Cyclodextrins in peptide and protein delivery Tetsumi Irie ...juliet84.free.fr/These/these/bibliographie... · Advanced Drug Delivery Reviews 36 (1999) 101–123 L Cyclodextrins in

LAdvanced Drug Delivery Reviews 36 (1999) 101–123

Cyclodextrins in peptide and protein deliverya b ,*Tetsumi Irie , Kaneto Uekama

a

Graduate School of Pharmaceutical Sciences, Kumamoto University, 5 –1, Oe-honmachi, Kumamoto 862-0973, JapanbFaculty of Pharmaceutical Sciences, Kumamoto University, 5 –1, Oe-honmachi, Kumamoto 862-0973, Japan

Abstract

The objective of this contribution is to summarize recent findings on the potential of cyclodextrins (CDs) and theirderivatives as carriers for therapeutically important peptides, proteins and oligonucleotides. As one of the indices relevant tobioadaptability of CDs in pharmaceutical uses, their interaction with cellular membranes in vitro is outlined. CDs enable thecreation of advanced dosage forms for the next generation of drugs that are difficult to formulate and deliver with theexisting pharmaceutical excipients. Furthermore, the diagnostic uses of CDs for the direct measurement of cholesterol inhigh-density and low-density lipoproteins in serum are discussed on the basis of their ability to recognize the surfaceproperties of each lipoprotein particle. 1999 Elsevier Science B.V. All rights reserved.

Keywords: Cyclodextrins; Inclusion complex; Peptides and proteins; Oligonucleotides; Solubilizing and stabilizing agents;Artificial chaperones; Absorption enhancers; Sustained-release carriers; Heparinoids

Contents

1. Introduction ............................................................................................................................................................................ 1012. Bioadaptability of cyclodextrins ............................................................................................................................................... 1023. Interaction of cyclodextrins with amino acids and polypeptides .................................................................................................. 1044. Hydrophilic cyclodextrins as solubilizers and stabilizers............................................................................................................. 1065. Hydrophilic cyclodextrins as artificial chaperones ...................................................................................................................... 1086. Hydrophilic cyclodextrins as absorption enhancers .................................................................................................................... 1097. Hydrophobic cyclodextrins as sustained-release carriers ............................................................................................................. 1128. Sulfated cyclodextrins as heparinoids........................................................................................................................................ 1149. Cyclodextrins as oligonucleotide carriers .................................................................................................................................. 11510. Use of cyclodextrins for lipoprotein measurements .................................................................................................................. 11611. Conclusions .......................................................................................................................................................................... 118References .................................................................................................................................................................................. 118

1. Introduction economical and large-scale production of therapeu-tically important complex polymers of amino acids

Advances in biotechnology have allowed the (peptides and proteins) and nucleosides (antisensemolecules) to be used to combat poorly controlled

* diseases. This rapid progress in molecular biology,Corresponding author. Tel.: 1 81 96 3714160; fax: 1 81 963727023; e-mail: [email protected] however, has not been matched by the progress in

0169-409X/99/$ – see front matter 1999 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 98 )00057-X

102 T. Irie, K. Uekama / Advanced Drug Delivery Reviews 36 (1999) 101 –123

the formulation and development of delivery systems erythrocytes, which have no nucleus, mitochondria,for the next generation of drugs. Rational drug endoplasmic reticulum and other organelles, maydesign does not necessarily mean rational drug provide a simple and reliable vehicle to classify thedelivery, which strives to incorporate into a molecule CDs in regard to their cytotoxicity, because thethe properties necessary for optimal transfer between interaction of CDs with plasma membranes must bethe site of administration and the pharmacological the initial step of cell damage. CDs are known totarget site in the body [1]. induce shape changes of membrane invagination on

There are considerable hurdles to be overcome human erythrocytes, and at higher concentrationsbefore practical use can be made of therapeutic induce lysis [6,7]. The hemolytic activity of thepeptides and proteins due to chemical and enzymatic parent CDs is reported to be in the order of b-CD .

instability, poor absorption through biological mem- a-CD . g-CD . d-CD [8]. These differences arebranes, rapid plasma clearance, peculiar dose–re- ascribed to the differential solubilization rates ofsponse curves, and immunogenicity. Many attempts membrane components by each CD. The acyl chainhave addressed these problems by chemical modi- of phospholipid fits tightly into the hydrophobicfications or by coadministration of adjuvants to cavity of the smallest a-CD and more loosely intoeliminate undesirable properties of peptides and the larger inner space of b- and g-CDs, whereas theproteins [2]. Cyclodextrin (CD) complexation seems side chain of cholesterol is preferably included in theto be an attractive alternative to these approaches [3]. b-CD cavity. Of the three parent CDs tested, g-CD

In this contribution, we focus primarily on recent has the least lipid selectivity.findings on the potential of CDs and their derivatives The process of solubilization occurs without entryas novel bioadaptable carriers for peptides, proteins of CDs into the membranes, a mechanism ofand oligonucleotides with emphasis on the inter- solubilization / lysis different from that of detergents,action between CDs, these drug candidates and which first incorporate themselves into the mem-various endogenous molecules. In addition, the diag- branes then extract membrane components intonostic uses of CDs for measuring lipoprotein choles- micelles. CDs form a new lipid-containing compart-terol in serum without the need for prior separation ment (or pool) in the aqueous phase into whichare described on the basis of their ability to recog- components of the erythrocyte surface are extracted.nize differences in surface properties of each lipopro- This new pool equilibrates freely and reversibly withtein fraction. the cell surface. The removal of cholesterol from the

cells may result in an increase in membrane fluidity,which would induce the membrane invaginationthrough a loss of bending resistance, and conse-

2. Bioadaptability of cyclodextrins quently lead to the lysis of the cells. In addition, ourprevious studies have shown that CDs remove

Since safety is a primary concern when consider- phospholipids especially phosphatidylcholine anding new excipients intended for use in pharma- sphingomyelin from the outer half of the membraneceutical formulations, the toxicological issues to- bilayer, leading to imbalance of the bilayer andgether with their biological fates must be thoroughly which may contribute in part to formation ofinvestigated prior to their practical use. The safety stomatocytes through an inward bending of thestatus of pharmaceutically useful CDs, including membranes. A similar solubilization process waspharmacokinetics and toxicological profiles follow- found for the CD-induced lysis of the artificialing administration by a variety of routes, has been membranes composed of lecithin and cholesterol [9].recently reviewed [4,5]. This contribution focuses on b-CD also removes proteins from erythrocytethe interaction of CDs with biological membranes membranes [7]. Analysis of extracts by electropho-and discusses its toxicological implications. resis indicated that polypeptides of bands V and VI

One of the most substantial requirements for drug were preferentially extracted. b-CD also extractscarriers is that they have either no or acceptably low bands III and IV to a lesser extent. This indicateslevels of intrinsic cytotoxicity. Studies using isolated that b-CD causes profound changes in membranes,

T. Irie, K. Uekama / Advanced Drug Delivery Reviews 36 (1999) 101 –123 103

since bands III and IV are tightly bound and cannot acetyl)-b-CD (DMA-b-CD (7.0)) and evaluated itsbe eluted using simple procedures. Band III is an pharmaceutical properties such as solubilizing powerintegral protein (ectoprotein), which interacts with and hemolytic activity [19]. As shown in Fig. 1-A,the hydrocarbon core of the lipid bilayer and is the hemolytic activity of DMA-b-CD (7.0) wasexposed to aqueous medium on both extracytoplas- significantly lower than those of b-CD, heptakis(2,6-mic and cytoplasmic sides. di-O-methyl)-b-CD (DM-b-CD), heptakis(2,3,6-tri-

