Mucoadhesive polymers in peroral peptide drug delivery. I. Influence of mucoadhesive excipients on the proteolytic activity of intestinal enzymes

EUIOPlAN

ELSEVIER

European Journal of Pharmaceutical Sciences 4 (1996) 117-128

JOURNAL

OF

PHARMACEUTICAL SCIENCES ]

Mucoadhesive polymers in peroral peptide drug delivery. I. Influence of mucoadhesive excipients on the proteolytic activity of intestinal enzymes 1 Henrik L. Luegen a, Bas J. de Leeuw a, David P6rard a, Claus-Michael Lehr b, A. (Bert) G. de Boer a, J. Coos Verhoef", Hans E. Junginger a'* ~Leiden/Amsterdam Center for Drug Research, Leiden University, Divisions of Pharmaceutical Technology and Pharmacology L, P.O. Box 9502, NL-2300 RA Leiden, The Netherlands ~'Saarland University, Department o f Pharmaceutical Technology, P.O. Box 151150, D-66041 Saarbriicken, Germany

Received 20 June 1995; accepted 11 September 1995

Abstract

In the present study the potency of mucoadhesive excipients to inhibit intestinal proteases has been evaluated. Among the different mucoadhesive polymers investigated, uniquely the poly(acrylates) polycarbophil and carbomer 934P were able to inhibit the activities of trypsin, a-chymotrypsin, carboxypeptidase A and cytosolic leucine aminopeptidase. However, they failed to inhibit microsomal leucine aminopeptidase and pyroglutamyl aminopeptidase. Carbomer was found to be more efficient to reduce proteolytic activity than polycarbophil. The pronounced binding properties of polycarbophil and carbomer for bivalent cations such as zinc and calcium was demonstrated to be a major reason for the observed inhibitory effect. These polymers were able to deprive Ca 2+ and Zn 2+, respectively, from the enzyme structures, thereby inactivating their activities. Carboxypeptidase A and a-chymotrypsin activities were observed to be reversible upon addition of Zn 2÷ and C a 2~ ions, respectively. It is concluded that poly(acrylates) may be promising excipients to protect peptide drugs from intestinal degradation. In combination with their low toxicity risk they are expected to be suitable excipients for improved peroral delivery of peptide drugs. K e y w o r d s : Trypsin; a-Chymotrypsin; Carboxypeptidase A; Leucine aminopeptidase M; Pyroglutamic acid aminopeptidase;

Poly(acrylates); Carbomer; Polycarbophil; Mucoadhesives; Enzyme inhibition; Peroral peptide drug delivery

1. Introduction

Besides the unfavourable mucosal barrier conditions peroral application of peptide drugs is also strongly hampered by the activity of the proteases in the GI-tract. According to their location, intestinal proteases can be divided into three main groups: luminal, membrane-bound and cytosolic enzymes. Luminal enzymes such as * Corresponding author. 1Dedicated to Prof. Dr. Ernst Mutschler, Frankfurt/ Main, in honour of his 65th birthday. 0928-0987/96/$15.1)0 © 1996 Elsevier Science B.V. All rights reserved S S D 1 0928-0987(95)00042-9

the endopeptidases trypsin and a-chymotrypsin often initiate the degradation of perorally administered peptides. The resulting fragments are further digested by a variety of exopeptidases, as carboxypeptidases, aminopeptidases, di- and oligopeptidases, which are mainly embedded in the brush border membrane of the intestinal epithelium but are also present in the lumen of the gut (Bai, 1994; Lee et al., 1991). It has been shown that ligation of the pancreatic duct or the use of protease inhibitors like aprotinin and soybean trypsin inhibitor can lead to increased peptide drug absorption (Lee et al.,

