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Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N
Thiolation of Polycarbophil Enhances its Inhibition of Intestinal Brush Border Membrane Bound Aminopeptidase N È RCH, HUDA ZARTI, GREG F. WALKER ANDREAS BERNKOP-SCHNU Center of Pharmacy, Institute of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria Received 19 March 2001; revised 24 June 2001; accepted 11 July 2001
ABSTRACT: The purpose of this study was to evaluate the potential of polycarbophil± cysteine conjugates (PCP±Cys) as an oral excipient to protect leucine enkephalin (leuenkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 mM (pH 6) signi®cantly (p < 0.05) inhibited aminopeptidase N activity, and PCP±Cys (0.25% w/v, pH 6) had a signi®cantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP±Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 4 and 60 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP± Cys (0.25% w/v, pH 6) 11 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1907±1914, 2001
Keywords: inhibition
polycarbophil; thiomers; aminopeptidase N; peptide delivery; enzyme
INTRODUCTION Oral bioavailability of peptide and protein drugs is generally very low (< 1.0%) because of the extensive luminal and mucosal proteolytic activity of the gut and the low permeability of this class of molecules through the intestinal epithelium.1 To overcome this metabolic barrier, a number of investigators have co-administered enzyme inhibitors with the peptide drug, improving its Correspondence to: A. Bernkop-SchnuÈrch (Telephone: 43 1 4277 55413; Fax: 43 1 4277 9554; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 1907±1914 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
stability against proteolytic attack and thereby increasing the possibility to reach the systemic circulation intact. A broad range of peptidases can be inhibited by chelating essential metal ions from the enzyme structure, resulting in protein denaturation and loss of activity.2±4 Metal ions can be chelated from the enzyme structure by the so-called ``multi-functional'' mucoadhesive polymers of acrylic acid, which are comparatively cheap and nontoxic excipients used for oral drug delivery. More recently, a new class of mucoadhesive polymers called thiomers, in which cysteine is covalently linked to the mucoadhesive polymer, have been developed.5 Thiomers of poly(acrylic) acid were shown to have greater af®nities for zinc
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ions in comparison to the unmodi®ed polymer.6 Subsequently, the polymer±cysteine conjugates had greater inhibitory activity, compared with the unconjugated polymer, towards the zinc requiring carboxypeptidase A and B.6 Thiolated polymers offer additional advantages for oral peptide delivery, including enhanced mucoadhesive and cohesive strengths5 and intestinal permeation enhancement of peptides.7 Because a large proportion of the peptidases associated with the brush-border membranes are metallopeptidases, requiring divalent metal ions for their activity,1 thiolated polymers should provide protection for peptides that are susceptible to such activity. Dodda-Kashi and Lee8 demonstrated that aminopeptidases appear to play a major role in the digestion of the enkephalins, and dipeptidyl carboxypeptidase and dipeptidyl peptidase, also known as enkephalinase A and B, respectively, play a lesser role in the digestion. These enzymes involved in the digestion of enkephalins are all metallopeptidases and therefore potentially can be inhibited by the chelator activity of polycarbophil±cysteine conjugates (PCP±Cys). The aim of this study was to determine the inhibitory activity of thiolated PCP towards isolated aminopeptidase N (EC 3.4.11.2) and the aminopeptidases present on the intestinal mucosa surface and to determine its protective effect for leu-enkp. The inhibitory activity of the polymers towards aminopeptidase N and pig intestinal mucosal enzymes was measured using the synthetic aminopeptidase substrate leu-pNA and the peptide leu-enkp. For high molecular weight polymers it is expected that the mucus layer covering the mucosal membrane-bound peptidases will limit the accessibility of polymer to these peptidases for chelation of their divalent ions. To determine this potential barrier, polymer peptidase inhibition was tested on intact intestinal mucosal membranes with native mucus excised from pig. We discuss the potential of thiolated PCP for the enhanced oral delivery of peptides susceptible to the activity of metallopeptidases.
EXPERIMENTAL SECTION Materials Polycarbophil (PCP) was a gift from BF Goodrich (Cleavland, OH). Aminopeptidase N (EC 3.4.11.2, 12 U/mg of solid), L-leucine p-nitroanilide
(Leu-pNA), leucine enkephalin (leu-enkp), p-nitroaniline, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), and L-cysteine were all obtained from Sigma Chemical Company (St. Louis, MO). For chromatography, HPLC grade solvents were used. All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany). Synthesis of Polycarbophil±Cysteine Conjugates The PCP±Cys conjugate was synthesized according to the method previously described by our research group.9 In brief, the covalent attachment of cysteine to neutralized PCP was achieved by the formation of amide bonds between the primary amino group of cysteine and the carboxylic acid group of the polymer. PCP (10g) was hydrated in 1 L of deionized water. The carboxylic acid moieties of the polymer were activated by EDAC (50 mM) coupling. The pH of the reaction mixture was adjusted to 6 by the addition of 1 M HCl, and L-cysteine was added to give a weight ratio of 2:1 (polymer±cysteine). The pH of the reaction mixture was maintained at 6 over the 3-h incubation period, with constant mixing at room temperature. The resulting polymer±cysteine conjugate was dialyzed in the dark at 108C, to avoid oxidation of the cysteine moieties. Polymers were dialyzed once against 1 mM HCl, two times against the same medium also containing 1% NaCl,and then exhaustively against 0.5 mM HCl. Control polymer was prepared and isolated in the same way as the polymer±cysteine conjugate but EDAC was omitted during the coupling reaction. Samples were lyophilized by drying frozen aqueous polymer solutions at ÿ 308C at 0.01 mbar (Christ Beta 1-8K; Osterode am Harz, Germany). The polymer±cysteine conjugate and control were stored at 48C until further use. Determination of the Thiol Group Content The degree of modi®cation was determined by quantifying the amount of thiol moieties on the polymer by iodometric titration at pH 3.0 (1 mM iodine, indicator, starch).9 Excised Pig Gut Mucosa The ®rst 70 cm of the small intestine (duodenum) of pig was excised immediately after sacri®cing the animal, and the luminal contents were removed by gentle washing with deionized water.
