Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 305

Models for the Transfer of Drugsfrom the Nasal Cavity to theCentral Nervous System

BY

BJÖRN JANSSON

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004

3

Contents

1 Introduction ............................................................................................... 71.1 The effects of physiological parameters

on the nasal absorption of drugs ...................................................81.1.1 The nasal respiratory mucosa ........................................91.1.2 The olfactory mucosa ....................................................91.1.3 The blood-brain barrier (BBB).................................... 10

1.2 Nose-to-brain transfer of drugs ................................................... 11

2 Models for nose-to-brain transfer of drugs ............................................. 132.1 Human studies ............................................................................. 13

2.1.1 Monitoring CNS effects .............................................. 132.1.2 Visualizing the transfer ............................................... 132.1.3 Measuring drug concentrations in the CNS ................ 14

2.2 Animal models ............................................................................ 142.2.1 Monitoring CNS effects .............................................. 152.2.2 Visualizing the transfer ............................................... 152.2.3 Measuring drug concentrations in the CNS ................ 17

2.3 In vitro models ............................................................................ 20

3 Aims of the thesis .................................................................................... 21

4 Quantitative studies of the olfactory transfer of dopamine .................... 224.1 Nasal administration to mice ...................................................... 224.2 Olfactory mucosa in vitro ........................................................... 24

5 Visualizing the olfactory transfer ofdopamine and fluorescein dextran .......................................................... 28

5.1 Transfer across the olfactory epithelium .................................... 285.2 Later stages of the transfer ......................................................... 295.3 Methodology for visualization of olfactory drug transfer .......... 325.4 Effect of an in situ gel on the epithelial transfer of FD3 in rats . 33

6 Conclusions ............................................................................................. 37

Acknowledgements ...................................................................................... 38

References .................................................................................................... 39

Sammanfattning ............................................................................................ 44

4

5

Papers discussed

I Dahlin, M., Bergman, U., Jansson, B., Björk, E., Brittebo, E. (2000).Transfer of dopamine in the olfactory pathway following nasaladministration in mice. Pharm Res 17(6): 737-742.http://www.kluweronline.com/issn/0724-8741/Reprinted by permission of Kluwer Academic Publishers.

II Jansson, B., Chemuturi, N., Donovan, M., Björk, E.Dopamine transfer across olfactory mucosa: species and diffusionchamber comparisons. In manuscript.

III Jansson, B., Björk, E. (2002). Visualization of in vivo olfactoryuptake and transfer using fluorescein dextran.J Drug Target 10(5): 379-386.http://www.tandf.co.uk/journals/titles/1061186x.htmlReprinted by permission of Taylor & Francis.

IV Jansson, B., Hägerström, H., Edsman, K., Björk, E.Gellan gum increases the uptake and transfer of fluoresceindextran in rat nasal epithelium. Submitted.

This thesis is based on the following papers, which will be referred to in thetext by their roman numerals.

6

Abbreviations

AUC Area under the curveBBB Blood-brain barrierCLSM Confocal laser scanning microscopyCNS Central nervous systemCSF Cerebrospinal fluidDOPAC 3,4-(Dihydroxyphenyl)acetic acidECF Extracellular fluidERP Event-related potentialFD3 Fluorescein-labeled dextran, 3 kDaGI GastrointestinalHPLC High performance liquid chromatographyHRP Horseradish peroxidaseIsc Short circuit currentm-s Mucosal-to-serosalPBS Phosphate buffered salinePD Mucosal resting potential differencePapp Apparent permeability coefficientR Transmucosal electrical resistanceS.D. Standard deviations-m Serosal-to-mucosalTLC Thin layer chromatography

7

There are approximately thirty nasally applied medical products available onthe Swedish market. Two-thirds of these are locally acting decongestants, anti-histamines or cortisones, and one-third comprise a number of anti-migrainedrugs, peptide hormone analogs, and a nicotine nasal spray, all with systemiceffects. They are all available as nasal sprays, but a few of these products arealso formulated as nasal powders or nasal drops. In addition to these, a cyano-cobalamine nasal gel and an estradiol nasal spray are registered1 but are notyet on the market.

The extensive first-pass metabolism affecting orally administered peptidehormones and nicotine is avoided with nasal administration. Nicotine is alsomore rapidly absorbed from the nasal spray formulation than from any of theother available nicotine dosage forms. Oral administration is also unsuitablein some patients for reasons related to the illness in question. Patients withmigraine, for example, often suffer from vomiting which can affect thebioavailability of oral antimigraine products.

The field of nasal administration of drugs appears to have potential forgrowth, as there are many conditions in which a rapid effect, unattainablewith oral administration, is desired. For example, many human studies ofnasal opioid formulations for pain relief have been carried out (see review byDale et al., 2002). It is also likely that nasal vaccines will be available in thenear future. There are several studies showing that mucosal administration ofvaccines offers better protection against pathogens that enter the body viamucosal tissues than that offered by conventional vaccines (Davis, 2001). Nasaladministration also lessens the need for trained medical personnel and sterilesyringes.

The blood-brain barrier restricts the access of many compounds to the brain.As discussed in more detail later, there are several studies indicating that someagents which act in the central nervous system are more effective when givennasally than when given by other routes. New models were developed to studythis phenomenon during the work outlined in this thesis.

1 Introduction

1http://www.mpa.se/, Nov 10, 2003

8

1.1 The effects of physiological parameterson the nasal absorption of drugs

Perhaps the most obvious function of the nose is to perceive scents and odors.The olfactory receptors, to which odorants will bind, thus initiating the olfac-tory signal, are positioned on the dendrites of the olfactory nerve cells (Jonesand Rog, 1998), present in the superior part of the human nasal cavity (Fig. 1).These cells are unique in that they directly interface both the external sur-roundings and the central nervous system (CNS). The axons of the olfactorynerve cells form bundles, which terminate in the olfactory bulb, after passingthrough the thin sieve-like cribriform plate. As will be discussed later, thisconnection has potential for administration of drug compounds.

In humans, the olfactory area occupies only a small fraction of the nasalcavity. The major function of the remaining nasal cavity is to condition theinspired air, to protect the lungs from pathogens or environments that are toocold, too dry or too dusty. In order to provide efficient heating, moisturizingand filtering properties, the nasal cavity requires a fairly large surface areaand narrow air passages. This is accomplished by mucosa-lined bone-and-cartilage protrusions, the turbinates (conchae). The functions of the nasal cav-ity also demand a high blood flow which, along with the large surface area,enables a relatively high rate of absorption of nasally administered compounds.Finally, the air flow pattern in the nasal cavity results in the efficient deposi-

Figure 1. Sagittal and frontal sections of the human nasal cavity.ob olfactory bulb, st superior turbinate (concha nasalis superior), mt middle turbinate(concha nasalis media), it inferior turbinate (concha nasalis inferior). Areas in lighter grayindicate sinuses.

9

tion of dust (Andersen and Proctor, 1982), and therefore also of drug particlesor drops of drug solutions.

However, the olfactory epithelium is a potential entry point to the CNS fora number of pathogens and toxic substances (Olitsky and Cox, 1934; Johnson,1964; Tjälve and Henriksson, 1999; Sunderman, 2001) and the physical struc-tures of the nose protect this specialized epithelium by directing most of theair flow away from this area (Cole, 1982). This may be a challenge for futuredesigners of spray devices for nose-to-brain transfer of drugs.

In the respiratory region, where most particles are deposited, mucociliaryclearance transports the particles from the nasal cavity. The mucus, in whichthe particles are trapped, is transported (mostly towards the pharynx, afterwhich it will be swallowed) by coordinated ciliary motions. This limits thetime available for absorption (Merkus et al., 1998).

1.1.1 The nasal respiratory mucosaThe anterior third of the nasal cavity is lined with a stratified squamous epi-thelium, gradually transforming to regular skin at the nostrils. The middle andposterior thirds are lined with a pseudostratified ciliated columnar epithelium(the nasal respiratory epithelium), which consists of columnar cells – dividedinto ciliated and non-ciliated cells, goblet cells and basal cells (Mygind et al.,1982). As in most other epithelial tissues, tight junctions between the cellslimit the transfer of solutes across the epithelium. The mucus is produced bythe goblet cells, along with the serous and seromucous glands in the underly-ing lamina propria.

1.1.2 The olfactory mucosaThe olfactory epithelium (Fig. 2) is markedly higher (60-80 µm) than the na-sal respiratory epithelium (around 25 µm). The cell bodies of the olfactorycells are typically located in the middle and deeper regions of the epithelium.The nuclei of the second major cell type, the supporting cells, are organized ina single layer closer to the mucosal surface. These supporting cells enclosethe olfactory cells throughout the entire depth of the epithelium; tight junc-tions join the supporting cells and also appear at the junction with the olfac-tory nerve cells (Morrison and Costanzo, 1992). The single dendrites of theolfactory cells terminate in olfactory knobs (with a slightly larger diameterthan the dendrites), which extend above the epithelial surface and exhibit about10-25 immobile cilia each.

The olfactory axons are 0.1-0.7 µm in diameter and form the olfactory nervebundles after passing into the lamina propria. The bundles are surrounded by

10

Schwann cells and perineural cells (Jackson et al., 1979). The extracellularspace inside the perineural cells (the perineural space) is believed to be con-tinuous with the subarachnoidal space, surrounding the brain. This comprises,along with axonal transport, the suggested pathway taken by compounds ex-hibiting direct nose-to-brain transfer. The lamina propria also contains Bow-man’s glands, responsible for the mucus production in the olfactory mucosa(Getchell and Getchell, 1992).

High activities of both phase I and phase II enzymes have been found in theolfactory mucosa (Minn et al., 2002). It has been suggested that the physi-ological functions of this high metabolizing capacity are to terminate the ol-factory signals and to act as a nose-brain barrier.

1.1.3 The blood-brain barrier (BBB)As reviewed by Paulson (2002), the BBB effectively restricts the transfer ofhydrophilic compounds from the vascular compartment to the brain tissue. Incontrast to other tissues, no bulk flow occurs across the capillary walls, due totight junctions between the cells. Active efflux proteins, such as p-glycopro-tein, also contribute to the barrier properties.

This leaves two pathways accessible for transfer of drugs across the BBB(Pardridge, 1996): 1. lipid-mediated transport, restricted to small molecules

Figure 2. The olfactory mucosa.a) Micrograph of the rat olfactory mucosa.b) Schematic representation of the apical surface of olfactory epithelium. Olfactory cells

with cilia are shown in dark gray.bg Bowman’s gland, oc nuclei of olfactory cells (multiple layers), on olfactory nervebundles, sc nuclei of supporting cells (single layer)

11

(less than around 700 Da) and generally dependent on the lipophilicity of thecompound, and 2. carrier- or receptor-mediated transport, restricted to com-pounds that resemble nutrients and other substances vital to the brain.

