
activity, none of the four conditions, even when combined, resulted in HMIT activation.
In order to rule out cell-dependent induction of HMIT activity, these tests were conducted in a
number of cell lines including: 1321N1 human neuroblastoma cell-line, SH-SY5Y human
astrocytoma cell-line, rat primary cortical neurons, as well as other cell-lines which, using
QPCR were found to express HMIT mRNA. In all the cells studied, no HMIT transport activity
was ever observed. For example, in mouse cortical primary neurons, incubated in sodium-free
medium (pH 6.0, 80 mM KCl) supplemented with myo-inositol-(2-3H) for 3 hours, only a small
current, probably attributable to residual SMIT1/2 activity was observed (Figure 6.6a). When
these cells were tested for competitive transport, again only background noise levels of
radioactivity were observed and no competitive transport was found (Figure 6.6b). In
agreement with my studies, a recent report by Di Daniel and colleagues (2009), conducted on
both rat primary cortical neurons in culture and rat cortical slice cultures, also failed to observe
HMIT translocation to the cell surface following changes in pH or depolarization, despite initial
reports to the contrary (Uldry et al, 2001; 2004). Therefore, these findings remove HMIT as a
viable candidate for drug transport studies, despite the high levels of mRNA expression in the
brain. Focus now shifted to SMIT1 and SMIT2 transport.
121
Figure 6.6 Measurement of HMIT myo-inositol transport in primary cells.
An examination of HMIT transport in rat cortical primary neurons grown in culture for 3 weeks
was conducted in sodium-free medium, supplemented with 80 mM of KCl to induce cell surface
depolarization. No appreciable myo-inositol-(2-3H) transport was observed. A myo-inositol-(2-3H) concentration curve showed only a small level of transport, likely due to residual SMIT1/2
activity (A) and no measurable competitive transport was observed in these cells (B). (n = 3
wells per variable).
122SMIT1 and SMIT2 substrate requirements
To examine SMIT1/2 substrate structural requirements, competition assays were conducted in
the human endothelial kidney cell-line (HEK293), which was found, using QPCR, to express
SMIT1 and SMIT2 but not HMIT (Figure 6.7). The transport properties of both myo- and
scyllo-inositol-(2-3H) were individually examined in these cells, in the presence or absence of 23
potential substrate competitors (Figure 6.1). Using this technique, different substrates were
found to compete with myo- and scyllo-inositol-(2-3H) for transport. The substrates myo-,
scyllo-, D-chiro-inositol, L-fucose, viburnitol, D-glucose, D-mannose, sequoyitol and D-pinitol
competed with myo-inositol-(2-3H) transport, while only myo-, scyllo-, D-chiro-inositol, L-
fucose and viburnitol competed with scyllo-inositol-(2-3H) transport (Figure 6.8). The literature
reports that SMIT1 cotransports myo- and scyllo-inositol with equal preference (Hager et al,
_____________________________________________________________________________
Figure 6.7 QPCR quantification of inositol transporter expression levels in HEK293 cells.
HMIT, SMIT1 and SMIT2 expression was analyzed in HEK293 cells using QPCR. Actin was
used as an internal control for the QPCR assay. While SMIT1 and SMIT2 were present in these
cells, no quantifiable levels of HMIT were found, as determined by tracking gene sequence
amplification (Delta Rn) as a function of time (cycle number).
123
Figure 6.8 myo-Inositol-(2-3H) and scyllo-inositol-(2-3H) transport in HEK 293 cells.
Transporter substrate specificity was examined by measuring myo-inositol-(2-3H) (A) and
scyllo-inositol-(2-3H) (B) transport in the absence or presence of 23 competiting substrates.
Since SMIT1, but not SMIT2 transports scyllo-inositol, a comparison of these two transport
assays allowed for a comparison of compounds transported by SMIT1 alone, versus those
transported by a combination of SMIT1 and SMIT2. Using this technique, basic transport
models for each transporter were generated. (n = 3 wells per variable).
1241995), while SMIT2 has never been shown to transport scyllo-inositol. This would explain the
discrepancy between the myo- and scyllo-inositol-(2-3H) results and allowed us to isolate the
individual substrate transport requirements of SMIT1 and SMIT2. In addition to only SMIT1
transporting scyllo-inositol, the literature reports that SMIT1, but not SMIT2, transports L-
fucose, therefore allowing us to attribute its competitor activity to transport through SMIT1
(Hager et al, 1995; Coady et al, 2002). In addition, both SMIT1 and SMIT2 have been
independently reported to transport D-chiro-inositol (Rubin and Hale, 1993; Ostlund et al,
1996). In bovine cardiac sarcolemmal vesicles, both a high-affinity and a low-affinity transport
system for myo-inositol were observed (Rubin and Hale, 1993). The low-affinity transport
system was unsaturable, suggesting diffusion, but the high-affinity system was Na+-dependent
and showed an equal transport preference for myo- and scyllo-inositol, identifying it as SMIT1.
They showed that D-chiro-inositol could compete for this high-affinity transport, identifying it
as a SMIT1 substrate (Rubin and Hale, 1993). In contrast, SMIT2 has been shown to transport
both myo- and D-chiro-inositol with equal preference (Ostlund et al, 1996). This was
determined in HepG2 cells, a human liver cell-line, using tritiated D-chiro-inositol. In the same
set of experiments, tritiated D-chiro-inositol transport was also inhibited 30% by D-pinitol
(Ostlund et al, 1996). Therefore, with this additional information, it can be concluded that
SMIT1 transports myo- and scyllo-inositol with equal preference, followed by D-chiro-inositol
and viburnitol. L-fucose, a competitive substrate for SMIT1 transport is theorized to be a
putitive inhibitor for SMIT1 (see below). In contrast, SMIT2 transports myo-inositol and D-
chiro-inositol with equal preference, followed by D-glucose, D-mannose, viburnitol, sequoyitol
and D-pinitol. Based on these observations, two models of substrate structural requirements
have been drawn (Figure 6.9, 6.10). For active transport SMIT1 requires that inositol
derivatives contain four adjoining equatorial hydroxyl groups, while hydroxyl groups at the
125
Figure 6.9 Basic structural model for SMIT1 transport.
The structures of the substrates transported by SMIT1 (A), along with the structures of
substrates not transported by SMIT1, such as the putative inhibitor L-fucose (B), were used to
design a basic structural model for SMIT1 transport (C).
126
Figure 6.10 Basic structural model for SMIT2 transport.
The structures of the substrates transported by SMIT2 (A), along with the structures of
substrates not transported by SMIT1 were used to design a basic structural model for SMIT2
transport (B).
127remaining two carbons can be positioned in either an axial or equatorial orientation. The
equatorial hydroxyl groups at positions 1 and 2 cannot be substituted with a methoxy group and
still be transported. The hydroxyl group at position 5 can be substituted for a hydrogen group.
SMIT2, in contrast, has a different set of substrate structural requirements. The hydroxyls
attached to carbons 1, 2, 5 and 6 must be in an equatorial orientation, while the hydroxyl group
at position 4 must be in an axial orientation. The hydroxyl group at position 3 can be positioned
either in an axial or equatorial orientation. The hydroxyl group at position 2 cannot be
substituted for a methoxy group. The hydroxyl groups at positions 1, 5 and 6 can be substituted
with other groups. At position 1, the hydroxyl group can be substituted with a methoxy group
and still be transported. The hydroxyl group at position 5 can be substituted with a hydrogen
group or the carbon with attached hydroxyl group can be substituted with an oxygen atom. At
position 6, the hydroxyl group can be substituted with a hydroxymethyl group (Figure 6.10). To
further test the proposed substrate transport models, substrates not transported by SMIT1 or
SMIT2 were analyzed to determine whether a structural feature could be identified to explain
why each compound would not be transported based on the current models (Figure 6.11, 6.12).
This analysis further confirmed the proposed structural requirements for active transport through
SMIT1 and SMIT2. For example, a comparison of transported versus non-transported inositols,
helps to determine which hydroxyl groups of inositol need to be in a fixed orientation for active
transport to occur and which hydroxyl groups can be positioned either equatorially or axially. In
addition, by comparing the structures of other compounds to these models, for example the
structure of sequoyitol to the model for SMIT1, the model can be further refined to account for
the importance of side chain composition for the maintenance of active transport. In the case of
sequoyitol, transport through SMIT1 was abolished because the hydroxyl group at position 1
was substituted for a methoxy group.