When the character of the lipophilic cavity of CDs O-methyl)-b-CD (TM-b-CD), 2-hydroxypropyl-b-is modified by chemical derivatization, the effects on CD (HPb-CD) and sulfobutyl ether of b-CD (SBE-cell membranes can be dramatically changed [10– b-CD). The hemolytic activity of DM-b-CD was17]. The hemolytic activity of CDs correlates with drastically decreased by introducing acetyl groupstheir inclusion ability toward membrane lipids rather into the secondary hydroxyl groups at the 3-positionthan their intrinsic solubility or surface activity. This of DM-b-CD, with increasing the average degree ofview is supported by the fact that a positive correla- substitution of the acetyl groups (Fig. 1-B). DMA-b-tion is found between the hemolytic activity of CD showed neither measurable hemolysis nor mor-several CDs and their capacity to solubilize choles- phological changes of erythrocytes even at higherterol, which acts as the main rigidifier in lipid concentrations of more than 100 mM, while itbilayers, in spite of their quite different physico- maintained certain inclusion ability comparable tochemical properties. It should be noted that CDs lose TM-b-CD.their abilities to interact with the membranes, when Of the CDs tested, the effects of sulfated CDstheir cavities are occupied with the guest molecules. (S-CDs) on the cell membranes differ from those ofThis fact further supports the aforementioned mecha- the other hydrophilic CDs [15–17]. For instance,nism for CD-induced hemolysis. On the other hand, S-b-CD shows biphasic effect on the shape ofsome efforts have been made for designing CD erythrocytes; i.e. the crenation at relatively lowderivatives with enhanced inclusion ability and lower concentrations and the invagination at higher con-toxicity, including methyl ethers, glycerol cyclic centrations. The S-b-CD-induced membrane crena-ethers and tertiary butyl ethers of CDs [18]. Recent- tion arises from a direct action on the membranesly, we prepared heptakis(2,6-di-O-methyl-3-O- rather than cell metabolism-mediated effects. Unlike

Fig. 1. Hemolytic effects of b-CDs on rabbit erythrocytes in phosphate buffered saline (pH 7.4) at 378C (see [19]). s: DM-b-CD, d:a bTM-b-CD, n: b-CD, m: HP-b-CD , h: SBE-b-CD , j: DMA-b-CD (7.0), ,: DMA-b-CD (1.5), .: DMA-b-CD (3.8), x: DMA-b-CD

a(6.3). Each value in parenthesis represents the average degree of substitution of acetyl groups in DMA-b-CD. The average degree ofbsubstitution of 2-hydroxypropyl groups was 4.8.; The average degree of substitution of sulfobutyl groups was 3.5.


13b-CyD, S-b-CD binds to the erythrocytes and may nuclear magnetic resonance ( C-NMR) chemicalbe confined to the outer surface of the membrane shifts of buserelin acetate (20 mM) in D O, where2

bilayer, which may expand the exterior layer relative an absolute value for the chemical shift displacementto the cytoplasmic half, and thereby induce the cells (ppm) for each carbon of the peptide was plottedto crenate. The lack of hemolytic activity of S-b-CD against the concentration of DM-b-CD added. The

1 3may be due to the minimal capacity to solubilize the C-NMR chemical shift displacements indicate thatmembrane lipids, together with a protective inter- in addition to the two aromatic side chains, a tertiaryaction with the membranes and an increase in butyl D-serine residue is inserted into the DM-b-CDosmotic pressure in the medium. cavity. Based on the two-dimensional rotating frame

Furthermore, the effects of CDs on other types of nuclear Overhauser effect spectroscopy and othercells have been studied using human skin fibroblasts spectral data, the most probable location of the three[20,21], intestinal cells [21,22] and brush border possible binding sites of buserelin acetate within themembrane vesicles [23], P388 murine leukaemic DM-b-CD cavity is shown in Fig. 3. Furthermore,cells [24], and E. coli bacterial cells [25]. All these upon the addition of DM-b-CD at concentrations ofstudies suggest that phenomena involved in the CD- more than 40 mM, most of the carbonyl carbons ininduced cytotoxicity are not specific to the cell type, the peptide backbone were shifted upfield, indicatingand are determined primarily by a loss of vital global conformational changes of the peptide.cellular components through solubilization by CDs On the other hand, the continuous variation plots[24]. for the buserelin acetate DM-b-CD system showed a

1:1 stoichiometry of the complex. Therefore, thecomplexation should be initiated by the inclusion of

3. Interaction of cyclodextrins with amino acids one of the three binding sites on the buserelinand polypeptides molecule into DM-b-CD, which may in turn prevent

further access of the second CD to the other bindingCDs can recognize not only the size and shape but sites, probably due to steric hindrance and/or con-

also the chirality of amino acids [26,27]. However, formational changes of the peptide. Consequently,molecules of many peptides and proteins are too the three buserelin acetate complexes with DM-b-hydrophilic and bulky to be wholly included in the CD at a 1:1 molar ratio with a difference in theCD cavity and the topological constraints of the binding site may coexist in the solution. This hetero-peptide backbone may reduce the formation of geneity in the structure of the complex formed isinclusion complexes, thus their interaction with CDs consistent with the complexity in the near-ultravioletcould be only local, that is, accessible hydrophobic circular dichroism spectrum, indicating that uponside chains may form inclusion complexes with CDs binding to DM-b-CD, the local environment around[28–30]. Such interaction possibly affects the overall the aromatic groups in the peptide differs distinctly.three-dimensional structure of peptides and proteins Hydrophilic CDs affect the tertiary structure ofor inhibits their intermolecular association and thus recombinant human growth hormone (hGH) in aque-changes their chemical and biological properties. ous solution [32]. The electrospray ionization mass

A synthetic nonapeptide buserelin acetate, defined spectrum of hGH obtained in an acidic medium gaveas pyroGlu-His-Trp-SerTyr-D-Ser(tert-butyl)-Leu- a broad charge distribution. The higher charge stateArg-PrO-ethylamide, is a highly potent agonist of distribution was observed for hGH with 6-O-mal-luteinizing hormone-releasing hormone. Ultraviolet tosyl-b-CD (G -b-CD), suggesting less compact2

absorption and circular dichroism spectroscopies conformation of the protein (Fig. 4). Furthermore, inindicate that the aromatic side chains of buserelin the presence of G -b-CD, new signals were ob-2

acetate, L-tryptophan and L-tyrosine residues, are served, which correspond to the 1:1 and 1:2 adductsincorporated into the hydrophobic environment of of ionized hGH with the CD. Fluorescence andthe DM-b-CD cavity [31]. circular dichroism spectroscopies revealed that CDs

Fig. 2 shows the effects of DM-b-CD at various changed the tertiary structure of hGH but had noconcentrations up to 100 mM on the carbon-13 effect on the secondary structure of the protein,


1 3Fig. 2. C-NMR chemical shift displacements of buserelin acetate (20 mM) as a function of DM-b-CD concentrations in D O (see [31]).2

Fig. 3. Proposed structures of inclusion complexes for three side chains in buserelin acetate with DM-b-CD in solution (see [31]). A:L-tryptophan residue, B: L-tyrosine residue, C: tertiary butyl-D-serine residue.

