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Hyaluronan in drug delivery
Advanced Drug Delivery Reviews, 7 (1991) 295-308
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Elsevier Science Publishers B.V. ADONIS 0169409X9100256W ADR 00090
Hyaluronan in drug delivery Jaroslav Drobnik Institute of Biotechnology, Faculty of Science, Charles University, Prague, Czechoslovakia (Received January 22, 1991) (Accepted February 6, 1991)
Key words: Hyaluronan; Macromolecular drug; Drug carrier; Drug targeting; Polymeric drug
Contents Summary ................................................................................................................. The macromolecular drug idea ........................................................................... 1. The carrier ............................................................................................... 2. The polymer-drug link ............................................................................... II. How hyaluronan fits in the idea .......................................................................... 1. Immunological considerations ...................................................................... 2. The size of the carrier ................................................................................. (a) Small molecules ................................................................................... (b) Large molecules ................................................................................... 3. Degradation ............................................................................................. 4. Targeting ................................................................................................. 5. The build-up chemistry with hyaluronan carrier ............................................... III. Conclusions ................................................................................................... References ...............................................................................................................
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Summary Biological and chemical properties of hyaluronan qualify this macromolecule as Abbreviations: Ab, antibody; BHK, baby hamster kidney; GPC, gel permeation chromatography; HABP, hyaluronan-binding protein; i.p., intraperitoneal; i.v., intravenous; CFA, complete Freund's adjuvant; LEC, liver endothelial cell; MAb, monoclonal antibody. Correspondence: J. Drobnik, Institute of Biotechnology, Faculty of Science, Charles University, Prague 2, 128 44, Czechoslovakia. Fax: (42) (2) 291958.
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a prospective carrier of drugs particularly for local application and/or targeting to lymphatic system. It is immunologically inert and safely degraded in lysosomes of many cells. Thus, it can be used in broad selection of molecular sizes. It also provides suitable chemical groups for spacer-drug side chain attachment. Nevertheless, several points call for experimental checking, viz the effect of substituents on the immunology, receptor affinity and degradation. Pharmacokinetics as affected by Mr and degree of substitution should be also monitored after various ways of administration. I. The macromolecular drug idea
The idea of soluble macromolecular drug carriers was a side product of early studies of polymer pharmacokinetics [1-3]. Physiologically inert polymers exhibited residential time which was but a dream for pharmacologists seeking long-lasting drug effects. Jatzkewitz [4,5] demonstrated its plausibility by the attachment of mescaline to the carrier copolymer vinylpyrrolidone comethacrylic acid. Since then the idea has developed in a well-populated branch of macromolecular science (see reviews, e.g., Refs. 6-8). Scientific excitement and promise of economic reward stimulated many studies which gave rise to the crystallization of our recent image of an ideal macromolecular drug form [9]. Macromolecular drug forms are composed basically of three components: (i) the carrier; (ii) the drug; and (iii) a link between them. Targeting moiety may be added to direct the deposition of the complex preferentially in certain compartments [10]. 1.1. The carrier
The main role of the carrier consists in the retardation of processes which eliminate the complex from the body. When glomerular filtration is the main elimination route, Mr about 20 000--40000 is considered sufficient to meet this demand. This rule is based on many studies which compare the renal clearance of macromolecules of various sizes. Using dextran Arturson and Wallenius [11] estimated the clearance in man and found half-times of 0.33, 1.1 and 12 h for Mr 18000-23000, 36 000-44 000 and 55 000-69 000, respectively. By a more advanced method Hardwicke et al. [12] studied the renal clearance of various polymers in rabbit and came to the conclusion that this process was isomorphic with ultrafiltration across a membrane with normal distribution of pore sizes around the mean 2.4 nm with practically zero incidence of pores above 6.5 nm. However, the reality is always far from this ideal model because glomerular filtration is not the only route of polymer clearance. Endocytosis [7] is another way of polymer elimination from circulation, fluid phase endocytosis being much less efficient than the specific receptor-mediated process [13]. Therefore the latter should be prevented when long residential time of the polymeric drug form in the central compartment (circulation) has to be reached. This goal together with immunological considerations asks for 'invisible', i.e., physiologically inert carrier polymers. However, in some special cases the structure
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of the polymer, when designed to react with receptors, can take over the targeting role. It is appreciated if the carrier also protects the attached drug moiety from metabolic inactivation such as acylation, oxidation, etc., extending the persistence of the drug-active form. After the detachment of the drug the carrier must disappear from the body leaving no residues or toxic products [9]. This point is the matter in many disputes. The conception backing the use of non-degradable carriers [14,15] argues that macromolecules of which the size is below the renal filtration limit are not accumulated in the body even if they are not depolymerized. A serious weak point is typical for this school: the absence of any theory suggesting how such molecules can escape from lysosomes since it is generally accepted that the permeation limit of lysosomal membrane is about Mr 200. This shortcoming becomes even more apparent when such conjugates of drugs with non-degradable polymers are presented as lysosomotropic drugs, i.e., drug forms which have to enter lysosomes in order to release the drug in its active form [16,17]. The opposite conviction can be formulated as follows [18]: 'in instances where the polymeric carrier is taken up by cells the use of non-degradable polymers is precluded'. It reflects the increasing concern of public and authorities in most countries about the drug safety. Therefore more and more the claim is sustained that only total catabolism of the carrier - i.e., its disassembling resulting in molecules which are then incorporated into normal metabolic pathways - provides an assurance of absolute elimination of the carrier.
1.2. The polymer-drug link The side chain between the polymer and the drug should keep the drug attached during the time required for prolonged effect but must not prevent the drug from accomplishing its physiological function. In the case of the drug exhibiting its effect even when bound to the carrier (e.g., enzyme inhibitors [19] and perhaps some hormones [20,21]) the link may be permanent. However, in most cases the drug can exert its role only in the cytoplasmic or nuclear compartments which are not accessible for xenobiotic macromolecules. Then the detachment of the drug from the carrier is obligatory. This process which should release the drug in its active form can proceed either in the extracellular or lysosomal compartments being chemical or enzymic in nature. Thus, two degradation processes come into play and their coordination represents a difficult but crucial task. If the carrier degradation starts before the drug is released, new drug modification(s) arise, the physiological activity of which should be considered separately. When such a process occurs in the extracellular compartment or the fragment carrying the drug is released from the intercellular (lysosomal) compartment the pharmacokinetics of it may be different from that of free drug and the fate of the drug in the body becomes hardly predictable. Therefore both steps of hydrolysis - the detachment of the drug and the degradation of the carrier - should be catalyzed by different enzymes and preferentially should occur in separate compartments. The drug release should be complete
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before the carrier degradation starts. In many cases the complete degradation of the carrier is catalyzed by specific enzymes which do not tolerate any side chains [18]. Therefore their safe and total removal is a necessary condition for carrier disassembling. Since the first work by Jatzkewitz [5] who demonstrated the effect of the degradable dipeptide chain Gly-Leu on the availability of attached mescaline, oligopeptides are favored in the role of the degradable link between the carrier and the drug. Rules of their hydrolysis were studied [22] and very sophisticated chains have been constructed to modulate the detachment rate due to different affinity to proteolytic enzymes [23]. Thus, a general construction scheme of a polymeric drug form may be summarized as follows. (a) The so-called 'biologically inert' polymer is selected having an Mr between 10 000 and 40 000 which represents Stokes radius big enough to retard its filtration through glomerular membrane but not as big to prevent it [24]. (b) A reactive group is formed in the polymer structure and a prepared side chain with or without a drug as a terminal moiety is attached to the reactive group by a covalent bond. Alternatively, the side chain may be built up step by step on the carrier. (c) If the drug was not part of the side chain it is bound to it - mostly by a cleavable bond - in the last step. (d) This macromolecular form is isolated (usually by GPC), analyzed and applied to a selected compartment, i.v. or i.p. in most cases. (e) Pharmacokinetics of both the drug and the carrier should be monitored. Unfortunately, in many studies only the 'therapeutic' and/or 'toxic' effects in some model systems are reported. Another critical point is missing as a rule in the evaluation of macromolecular drug forms - the control of possible immunological response after repeated administrations or (better) the investigation about the presence of antibodies either against the carrier or the drug. Absence of this immunological activity and complete elimination of the carrier from the body are a conditio sine qua non for the practical application of any macromolecular drug form. II. How hyaluronan fits in the idea
The data presented by contributors to this issue provide the necessary basis for the evaluation how suitable hyaluronan would be as a carrier for a construction of a polymeric drug form.