128
Figure 6.11 Substrates not transported by SMIT1.
The SMIT1 basic substrate model for transport (A) was compared to the structures of
compounds not transported by SMIT1 (B). Structural differences, between the compounds not
transported and the model, are highlighted using red boxes.
129
Figure 6.12 Substrates not transported by SMIT2.
The SMIT2 basic substrate model for transport (A) was compared to the structures of
compounds not transported by SMIT2 (B). Structural differences, between the compounds not
transported and the model, are highlighted using red boxes.
130Substrate saturation kinetics and testing of the model
The two compounds most readily transported by SMIT1/2 are myo- and scyllo-inositol, with
SMIT1 transporting both myo- and scyllo-inositol and SMIT2 only transporting myo-inositol.
To examine myo- and scyllo-inositol transport kinetics in greater detail, the effects of increasing
concentrations of myo- and scyllo-inositol were examined in HEK293 cells (Figure 6.13). myo-
Inositol-(2-3H) transport was measured after 15 minutes of incubation, rather than at 3 hours as
in the initial tests, to more accurately examine the rate of myo-inositol transport. In this study,
10 mM of cold myo-inositol, the concentration used in the competitive substrate transport
studies, was found to produce a near maximal inhibition of myo-inositol-(2-3H) transport, while
half-maximal inhibition (Km) was observed following the addition of approximately 200 µM of
cold myo-inositol (Figure 6.13a). scyllo-Inositol-(2-3H) transport was examined in the presence
of increasing concentrations of cold scyllo-inositol (Figure 6.13b). In contrast to myo-inositol
transport, a 3 h incubation window was needed to obtain a similar level of radioactivity using
scyllo-Inositol-(2-3H). This indicates a much slower transport rate for scyllo-inositol, likely due
to the fact that it is only transported by SMIT1 and not by SMIT2. Once again, 10 mM of cold
scyllo-inositol produced near maximal inhibition of scyllo-inositol-(2-3H) transport and a Km of
approximately 50 µM was observed. These Km values for myo- and scyllo-inositol were within
the range previously reported in the literature; ranging for myo-inositol from 55-117 µM for
SMIT1 to 120-348 µM for SMIT2 (Hager et al, 1995; Ostlund et al, 1996; Hakvoort et al, 1998;
Coady et al, 2002; Bissonnette et al, 2004; Aouameur et al, 2007; Klaus et al, 2008; Lin et al,
2009), while for scyllo-inositol, a Km of 53 µM had been previously reported (Hager et al,
1995). In Xenopus oocytes, transfected with canine SMIT1, a myo-inositol Km value of 55-59
µM was observed (Hager et al, 1995; Kaus et al, 2008). A similar scyllo-inositol Km value of 53
µM was also documented in these cells (Hager et al, 1995). In porcine choroid plexus cells in
131
Figure 6.13 myo-Inositol and scyllo-inositol transport kinetics in HEK293 cells.
myo-Inositol testing was conducted in HEK293 cells, by incubating cells for 15 minutes in
medium containing 100 µM of myo-inositol-(2-3H) in the presence of increasing concentrations
of cold myo-inositol (A). Addition of 10 mM of cold myo-inositol, the concentration used in the
competitive transport assays, resulted in a near maximal inhibition of myo-inositol-(2-3H)
transport in these cells. scyllo-Inositol testing was conducted over a period of 3 hours in these
cells, due to slower uptake rates (B). Once again, 10 mM of cold scyllo-inositol produced near
maximal inhibition of scyllo-inositol-(2-3H). (n = 3 wells per concentration).
132culture, which endogenously express SMIT1, a Km value of 117 µM for myo-inositol-(2-3H) was
reported (Hakvoort et al, 1998). Electrophysiological studies in Xenopus oocytes, transfected
with rabbit SMIT2, showed a Km value of 120 µM for myo-inositol (Coady et al, 2002). Also
using voltage-clamp recordings, a SMIT2 Km value for myo-inositol of 150 + 40 µM was
observed in rat kidney brush border membrane vesicles (Aouameur et al, 2007). In contrast,
Xenopus oocytes, transfected with rat SMIT2, showed a Km for myo-inositol of 270 + 19 µM
(Aouameur et al, 2007). myo-Inositol competition against tritiated D-chiro-inositol transport, in
L6 myoblasts transfected with human SMIT2, showed a Km for myo-inositol of 158 µM (Lin et
al, 2009). In the HepG2 human liver cell-line, a Km for myo-inositol of 348 µM was observed in
competition against tritiated D-chiro-inositol (Ostlund et al, 1996) and finally, Km values of 120
µM and 334 µM, respectively, were found in Xenopus oocytes and in Madin-Darby canine
kidney cells, both transfected with rabbit SMIT2 (Bissonnette et al, 2004). Therefore, our
observed substrate saturation kinetics for myo- and scyllo-inositol are comparable to the range of
values that has been reported in the literature.
The third most readily transported inositol stereoisomer, D-chiro-inositol, appears to be
transported by both SMIT1 and SMIT2, however, its enantiomer, L-chiro-inositol, was not. We
wanted to examine this discrepancy in the transport of these two compounds more closely, by
examining the effects of increasing concentrations of these substrates on myo-inositol-(2-3H)
transport. As expected, L-chiro-inositol was not transported by SMIT1/2, while D-chiro-
inositol was transported (Figure 6.14). L-chiro-inositol transport was not observed even when
its levels were increased 8-fold (Figure 6.14a). A drop in radioactivity was observed at the
highest concentration (160 mM), likely as a result of L-chiro-inositol acting as a nonspecific
blocking agent, since at that concentration a 1600-fold difference would exist between L-chiro-
133inositol and tritiated myo-inositol (160 mM of L-chiro-inositol versus 100 µM of myo-inositol-
(2-3H)). In contrast, D-chiro-inositol competition for myo-inositol-(2-3H) transport did increase
with an increase in its concentrations (Figure 6.14b). These results validate the conclusions
made in the competitive transport studies and further point to the stereo-selectivity of the
transporters.
Based on the models generated, SMIT1 has the most stringent substrate structural requirements.
Of the 23 compounds examined, the most structurally altered one apparently transported by
SMIT1 was L-fucose, in which two carbons are altered: one carbon with attached hydroxyl
group is substituted with an oxygen atom and a second has a methyl group attached in place of
its hydroxyl group (Figure 6.9). What makes L-fucose particularly intriguing however is that
according to the SMIT1 model presented, L-fucose should not be transported by SMIT1, since
one of the four equatorial hydroxyl groups, theorized to be needed for active transport, is
oriented from an equatorial to an axial position. This would suggest that L-fucose might, in
fact, be a SMIT1 transport blocker. Therefore, L-fucose transport activity was examined more
closely. The initial experiments showed that 10 mM of L-fucose competed for myo-inositol-(2-
3H) transport, this was further examined in HEK-293 cells, using increasing concentrations of L-
fucose. In agreement with the initial studies, increasing the concentration of L-fucose resulted
in a reduction in myo-inositol-(2-3H) transport in these cells (Figure 6.15a). L-fucose and myo-
inositol competitive transport was examined in a second set of experiments, in which myo-
inositol inhibition of L-fucose-(5,6-3H) transport was examined. The theory behind this
experiment was that if L-fucose was an actively transported substrate through SMIT1, then myo-
inositol would be expected to compete for this transport. However, in contrast to this
hypothesis, no prominent reductions in L-fucose-(5,6-3H) activity levels were observed upon the
134
Figure 6.14 A comparison of D- and L-chiro-inositol transport in HEK293 cells.
A comparison of D- and L-chiro-inositol transport in HEK293 cells. Based on competitive
substrate assays, D-chiro-inositol is transported by SMIT1/2, while L-chiro-inositol is not. This
finding was examined more closely in HEK293 cells, by comparing the transport of myo-
inositol-(2-3H) in the presence of increasing concentration of D-chiro-inositol (A), to transport
observed in the presence of increasing concentrations of L-chiro-inositol (B). As expected myo-
inositol-(2-3H) transport was inhibited by D-chiro-inositol in a concentration dependent manner.
In contrast, L-chiro-inositol did not inhibit myo-inositol-(2-3H) transport, except at the highest
concentration, 160 mM. This might result from L-chiro-inositol acting as a nonspecific
blocking agent at this concentration. (n = 3 wells per variable).