1.2 Nose-to-brain transfer of drugsFigure 3 outlines the fates of a substance after nasal administration. As isevident from review articles in this field (Mathison et al., 1998; Illum, 2000),most compounds shown to be transferred directly from the nasal cavity to thebrain are fairly hydrophilic. This is likely to be not so much an exclusion oflipophilic compounds but rather a manifestation of the fact that many lipophiliccompounds are normally rapidly absorbed across the nasal mucosa (Illum,2003) and efficiently transferred across the BBB (van de Waterbeemd et al.,1998). Hence, olfactory transfer will be insignificant for these compounds.

Given the large number of compounds that have been shown in animalmodels to be directly transferred from the nasal cavity to the olfactory bulb(Mathison et al., 1998; Illum, 2000), there should be no doubt that this type of

Figure 3. Schematic representation of the fates of a nasally administered drug.

12

transfer occurs. The question of whether this form of transfer is sufficientlyextensive to result in therapeutically effective concentrations at the site ofaction in humans, however, remains to be answered. It has been shown thatnerve growth factor is taken up into the brain after nasal administration to ratsto a degree that exceeds the physiological concentrations, also in regions out-side the olfactory bulb (Chen et al., 1998). There are also indications thatnasal administration of peptides to humans results in CNS effects, where in-travenous administration fails to do so (Pietrowsky et al., 1996), and that cer-ebrospinal fluid (CSF), but not plasma, concentrations of some peptides areelevated after nasal administration (Born et al., 2002).

Nonetheless, in addition to offering potential new therapeutics, there couldalso be risks involved with direct nose-to-brain transfer of drugs. A drug thathas been administered for either a local nasal effect or for a systemic effectcould potentially reach toxic concentrations in the olfactory bulb of the brain.For example, the investigational mucosal vaccine adjuvants cholera toxin andits subunit CT-B, accumulate in the olfactory epithelium and the olfactorybulb (van Ginkel et al., 2000). Thus, the deposition of drug in the nasal cavityafter nasal administration should be optimized for the therapeutic goal, whetherit may be highly concentrated deposition at the olfactory mucosa for a CNSeffect or low deposition in that area when a systemic effect is desired.

In addition to transfer via the olfactory pathway, there is a theoretical pos-sibility of transfer via the trigeminal nerve. Some viruses enter the CNS viathis pathway, but others only enter the CNS via the olfactory nerve (Perlmanet al., 1995). To date, no published studies have demonstrated significant trans-fer of drugs or related compounds along the trigeminal nerve. However, thisnerve may still be relevant in this context. For example, the anti-migraineagent civamide is thought to affect the trigeminal nerve locally in the nasalcavity, leading to CNS effects (Diamond et al., 2000).

Some compounds, even when administered non-nasally, are preferentiallydistributed to the olfactory mucosa and the olfactory bulbs (Bergström et al.,2002). This highlights the need for appropriate controls when designing stud-ies in this field.

13

2.1 Human studies

2.1.1 Monitoring CNS effectsThe obvious difficulties in measuring CNS concentrations of drugs in humansand the limitations associated with the resolution of imaging techniques (suchas emission computed tomography) have meant that most human nose-to-brainstudies rely on measurement of the CNS effects from the drug in order toestimate the extent of its uptake. A group in Lübeck, Germany, has publishedthe results of several human studies in which peptides were administered na-sally to volunteers, and electroencephalographic (EEG) measurements weremade. Changes in the event-related potential (ERP) were investigated whilethe subject was performing an auditory oddball task. Many of these studies(Kern et al., 1997; Derad et al., 1998; Kern et al., 1999; Pietrowsky et al.,2001) used plasma drug concentrations after nasal administration and/or asystemic response to the drug as a control, rather than effects after intravascu-lar administration. If a CNS response was detected while the plasma concen-tration or the systemic response remained unchanged, this was interpreted asan indication of direct nose-to-brain transfer. Arginine-vasopressin was, how-ever, administered both nasally and intravenously in one study (Pietrowskyet al., 1996). Despite significantly higher plasma concentrations after intrave-nous administration (dose: 1.5 IU) than after nasal administration (dose: 20 IU)during the time the effect was monitored, the central effect (estimated usingthe ERP measurements) was more prominent after nasal administration.

A similar methodology was used by Lindhardt et al. (2001), who studieddiazepam. Intravenous administration resulted in stronger CNS effects thannasal administration, as expected because of the lipophilic character of thecompound, leading to an extensive BBB transfer. However, the onset of effectappeared to be more rapid after nasal administration, a phenomenon that maybe explained by rapid olfactory transfer of the drug.

2.1.2 Visualizing the transferThe nose-to-brain transfer of compounds in humans was demonstrated in onestudy using scintigraphy (Okuyama, 1997). A hydrophilic radioactively labeled

2 Models for nose-to-brain transfer of drugs

14

marker compound, [99mTc]-DTPA1 was administered intranasally to two sub-jects. No scintigrams could be produced because of the low levels of radioac-tivity in the brain, but the radioactivity recorded just inside the cribriformplate of an anosmic patient exceeded that in a remote brain area. In contrast,no significant differences were observed in the healthy control subject.

2.1.3 Measuring drug concentrations in the CNSThe Lübeck group also directly monitored the lumbar CSF and blood serumconcentrations of three peptides, melanocortin (4-10) [MSH/ACTH (4-10)],vasopressin and insulin, after nasal administration to healthy volunteers (Bornet al., 2002). The serum concentrations of MSH/ACTH (4-10) and insulinwere no higher than the physiological background concentrations. In the CSF,however, the concentrations of the peptides were higher than in placebo treatedsubjects.

Merkus et al. (2002) compared the CSF concentrations of melatonin in threeneurosurgery patients (with a cerebrospinal drain) after intravenous and nasaladministration. The area under the concentration-time curves (AUC) in CSFwere similar after the two administration routes, most probably due to thehigh nasal bioavailability and efficient transfer of melatonin across the BBB.

2.2 Animal modelsAlmost all reported animal models for the nose-to-brain transfer of drugs arerodent models. Since the olfactory area in rodents is approximately 50% ofthe total nasal area (Gross et al., 1982), while it is only 2-6% of the total inhumans (Morrison and Costanzo, 1990) the extent of direct nose-to-brain trans-fer in humans is likely to be overestimated. Hence, conclusions regarding thebioavailability of a compound and the extent of its delivery to the brain inhumans cannot be drawn from results obtained in animal models. Issues in-volving the relative influence of different formulations on the uptake of drugs,or comparison of the uptake of different compounds may, however, be use-fully explored in animal models.

Typically, administrations are performed by either placing small amountsof the formulation at the nostrils, or through thin tubing or a blunt needleinserted into the nasal cavity from the nostrils. Also, rodents exhibit a directconnection between the oral and nasal cavities, the nasopalatine or incisive

1 Diethylenetriaminepenta-acetic acid

15

duct. The delivery of insulin to the brain in mice after administration throughthis connection (intraolfactory administration) has been compared to that af-ter administration by the previously mentioned procedures (Gizurarson et al.,1996). It was demonstrated that the brain/blood radioactivity ratio was higherafter the intraolfactory administration than after the other methods of admin-istration.

2.2.1 Monitoring CNS effectsAs demonstrated in Table 1, most animal studies monitoring a central effectafter nasal administration have not included any controls in which the com-pound was given by other routes. It is, therefore, difficult to draw conclusionsfrom these experiments, since systemic absorption followed by transfer acrossthe BBB may have taken place. In a study performed by De Souza Silva et al.(1997), however, unilateral nasal administration of L-dopa methyl ester to ratsresulted in a unilateral increase in dopamine concentration in the neostriatum.Such responses are likely to be the result of the unilateral olfactory transfer ofa compound.

A few studies included control animals which received the drug by alterna-tive routes. Jagannadha Rao et al. (1986) showed that the nasal administrationof β-endorphin to monkeys resulted in a more rapid increase in serum prolac-tin concentrations than intravenous administration of the same dose. In a studyby Dluzen and Kefalas (1996), the concentrations of norepinephrine in themouse olfactory bulb and the corpus striatum were lower 7 days after nasaladministration of the neurotoxin MPTP1 than after intraperitoneal administra-tion.

2.2.2 Visualizing the transferThe animal models used in this thesis for visualizing the nose-to-brain trans-fer of model compounds will be discussed later, in relation to the studies per-formed in Papers I and III. This section presents a review of earlier studies inwhich attempts were made to visually demonstrate drug transfer.

Faber (1937) and Rake (1937) studied the olfactory transfer of Prussianblue (an iron ferrocyanide suspension) after nasal administration to rabbitsand mice, respectively. Absorption across the epithelium was rapid, with dyeobserved in the submucosa at the first measured time point at 2 minutes. Ab-sorption through the epithelium took place mainly in relation to the olfactory

11-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine

16

Syst

emic

Com

poun

dEf

fect

par

amet

erSp

ecie

sco

ntro

lFo

rmul

atio

n

Jaga

nnad

ha R

ao e

t al.,

198

6β-

Endo

rphi

nSe

rum

pro

lact

inB

onne

tye

sEt

hano

l: pr

opyl

ene

conc

entra

tion

mon

keys

glyc

ol: w

ater

(3:3

:4)

Dlu

zen

and

Kef

alas

, 199

6M

PTP

Olfa

ctor

y bu

lb a

ndM

ice

yes

10%

eth

anol

in s

alin

eco

rpus

stria

tum

cat

echo

l-am

ine

conc

entra

tion

Goz

es e

t al.,

199

6Va

soac

tive

inte

stin

alW

ater

maz

e pe

rfor

man

ceR

ats

no40

% is

opro

pano

l,pe

ptid

e an

alog

10%

pro

pyle

ne g

lyco

l-fa

tty a

cid

este

rD

e So

uza

Silv

a et

al.,

199

7L-

dopa

met

hyl e

ster

Neo

stria

tum

cat

echo

lam

ine

Rat

sno

Salin

eC

ocai

neco

ncen

tratio

n (m

icro

dial

ysis

)A

mph

etam

ine

Bet

bede

r et a

l., 2

000

Mor

phin

eN

ocic

eptiv

e re

spon

seM

ice

yes

Poly

sacc

harid

icna

nopa

rticl

es in

PB

SG

ozes

et a

l., 2

000

Act

ivity

-dep

ende

ntW

ater

maz

e pe

rfor

man

ceR

ats

no20

% is

opro

pano

l,ne

urot

roph

ic fa

ctor

5% p

ropy

lene

gly

col-

anal

ogs

fatty

aci

d es

ter

Liu

et a

l., 2

001

Insu

lin-li

ke g

row

thSe

nsor

y an

d m

otor

Rat

sno

Succ

inat

e bu

ffer

edfa

ctor

-1pe

rfor

man

cesa

line

pH 6

Cap

soni

et a

l., 2

002

Ner

ve g

row

thH

isto

logi

cal s

igns

inM

ice

noPB

Sfa

ctor

an A

lzhe

imer

mod

elC

hen

et a

l., 2

002

Kai

nic

acid

Seiz

ures

Mic

eno

Wat

erJi

n et

al.,

200

3Fi

brob

last

gro

wth

fact

or-2

,N

euro

gene

sis

Mic

eno

— a

Hep

arin

-bin

ding

epi

derm

algr

owth

fact

or

Tabl

e 1.