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The intestinal section was cut into 10-cm lengths and stored in saline at ÿ 208C. Metabolism by Aminopeptidase N Enzyme activity of puri®ed aminopeptidase N was determined against leu-pNA and leu-enkp in 50 mM sodium phosphate buffer (pH 6.0) containing 2 % NaCl. Aminopeptidase N was dissolved in 50 mM phosphate buffer (pH 6.0) to give a ®nal concentration of 62.5 mg mLÿ1. Polymer (12.5 mg) was hydrated in 4.9 mL of 50 mM phosphate buffer (pH 6.0) and equilibrated at 378C. Prior to the inhibition assay, the pH of the polymer solution was determined and adjusted to pH 6 with 1 M NaOH, and 50 mM phosphate buffer (pH 6.0) was added to give a total volume 5 mL. The enzyme stock solution (10 mL) was mixed with either 1 mL of the polymer solution or 1 mL of phosphate buffer (pH 6.0) and incubated at 378C for 30 min. For aminopeptidase N activity towards leu-pNA, 0.25 mL of the incubate was transferred to a microtitre plate and 50 mL of the substrate leu-pNA was added to give a ®nal concentration of 1.0 mM. The release of pnitroaniline was measured every 1 min at room temperature by the increase in absorbance at 405 nm using a Anthos reader.2001 (Anthos Labtec Instruments, Austria). Activity of the incubate towards leu-enkp was determined by the addition of 50 mL of leu-enkp, prepared in 50 mM phosphate buffer (pH 6.0), giving a ®nal substrate concentration of 0.1 mM. The incubates were maintained at 378C. Samples were taken at predetermined times and added to 20 mL of 20% (v/v) tri¯uoroacetic acid (TFA) to stop the reaction. Preliminary studies showed that the concentration of TFA used was suf®cient to precipitate the polymer and proteins entirely (data not shown). The resulting mixture was centrifuged (20000g for 5 min), and the supernatants were analyzed by HPLC.
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reaction cylinder. After 30 min of incubation, 1 mL of the either leu-pNA (2 mM) or leu-enkp (0.2 mM) prepared in control buffer at 378C was added to the incubate. At various times after the start of the reaction, samples of 300 mL were taken. LeupNA samples were placed on ice and centrifuged at 20000g at 48C for 5 min, a 200-mL sample of the supernatant was transferred to a microtitre plate, and the absorbance was measured at 405 nm using a Anthos reader.2001 (Anthos Labtec Instruments, Austria). Leu-enkp samples were added to 20 mL of 20% (v/v) TFA to stop the reaction. The resulting mixture was centrifuged (20000g) for 5 min, and the supernatants were analyzed by HPLC. HPLC of Leucine-Enkephalin Analysis of leu-enkp reaction samples by reversed-phase HPLC was conducted using a Perkin-Elmer (Norwalk, CT) series 200 LC pump, Perkin-Elmer 200 series auto sampler with a 20-mL injection loop, and a diode array detector (Perkin-Elmer 235C). Samples were eluted from a Nucleosil 100 ± 5 C18 column (250 4 mm), with mobile phases of (A) 0.1% TFA and (B) 90% acetonitrile, 0.1% TFA at a ¯ow rate of 1.0 mL minÿ1. Detection was at 220 nm. A linear gradient was applied from 90% A to 10% A over a 22 min period. Preliminary studies showed that the concentration of TFA used was suf®cient to precipitate the polymer and proteins (data not shown). Statistical Analysis To determine signi®cance of enzyme inhibition, statistical analysis was performed using a nonpaired Students t test, and a p value of 0.05 or less was considered to be signi®cant.
RESULTS
Metabolism by Intact Pig Intestinal Mucosa A plastic cylinder with an internal surface area of 1.77 cm2 was placed vertically on top of the mucosal side of thawed intestinal tissue and clamped, as described previously.10 Polymers were prepared in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl as described for the aminopeptidase N inhibition studies. Control buffer (1 mL) with or without inhibitor equilibrated at 378C was added into the
Free Cysteine Studies Preliminary studies demonstrated the high af®nity of cysteine for zinc ions. The addition of Lcysteine to an aqueous 1 M ZnCl2 solution, for instance, resulted in precipitation. Hence, Lcysteine may also interact with the zinc ion of aminopeptidase N, consequently leading to the inhibition of this peptidase. To verify this hypothesis, enzyme inhibition studies were carried out
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Figure 1. Time-course of the formation of p-nitroaniline from leu-p-nitroanilide by aminopeptidase N after incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing 0.63 mM L-cysteine (~), control buffer containing 6.3 mM L-cysteine (*), and control buffer containing 63 mM L-cysteine (~). Each point represents the mean SD (n 3).
with isolated aminopeptidase N in the presence of increasing concentrations of free L-cysteine. The results of the following inhibition studies with free cysteine are shown in Figure 1. The higher the cysteine concentration, the higher the inhibitory effect towards aminopeptidase N; L-cysteine at 0.63 mM did not signi®cantly inhibit aminopetidase N activity, however, at 6.3 and 63 mM, there was signi®cant inhibition of enzyme activity. Additionally, inhibition studies performed with brush border membrane-bound aminopeptidase N also demonstrated an inhibitory effect of Lcysteine. Results, as shown in Figure 2, however, revealed that comparatively higher concentrations of cysteine were required to achieve the same inhibitory effect. Under similar assay conditions, the aminopeptidase N activity of the intact mucosa was only signi®cantly inhibited by L-cysteine at the highest L-cysteine concentration of 63 mM. Polymer Inhibition of Aminopeptidase N To compare the inhibitory effect of PCP and thiolated PCP towards aminopeptidase N, ®rst
Figure 2. Time-course of the formation of p-nitroaniline from leu-p-nitroanilide by intact mucosa after incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing 0.63 mM L-cysteine (~), control buffer containing 6.3 mM L-cysteine (*), and control buffer containing 63 mM L-cysteine (~). Each point represents the mean SD (n 3).
