Drug Delivery System Using Biodegradable Carrier - American

Chapter 25

Drug Delivery System Using Biodegradable Carrier S. Tokura , Y. Miura , Y. Kaneda , and Y. Uraki 1

Downloaded by YORK UNIV on October 29, 2012 | http://pubs.acs.org Publication Date: March 5, 1993 | doi: 10.1021/bk-1993-0520.ch025

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Department of Polymer Science, Faculty of Science, and Department of Forest Science, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan 2

6-O-Carboxymethyl-chitin (CM-chitin), one of the biodegradable chitin derivatives, shows several specificities such as chelating ability with calcium ion, specific adsorption of benzyl group following to the calcium chelation, and gel formation with trivalent iron ion. A prodrug was found to be released slowly into blood following the subcutaneous injection of polymeric drugs, in which the prodrug was either pendanted through a covalent bond to CM-chitin or entrapped within CM-chitin matrix in the presence of Fe . The prodrug was then hydrolyzed, to become the active form, by enzymes in the blood. 3+

Recently, a number of novel drug delivery approaches have been developed. These approaches include drug modification by chemical means, drug entrapment in small vesicles or within polymeric matrices. These arose from the fact that the conventional dosage forms of drug are not sufficiently effective. The potential use of macromolecules or macromolecular prodrugs as means to improve the efficiency of drugs has been studied, but a superior macromolecule for this is not yet developed because of the need for highly sophisticated materials. Chitin seems to have some advantageous properties to fulfill those requirements. Chitin is known to be hydrolyzed by lysozyme, a glycosidase distributed extensively in animals relating to bacteriolysis. Because it consists of naturally occurring substance, N-acetylglucosamine, chitin is non-toxic or has very low toxicity and immunogenicity. 6-O-Carboxymethyl-chitin (CM-chitin) is a chitin derivative which has high susceptibility to lysozyme, high water solubility, a reactive functional group, and chelating ability with calcium ions. CM-Chitin has known to chelate with calcium ion specifically (1) and form a gel-like matrix with ferric ion (2). Furthermore, it has been found that CM-chitin and its lysozymic hydrolysate showed little immunogenicity except slight mitogenic activity (3). These properties make it a very suitable material for a drug carrier. Several proposed types of CM-chitin drug carriers are shown in Figure 1. Lysozyme Susceptibility of C M - c h i t i n Lysozyme hydrolyzes not only the pi,4-bond between N-acetylmuramic acid and Nacetyl-β-D-glucosamine (MurNAcβl>4GlcNAc) but also the βl,4-bond of chitin. 0097-6156/93/0520-0351$06.00/0 © 1993 American Chemical Society

In Polymeric Delivery Systems; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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However, the latter hydrolysis proceeds very slowly owing to the high crystallinity, thus, chitin is degraded gradually i n animals. The lysozyme susceptibility of chitin was found to be remarkably enhanced by the carboxymethylation of the C-6 hydroxyl group unless the C-3 hydroxyl group was substituted (3). A viscosity change, which was monitored using Ubbelohde viscometer, of C M - c h i t i n aqueous solutions after the a d d i t i o n o f l y s o z y m e is s h o w n i n F i g u r e 2. It is o b v i o u s that the further c a r b o x y m e t h y l a t i o n of the C - 3 p o s i t i o n s i g n i f i c a n t l y reduced the l y s o z y m e susceptibility of CM-chitin. The rate of hydrolysis seems to be influenced by the degree of substitution which depends on the reaction conditions such as alkaline concentration, temperature, etc. Molecular Weight Distribution of Lysozymic Hydrolysate. CM-chitin was hydrolyzed with lysozyme to investigate the molecular weight distribution of lysozymic hydrolysate by gel permeation chromatography (GPC). A 500 m l of 0.1% (w/v) C M chitin (D.S.= 0.8) phosphate buffer solution (pH 6.2, M/15) was hydrolyzed at 37 - G l y - P h e - ρ Ν Α » -Phe-pNA. This might be due to the increasing mobility of the side chain. The higher rate for chymotryptic hydrolysis of CM-chitin-Ala-Phe-pNA than those of other conjugates is regarded as a synergism of its higher susceptibility and the flexibility of the side chain for chymotrypsin-catalyzed hydrolysis. c c - C h y m o t r y p s i n H y d r o l y s i s o f O l i g o m e r i c P r o d r u g s . Figure 7 shows the dependence of the rate of α-chymotrypsin-catalyzed hydrolysis (v ) on the number0



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Time after Injection (hr) Figure 5. Time course of release of methamphetamine derivatives into serum after subcutaneous injection of M A E A - C M - c h i t i n saline solution into rabbits; 4.2 mg o f MA-containing polymer was injected; B o t h - O - and - D - : 2.8 mg o f M A containing polymer was injected. ο Is

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Reaction Time (hr) Figure 6. Chymotryptic hydrolysis of polymeric prodrugs in the presence o f lysozyme (5,000 unit/ml). O; CM-chitin-Phe-pNA, Δ ; CM-chitin-Gly-Phe-pNA, · ; CM-chitin-Ala-Phe-pNA, • ; CM-chitin-Abu-Phe-pNA.