Pub

licat

ions

whe

re C

NS

effe

cts

have

bee

n m

onito

red

in a

nim

al m

odel

s af

ter

nasa

l adm

inis

trat

ion

of v

ario

us c

ompo

unds

.

a not

sta

ted

17

cells (Rake, 1937). The dye was observed in the perineural space and also inthe subarachnoid space.

De Lorenzo (1970) and Gopinath et al. (1978) demonstrated, using elec-tron microscopy, the olfactory epithelial absorption of colloidal gold particlesafter nasal administration to squirrel monkeys and rhesus monkeys. Gopinathet al. observed uptake by both supporting and olfactory cells and subsequentdistribution inside the olfactory neurons. De Lorenzo studied the transfer ofcolloidal gold to the brain and demonstrated that colloidal gold was present inthe glomeruli in the olfactory bulb within 1 hour after administration.

Horseradish peroxidase (HRP) has been used as a model compound by sev-eral investigators. Kristensson and Olsson (1971) detected HRP in vesicles ofboth olfactory and supporting cells 5–15 minutes after nasal administration of1.0% HRP to mice. HRP was also observed in what was believed to be theBowman’s ducts. A smaller amount was found between the epithelial cells.After 1–24 hours, HRP was also detected in the olfactory nerve bundles andthe pia mater and arachnoid meninges covering the olfactory bulb.

Balin et al. (1986), in contrast, observed predominately paracellular ab-sorption of 0.5-3.0% HRP given nasally to rats, mice and squirrel monkeys.Bannister and Dodson (1992) observed a dose-dependent uptake mechanismin mice; with lower concentrations (0.08%) of HRP solution, the compoundwas found in intracellular vesicles but, with higher concentrations (1.0%),absorption was mainly paracellular, with some cellular uptake. An associatedcompound, wheat germ agglutinin-HRP was endocytosed in the epitheliumand underwent axonal transport to the CNS in the olfactory neurons (Balinet al., 1986).

15– 60 minutes after nasal administration of Evans blue-labeled albumin tomice (Kristensson and Olsson, 1971), fluorescence was exhibited in olfactoryand supporting cells, as well as in the perineural space. Bannister and Dodson(1992) studied the cellular distribution of ferritin (10% suspension in dex-trose) and thorium dioxide (25% suspension in dextrose) after nasal adminis-tration to mice. These were distributed to approximately the same areas asHRP, as reported by the same authors. Thus, they were endocytosed by botholfactory and supporting cells.

2.2.3 Measuring drug concentrations in the CNSTable 2 lists recent studies where drug concentrations have been measured inthe CSF and the brain tissue. With the exception of a few studies involvingrhesus monkeys (Anand Kumar et al., 1974; Anand Kumar et al., 1982) andrabbits (Czerniawska, 1970), studies investigating nose-to-brain transfer by

18

Com

poun

dD

etec

tion

Spec

ies

Sur

gery

of a

irway

sFo

rmul

atio

n

CSF

Dah

lin a

nd B

jörk

, 200

0S-

UH

-301

HPL

CR

ats

Trac

hea

cann

ulat

ion

Salin

eK

ao e

t al.,

200

0L-

dopa

est

ers

HPL

CR

ats

Hira

i pro

cedu

rea

—b

Dah

lin e

t al.,

200

1[3 H

]-D

opam

ine

Rad

ioac

tivity

Rat

sTr

ache

a ca

nnul

atio

nPB

SD

ahlin

and

Bjö

rk, 2

001

NX

X-0

66H

PLC

Rat

sTr

ache

a ca

nnul

atio

nA

ceta

te b

uffe

rva

n de

n B

erg

et a

l., 2

002

Hyd

roco

rtiso

neR

adio

imm

uno-

Rat

sno

neR

ando

mly

met

hyla

ted

assa

yβ-

cycl

odex

trin

in sa

line

Wan

g et

al.,

200

3M

etho

trexa

teH

PLC

Rat

sM

odifi

ed H

irai

Phos

phat

e bu

ffer

with

proc

edur

eN

a 2SO

3an

d be

nzyl

alc

ohol

Tissue

Dah

lin e

t al.,

200

0[3 H

]-D

opam

ine

Rad

ioac

tivity

,M

ice

none

Phos

phat

e bu

ffer

TLC

van

Gin

kel e

t al.,

200

0[12

5 I]-C

hole

ra to

xin,

Rad

ioac

tivity

Mic

e—

bPB

SC

T-B

sub

unit

Goz

es e

t al.,

200

0A

ctiv

ity-d

epen

dent

Rad

ioac

tivity

,R

ats

none

20%

isop

ropa

nol,

neur

otro

phic

fact

orH

PLC

5% p

ropy

lene

gly

col-

anal

ogs

fatty

aci

d es

ter

Cho

w e

t al.,

200

1B

enzo

ylec

goni

neH

PLC

Rat

sH

irai p

roce

dure

Salin

eB

ergs

tröm

et a

l., 2

002

[3 H]-

Pico

linic

aci

dR

adio

activ

ity,

Mic

eno

nePh

osph

ate

buff

erG

C-M

Sc

Duf

es e

t al.,

200

3[12

5 I]-V

asoa

ctiv

eR

adio

activ

ity,

Rat

sH

irai p

roce

dure

Buf

fers

with

var

ying

pH

inte

stin

al p

eptid

eH

PLC

and

osm

olar

ity.

0, 0

.1 o

r 1%

laur

oylc

arni

tine

Tabl

e 2.

Rec

ent p

ublic

atio

ns w

here

CSF

or

brai

n tis

sue

conc

entr

atio

ns w

ere

mea

sure

d in

ani

mal

mod

els.

a Hira

i et a

l., 1

981

b not

sta

ted

c Gas

chr

omat

ogra

phy,

mas

s spe

ctro

met

ry

19

measuring CSF concentrations have been performed in rats. Most of thesestudies involved various surgical procedures, ranging from just cannulatingthe trachea to a series of procedures described by Hirai et al. (1981): cannula-tion of the trachea, closing the esophagus (either by inserting a sealed poly-ethylene tube, or by tying a piece of suture around it) and sealing the naso-palatine duct with glue. A few of these studies (Sakane et al., 1991a; Sakaneet al., 1991b; Sakane et al., 1999; Dufes et al., 2003) used either single-passor recirculating perfusion techniques. The additional stress on the mucosacaused by this procedure should be carefully considered, especially when evalu-ating the effects of absorption enhancers.

Ideally, these studies should include frequent sampling of both plasma andbrain tissue/CSF after nasal and intravenous administration, to enable calcu-lation of the AUC in both brain tissue/CSF and in plasma. An indication ofdirect nose-to-brain transfer is then obtained when AUCnasal /AUCintravenous inthe brain samples is larger than the plasma bioavailability after nasal adminis-tration. The importance of frequent, and early, sampling is highlighted by astudy performed by Chow et al. (1999) in which cocaine concentrations in ratbrain tissue were analyzed after nasal and intravenous administration. TheAUC of cocaine in the olfactory bulb after nasal administration exceeded thatobtained after intravenous administration only when calculated for times upto 4 min. The AUC0-60 was lower after nasal than after intravenous administra-tion. This indication of direct nose-to-brain transfer could easily have beenoverlooked if samples had not been taken as early as 1, 2 and 4 min.

Similar approaches have been used when analyzing CSF concentrations(Chou and Donovan, 1997; Chou and Donovan, 1998a; Wang et al., 2003). Inthese studies, a CSF replacement procedure was used, enabling repeated sam-pling in the same animals. These procedures, along with most of the proce-dures used in this field of research, are terminal for the experimental animals.In contrast, van den Berg et al. (2002) developed a procedure for serial CSFsampling without replacing the withdrawn CSF, which raised the possibilityof repeated use of the same rat for additional experiments. These authors alsoinvestigated the nasal absorption of compounds when the rat heads were heldin different positions. It was demonstrated that, in addition to influencing theCSF sampling flow rate, the extent of absorption into the plasma and CSFvaried depending on which position was used.

When planning models of nose-to-brain transfer, it is also necessary to con-sider the possible uneven distribution of the compound in the various parts ofthe CSF (Seki et al., 1994; Yajima et al., 1998; Kumbale et al., 1999). Whenthe CSF, sampled from the cisterna magna, was divided into a number of

20

fractions, the last fraction to be sampled (i.e. from the vicinity of the olfactorybulb) exhibited higher concentrations.

Chou and Donovan (1998b) compared the CSF concentrations of lidocaineobtained after cisternal puncture with those obtained by implanting a micro-dialysis probe in the cisterna magna, and observed slightly different time-concentration profiles after nasal administration. This phenomenon might berelated to uneven distribution throughout the CSF, as mentioned above, in thatthe microdialysis method gives an estimation of the concentration in the cis-terna magna, and the cisternal puncture method samples CSF from other re-gions as well. In the same study, microdialysis was also used to measure brainextracellular fluid (ECF) concentrations of lidocaine. Despite the fact that thismethod allows for cross-over studies, and that the unbound ECF concentra-tions most likely are more relevant than CSF concentrations (Hammarlund-Udenaes, 2000), this report is at present the only example where microdialysishas been used to measure brain uptake after nasal administration.

Although mice, because of their small size, are unsuitable for CSF sam-pling and frequent blood sampling, they can be used in studies estimatingdrug concentrations in brain tissue. In the mouse model, the amount trans-ferred is typically assessed by administering a radioactively labeled compoundand measuring the transferred radioactivity (Gizurarson et al., 1996; Sigurdssonet al., 1997; Eriksson et al., 1999; van Ginkel et al., 2000; Bergström et al.,2002). The relative absence of sample workup makes this a suitable and rapidmethod for initial experiments. As metabolism in the olfactory mucosa is knownto be extensive (Minn et al., 2002), the radioactivity measurements should,however, be supported by some means of identifying the compound in ques-tion.