135addition of cold myo-inositol (Figure 6.15b). Even when L-fucose-(5,6-3H) transport was
examined in the presence of increasing concentrations of cold L-fucose, no changes in the
radioactivity levels were observed (Figure 6.15c). Therefore, we propose that L-fucose is not a
SMIT1 substrate competitor but that it is, in fact, a SMIT1 transport inhibitor/blocker. We
hypothesize that L-fucose is only partially transported by SMIT1, whereupon it becomes lodged
in the channel, thus preventing myo-inositol-(2-3H) transport and reducing radioactivity levels.
Inhibition of myo-inositol transport by L-fucose has been previously reported in the literature
(Yorek et al, 1992; Rubin and Hale, 1993; Hager et al, 1995). Yorek and colleagues (1992) also
reported that myo-inositol competed for L-fucose-(3H) transport in neuroblastoma cells.
Neuroblastoma cells would be expected to express SMIT1 and possibly SMIT2, however in the
study the authors only concluded that L-fucose was a competitive inhibitor of sodium-
dependent, high-affinity myo-inositol transport, as SMIT1/2 had not yet been isolated when the
study was published (Yorek et al, 1992). In contrast, Hager and colleagues (1995) specifically
showed that L-fucose was a substrate competitor for myo-inositol transport in Xenopus oocytes
transfected with canine SMIT1. However, they incorrectly identified L-fucose as a substrate for
SMIT1, rather than as a competitive inhibitor (Hager et al, 1995). Rubin and Hale (1993)
labeled any compounds that competed for myo-inositol transport in cardiac sarcolemmal
vesicles as transport inhibitors and did not specify whether each compound studied was a
competitive substrate for inositol transport, or a transport channel blocker.
The effects of increasing concentrations of L-fucose on L-fucose-(5,6-3H) transport, not
previously examined in the literature, was also determined in HEK-293 cells. No concentration
dependent inhibition of L-fucose-(5,6-3H) transport was observed when cells were incubated in
L-fucose-(5,6-3H) with increasing concentrations of cold L-fucose (Figure 6.15c). This supports
136
Figure 6.15 L-fucose-(5,6-3H) transport.
An analysis of competitive substrate transport found L-fucose to be a competitive inhibitor of
myo- and scyllo-inositol-(2-3H) transport through SMIT1. However, a comparison of the
structure of this compound to the SMIT1 basic structural transport model would indicate that L-
fucose should not be transported by this system. This finding was further examined by: (A)
examining myo-inositol-(2-3H) transport in the presence of increasing concentrations of L-
fucose. A concentration-dependent reduction of myo-inositol-(2-3H) transport was observed.
(B) An examination of L-fucose-(5,6-3H) transport in these cells, in the absence or presence of
potential competitive substrates. Only background radioactivity was observed, with not active
transport. (C) L-fucose-(5,6-3H) transport was examined in the absence or presence of D-
fucose and increasing concentrations of cold L-fucose and again only backgroud radioactivity
was observed. (n = 3 wells per variable).
137the hypothesis that L-fucose is blocking SMIT1 activity in these cells. These experiments
demonstrate that fact that substrate transport kinetic studies only suggest the features required
for active transport into cells and the transport of any potential drug candidates must be
confirmed through additional experiments.
The SMIT1 basic substrate transport model was further tested, using a set of scyllo-inositol
derivatives, initially designed for an unrelated study, undertaken to examine the effects of
scyllo-inositol derivatives on Aβ fibrillogenesis (Sun et al, 2008). Use of these compounds
allowed a further testing and refinement of the SMIT1 basic transport model. In addition, it
allowed an examination of the degree of change tolerated by this transporter system. Seven
compounds were tested, in which either: one or two hydroxyl groups were removed, one
hydroxyl group was exchanged for either a fluorine, azide, chlorine, or a hydroxy methyl group,
or in the last compound, two hydroxyl groups were substituted for hydroxy methyl groups
(Figure 6.16a). Overall, each alteration resulted in very different degrees of transport into the
cells (Figure 6.16b, Table 6.2). Transport was still observed following the removal of one
hydroxyl group from scyllo-inositol, however, the removal of two hydroxyl groups stopped
transport. This is in agreement with the SMIT1 model, which found that four adjoining
hydroxyl groups, all in an equatorial position, are necessary for active transport to occur.
Similarly, one substitution of a hydroxyl group for a hydroxy methyl group still resulted in
active transport, but two substitutions did not. When the effects of substituting one hydroxyl
group with a fluorine, azide, chloride or hydroxy methyl group were examined, a hierarchy of
138
Figure 6.16 Transport of scyllo-inositol derivatives via SMIT1.
Further testing of SMIT1 transport was conducted using derivatives of scyllo-inositol, in which
one or two hydroxyl groups were removed or substituted (A). An analysis of myo-inositol-(2-3H) transport in the presence of each of these compounds, found monodeoxy- but not dideoxy-
scyllo-inositol competed for myo-inositol-(2-3H) transport. Single hydroxyl group substitutions
were tolerated in the order chlorine > fluorine > azide > hydroxy methyl. (n = 3 wells per
variable).
139
Table 6.2 Analysis of the SMIT1 transport model based on the structure of the scyllo-inositol
derivatizes that were transported or not.
140transport inhibition was observed in the following order: chlorine > fluorine > azide > hydroxy
methyl. Based on how drastic the reduction in transport was following these relatively simple
substitutions to scyllo-inositol, active transport through the SMIT1 transporter system appears to
be very sensitive to any changes to the structure of the inositol backbone.
141Discussion
There are three inositol transporters that have been reported in the literature: HMIT, SMIT1 and
SMIT2. Protein alignment between the human, rat and mouse sequences for each of these
proteins found a high degree of interspecies homology. This finding points to the importance of
each of these proteins for the maintenance of myo-inositol levels in the body. An analysis of
intertransporter protein homology in the same three species, showed how distinct the three
protein structures are from each other, with HMIT showing 11% or less homology to the two
sodium myo-inositol transporters and SMIT1/2 showing only 43% or less homology with each
other. Therefore, these three transporters are likely to have very distinct functions in the body.
My QPCR findings found that in the brain, HMIT mRNA was present at significantly higher
concentrations than SMIT1 or SMIT2 mRNA, in nearly all the regions studied (Chapter 5).
However, while this is true, the present results suggest that HMIT is not active in cells. HMIT
was first reported by Uldry et al (2001) to be a proton myo-inositol transporter, localized in the
cytoplasm because of 3 internalization sequences in its protein structure, but reported to
translocate to the cell surface under conditions that occur following synaptic activity. They
reported that both cell depolarization and changes in extracellular pH would result in HMIT
translocation to the cell surface and activation, resulting in myo-inositol transport (Uldry et al,
2001). Based on these findings, I tried a combination of four conditions to translocate HMIT to
the cell surface, with the goal of studying substrate transporter kinetics in this system. These
conditions included depolarization using elevated potassium levels, acidification of the
extracellular environment, as well as two techniques to silence SMIT1/2 transport: phloridzin
treatment and the use of Na+-free medium. However, none of these conditions resulted in
HMIT activation in any of the cells studied. A recent paper by Di Daniel et al (2009) supports
142these findings. In both rat primary cortical neurons and brain slices, changes in pH or cell
surface depolarization by either running a depolarizing current, increasing potassium or
increasing glutamate levels, did not result in HMIT translocation to the cell surface (Di Daniel et
al, 2009). Only mutation of the three internalization sequences resulted in HMIT translocation
and activity at the cell surface (Uldry et al, 2001; Di Daniel et al, 2009). Therefore, an
alternative function for HMIT has been suggested, as an inositol triphosphate transporter within
the cell (Di Daniel et al, 2009). While interesting, these observations eliminate HMIT as a
viable option for drug transport studies.