11.1. Immunological considerations Hyaluronan is a linear/3-1,4-homopolymer of a disaccharide composed from glucuronic acid and N-acetylglucosamine, more exactly from D-glucopyranoside uronic acid bond by /3--1,3-linkage to 2-acetamido-2-deoxy-D-glucopyranose, so that their name according to I U P A C [25] is poly(2-acetamido-2-deoxy-D-glucano-
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D-glucan). Hyaluronan persists in an unordered structure in water but adopts an ordered structure in water-organic mixed solvents [26]. In water solutions of sufficient ionic strength shorter segments tend to associate and longer segments form hair-pin structures [27]. The coil in water assumes an extraordinary big volume. Molecules of Mr 3"106 have gyration radius of 200 nm [28] which means that the sphere contains 99.9% of water immobilized in the polymer structure. As a polyanion the shape of the molecule is very sensitive to pH and ionic strength [29,30]. In water the molecules are separated only at concentrations below 0.1%. Above this value the solution adopted rather a network character of entangled chains [31]. Such a structure provides only a limited number of specific epitopes. Klaus et al. [32] used hyaluronan as a non-immunogenic hapten carrier. Unfortunately, the purity of the sample was not satisfactory (1% of protein and 12% of chondroitin sulfate). Fillit et al. [33] were not able to raise anti-hyaluronan antibody by immunization with hyaluronan degradation products and CFA, but positive response was obtained with hyaluronan covalently bound to liposomes. Two epitopes were distinguished by MAb: terminal glucuronic acid was dominating whereas the other was uncertain because the MAb cross reacts with heparan sulfate and electrostatic effects played a role in this reaction. This is in agreement with a previous result of this group [34], viz, the detection of an Ab against terminal glucuronic acid moiety after immunization with formalized Streptococcus. It has to be kept in mind that these results were obtained after some chemical modification which can generate epitopes not present in native hyaluronan. The occurrence of natural anti-hyaluronan Ab was referred by Underhill [35]. This finding is surprising in the light of the constant presence of endogenous hyaluronan in the body fluids which makes the occurrence of such antibodies improbable in healthy conditions. This fact with the above-mentioned high hydrophilic nature qualifies hyaluronan as a prospective 'invisible' inert polymer. As in other 'inert' carriers this advantage may be invalid as soon as a n y t h i n g the drug or the residual linking oligopeptide - is attached to it since such side chain introduces an epitope in the structure. Particularly the oligopeptides containing aromatic amino acids may be immunogenic. However, as a rule an immunogenic carrier is needed for induction of anti-hapten Ab which is definitely not the case with hyaluronan. Nevertheless, experimental verification is necessary.
11.2. The size of the carrier Optimal size of hyaluronan as a carrier may be discussed from several points: H.2(a) Small molecules. Molecules of Mr up to 4.10 4 may pass glomerular filtration. We have described [36] the resorption of a soluble polymer in tubular epithelial cells and many data about hyaluronan pharmacokinetics (Refs. 37 and 38, and Lebel (Ref. 89) in this issue) indicate that this might be the case with hyaluronan as well. Therefore using small hyaluronan molecules the drug release in the kidney cortex may be expected. It was demonstrated [39,40] that small hyaluronan molecules are less bound to cells than big ones. The association constant kd for octasaccharides in liver endothelial cells (LEC) was found to be 4.6-10 6 mol.1 1,
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whereas for hyaluronan of Mr 6.4"106 its value was 9-10 -12 mol.1-1. These results correspond with longer half-lives observed in some cases (rabbit) with small molecules after application in the circulation [41].
lI.2(b)
Large molecules. Molecules above the renal filtration limit are usually out of consideration as carriers because the kidney excretion is believed to be the best guarantee of complete elimination of the carrier from the body. We have shown [7] that such an argument is not valid with non-degradable polymers, since they are subjected to endocytosis during the circulation and resorption of the polymer may occur even after the passage through glomerulus [36]. From the data presented in this issue and mentioned above it is evident that the fate of small and large hyaluronan molecules is different mainly due to strong binding of the latter (probably because of multi-point contacts and thermodynamic forces). As a result the clearance of large molecules from circulation is very fast (halflives about 1.8 rain) [41]. Nevertheless, thanks to very high viscosity of solutions large molecules may be useful for local, e.g., subcutaneous, intrasinovial or intramuscular applications. As given by Laurent and Reed in this issue and others [42] the turnover of hyaluronan with Mr > 106 extends over 2--4 days in skin and 0.5-2 days in joints. H.3. Degradation In principle there are two ways of hyaluronan degradation [43]. (a) Coordinated activity of two exoglycosidases, viz ~D-glucuronidase and /~Nacetyl-D-hexosamidase [44]. Rod6n et al. [43] pointed out that the Km = 3"10 -4 mol [45] of the former enzyme is too high to explain the degradation of hyaluronan in vivo where the Mr of around 2.5.106 is supposed. However, when using a carrier of Mr about 1 0 4 the above K~ represents 0.3% solution which may sound realistic for, e.g., lysosomal compartment where both enzymes are present. Fraser (see Discussion in Ref. 46) reported 30 saccharide units as a possible substrate for these two exoglycosidases and Orkin [46] showed larger molecules to be resistant. (b) Hyaluronidase is considered as a most powerful degradation enzyme for hyaluronan but its role is not universal. (i) In adult humans its presence was referred only in a limited set of tissues [47]. (ii) mammalian hyaluronidase is strictly a lysosomal enzyme with optimal pH below 4, whereas above 5 its activity is negligible [48]. Neutral hyaluronidase was detected only during 'very narrow window of time' in the embryonic development [49], in wounds [50] and in testes. The hyaluronidase in serum, if any, is of lysosoreal type. (iii) The smallest susceptible substrate is represented by a hexasaccharide which cannot escape from lysosomes. High activity of lysosomal hyaluronidase was detected in liver [51], rat and bovine brain [52] whereas its presence in skin is still questionable [43], since it was not detected in skin fibroblasts [53]. Even immunological methods failed to detect hyaluronidase in various human cell lines except for hepatoma [54]. Hyaluronidase inhibitors were found in serum [43,55,56].
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Binding to the cell surface and internalization are the first steps in hyaluronan degradation in the body. On the LEC surface about 105 receptors [46] were estimated. The binding domain occupies probably the size of the octasaccharide; however, longer chains showed higher affinity. Since the gyration radius of molecules having Mr 6.4"105 is about 300 nm the number of such molecules which can be arranged over the cell surface is limited for steric reasons (about 900 coils per cell). The internalization proceeds through both the receptor-mediated and fluid-phase endocytosis [57]. At the concentration of 50 Izg/1 each LEC deposited on average 0.1 fg of hyaluronan in lysosomes. About 30 min-1 h after the internalization low-molecular-weight degradation products occurred from LEC. Uptake is the rate-limiting step in hyaluronan degradation by LEC and does not go parallel with the uptake of other glycosaminoglycans according to SmedsrCd et al. [57]. These authors claimed that neither Kupffer cells nor hepatocytes accumulated and degraded hyaluronan [58]. Contrary to this statement is the study of Frost et al. [59] who quantitatively assessed the membrane-associated hyaluronan receptors in isolated hepatocytes. Uptake of hyaluronan by hepatocytes was also observed by Truppe et al. [60]. Summarizing the data from the coordinated hydrolysis point of view, the time sequence - first the drug release and then the carrier disassembling - could be expected with the macromolecular drug form when hyaluronan serves as a carrier and oligopeptide as a side arm: (a) in the central compartment - because at a neutral reaction in blood and lymph proteases but not hyaluronidase can be active. This is valid for other compartments as well, except for lysosomes. (b) in lysosomes - since hyaluronidase will hardly hydrolyse the substrate substituted with bulky side chains carrying the drug moiety. Even if it does, big chunks cannot escape across the lysosomal membrane. Nevertheless, these assumptions have to be verified by experiment. On the other hand all published data put the elimination of hyaluronan carrier out of any doubt provided all side chains are removed. The structure which would grant this condition is another problem waiting for experimental testing.