showing a molten globule-like state. The proton reduces the intramolecular hydrophobic interaction1( H-) NMR signals of tyrosine residues in the hGH of the protein.

molecule were largely shifted with a rise in the a-CD is known to be a competitive inhibitor forG -b-CD concentrations, indicating that the CD starch hydrolysis by b-amylase, a fact which sup-2


resulting b-CD-protein complex is incapable ofinteracting properly with the inner membrane-associ-ated transporter complex. Other studies have shownthat b-CD binds to the starch-binding domain ofAspergillus niger glucoamylase, but does not inhibitthe enzyme activity, indicating that there is nointeraction between the catalytic and the starch-bind-ing domains [36,37]

4. Hydrophilic cyclodextrins as solubilizers andstabilizers

CDs can be used to solubilize and stabilize variousbiomedically-important peptides and proteins includ-ing growth hormones [38,39], interleukin-2 [38],monoclonal antibody MN12 [40], aspartame [41],tumor necrosis factor [42], albumin [43], g-globulin[43], lactate dehydrogenase [44], etc. For instance,a-CD increases the solubility of cyclosporin A, animmunosuppressive agent, in eyedrop form, andhelps the drug to penetrate into the cornea with theleast local toxicity [45,46]. Cyclosporin A exhibits a

Fig. 4. Electrospray mass spectra of hGH (0.1 mM) in positive ionlow therapeutic index and a poor oral bioavailabilitymode in the absence (A) and presence (B) of G -b-CD (10 mM)2with large intra- and inter-individual variations,in water–methanol–acetic acid (49:49:2) solution (pH 3.0) (seebecause of the limited solubility, the influence of[32]).

foods and bile flow, the poor intestinal membraneports the induced fit theory of catalysis. On the other permeability, the intestinal and hepatic first-passhand, a crystallographic study has shown that a-CD metabolism and P-glycoprotein counter-transportbinds to d-methyl groups of leucine 383 at a cleft processes. Of the hydrophilic CDs tested, DM-a-CDentrance of soybean b-amylase and occupies much is the most potent solubilizer for cyclosporin Aof the entrance [33]. Since a-CD does not extend [47,48]. DM-a- and DM-b-CDs enhance the extentdeeply enough into the cleft to make contact with the of oral bioavailability of cyclosporin A about 5-fold,enzyme’s catalytic center, it should inhibit catalysis reaching | 25% of that of the intravenous adminis-by physically blocking the access of substrates to the tration in rats. On the other hand, both DM-CDs doreaction center. This finding would provide a novel not affect the lymphatic transfer of cyclosporin Amechanism whereby a-CD inhibits b-amylase, dif- (Fig. 5). It is noteworthy that DM-CDs decrease thefering from the induced fit theory. inter-individual variability in plasma levels of

b-CD is known to bind to the substrate binding cyclosporin A after oral administration, as the de-domain of maltodextrin binding protein from Es- crease in the coefficient of variation for the plasmacherichia coli with the same stability order as those drug level from 63% to 22% [49].of the linear maltodextrins, although it is not a Interactions of CDs with side chains on oligomericphysiological ligand for that protein [34]. An elec- peptides can dissociate the oligomers, especially iftron spin resonance study has revealed that the the complexation occurs at sites in the peptide-binding of physiological ligands (e.g., maltose and peptide interface. The propensity of insulin to formmaltotetraose) induces a considerable closure of the both reversible and irreversible aggregates in solu-N- and C-terminal lobes of the protein, while little tion leads to complications in the development ofclosure occurs upon the binding of b-CD [35]. The long-term insulin therapeutic systems and limits the


Fig. 5. Plasma and lymph levels of cyclosporin A (CsA) after oral administration of CsA (10 mg/kg) suspensions with or without CDs(molar ratio of 1:10 (CsA:CD)) to rats (see [49]). s: CsA alone, n: with a-CD, m: with DM-a-CD, d: with DM-b-CD.

rate of subcutaneous absorptions, a process which is where the peptide is predominantly in a monomertoo slow to mimic the physiological plasma insulin state, and the rate was unchanged by the addition ofprofile at the time of meal consumption. These G -b-CD. However, the exchange rate significantly2

problems are further complicated by the tendency for slowed down in pH 2.0 solution where insulin isinsulin to adsorb on to the surfaces of containers and predominantly in a dimer state, and the rate in-devices, perhaps by mechanisms similar to those creased with increasing G -b-CD concentrations,2

inducing aggregation [50]. indicating that G -b-CD shifts the monomer–dimer2

Some hydrophilic CDs, including HP-b-CD and equilibrium of insulin in favour of the dissociatedG -b-CD, significantly inhibit the adsorption of form (Fig. 6) [54].2

insulin to hydrophobic surfaces of containers and its Dilution microcalorimetric study indicates a se-aggregation in neutral solutions [51]. HP-b-CD is quential binding of CDs to at least two possible sitesalso found to prevent the shaking-induced formation on the insulin monomer at acidic condition [55].

1of insoluble aggregates of insulin in neutral solutions Based on the two-dimensional H-NMR measure-[52,53]. Both b-CDs facilitate the permeation of ments, G -b-CD may include accessible hydropho-2

insulin through the ultrafiltration membranes, and bic amino acid residues of insulin such as phenylala-increase the surface tension of insulin solutions. In nine and tyrosine at the N-terminal end (B1) and inthe circular dichroism spectrum of insulin, the b- the C-terminal region (B25 and B26) of the B-chain,CDs increase the negative band intensity around 208 these side chains having a high motional freedom,nm assigned to a-helix structure of insulin, while while the side chains in the a-helices are notdecrease that around 275 nm assigned to the an- significantly perturbed in the presence of G -b-CD2

tiparallel b-structure of insulin oligomers. These [56]. Thus, G -b-CD should perturb the intermolecu-2

spectral changes are in close agreement with those lar hydrophobic contacts between aromatic sideobserved when insulin aggregates are dissociated to chains across the monomer–monomer interfaces,monomer or lower-order aggregates. eventually leading to the inhibition of self-associa-

Hydrogen–deuterium exchange measurements tion of the peptide.coupled with electrospray ionization mass spec- By contrast, SBE-b-CD shows varying effects ontrometry have shown that the exchange rate of insulin aggregation, depending on the degree ofinsulin was rapid in 30% v/v acetic acid solution substitution of the sulfobutyl group, i.e., the inhibi-


the amyloid deposits has been controversial. Studiesusing electrospray ionization mass spectrometry haverevealed that b-CD interacts with a synthetic 40amino acid b-amyloid peptide, presumably with thehydrophobic aromatic residues on the peptide. b-CDinhibits the fibrillization of the amyloid peptide andreduces its neurotoxic effects on rat phaeoch-romocytoma cells [58]. However, the available dataare still too preliminary to be applied to preventamyloid accumulation in vivo. On the other hand,HP-b-CD can be used to inject high concentrationsof the amyloid peptide into the brain without markedacute toxicity and thereby may provide a physiologi-cally relevant model for the amyloid deposits ob-served in the human brain [59].