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1991). A major drawback of these inhibitors, however, is their high toxicity, especially in chronic drug therapy. Also the non site-specific intestinal application of such compounds will markedly change the metabolic pattern in the gastro-intestinal tract due to a reduced digestion of food proteins. Furthermore, their activity is mainly limited to luminal enzymes with a preference to endopeptidases. Proteases embedded in the mucus layer or located to the apical membrane of the epithelial cells are not easily affected, since a direct interaction between enzyme and inhibitor is difficult to achieve. This particularly holds for high molecular weight structures such as soybean trypsin inhibitor, aprotinin and Bowman-Birk inhibitor, for which diffusion is hampered by the mucus layer (MacAdam, 1993; Matthes et al., 1992; Strous and Dekker, 1992). A locally acting drug delivery system, which is able to change the physiological surroundings of the intestine in a small restricted area, however, will be advantageous for chronic peptide drug delivery. One approach to design such a dosage form may be the use of mucoadhesive excipients. Their sticking capability is rather limited under physiological conditions due to the high turn-over rate of the mucus layer (Lehr et al., 1991), but they may be applicable for short-term delay of transit time and for intensifying contact between the dosage form and the site of peptide drug absorption. In previous studies improved intestinal absorption of the peptide drug 9-desglycinamide, 8arginine vasopressin (DGAVP) was observed in rats in vitro as well as in vivo using the weakly crosslinked poly(acrylate) derivative polycarbophil dispersed in physiological saline (Lehr et al., 1992). A similar effect was shown with another class of mucoadhesive polymers, the chitosans, in a vertically perfused intestinal loop model of the rat (Rentel et al., 1993). The improved DGAVP absorption could not be explained by mucoadhesion alone. Additionally, an influence on the physiological absorption barriers, such as inhibition of proteolytical enzyme activities and enhanced paracellular permeability, has also been discussed. The aim of the present study was to investigate

the mucoadhesive poly(acrylates) polycarbophii and carbomer on their capability to reduce the activity of the endopeptidases trypsin and achymotrypsin, and the exopeptidases carboxypeptidase A, microsomal and cytosolic leucine aminopeptidase and pyroglutamyl aminopeptidase.

2. Experimental procedures 2. i. Materials

Trypsin (TPCK treated, Type XIII), ~-chymotrypsin, carboxypeptidase A, microsomal leucine aminopeptidase (Type IV-S), cytosolic leucine aminopeptidase (Type III-CP, from kidney), pyroglutamyl aminopeptidase, 2-[N-morpholino]ethane-sulfonic acid] (MES), N-c~-benzoyl-L-arginine-ethylester (BAEE), N-o~-benzoylarginine (BA), N-acetyl-e-tyrosine ethylester (ATEE), N-acetyl-L-tyrosine (AT), hippuryl-Lphenylalanine (HPA), hippuric acid (Hipp), Lleucine-p-nitrophenylanilide (LNA), e-pyroglutamic acid p-nitroanilide (PNA), p-nitroaniline and zinc chloride were obtained from Sigma Chemie, Bornem, Belgium. All other chemicals used were at least of analytical grade. The following mucoadhesive polymers were used: polycarbophil (PCP, Noveon ® AA1, BF Goodrich, Cleveland, Ohio, USA), carbomer (C934P, Carbopol ® 934P, BF Goodrich, Cleveland, Ohio, USA), chitosan-glutamate (SeaCure ® G210, Pronova Biopolymers AS, Drammen, Norway), chitosan-lactate (Pronova Biopolymers AS, Drammen, Norway), poly(methyl vinyl ether/maleic anhydride) (linear interpolymer with 1:1 molar ratio, MW approximately 20000, Gantrez ® AN-119, GAF Chemicals Corporation, Schiedam, The Netherlands) and methylcellulose (medium viscosity grade, lot: 111762/80H26, Brocacef, Maarssen, The Netherlands). 2.2. H P L C equipment

HPLC analysis was performed with a ThermoSeparations system consisting of a P 200 gradient

H.L. Lueflen et al. / European Journal of Pharmaceutical Sciences 4 (1996) 117-128

pump, AS 100 autosampler, AS 200 UV/VIS detector and a datajet integrator. Data were calculated with the software package 'Winner on Windows' (Thermo-Separations, Breda, The Netherlands). For all HPLC methods a Lichrosorb 7 RP 18 column 100 x 3.0 mm (Chrompack, Middelburg, The Netherlands) equipped with a RP 18 precolumn was used as stationary phase. Following eluents were used as mobile phases: eluent A: 86% (V/V) 10 mM ammonium acetate buffer pH 4.2 and 14% (v/v) methanol, eluent B: 50% (v/v) 10 mM ammonium acetate buffer pH 4.2 and 50% (v/v) methanol. The injection volume was 20/xl.