2.3 In vitro modelsIn vitro models of the systemic nasal absorption of drugs, comprising rabbitnasal respiratory mucosa (Bechgaard et al., 1992), bovine nasal respiratorymucosa (Schmidt et al., 2000), porcine nasal respiratory mucosa (Wadell etal., 1999) and human nasal primary cell culture (Werner and Kissel, 1995),have been developed previously. However, no in vitro studies of the olfactorytransfer of drugs have been published to date. The use of rabbit olfactorymucosa is probably not an option for this undertaking, since a sufficientlylarge area of mucosa to allow easy isolation is not available. In this thesis,experiments with porcine and bovine olfactory mucosa are presented.

21

The aims of this thesis were to identify model compounds suitable forinvestigation of the transfer of drugs from the nasal cavity to the CNS andto develop and refine models for testing these compounds. The mechanismsinvolved in drug transport and the influence of the formulation of themodel compound on its uptake were considered parameters of particularimportance.

The objectives of the individual studies were to:

• Investigate the transfer of dopamine to the brain following nasaladministration to mice.

• Compare the bidirectional transfer of dopamine across olfactorymucosa in two diffusion chamber models.

• Visualize the transfer of fluorescein-labeled dextran in the olfactorypathway after nasal administration to rats.

• Investigate the influence of deacetylated gellan gum on the uptakeand transfer of fluorescein-labeled dextran across nasal epithelia inrats.

3 Aims of the thesis

22

4.1 Nasal administration to miceIn Paper I of this thesis, [3H]-dopamine in 5 µl phosphate buffer (pH 7.4) wasadministered to mice through polyethylene tubing (outer diameter 0.61 mm)inserted into the right nostril. Unilateral administration allows the contralat-eral olfactory bulb to be used as a control. The mice were euthanized and thebrain was dissected into several regions and analyzed for radioactivity by liq-uid scintillation counting. There were significantly higher radioactivity levelsin the right olfactory bulb than in the left bulb at 30 min, and at 1, 2 and4 hours (Fig. 5). Eight hours after administration, radioactivity levels did notdiffer significantly between the two sides. The maximum radioactivity level

4 Quantitative studies of the olfactorytransfer of dopamine

Distribution of dopamine (Fig. 4) to the CNS is low after oral or parenteraladministration (Oldendorf, 1971). Anand Kumar et al. (1976) reported resultsfrom preliminary studies involving administration of [3H]-dopamine nasallyand intravenously to rhesus monkeys. After intravenous administration, radio-activity was present systemically, but only in very low levels in the CSF. Afternasal administration in the same study, high levels of radioactivity were foundin both the CSF and serum. Later, Ikeda et al. (1992) investigated thebioavailability of dopamine in beagle dogs after administration by variousroutes. The average bioavailability after nasal administration was 11.7%, com-pared with 3.0, 1.4, 1.1 and 0% after oral, rectal, dermal and buccal adminis-tration, respectively.

Figure 4. Dopamine and 3,4-(dihydroxyphenyl)-acetic acid (DOPAC).

23

Figure 5. The radioactivity in brain tissue samples after unilateral nasal administration(right side) of [3H]-dopamine to mice.a) Right and left olfactory bulbs after nasal administration (n =5-7) and in whole brain

tissue samples 30 min after intravenous administration (n=3) of [3H]-dopamine.b) Radioactivity in various brain tissues after nasal administration.Data are expressed as means+S.D. (* p < 0.05, ** p < 0.01)

in the olfactory bulb was observed 4 hours after nasal administration. If drugtransport is entirely passive, occurring through the perineural space surround-ing the olfactory nerves rather than through the nerve cells themselves, peakconcentrations are normally reached more rapidly than this (as is evident fromPaper III). It is therefore possible that active processes such as axonal trans-port are involved in the olfactory transfer of dopamine in mice. Axonal trans-port of dopamine has previously been observed in the feline peroneal nerve(Ben-Jonathan et al., 1978).

In other brain regions, the radioactivity levels were 10- to 50-fold lowerthan in the olfactory bulbs. The only region exhibiting higher radioactivitywas the lateral olfactory tract, 4 hours after administration (Fig. 5).

Samples from the olfactory mucosa and from the olfactory bulb wereanalyzed by thin layer chromatography (TLC) to investigate the origin of theradioactivity. Metabolism of dopamine in the olfactory mucosa was extensivebut, in the samples from the olfactory bulb, the radioactivity levels indicatedalmost exclusively transfer of unchanged dopamine. The levels of radioactiv-ity co-eluting with DOPAC (Fig. 4), one of the main metabolites of dopamine,were no higher than the background radioactivity.

The nasal absorption of [3H]-dopamine has also been investigated in rats(Dahlin et al., 2001). Radioactivity levels in the CNS were significantly higher

a) b)

24

30 min after unilateral nasal administration than after intravenous administra-tion in these animals. However, since there were no significant differences inthe levels of radioactivity between the left and right olfactory bulbs, it was feltthat evidence of olfactory transfer of dopamine in rats was inconclusive. TLCagain showed that most of the radioactivity in the olfactory bulbs originatedfrom unchanged [3H]-dopamine. However, high levels of radioactivity co-elut-ing with DOPAC (30-40%) were also found. In the CSF, 83% of the radioac-tivity co-eluted with DOPAC and only 14% co-eluted with dopamine.

4.2 Olfactory mucosa in vitroThe complexity of animal models makes it difficult to draw conclusions regard-ing individual stages and barriers in the olfactory transfer process. A concen-tration-time profile in the olfactory bulb gives no clear information regardingthe extent or rate of transfer across the distinct tissue barriers encounteredduring drug transfer to the bulb. The focus of Paper II was the first of thesebarriers in the transfer process, the olfactory epithelium, which was studiedusing in vitro methodology. This stage is important, since the epithelial trans-port pathways preferably should be identified before methods of enhancingabsorption are developed, and this is the only stage of the transfer process thatcan be influenced by drug formulation measures such as optimization of vis-cosity or addition of permeation enhancers.

In the most commonly used diffusion chamber setup, the mucosal tissue ismounted vertically between symmetrical chambers, which allows efficientoxygenation and stirring by bubbling oxygen through the medium (Grass andSweetana, 1988). However, an alternative chamber system in which the mucosal

Table 3. Dopamine permeability coefficients (mean±S.D.) of bovine olfactory mucosa invertical diffusion chambers and porcine olfactory mucosa in horizontal diffusion chambers(n=3-5).

Papp (cm·s–1) (·106)C0 (mg/ml) m-s s-m

1.0 29 ±11 16 ± 3.7 p<0.054.0 14 ± 1.4 19 ± 2.7 n.s.

1.0 3.9± 2.4 2.0± 1.1 p<0.054.0 2.6± 0.95 2.6± 0.63 n.s.

m-s mucosal-to-serosal direction, s-m serosal-to-mucosal direction,C0 initial donor concentration, n.s. not significant.

Bovine mucosa invertical chambers

Porcine mucosa inhorizontal chambers

25

tissue is mounted horizontally, with an air interface at the mucosal surface(Östh et al., 2002a), is more physiologically realistic for the nasal mucosa,and also enables the study of semisolid and solid formulations (Östh et al.,2002b; Östh et al., 2003).

In Paper II, bidirectional dopamine permeability coefficients were deter-mined using two in vitro models of olfactory drug absorption, namely bovineolfactory mucosa in the vertical diffusion chamber and porcine olfactory mu-cosa in the horizontal chamber.

With both models, there was a significant difference between the apparentpermeability coefficient in the mucosal-to-serosal direction (Papp m-s) and thatin the serosal-to-mucosal direction (Papp s-m) when the initial donor concentra-tion was 1 mg/ml (Table 3). The Papp m-s was approximately twice as high as thePapp s-m, both for bovine olfactory mucosa in vertical chambers and porcineolfactory mucosa in horizontal chambers. At a higher donor concentration(4 mg/ml), the Papp was similar in both directions. Also, the difference in Papp m-s

between donor concentrations of 1 mg/ml and 4 mg/ml was significant for thebovine mucosa. This indicates the presence of an active dopamine transportsystem in the olfactory epithelium.

The Papp in porcine olfactory mucosa was negatively correlated with thetransmucosal electrical resistance (R) for both directions studied (Fig. 6). SinceR is believed to reflect the integrity of the tight junctions, this correlation islikely to indicate that the transfer of dopamine across porcine olfactory mu-cosa involves the paracellular route. Further support for this idea was ob-tained by plotting Papp for testosterone and mannitol vs R in porcine nasal

Figure 6. The influence of the transmucosal electrical resistance on the permeabilitycoefficients of dopamine for the (a) mucosal-to-serosal and (b) serosal-to-mucosaldirections in porcine olfactory mucosa in horizontal diffusion chambers. The slopes aresignificantly non-zero (dashed lines show 95% confidence intervals).

a) b)

26

respiratory mucosa (Fig. 7). The data used as the basis for these calculationswere obtained from Östh et al. (2002a). A significant correlation was obtainedfor mannitol, a widely used marker compound for paracellular transport, butnot for testosterone, a lipophilic compound that is believed to cross the epithe-lium by transcellular diffusion.

The change in resting potential difference over the mucosa (PD) during thetransport experiments depended on the direction (m-s or s-m) of the experi-ment (Fig. 8). The PD increased when the mucosal surface was on the donorside and decreased when the serosal surface was on the donor side. This sug-gests that the absence of serosal buffer caused unwanted effects on the mu-cosa, such as complications with nutrition of the epithelial cells, and indicatesthat the horizontal chamber setup with an air interface is unsuitable for bidirec-tional experiments. R, however, increased slightly in both cases (data notshown); it is relatively common in experimental work of this kind to onlymonitor R, which in this case would have lead to the erroneous conclusionthat the electrophysiology was unchanged.

The dopamine Papp values were 5-8 times higher for bovine mucosa in thevertical diffusion chamber than for porcine mucosa in the horizontal diffusionchamber (p<0.05 for all relevant comparisons). While species differences werelikely to have played a part in this large difference between the models, thethicker unstirred water layer in the horizontal diffusion chamber may alsohave affected the outcome. The stirring produced by bubbling oxygen in thevertical diffusion chamber will be more efficient than that produced by thecircular shaking used for the horizontal chamber. Because of the very small

Figure 7. The influence of the transmucosal electrical resistance on the permeabilitycoefficients of (a) mannitol and (b) testosterone in porcine nasal respiratory mucosa inhorizontal diffusion chambers (Östh et al., 2002a). The slope is significantly non-zero formannitol but not for testosterone.

a) b)

27

donor volume (50 µl) in the horizontal diffusion chambers, stirring will beclose to non-existent on the donor side, reflecting the actual situation in thenasal cavity. The influence of the unstirred water layer upon Papp has previ-ously been investigated in the Caco-2 cell model, and was shown to be impor-tant for some drugs (Karlsson and Artursson, 1991).