5. Hydrophilic cyclodextrins as artificialchaperones

The process by which protein molecules achievetheir native compact conformations is a subject ofboth fundamental and practical importance. In par-ticular, the practical interest in the protein refoldingproblem stems from the fact that proteins are over-produced by genetically engineered cells in the formof cytoplasmic aggregates or inclusion bodies, inwhich the proteins are misfolded and thereforefunctionally inactive. Weak interactions of CDs with

Fig. 6. Deuterated ratio of insulin versus time of exposure to D O unfolded proteins may enhance the solubility of2

in the absence and presence of G -b-CD in 30% v/v acetic acid2 denaturated proteins by masking the exposed hydro-(A) and pH 2.0 solution (B) at 258C (see [54]). s: insulin alone,

phobic residues, thereby possibly assisting the re-d: with 0.05 M G -b-CD, n: with 0.1 M G -b-CD.2 2

folding of the proteins. In this way CDs might act assmall chaperone-mimics in the protein folding pro-cess in cases where refolding is inhibited by poorly-

tion at relatively low substitution and acceleration at reversible aggregation or entanglement [60,61].higher substitution. In such cases, the electrostatic Calorimetric studies for thermal unfolding of hGHinteraction between the positive charges of insulin at a neutral pH have demonstrated that the ratio ofand the negative charges of the sulfonate group of the van’t Hoff enthalpy to the calorimetric enthalpySBE-b-CD seems to be more of a factor than the (DH /DH ) is more than one, indicating the aggrega-v c

inclusion effects. These results suggest that proper tion of the protein during the unfolding process. Theuse of hydrophilic CDs could be effective in design- hydrophilic CDs increase the unfolding temperatureing rapid or long-acting insulin preparations [57]. for hGH and decrease the DH /DH ratio. Thesev c

Alzheimer’s disease is characterized pathological- results suggest that the interaction of the hydrophilicly by extracellular amyloid deposits in the brain, CDs with accessible hydrophobic side chains in thecontaining a b-A4 amyloid peptide, derived from a hGH molecule leads to the less compact conforma-larger amyloid precursor protein. Although a princi- tion of the protein and reduces its aggregation duringpal morphological marker of the disease, the role of the unfolding process [62].


When hGH is refolded from its molten globule- state. In the second step, removal of the detergentlike intermediates produced by a denaturant from the protein is triggered by the addition of a CD,guanidium hydrochloride, insoluble aggregates are allowing the protein to refold.formed, as indicated by an increase in absorbance ofthe solution at 350 nm. As shown in Fig. 7, 6-O-glucosyl-b-CD (G -b-CD) and G -b-CD, as well as 6. Hydrophilic cyclodextrins as absorption1 2

non-ionic surfactant Tweens, significantly reduced enhancersthe aggregation of hGH during refolding from theintermediates, while G -a-CD, HP-CDs and linear The systemic delivery of peptide- and protein-2

saccharides showed no noticeable inhibitory effect. based drugs via various mucosal routes is receivingFurthermore, the size exclusion chromatographic extensive scrutiny as an alternative to the oral andanalysis revealed that the concentration of the hGH parenteral routes. The transmucosal delivery hasmonomer remaining in solution after refolding was advantages of being noninversive and of bypassinghighest in the presence of G -b-CD. This indicates gastrointestinal and hepatic clearances. Among them2

that the b-CD cavity with a branched sugar moiety is the peptide delivery through nasal mucosa seems tomost preferable to prevent the aggregation of hGH be most successful and practical; nasal sprays for[62]. some therapeutic peptides are already available

More efficient protein refolding is established commercially [2]. However, even with the intranasalwhen CDs are used in combination with detergents route of delivery, the nasal epithelium presents both[63–66]. In the first step, the non-native target a physical and a metabolic barrier to the absorptionprotein is captured by a detergent under conditions of peptides and proteins. Therefore, the use ofthat would normally lead to irreversible protein absorption-promoting agents is necessary to achieveaggregation, in which the substrate protein cannot sufficient intranasal absorption of most peptides andspontaneously refold from the detergent-complexed proteins. The potential of CDs, especially the

methylated CDs, as nasal absorption enhancers hasbeen demonstrated for luteinizing hormone-releasinghormone agonists [67,68], insulin [69–72], adreno-corticotropic hormone analogue [73], calcitonin [74],granulocyte colony-stimulating factor [75], insulin-like growth factor-I [76], etc. The absorption en-hancement afforded by CDs can be attributed pri-marily to their ability to reduce the physical and/ormetabolic barriers to these peptides and proteins.Further details on the uses of CDs in nasal drugdelivery have been described in chapter 6.

The limited systemic bioavailability of peptidesand proteins is partly due to the existence of asubstantial enzymatic barrier in the epithelial cells.CDs can protect peptides and proteins against en-zymatic as well as chemical degradation [77,78]. Forexample, CDs, especially G -b-CD, significantly2

inhibited the enzymatic degradation of buserelinFig. 7. Effects of additives (50 mM) on aggregation of hGH acetate in rat nasal mucosa [68]. On the basis of theduring refolding from molten globule-like intermediates (the inclusion mode of buserelin acetate with CDs asinitial concentration of hGH: 500 mM) in phosphate buffer (pH described in Section 2, they may protect buserelin5.0) containing 4.5 M guanidium hydrochloride at 258C (see [62]).a b acetate sterically from proteolytic enzymes, by in-The average degree of substitution was 4.1; The average degree

c cluding the hydrophobic side chains of the peptideof substitution was 4.8; The average degree of substitution was4.8. within the CD cavity, because these binding sites are


located near the enzymatic cleavage sites of the thermal stabilization arising from the branched sugarpeptide [68]. moiety. Rather small DH value of a-chymotrypsinc

The possibility for the CDs to directly deactivate with G -b-CD may be ascribable to the binding of2

the proteolytic enzymes, however, should not be G -b-CD to aromatic amino acid residues of the2

totally dismissed [71,79,80]. For example, G -b-CD protease, a process which is known to be endo-2

decelerated the hydrolysis of buserelin acetate cata- thermic. These results indicate that G -b-CD reduces2

lyzed by a-chymotrypsin, a typical serine protease. the catalytic activity of a-chymotrypsin in such aBased on the kinetic studies, this deceleration can be way that the accessible hydrophobic side chains ofexplained solely by a non-productive encounter the protease may be incorporated into the CD cavity,between a complex of the substrate with G -b-CD a situation which should produce some localized2

and the protease at relatively low CD concentrations, distortion and/or steric hindrance near the catalyticwhile the direct inhibitory effect of G -b-CD on the site of the protease.2

proteolytic activity made a considerable contribution Another potential barrier to the nasal absorption ofto the overall deceleration of the hydrolysis at higher peptides and proteins is the limitation in the size ofCD concentrations [80]. hydrophilic pores through which they are thought to

Further insight into the direct interaction between pass. The hydrophilic CDs can solubilize someG -b-CD and a-chymotrypsin was gained by dif- specific lipids from biological membranes through2

ferential scanning calorimetry (Table 1). In the case the rapid and reversible formation of inclusionof a-chymotrypsin alone, the DH value was about complexes, leading to an increase in the membranev

half the DH value, suggesting the presence of permeability [7]. CDs may affect nasal mucosalc

intermediate states in the unfolding process, pre- membranes in the same manner, thus allowing theirsumably due to the autolysis [80]. By contrast, these extended use as adjuvants to improve the nasalquantities for the unfolding of a-chymotrypsin with absorption of poorly absorbable peptide and proteinG -b-CD were nearly the same, indicating that the drugs. The lipid solubilization mediated by CDs may2

denaturation is very close to a two-state process. A cause changes in transcellular processes, and thesesimilar two-state denaturation of a-chymotrypsin has changes are believed to be transmitted to the paracel-been observed in the pH range 2–4, in which the lular region, which appears to be the most likelyprotease is no longer reactive. It is well known that route for the transport of polypeptides [71,81].maltose stabilizes proteins against thermal denatura- Nasal preparations must be critically evaluated fortion through its structure-making effect on the sur- their possible effect on the nasal mucociliary func-rounding water molecules. In fact, maltose increased tions, which are known to defend the respiratorythe T value of a-chymotrypsin by approximately tract against noxious inhaled materials such as dust,m