2.3. Buffer The buffer system used in all enzyme inhibition experiments was a 50 mM 2-[N-morpholino]ethane-sulfonic acid ( M E S ) / K O H buffer, pH 6.7, containing 250 mM mannitol.

3. Methods

3.1. Enzyme inhibition studies Trypsin (EC 3.4.21.4). Amounts of 1.5 mmol N-o~-benzoyl-L-arginine ethylester ( B A E E ) / I were dissolved in the polymer preparations. After adding 23 I U / m l trypsin, the solutions were incubated at 37°C. Samples were withdrawn at predetermined time intervals and diluted with 0.1 M HCI to stop trypsin activity. The degradation of the substrate B A E E was studied by determining the formation of the metabolite N-~-benzoyl-L-arginine (BA) by H P L C with UV detection at 253 nm. Eluent A was used as an isocratic eluent. The retention time of the metabolite peak was detected 3.9 rain after injection at a flow rate of 0.75 ml/min. The degree of trypsin inhibition was expressed by the inhibition factor IF: IF = AUCcontro I/AUCpolyme r in which A U C is the area under the metabolite versus time curve (calculated by the trapezoidal

119

rule up to 4 h) without any polymer (AUCco,tro~) and with polymer (AUCpolymer).

a-Chymotrypsin (EC 3.4.21.1). The conversion of N-acetyl-L-tyrosine ethylester (ATEE) by a-chymotrypsin to N-acetyl-Ltyrosine (AT) and ethanol was measured as follows: a concentration of 4 /zmol of the substrate was dissolved in 1 ml of different polycarbophil and carbomer preparations. The degradation experiment was started by adding 80 mU a-chymotrypsin per ml (related to N-benzoyl-Ltyrosine ethylester at pH 7.8 and 25°C as indicated by Sigma Chemie, Bornem, Belgium). Incubation temperature was 37°C. Sample volumes of 50 txl were taken at predetermined time points from the incubation medium into 1 ml of the stop solution (phosphoric acid pH 2.0). Both the substrate A T E E and the metabolite AT were detected by HPLC-UV232. Gradient elution was performed as follows: 0-1 min: 100% A, isocratic (flow: 0.75 ml/min); 1-5 min: 10% A / 9 0 % B, linear gradient (flow: 0.75 ml/min); 5-8 min: 10% A / 9 0 % B, isocratic (flow: 0.75 ml/min); 8-9 min: 100% A, linear gradient (flow: 0.75 ml/min). The retention times of the substrate A T E E and the metabolite AT were 7.3 and 2.0 min, respectively. The activity of the enzyme was determined by calculating the area under the substrate concentration-time curve up to 4 h, using the trapezoidal rule. The effect of the polymers was expressed as percentage of the control.

Carboxypeptidase A (EC 3.4.17.1). Degradation studies with carboxypeptidase A were performed using hippuryl-L-phenylalanine (HPA) as enzyme substrate. After dissolving 3 /xmol substrate/ml polymer preparation, 0.11 IU carboxypeptidase A / m l were added and incubated at 37°C. Sample volumes of 50 /zl were withdrawn at predetermined time points and diluted in 1 ml stop solution consisting of 50% (v/v) methanol saturated with E D T A . Both substrate and metabolite hippuric acid were determined by HPLC-UV231 using a binary gradient method. Gradient elution was performed as follows: 0-2 min: 100% A, isocratic (flow: 0.7 ml/ min); 2-9 min: 20% A / 8 0 % B, linear gradient

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(flow: 0.7 ml/min); 9-11 min: 20% A / 8 0 % B, isocratic (flow: 0.7 ml/min); 11-12 min: 100% A, linear gradient (flow: 0.7 ml/min). The retention times of the substrate HPA and the metabolite hippuric acid were 2.3 min and 11.5 min, respectively. The enzyme activity values were determined as described for a-chymotrypsin.