H.4. Targeting Many data have accumulated on hyaluronan receptors [61,39] and hyaluronanbinding proteins (HA-BP) (see Turley [90] in this issue). Underhill [62] suggested two types of them: one as a tool of hyaluronan internalization by means of coated pits and the other one on those sites where hyaluronan 'can act as an adhesive' between, e.g., epithelial cells and the basement membrane. A similar role of 'glue' is suggested for hyaluronan in macrophages aggregation [63,64] because this effect depends on the presence of hyaluronan, receptors and receptor-bound hyaluronan. Hexasaccharide [43] or octasaccharide [41] are the smallest structural units recognized by receptors. LeBoeuf et al. [65] using Sepharose immobilized hyaluronan found by displacement measurements 200 monosaccharide units and more as the optimal size for binding to fibrinogen. The affinity constant was estimated to be 2.10 -7 mol.1-1.
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Underhill and Toole [39,61] studied extensively the binding of hyaluronan to the surface of 3T3 cells. An optimal pH was found around neutrality and binding was increased by increasing ionic strength and lowering the temperature. Monoclonal antibody (MAb) revealed that the hyaluronan receptor on BHK cells was identical with the gp85 membrane glycoprotein [66] which is associated with actin filaments of cytoskeleton. Raja et al. [67] using radioiodinated non-metabolizable tag for hyaluronan studied hyaluronan receptors on intact and digitonin permeabilized rat LEC and concluded that 50-80% of receptors were intracellular. However, their nature is not fully understood. Frost et al. [59] estimated about 9000 surface receptors per hepatocyte. After opening the cells by digitonin the binding capacity corresponded to 1.3.106 binding sites with ka = 3"10 7 mol'l I for hyaluronan of Mr 30000. Many studies were performed with MAb as an indicator of hyaluronan receptor localization [68,69]. However, some of these results were opposed with the argument that receptor already occupied by hyaluronan need not react with MAb [70]. The relationship between cell transformation and hyaluronan receptors and/or HA-BP is rather a controversial issue. When HA-BP were added to non-transformed 3T3 cells their binding capacity for hyaluronan was increased and after the addition of external hyaluronan the cells adopted a transformed character, viz., aggregation, reduced cell spreading and increased nuclear overlap [71]. Underhill and Toole [72,73] studied hyaluronan receptors and hyaluronan binding in pairs of cell lines: non-transformed 3T3 and BHK and transformed SV3T3 and PyBHK. The association constant was lower and the saturation capacity higher with transformed cells. Goldberg et al. [74] reported 30 times more hyaluronan in the coat of non-transformed 3T3 cells than in SV3T3 cells. However, the latter cells showed a much higher affinity for exogenous hyaluronan. Also the response to hyaluronidase treatment was different. It was suggested that in normal cells the lowaffinity receptors are located within the rich hyaluronan coat, whereas the highaffinity sites are very scarce. Transformed cells with rapid turnover of hyaluronan expose many binding siJes which are associated with hyaluronan endocytosis. Rapid hyaluronargturnover by transformed cells may explain also other data: Turley and Tretiak [75] and Angelo et al. [76] reported more hyaluronan production in more invasive tumor lines. This was also supported by experiments of Kimata et al. [77] based on the labelling of newly synthesized hyaluronan. It is possible that the protective effect of hyaluronan coat suggested for viral infection [73] may provide similar benefit to malignant cells during tumor invasion. Another hypothesis may relate the role played by hyaluronan in the tumor development to its regulatory function in cell behavior, particularly during embryogenesis. Kawatsu et al. [78], using lymphoma cells KE-5 which are lacking estradiol receptors, concluded that the stimulation of hyaluronan synthesis was most probably the way this tumor is promoted by estradiol. Turley [79] described in the chick embryonic heart fibroblast hyaluronan receptors formed by a complex of phosphoproteins which show protein kinase activity distinct from other protein kinases. Phosphorylation of tyrosine, serine and threonine was stimulated specifically by hyaluronan.
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The data on hyaluronan receptors and HA-BP indicate that there are several of these classes. (a) The first class is represented by a linking protein serving as a joint in the building of hyaluronan into the structure of connective tissue, particularly cartilage. (b) The second class may be characterized by a similar predominantly mechanical function. It includes surface receptors which are an implement of cell adherence and aggregation. However, this class may also play some sophisticated role in the cell behavior, particularly during the embryo development. (c) Well-defined is a class of receptors initiating the internalization of hyaluronan via receptor-mediated endocytosis as a first step in hyaluronan catabolism in LEC, lymph nodes, etc. (d) The most complex is the class of receptors participating in cell regulations, viz, protein kinases, cell transformation, invasiveness of tumors, triggering of vascularization, etc. Thus, the role of a carrier in the targetting of drugs carried by hyaluronan can be hardly predicted. Nevertheless, it is firmly established that most of circulating hyaluronan is metabolized in LEC and lymph nodes where it is removed from circulation, internalized and hydrolyzed. Consequently, both sites will be natural targets for hyaluronan-carried drugs. The question may arise which density of substitution with bulky side chains is tolerated before the receptors affinity for the hyaluronan-part of the drug complex is abolished. We know the limits: the experiments by Raja et al. [80] with iodinated hydroxyphenylpropionyl tag and by Dahl et al. [81] with iodinated tyramine-cellobiose group set the lowest limit because hyaluronan macromolecules labelled in this way seem to follow the distribution pattern observed with chemically unaltered macromolecules. The highest limit is given by the above-mentioned octasaccharide unit which should be free for the affinity binding. Optimal substitution degree for a polymer-drug complex will be found by experiment somewhere in between.
H.5. The build-up chemistry with hyaluronan carrier As discussed above the carrier-drug attachment must facilitate the drug release and the removal of the side chain. Three types of groups on hyaluronan macromolecule - carboxyl, hydroxyl and acetamido - are available for the construction. Advantageous esterification of hyaluronan carboxyl groups was described [82]. It may form a bound hydrolyzable either by enzymes or by pure chemical process, e.g., acid hydrolysis in lysosomes. However, the above esterification reaction requires the alcoholic component to be prepared in the form of alkyliodide, which may cause some problems. Hydroxyl groups may be oxidized to aldehydes [80] which then enter the binding reaction. An aldehyde group only on the reducing end may be formed by limited oxidation. The reactivity with epoxy groups was demonstrated by crosslinking hyaluronan with epichlorhydrine [83], 1,2,3,4,-diepoxybutane [84] or epoxy-substituted pentaerythritol [85]. The acetamido group is not reactive, but a highly reactive amino group may be
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obtained by deacetylation. Hydrazinolysis was recommended for this purpose [86]; however, Shaklee and Conrad [87] found the formation of hydrazides of uronic acid as a side-product of this procedure. Treatment with HNO2 at low pH or by HIO4 bring back the hydrazides to uronic acids. This technique was used by H66k et al. [88] and by Dahl et al. [81]. The latter authors reported 70% of acetyl groups removed after 240 min of hydrazinolysis, but with a considerably reduced degree of polymerization. However, hyaluronan of much higher Mr than required for the carrier is available and narrow fraction may be selected after modification. Partially deacetylated hyaluronan when treated with [3H]- or [14C]acetanhydride yields radiolabelled hyaluronan with unchanged chemical structure. III. Conclusions
The requirements applied to a polymer designed as a carrier in polymeric drug forms are very controversial: it has to retard the elimination of the conjugate; however, it must also assure safe and complete elimination of the polymer after its role as a carrier is over. It should avoid non-specific endocytosis and induction of immunologic reactions. Hyaluronan in certain applications can meet these demands: it is 'invisible' for the immunity system, since it is a constant component of body fluids. It is safely metabolized in lysosomes of certain cells. Its structure provides reasonable ways of chemical binding procedures. On the other hand, specific receptors on some cells cut short their residential time in the circulation and make these cells a natural target after systemic administration. Thus, a preliminary suggestion may qualify hyaluronan as a prospective carrier for high-molecular-weight conjugates designed for local, e.g., intramuscular, intrasinovial or subcutaneous application. Transport of unchanged or partially disassembled conjugates to regional lymph nodes may be expected where most of them will be perished. In this way the drug distribution will occur preferentially in the lymphatic system. The fraction of the complexes escaping in the circulation will be trapped in liver endothelial cells and smaller molecules may even pass glomerular filtration being resorbed in tubular epithelium. Therefore liver and kidney cortex might be minority sites of drug release. This contemplation needs several experiments to answer open questions: first, the way in which substituents - the side chain carrying the drug - affects the known behavior of hyaluronan in the body, viz immunological 'invisibility', degradation in lysosomes and receptor affinity; second, using complexes with various Mr and degree of substitution, the biodistribution of the complex and the pharmacokinetics of the drug should be checked testing various application routes. Only results of all these studies can substantiate a conclusion about the suitability of hyaluronan as a drug carrier.