of all, a PCP±Cys conjugate was synthesised and characterized. Iodometric titration of the isolated PCP±Cys conjugate showed that 148 mmol 42 (mean SD; n 3) of sulfhydryl groups were conjugated per gram of polymer. Negligible amounts of sulfhydryl groups were detected for the control polymer, where the coupling reagent EDAC was omitted. The level of cysteine coupling and the features of the modi®ed polymer were consistent with previous preparations by our group.5 Following the incubation of aminopeptidase N in the presence of either PCP or cysteine-conjugated PCP at 0.25% (w/v), pH 6.0, the amount of leu-pNA hydrolyzed was signi®cantly reduced compared with the buffer only control (Figure 3). The polymer conjugate demonstrated a much stronger inhibitory effect than the unconjugated polymer, almost completely inhibiting the hydrolysis of leu-pNA. Under similar assay conditions for leu-pNA, the polymers were tested for their ability to protect the peptide leu-enkp from the hydrolytic activity of aminopeptidase N. For leupNA, both conjugated and unconjugated polycarbophil signi®cantly improved the stability of the
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Figure 3. Time-course of the formation of p-nitroaniline from leu-p-nitroanilide by aminopeptidase N after incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing (0.25% w/v) polycarbophil (~), and in control buffer containing (0.25% w/v) cysteine-conjugated polycarbophil (*). Each point represents the mean SD (n 3).
Figure 4. Time-course of the percentage remaining of leu-enkephalin in the presence of aminopeptidase N after the incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing (0.25% w/v) polycarbophil (~), and in control buffer containing (0.25% w/v) cysteineconjugated polycarbophil (*). Each point represents the mean SD (n 3).
substrate (Figure 4). Furthermore, the thiolated polymer had a signi®cantly greater inhibitory effect compared with the unconjugated polymer.
nilide was slightly lower than for the fresh intestine.10 Both conjugated and unconjugated polymer signi®cantly inhibited the hydrolysis of leu-pNA by aminopeptidases present on the intact intestinal mucosa (Figure 5). Thiolated PCP was thereby signi®cantly more effective than unconjugated polymer at protecting leu-pNA from enzymatic hydrolysis. In comparison to the studies with soluble aminopeptidase N, as shown in Figure 3, both polymers appear to be less effective at inhibiting the aminopeptidase N activity of the intact intestinal mucosa. The degradation of leu-enkp by intact intestinal mucosa and in the presence of conjugated or unconjugated PCP is shown in Figure 6. The HPLC spectrum of the degradation products of leu-enkp obtained by incubation with intestinal mucosa was different from that obtained for the isolated enzyme (data not shown). This result suggests that there are also other enzymes on the mucosa involved in the degradation of the peptide drug. Hydrolysis of leu-enkp was very rapid, the peptide was almost completely degraded after
Polymer Inhibition of Intact Intestinal Mucosa The inhibitory effect of the polymers was tested on the structurally more relevant intact intestinal mucosa of pig with native mucus. Microscopic investigations of the mucosa showed that a mucus gel layer was still covering the tissue after rinsing it with buffer solution. A preliminary histological study indicated that the damage to the mucosal surface from freezing and thawing was minimal and a layer of mucus was present on the mucus surface.10,11 Studies have shown that the storage of the pig intestine at ÿ208C does not in¯uence the structure of the mucus.12 It was shown that ionic strength, NaCl, at 1.2 and 11%, had no effect on the structure of native mucus from the stomach and the duodenum.13 Furthermore, studies demonstrated that the hydrolytic activity of the frozen pig intestine towards L-leucine±p-nitroa-
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Figure 5. Time-course of the formation of p-nitroaniline from leu-p-nitroanilide by intact mucosa after incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing (0.25% w/v) polycarbophil (~), and in control buffer containing (0.25% w/v) cysteine-conjugated polycarbophil (*). Each point represents the mean SD (n 3).
Figure 6. Time-course of percentage remaining of leu-enkephalin by intact mucosa after incubation control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing (0.25% w/v) polycarbophil (~), and in control buffer containing (0.25% w/v) cysteine-conjugated polycarbophil (*). Each point represents the mean SD (n 3).
30 min. In the presence of unconjugated polymer, a signi®cant amount (12.3 4 %) of the peptide was remaining after 60 min. However, no peptide was detected after 90 min, whereas for the polymer±cysteine conjugate, a signi®cant amount (11 3.5%) of the peptide was remaining after 90 min.
aminopeptidase N. This inhibition could be due to either the cysteine competitively binding to Zn2 in the active site of the enzyme, the cysteine acting as a divalent ion chelator or a combination of both mechanisms. Free cysteine was less effective in inhibiting the enzymatic activity on the intact intestinal mucosa. The lower potency of free cysteine may be due to thiol/disul®de exchange reactions with the native mucin present on the mucosa surface, resulting in the cysteine moiety being covalently bound to the mucin.5 Iodometric titration showed that 148 mmol 42 (mean SD; n 3) of cysteine was conjugated to 1g of PCP in the presence of EDAC. For the inhibitory studies, polymers were hydrated in buffer at pH 6 to give a polymer concentration of 0.25% (w/v; i.e., a cysteine concentration of 370 mM). In experiments with free cysteine (Figures 1 and 2), the reaction kinetics are different than those in which cysteine is immobilized onto the polymer (Figures 3 and 4). The greater inhibitory activity of the free cysteine is most likely due to its greater diffusion in comparison to the immobilized cysteine. However,
DISCUSSION The results demonstrate that thiolation of PCP enhances the inhibitory potency of PCP towards aminopeptidase N and membrane-bound peptidase(s) involved in the digestion of leu-pNA and leu-enkp. It is well established for aminopeptidase N that the complexation of the divalent ion Zn2 within the enzyme structure is essential for its activity.14 Studies with the strong divalent ion chelator EDTA strongly inhibited the activity of aminopeptidase N,14 whereas the comparatively weak divalent ion chelator PCP was a poor inhibitor of aminopeptidase N activity.4 Figures 1 and 2 show that free cysteine at 6.3 and 63 mM signi®cantly inhibited the activity of soluble