Next, I conducted substrate transport studies for SMIT1. QPCR tests showed SMIT1 to have
the second highest brain mRNA expression levels of the three transporters studied (Chapter 5)
and in contrast to HMIT, SMIT1 is constitutively expressed at the cell surface (Uldy et al,
2004), making it a better candidate as a drug transport system. Based on the competition assay
results, SMIT1 transported myo- and scyllo-inositol with equal preference, followed by L-
fucose, D-chiro-inositol, and viburnitol. These results differ from those found in Xenopus
oocytes transfected with canine SMIT1, which found the following compounds transported:
myo-inositol = scyllo-inositol > L-fucose > L-xylose > L-glucose = D-glucose = α-methyl-D-
glucopyranoside > D-galactose = D-fucose = 3-O-methyl-D-glucose = 2-deoxy-D-glucose > D-
xylose (Hager et al, 1995). This difference in substrate selectivity between the two studies
could result from the current study being conducted on human SMIT1, while the Xenopus
oocytes were transfected with canine SMIT1 (Hager et al, 1995). The human and canine SMIT1
amino acid sequences are more than 92% homologous (Kwon et al, 1992; Berry et al, 1995;
Lubrich et al, 2000; McVeigh et al, 2000). This level of homology is high, but since the
sequences are not identical, this might account for the differences observed.
143
Strengthening this argument is the fact that in both studies, the top three compounds that
competed for myo- and scyllo-inositol-(2-3H) transport remained the same, with myo- and
scyllo-inositol being transported the most readily, and with equal preference, in both studies.
The ability of scyllo-inositol to strongly compete out myo-inositol transport has also been
previously shown in murine neuroblastoma, murine cerebral microvessel endothelial and bovine
aortic endothelial cell-lines (Wiese et al, 1996), indicating that this is a feature common to
multiple species.
The present study found the third most competitive substrate for SMIT1 transport was L-fucose.
L-fucose might actually be a SMIT1 transport blocker, a conclusion reached based on the fact
that myo-inositol and increasing concentrations of cold L-fucose did not reduce L-fucose-(5,6-
3H) radioactivity levels. Therefore any drug compounds, initially interpreted as being
transported in cell-lines, need to be examined closely to ensure that they are in fact, transported
by the shuttling system and not lodged within it. In addition, this finding that L-fucose,
containing of a few minor substitutions in comparison to the structure of myo-inositol, becomes
lodged in the SMIT1 transport channel, points to how rigid the SMIT1 transporter is to changes
to the structure of myo-inositol.
Derivatives of inositol containing hydroxyl group deletions or substitutions, were used to further
define SMIT1 transport selectivity. A preference of chlorine > hydroxyl > fluorine > azide >
hydroxyl methyl, in place of a single hydroxl group was observed for SMIT1 transport.
Therefore, in agreement with the L-fucose observations, this indicates that SMIT1 is very
144sensitive to changes to the structure of myo-inositol and that only drugs based on conservative
substitutions to the structure of myo- or scyllo-inositol will likely be tolerated by this system.
The final inositol transporter, SMIT2, is also constitutively expressed at the cell surface, as was
seen for SMIT1 (Uldry et al, 2004). Similar to SMIT1, expression of SMIT2 is observed in
both the brain and the kidney, although expression in the kidney is significantly higher than
expression in the brain (Chapter 5). My study found that SMIT2 transports a distinct group of
compounds from SMIT1, specifically myo-inositol, D-chiro-inositol, D-glucose, D-mannose,
viburnitol, sequoyitol and D-pinitol. The preference of SMIT2 for D-stereoisomers over L-
stereoisomers has been previously reported in the literature (Ostlund et al, 1996). The HepG2
human liver cell-line was shown to prefer D-chiro-inositol and D-glucose over the L-
stereoisomers (Ostlund et al, 1996). In the present study, both SMIT1 and SMIT2 showed a
preference for D-chiro-inositol, over L-chiro-inositol, both in the initial substrate transport tests
and following an examination of the effects of increasing concentrations of D- and L-chiro-
inositol on myo-inositol-(2-3H) transport in HEK293 cells. Also in agreement with previously
publications (Ostlund et al, 1996), SMIT2 showed a preference for D-glucose over the L-
stereoisomer in HEK293 cells. Given the increased number of compounds transported by
SMIT2, this transporter would appear to be less stringent in substrate requirements than SMIT1.
However, given the expression pattern of SMIT2, drugs designed to target this transport system
would preferentially be funneled out of the body, based on the high SMIT2 expression observed
in the kidney.
In conclusion, the use of HMIT, SMIT1 or SMIT2 as a drug shuttling system appears unlikely
to be effective, based on the results presented in my thesis. HMIT, while highly expressed in
145the brain, is internalized in cells and therefore, unavailable as a drug shuttling system. SMIT1
and SMIT2 are both constitutively located at the cell surface membrane and are expressed in the
brain, therefore, they are available for drug transport. However, they are both very selective in
the compounds they will actively transport. SMIT1 especially appears to have very rigid
substrate structural requirements for active transport and it is very sensitive to changes to the
structure of myo- and scyllo-inositol, which are its preferred substrates. SMIT2 is slightly less
stringent than SMIT1, but it is still only likely to act as an effective shuttling system for
conservative derivatives of myo- or D-chiro-inositol, its two preferred substrates (Ostlund et al,
1996; Coady et al, 2002). Therefore, based on these observations, the inositol transporters
would not make an effective shuttling system for drug transport into the brain, thus disproving
my initial hypothesis.
145
CHAPTER 7
Discussion, Conclusions and Future Directions
146Discussion
One significant challenge to the treatment of CNS diseases is shuttling drugs across the blood-
brain and/or blood-CSF barriers to where they are needed. These barriers are very selective in
the types of compounds they will let through. The BBB consists of a monolayer of endothelial
cells joined together by tight junctions, separating the blood from the brain, supported on the
brain side by astrocytic endfeet, pericytes, a basal lamina and neurons (reviewed in Hawkins and
Davis, 2005). The blood-CSF barrier, mainly consists of choroid plexuses located in each of the
brain ventricles, which are composed of epithelial cells, joined together by tight junctions,
separating a network of capillaries and surrounding interstitial fluid, from the CSF (reviewed in
Johanson et al, 2005). The main purposes of both these structures are to act as physiological
barriers in order to protect the brain from any harmful substances that might be present in the
blood stream, to transport any substances, such as glucose and amino acids, which are needed
for proper brain function and to maintain homeostasis (Stern, 1921). A side effect of the
rigorous control imposed by these barriers is that compounds that would prove beneficial to
stopping CNS disease pathology, such as that which occurs in neurodegenerative diseases, are
usually unable to access their drug targets in the brain. Therefore, strategies are needed to
shuttle any drugs that cannot cross the brain barriers naturally, into the brain. Two strategies
that are often employed in a laboratory setting to avoid these brain barriers are to either inject
the potential drugs directly into the brain or implant a pump to slowly release drugs into the
brain ventricles. However, both these methods are highly invasive and not ideal as long-term
drug delivery strategies in humans. Therefore, alternative methods for drug delivery into the
brain are being examined. One set of strategies use less invasive barrier circumvention
strategies to bypass the brain barriers: these include avoiding the barriers entirely through the
use of intranasal delivery or temporarily opening the barriers, through the use of techniques such
147as osmotic disruption to allow the drug access (reviewed in Patel et al, 2009). Barrier
navigation strategies are also being developed, such as the use of nanoparticles or a Trojan horse
system for the delivery of compounds into the brain (reviewed in Patel et al, 2009). Both barrier
circumvention and navigation strategies center on converting compounds that cannot enter the
brain, into ones that can. An alternative technique that can be employed is to identify
compounds that already enter the brain naturally and try to either use these compounds as
disease therapeutics or attach other therapeutics to these compounds for transport into the brain.
myo-Inositol and scyllo-inositol are examples of compounds that are present in the body, enter
the brain naturally and appear to act as disease therapeutics (Hallman et al, 1986; Hallman et al,
1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al, 1996; Chengappa et al, 2000; Gelber
et al, 2001; Palatnik et al, 2001; Eden et al, 2006; McLaurin et al, 2006). myo-Inositol has been
successfully used in human studies to treat psychiatric disorders, such as depression (Levine et
al, 1995), bipolar/affective disorder (Chengappa et al, 2000; Eden et al, 2006), obsessive-
compulsive disorder (Fux et al, 1996), eating disorders (Gelber et al, 2001) and panic disorder
(Benjamin et al, 1995; Palatnik et al, 2001), as well as for the treatment of respiratory distress
syndrome in premature infants (Hallman et al, 1986; 1992). scyllo-Inositol has been
successfully used as an effective prophylactic and therapeutic for the treatment of AD in the
TgCRND8 mouse model of AD (McLaurin et al, 2006). Therefore, they are both naturally
occurring compounds that show promise as disease therapeutics and it might be possible to alter
or adapt them to either successfully treat other diseases or to transport other drug compounds
into the brain.