In the diffusion chamber models, DOPAC appeared to be the main metabolite.The final DOPAC concentrations were negligible on the donor side of thevertical diffusion chambers, but was about 5-25% of the concentration ofdopamine on the donor side of the horizontal diffusion chambers. Since thisleaves less dopamine to be transferred to the receiver side, this may in partexplain the lower permeabilities obtained with the horizontal diffusion cham-bers. No differences between donor or receiver DOPAC concentrations be-tween the m-s and s-m directions that could explain the higher Papp m-s could beobserved.

Taken together, the results from Papers I and II indicate that both the epi-thelial stage and the later stages of the olfactory transfer of dopamine haveactive components and that the epithelial stage has a significant contributionof paracellular transfer.

Figure 8. Average (–S.D.) electrical potential differences over the porcine olfactory mucosabefore (empty bars) and after (solid bars) the dopamine transport experiments (n=7-8).

28

5.1 Transfer across the olfactory epitheliumThe transfer of 3 kDa fluorescein-labeled dextran (FD3) through the rat olfac-tory epithelium was studied in Paper III. This particular fluorescein dextran ismade aldehyde-fixable, a prerequisite for the methods used, by covalentlybound lysine residues. The molecular weight of 3 kDa was selected to com-plement earlier studies in the field, which used suspensions and considerablylarger molecules. Further, FD3 was seen as a possible model for peptides withtherapeutic potential (Fehm et al., 2000; Gozes, 2001).

A solution of FD3 (50 µl in saline) was administered unilaterally to anes-thetized rats through polyethylene tubing (outer diameter 0.965 mm) insertedinto the nasal cavity. At set intervals after administration, the rats were perfusionfixed via the heart. The nasal cavities, including the olfactory bulbs, wereisolated. After embedding them in plastic, sections from various levels (Fig. 9)of the nasal cavity were studied with fluorescence microscopy.

In Paper III, two distinct modes of distribution across the olfactory epithe-lium were observed. In mucosa from the nasal septum, many olfactory cells,

5 Visualizing the olfactory transfer ofdopamine and fluorescein dextran

Figure 9. Schematic representation of the levels (I -V) of the nasal cavity, referred to inPaper III and IV.e endo- and ectoturbinates, m maxilloturbinate, n nasoturbinate, ob olfactory bulb

29

but few supporting cells, exhibited FD3 fluorescence. In mucosa from theecto- and endoturbinates, both the supporting cells and the olfactory cellsshowed high levels of FD3 fluorescence (Fig. 10). The lamina propria alsoexhibited FD3 fluorescence, indicating that FD3 was also transferred acrossthe olfactory epithelium. The uptake process was rapid, with higher degreesof FD3 fluorescence observed after 2 and 15 min than after 1, 4 and 24 h.

Despite the large differences in molecular weight compared with Prussianblue (a suspension) (Faber, 1937; Rake, 1937), Evans blue-labeled albumin(appr. 66 kDa) and HRP (47 kDa) (Kristensson and Olsson, 1971), the resultsobtained for these compounds were similar. The most probable mechanism ofabsorption of such large hydrophilic compounds is endocytosis. No paracellularFD3 fluorescence was discerned, and transcellular diffusion was consideredmost unlikely to have occurred. Interestingly, olfactory epithelial cells havebeen shown to contain large amounts of endocytic vesicles (Bannister andDodson, 1992).

In the lamina propria, FD3 fluorescence was observed in the perineuralspace surrounding the olfactory nerve bundles, and was not seen inside thenerves. This implies that the later stages of transfer of FD3 are passive, occur-ring in the perineural space.

5.2 Later stages of the transferIn Paper I, the distribution of radioactivity in the nasal cavities and brains ofmice was also illustrated using tape section autoradiography, after unilateralnasal administration of [3H]-dopamine. High levels of radioactivity were ob-served in the ipsilateral (right) side but not in the contralateral side of thenasal cavity (Fig. 11). Thus, Paper I demonstrated little or no communicationbetween the two sides of the mouse nasal cavity. Similarly, radioactivity wasonly observed in the ipsilateral olfactory bulb. The radioactivity was localizedto the olfactory nerve layer and the glomerular layer, and decreased in inten-sity towards the center of the olfactory bulb. 24 hours after administration, noradioactivity was observed in the olfactory bulb.

FD3 was shown in Paper III to be distributed similarly to dopamine in therat olfactory bulb. As demonstrated in Figure 12, the ipsilateral olfactory bulbexhibited FD3 fluorescence in animals euthanized as early as 2 min after ad-ministration. Here, the model compound was detected in the olfactory nervelayer, the glomerular layer and the external plexiform layer. FD3 fluorescencewas also observed in the perineural space surrounding the olfactory nervebundles penetrating the cribriform plate. These results are supported by previ-

30

ous findings by Sakane et al. (1995) who detected higher levels of FITC-labeled dextrans in the CSF after nasal than after intravenous administrationin rats.

Thus, the transfer of FD3 through olfactory tissue appears to be intracellu-lar across the outer epithelial layer and extracellular through the later stages.Mathison et al. (1998) have suggested that there may be two pathways in-volved in olfactory transfer: the olfactory nerve pathway, which involves up-take of the agent into the olfactory cells and subsequent axonal transport, andthe olfactory epithelial pathway, which involves epithelial transfer by othermeans (across supporting cells, through Bowman’s ducts or paracellularly)

Figure 11. The distribution of radioactivity in the mouse nasal cavity and olfactory bulbafter nasal administration of [3H]-dopamine.a) Autoradiogram of a mouse skull (transversal section) 2 h after unilateral nasal

administration (right side) of [3H]-dopamine.b) Corresponding hematoxylin-eosin stained tissue section.There is a high level of radioactivity in the right olfactory mucosa, the right axonal nervelayer and the glomerular layer of the olfactory bulb. Radioactivity is present only at lowlevels in the other brain regions. The arrowheads in both figures point at the border betweenthe olfactory mucosa in the nasal cavity and the olfactory bulb.b brain, e eye, n nasal cavity, o olfactory bulbs

Figure 12. The distribution of fluorescence in the rat nasal cavity and olfactory bulb 2 minafter administration of FD3. Ipsilateral to administration (right side). A high level of FD3fluorescence is located in the olfactory mucosa adjacent to the cribriform plate. FD3fluorescence can also be observed surrounding the olfactory nerve bundles crossing thecribriform plate, in the olfactory nerve layer in the olfactory bulb, and in the glomerular andexternal plexiform layers. Scalebar 50 µm.cp cribriform plate, epl, gl, onl external plexiform, glomerular and olfactory nerve layers ofthe olfactory bulb, oe olfactory epithelium, on olfactory nerve bundle

Figure 10. The distribution of fluorescence in the rat olfactory mucosa after nasaladministration of FD3.a) Olfactory mucosa on the septum in level II of a rat euthanized 2 min after administration.

Many olfactory cells and a few supporting cells have taken up FD3. The olfactoryknobs, dendrites and cell bodies of the olfactory cells exhibit a high intensity ofFD3 fluorescence. Scalebar 50 µm.

b) Olfactory mucosa on ethmoid turbinate in level III of a rat euthanized 2 min afteradministration. The supporting cells of the olfactory mucosa and some olfactory cellshave taken up FD3. The lamina propria also exhibits a high level of FD3 fluorescence.Scalebar 20 µm.

bg Bowman’s gland, oc nuclei of olfactory cells, on olfactory nerve bundle, sc nuclei ofsupporting cells

31

a) b)Figure 10.

Figure 11.

Figure 12.

32

followed by transfer along the perineural space. Our results indicate that,although part of the epithelial transfer of the FD3 occurred across olfactorycells, the main mechanism involved in the later stages was extracellular in itsnature. It thus seems more accurate to address the epithelial stage and the laterstages of olfactory transfer individually.

5.3 Methodology for visualization ofolfactory drug transfer

The use of fixable fluorescein dextran for the visualization of epithelial trans-fer was first reported by Marttin et al. (1997). They used confocal laser scan-ning microscopy (CLSM) to visualize the transfer across rat nasal respiratorymucosa. CLSM was also used in preliminary experiments for this thesis. Themain reason for shifting the focus to the more time consuming fluorescencemicroscopy of plastic sections was that this latter method offered the opportu-nity to obtain single images of epithelial, submucosal, and in some cases evenolfactory bulb distribution of the model compound. Also, with this method, itwas possible to obtain information from the entire nasal cavity, not just thenasal septum.

Tape section autoradiography, which was used in Paper I, provides a roughidea of the distribution of a radiolabeled compound in the nasal cavity and theolfactory bulb. However, information regarding cellular distribution is diffi-cult to obtain with this method, because of the poor quality of the cryosections.Microautoradiography of paraffin sections is an alternative but is, just as theapproach in Papers III and IV, limited to compounds that can be fixed to thetissue.

Figure 13. The structure of deacetylated gellan gum.

33

5.4 Effect of an in situ gel on the epithelialtransfer of FD3 in rats

The methodology of Paper III was applied to the investigation of the effects ofthe in situ gelling agent, deacetylated gellan gum, on the transfer of FD3 acrossnasal epithelia in paper IV. Deacetylated gellan gum (Fig. 13) is a polysaccha-ride that readily forms strong gels in physiological concentrations of cations(Paulsson et al., 1999). In an ion-free environment, solutions of gellan gumexhibit low viscosity. In Paper IV, the epithelial uptake of FD3 after nasaladministration of a formulation containing 0.5% deacetylated gellan gum wascompared with that after administration of a formulation without the gum.The formulations were made isotonic by the addition of mannitol.

In addition to comparing the distribution of the fluorescence, its intensitywas scored (0-5) in various regions in order to facilitate comparison betweenthe formulations. Figure 14 illustrates the average ranges of these scores at

Figure 14. The average ranges of the fluorescence scoring after nasal administration of theFD3 mannitol solution (dashed bars) and the FD3 gellan gum formulation (solid bars).a) The nasal respiratory epithelium at level I.b) The lamina propria at level I.c) The olfactory epithelium and lamina propria at level IV.

a) b)

c)

34

Figure 15. Representative fluorescence micrographs for the nasal respiratory region (a) andthe olfactory region (b), from rats euthanized 120 min after nasal administration of the FD3formulations.a) At level I, the septum at the level of the inferior part of the nasoturbinate (square) was

defined as a region with a high probability of deposition of both the mannitol solutionand the gellan gum formulation.

b) At level III, the defined region was the superior, medial part of ectoturbinate 1' (square).c) FD3 gellan formulation in the nasal respiratory region. Gel is visible to the lower left.d) FD3 gellan formulation in the olfactory region. Gel is visible over the epithelium

throughout the image.e) FD3 mannitol solution in the same region as c).f) FD3 mannitol solution in the same region as d).Scalebars 50 µm; s septum, n nasoturbinate, m maxilloturbinate, 1', 2' ectoturbinates,1, 2, 3 endoturbinates, np nasopharynx.

levels I and IV (as described in Fig. 9) at different times after administration.The gel was only occasionally observed to have reached as far back in thenasal cavity as level IV and, consequently, the extent of FD3 fluorescence inthe olfactory mucosa after administration of the gellan gum formulation waslow. At level I, however, the gel was always observed over large areas of thenasal cavity. Here, the scores with or without gellan gum were approximatelyequal at 15 min but, at later times, the scores after administration of the gellangum formulation were clearly higher.