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26 Staskus, P.V. and Johnson Jr., W.C. (1988) Conformational transition of hyaluronic acid in aqueous-organic solvent monitored by vacuum ultraviolet circular dichroism, Biochemistry 27, 15221527. 27 Turner, E.A., Lin, P. and Cowman, N.K. (1988) Self-association of hyaluronate segments in aqueous sodium chloride solution, Arch. Biochem. Biophys. 265, 84-95. 28 Laurent, T.C. and Gergely, J. (1955) Light scattering studies on hyaluronic acid, J. Biol. Chem. 212, 325-333. 29 Laurent, T.C., Ryan, M. and Pietruszkiewicz, A. (1960) Fractionation of hyaluronic acid. The polydispersity of hyaluronic acid from the bovine vitreous body, Biochim. Biophys. Acta 42,476-485. 30 Cleland, R.L. (1968) Ionic polysaccharides. II. Comparison of polyelectrolyte behavior of hyaluronate with that of carboxymethyl cellulose, Biopolymers 6, 1519-1529. 31 Bother, H. and Wik, O. (1987) Rheology of hyaluronate, Acta Otolaryngol. Suppl. 442, 25-30. 32 Klaus, G.G.B., Janossy, G. and Humphrey, J.H. (1975) The immunological properties of haptens coupled to thymus-independent carrier molecules. III. The role of immunogenicity and mitogenicity of the carrier in the induction of primary IgM antihaptens responses, Eur. J. Immunol. 5,105-111. 33 Fillit, H., Blake, M., McDonald, Ch. and McCarty, M. (1988) Immunogenicity of liposome-bound hyaluronate in mice. At least two different antigenic sites on hyaluronate are identified by mouse monoclonal antibodies, J. Exp. Med. 168, 971-982. 34 Fillit, H., McCarty, M. and Blake, M. (1981) Induction of antibodies to hyaluronic acid by immunization of rabbits with encapsulated streptococci, J. Exp. Med. 164, 762-776. 35 Underhill, C.B. (1982) Naturally occurring antibodies which bind hyaluronate, Biochem. Biophys. Res. Commun. 108, 1488-1494. 36 Rypfi6ek, F., Drobn[k, J., Chmela~, V. and K~lal, J. (1982) The renal excretion and retention of macromolecules. The chemical structure effect, Pfltigers Arch. 392, 211-217. 37 Fraser, J.R.E., Engstr6m-Laurent, A., Nyberg, A. and Laurent, T.C. (1986) Removal of hyaluronic acid from the circulation in rheumatoid disease and primary biliary cirrhosis, J. Lab. Clin. Med. 107, 79-85. 38 Henriksen, J.H., Bentsen, K.D. and Laurent, T.C. (1988) Splanchnic and renal extraction of circulating hyaluronan in patients with alcoholic liver disease, J. Hepatol. 6, 158-166. 39 Underhill, C.B. and Toole, B.P. (1986) Physical characteristic of hyaluronate binding to the surface of the simian virus 40-transformed 3T3 cells, J. Biol. Chem. 255, 4544-4549. 40 Laurent, T.C., Fraser, J.R.E., Pertoft, H. and Smedsr0d, B. (1986) Binding of hyaluronate and chondroitin sulphate to liver endothelial cells, Biochem: J. 234,653-658. 41 Fraser, J.R.E. and Laurent, T.C. (1989) Turnover and metabolism of hyaluronan. In: The Biology of Hyaluronan (Ciba Found. Syrup. 143), Wiley, Chichester, pp. 41-59. 42 Schiller, S. and Dorfman, A. (1957) The metabolism of mucopolysaccharides in animals, J. Biol. Chem. 227,625-632. 43 Rod6n, L., Campbell, P., Fraser, J.R.E., Laurent, T.C., Pertoft, H. and Thompson, J.N. (1989) Enzymic pathways in hyaluronan catabolism. In: The Biology of Hyaluronan (Ciba Found. Symp. 143), Wiley, Chichester, pp. 60--86. 44 Longas, M.O. and Meyer, K. (1981) Sequential hydrolysis of hyaluronate by/3-glucuronidase and/3N-acetylhexosamidase, Biochem. J. 197,275-282. 45 Niemann, R. and Buddecke, E. (1982) Substrate specificity and regulation of activity of rat liver/3glucuronidase, Hoppe-Seyler's Z. Physiol. Chem. 363,551-598. 46 Orkin, R.W. (1989) Discussion. In: The Biology of Hyaluronan (Ciba Found. Symp. 143), Wiley, Chichester, p. 84. 47 Orkin, R.W. and Toole, B.P. (1980) Isolation and characterization of hyaluronidase from cultures of chick embryo skin- and muscle-derived fibroblasts, J. Biol. Chem. 255, 1036-1042. 48 McGuire, P.G., Castellot Jr., J.J. and Orkin, R.W. (1987) Size-dependent hyaluronate degradation by cultured cells, J. Cell. Physiol. 133,267-276. 49 Bernfield, M., Banerjee, S.D., Koda, J.E. and Rapraeger, A.C. (1948) Remodelling of the basement membrane as a mechanism of morphogenetic tissue interaction. In: R.L. Trelstad (Ed.), The Role of Extracellular Matrix in Development, Alan R. Liss, New York, pp. 545-572. 50 Bertolami, C.N., Day, R.H. and Ellis, D.G. (1986) Separation and properties of rabbit buccal mucosal wound hyaluronidase, J. Dent. Res. 65,939-944.