148In order to use these inositols as disease therapeutics or drug delivery systems, it is important to
understand how carefully their concentrations are regulated within the brain, to determine to
what extent additional transport of these compounds into the brain is possible. For example, in
the TgCRND8 mouse model of AD, while scyllo-inositol was effective for the treatment of AD,
both prophylactically, before the visible onset of symptoms, and therapeutically at 5 months of
age, once symptoms had fully developed, myo-inositol did not significantly improve disease
outcome measures in these animals (McLaurin et al, 2006). This is despite the fact that both
compounds were equally effective at reducing Aβ aggregation, in vitro (McLaurin et al, 2000).
This discrepancy between in vitro and in vivo findings led us to examine myo- and scyllo-
inositol regulation in the brain and to determine whether this regulation would change with
disease pathology in TgCRND8 mice. Based on the study by McLaurin and colleagues (2006)
on the effectiveness of the two compounds as therapeutic agents for AD, scyllo-inositol
administration would be expected to reach higher levels in the brain, than myo-inositol
administration. This was proven using GC/MS, which found that while oral ad libitum
administration of either compound caused a significant increase in its corresponding levels in
the brain, ad libitum scyllo-inositol administration resulted in a more significant increase in
brain levels, 7-fold compared to 0.3-fold for myo-inositol. Therefore a tighter control of myo-
inositol levels by the body appears to be in place. This could be because myo-inositol is critical
for maintaining osmolarity in the body and it is a constituent of phosphatidylinositol, which is
an important phospholipid in membranes and second messenger systems (Fisher et al, 2002). In
contrast, scyllo-inositol has no known function either within the brain or in the periphery
therefore, perhaps explaining the lack of tight regulation (Fisher et al, 2002).
149Given the importance of myo-inositol and the presence of scyllo-inositol in the body, the inter-
regulation of the two inositol isomers was tracked, by examining the effects of ad libitum myo-
or scyllo-inositol administration on the other inositol stereoisomer. Rat, rabbit and bovine
brains have been shown to contain an epimerase that can convert myo- to scyllo-inositol and
vice versa (Sherman et al, 1968a; b; Hipps et al, 1977). In women a 1% conversion of myo- to
scyllo-inositol within plasma has been reported and in rats a 0.06% conversion of myo- to scyllo-
inositol has been observed (Groenen et al, 2003). These studies suggest that inter-regulation
between the two isomers exists. This theory is supported by the present research, which found a
reduction in scyllo-inositol levels following myo-inositol administration and vice versa. This
decrease in the concentration of the non-administered isomer following either myo- or scyllo-
inositol administration, might result from a shift in inositol equilibrium toward degradation
pathways, in an effort to reestablish/maintain brain homeostasis. Degradation of both inositols
occurs by the same pathway, therefore a shift towards degradation would occur for both
inositols. The extent of this degradation becomes evident when the levels of the non-
administered inositol are quantified between untreated and treated animals. A 5-fold decrease in
scyllo-inositol levels was observed following myo-inositol administration, while only a 0.3-fold
decrease in myo-inositol occurred following scyllo-inositol administration. This severe drop in
scyllo-inositol levels observed following myo-inositol administration, compared to the smaller
drop in myo-inositol levels observed following scyllo-inositol administration, supports the idea
that myo-inositol levels are more tightly regulated. Therefore, these findings indicate that the
body regulates both elevations in myo-inositol following ad libitum administration, but also
150reductions in myo-inositol levels as a consequence of ad libitum scyllo-inositol administration.
This is an important finding, because it reduces the odds of ad libitum scyllo-inositol
administration inadvertently causing a detrimental shift in either osmolarity or
phosphatidylinositol levels in the body.
myo-Inositol is the most abundant inositol stereoisomer found in nature and it is a ubiquitous
component of all eukaryotic cells (Fisher et al, 2002). scyllo-Inositol is the next most abundant
inositol stereoisomer and its levels are usually 10% the levels of myo-inositol (Michaelis et al,
1993). While scyllo-inositol has no known function in the body, we wished to determine
whether a 7-fold increase in its levels in the brain, as was observed following ad libitum
administration, would result in its incorporation into phosphatidylinositol. This could alter
second messenger systems and lead to undesired side effects. Incorporation of scyllo-inositol
into phosphatidylinositol has been reported to occur in lower organisms, such as mycobacteria,
tetrahymena cells and barley seeds (Kinnard et al, 1995; Ryals et al, 1999; Salman et al, 1999;
Riggs et al, 2007). However, scyllo-inositol incorporation into phosphatidylinositols has not
been observed in higher organisms (Takenawa and Egawa, 1977). Therefore, the levels of
scyllo-inositol incorporation into phosphatidylinositol, in both untreated mice and mice treated
with ad libitum scyllo-inositol, were examined. No incorporation of scyllo-inositol into
phosphatidylinositol was observed in either group. This could result because of a reduced
binding affinity of phosphatidylinositol synthesizing enzymes for scyllo-inositol, which has
been observed even when scyllo-inositol levels are elevated (Takenawa and Egawa, 1977;
Salman et al, 1999). For example, the final step in the de novo synthesis of phosphatidylinositol
is catalyzed by CDP-diglyceride:inositol transferase and while it was found to bind myo-inositol
with a Km of 2.5 mM, binding to scyllo-inositol was not observed (Takenawa and Egawa, 1977).
151When the activity of another enzyme, phosphatidylinositol:inositol phosphatidyl transferase,
was examined in rat liver microsomes, scyllo-inositol inhibition of myo-inositol-(3H)
incorporation into phosphatidylinositol was two orders of magnitude lower than that of cold
myo-inositol and nonexistent when examined using the micromolar concentrations of scyllo-
inositol found endogenously in most tissues (Irvine, 1998). Therefore, an increase in the levels
of scyllo-inositol should not result in any significant incorporation and increase in the levels of
phosphatidylinositol, which could have deleterious effects on cell membrane composition and
secondary messenger systems. In support of this hypothesis, significantly increased levels of
scyllo-inositol have been previously observed in a human subject using MRS, with no apparent
neurological problems, despite having twice the average concentration of scyllo-inositol in the
brain (Seaquist and Gruetter, 1998).
The 7-fold increase in brain scyllo-inositol levels, observed following ad libitum oral
administration to mice, would suggest a high degree of CNS bioavailability for scyllo-inositol.
This is an important factor when selecting or designing drug therapeutics. We examined the
effects of once-daily administration of three different doses of scyllo-inositol versus ad libitum
scyllo-inositol treatment, in order to further study scyllo-inositol CNS bioavailability. Once-
daily administration of scyllo-inositol did not result in a sustained increase in brain scyllo-
inositol, suggesting that a multiple dosing regiment would be more effective than a once-daily
dose. This theory is supported by observations made using TgCRND8 mice, which found a
twice-daily dose of scyllo-inositol more effective than a once-daily dose at treating AD
152pathology (McLaurin et al, 2006). Therefore, while ad libitum scyllo-inositol administration
resulted in a significant increase in brain scyllo-inositol levels, twice-daily scyllo-inositol
administration was sufficient to achieve therapeutic doses of scyllo-inositol in the brain for
effective disease modification.
While scyllo-inositol was an effective disease therapeutic against AD in TgCRND8 mice, myo-
inositol was not (McLaurin et al, 2006). This finding was unexpected given, both the
effectiveness of myo-inositol in vitro (McLaurin et al, 2000) and in other diseases in vivo
(Hallman et al, 1986; Hallman et al, 1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al,
1996; Chengappa et al, 2000; Gelber et al, 2001; Palatnik et al, 2001; Eden et al, 2006). One
possible reason why myo-inositol would be ineffective as a drug treatment in TgCRND8 mice
could be because these animals display different myo-inositol regulation parameters than control
animals. To test this theory, the effects of ad libitum myo-inositol administration on brain myo-
and scyllo-inositol levels were compared between TgCRND8 mice and their wild-type
littermates. No significant differences were observed between the two groups on the effects of
myo-inositol administration on brain inositol levels. Both groups showed a similar, significant
increase in brain myo-inositol levels and a similar, significant decrease in scyllo-inositol levels
following ad libitum myo-inositol administration. Therefore, regulation of inositol levels in the
brain appears to be comparable between the two groups and not quantifiably altered by disease
pathology using this technique.