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average molecular weight ( M W ) of oligomeric prodrugs, which might correspond to the steric hindrance of CM-chitin backbone. The release of chromophore from C M chitin conjugate with a spacer of -Ala-Phe-(CM-chitin-Ala-Phe-pNA) was not clearly shown i n the range of 4,000 to 50,000 of molecular weight, but a significant increase of the rate of α-chymotryptic hydrolysis was observed by the reduction of the M W to less than 4,000. This suggests that the susceptibility of polymeric substrates ( M W = 4,000-50,000) for α-chymotrypsin is significantly affected by the molecular conformation of polymer chain, but the susceptibility of oligomeric prodrugs of molecular weight less than 4000 to α-chymotrypsin seems to be similar to that of low molecular weight prodrugs, because the spacer-chromophore bond might no longer be protected by the CM-chitin molecule. O n the other hand, i n case of CM-chitin-AbuPhe-pNA, there is no significant change of v i n the M W range of 2,000-50,000. The independence of v for CM-chitin-Abu-Phe-pNA seems to reflect the lower substrate specificity of the side chain for chymotryptic hydrolysis, i.e. the smaller the carrier chain, the higher the value of L or k JK for CM-chitin-Ala-Phe-pNA but not for C M - c h i t i n - A b u - P h e - p N A . It is l i k e l y that chymotrypsin recognizes the natural occurring A l a residue more readily than A b u residue. The results of CM-chitin-AlaPhe-pNA and CM-chitin-Abu-Phe-pNA shows that both a difference i n the structure of spacer and the molecular weight of drug carrier affects the α-chymotryptic hydrolysis. In other words, the structure of spacer is one of the factors that can be used to control the rate of drug release. 0

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Entrapment of Drugs into CM-chitin Gel Formed by Ferric Ion Since the addition of iron (III) chloride into CM-chitin solution induces gel formation, the anticancer agent doxorubicin or protein was incorporated into the C M - c h i t i n gel during the gelation with iron (III) ion (2). In the present study, the peptidyl anticancer agent, neocarzinostatin (NCS), was incorporated into the gel and the release of N C S from the gel was investigated both in vitro and in vivo. Preparation of C M - c h i t i n Gel Containing N C S . CM-chitin gel was prepared by the method described previously (8). When calcium and ferric ion were added to the mixture of CM-chitin and N C S , more than 8 0 % gel formation was observed. The use of calcium ion i n addition to the ferric ion increased the amount of incorporation of N C S . Thus, a final concentration of 50 m M calcium chloride and 30 m M of iron (III) chloride was used for the preparation of NCS-containing gel with more than 5 0 % recovery of N C S . The average size of the gel obtained was smaller than human platelets (2-3 μπι i n diameter) and showed a uniform distribution i n gel size. The average size of a gel particle was estimated to be 0.70 μπι by a Coulter Multisizer. Release of N C S from the gel by lysozymic hydrolysis. CM-chitin gel containing N C S was incubated with 500 or 5,000 unit/ml of lysozyme (Figure 8). The release of N C S resulting from lysozymic digestion of the gel was observed i n the presence of 500 or 5,000 unit/ml of lysozyme i n a time- and dose-dependent manner. O n day 7 after the incubation, about 8 0 % of N C S incorporated was released by the exposure to 500 unit/ml of lysozyme, which is approximately equivalent to the physiological concentration in the human plasma. A spontaneous release of N C S from the gel, approximately 5 0 % during four days, was also observed when the gel was incubated without lysozyme. However, when 500 unit/ml of lysozyme was added fresh on day 4, as indicated by an arrow i n Figure 8, about 2 0 % of N C S incorporated was additionally released from the gel during the last 3 days. Release of N C S from the G e l in vivo. The N C S level i n plasma after subcutaneous (s.c.) administration of C M - c h i t i n gel containing N C S into mice was investigated to examine the potential of CM-chitin gel for sustained release of drugs.