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in vivo this greater diffusion will mean that the excipient does not remain localized with the dosage form. Figures 3±6 show that conjugation of cysteine to PCP improves the enzyme inhibition in comparison to the unconjugated PCP. Because cysteine is attached to a high molecular weight cross-linked polymer it is unlikely that the conjugated cysteine will enter the active site of the enzyme. Therefore, it is likely that the mechanism of enzyme inhibition by the conjugated polymer is by chelation of the Zn2 from the protein structure. This mechanism is supported by the observation that the thiolated PCP has a greater af®nity for Zn26 and a corresponding greater inhibitory potency towards aminopeptidase N activity (Figures 3 and 4) in comparison with the control polymer. An enhanced stability of leu-enkp based on interactions of the peptide with the thiolated polymer, as shown for other peptides,6 can thereby be excluded because leu-enkp doesn't exhibit any thiol moieties. These studies showed that both free and conjugated cysteine had a lower inhibitory effect on the intact mucosa in comparison to aminopeptidase N. Due to cell lysis, it is possible that the activity observed on the mucosa is of cytosolic origin. This activity, however, is expected to be low in comparison with the membrane peptidase activity because 80% of the aminopeptidase activity of the ileal mucosa is membrane bound.15 The lower inhibitory activity in the presence of the mucus layer suggests it is acting as a barrier to the polymers ability to chelate metal ions from the surface membrane-bound peptidases. Previous studies have shown that the intestinal mucous layer is a barrier to permeation enhancing effect of the mucoadhesive polymer chitosan.16 For the thiolated polymer, the mucus layer could be limiting by the physical interaction between thiolated polymer and the intestinal mucus or by the diffusion of the polymer through the mucus. Indeed interaction between the thiolated polymer and the mucus could occur by the ability of cysteine to act as a mucolytic agent.17,18 This process involves disul®de exchange reactions between the cysteine moiety and the mucin glycoproteins, resulting in the formation of disul®de bonds between the cysteine and the glycoprotein.17 As a result of these disul®de exchange reactions, the concentration of free thiol groups for chelation is reduced. These disul®de exchange reactions have been postulated for the enhanced
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mucoadhesive interaction observed between the thiolated polymers and the intestinal mucus.5 The lower inhibitory activity of both thiolated and unthiolated PCP on intact mucosa may also be explained by inability of chelators to completely inhibit all intestinal membrane-bound peptidase activity towards enkephalin. Inhibition studies on the stability of methionine enkephalin in nasal, rectal, and vaginal mucosal homogenates from rabbit showed that although EDTA alone gave the strongest inhibitory activity, a combination of amastatin, thimerosal, and EDTA was required to almost completely stabilize methionine enkephalin.19 Therefore, inhibition by chelation is not suf®cient to inhibit all intestinal membrane-bound peptidases involved in the digestion of enkephalins. Furthermore, dosage forms of thiolated polymers should maintain their inhibitory effect at the site of peptide delivery. Studies by Chun and Chien19 suggested that high concentrations of the inhibitors amastatin, thimerosal, and EDTA are required to protect enkephalins from proteolytic attack in vitro. These concentrations would be dif®cult to maintain in vivo. A drug delivery system of the thiolated polymer described here would not be affected by inhibitor dilution and would offer several additional formulation advantages. The peptide drug would be localized with the inhibitory activity of the cysteine conjugated polymer.2 The higher mucoadhesive properties of the thiolated polymer5 would provide (i) prolonged residence time with the absorbing intestinal mucosal membrane, (ii) increased contact to the absorbing mucosa resulting in a steep concentration gradient to favor drug absorption, and (iii) reduce the diffusional distance for the peptide drug to reach the absorbing membrane, reducing the opportunity for metabolism by luminal proteases and localize the inhibitory activity of the polymer close to the membrane bound peptidases. In conclusion, the thiolation of PCP increases the inhibitory potency of PCP towards aminopeptidase N and intestinal membrane-bound peptidases, increasing the stability of leu-enkp. Thereby another mechanism was identi®ed by which PCP±Cys can improve the oral bioavailability of peptide drugs. Because similar peptidase activities towards enkephalins have been identi®ed in nasal, rectal, vaginal, and buccal mucosal homogenates,8 drug delivery systems of thiolated PCP should also provide enzymatic protection at these mucosal sites. Furthermore, because there are a large number of peptide drugs
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susceptible to intestinal luminal and membranebound metallopeptidases,1 thiolated PCP drug delivery systems should also provide protection towards enzymatic attack for a wide range of peptide drugs. It has also been shown that the thiolation of PCP increases the transport of model peptide drugs across the intestine. The enhanced transport is believed to be due to the opening of the tight junctions.7 The mucus layer is expected to act as a diffusional barrier, preventing the polymer from interacting with the epithelial surface, thereby reducing its enzyme inhibitory and permeation enhancing effect. This barrier, however, may be overcome in vivo because of to the higher effective concentration of the polymer conjugate when delivered in an appropriate drug delivery system.
ACKNOWLEDGMENTS This work was supported by a grant from the Fonds zur FoÈrderung der wissenschaftlichen Forschung (FWF) to A. Bernkop-SchnuÈrch.