The similarity between TgCRND8 mice and their wild-type littermates was further examined by
comparing the mRNA expression levels of the three inositol transporters, HMIT, SMIT1 and
SMIT2 between the two groups. Basal CNS myo- and scyllo-inositol levels are normally almost
153100-fold higher than circulating plasma inositol levels (Palmano et al, 1977). This significant
elevation in brain inositol levels in comparison to the periphery, points to the presence of active
transport systems, in addition to simple diffusion, for the transport of inositol stereoisomers
across the brain barriers (Spector, 1988; Wiesinger, 1991; Rubin and Hale, 1993; Uldry et al,
2001; Coady et al, 2002). There are three active inositol transporters that have been reported in
the literature, HMIT, SMIT1 and SMIT2 (Kwon et al, 1992; Uldry et al, 2001; Roll et al, 2002)
and all three are expressed in the brain (Berry et al, 1995; Inoue et al, 1996; Uldry et al, 2001;
Roll et al, 2002). In addition, because the endothelial cells located at the BBB are the only cells
in the brain capable of synthesizing inositol from glucose (Wong et al, 1987), the majority of
cells in the brain also require active transport to maintain their inositol levels. These inositol
transporters function by using the cotransport of either a hydrogen atom or two sodium atoms
along their concentration gradients, to generate enough energy to actively transport inositol
across concentration gradients (Hager et al, 1995; Uldry et al, 2001; Coady et al, 2002;
Bourgeois et al, 2005). The mRNA expression of these three transporters was compared
between TgCRND8 mice and their wild-type littermates in four brain regions: the cortex,
hippocampus, septum and cerebellum, using QPCR. These four brain regions were chosen
because they are regions that are affected to varying degrees by Aβ plaque deposition in AD
(Thal et al, 2002). The earliest plaque deposition in AD is observed in the cortex and
hippocampus, followed by the septum and finally the cerebellum (Thal et al, 2002). Deposition
in the cerebellum is only observed in advanced stages of AD (Thal et al, 2002). In addition to
comparing transporter mRNA expression these two groups in the four brain regions listed,
inositol transporter expression was also examined as a function of age, by examining expression
at 2, 4 and 6 months of age. These time points were selected because they correspond to
different stages in Aβ plaque deposition in TgCRND8 mice: 2 months of age corresponds to
154pre-plaque deposition, 4 months of age corresponds to mid-stage AD and 6 months of age
corresponds to advanced stage AD (Chishti et al, 2001). In agreement with the non-significant
difference between TgCRND8 mice and wild-type animals observed in the GC/MS study
following myo-inositol administration, disease pathology did not significantly change inositol
transporter mRNA expression levels in any of the four brain regions examined, at any of the
three ages selected. Inositol transporter mRNA expression was also not significantly changes as
a function of age in either group of animals, indicating that transporter expression is stable with
age, at least in the range of ages tested in this study. A stable expression pattern, irrespective of
disease pathology or age, is an important factor for successful CNS drug design, because it
removes potential confounding variables to successfully drug shuttling into the brain using these
transporter systems.
It is known that all three inositol transporters are expressed in the brain (Berry et al, 1995; Inoue
et al, 1996; Uldry et al, 2001; Roll et al, 2002), however, the relative expression of the three
transporters to each other and their subregional expression in the brain have not been
extensively studied. This information would provide a better understanding of active inositol
transport within the brain and between subregions. Based on their relative expression and
subregional distribution, individual transporters could be targeted in drug design studies to
direct transport and subregional drug delivery. This study found that overall, relative expression
of the transporters in the brain occurred in the order HMIT > SMIT1 > SMIT2. In addition,
each transporter showed a distinct regional expression profile. HMIT mRNA subregional
expression occurred in the order of concentration: cortex = septum > hippocampus >
cerebellum. SMIT1 expression occurred in the order septum > cerebellum ≥ cortex =
hippocampus and SMIT2 expression occurred in the order cerebellum ≥ septum = cortex ≥
155hippocampus. If drug targeting to the cerebellum was required, than SMIT2 would be the best
transporter to target, because its expression levels were highest in the cerebellum, in comparison
to the other three brain regions examined. In contrast, if targeted drug transport to the septum
was required, then SMIT1 would be the better choice, based on its expression profile.
Therefore, depending on the transporter chosen, drug accumulation could theoretically be
preferentially directed to selected subregions of the brain.
In addition to examining CNS bioavailability, an additional variable that can be considered for
CNS drug design is the rate at which a drug will be excreted from the body. The kidney is
where inositol excretion from the body occurs (Arner et al, 2006) and all three inositol
transporters are expressed there (Berry et al, 1995; Uldry et al, 2001; Roll et al, 2002). An
examination of mRNA expression of the three transporters in the kidney, which was used as a
positive control tissue in QPCR experiments, found no changes in expression in any of the
groups examined, therefore indicating that age and disease pathology do not affect inositol
transporter expression in this tissue. A comparison of the relative expression levels of the
inositol transporters in the kidney, found expression occurred in the order SMIT2 > SMIT1 >
HMIT, which is the opposite of what was observed in the brain. SMIT2 expression in the
kidney was 80-fold higher than its expression in the brain. A higher level of SMIT2 expression
in the kidney, relative to the brain, has been previously reported in the literature (Roll et al,
2002). This indicates that SMIT2 is particularly important for regulating inositol excretion in
mice. In contrast, HMIT levels were much lower in the kidney, relative to the brain, which has
also been previously reported in the literature (Uldry et al, 2001). This suggests that targeting
drugs to SMIT1, rather than SMIT2, might result in higher CNS bioavailability, by reducing the
rate of uptake and excretion by the kidney.
156
GC/MS found ad libitum myo-inositol and especially scyllo-inositol administration resulted in a
sustained increase in their levels in the brain (Fenili et al, 2007). These findings indicate that
scyllo-inositol has excellent CNS bioavailability and suggests that derivatives of scyllo-inositol
or compounds attached to scyllo-inositol might show similar rates of CNS bioavailability. In
order for these compounds to be successfully transported into the brain, they need to cross the
brain barriers. The inositol transporters HMIT, SMIT1 and SMIT2 are expressed in the brain
(Berry et al, 1995; Inoue et al, 1996; Uldry et al, 2001; Roll et al, 2002) and QPCR found
mRNA expression for all three transporters, in all four of the brain regions examined. This
potentially creates three different avenues through which drugs can travel into the brain. By
comparing the transport of tritiated myo- or scyllo-inositol in the absence or presence of 23
potential substrate competitors, the structural features required for active transport through each
transporter can be characterized. This assay provides information on the basic structural
features of inositol required for active transport and suggests how flexible the three transporters
are to alterations to the base structure. This research helps determine whether derivatives of
inositol can be transported or whether compounds can be attached to inositol and successfully
shuttled into and within the brain, using these transporters.