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51 Aronson Jr., N.N. and Davidson, E.A. (1967) Lysosomal hyaluronidase from rat liver, J. Biol. Chem. 242,437-444. 52 Margolis, R.U., Margolis, R.K., Santella, R. and Atherton, D.M. (1972) The hyaluronidase of brain, J. Neurochem. 15, 2325-2332. 53 Argoblast, B., Hopwood, J.J. and Dorfman, A. (1975) Absence of hyaluronidase in cultured human skin fibroblasts, Biochem. Biophys. Res. Commun. 67, 376-382. 54 Delpech, B., Betrand, P. and Chauzy, C. (1987) An indirect enzymo-immunological assay for hyaluronidase, J. Immunol. Methods 104, 223-229. 55 Fiszer-Szafarz, H. (1968) Demonstration of new hyaluronidase inhibitor in serum of cancer patients, Proc. Soc. Exp. Biol. Med. 125,300-302. 56 Chakraborti, A.S., Basu, A. and Mitra, S. (1982) Hyaluronidase activity in murine leukemia and lymphoma, Ind. J. Exp. Biol. 20, 423-424. 57 Smedsrod, B., Pertoft, H., Eriksson, S., Fraser, J.R.E. and Laurent, T.C. (1984) Studies in vitro on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells, Biochem. J. 223, 617-626. 58 Eriksson, S., Fraser, J.R.E., Laurent, T.C., Pertoft, H. and Smedsrod, B. (1983) Endothelial cells are a site of uptake and degradation of hyaluronic acid in the liver, Exp. Cell Res. 144, 223-228. 59 Frost, S.J., McGary, C.T., Raja, R.H. and Weigel, P.H. (1988) Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated, Biochim. Biophys. Acta 946, 66-74. 60 Truppe, W., Basner, R., Figura, K. and Kresse, H. (1977) Uptake of hyaluronate by cultured cells, Biochem. Biophys. Res. Commun. 108, 1016--124. 61 Underhill, C.B. and Toole, B.P. (1979) Binding of hyaluronate to the surface of cultured cells, J. Cell. Biol. 82,475-484. 62 Underhill, C.B (1989) The interaction of hyaluronate with the cell surface: the hyaluronate receptor and the core protein. In: The Biology of Hyaluronan (Ciba Found. Symp. 143), Wiley, Chichester, pp. 87-106. 63 Shannon, B.T. Love, S.H. and Myrvik, Q.N. (1980) Participation of the hyaluronic acid in the macrophage disappearance reaction, Immunol. Commun. 9,357-370. 64 Wright, T.C., Underhill, C.B., Toole, B.P. and Karnovsky, M.J. (1981) Divalent cation-independent aggregation of rat-1 fibroblasts infected with a temperature-sensitive mutant of Rous-sarcoma virus, Cancer Res. 41, 5107-5113. 65 LeBoeuf, R.D., Raja, R.H., Fuller, G.M. and Weigel, P.H. (1986) Human fibrinogen specifically binds hyaluronic acid, J. Biol. Chem. 261, 12,586-12,592. 66 Underhill, C.B., Green, S.J., Comoglio, P.M. and Tarone, G. (1987) The hyaluronate receptor is identical to glycoprotein Mr 85000 (gp85) as shown by monoclonal antibody which interferes with binding activity, J. Biol. Chem. 252, 13,142-13,146. 67 Raja, R.H., McGary, T.C. and Weigel, P.H. (1988) Affinity and distribution of surface and intracellular hyaluronic acid receptors in isolated rat liver endothelial cells, J. Biol. Chem. 263, 1666116668. 68 Green, S,J., Tarone, G. and Underhill, C.B. (1988) Aggregation of macrophages and fibroblasts inhibited by a monoclonal antibody to the hyaluronate receptor, Exp. Cell. Res. 178, 224-232. 69 Alho, A.M. and Underhill, C.B. (1989) The hyaluronate receptor is preferentially expressed on proliferating epithelial cells, J. Cell Biol. 108, 1557-1565. 70 Weigel, P.H. Discussion. In: The Biology of Hyaluronan (Ciba Found. Symp. 143), Wiley, Chichester, pp. 8%106. 71 Turley, E.A. (1982) Purification of hyaluronate binding protein fraction that modifies cell social behavior, Biochem. Biophys. Res. Commun. 108, 1016-1024. 72 Underhill, C.B. and Toole, B.P. (1981) Receptors for hyaluronate on the surface of parent and virus-transformed cell lines, Exp. Cell Res. 131,419-423. 73 Underhill, C.B. and Toole, B.P. (1982) Transformation-dependent loss of the hyaluronate°containing coats of cultured cells, J. Cell. Physiol. 110, 123-128. 74 Goldberg, R.L., Siedman, J.D., Chi-Rosso, G. and Toole, B.P. (1984) Endogenous hyaluronatecell surface interactions in 3T3 and simian virus-transformed 3T3 cells, J. Biol. Chem. 259, 94409446. 75 Turley, E.A and Tretiak, M. (1985) Glycosaminoglycan production by murine melanoma variants in
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vivo and in vitro, Cancer Res. 45, 5098-5105. 76 Angelo, J.C., Danielson, K.G., Anderson, L.W. and Hosick, H.L. (1982) Glycosamin0glycan synthesis by subpopulations of epithelial cells from mammary adenocarcinoma, Cancer Res, 42, 22072210. 77 Kimata, K., Honma, Y., Okayama, M. and Oguri, K. (1983) Increased synthesis of hyaluronic acid by mouse mammary carcinoma cells variants with high metastatic potential, Cancer Res. 43, 13471354. 78 Kawatsu, R., Ezaki, T., Kotani, M. and Akagi, M. (1989) Growth-promoting effect of estriol in a lymphoma lacking estrogen receptor, Br. J. Cancer 59, 563-568. 79 Turley, E.A. (1989) Hyaluronic acid stimulates protein kinase activity in intact cells and in isolated protein complex, J. Biol. Chem. 264, 8951-8955. 80 Raja, R.H., LeBoeuf, R.D., Stone, G.W. and Weigel, P.H. (1984) Preparation of alkylamine and ~25I-radiolabelled derivatives of hyaluronic acid uniquely modified at the reducing end, Anal. Biochem. 139, 168-177. 81 Dahl, L.B., Laurent, T.C. and Smedsr0d, B. (1988) Preparation of biologically intact radioiodinated hyaluronan of high specific radioactivity, Anal. Biochem. 175,397-405. 82 DellaValle, F. and Romeo, A. (1982) New polysaccharide esters and their salts, EP 216,453. 83 Sakurai, K., Ueno, Y. and Okuyama, T. (1985) Crosslinked hyaluronic acid and its use, EP 161,887. 84 Laurent, T.C., Helsing, K. and Gelotte, B. (1964) Cross-linked gels of hyaluronic acid, Acta Chem. Scand. 18,274-275. 85 Melson, T. (1987) Process for the manufacture of crosslinked carboxy group-containing polysaccharides, WO 87/07898 A1. 86 Dimitriev, B.A., Knirel, Yu.A. and Kochetkov, N.K. (1973) Selective cleavage of glycosidic linkages. Studies with the model compound benzyl 2-acetamido-2-deoxy-3-O-fl-D-galactopyranosyl-a-oglucopyranoside, Carbohydrate Res. 29,451-457. 87 Shaklee, P.N. and Conrad, H.E. (1984) Hydrazinolysis of heparin and other glycosaminoglycans, Biochem. J. 217, 187-197. 88 Ho6k, M., Reisenfeld, J. and Lindahl, U. (1982) N-[3H]Acetyllabeling, a convenient method of radiolabeling of glycosaminoglycans, Anal. Biochem. 119,236-245. 89 Lebel, L. (1991) Clearance of hyaluronan from the circulation, Adv. Drug Deliv. Rev. 7, 221-235. 90 Turley, E.A. (1991) Hyaluronan-binding proteins and receptors, Adv. Drug Deliv. Rev. 7,257-264.
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Elsevier Science Publishers B.V. ADONIS 0169409X9100256W ADR 00090
Hyaluronan in drug delivery Jaroslav Drobnik Institute of Biotechnology, Faculty of Science, Charles University, Prague, Czechoslovakia (Received January 22, 1991) (Accepted February 6, 1991)
Key words: Hyaluronan; Macromolecular drug; Drug carrier; Drug targeting; Polymeric drug
Contents Summary ................................................................................................................. The macromolecular drug idea ........................................................................... 1. The carrier ............................................................................................... 2. The polymer-drug link ............................................................................... II. How hyaluronan fits in the idea .......................................................................... 1. Immunological considerations ...................................................................... 2. The size of the carrier ................................................................................. (a) Small molecules ................................................................................... (b) Large molecules ................................................................................... 3. Degradation ............................................................................................. 4. Targeting ................................................................................................. 5. The build-up chemistry with hyaluronan carrier ............................................... III. Conclusions ................................................................................................... References ...............................................................................................................
295 296 296 297 298 298 299 299 300 300 301 303 304 305
Summary Biological and chemical properties of hyaluronan qualify this macromolecule as Abbreviations: Ab, antibody; BHK, baby hamster kidney; GPC, gel permeation chromatography; HABP, hyaluronan-binding protein; i.p., intraperitoneal; i.v., intravenous; CFA, complete Freund's adjuvant; LEC, liver endothelial cell; MAb, monoclonal antibody. Correspondence: J. Drobnik, Institute of Biotechnology, Faculty of Science, Charles University, Prague 2, 128 44, Czechoslovakia. Fax: (42) (2) 291958.