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polypeptide delivery systems. J Pharm Sci 89:901± 909. Clausen AE, Bernkop-SchnuÈrch A. 2000. In vitro evaluation of the permeation-enhancing effect of thiolated polycarbophil. J Pharm Sci 89:1253± 1261. Dodda-Kashi SD, Lee VHL. 1986. Enkephalin hydrolysis in homogenates of various absorptive mucosae of the albino rabbit: Similarities in rates and involvement of aminopeptidases. Life Sci 38: 2019±2028. Bernkop-SchnuÈrch A, Steininger S. 2000. Synthesis and characterization of mucoadhesive thiolated polymers. Int J Pharm 25:239±247. Bernkop-SchnuÈrch A, Paikl C, Valenta C. 1997. Novel bioadhesive chitosan-EDTA conjugate protects leucine enkephalin from degradation by aminopeptidase N. Pharm Res 14:917±922. Mortazavi SA, Smart JD. 1995. An investigation of some factors in¯uencing the in vitro assessment of mucoadhesion. Int J Pharm 116:223±230. Matthes I, Nimmerfall F, Sucker H. 1992. Mucus models for investigation of intestinal absorption mechanisms. 1. Validation and optimization of the model. Pharmazie 47:505±515. Bell AE, Sellers LA, Allen A, Cunliffe WJ, Morris ER, Ross-Murphy SB. 1985. Properties of gastric and duodenal mucus: Effect of proteolysis, disul®de reduction, bile acid, ethanol, and hypertonicity on mucus gel structure. Gastroenterology 88:269± 280. Garner CW, Behal FJ. 1974. Human liver aminopeptidase. Role of metal ions in mechanism of action. Biochemistry 13:3227±3233. Lee VHL, Yamamoto A. 1990. Penetration and enzymatic barriers to peptide and protein absorption. Adv Drug Del Rev 4:171±207. Schipper NG, Varum KM, Stenberg P, Lennernas G, Art H. 1999. Chitosans as absorption enhancers of poorly absorbable drugs. 3: In¯uence of mucus on absorption enhancement. Eur J Pharm Sci 8:335± 343. Snyder GH, Reddy MK, Cennerazzo MJ, Field D. 1983. Use of local electrostatic environments of cysteine to enhance formation of a desired species in a reversible disul®de exchange reaction. Biochim Biophys Acta 749:219±226. Snyder GH. 1987. Intramolecular disul®de loop formation in a peptide containing two cysteines. Biochemistry 26:688±694. Chun IK, Chien YW. 1995. Stabilization of methionine enkephalin in various rabbit mucosal extracts by enzyme inhibitors. Int J Pharm 121:217±231.
ABSTRACT: The purpose of this study was to evaluate the potential of polycarbophil± cysteine conjugates (PCP±Cys) as an oral excipient to protect leucine enkephalin (leuenkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 mM (pH 6) signi®cantly (p < 0.05) inhibited aminopeptidase N activity, and PCP±Cys (0.25% w/v, pH 6) had a signi®cantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP±Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 4 and 60 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP± Cys (0.25% w/v, pH 6) 11 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1907±1914, 2001
Keywords: inhibition
polycarbophil; thiomers; aminopeptidase N; peptide delivery; enzyme
INTRODUCTION Oral bioavailability of peptide and protein drugs is generally very low (< 1.0%) because of the extensive luminal and mucosal proteolytic activity of the gut and the low permeability of this class of molecules through the intestinal epithelium.1 To overcome this metabolic barrier, a number of investigators have co-administered enzyme inhibitors with the peptide drug, improving its Correspondence to: A. Bernkop-SchnuÈrch (Telephone: 43 1 4277 55413; Fax: 43 1 4277 9554; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 1907±1914 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
stability against proteolytic attack and thereby increasing the possibility to reach the systemic circulation intact. A broad range of peptidases can be inhibited by chelating essential metal ions from the enzyme structure, resulting in protein denaturation and loss of activity.2±4 Metal ions can be chelated from the enzyme structure by the so-called ``multi-functional'' mucoadhesive polymers of acrylic acid, which are comparatively cheap and nontoxic excipients used for oral drug delivery. More recently, a new class of mucoadhesive polymers called thiomers, in which cysteine is covalently linked to the mucoadhesive polymer, have been developed.5 Thiomers of poly(acrylic) acid were shown to have greater af®nities for zinc
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ions in comparison to the unmodi®ed polymer.6 Subsequently, the polymer±cysteine conjugates had greater inhibitory activity, compared with the unconjugated polymer, towards the zinc requiring carboxypeptidase A and B.6 Thiolated polymers offer additional advantages for oral peptide delivery, including enhanced mucoadhesive and cohesive strengths5 and intestinal permeation enhancement of peptides.7 Because a large proportion of the peptidases associated with the brush-border membranes are metallopeptidases, requiring divalent metal ions for their activity,1 thiolated polymers should provide protection for peptides that are susceptible to such activity. Dodda-Kashi and Lee8 demonstrated that aminopeptidases appear to play a major role in the digestion of the enkephalins, and dipeptidyl carboxypeptidase and dipeptidyl peptidase, also known as enkephalinase A and B, respectively, play a lesser role in the digestion. These enzymes involved in the digestion of enkephalins are all metallopeptidases and therefore potentially can be inhibited by the chelator activity of polycarbophil±cysteine conjugates (PCP±Cys). The aim of this study was to determine the inhibitory activity of thiolated PCP towards isolated aminopeptidase N (EC 3.4.11.2) and the aminopeptidases present on the intestinal mucosa surface and to determine its protective effect for leu-enkp. The inhibitory activity of the polymers towards aminopeptidase N and pig intestinal mucosal enzymes was measured using the synthetic aminopeptidase substrate leu-pNA and the peptide leu-enkp. For high molecular weight polymers it is expected that the mucus layer covering the mucosal membrane-bound peptidases will limit the accessibility of polymer to these peptidases for chelation of their divalent ions. To determine this potential barrier, polymer peptidase inhibition was tested on intact intestinal mucosal membranes with native mucus excised from pig. We discuss the potential of thiolated PCP for the enhanced oral delivery of peptides susceptible to the activity of metallopeptidases.
EXPERIMENTAL SECTION Materials Polycarbophil (PCP) was a gift from BF Goodrich (Cleavland, OH). Aminopeptidase N (EC 3.4.11.2, 12 U/mg of solid), L-leucine p-nitroanilide
(Leu-pNA), leucine enkephalin (leu-enkp), p-nitroaniline, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), and L-cysteine were all obtained from Sigma Chemical Company (St. Louis, MO). For chromatography, HPLC grade solvents were used. All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany). Synthesis of Polycarbophil±Cysteine Conjugates The PCP±Cys conjugate was synthesized according to the method previously described by our research group.9 In brief, the covalent attachment of cysteine to neutralized PCP was achieved by the formation of amide bonds between the primary amino group of cysteine and the carboxylic acid group of the polymer. PCP (10g) was hydrated in 1 L of deionized water. The carboxylic acid moieties of the polymer were activated by EDAC (50 mM) coupling. The pH of the reaction mixture was adjusted to 6 by the addition of 1 M HCl, and L-cysteine was added to give a weight ratio of 2:1 (polymer±cysteine). The pH of the reaction mixture was maintained at 6 over the 3-h incubation period, with constant mixing at room temperature. The resulting polymer±cysteine conjugate was dialyzed in the dark at 108C, to avoid oxidation of the cysteine moieties. Polymers were dialyzed once against 1 mM HCl, two times against the same medium also containing 1% NaCl,and then exhaustively against 0.5 mM HCl. Control polymer was prepared and isolated in the same way as the polymer±cysteine conjugate but EDAC was omitted during the coupling reaction. Samples were lyophilized by drying frozen aqueous polymer solutions at ÿ 308C at 0.01 mbar (Christ Beta 1-8K; Osterode am Harz, Germany). The polymer±cysteine conjugate and control were stored at 48C until further use. Determination of the Thiol Group Content The degree of modi®cation was determined by quantifying the amount of thiol moieties on the polymer by iodometric titration at pH 3.0 (1 mM iodine, indicator, starch).9 Excised Pig Gut Mucosa The ®rst 70 cm of the small intestine (duodenum) of pig was excised immediately after sacri®cing the animal, and the luminal contents were removed by gentle washing with deionized water.