Microsomal leucine aminopeptidase (EC 3.4.11.2). u-Leucine-p-nitrophenylanilide(LNA) was used as a substrate for leucine aminopeptidase M. A concentration of 4 ~ m o l substrate/ml was dissolved in various polycarbophil and carbomer preparations, respectively. The degradation experiment was started after adding 0.09 IU leucine aminopeptidase M/ml to the incubation media. Incubation temperature was kept at 37°C. Samples of 50 >1 were withdrawn at predetermined time points and diluted in 1 ml stop solution consisting of phosphoric acid (pH 2). Both the substrate (LNA) and the metabolite p-nitroaniline (NA) were determined by HPLC using a binary gradient method. Gradient elution with a flow rate of 0.75 ml/min was performed as follows: 0-1 min: 40% A / 6 0 % B, isocratic; 1-5 min: 100% B, linear gradient; 5-10 min: 100% B, isocratic. Following wavelength program was used: 0-6 min, A = 366 nm; 6-10 min, a = 311 nm. The retention times of the substrate LNA and the metabolite NA were 7.4 min and 2.3 min, respectively.

Cytosolic leucine aminopetidase (EC 3.4.11.1). An amount of 18.5 IU cytosolic aminopeptidase was added to 500 /xl of the blank/polymer preparations containing 4 mM LNA to start the degradation experiment. The experimental procedure and subsequent HPLC analysis were then performed as described for microsomal leucine aminopeptidase. The enzyme activity values were determined as mentioned for a-chymotrypsin.

Pyroglutamyl aminopeptidase (EC 3.4.19.3). Degradation studies with pyroglutamyl aminopeptidase were performed using e-pyroglutamic acid p-nitroanilide (PNA) as enzyme substrate. P N A was dissolved in a concentration of 0.2 /zmol/ml in different polycarbophil and carbomer

preparations. The incubation was started by adding 80 IU pyroglutamyl aminopeptidase/ml (units related to e-pyroglutamyl/3-naphthylamide at pH 8 and 37°C). Temperature of the incubation was 37°C. Samples of 20 /zl were taken at predetermined time points and diluted in 400/xl stop solution (phosphoric acid, pH 2). Both the substrate PNA and the metabolite NA were detected by isocratic HPLC using 35% (v/v) methanol in 10 mM ammonium-acetate buffer pH 4.2 as mobile phase. The following wavelength program was used: 0-4.4 min, A = 366 nm; 4.4-7 min, A = 312 nm. Autozero was performed at t = 4.8 min after sample injection. The retention times of the substrate PNA and the metabolite NA were 5.4 min and 3.7 min, respectively, using a flow rate of 0.75 ml/min.

3.2. Effect of inhibition

C a 2+

and

Z n 2+

ions on enzyme

Reversibility of ~-chymotrypsin by time dependent addition of calcium. The effect of C a 2+ ions on the degradation activity of ~-chymotrypsin was studied in the following incubation media: M e s / K O H buffer pH 6.7 (control), M e s / K O H buffer pH 6.7 containing 40 mM calcium (control with Ca 2+) and 0.5% (w/v) carbomer in M e s / K O H buffer pH 6.7. In addition to the above described degradation study with c~-chymotrypsin in the presence of carbomer, 40 mM Ca 2+ was added just before, 10 and 240 min after starting the incubation experiment. To ensure that pH did not decrease more than 0.4 units below pH 6.7, 35 p~l of 0.05 M K O H was added per 1 ml of the remaining incubation fluid. HPLC analysis of substrate and metabolite were performed as described above.

Reversibility of carboxypeptidase A activity by time dependent addition of zinc. The effect of Zn 2+ ions on the degradation activity of carboxypeptidase A was studied in the following incubation media: (a) M E S / K O H buffer pH 6.7 (control); (b) M E S / K O H buffer pH 6.7 containing 10 mM zinc (control with ZnZ+); (c) 0.1% (w/v) carbomer in M E S / K O H buffer pH 6.7. Amounts of 3.06 mmol H P A / m l were dissolved in the incubation media. Volumes of 1

H.L. Lueflen et al. / European Journal o f Pharmaceutical Sciences 4 (1996) 117-128

ml of the different substrate dilutions were used for one degradation experiment. The degradation experiment was started by adding 0.22 IU carboxypeptidase A / m l to the different incubation media. An appropriate volume of a 0.25 M ZnCI 2 dilution in 50 mM M e s / K O H buffer pH 6.0 was added either 10 min or 240 min after starting the degradation experiment to the polymer preparations, to yield a final concentration of 10 mM Z n 2~ . The preparations were incubated at 37°C and samples were taken and analysed as described above.