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a prospective carrier of drugs particularly for local application and/or targeting to lymphatic system. It is immunologically inert and safely degraded in lysosomes of many cells. Thus, it can be used in broad selection of molecular sizes. It also provides suitable chemical groups for spacer-drug side chain attachment. Nevertheless, several points call for experimental checking, viz the effect of substituents on the immunology, receptor affinity and degradation. Pharmacokinetics as affected by Mr and degree of substitution should be also monitored after various ways of administration. I. The macromolecular drug idea
The idea of soluble macromolecular drug carriers was a side product of early studies of polymer pharmacokinetics [1-3]. Physiologically inert polymers exhibited residential time which was but a dream for pharmacologists seeking long-lasting drug effects. Jatzkewitz [4,5] demonstrated its plausibility by the attachment of mescaline to the carrier copolymer vinylpyrrolidone comethacrylic acid. Since then the idea has developed in a well-populated branch of macromolecular science (see reviews, e.g., Refs. 6-8). Scientific excitement and promise of economic reward stimulated many studies which gave rise to the crystallization of our recent image of an ideal macromolecular drug form [9]. Macromolecular drug forms are composed basically of three components: (i) the carrier; (ii) the drug; and (iii) a link between them. Targeting moiety may be added to direct the deposition of the complex preferentially in certain compartments [10]. 1.1. The carrier
The main role of the carrier consists in the retardation of processes which eliminate the complex from the body. When glomerular filtration is the main elimination route, Mr about 20 000--40000 is considered sufficient to meet this demand. This rule is based on many studies which compare the renal clearance of macromolecules of various sizes. Using dextran Arturson and Wallenius [11] estimated the clearance in man and found half-times of 0.33, 1.1 and 12 h for Mr 18000-23000, 36 000-44 000 and 55 000-69 000, respectively. By a more advanced method Hardwicke et al. [12] studied the renal clearance of various polymers in rabbit and came to the conclusion that this process was isomorphic with ultrafiltration across a membrane with normal distribution of pore sizes around the mean 2.4 nm with practically zero incidence of pores above 6.5 nm. However, the reality is always far from this ideal model because glomerular filtration is not the only route of polymer clearance. Endocytosis [7] is another way of polymer elimination from circulation, fluid phase endocytosis being much less efficient than the specific receptor-mediated process [13]. Therefore the latter should be prevented when long residential time of the polymeric drug form in the central compartment (circulation) has to be reached. This goal together with immunological considerations asks for 'invisible', i.e., physiologically inert carrier polymers. However, in some special cases the structure
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of the polymer, when designed to react with receptors, can take over the targeting role. It is appreciated if the carrier also protects the attached drug moiety from metabolic inactivation such as acylation, oxidation, etc., extending the persistence of the drug-active form. After the detachment of the drug the carrier must disappear from the body leaving no residues or toxic products [9]. This point is the matter in many disputes. The conception backing the use of non-degradable carriers [14,15] argues that macromolecules of which the size is below the renal filtration limit are not accumulated in the body even if they are not depolymerized. A serious weak point is typical for this school: the absence of any theory suggesting how such molecules can escape from lysosomes since it is generally accepted that the permeation limit of lysosomal membrane is about Mr 200. This shortcoming becomes even more apparent when such conjugates of drugs with non-degradable polymers are presented as lysosomotropic drugs, i.e., drug forms which have to enter lysosomes in order to release the drug in its active form [16,17]. The opposite conviction can be formulated as follows [18]: 'in instances where the polymeric carrier is taken up by cells the use of non-degradable polymers is precluded'. It reflects the increasing concern of public and authorities in most countries about the drug safety. Therefore more and more the claim is sustained that only total catabolism of the carrier - i.e., its disassembling resulting in molecules which are then incorporated into normal metabolic pathways - provides an assurance of absolute elimination of the carrier.
1.2. The polymer-drug link The side chain between the polymer and the drug should keep the drug attached during the time required for prolonged effect but must not prevent the drug from accomplishing its physiological function. In the case of the drug exhibiting its effect even when bound to the carrier (e.g., enzyme inhibitors [19] and perhaps some hormones [20,21]) the link may be permanent. However, in most cases the drug can exert its role only in the cytoplasmic or nuclear compartments which are not accessible for xenobiotic macromolecules. Then the detachment of the drug from the carrier is obligatory. This process which should release the drug in its active form can proceed either in the extracellular or lysosomal compartments being chemical or enzymic in nature. Thus, two degradation processes come into play and their coordination represents a difficult but crucial task. If the carrier degradation starts before the drug is released, new drug modification(s) arise, the physiological activity of which should be considered separately. When such a process occurs in the extracellular compartment or the fragment carrying the drug is released from the intercellular (lysosomal) compartment the pharmacokinetics of it may be different from that of free drug and the fate of the drug in the body becomes hardly predictable. Therefore both steps of hydrolysis - the detachment of the drug and the degradation of the carrier - should be catalyzed by different enzymes and preferentially should occur in separate compartments. The drug release should be complete
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before the carrier degradation starts. In many cases the complete degradation of the carrier is catalyzed by specific enzymes which do not tolerate any side chains [18]. Therefore their safe and total removal is a necessary condition for carrier disassembling. Since the first work by Jatzkewitz [5] who demonstrated the effect of the degradable dipeptide chain Gly-Leu on the availability of attached mescaline, oligopeptides are favored in the role of the degradable link between the carrier and the drug. Rules of their hydrolysis were studied [22] and very sophisticated chains have been constructed to modulate the detachment rate due to different affinity to proteolytic enzymes [23]. Thus, a general construction scheme of a polymeric drug form may be summarized as follows. (a) The so-called 'biologically inert' polymer is selected having an Mr between 10 000 and 40 000 which represents Stokes radius big enough to retard its filtration through glomerular membrane but not as big to prevent it [24]. (b) A reactive group is formed in the polymer structure and a prepared side chain with or without a drug as a terminal moiety is attached to the reactive group by a covalent bond. Alternatively, the side chain may be built up step by step on the carrier. (c) If the drug was not part of the side chain it is bound to it - mostly by a cleavable bond - in the last step. (d) This macromolecular form is isolated (usually by GPC), analyzed and applied to a selected compartment, i.v. or i.p. in most cases. (e) Pharmacokinetics of both the drug and the carrier should be monitored. Unfortunately, in many studies only the 'therapeutic' and/or 'toxic' effects in some model systems are reported. Another critical point is missing as a rule in the evaluation of macromolecular drug forms - the control of possible immunological response after repeated administrations or (better) the investigation about the presence of antibodies either against the carrier or the drug. Absence of this immunological activity and complete elimination of the carrier from the body are a conditio sine qua non for the practical application of any macromolecular drug form. II. How hyaluronan fits in the idea
The data presented by contributors to this issue provide the necessary basis for the evaluation how suitable hyaluronan would be as a carrier for a construction of a polymeric drug form.