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The intestinal section was cut into 10-cm lengths and stored in saline at ÿ 208C. Metabolism by Aminopeptidase N Enzyme activity of puri®ed aminopeptidase N was determined against leu-pNA and leu-enkp in 50 mM sodium phosphate buffer (pH 6.0) containing 2 % NaCl. Aminopeptidase N was dissolved in 50 mM phosphate buffer (pH 6.0) to give a ®nal concentration of 62.5 mg mLÿ1. Polymer (12.5 mg) was hydrated in 4.9 mL of 50 mM phosphate buffer (pH 6.0) and equilibrated at 378C. Prior to the inhibition assay, the pH of the polymer solution was determined and adjusted to pH 6 with 1 M NaOH, and 50 mM phosphate buffer (pH 6.0) was added to give a total volume 5 mL. The enzyme stock solution (10 mL) was mixed with either 1 mL of the polymer solution or 1 mL of phosphate buffer (pH 6.0) and incubated at 378C for 30 min. For aminopeptidase N activity towards leu-pNA, 0.25 mL of the incubate was transferred to a microtitre plate and 50 mL of the substrate leu-pNA was added to give a ®nal concentration of 1.0 mM. The release of pnitroaniline was measured every 1 min at room temperature by the increase in absorbance at 405 nm using a Anthos reader.2001 (Anthos Labtec Instruments, Austria). Activity of the incubate towards leu-enkp was determined by the addition of 50 mL of leu-enkp, prepared in 50 mM phosphate buffer (pH 6.0), giving a ®nal substrate concentration of 0.1 mM. The incubates were maintained at 378C. Samples were taken at predetermined times and added to 20 mL of 20% (v/v) tri¯uoroacetic acid (TFA) to stop the reaction. Preliminary studies showed that the concentration of TFA used was suf®cient to precipitate the polymer and proteins entirely (data not shown). The resulting mixture was centrifuged (20000g for 5 min), and the supernatants were analyzed by HPLC.
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reaction cylinder. After 30 min of incubation, 1 mL of the either leu-pNA (2 mM) or leu-enkp (0.2 mM) prepared in control buffer at 378C was added to the incubate. At various times after the start of the reaction, samples of 300 mL were taken. LeupNA samples were placed on ice and centrifuged at 20000g at 48C for 5 min, a 200-mL sample of the supernatant was transferred to a microtitre plate, and the absorbance was measured at 405 nm using a Anthos reader.2001 (Anthos Labtec Instruments, Austria). Leu-enkp samples were added to 20 mL of 20% (v/v) TFA to stop the reaction. The resulting mixture was centrifuged (20000g) for 5 min, and the supernatants were analyzed by HPLC. HPLC of Leucine-Enkephalin Analysis of leu-enkp reaction samples by reversed-phase HPLC was conducted using a Perkin-Elmer (Norwalk, CT) series 200 LC pump, Perkin-Elmer 200 series auto sampler with a 20-mL injection loop, and a diode array detector (Perkin-Elmer 235C). Samples were eluted from a Nucleosil 100 ± 5 C18 column (250 4 mm), with mobile phases of (A) 0.1% TFA and (B) 90% acetonitrile, 0.1% TFA at a ¯ow rate of 1.0 mL minÿ1. Detection was at 220 nm. A linear gradient was applied from 90% A to 10% A over a 22 min period. Preliminary studies showed that the concentration of TFA used was suf®cient to precipitate the polymer and proteins (data not shown). Statistical Analysis To determine signi®cance of enzyme inhibition, statistical analysis was performed using a nonpaired Students t test, and a p value of 0.05 or less was considered to be signi®cant.
RESULTS
Metabolism by Intact Pig Intestinal Mucosa A plastic cylinder with an internal surface area of 1.77 cm2 was placed vertically on top of the mucosal side of thawed intestinal tissue and clamped, as described previously.10 Polymers were prepared in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl as described for the aminopeptidase N inhibition studies. Control buffer (1 mL) with or without inhibitor equilibrated at 378C was added into the
Free Cysteine Studies Preliminary studies demonstrated the high af®nity of cysteine for zinc ions. The addition of Lcysteine to an aqueous 1 M ZnCl2 solution, for instance, resulted in precipitation. Hence, Lcysteine may also interact with the zinc ion of aminopeptidase N, consequently leading to the inhibition of this peptidase. To verify this hypothesis, enzyme inhibition studies were carried out
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Figure 1. Time-course of the formation of p-nitroaniline from leu-p-nitroanilide by aminopeptidase N after incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing 0.63 mM L-cysteine (~), control buffer containing 6.3 mM L-cysteine (*), and control buffer containing 63 mM L-cysteine (~). Each point represents the mean SD (n 3).