58C, but did not affect the DH /DH ratio. allergens and bacteria. Since most of the enhancersv c

In the case of G -b-CD, the conformational including CDs may promote the systemic absorption2

energy of the unfolded a-chymotrypsin is likely to of peptides and proteins by perturbing membranebe reduced by incorporating the exposed hydro- integrity in a rather non-specific manner, it is inevit-phobic groups in the unfolding protein into the CD able that varying extents of insult would occur to thecavity [28], which may compensate fully for the mucosal tissue in intimate contact with the enhan-

Table 1Thermodynamic parameters of a-chymotrypsin (80 mM) in the absence and presence of G -b-CD (0.1 M) and maltose (1 M) in isotonic2

phosphate buffer (pH 7.4) (see [80])

System T DH DH DH /DHm c v v c

(8C) (kcal /mol) (kcal /mol)

a-Chymotrypsin alone 48.960.2 16762 8161 0.4960.02With G -b-CD 48.760.1 12666 11662 0.9360.062

With maltose 54.260.4 188614 8663 0.4660.05

Each value represents the mean6S.D. of three experiments.


cers. When compared with other absorption-promot- Upon further addition of HP-b-CD beyond theing agents and preservatives used commonly in nasal critical concentration necessary for completeformulations, CDs exert a rather mild and reversible solubilization of HPE-101, the activity of the en-effect on the surface morphology of nasal mucosa hancer decreased. The excess amount of HP-b-CD inand the ciliary beating [82–84]. To evaluate the solution may reduce the free fraction of HPE-101,nasal tissue tolerability to CDs, the release behaviors which is in an equilibrium with the complexed form,of several biochemical markers from nasal mucosa and thereby reduce the thermodynamic activity of thewere measured using an in-situ recirculating perfu- enhancer at the mucosal surface.sion technique in rats [85]. Five percent solutions of The sole use of HPE-101 in emulsion showed theCDs released the biochemical markers from the nasal maximal enhancing effect on the nasal absorption ofmucosa with the efficacy increasing in the order: FD with an average molecular mass of | 40000. OnSBE-b-CD # 2-hydroxyethyl-b-CD , HP-b-CD , the other hand, the combination of HPE-101 withheptakis(3-mono-O-methyl)-b-CD , DM-b-CD, a HP-b-CD provided the prominent enhancement ofsequence which is almost proportional to their the nasal absorption of FDs with average molecularhemolytic activity or ciliotoxicity. Other studies masses of less than 20 000, compared with thatusing an in-vivo lavage technique have shown that obtained for the sole use of the enhancer (Fig. 8).the rat nasal mucotoxicity increased in the order: With increasing molecular masses of FDs, the per-HP-b-CD , randomly methylated b-CD , DM-b- meation enhancing effect of this combination de-CD , sodium glycocholate , sodium taurodihydro- creased steeply. The rate of overall processes involv-fusidate , L-a-lysophosphatidylcholine , laureth-9 ing the dissociation of HPE-101 from its HP-b-CD[86]. This rank order correlates well with those complex and subsequent uptake of the free form ofobserved in morphological as well as ciliotoxicity the enhancer into the nasal mucosa was much fasterstudies. Based on the results of these studies, the than that of the enhancer from oil droplets into theminimal concentration of DM-b-CD necessary to mucosa. Therefore, the rapid onset and short durationachieve substantial absorption enhancement in rats of the enhancing effect of this combination wouldis considered to be | 2%, showing only a mild effecton the nasal ciliary function. It should be also notedthat the efficacy and safety of CDs differ largelybetween species [87–89] and is also greatly depend-ent upon the dosage form [90–93].

In an attempt to design a more effective and lessirritating nasal formulation, combinations of CDswith absorption enhancers have received increasingattention [94–98]. For instance, HP-b-CD solubil-ized a lipophilic absorption enhancer, HPE-101 andpotentiated its action at an appropriate combinationratio without causing severe local irritation on thenasal application [99–101]. When the concentrationof HPE-101 was kept constant at 1% (w/v), the nasalmembrane permeability of fluorescein isothiocyanatedextran with an average molecular mass of 4400 Da(FD-4) increased with a rise in the HP-b-CD con-centration, reaching a maximum when just enough

Fig. 8. Enhancing effects of HPE-101 (1% w/v) and HP-b-CDHP-b-CD ( | 15%, w/v) was used to keep all the(15% w/v) on nasal absorption of FDs (7.5 mg/kg) with variousenhancer in solution. HP-b-CD may potentiate themolecular weights to rats, where the extent of nasal absorption of

activity of HPE-101 by solubilizing, thus making it FDs without the enhancer up to 3 h post-administration was takenmore available at the mucosal surface for subsequent as unit (see [102]). s: with HPE-101, d: with HPE-101 andpenetration into the nasal epithelium, a site of action. HP-b-CD (the average degree of substitution of 5.8).


not be enough to allow the slow diffusion of FDs use of CDs as rectal absorption enhancers needswith higher molecular masses through the nasal great care as to their irritating effects on rectalmucosa [102]. These results indicate that HPE-101 mucosa and also the potential for pathogenic sub-solubilized in HP-b-CD at the appropriate combina- stances and the CDs themselves to be systemicallytion ratio could cause just enough and transient absorbed. In vitro permeation studies using an iso-perturbation of the nasal mucosa to allow the absorp- lated rectal mucosal preparation of rabbits havetion of the permeation marker, without widespread revealed that a-CD at higher concentrations ( | 100damage of the epithelium. The approach described mM) reduced the basal transepithelial potentialhere would be extended to the optimal use of other difference and short-circuit current and increasedlipophilic absorption enhancers particularly in the transepithelial conductance, a circumstance whichenvironment of the mucosal absorption site. facilitated its own mucosal permeation as well as the

As described in chapter 8, CDs have been applied drug permeation in a rather non-specific mannerto optimize the transdermal delivery of drugs in- [108]. DM-b-CD is reported to enhance the rectaltended either for local or systemic use. CDs may absorption of insulin from the hollow-type oleagin-interact with some components of the skin. For ous suppository in rabbits, in which both the peptideinstance, DM-b-CD is known to extract cholesterol and the CD are dissolved in citrate buffered solutionand triglyceride from powdered hide [103] and from (pH 3.0) and loaded into a cavity of the suppository.rabbit skin in vitro [104], a process which may This formulation seems to be less irritating to thereduce the function of skin as a barrier and eventual- rectal mucosa, as indicated by the fact that thely may contribute in part to the absorption enhance- hyperpermeable state of the rectal mucosa mediatedment of poorly absorbable drugs, including peptide- by DM-b-CD returned to a normal physiologicaland protein-based drugs. In such a case, particular level within 24 h after rectal administration [109].attention should be directed toward the possibleirritation effects of CDs on the skin. Our previousstudies have demonstrated that the parent CDs at 7. Hydrophobic cyclodextrins as sustained-sufficiently higher concentrations caused skin irrita- release carrierstion in guinea pigs in the order of g- , a- , b-CD, aresult which depends largely on their abilities to Chronic treatment with peptide and protein drugsextract lipids from the skin [105]. has disadvantages; the short biological half-lives of