The uptake of fluorescence was directly compared in two of the regions inwhich it was highly probable that both formulations would be deposited: Oneof the regions comprised respiratory epithelium, and the other olfactory epi-thelium. Figure 15 provides a description of the regions and representativemicrographs.

In the defined olfactory region, gel was visible in 5 out of the 8 studiedimages from rats which received the FD3 gellan formulation. In all of thesecases, the degree of FD3 fluorescence was higher than in the correspondingimages from rats which received the gellan-free FD3 solution. At 120 and240 min, some of the intracellular FD3 fluorescence in the olfactory epithe-lium had a point-shaped distribution. No paracellular fluorescence was dis-cerned.

In the defined respiratory region, the extent of FD3 fluorescence was ap-proximately the same 15 min after administration, irrespective of the formula-tion. At later time points, the degree of FD3 fluorescence with the gellan gumformulation exceeded that obtained with the gellan-free solution. Both intrac-ellular and paracellular fluorescence was seen in the respiratory epithelium.

The reason for the higher degree of FD3 fluorescence after administrationof the gellan gum formulation was not investigated, but it is likely to be related

35

e) f)

c) d)

a) b)

36

to the slower clearance of the gel from the nasal cavity. This was confirmed bythe observation that although the degree of fluorescence was different for thegellan gum formulation and the gellan-free solution, the distribution of FD3fluorescence in the epithelial cells was the same, irrespective of formulation.

The deposition of the gel in the nasal cavity differed from that of the solu-tion. This made direct comparisons of specific nasal cavity regions difficult.Issues such as these require consideration when evaluating results from quali-tative biopharmaceutical studies.

37

Dopamine and fluorescein-labeled dextran were identified as suitable modelcompounds for the study of nose-to-brain drug transfer mechanisms and theinfluence of drug formulation. These compounds have low CNS distributionafter systemic absorption and are transferred along the olfactory pathway inanimal models. Two new in vitro models for olfactory transfer were com-pared. Also, a rat model, which enabled the visualization of the entire olfac-tory transfer, was developed.

[3H]-Dopamine was transferred from the nasal cavity to the olfactory bulbin mice, with a maximum radioactivity in the olfactory bulb of 4 h. This latepeak may be an indication of active transport processes in the later stages ofnose-to-brain transfer. In addition, in vitro experiments showed that the mu-cosal-to-serosal transfer of dopamine across olfactory mucosa was higher thanthat in the reverse direction. This is an indication of active transport processesbeing involved in the epithelial stage of olfactory dopamine transfer. The per-meability coefficients were negatively correlated to the transmucosal electri-cal resistance, which indicates a contribution of paracellular transfer.Electrophysiological measurements also indicated that using horizontal diffu-sion chambers with an air interface may be unsuitable for bidirectional stud-ies. Both in vivo and in vitro, metabolism of dopamine took place; this shouldbe considered along with the active transport processes when choosing modelcompounds.

The transfer of fluorescein-labeled dextran across rat olfactory epitheliumwas primarily transcellular, whereas later stages of the transfer were mainlyextracellular. Epithelial uptake of the compound was enhanced by the use ofthe in situ gelling agent deacetylated gellan gum. Since only the extent, andnot the route, of transfer was altered with this agent, the enhancing effect waslikely to be the result of an increased contact time.

6 Conclusions

38

Acknowledgements

The studies in this thesis were carried out at the Department of Pharmacy,Uppsala University.I wish to express my sincere gratitude to:My supervisor Dr. Erik Björk for having the courage to accept me as a PhD student and togive me the freedom to find my own way, for knowing when I needed guidance, and forproviding the proper atmosphere for allowing me to mature as a researcher.Prof. Göran Alderborn, head of Department and Prof. Christer Nyström, former head ofDepartment, for providing and developing the necessary research infrastructure.Coauthors Prof. Eva Brittebo, Dr. Maria Dahlin and Dr. Ulrika Bergman for giving me agood start.Coauthors: Dr. Maureen Donovan, especially for generously giving me the opportunity tovisit College of Pharmacy, University of Iowa, and for giving excellent comments on ourpaper despite the tight schedule. Nagendra Chemuturi, especially for excellent practicaladvice and help.Coauthors Dr. Katarina Edsman and Dr. Helene Hägerström for introducing formulationsother than saline and phosphate buffers to my world. Special thanks to Helene for valuablecomments on this thesis.IF:s stiftelse för farmacevtisk forskning, Apotekare CD Carlssons stiftelse, Apotekar-societeten för stipendier som gjorde det ekonomiskt möjligt att genomföra projektet vidCollege of Pharmacy i Iowa och besöka givande kongresser.Examensarbetare: Cecilia Påhlstorp (tidig bakgrund till Paper II), Holger Petry (back-ground to methods in Papers III and IV) och Nelly Fransén (stora delar av Paper IV).Sommarjobbare: Mats Jönsson (diverse snittning och färgning), Hedda Magnusson(HPLC-metodvalidering) och Marcus Söderberg (HPLC-körningar).Antona Wagstaff and Suzanne Lidström (Paper IV) for excellent linguistic revision of thethesis and manuscripts.Agneta Bergström (Institutionen för farmakologi och toxikologi, SLU) och Raili Engdahl(Avdelningen för toxikologi) för assistans med autoradiografi och mycket annat.Johan Gråsjö, för kunnig elektrofysiologisk hjälp, både teoretisk och handgriplig.Dr. Göran Ocklind för stor mikroskopisk hjälp, datortips och histologiknep.Tomas, Bert, Erik och andra på Swedish Meats som försörjt mig med nässlemhinna.Tidigare och nuvarande näsgruppskolleger för givande vetenskapliga diskussioner och föratt ni alla är så positiva och trevliga: Dr. Maria Dahlin (även tack för gott samarbete på laboch vid skrivande), Dr. Karin Östh, Dr. Cecilia Wadell, Ulrika Westin, Nelly Fransén.Speciellt tack till Ulrika och Nelly för bra kommentarer till avhandlingen.Eva Nises-Ahlgren, Harriet Pettersson och Ulla Wästberg-Galik, för att ni håller ordningpå oss. Speciellt tack till Ulla för all hjälp till en lite rörig Björn nu på slutet.Kicki, min rumbo, för smittande skratt och intressanta diskussioner. Ibland om forskning.Alla andra vid Avdelningen för galenisk farmaci/Institutionen för farmaci de senaste sexåren, för att ni gör lokalerna till en fungerande, trevlig och stimulerande arbetsplats.Everyone who made my stay in Iowa City an educational and fun experience, both insideand outside the lab, especially Lily, Nagendra, Karunya, Ankur, Mow-Yee and Kia-Joo.Alla som distraherat mig från avhandlingsarbetet (med usel verkningsgrad det senastehalvåret; jag bättrar mig nog), både kolleger och andra.Min släkt, speciellt mina syskon Barbro, Lasse, Rune och Arne med familjer.Mina föräldrar Kerstin och Wincent för att ni står bakom mig oavsett vad jag hittar på förkonstigheter.Helene. Tack för att du finns.

39

References

Anand Kumar, T. C., David, G. F., Umberkoman, B., Saini, K. D. (1974). Uptake ofradioactivity by body fluids and tissues in rhesus monkeys after intravenous injection orintranasal spray of tritium-labelled oestradiol and progesterone.Curr Sci 43(14): 435-439.

Anand Kumar, T. C., David, G. F. X., Kumar, K., Umberkoman, B., Krishnamoorthy, M. S.(1976). A new approach to fertility regulation by interfering with neuroendocrinepathways. Neuroendocrine Regulation of Fertility. T. C. A. Kumar. Basel, Karger:314-322.

Anand Kumar, T. C., David, G. F. X., Sankaranarayanan, A., Puri, V., Sundram, K. R.(1982). Pharmacokinetics of progesterone after its administration to ovariectomizedrhesus monkeys by injection, infusion, or nasal spraying. Proc Natl Acad Sci USA79: 4185-4189.

Andersen, I., Proctor, D. F. (1982). The fate and effects of inhaled materials. The Nose.Upper airway physiology and the atmospheric environment. D. F. Proctor andI. Andersen. Amsterdam, Elsevier Biomedical Press B.V.: 423-455.

Balin, B. J., Broadwell, R. D., Salcman, M., El-Kalliny, M. (1986). Avenues for entry ofperipherally administered protein to the central nervous system in mouse, rat andsquirrel monkey. J Comp Neurol 251: 260-280.

Bannister, L. H., Dodson, H. C. (1992). Endocytic pathways in the olfactory andvomeronasal epithelia of the mouse: ultrastructure and uptake of tracers.Microsc Res Tech 23(2): 128-41.

Bechgaard, E., Gizurarson, S., Jorgensen, L., Larsen, R. (1992). The viability of isolatedrabbit nasal mucosa in the Ussing chamber, and the permeability of insulin across themembrane. Int J Pharm 87(1-3): 125-132.

Ben-Jonathan, N., Maxson, R. E., Ochs, S. (1978). Fast axoplasmic transport ofnoradrenaline and dopamine in mammalian peripheral nerve. J Physiol 281: 315-24.

Bergström, U., Franzen, A., Eriksson, C., Lindh, C., Brittebo, E. B. (2002). Drug targetingto the brain: transfer of picolinic acid along the olfactory pathways. J Drug Target10(6): 469-78.

Betbeder, D., Sperandio, S., Latapie, J. P., de Nadai, J., Etienne, A., Zajac, J. M.,Frances, B. (2000). Biovector nanoparticles improve antinociceptive efficacy ofnasal morphine. Pharm Res 17(6): 743-8.

Born, J., Lange, T., Kern, W., McGregor, G. P., Bickel, U., Fehm, H. L. (2002). Sniffingneuropeptides: a transnasal approach to the human brain. Nat Neurosci 5(6): 514-6.

Capsoni, S., Giannotta, S., Cattaneo, A. (2002). Nerve growth factor and galantamineameliorate early signs of neurodegeneration in anti-nerve growth factor mice.Proc Natl Acad Sci USA 99(19): 12432-7.

Chen, X.-Q., Fawcett, J. R., Rahman, Y.-E., Ala, T. A., Frey II, W. H. (1998). Delivery ofnerve growth factor to the brain via the olfactory pathway. J Alzheimers Dis 1: 35-44.