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Figure 7. Dependence of the rate of α-chymotryptic hydrolysis (v ) on the number-average molecular weight of polymeric prodrugs (MW); · : CM-chitinAia-Phe-pNA, O: CM-chitin-Abu-Phe-pNA. 0

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Figure 8. Release of I-labeled N C S from CM-chitin gel by lysozyme digestion. CM-chitin gel containing I-labeled N C S (1.5 x 1 0 cpm/15 μ% NCS/45 μ% gel) was incubated without (O) or with lysozyme of 500 unit/ml ( · ) or 5,000 unit/ml ( Δ ) . A r r o w ; see text. 125

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Figure 9 shows the plasma concentration of N C S and demonstrates that the clearance of N C S from the circulation was very rapid over a 6 h period after the s.c. injection of N C S alone. The N C S concentration i n plasma was calculated from the standard curve by the growth inhibition of B 1 6 - B L 6 melanoma cells in vitro. A t 24 h after the injection of N C S alone, N C S i n the plasma was undetectable (less than 1 ng/ml) as assessed by the growth inhibition of B 1 6 - B L 6 melanoma cells by the plasma. Whereas, the N C S containing gel gave a much smaller maximum plasma level at 6 h after the s.c. injection and N C S was gradually cleared from the circulation. N C S i n the plasma was still detectable 48 h after the injection.


Stabilization of Peptide Drug by Adsorption to CM-chitin Since a biodegradable CM-chitin has shown ability to adsorb neutral amino acids or peptides, especially phenylalanine (Phe), when calcium ion was chelated onto C M chitin (9), stabilization of substances containing a hydrophobic moiety or Phe by C M chitin would be expected. Thus, the stabilization of a biologically active peptide to enzymatic degradation through the adsorption on CM-chitin was studied in vitro for the sustained release of biologically active or drug-bound peptides. The chromophore-bearing model peptide; enkephalin analogue ( H C l H - T y r - G l y - G l y - P h e - p N A ; [pNA]-Enk), was synthesized by the liquid phase peptide synthesis and the susceptibility of peptides adsorbed on C M chitin to proteolytic hydrolysis was investigated to isolate the effect of CM-chitin on the stabilization of peptide from degradation. cc-Chymotrypsin-catalyzed H y d r o l y s i s of Peptide A d s o r b e d on C M chitin. The peptide, [pNA]-Enk, was used to investigate the entrapping effect of C M chitin on α-chymotryptic degradation. Profiles of α-chymotryptic hydrolysis of [pNA]-Enk (0.48 m M ) preincubated with or without CM-chitin were shown i n Figure 10 as time-dependent curves. p-Nitroaniline liberation was monitored at 410 nm. The rates of α-chymotryptic hydrolyses were influenced by CM-chitin concentration and the depression was maintained for a fairly long period i n the absence of lysozyme probably due to the entrapping effect of CM-chitin because no marked difference i n occhymotrypsin-catalyzed hydrolysis of peptide was observed in the presence of lower concentration of CM-chitin. The C M - c h i t i n - C a complex data showed any influence on the α-chymotryptic hydrolysis when the peptide was not pretreated with CM-chitin prior to an exposure to the α-chymotrypsin. Thus, the depression of α-chymotryptic hydrolysis seems to be due to the decrease of free substrate by the adsorption onto C M c h i t i n - C a complex. It seems that the peptides pretreated with CM-chitin were not bared to the proteolytic hydrolysis. The prevention of proteolytic hydrolysis through the adsorption on CM-chitin might have potential to stabilize the biologically active peptides and sustain the release of them followed by lysozymic hydrolysis of C M chitin backbone. 2+

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Conclusion Several types of drug delivery system was studied using CM-chitin as a carrier. In each case, the potential use of biodegradable and water soluble CM-chitin was indicated to be useful as the drug carrier of sustained release. Pendant and entrapping (including adsorption) type of drug delivery seems to fit for the sustained release of low molecular weight drugs. Although ferric ion entrapping seems to be favorable for the molecule of medium size, effective entrapping of drug of high molecular weight, such as proteins, was achieved by the use of ferric and calcium ions. The biodegradability of CM-chitin would contribute to effective sustained release of drug i n each case.


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Figure 9. Plasma concentration of N C S after the s.c. administration of N C S alone or NCS-containing CM-chitin gel. Mice were administered s.c. with N C S (10μg) (O) or CM-chitin gel containing N C S ( 10/

Drug Delivery System Using Biodegradable Carrier - American

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