Differential scanning calorimetric studies have the drugs require long-term daily injection or fre-shown that DM-b-CD affected the endothermic quent nasal application in order to maintain atransition of an isolated human stratum corneum, therapeutic concentration of the drugs. Therefore,while no noticeable changes were observed for the attention has been directed toward the developmentstratum corneum treated with HP-b-CD [106]. Other of drug delivery systems with controlled-releasestudies have shown that randomly methylated b-CD features so as to realize their potential and efficacy.extracted all the major lipid classes from an isolated Several approaches have been proposed including thestratum corneum of hairless rats and reduced the use of implants or injectable microcapsules of bio-barrier function of the skin, while HP-b-CD had the degradable copolymers or gel-forming agents.limited specificity for cholesterol and triglycerides Injectable oily suspensions of buserelin acetateand to a small extent, cholesterol esters. Neverthe- with sustained-release feature can be obtained byless, neither CD induces any major modification of using hydrophobic CDs such as heptakis(2,6-di-O-the differential scanning calorimetric profile and the ethyl)-b-CD (DE-b-CD) [110,111] andFourier-transformed infrared spectrum of the stratum heptakis(2,3,6-tri-O-acethyl)-b-CD (TA-b-CD) andcorneum, suggesting the CDs do not penetrate the octakis(2,3,6-tri-O-acethyl)-g-CD (TA-g-CD) [112].lipid structure of the stratum corneum [107]. The interfacial transfer of buserelin from the peanut

The practical use of CDs to increase the drug oil suspension into the aqueous phase was signifi-solubility and stability and to improve the drug cantly retarded by the hydrophobic CDs in the orderdelivery from the rectal formulations has been of TA-b-CD , TAy-CD , DE-b-CD. The drug re-extensively reviewed in chapter 8. In contrast, the lease from a vehicle is influenced by various factors


increased by the complexation with DE-b-CD orTA-CDs. In contrast, the solubility of the CDs in thevehicle increased in the order of TA-b-CD , TA-g-CD , DE-b-CD corresponding with the retardationorder of buserelin release. The above results suggestthat the drug might be dispersed within an oilymatrix through a weak interaction of the drug withthe CDs.

A single subcutaneous injection of the oily suspen-sion of buserelin acetate containing TA-b-CD, TA-g-CD and DE-b-CD in rats provided retardation ofplasma levels of buserelin, with giving 25, 39 and 70times longer mean residence time, respectively, thanthat of the drug alone (Fig. 9). Simultaneously withthe suppression of plasma testosterone to castratelevel, the antigonadal effect of buserelin continuedfor 1, 2, and 4 weeks and a significant weightreduction on genital organs was observed (Fig. 10).For example, when the DE-b-CD complex wasadministered subcutaneously to rats, the weight of

Fig. 9. Plasma levels of buserelin (A) and testosterone (B) after genital organs decreased on week 1 due to ansubcutaneous administrations of buserelin acetate and its CD antigonadal effect, and the weight reduction wascomplexes in oily suspension (1 mg/kg as buserelin acetate) to

maintained for 8 weeks, while the drug alone showedrats (see [110–112]). s: buserelin acetate alone, d: DE-b-CDno significant effect.complex, m: TA-b-CD complex, n: TA-g-CD complex.

Since TA-CDs are ester-type derivatives, they areincluding drug–vehicle interactions, solubility, parti- susceptible to alkaline hydrolysis resulting in re-tion coefficient and particle size of the drug in the formation of corresponding parent CDs and aceticvehicle. Buserelin acetate was practically insoluble acid in a 1:3 molar ratio in a fashion of the first-orderin peanut oil and the solubility was only slightly kinetics. TA-CDs are also degraded enzymatically

Fig. 10. Weight changes of genital organs in rats after subcutaneous administrations of buserelin acetate and its CD complexes in oilysuspension (1 mg/kg as buserelin acetate) (see [110–112]). s: buserelin acetate alone, d: DE-b-CD complex, m: TA-b-CD complex, n:TA-g-CD complex.


with the rat skin homogenates. For example, the anticoagulant activity. The anticoagulant activity ofresidual amounts of TA-b-CD and TA-g-CD were S-CDs was about 100 times weaker than that of72% and 60% after the 8 h incubation, respectively, heparin on a weight basis and was comparable to thatwhile DE-b-CD remained intact under the ex- of dextran sulfate with a similar sulfur content.perimental conditions because of the ether-type Basic fibroblast growth factor (bFGF) is a potentderivative. Although there is no in-vivo kinetic mitogen that stimulates the proliferation of a wideevidence on TA-CDs subcutaneously administered, variety of cells and could play a crucial role inonce they are hydrolyzed into the respective parent wound healing processes. The therapeutic potentialCDs at the injection site, the resulting CDs are of bFGF, however, has not been fully realizedsupposed to be easily absorbed and excreted into because of its susceptibility to proteolytic inactiva-urine. Since the enzymatic hydrolysis of TA-CDs at tion and short duration of retention at the site ofthe injection site may proceed gradually as described action. Recent studies have demonstrated that sul-above, it may be free from nephrotoxicity. In fact, no fated oligosaccharides, including a sodium salt ofabnormality was found in the hemodiagnosis during S-b-CD (Na ? S-b-CD), have a high affinity forthe experiments through week 0 to week 4 after the bFGF and protect it from heat, acid, and proteolyticsubcutaneous administration of oily suspension con- degradation. These sulfated oligosaccharides maytaining TA-CDs. Furthermore, it is likely that TA- bind close to the putative heparin binding domain, aCDs, in addition to controlling the release of cluster of several basic amino acid residues, on thebuserelin acetate from the oil vehicle, may also act surface of the bFGF molecule, probably through anas stabilizers for the peptide against the enzymatic electrostatic interaction [122,123]. Unfortunately, thedegradation at the site of administration. These facts highly hydrophilic nature of Na ? S-b-CD is notsuggest that TA-CDs are preferable drug carriers for suited to the design of bFGF formulations withsubcutaneous injection of peptides and proteins than controlled-release features.DE-b-CD due to their possible bioabsorbable charac- A water-insoluble aluminium salt of S-b-CD (Al ?

teristics. S-b-CD) was prepared, and its possible utility as astabilizer and sustained-release carrier for bFGF wasevaluated [124,125]. An adsorbate of bFGF with

8. Sulfated cyclodextrins as heparinoids Al ? S-b-CD was prepared by incubating the proteinwith a suspension of Al ? S-b-CD in water. The

The introduction of sulfate groups onto the hy- mitogenic activity of bFGF released from the adsor-droxyl groups of CDs confer biological activities, bate, as indicated by the proliferation of kidney cellssuch as anti-inflammatory and antilipemic activities, of baby hamsters (BHK-21), was almost comparablesimilar and sometimes superior to those of heparin with that of the intact protein. Al ? S-b-CD sig-on such derivatives [113]. Recently, sulfated CDs nificantly protected bFGF from the proteolytic degra-(S-CDs) have been found to be effective in inhibiting dation by pepsin of a-chymotrypsin compared withcellular invasion by human immunodeficiency re- their sodium salts and other oligosaccharides. Thetrovirus [114–117] and to have antiangiogenic activi- in-vitro release of bFGF from the adsorbate wasty in combination with appropriate angiostatic ster- sustained in proportion to a rise in the ratio ofoids [118–120]. Al ? S-b-CD to the protein.