Chen, Z., Ljunggren, H. G., Bogdanovic, N., Nennesmo, I., Winblad, B., Zhu, J. (2002).Excitotoxic neurodegeneration induced by intranasal administration of kainic acid inC57BL/6 mice. Brain Res 931(2): 135-45.

Chou, K. J., Donovan, M. D. (1997). Distribution of antihistamines into the CSF followingintranasal delivery. Biopharm Drug Dispos 18(4): 335-46.

Chou, K. J., Donovan, M. D. (1998a). The distribution of local anesthetics into the CSFfollowing intranasal administration. Int J Pharm 168: 137-145.

Chou, K. J., Donovan, M. D. (1998b). Lidocaine distribution into the CNS following nasaland arterial delivery: a comparison of local sampling and microdialysis techniques.Int J Pharm 171: 53-61.

40

Chow, H. H., Anavy, N., Villalobos, A. (2001). Direct nose-brain transport ofbenzoylecgonine following intranasal administration in rats.J Pharm Sci 90(11): 1729-35.

Chow, H. S., Chen, Z., Matsuura, G. T. (1999). Direct transport of cocaine from the nasalcavity to the brain following intranasal cocaine administration in rats.J Pharm Sci 88(8): 754-758.

Cole, P. (1982). Upper respiratory airflow. The Nose. Upper airway physiology and theatmospheric environment. D. F. Proctor and I. Andersen. Amsterdam, ElsevierBiomedical Press B.V.: 163-189.

Czerniawska, A. (1970). Experimental investigations on the penetration of 198Au fromnasal mucous membrane into cerebrospinal fluid. Acta Otolaryngol (Stockh)70(1): 58-61.

Dahlin, M., Bergman, U., Jansson, B., Björk, E., Brittebo, E. (2000). Transfer of dopaminein the olfactory pathway following nasal administration in mice.Pharm Res 17(6): 737-42.

Dahlin, M., Björk, E. (2000). Nasal absorption of (S)-UH-301 and its transport into thecerebrospinal fluid of rats. Int J Pharm 195(1-2): 197-205.

Dahlin, M., Björk, E. (2001). Nasal administration of a physostigmine analogue (NXX-066)for Alzheimer’s disease to rats. Int J Pharm 212(2): 267-74.

Dahlin, M., Jansson, B., Björk, E. (2001). Levels of dopamine in blood and brain followingnasal administration to rats. Eur J Pharm Sci 14(1): 75-80.

Dale, O., Hjortkjaer, R., Kharasch, E. D. (2002). Nasal administration of opioids for painmanagement in adults. Acta Anaesthesiol Scand 46(7): 759-70.

Davis, S. S. (2001). Nasal vaccines. Adv Drug Deliv Rev 51(1-3): 21-42.de Lorenzo, A. J. D. (1970). The olfactory neuron and the blood-brain barrier. Taste and

smell in vertebrates. G. E. W. Wolstenholme and J. Knight. London,J & A Churchill Ltd.: 151-176.

De Souza Silva, M. A., Mattern, C., Hacker, R., Nogueira, P. J., Huston, J. P., Schwarting,R. K. (1997). Intranasal administration of the dopaminergic agonists L-DOPA,amphetamine, and cocaine increases dopamine activity in the neostriatum: amicrodialysis study in the rat. J Neurochem 68(1): 233-9.

Derad, I., Willeke, K., Pietrowsky, R., Born, J., Fehm, H. L. (1998). Intranasal angiotensinII directly influences central nervous regulation of blood pressure.Am J Hypertens 11(8 Pt 1): 971-7.

Diamond, S., Freitag, F., Phillips, S. B., Bernstein, J. E., Saper, J. R. (2000). Intranasalcivamide for the acute treatment of migraine headache. Cephalalgia 20(6): 597-602.

Dluzen, D. E., Kefalas, G. (1996). The effects of intranasal infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon catecholamine concentrations within olfactorybulbs and corpus striatum of male mice. Brain Res 741(1-2): 215-9.

Dufes, C., Olivier, J. C., Gaillard, F., Gaillard, A., Couet, W., Muller, J. M. (2003). Braindelivery of vasoactive intestinal peptide (VIP) following nasal administration to rats.Int J Pharm 255(1-2): 87-97.

Eriksson, C., Bergman, U., Franzen, A., Sjöblom, M., Brittebo, E. B. (1999). Transfer ofsome carboxylic acids in the olfactory system following intranasal administration.J Drug Target 7(2): 131-42.

Faber, W. F. (1937). The nasal mucosa and the subarachnoid space. Am J Anat 62: 121-148.Fehm, H. L., Perras, B., Smolnik, R., Kern, W., Born, J. (2000). Manipulating

neuropeptidergic pathways in humans: a novel approach to neuropharmacology?Eur J Pharmacol 405(1-3): 43-54.

Getchell, M. L., Getchell, T. V. (1992). Fine structural aspects of secretion and extrinsicinnervation in the olfactory mucosa. Microsc Res Tech 23(2): 111-27.

Gizurarson, S., Thorvaldsson, T., Sigurdsson, P., Gunnarsson, E. (1996). Selective deliveryof insulin into the brain: intraolfactory absorption. Int J Pharm 140: 77-83.

41

Gopinath, P. G., Gopinath, G., Kumar, T. C. A. (1978). Target site of intranasally sprayedsubstances and their transport across the nasal mucosa: a new insight into the intranasalroute of drug-delivery. Curr Ther Res 23(5): 596-607.

Gozes, I. (2001). Neuroprotective peptide drug delivery and development: potential newtherapeutics. Trends Neurosci 24(12): 700-5.

Gozes, I., Bardea, A., Reshef, A., Zamostiano, R., Zhukovsky, S., Rubinraut, S., Fridkin,M., Brenneman, D. E. (1996). Neuroprotective strategy for Alzheimer disease:intranasal administration of a fatty neuropeptide. Proc Natl Acad Sci USA 93(1):427-32.

Gozes, I., Giladi, E., Pinhasov, A., Bardea, A., Brenneman, D. E. (2000). Activity-dependentneurotrophic factor: intranasal administration of femtomolar-acting peptides improveperformance in a water maze. J Pharmacol Exp Ther 293(3): 1091-8.

Grass, G. M., Sweetana, S. A. (1988). In vitro measurement of gastrointestinal tissuepermeability using a new diffusion cell. Pharm Res 5(6): 372-6.

Gross, E. A., Swenberg, J. A., Fields, S., Popp, J. A. (1982). Comparative morphometry ofthe nasal cavity in rats and mice. J Anat 135: 83-88.

Hammarlund-Udenaes, M. (2000). The use of microdialysis in CNS drug delivery studies.Pharmacokinetic perspectives and results with analgesics and antiepileptics.Adv Drug Deliv Rev 45(2-3): 283-94.

Hirai, S., Yashiki, T., Matsuzawa, T., Mima, H. (1981). Absorption of drugs from nasalmucosa of rat. Int J Pharm 7: 317-325.

Ikeda, K., Murata, K., Kobayashi, M., Noda, K. (1992). Enhancement of bioavailability ofdopamine via nasal route in beagle dogs. Chem Pharm Bull 40(8): 2155-8.

Illum, L. (2000). Transport of drugs from the nasal cavity to the central nervous system.Eur J Pharm Sci 11(1): 1-18.

Illum, L. (2003). Nasal drug delivery - possibilities, problems and solutions. J ControlRelease 87: 187-198.

Jackson, R. T., Tigges, J., Arnold, W. (1979). Subarachnoid space of the CNS, nasalmucosa, and lymphatic system. Arch Otolaryngol 105(4): 180-4.

Jagannadha Rao, A., Moudal, N. R., Li, C. H. (1986). beta-Endorphin: intranasaladministration increases the serum prolactin level in monkey. Int J Pept Protein Res28: 546-548.

Jin, K., Xie, L., Childs, J., Sun, Y., Mao, X. O., Logvinova, A., Greenberg, D. A. (2003).Cerebral neurogenesis is induced by intranasal administration of growth factors. AnnNeurol 53(3): 405-9.

Johnson, R. T. (1964). The pathogenesis of herpes virus encephalitis. J Exp Med119: 343-356.

Jones, N., Rog, D. (1998). Olfaction: a review. J Laryngol Otol 112(1): 11-24.Kao, H. D., Traboulsi, A., Itoh, S., Dittert, L., Hussain, A. (2000). Enhancement of the

systemic and CNS specific delivery of L-dopa by the nasal administration of its watersoluble prodrugs. Pharm Res 17(8): 978-84.

Karlsson, J., Artursson, P. (1991). A method for the determination of cellular permeabilitycoefficients and aqueous boundary-layer thickness in monolayers of intestinal epithelial(Caco-2) cells grown in permeable filter chambers. Int J Pharm 71(1-2): 55-64.

Kern, W., Born, J., Schreiber, H., Fehm, H. L. (1999). Central nervous system effects ofintranasally administered insulin during euglycemia in men. Diabetes 48(3): 557-63.

Kern, W., Schiefer, B., Schwarzenburg, J., Stange, E. F., Born, J., Fehm, H. L. (1997).Evidence for central nervous effects of corticotropin-releasing hormone on gastric acidsecretion in humans. Neuroendocrinology 65(4): 291-8.

Kristensson, K., Olsson, Y. (1971). Uptake of exogenous proteins in mouse olfactory cells.Acta Neuropathol (Berl) 19: 145-154.

Kumbale, R., Frey, W. H., Wilson, S., Rahman, Y. E. (1999). GM1 Delivery to the CSF viathe olfactory pathway. Drug Deliv 6: 23-30.

42

Lindhardt, K., Gizurarson, S., Stefansson, S. B., Olafsson, D. R., Bechgaard, E. (2001).Electroencephalographic effects and serum concentrations after intranasal andintravenous administration of diazepam to healthy volunteers. Br J Clin Pharmacol52(5): 521-7.

Liu, X. F., Fawcett, J. R., Thorne, R. G., DeFor, T. A., Frey, W. H., 2nd (2001). Intranasaladministration of insulin-like growth factor-I bypasses the blood-brain barrier andprotects against focal cerebral ischemic damage. J Neurol Sci 187(1-2): 91-7.

Marttin, E., Verhoef, J. C., Cullander, C., Romeijn, S. G., Nagelkerke, J. F., Merkus, F. W.(1997). Confocal laser scanning microscopic visualization of the transport of dextransafter nasal administration to rats: effects of absorption enhancers.Pharm Res 14(5): 631-7.

Mathison, S., Nagilla, R., Kompella, U. B. (1998). Nasal route for direct delivery of solutesto the central nervous system: fact or fiction? J Drug Target 5(6): 415-441.