11.1. Immunological considerations Hyaluronan is a linear/3-1,4-homopolymer of a disaccharide composed from glucuronic acid and N-acetylglucosamine, more exactly from D-glucopyranoside uronic acid bond by /3--1,3-linkage to 2-acetamido-2-deoxy-D-glucopyranose, so that their name according to I U P A C [25] is poly(2-acetamido-2-deoxy-D-glucano-
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D-glucan). Hyaluronan persists in an unordered structure in water but adopts an ordered structure in water-organic mixed solvents [26]. In water solutions of sufficient ionic strength shorter segments tend to associate and longer segments form hair-pin structures [27]. The coil in water assumes an extraordinary big volume. Molecules of Mr 3"106 have gyration radius of 200 nm [28] which means that the sphere contains 99.9% of water immobilized in the polymer structure. As a polyanion the shape of the molecule is very sensitive to pH and ionic strength [29,30]. In water the molecules are separated only at concentrations below 0.1%. Above this value the solution adopted rather a network character of entangled chains [31]. Such a structure provides only a limited number of specific epitopes. Klaus et al. [32] used hyaluronan as a non-immunogenic hapten carrier. Unfortunately, the purity of the sample was not satisfactory (1% of protein and 12% of chondroitin sulfate). Fillit et al. [33] were not able to raise anti-hyaluronan antibody by immunization with hyaluronan degradation products and CFA, but positive response was obtained with hyaluronan covalently bound to liposomes. Two epitopes were distinguished by MAb: terminal glucuronic acid was dominating whereas the other was uncertain because the MAb cross reacts with heparan sulfate and electrostatic effects played a role in this reaction. This is in agreement with a previous result of this group [34], viz, the detection of an Ab against terminal glucuronic acid moiety after immunization with formalized Streptococcus. It has to be kept in mind that these results were obtained after some chemical modification which can generate epitopes not present in native hyaluronan. The occurrence of natural anti-hyaluronan Ab was referred by Underhill [35]. This finding is surprising in the light of the constant presence of endogenous hyaluronan in the body fluids which makes the occurrence of such antibodies improbable in healthy conditions. This fact with the above-mentioned high hydrophilic nature qualifies hyaluronan as a prospective 'invisible' inert polymer. As in other 'inert' carriers this advantage may be invalid as soon as a n y t h i n g the drug or the residual linking oligopeptide - is attached to it since such side chain introduces an epitope in the structure. Particularly the oligopeptides containing aromatic amino acids may be immunogenic. However, as a rule an immunogenic carrier is needed for induction of anti-hapten Ab which is definitely not the case with hyaluronan. Nevertheless, experimental verification is necessary.
11.2. The size of the carrier Optimal size of hyaluronan as a carrier may be discussed from several points: H.2(a) Small molecules. Molecules of Mr up to 4.10 4 may pass glomerular filtration. We have described [36] the resorption of a soluble polymer in tubular epithelial cells and many data about hyaluronan pharmacokinetics (Refs. 37 and 38, and Lebel (Ref. 89) in this issue) indicate that this might be the case with hyaluronan as well. Therefore using small hyaluronan molecules the drug release in the kidney cortex may be expected. It was demonstrated [39,40] that small hyaluronan molecules are less bound to cells than big ones. The association constant kd for octasaccharides in liver endothelial cells (LEC) was found to be 4.6-10 6 mol.1 1,
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whereas for hyaluronan of Mr 6.4"106 its value was 9-10 -12 mol.1-1. These results correspond with longer half-lives observed in some cases (rabbit) with small molecules after application in the circulation [41].
lI.2(b)
Large molecules. Molecules above the renal filtration limit are usually out of consideration as carriers because the kidney excretion is believed to be the best guarantee of complete elimination of the carrier from the body. We have shown [7] that such an argument is not valid with non-degradable polymers, since they are subjected to endocytosis during the circulation and resorption of the polymer may occur even after the passage through glomerulus [36]. From the data presented in this issue and mentioned above it is evident that the fate of small and large hyaluronan molecules is different mainly due to strong binding of the latter (probably because of multi-point contacts and thermodynamic forces). As a result the clearance of large molecules from circulation is very fast (halflives about 1.8 rain) [41]. Nevertheless, thanks to very high viscosity of solutions large molecules may be useful for local, e.g., subcutaneous, intrasinovial or intramuscular applications. As given by Laurent and Reed in this issue and others [42] the turnover of hyaluronan with Mr > 106 extends over 2--4 days in skin and 0.5-2 days in joints. H.3. Degradation In principle there are two ways of hyaluronan degradation [43]. (a) Coordinated activity of two exoglycosidases, viz ~D-glucuronidase and /~Nacetyl-D-hexosamidase [44]. Rod6n et al. [43] pointed out that the Km = 3"10 -4 mol [45] of the former enzyme is too high to explain the degradation of hyaluronan in vivo where the Mr of around 2.5.106 is supposed. However, when using a carrier of Mr about 1 0 4 the above K~ represents 0.3% solution which may sound realistic for, e.g., lysosomal compartment where both enzymes are present. Fraser (see Discussion in Ref. 46) reported 30 saccharide units as a possible substrate for these two exoglycosidases and Orkin [46] showed larger molecules to be resistant. (b) Hyaluronidase is considered as a most powerful degradation enzyme for hyaluronan but its role is not universal. (i) In adult humans its presence was referred only in a limited set of tissues [47]. (ii) mammalian hyaluronidase is strictly a lysosomal enzyme with optimal pH below 4, whereas above 5 its activity is negligible [48]. Neutral hyaluronidase was detected only during 'very narrow window of time' in the embryonic development [49], in wounds [50] and in testes. The hyaluronidase in serum, if any, is of lysosoreal type. (iii) The smallest susceptible substrate is represented by a hexasaccharide which cannot escape from lysosomes. High activity of lysosomal hyaluronidase was detected in liver [51], rat and bovine brain [52] whereas its presence in skin is still questionable [43], since it was not detected in skin fibroblasts [53]. Even immunological methods failed to detect hyaluronidase in various human cell lines except for hepatoma [54]. Hyaluronidase inhibitors were found in serum [43,55,56].
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Binding to the cell surface and internalization are the first steps in hyaluronan degradation in the body. On the LEC surface about 105 receptors [46] were estimated. The binding domain occupies probably the size of the octasaccharide; however, longer chains showed higher affinity. Since the gyration radius of molecules having Mr 6.4"105 is about 300 nm the number of such molecules which can be arranged over the cell surface is limited for steric reasons (about 900 coils per cell). The internalization proceeds through both the receptor-mediated and fluid-phase endocytosis [57]. At the concentration of 50 Izg/1 each LEC deposited on average 0.1 fg of hyaluronan in lysosomes. About 30 min-1 h after the internalization low-molecular-weight degradation products occurred from LEC. Uptake is the rate-limiting step in hyaluronan degradation by LEC and does not go parallel with the uptake of other glycosaminoglycans according to SmedsrCd et al. [57]. These authors claimed that neither Kupffer cells nor hepatocytes accumulated and degraded hyaluronan [58]. Contrary to this statement is the study of Frost et al. [59] who quantitatively assessed the membrane-associated hyaluronan receptors in isolated hepatocytes. Uptake of hyaluronan by hepatocytes was also observed by Truppe et al. [60]. Summarizing the data from the coordinated hydrolysis point of view, the time sequence - first the drug release and then the carrier disassembling - could be expected with the macromolecular drug form when hyaluronan serves as a carrier and oligopeptide as a side arm: (a) in the central compartment - because at a neutral reaction in blood and lymph proteases but not hyaluronidase can be active. This is valid for other compartments as well, except for lysosomes. (b) in lysosomes - since hyaluronidase will hardly hydrolyse the substrate substituted with bulky side chains carrying the drug moiety. Even if it does, big chunks cannot escape across the lysosomal membrane. Nevertheless, these assumptions have to be verified by experiment. On the other hand all published data put the elimination of hyaluronan carrier out of any doubt provided all side chains are removed. The structure which would grant this condition is another problem waiting for experimental testing.
H.4. Targeting Many data have accumulated on hyaluronan receptors [61,39] and hyaluronanbinding proteins (HA-BP) (see Turley [90] in this issue). Underhill [62] suggested two types of them: one as a tool of hyaluronan internalization by means of coated pits and the other one on those sites where hyaluronan 'can act as an adhesive' between, e.g., epithelial cells and the basement membrane. A similar role of 'glue' is suggested for hyaluronan in macrophages aggregation [63,64] because this effect depends on the presence of hyaluronan, receptors and receptor-bound hyaluronan. Hexasaccharide [43] or octasaccharide [41] are the smallest structural units recognized by receptors. LeBoeuf et al. [65] using Sepharose immobilized hyaluronan found by displacement measurements 200 monosaccharide units and more as the optimal size for binding to fibrinogen. The affinity constant was estimated to be 2.10 -7 mol.1-1.