In our previous study a single intravenous ad- Of the bFGF preparations tested, the adsorbate ofministration of S-a-, S-b- or S-g-CD at a dose of 1 bFGF with Al ? S-b-CD, when given subcutaneouslyg/kg was tolerated well in rats without conspicuous to rats, showed the most prominent increase in thechanges in blood chemistry values, while several formation of granulation tissues, probably due to theparameters in rats receiving other polyanions includ- stabilization and sustained delivery of the mitogening heparin, dextran sulfate and poly-L-aspartic acid (Fig. 11). These results suggest that the adsorbate ofat the same doses provoked renal or hepatic disorders bFGF with Al ? S-b-CD has a potent therapeutic[121]. In clinical practice, higher doses of efficacy for wound healing, and can be applicable toheparinoids are sometimes associated with untoward oral protein formulation for the treatment of intesti-reactions such as bleeding episodes due to their nal mucosal erosions.


for bFGF than sucralfate and other sulfated sac-charides (Table 2) [125]. The rigid macrocyclicstructure of S-b-CD imposes spatial constraints onthe sulfate groups, which enhances the charge den-sity and the affinity for bFGF. The oral administra-tion of Al ? S-b-CD tended to enhance the healingrate of acetic acid-induced gastric ulcers andcysteamine-induced duodenal ulcers, probably in asimilar manner to sucralfate. In particular, the oraladministration of Al ? S-b-CD loaded with bFGF hadthe most prominent healing effects on the ulcers[125]. In addition, the heparin-mimicking activity ofFig. 11. Effects of bFGF preparations (10 mg/disc as bFGF) onS-CDs has been successfully applied to the inhibitiongranulation tissue formation in rats (see [124]). Empty bars:

without bFGF, filled bars: with bFGF. of restenosis after the surgical approaches to thetreatment of atherosclerosis [127–129], the chro-matographic separation of heparin binding proteins

An hypothesis is proposed to provide a common [130,131].mechanism for conventional antiulcer therapy, inwhich endogenous growth factors such as basicfibroblast growth factor (bFGF) play a central role 9. Cyclodextrins as oligonucleotide carriers[126]. An aluminium salt of sucrose sulfate (sucral-fate) has a high affinity for bFGF and protects it from Antisense oligonucleotides are widely used asacid degradation and inactivation. Oral administra- research tools for inhibiting specific gene expressiontion of sucralfate elevates local levels of bFGF in the and are under investigation for possible use asulcer bed, indicating that sucralfate acts as a potent therapeutic agents for the treatment of diseasesangiogenesis stimulator, primarily on the basis of its caused by several viruses, including herpes simplexability to stabilize and slowly release locally avail- virus, cytomegalovirus and especially human im-able bFGF. As described previously, Al ? S-b-CD is a munodeficiency virus [132]. Cellular uptake, inter-more potent stabilizer and sustained-release carrier nalization and nuclease resistance of oligonucleotides

are important factors in determining the effectivenessof antisense therapies. To improve the cellularTable 2

Effects of additives (25 mg/ml) on stability of bFGF (250 mg/ml) uptake of oligonucleotides, several approaches havein the presence of pepsin (50 mg/ml, pH 1.6) for 3 h at 378C (see been proposed, including the encapsulation into[125]). liposomes or nanoparticles, the use of membrane-

aCDs pH Remaining bFGF (%) perturbing agents and the conjugation with choles-terol or poly-L-lysine [133–135]. Although theseWithout additives 1.67 N.D.

a-CD 1.67 N.D. strategies have resulted in increases in cellularb-CD 1.60 N.D. uptake of oligonucleotides and/or increases in theirg-CD 1.66 N.D. stability against nucleases, cytotoxicity and non-se-HP-b-CD 1.65 N.D.

quence specific activities may also increase.Na?S-a-CD 1.74 1.661.3A recent study using charge-transfer chromatog-Na?S-b-CD 1.74 3.361.4

Na?S-g-CD 1.73 5.161.0 raphy indicates that HP-b-CD interacts with nu-Na?S-HP-b-CD 1.63 4.563.2 cleoside derivatives bearing different alkyl chains inAl?S-a-CD 3.81 96.461.2 such a manner as both sterical and hydrophobicityAl?S-b-CD 3.88 99.262.6

parameters of the nucleosides affect the stability ofAl?S-g-CD 3.82 96.563.1the complexes [136]. Hydroxyalkylated b-CDs canAl?S-HP-b-CD 3.86 93.662.1

Dextran sulfate 1.84 20.269.6 increase the uptake of phosphorothioate oligodeoxy-Sucralfate 3.78 59.666.1 nucleotide in human T cell leukemia H9 cell line bya, Each value represents the mean6S.D. of three determinations. two- to three-fold [137]. Confocal microscopic ob-


servations confirmed that the increase in the oligo- complex with T3 DNA. This strategy could be usefulnucleotide uptake observed with the CDs was indeed in designing novel sequence-specific DNA bindingdue to an increase in intracellular uptake, rather than peptides [142].an increase in surface binding of the oligonucleotide.Since the CDs neither affected the efflux of theoligonucleotide from the cells nor stabilized it 10. Use of cyclodextrins for lipoproteinagainst endogenous cellular nucleases, they should measurementsmediate its influx into the cells, probably through thecomplexation or the direct effect on the cellular In diagnostic preparations, CDs have been suc-surface. Furthermore, the CDs can suppress the cessfully utilized as substrates, stabilizers, solubiliz-immune stimulation induced with the oligonucleo- ers and scavengers of interfering substances [143].tides in vitro and in vivo. It is possible that the CDs are reported to interact with serum lipoproteins,complexation of the oligonucleotides with the CDs the efficacy increasing in the order: very-low-densityreduces the non-specific binding of the oligonucleo- lipoprotein (VLDL) , high-density lipoproteintides with proteins, thereby reducing their immune (HDL) , low-density lipoprotein (LDL) [144]. CDsstimulation. Additional advantages of the CDs are may form complexes with accessible hydrophobictheir abilities to reduce certain other undesirable regions in the lipoproteins, and consequently precipi-side-effects of oligonucleotides such as reduction of tate them in a manner different from those describedplatelet counts in vivo [138]. for polyanion-metal combinations [145].

Introduction of a thiogalactose residue onto the Of the CDs tested, DM-b-CD shows the mostprimary hydroxyl group of b-CD significantly im- prominent effect on the electrophoretic pattern of theproves the cellular uptake of phosphodiester oligo- serum lipoproteins [146]. In particular, an anodicnucleotide probably through a ligand-carbohydrate mobility of the band assigned to HDL is significantlycell receptor interaction. In addition, the complex- decreased with increasing concentrations of DM-b-ation could modify the intracellular distribution of CD. The interaction of DM-b-CD with the fourthe antisense oligonucleotide, leading to the en- major lipoprotein classes in the serum (chylomicron,hanced antiviral activity [139]. A recent study has VLDL, LDL and HDL) was investigated by combin-shown that CDs enhance adenoviral-mediated gene ing two methods: separation by high-performancedelivery to the intestinal epithelium with minimal liquid chromatography with a gel permeation columncytotoxicity. A tertiary amino derivative of b-CD is and selective detection of lipid constituents of eachthe most effective in improving the adenoviral lipoprotein fraction including cholesterol, phos-transduction efficiency in differentiated Caco-2 cells pholipids, and triglycerides by enzymatic reaction.[140]. When the relatively low concentrations of DM-b-CD