The first transporter, HMIT, is mainly expressed in the brain, with limited expression in the
kidney and adipose tissue (Uldry et al, 2001). In the brain, mRNA expression is observed in
both neurons and glia, with high levels found in all the brain regions examined: cerebral cortex,
hippocampus, hypothalamus, cerebellum and brainstem (Uldry et al, 2001). When the mRNA
expression levels of the three transporters were compared in the brain and the kidney, HMIT
showed the highest levels of expression in the brain and the lowest levels of expression in the
157kidney. Based on these findings, HMIT would appear to have all the features required to be an
effective drug shuttling system into the brain. One challenge to this conclusion is the fact that
HMIT is normally internalized in the cell, because of three internalization sequences located in
the protein structure (Uldry et al, 2001). However, cell surface translocation of HMIT has been
reported to occur following membrane depolarization or a decrease in extracellular pH (Uldry et
al, 2001; 2004), conditions that are typical byproducts of synaptic activity (Chesler and Kaila,
1992). Since, HMIT might still be usable as a drug delivery system in regions where synaptic
activity is present, an effort was made to study the transporter activity in vitro. Based on the cell
surface translocation information published in the literature, two conditions: an elevation in
external potassium levels and acidification of the extracellular environment were used to induce
HMIT translocation to the cell surface and activation. However, neither condition resulted in
HMIT activation, in any of the cells studied, which included the SH-SY5Y human
neuroblastoma cell-line, the 1321N1 human astrocytoma cell-line and mouse primary cortical
neurons. A recent study by Di Daniel and colleagues (2009), also did not observe HMIT
translocation to the cell surface following changes in pH or depolarization, either in rat primary
cortical neurons in culture or rat cortical slice cultures, despite initial reports to the contrary
(Uldry et al, 2001; 2004). Two additional conditions, centered on inducing HMIT translocation
and activity through SMIT1/2 silencing, were also tested. Since both SMIT1 and SMIT2
cotransport inositol with two sodium ions, they are inhibited by phlorizin, a sodium-dependent
transport inhibitor (Hager et al, 1995; Coady et al, 2002). Therefore, the effect of phlorizin-
induced SMIT1/2 inhibition on HMIT activity was examined. In addition, prewashing cells in
Na+-free medium, and using Na+-free medium during the competition transport assay was tried
as another method of silencing SMIT1/2 activity. Both of these techniques were effective in
silencing SMIT1/2 activity, but they did not result in HMIT activation. Only mutation of the
158three internalization sequences, located in the protein structure of HMIT, was found to cause
translocation to the cell surface and activation (Uldry et al, 2001; Di Daniel et al, 2009). If
HMIT is not a cell surface inositol transporter, what is its function inside the cell? Di Daniel
and colleagues (2009) suggest that HMIT regulates inositol triphosphate levels by acting as an
inositol triphosphate transporter within the cytoplasm. Therefore, despite high levels of mRNA
expression in the brain, HMIT does not appear to be a viable candidate for drug transport
studies.
The next candidate for drug transport, SMIT1, is constitutively expressed at the cell surface
(Uldry et al, 2001). It has the second highest brain mRNA expression levels of the inositol
transporters, with expression found in all four of the brain regions tested: the cortex,
hippocampus, septum and cerebellum, using QPCR. Expression in the brain has also been
observed in the locus coeruleus, the suprachiasmatic nucleus and the olfactory bulb (Inoue et al,
1996). However, the highest levels of SMIT1 in the brain are in the choroid plexus (Inoue et al,
1996), therefore, SMIT1 is very important for inositol transport across the blood-CSF barrier.
SMIT1 knockout studies would suggest that it is the main transporter responsible for
maintaining inositol levels both in the brain and in the periphery (Berry et al, 2003).
Homozygous knockout mice showed a 92% and an 84% reduction in inositol levels in the brain
and in the periphery, respectively (Berry et al, 2003). Heterozygous mice showed a 15% and a
25% reduction in frontal cortex and hippocampal inositol levels, respectively, suggesting these
regions are particularly dependent on SMIT1 activity (Shaldubina et al, 2007). This high
dependence of inositol levels on SMIT1, suggests that designing drugs to target this transporter
might be an effective strategy for maximizing drug transport into the brain, particularly to the
frontal cortex and hippocampus. Competition assay results found that SMIT1 transports myo-
159and scyllo-inositol with equal preference, followed by L-fucose, D-chiro-inositol and viburnitol.
These findings contrast those of canine SMIT1 transfected into Xenopus oocytes, which
transported the following compounds, in the order of preference: myo-inositol = scyllo-inositol >
L-fucose > L-xylose > L-glucose = D-glucose = α-methyl-D-glucopyranoside > D-galactose =
D-fucose = 3-O-methyl-D-glucose = 2-deoxy-D-glucose > D-xylose (Hager et al, 1995). This
difference in substrate selectivity between the two studies is likely due to differences in the
structures of the human and canine SMIT1 transporters. Despite the 92% homology between
the human, mouse, rat, canine and bovine SMIT1 amino acid sequences (Kwon et al, 1992;
Berry et al, 1995; Lubrich et al, 2000; McVeigh et al, 2000), the differences that exist are
reflected in the substrates transported by SMIT1 in each species. However, in both species, the
top three compounds competing for myo-inositol-(2-3H) transport remained the same, with myo-
and scyllo-inositol transported the most readily and with equal preference. This ability of
scyllo-inositol to strongly compete out myo-inositol transport has also been shown in murine
neuroblastoma, murine cerebral microvessel endothelial and bovine aortic endothelial cell-lines
(Wiese et al, 1996), indicating that this feature is common across multiple species.
Derivatives of inositol containing hydroxyl group omissions or substitutions were used to
further define SMIT1 transport selectivity. Alterations in a single hydroxyl group resulted in
substrate transport in the following order of substitution preference: chlorine > fluorine >
hydroxyl > deoxy omission > azide > hydroxylmethyl. Based on the marked decrease in
transport observed following these conservative changes to the structure of scyllo-inositol, it can
be concluded that SMIT1 is very sensitive to alterations in the structure of inositol. Given this
information, in addition to the initial observations that four neighboring hydroxyl groups need to
160be in an equatorial position for transport and only the remaining two hydroxyl groups are
flexible to reorientation, it would appear that only drugs resulting from very conservative
substitutions to the structure of myo- or scyllo-inositol would be tolerated by this system.
This conclusion is supported by competitive transport studies using L-fucose. L-fucose contains
three minor substitutions to the structure of myo-inositol: one carbon with its attached hydroxyl
group is substituted with an oxygen, a second hydroxyl group is substituted with a methyl group
and a third hydroxyl is reoriented from an equatorial to an axial position. These substitutions
resulted in the partial transport of L-fucose by SMIT1, before becoming lodged in the transport
channel. This conclusion was reached based on the observation that myo-inositol and increasing
concentrations of cold L-fucose were not successful in reducing tritiated L-fucose radioactivity
levels, suggesting that SMIT1 transport was blocked by L-fucose. Therefore, it can be
concluded that carefully designed derivatives of myo- or scyllo-inositol will be transported by
this system, but not complex compounds attached to inositol.
The third transporter, SMIT2, is also expressed at the cell surface (Uldry et al, 2004). Also like
SMIT1, it’s a member of the solute carrier family 5, sodium/glucose cotransporter superfamily,
but the two transporters are only 40-43% homologous to each other. Therefore, the two would
be expected to have very different substrate structural requirements. While SMIT2 is expressed
in the brain, QPCR results show that expression in the brain is much lower than expression in
the kidney. This lowers the chances of being an effective shuttling system for drugs to the
brain, but its substrate structural requirements were examined, regardless. As expected, SMIT2
was found to transport a distinct group of compounds from SMIT1, specifically myo-inositol, D-
chiro-inositol, D-glucose, D-mannose, viburnitol, sequoyitol and D-pinitol. A preference of
161SMIT2 for D-stereoisomers over L-stereoisomers has been previously reported in the literature
(Ostlund et al, 1996). The HepG2 human liver cell-line was shown to prefer D-chiro-inositol
and D-glucose rather than the L-stereoisomers (Ostlund et al, 1996). In the present study, both
SMIT1 and SMIT2 showed a preference for D-chiro-inositol, rather than L-chiro-inositol, both
in the initial substrate transport tests and following an examination of the effects of increasing
concentrations of D- and L-chiro-inositol on myo-inositol-(2-3H) transport in HEK293 cells.
Previously published data also observed a preference by SMIT2 for D-glucose over its L-
stereoisomer (Ostlund et al, 1996). Of the 23 potential substrate competitors, SMIT2
transported more substrates than SMIT1 and it would appear to have less stringent substrate
requirements. Like SMIT1, many of the hydroxyl groups on inositol need to be in a fixed
position, however unlike SMIT1 many of these hydroxyl groups can be successfully substituted
with other groups while still maintaining transport. Two hydroxyl groups can be substituted
with a methoxy or hydroxymethyl, respectively. A third hydroxyl group can be substituted for a
hydrogen or the carbon with attached hydroxyl group can be substituted for an oxygen. Overall,
this results in SMIT2 being able to transport a wider range of structures than SMIT1, however,
its substrate requirements are still relatively stringent and, like SMIT1, this system would
probably only transport carefully designed derivatives of inositol. In addition, while all three
transporters are expressed both in the brain and the kidney, the 80-fold higher SMIT2
expression levels observed in the kidney, relative to the brain, would suggest that drugs
designed to target this transporter would be more likely to be excreted from the body prior to
their transport into the brain.