3.3. Zinc binding studies Polycarbophil and carbomer were dispersed in a concentration of 0.25% (w/v) in following buffer systems: pH 3 - p H 5, 50 mM ammonium a c e t a t e / N a O H buffer; pH 6, 50 mM M E S / K O H buffer without mannitol. Due to the formation of Zn(OH)2 at higher pH values, 0.1% (w/v) polymer preparations in 500 mM ammonium acetate/ ammonia buffer were used to study Zn2+-poly mer binding at pH 7. Zinc chloride was added in a concentration of 17.7 mmol Zn2+/l polymer preparation. The pH was readjusted after adding Zn 2+. Following incubation at 37°C for 30 min, the preparations were centrifuged (800 g/20°/15 min). Zinc concentrations in the supernatants were determined by complexometric titration with 0.025 M E D T A (Titriplex ® I I I , Merck, Darmstadt, Germany) using a 3,3 dimethylnaphtidin/potassiumhexacyanoferrat III mix-indicator according to Merck. The amount of polymerbound zinc was calculated from the difference between total amount of Z n 2+ added and the amount of free Zn 2+ measured in the supernatant.

3.4. Calcium binding studies The capability of the poly(acrylates) to bind investigated at different pH values. The polymers were dispersed at a concentration of 0.25% (w/v) using the following solutions/buffer systems: (a) pH 3, 50 mM acetic acid; (b) pH 4-5, 50 mM acetate/NaOH buffer; (c) pH 6-9, 50 mM M e s / K O H buffer. After homogeneous dispersion of the polymer C a 2+ w a s

121

preparations, 13 mM CaC12 was added to each preparation and the samples were incubated for 30 min at 37°C. The polymers were spun down (800 g/20°C/15 rain). C a 2+ content in the supernatant was determined by complexometric titration with Titriplex ® III (Merck, Darmstadt, Germany), using calcein as an indicator. The amount of polymer-bound calcium was calculated as described above for the zinc binding studies.

3.5. Rheological studies The viscosity of the various mucoadhesive polymer preparations was measured at 37°C and 25°C, respectively, using a rotation viscosimeter (Haake CV 100/ME 30). Rheological studies at 37°C were carried out after prewarming the preparations and the rotation cylinder for 10 min in the measuring cup. Flow curves were recorded by applying continuously increasing shear rates (0-100 s 1, A D = 1 0 s-I/min).

4. Results

4.1. Enzyme inhibition studies Trypsin. In contrast to all other polymers investigated, uniquely polycarbophil and carbomer showed a strong concentration-dependent inhibitory effect on the hydrolytic activity of trypsin using B A E E as a model substrate (Fig. la,b and Table 1). In the first 20 min a strong non-linear decrease of trypsin activity was observed. Carbomer showed a more pronounced inhibitory effect than polycarbophil. The minimal polymer concentration resulting in complete inhibition of trypsin activity after a period of 20 min was 0.35% and 0.15% (w/v) for polycarbophil and carbomer, respectively. a-Chymotrypsin. a-Chymotrypsin, could also be inhibited by the poly(acrylates) polycarbophil and carbomer (Fig 2a,b). Inhibition was found to be dependent on the polymer concentration. Just as for trypsin inhibition, carbomer was more efficient to inhibit a-chymotrypsin than polycarbophil. Whereas a

122

H.L. Lueflen et al. / European Journal of Pharmaceutical Sciences 4 (1996) 117-128 1.50

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Fig. 1. (a) Formation of N-c~-benzoyl-L-arginine due to the degradation of N-a-benzoyl-L-arginine ethylester by trypsin in presence of polycarbophil. +, control; O, 0.25% (w/v) polycarbophil; II, 0.3% (w/v) polycarbophil; A, 0.35% (w/ v) polycarbophil (mean +- SD, N = 3). (b) Formation of N-abenzoyl-L-arginine due to the degradation of N-,~-benzoyl-Larginine ethylester by trypsin in presence of carbomer. +, control; O, 0.1% (w/v) carbomer; II, 0.15% (w/v) carbomer; A, 0.25% (w/v) carbomer (mean-+ SD, N = 3).

concentration of 0.25% (w/v) polycarbophil showed only weak inhibition (62% activity of the control), 0.25% (w/v) carbomer was already able to block markedly the hydrolytic activity of achymotrypsin towards ATEE (34% activity of the control).

incubation

time

[min]