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Underhill and Toole [39,61] studied extensively the binding of hyaluronan to the surface of 3T3 cells. An optimal pH was found around neutrality and binding was increased by increasing ionic strength and lowering the temperature. Monoclonal antibody (MAb) revealed that the hyaluronan receptor on BHK cells was identical with the gp85 membrane glycoprotein [66] which is associated with actin filaments of cytoskeleton. Raja et al. [67] using radioiodinated non-metabolizable tag for hyaluronan studied hyaluronan receptors on intact and digitonin permeabilized rat LEC and concluded that 50-80% of receptors were intracellular. However, their nature is not fully understood. Frost et al. [59] estimated about 9000 surface receptors per hepatocyte. After opening the cells by digitonin the binding capacity corresponded to 1.3.106 binding sites with ka = 3"10 7 mol'l I for hyaluronan of Mr 30000. Many studies were performed with MAb as an indicator of hyaluronan receptor localization [68,69]. However, some of these results were opposed with the argument that receptor already occupied by hyaluronan need not react with MAb [70]. The relationship between cell transformation and hyaluronan receptors and/or HA-BP is rather a controversial issue. When HA-BP were added to non-transformed 3T3 cells their binding capacity for hyaluronan was increased and after the addition of external hyaluronan the cells adopted a transformed character, viz., aggregation, reduced cell spreading and increased nuclear overlap [71]. Underhill and Toole [72,73] studied hyaluronan receptors and hyaluronan binding in pairs of cell lines: non-transformed 3T3 and BHK and transformed SV3T3 and PyBHK. The association constant was lower and the saturation capacity higher with transformed cells. Goldberg et al. [74] reported 30 times more hyaluronan in the coat of non-transformed 3T3 cells than in SV3T3 cells. However, the latter cells showed a much higher affinity for exogenous hyaluronan. Also the response to hyaluronidase treatment was different. It was suggested that in normal cells the lowaffinity receptors are located within the rich hyaluronan coat, whereas the highaffinity sites are very scarce. Transformed cells with rapid turnover of hyaluronan expose many binding siJes which are associated with hyaluronan endocytosis. Rapid hyaluronargturnover by transformed cells may explain also other data: Turley and Tretiak [75] and Angelo et al. [76] reported more hyaluronan production in more invasive tumor lines. This was also supported by experiments of Kimata et al. [77] based on the labelling of newly synthesized hyaluronan. It is possible that the protective effect of hyaluronan coat suggested for viral infection [73] may provide similar benefit to malignant cells during tumor invasion. Another hypothesis may relate the role played by hyaluronan in the tumor development to its regulatory function in cell behavior, particularly during embryogenesis. Kawatsu et al. [78], using lymphoma cells KE-5 which are lacking estradiol receptors, concluded that the stimulation of hyaluronan synthesis was most probably the way this tumor is promoted by estradiol. Turley [79] described in the chick embryonic heart fibroblast hyaluronan receptors formed by a complex of phosphoproteins which show protein kinase activity distinct from other protein kinases. Phosphorylation of tyrosine, serine and threonine was stimulated specifically by hyaluronan.
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The data on hyaluronan receptors and HA-BP indicate that there are several of these classes. (a) The first class is represented by a linking protein serving as a joint in the building of hyaluronan into the structure of connective tissue, particularly cartilage. (b) The second class may be characterized by a similar predominantly mechanical function. It includes surface receptors which are an implement of cell adherence and aggregation. However, this class may also play some sophisticated role in the cell behavior, particularly during the embryo development. (c) Well-defined is a class of receptors initiating the internalization of hyaluronan via receptor-mediated endocytosis as a first step in hyaluronan catabolism in LEC, lymph nodes, etc. (d) The most complex is the class of receptors participating in cell regulations, viz, protein kinases, cell transformation, invasiveness of tumors, triggering of vascularization, etc. Thus, the role of a carrier in the targetting of drugs carried by hyaluronan can be hardly predicted. Nevertheless, it is firmly established that most of circulating hyaluronan is metabolized in LEC and lymph nodes where it is removed from circulation, internalized and hydrolyzed. Consequently, both sites will be natural targets for hyaluronan-carried drugs. The question may arise which density of substitution with bulky side chains is tolerated before the receptors affinity for the hyaluronan-part of the drug complex is abolished. We know the limits: the experiments by Raja et al. [80] with iodinated hydroxyphenylpropionyl tag and by Dahl et al. [81] with iodinated tyramine-cellobiose group set the lowest limit because hyaluronan macromolecules labelled in this way seem to follow the distribution pattern observed with chemically unaltered macromolecules. The highest limit is given by the above-mentioned octasaccharide unit which should be free for the affinity binding. Optimal substitution degree for a polymer-drug complex will be found by experiment somewhere in between.
H.5. The build-up chemistry with hyaluronan carrier As discussed above the carrier-drug attachment must facilitate the drug release and the removal of the side chain. Three types of groups on hyaluronan macromolecule - carboxyl, hydroxyl and acetamido - are available for the construction. Advantageous esterification of hyaluronan carboxyl groups was described [82]. It may form a bound hydrolyzable either by enzymes or by pure chemical process, e.g., acid hydrolysis in lysosomes. However, the above esterification reaction requires the alcoholic component to be prepared in the form of alkyliodide, which may cause some problems. Hydroxyl groups may be oxidized to aldehydes [80] which then enter the binding reaction. An aldehyde group only on the reducing end may be formed by limited oxidation. The reactivity with epoxy groups was demonstrated by crosslinking hyaluronan with epichlorhydrine [83], 1,2,3,4,-diepoxybutane [84] or epoxy-substituted pentaerythritol [85]. The acetamido group is not reactive, but a highly reactive amino group may be
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obtained by deacetylation. Hydrazinolysis was recommended for this purpose [86]; however, Shaklee and Conrad [87] found the formation of hydrazides of uronic acid as a side-product of this procedure. Treatment with HNO2 at low pH or by HIO4 bring back the hydrazides to uronic acids. This technique was used by H66k et al. [88] and by Dahl et al. [81]. The latter authors reported 70% of acetyl groups removed after 240 min of hydrazinolysis, but with a considerably reduced degree of polymerization. However, hyaluronan of much higher Mr than required for the carrier is available and narrow fraction may be selected after modification. Partially deacetylated hyaluronan when treated with [3H]- or [14C]acetanhydride yields radiolabelled hyaluronan with unchanged chemical structure. III. Conclusions
The requirements applied to a polymer designed as a carrier in polymeric drug forms are very controversial: it has to retard the elimination of the conjugate; however, it must also assure safe and complete elimination of the polymer after its role as a carrier is over. It should avoid non-specific endocytosis and induction of immunologic reactions. Hyaluronan in certain applications can meet these demands: it is 'invisible' for the immunity system, since it is a constant component of body fluids. It is safely metabolized in lysosomes of certain cells. Its structure provides reasonable ways of chemical binding procedures. On the other hand, specific receptors on some cells cut short their residential time in the circulation and make these cells a natural target after systemic administration. Thus, a preliminary suggestion may qualify hyaluronan as a prospective carrier for high-molecular-weight conjugates designed for local, e.g., intramuscular, intrasinovial or subcutaneous application. Transport of unchanged or partially disassembled conjugates to regional lymph nodes may be expected where most of them will be perished. In this way the drug distribution will occur preferentially in the lymphatic system. The fraction of the complexes escaping in the circulation will be trapped in liver endothelial cells and smaller molecules may even pass glomerular filtration being resorbed in tubular epithelium. Therefore liver and kidney cortex might be minority sites of drug release. This contemplation needs several experiments to answer open questions: first, the way in which substituents - the side chain carrying the drug - affects the known behavior of hyaluronan in the body, viz immunological 'invisibility', degradation in lysosomes and receptor affinity; second, using complexes with various Mr and degree of substitution, the biodistribution of the complex and the pharmacokinetics of the drug should be checked testing various application routes. Only results of all these studies can substantiate a conclusion about the suitability of hyaluronan as a drug carrier.
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