162Conclusions
In this thesis, experiments were conducted to quantify myo- and scyllo-inositol levels in the
brain, to understand the effects of oral myo- or scyllo-inositol administration on these levels and
to determine the expression of the inositol transporters in the brain and the substrate structural
requirements for active transport using these transporters. These experiments were conducted to
test the hypothesis that inositol transporters would make effective shuttling systems for drug
delivery into the brain. A GC/MS study was conducted to quantify baseline brain myo- and
scyllo-inositol levels and the effects of oral ad libitum administration, of either myo- or scyllo-
inositol, on brain and CSF inositol concentrations. Using this technique, no differences were
observed in either baseline brain myo- and scyllo-inositol levels or those levels following myo-
inositol administration, when comparing TgCRND8 mice to their wild-type littermates. These
findings led to the conclusion that myo- or scyllo-inositol concentrations and uptake into the
brain are not significantly altered by disease pathology in these animals. An examination of
inositol transporter mRNA expression using QPCR, conducted in four regions of the brain as
well as the kidney, showed no significant changes in transporter expression as a function of
either age or disease pathology. These findings are in agreement with the GC/MS conclusions.
QPCR analysis showed SMIT2 expression was very high in the kidney, with much lower
expression levels in the brain, making it a less promising candidate for CNS drug transport. An
examination of inositol transporter activity found HMIT to be inactive, with no evidence of cell
surface transport and activity. HMIT was concluded to likely be an internal transporter for
inositol derivatives and therefore, not a viable shuttling system for drugs into the brain. These
findings placed SMIT1 as the primary candidate for drug transport into the brain. However,
competitive substrate transport assays for both SMIT1 and SMIT2 were conducted and both
were concluded to have very stringent requirements for the structural features required for
163substrate transport. Therefore, it is concluded that inositol transporters would not make
effective shuttling systems for drug transport into the brain, but only for the transport of certain
inositol stereoisomers and derivatives, thus disproving the initial thesis hypothesis.
Future Directions
scyllo-Inositol is an effective prophylactic and therapeutic against AD in TgCRND8 mice
(McLaurin et al, 2006) and it is currently in Phase II clinical trials for the treatment of AD
(www.clinicaltrials.gov). Additional research, to further understand how scyllo-inositol levels
are regulated in both AD mice and in human subjects with or without AD, would further our
understanding of the potential function of this compound and how it is transported into the
brain. Towards this purpose, three future direction studies have been proposed. First, to
compare basal myo- and scyllo-inositol concentrations and concentrations following scyllo-
inositol treatment, in human patients with AD and control subjects. Second, to quantify mRNA
levels for the inositol transporters in post-mortem tissue from both AD and control subjects, to
determine where the transporters are expressed in human tissue and whether their expression is
stable as a function of age and disease pathology (Chapter 5). Lastly, given the fact that SMIT1
appears primarily responsible for scyllo-inositol transport into the brain, to examine the effects
of decreasing or increasing SMIT1 expression, through gene knockout or knockin studies, on
scyllo-inositol transport into the brain of TgCRND8 mice and on its effectiveness as a
prophylactic and therapeutic for AD.
In order to examine the levels of myo- and scyllo-inositol in human subjects in vivo, MRS can be
used. Both myo- and scyllo-inositol have distinct resonance frequencies when examined using
MRS, allowing for the repetitive, non-invasive analysis of myo- and scyllo-inositol
164concentrations as a function of age and disease pathology. Elevated levels of both myo- and
scyllo-inositol have been reported when comparing human subjects with AD to age-matched
control subjects (Valenzuela and Sachdev, 2001; Griffith et al, 2007). Increases in myo- and
scyllo-inositol concentrations have also been reported in normal older subjects, compared to
younger subjects (Kaiser et al, 2005). Therefore, both age and disease pathology are correlated
with increases in brain myo- and scyllo-inositol concentrations (Valenzuela and Sachdev, 2001;
Kaiser et al, 2005; Griffith et al, 2007). An extensive examination of how myo- and scyllo-
inositol levels change with age in both healthy subjects and patients with AD would help to
clarify when these concentrations become altered. Sub-regional changes in myo- and scyllo-
inositol levels, as a function of age and disease pathology, could also be monitored to help
determine where changes in myo- and scyllo-inositol concentrations begin in these subjects and
how these changes progress over time. In addition, MRS data from the Phase I and Phase II
clinical trials for scyllo-inositol would provide information on the effects of twice-daily, oral
administration of scyllo-inositol on brain myo- and scyllo-inositol levels, in AD patients and in
older and younger healthy control subjects. These studies would provide additional information
on the stability of scyllo- inositol levels within the brain and when these levels become altered.
While myo- and scyllo-inositol concentrations are not significantly different between TgCRND8
mice and their wild-type littermates, in human AD patients, these concentrations are elevated
(Valenzuela and Sachdev, 2001; Griffith et al, 2007). One possible reason for this increase
might be a change in brain inositol transporter expression patterns in these patients, when
compared to control subjects. Therefore, it would be interesting to analyze sub-regional inositol
transporter expression in post-mortem tissue of AD patients and control subjects using QPCR.
In mice, no significant differences in SMIT1 or SMIT2 expression was observed in any of the
165tissues examined, either as a function of age or disease pathology however, these studies have
not been conducted on human tissue. This study would provide information on whether the
elevations in brain myo- and scyllo-inositol concentrations observed in human subjects with AD
and in older normal subjects, results from a corresponding elevation in SMIT1 and SMIT2
levels. These studies would help us gain greater insight into the process of inositol transport
into the brain and in interpreting clinical trial findings.
In this thesis, SMIT1 was shown using competitive transport assays, to be responsible for
scyllo-inositol transport across cellular membranes (Chapter 6). SMIT1 is expressed at both the
blood-brain and blood-CSF barriers (Spector, 1988; Inoue et al, 1996) and as a result, it appears
to be the main transporter responsible for myo- and scyllo-inositol entry into the brain. SMIT1-/-
mice show a 92% reduction and an 84% reduction in myo-inositol levels in the brain and
periphery, respectively, resulting in these mice expiring shortly after birth as a result of
hypoventilation (Berry et al, 2003). A similar reduction in scyllo-Inositol levels would be
expected to occur in the brains of these animals. In contrast, Ts65Dn mice, a mouse model of
Down syndrome, show a 38% increase in brain myo-inositol levels (Huang et al, 2000) and
presumably scyllo-inositol levels. myo-Inositol levels in these mice are elevated in the frontal
cortex, hippocampus and brainstem, but not in the cerebellum (Shetty et al, 2000). Therefore,
by crossing these animals with TgCRND8 mice, studies could be conducted on the effects of
decreasing or increasing SMIT1 expression, on scyllo-inositol transport into the brain and on its
ability to alter disease pathology in the TgCRND8 mice. SMIT1 homozygous knockout mice
expire soon after birth, however, TgCRND8 mice could be crossed with heterozygous SMIT1
knockout mice, which have a normal lifespan and show a more modest 15% and 25% reduction
in frontal cortex and hippocampus myo-inositol levels, respectively (Shaldubina et al, 2007).
166This would allow us to determine the effects of a reduction in SMIT1 activity on scyllo-inositol
transport and on the treatment of AD. These affects could be compared to the effects of
increasing the SMIT1 copy number scyllo-inositol transport and therapeutic activity.
Down syndrome mice have a triplication of a segment of chromosome 16, which corresponds to
the region on chromosome 21 that is triplicated in the human condition and contains the SMIT1
gene (Gardiner et al, 2003). One potential concern with using Down syndrome mice as a
method to triplicate the SMIT1 gene, is that this region of chromosome 16 also contains the
APP gene, which generates the amyloid precursor protein and following cleavage, the Aβ
peptide, which is aggregated in AD (Kang et al, 1987). However, Ts1Cje mice (Sago et al,
1998), unlike Ts65Dn mice and other mouse models of Down syndrome, have a triplication of a
shorter segment of chromosome 16 that does not include the APP gene, thus removing this
potential confounding variable (Gardiner et al, 2003). The effects of SMIT1 gene copy number
on baseline scyllo-inositol levels could be examined by comparing these two mouse models. In
addition, prophylactic and therapeutic scyllo-inositol studies could be conducted in both sets of
mice to examine the effects of changing the SMIT1 gene copy number on the effectiveness of
scyllo-inositol as an AD therapeutic.
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