Conjugates of nucleotides with adamantane at the ( , 10 mM) were added to each lipoprotein fraction,39-end of host nucleotides have increased nuclease it reduced the elution volume of the HDL fraction,resistance compared to their parent oligonucleotides. with a small change in the lipid composition, sug-HP-b-CD can enhance the cellular uptake of the gesting a change in the density or surface charge ofoligonucleotide-adamantane conjugates [141]. In a the HDL particles and/or its aggregation to mac-recent study a series of short peptides derived from roparticles. On the other hand, DM-b-CD at thethe basic region of the basic leucine zipper protein same concentration range only slightly affected theGCN4 was synthesized to examine the cooperative elution volume of the other lipoprotein fraction.DNA binding to direct repeat sequences. A modified Furthermore, DM-b-CD at higher concentrationslysine residue bearing an adamantyl group at the ( . 60 mM) increased the elution volume of all the´-amino group was incorporated at the N-terminal lipoprotein fractions in a non-selective way, probablyposition, and b-CD was attached at the C-terminal due to the partial solubilization and/or collapse ofcysteine residue of the parent basic region peptide. the particles. When DM-b-CD was added to eachThe resulting peptide formed an intramolecular lipoprotein fraction, an increase in turbidity of theinclusion complex in the absence of a specific DNA mixture is observed only for the HDL fraction.sequence, while it formed an a-helical trimer-DNA Additionally, the increment of turbidity in the whole


serum treated with DM-b-CD was well correlated tothe concentration of HDL in the serum determinedby the conventional precipitation-based method,suggesting the potential application of DM-b-CD tothe direct measurement of HDL particle in the serumwithout any separation procedures.

Recently, we have developed an automated meth-od for measuring HDL-cholesterol in serum withoutprior separation, using polyethylene glycol (PEG)-modified enzymes and S-a-CD [147]. When choles-terol esterase and cholesterol oxidase are modifiedwith PEG, they show selective catalytic activitiestowards lipoprotein fractions, with the reactivityincreasing in the order: LDL , VLDL ¯chylomicron , HDL. In the presence of magnesiumions, S-a-CD reduced the reactivity of cholesterol,especially in chylomicron and VLDL, without needfor precipitation of those lipoprotein fractions (Fig.12). A combination of PEG-modified enzymes withS-a-CD provides selectivity for the direct measure-ment of HDL-cholesterol in serum. Fig. 12. Effects of S-a-CD and MgCl on relative reactivities of2

It is well known that polyanions such as heparin cholesterol in various lipoprotein fractions with PEG-modifiedand dextran sulfate interact primarily with positive cholesterol esterase (1 kU/ l) and cholesterol oxidase (5 kU/ l) in

3-(N-morpholino)propanesulfonic acid buffer (pH 7.0) at 378Ccharges of the protein moiety and, in the presence of(see [147]). s: 0.5 mM MgCl , d: 1 mM MgCl , n: 2 mM2 2the divalent metal ions associated with the zwit-MgCI .2terionic polar heads of the phospholipids, may also

contribute to the interaction by forming insolubledetermination of LDL-cholesterol in serum are cum-complexes [145]. As described in Section 8 in thisbersome, time consuming and require specializedchapter, the heparin-like activity of S-a-CD may alsoinstrumentation, which limits their use in the clinicalenable lipoproteins to interact in a manner similar tolaboratory. We have established a direct method forthat reported for polyanions. S-a-CD used in thisdetermining the concentration of LDL-cholesterol inmethod has an average molecular mass of 2194 Da,serum in a convenient format with the combined usewhich is | 100 times smaller than those of theof a non-ionic surfactant, polyoxyethylene-polyox-polyanions used in precipitation-based methods.ypropylene triblock copolymer (POE-POP) and S-a-Furthermore, the charge density of S-a-CD appearsCD [151]. Only a very small sample volume (4 ml)to be higher than those of the polyanions. Theseis needed for this method, without the need fordifferences between S-a-CD and the polyanions mayisolation of LDL. LDL-cholesterol can be deter-explain the differential effects on lipoproteins. Un-mined in a short time (10 mm) by this homogenouslike the polyanions, S-a-CD forms water-solublemethod, which can be easily automated. The strategycomplexes selectively with chylomicron and VLDL,used for direct measurement of LDL-cholesterol inwhich are resistant to the PEG-modified enzymes,serum is based on the cooperative actions of POE-and thus exhibit reduced reactivities towards choles-POP as a quencher for HDL-cholesterol and S-a-CDterol in those lipoprotein fractions. The methodas a quencher for chylomicron-and VLDL-choles-described here is simple and reliable for measuringterol. Correlation between the proposed method andHDL-cholesterol in serum without the need for priorother methods (Friedewald formula, immunosepara-separation of other fractions and has been usedtion, high-performance liquid chromatographyclinically as a first-line test to monitor HDL-choles-(HPLC) and beta-quantification methods) are sum-terol in serum [148–150].marized in Table 3. In the serum samples fromMany of the current techniques used in the


Table 3Correlation between the proposed LDL assay and other methods with sera from healthy volunteers (I), patients with hyperlipemia

a)(triglycerides,4.5 mmol / l) (II), and with hyperlipemia (triglycerides$4.5 mmol / l) (III) (see [151]).

Method Group Slope (a) y-intercept (b) r n Mean6SD (mmol / l)

Proposed method I 86 2.75260.954bII 41 5.13162.333

III 34 2.97961.378Friedewald formula I 0.973 0.137 0.987 86 2.71560.975

bII 0.933 0.468 0.990 41 4.99462.483b cIII 0.395 2.338 0.552 34 2.02061.771

Immunoseparation I 0.936 0.140 0.972 86 2.71860.918bII 0.981 0.047 0.980 41 5.07662.312

III 0.988 20.044 0.974 34 2.90761.384HPLC I 0.995 0.003 0.987 86 2.74160.962

bII 1.023 20.202 0.989 41 5.04862.397III 0.932 0.059 0.980 34 2.83461.309

Beta quantification I 1.001 0.010 0.989 86 2.76460.965bII 1.008 20.065 0.992 41 5.16462.361

III 1.010 20.052 0.992 34 2.96461.381a In the form y5ax1b, y5the proposed method, x5other method.b P,0.01 vs group I.c P,0.01 vs the proposed method.

healthy volunteers, the results obtained by the pro- these undesirable properties of the drug candidatesposed method are well correlated with other meth- through inclusion complex formation, which conse-ods, but in those from patients with hyperlipemia quently improves drug delivery via various routes of(triglycerides $ 4.5 mmol / l), the correlation coeffi- administration. In this contribution, particular atten-cient increased in the order: Friedewald formula , tion was also paid to the potentials of S-CDs asimmunoseparation , beta-quantification method. The heparin-mimicking modulators for growth factorsproposed method for measuring LDL-cholesterol involved in repairing processes and as diagnosticwould include the contribution of intermediate den- agents for the direct measurement of lipoproteinsity lipoprotein and lipoprotein (a), as the Friedewald cholesterol in serum. Although the toxicologicalequation or beta-quantification method does. Because issues together with biological fates should be in-all the particles of this wide-density LDL population vestigated in detail, the CDs described here haveare atherogenic, the proposed method might be a many advantages as novel tools for the delivery ofmore sensitive indicator of risk for premature cor- peptides, proteins and oligonucleotides and should beonary artery diseases than a method that has the pursued.limited specificity for the narrow-density LDL popu-lation.

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