with isolated aminopeptidase N in the presence of increasing concentrations of free L-cysteine. The results of the following inhibition studies with free cysteine are shown in Figure 1. The higher the cysteine concentration, the higher the inhibitory effect towards aminopeptidase N; L-cysteine at 0.63 mM did not signi®cantly inhibit aminopetidase N activity, however, at 6.3 and 63 mM, there was signi®cant inhibition of enzyme activity. Additionally, inhibition studies performed with brush border membrane-bound aminopeptidase N also demonstrated an inhibitory effect of Lcysteine. Results, as shown in Figure 2, however, revealed that comparatively higher concentrations of cysteine were required to achieve the same inhibitory effect. Under similar assay conditions, the aminopeptidase N activity of the intact mucosa was only signi®cantly inhibited by L-cysteine at the highest L-cysteine concentration of 63 mM. Polymer Inhibition of Aminopeptidase N To compare the inhibitory effect of PCP and thiolated PCP towards aminopeptidase N, ®rst
Figure 2. Time-course of the formation of p-nitroaniline from leu-p-nitroanilide by intact mucosa after incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing 0.63 mM L-cysteine (~), control buffer containing 6.3 mM L-cysteine (*), and control buffer containing 63 mM L-cysteine (~). Each point represents the mean SD (n 3).
of all, a PCP±Cys conjugate was synthesised and characterized. Iodometric titration of the isolated PCP±Cys conjugate showed that 148 mmol 42 (mean SD; n 3) of sulfhydryl groups were conjugated per gram of polymer. Negligible amounts of sulfhydryl groups were detected for the control polymer, where the coupling reagent EDAC was omitted. The level of cysteine coupling and the features of the modi®ed polymer were consistent with previous preparations by our group.5 Following the incubation of aminopeptidase N in the presence of either PCP or cysteine-conjugated PCP at 0.25% (w/v), pH 6.0, the amount of leu-pNA hydrolyzed was signi®cantly reduced compared with the buffer only control (Figure 3). The polymer conjugate demonstrated a much stronger inhibitory effect than the unconjugated polymer, almost completely inhibiting the hydrolysis of leu-pNA. Under similar assay conditions for leu-pNA, the polymers were tested for their ability to protect the peptide leu-enkp from the hydrolytic activity of aminopeptidase N. For leupNA, both conjugated and unconjugated polycarbophil signi®cantly improved the stability of the
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Figure 3. Time-course of the formation of p-nitroaniline from leu-p-nitroanilide by aminopeptidase N after incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing (0.25% w/v) polycarbophil (~), and in control buffer containing (0.25% w/v) cysteine-conjugated polycarbophil (*). Each point represents the mean SD (n 3).
Figure 4. Time-course of the percentage remaining of leu-enkephalin in the presence of aminopeptidase N after the incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing (0.25% w/v) polycarbophil (~), and in control buffer containing (0.25% w/v) cysteineconjugated polycarbophil (*). Each point represents the mean SD (n 3).
substrate (Figure 4). Furthermore, the thiolated polymer had a signi®cantly greater inhibitory effect compared with the unconjugated polymer.
nilide was slightly lower than for the fresh intestine.10 Both conjugated and unconjugated polymer signi®cantly inhibited the hydrolysis of leu-pNA by aminopeptidases present on the intact intestinal mucosa (Figure 5). Thiolated PCP was thereby signi®cantly more effective than unconjugated polymer at protecting leu-pNA from enzymatic hydrolysis. In comparison to the studies with soluble aminopeptidase N, as shown in Figure 3, both polymers appear to be less effective at inhibiting the aminopeptidase N activity of the intact intestinal mucosa. The degradation of leu-enkp by intact intestinal mucosa and in the presence of conjugated or unconjugated PCP is shown in Figure 6. The HPLC spectrum of the degradation products of leu-enkp obtained by incubation with intestinal mucosa was different from that obtained for the isolated enzyme (data not shown). This result suggests that there are also other enzymes on the mucosa involved in the degradation of the peptide drug. Hydrolysis of leu-enkp was very rapid, the peptide was almost completely degraded after
Polymer Inhibition of Intact Intestinal Mucosa The inhibitory effect of the polymers was tested on the structurally more relevant intact intestinal mucosa of pig with native mucus. Microscopic investigations of the mucosa showed that a mucus gel layer was still covering the tissue after rinsing it with buffer solution. A preliminary histological study indicated that the damage to the mucosal surface from freezing and thawing was minimal and a layer of mucus was present on the mucus surface.10,11 Studies have shown that the storage of the pig intestine at ÿ208C does not in¯uence the structure of the mucus.12 It was shown that ionic strength, NaCl, at 1.2 and 11%, had no effect on the structure of native mucus from the stomach and the duodenum.13 Furthermore, studies demonstrated that the hydrolytic activity of the frozen pig intestine towards L-leucine±p-nitroa-
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Figure 5. Time-course of the formation of p-nitroaniline from leu-p-nitroanilide by intact mucosa after incubation in control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing (0.25% w/v) polycarbophil (~), and in control buffer containing (0.25% w/v) cysteine-conjugated polycarbophil (*). Each point represents the mean SD (n 3).
Figure 6. Time-course of percentage remaining of leu-enkephalin by intact mucosa after incubation control buffer (50 mM sodium phosphate buffer, pH 6.0) containing 2% NaCl (*), control buffer containing (0.25% w/v) polycarbophil (~), and in control buffer containing (0.25% w/v) cysteine-conjugated polycarbophil (*). Each point represents the mean SD (n 3).
30 min. In the presence of unconjugated polymer, a signi®cant amount (12.3 4 %) of the peptide was remaining after 60 min. However, no peptide was detected after 90 min, whereas for the polymer±cysteine conjugate, a signi®cant amount (11 3.5%) of the peptide was remaining after 90 min.