Merkus, P., Guchelaar, H. J., Rijn-Bikker, P. C., Torano, J. S., Bosch, D. A., Merkus, F. W.(2002). Nasal drug delivery to the cerebrospinal fluid: transport of a lipophiliccompound. Br J Clin Pharmacol 54(5): 560.

Merkus, F. W., Verhoef, J. C., Schipper, N. G., Marttin, E. (1998). Nasal mucociliaryclearance as a factor in nasal drug delivery. Adv Drug Deliv Rev 29(1-2): 13-38.

Minn, A., Leclerc, S., Heydel, J. M., Minn, A. L., Denizcot, C., Catrarelli, M., Netter, P.,Gradinaru, D. (2002). Drug transport into the mammalian brain: the nasal pathway andits specific metabolic barrier. J Drug Target 10(4): 285-96.

Morrison, E. E., Costanzo, R. M. (1990). Morphology of the human olfactory epithelium.J Comp Neurol 297(1): 1-13.

Morrison, E. E., Costanzo, R. M. (1992). Morphology of olfactory epithelium in humansand other vertebrates. Microsc Res Tech 23(1): 49-61.

Mygind, N., Pedersen, M., Nielsen, M. H. (1982). Morphology of the upper airwayepithelium. The Nose. Upper airway physiology and the atmospheric environment.D. F. Proctor and I. Andersen. Amsterdam, Elsevier Biomedical Press B.V.: 71-97.

Okuyama, S. (1997). The first attempt at radioisotopic evaluation of the integrity of thenose-brain barrier. Life Sci 60(21): 1881-4.

Oldendorf, W. H. (1971). Brain uptake of radiolabeled amino acids, amines, and hexosesafter arterial injection. Am J Physiol 221(6): 1629-39.

Olitsky, P. K., Cox, H. R. (1934). Temporary prevention by chemical means of intranasalinfection of mice with equine encephalomyelitis virus. Science 80(2085): 566-567.

Pardridge, W. M. (1996). Brain drug delivery and blood-brain barrier transport. DrugDelivery 3: 99-115.

Paulson, O. B. (2002). Blood-brain barrier, brain metabolism and cerebral blood flow.Eur Neuropsychopharmacol 12(6): 495-501.

Paulsson, M., Hagerstrom, H., Edsman, K. (1999). Rheological studies of the gelation ofdeacetylated gellan gum (Gelrite((R))) in physiological conditions. Eur J Pharm Sci9(1): 99-105.

Perlman, S., Sun, N., Barnett, E. M. (1995). Spread of MHV-JHM from nasal cavity towhite matter of spinal cord. Transneuronal movement and involvement of astrocytes.Adv Exp Med Biol 380: 73-8.

Pietrowsky, R., Claassen, L., Frercks, H., Fehm, H. L., Born, J. (2001). Time course ofintranasally administered cholecystokinin-8 on central nervous effects.Neuropsychobiology 43(4): 254-9.

Pietrowsky, R., Struben, C., Molle, M., Fehm, H. L., Born, J. (1996). Brain potentialchanges after intranasal vs. intravenous administration of vasopressin: evidence for adirect nose-brain pathway for peptide effects in humans. Biol Psychiatry 39(5): 332-40.

Rake, G. (1937). The rapid invasion of the body through the olfactory mucosa.J Exp Med 65: 303-315.

43

Sakane, T., Akizuki, M., Taki, Y., Yamashita, S., Sezaki, H., Nadai, T. (1995). Direct drugtransport from the rat nasal cavity to the cerebrospinal fluid: the relation to themolecular weight of drugs. J Pharm Pharmacol 47: 379-381.

Sakane, T., Akizuki, M., Yamashita, S., Nadai, T., Hashida, M., Sezaki, H. (1991a). Thetransport of a drug to the cerebrospinal fluid directly from the nasal cavity: the relationto the lipophilicity of the drug. Chem Pharm Bull 39(9): 2456-2458.

Sakane, T., Akizuki, M., Yoshida, M., Yamashita, S., Nadai, T., Hashida, M., Sezaki, H.(1991b). Transport of cephalexin to the cerebrospinal fluid directly from the nasalcavity. J Pharm Pharmacol 43(6): 449-51.

Sakane, T., Yamashita, S., Yata, N., Sezaki, H. (1999). Transnasal delivery of 5-fluorouracilto the brain in the rat. J Drug Target 7(3): 233-40.

Schmidt, M. C., Simmen, D., Hilbe, M., Boderke, P., Ditzinger, G., Sandow, J., Lang, S.,Rubas, W., Merkle, H. P. (2000). Validation of excised bovine nasal mucosa as in vitromodel to study drug transport and metabolic pathways in nasal epithelium.J Pharm Sci 89(3): 396-407.

Seki, T., Sato, N., Hasegawa, T., Kawaguchi, T., Juni, K. (1994). Nasal absorption ofzidovudine and its transport to cerebrospinal fluid in rats. Biol Pharm Bull 17(8):1135-1137.

Sigurdsson, P., Thorvaldsson, T., Gizurarson, S. (1997). Olfactory absorption of insulin tothe brain. Drug Deliv 4: 195-200.

Sunderman, F. W., Jr. (2001). Nasal toxicity, carcinogenicity, and olfactory uptake of metals.Ann Clin Lab Sci 31(1): 3-24.

Tjälve, H., Henriksson, J. (1999). Uptake of metals in the brain via olfactory pathways.Neurotoxicology 20(2-3): 181-95.

Wadell, C., Björk, E., Camber, O. (1999). Nasal drug delivery—evaluation of an in vitromodel using porcine nasal mucosa. Eur J Pharm Sci 7(3): 197-206.

van de Waterbeemd, H., Camenisch, G., Folkers, G., Chretien, J. R., Raevsky, O. A. (1998).Estimation of blood-brain barrier crossing of drugs using molecular size and shape, andH-bonding descriptors. J Drug Target 6(2): 151-65.

van den Berg, M. P., Romeijn, S. G., Verhoef, J. C., Merkus, F. W. (2002). Serialcerebrospinal fluid sampling in a rat model to study drug uptake from the nasal cavity.J Neurosci Methods 116(1): 99-107.

van Ginkel, F. W., Jackson, R. J., Yuki, Y., McGhee, J. R. (2000). Cutting edge: the mucosaladjuvant cholera toxin redirects vaccine proteins into olfactory tissues. J Immunol165(9): 4778-82.

Wang, F., Jiang, X., Lu, W. (2003). Profiles of methotrexate in blood and CSF followingintranasal and intravenous administration to rats. Int J Pharm 263(1-2): 1-7.

Werner, U., Kissel, T. (1995). Development of a human nasal epithelial cell culture and itssuitability for transport and metabolism studies under in vitro conditions.Pharm Res 12(4): 565-571.

Yajima, T., Juni, K., Saneyoshi, M., Hasegawa, T., Kawaguchi, T. (1998). Direct transportof 2',3'-didehydro-3'-deoxythymidine (D4T) and its ester derivatives to the cerebrospinalfluid via the nasal mucous membrane in rats. Biol Pharm Bull 21(3): 272-7.

Östh, K., Gråsjö, J., Björk, E. (2002a). A new method for drug transport studies on pig nasalmucosa using a horizontal Ussing chamber. J Pharm Sci 91(5): 1259-1273.

Östh, K., Paulsson, M., Björk, E., Edsman, K. (2002b). Evaluation of drug release from gelson pig nasal mucosa in a horizontal Ussing chamber. J Control Release 83(3): 377-388.

Östh, K., Strindelius, L., Larhed, A., Ahlander, A., Roomans, G. M., Sjöholm, I., Björk, E.(2003). Uptake of ovalbumin-conjugated starch microparticles by pig respiratory nasalmucosa in vitro. J Drug Target 11(1): 75-82.

44

Sammanfattning

Temat för avhandlingen är modeller för transport av läkemedel från näshålantill hjärnan. Det finns ett trettiotal läkemedel för nasal administrering iSverige. De flesta är förkylnings- och allergipreparat avsedda att utöva sineffekt lokalt i näshålan. Ett tiotal verkar dock på andra ställen i kroppen.Några av dessa läkemedel förstörs i mage, tarm, eller lever när de ges somtablett. Den ofta snabbare effekten kan vara ett annat skäl att ge något som ennässpray istället för via munnen.

Läkemedelsbehandling av sjukdomar som påverkar hjärnan innebärspeciella problem. Hjärnan skyddas från främmande ämnen av blod-hjärn-barriären. De läkemedel på marknaden som är avsedda för effekt i hjärnanuppfyller kraven på fettlöslighet och molekylstorlek som ställs för att de skata sig genom barriären, men det finns många ämnen som skulle vara intres-santa, bara de tog sig in i hjärnan. Metoder för att lura barriären att släppagenom läkemedel eller sätt att helt kringgå den är därför välbehövliga.

Ett flertal experiment med försöksdjur visar att en del ämnen kan förflyttasdirekt från näshålan till hjärnan via luktnerverna, och mycket tyder på attdetta är möjligt även i människor. Avhandlingsarbetet har syftat till attutveckla och förfina modeller för att studera de inblandade mekanismerna,och för att studera hur upptaget av det aktuella ämnet påverkas av dessomgivning i läkemedelsberedningen (hur ämnet formulerats).

En direkt näs-hjärntransport av dopamin påvisades hos möss. Transportenundersöktes vidare i en modell där nässlemhinna från slaktsvin och ungnötanvändes. Det andra studerade ämnet, fluoresceinmärkt dextran, tillät ettvisuellt åskådliggörande av transporten hos råtta. Denna metod användes se-dan för att undersöka hur upptaget förändrades när dextranet formuleradesmed polymeren gellangummi, som bildar en gel när den kommer i kontaktmed slemhinnan i näsan. När vanliga vattenlösningar eller pulver ges nasalt,kommer tiden som läkemedlet är i kontakt med nässlemhinnan vara ganskakort, då det finns ett försvarssystem som bygger på att flimmerhår trans-porterar bort främmande ämnen. Genom gellangummits konsistens minskadeborttransporten. Sannolikt var detta anledningen till det ökade upptaget somiakttogs.

Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

from the Faculty of PharmacyEditor: The Dean of the Faculty of Pharmacy

Distribution:Uppsala University Library

Box 510, SE-751 20 Uppsala, Swedenwww.uu.se, acta@ub.uu.se

ISSN 0282-7484ISBN 91-554-5834-3

A doctoral dissertation from the Faculty of Pharmacy, Uppsala University,is usually a summary of a number of papers. A few copies of the completedissertation are kept at major Swedish research libraries, while the sum-mary alone is distributed internationally through the series Comprehen-sive Summaries of Uppsala Dissertations from the Faculty of Pharmacy.(Prior to July, 1985, the series was published under the title “Abstracts ofUppsala Dissertations from the Faculty of Pharmacy”.)