aminopeptidase N. This inhibition could be due to either the cysteine competitively binding to Zn2 in the active site of the enzyme, the cysteine acting as a divalent ion chelator or a combination of both mechanisms. Free cysteine was less effective in inhibiting the enzymatic activity on the intact intestinal mucosa. The lower potency of free cysteine may be due to thiol/disul®de exchange reactions with the native mucin present on the mucosa surface, resulting in the cysteine moiety being covalently bound to the mucin.5 Iodometric titration showed that 148 mmol 42 (mean SD; n 3) of cysteine was conjugated to 1g of PCP in the presence of EDAC. For the inhibitory studies, polymers were hydrated in buffer at pH 6 to give a polymer concentration of 0.25% (w/v; i.e., a cysteine concentration of 370 mM). In experiments with free cysteine (Figures 1 and 2), the reaction kinetics are different than those in which cysteine is immobilized onto the polymer (Figures 3 and 4). The greater inhibitory activity of the free cysteine is most likely due to its greater diffusion in comparison to the immobilized cysteine. However,
DISCUSSION The results demonstrate that thiolation of PCP enhances the inhibitory potency of PCP towards aminopeptidase N and membrane-bound peptidase(s) involved in the digestion of leu-pNA and leu-enkp. It is well established for aminopeptidase N that the complexation of the divalent ion Zn2 within the enzyme structure is essential for its activity.14 Studies with the strong divalent ion chelator EDTA strongly inhibited the activity of aminopeptidase N,14 whereas the comparatively weak divalent ion chelator PCP was a poor inhibitor of aminopeptidase N activity.4 Figures 1 and 2 show that free cysteine at 6.3 and 63 mM signi®cantly inhibited the activity of soluble
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in vivo this greater diffusion will mean that the excipient does not remain localized with the dosage form. Figures 3±6 show that conjugation of cysteine to PCP improves the enzyme inhibition in comparison to the unconjugated PCP. Because cysteine is attached to a high molecular weight cross-linked polymer it is unlikely that the conjugated cysteine will enter the active site of the enzyme. Therefore, it is likely that the mechanism of enzyme inhibition by the conjugated polymer is by chelation of the Zn2 from the protein structure. This mechanism is supported by the observation that the thiolated PCP has a greater af®nity for Zn26 and a corresponding greater inhibitory potency towards aminopeptidase N activity (Figures 3 and 4) in comparison with the control polymer. An enhanced stability of leu-enkp based on interactions of the peptide with the thiolated polymer, as shown for other peptides,6 can thereby be excluded because leu-enkp doesn't exhibit any thiol moieties. These studies showed that both free and conjugated cysteine had a lower inhibitory effect on the intact mucosa in comparison to aminopeptidase N. Due to cell lysis, it is possible that the activity observed on the mucosa is of cytosolic origin. This activity, however, is expected to be low in comparison with the membrane peptidase activity because 80% of the aminopeptidase activity of the ileal mucosa is membrane bound.15 The lower inhibitory activity in the presence of the mucus layer suggests it is acting as a barrier to the polymers ability to chelate metal ions from the surface membrane-bound peptidases. Previous studies have shown that the intestinal mucous layer is a barrier to permeation enhancing effect of the mucoadhesive polymer chitosan.16 For the thiolated polymer, the mucus layer could be limiting by the physical interaction between thiolated polymer and the intestinal mucus or by the diffusion of the polymer through the mucus. Indeed interaction between the thiolated polymer and the mucus could occur by the ability of cysteine to act as a mucolytic agent.17,18 This process involves disul®de exchange reactions between the cysteine moiety and the mucin glycoproteins, resulting in the formation of disul®de bonds between the cysteine and the glycoprotein.17 As a result of these disul®de exchange reactions, the concentration of free thiol groups for chelation is reduced. These disul®de exchange reactions have been postulated for the enhanced
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mucoadhesive interaction observed between the thiolated polymers and the intestinal mucus.5 The lower inhibitory activity of both thiolated and unthiolated PCP on intact mucosa may also be explained by inability of chelators to completely inhibit all intestinal membrane-bound peptidase activity towards enkephalin. Inhibition studies on the stability of methionine enkephalin in nasal, rectal, and vaginal mucosal homogenates from rabbit showed that although EDTA alone gave the strongest inhibitory activity, a combination of amastatin, thimerosal, and EDTA was required to almost completely stabilize methionine enkephalin.19 Therefore, inhibition by chelation is not suf®cient to inhibit all intestinal membrane-bound peptidases involved in the digestion of enkephalins. Furthermore, dosage forms of thiolated polymers should maintain their inhibitory effect at the site of peptide delivery. Studies by Chun and Chien19 suggested that high concentrations of the inhibitors amastatin, thimerosal, and EDTA are required to protect enkephalins from proteolytic attack in vitro. These concentrations would be dif®cult to maintain in vivo. A drug delivery system of the thiolated polymer described here would not be affected by inhibitor dilution and would offer several additional formulation advantages. The peptide drug would be localized with the inhibitory activity of the cysteine conjugated polymer.2 The higher mucoadhesive properties of the thiolated polymer5 would provide (i) prolonged residence time with the absorbing intestinal mucosal membrane, (ii) increased contact to the absorbing mucosa resulting in a steep concentration gradient to favor drug absorption, and (iii) reduce the diffusional distance for the peptide drug to reach the absorbing membrane, reducing the opportunity for metabolism by luminal proteases and localize the inhibitory activity of the polymer close to the membrane bound peptidases. In conclusion, the thiolation of PCP increases the inhibitory potency of PCP towards aminopeptidase N and intestinal membrane-bound peptidases, increasing the stability of leu-enkp. Thereby another mechanism was identi®ed by which PCP±Cys can improve the oral bioavailability of peptide drugs. Because similar peptidase activities towards enkephalins have been identi®ed in nasal, rectal, vaginal, and buccal mucosal homogenates,8 drug delivery systems of thiolated PCP should also provide enzymatic protection at these mucosal sites. Furthermore, because there are a large number of peptide drugs
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susceptible to intestinal luminal and membranebound metallopeptidases,1 thiolated PCP drug delivery systems should also provide protection towards enzymatic attack for a wide range of peptide drugs. It has also been shown that the thiolation of PCP increases the transport of model peptide drugs across the intestine. The enhanced transport is believed to be due to the opening of the tight junctions.7 The mucus layer is expected to act as a diffusional barrier, preventing the polymer from interacting with the epithelial surface, thereby reducing its enzyme inhibitory and permeation enhancing effect. This barrier, however, may be overcome in vivo because of to the higher effective concentration of the polymer conjugate when delivered in an appropriate drug delivery system.
ACKNOWLEDGMENTS This work was supported by a grant from the Fonds zur FoÈrderung der wissenschaftlichen Forschung (FWF) to A. Bernkop-SchnuÈrch.
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