Fig. 2. (a) Degradation of the substrate N-acetyl-L-tyrosine ethylester (ATEE) by c~-chymotrypsin in presence of polycarbophil. +, control; A, 0.1% (w/v) polycarbophil; O, 0.25% (w/v) polycarbophil; B, 0.5% (w/v) polycarbophil. (mean-+ SD, N = 3). (b) Degradation of the substrate Nacetyl-L-tyrosine ethylester (ATEE) by a-chymotrypsin in presence of carbomer. +, control; &, 0.1% (w/v) carbomer; O, 0.25% (w/v) carbomer; II, 0.5% (w/v) carbomer (mean -+ SD, N = 3).

of 0.005% (w/v) were already sufficient to reduce carboxypeptidase A activity to about 15% of the controls. The slopes of the curves revealed a reduction of the degradation rate of hippuryl-Lphenylalanine with time.

Carboxypeptidase A. Polycarbophil and carbomer were also able to inhibit carboxypeptidase A activity (Fig. 3), and both poly(acrylates) showed a quite similar concentration-dependency. Polymer concentrations

Microsomal leucine aminopeptidase. Leucine aminopeptidase M activity was not inhibited by the poly(acrylic acid) derivatives polycarbophil and carbomer (data not shown).

H.L. l.ueJ3en et al. / European Journal of Pharmaceutical Sciences 4 (1996) 117-128

123

Table 1 Viscosity and trypsin inhibition factor (IF) of a number of mucoadhesivepolymers (mean, N = 3) Mucoadhesive polymer Mes/KOH buffer pH 6.7 Polycarbophil Carbomer Methylcellulose Chitosan-glutamate (SeaCure® + 210) Chitosan-lactate (medium MW) Chitosan-lactate (high MW) Gantrez® 119 AN EDTA

Concentration [% (w/v)]

r/(mPa s]

0.25 0.35 0.1 0.25 0.5 1 2 0.5

0.70 4.66 18.11 1.62 7.62 12.39 29.68 312.63 2.87

1.1 7.2 3.7 1(I.4 0.8 1.1 3.9 (1.8

0.5 0.25 1 7.5

9.47 5.1)7 5.13 1.36

11.7 (I.8 (/.7 1.11

Cytosolic leucine aminopetidase. In contrast to the enzymes from microsomal origin, the cytosolic leucine aminopeptidase showed a strong reduction of its proteolytic activity in the presence of either polycarbophil or carbomer (Fig. 4). At a concentration of 0.5% (w/v) the proteolytic activity was reduced to 13% for the carbomer and to 22% for the polycarbophil preparation as compared to the control. Pyroglutamyl aminopeptidase. The cysteine protease pyroglutamyl aminopeptidase could not be inhibited by polycarbophil and carbomer (data not shown). 4.2. Effect of Ca 2÷ and Zn 2+ ions on enzyme inhibition a-Chymotrypsin. In both of the two control situations complete degradation of the substrate A T E E was observed within the first 30 min (Fig. 5a). Addition of 40 mM Ca 2+ to the control buffer did not lead to any change in a-chymotrypsin activity, demonstrating that observed effects by adding Ca 2+ to the incubation media during the experiment are not due to higher Ca 2+ concentrations alone. In 0.5% (w/v) carbomer preparation without any addition of Ca 2+ a strong reduction of enzyme activity was found. An amount of about 40% of

IF 1.11

the substrate was still present at 240 min. Addition of calcium at 10 min showed good recovery of a-chymotrypsin activity. When calcium was added to the carbomer preparation 240 min after starting the experiment, only a slight increase of a-chymotrypsin activity could be observed.

Carboxypeptidase A. The controls without and with 10 mM Z n 2+ showed a comparable and fast degradation of the substrate HPA. Within 30 min the substrate was nearly completely hydrolyzed by the enzyme. Thus, the used Zn 2+ concentration was not sufficient to affect the activity of carboxypeptidase A. A concentration of 0.25% (w/v) carbomer resulted in strong inhibition of carboxypeptidase A over 4 h (Fig. 5b). Addition of zinc to the carbomer preparations at 10 or 240 min after starting the degradation experiment, however, showed recovery of carboxypeptidase A activity. 4.3. Zinc binding studies As shown in Fig. 6, the zinc binding capacities of polycarbophil and carbomer were strongly pHdependent. The capability to bind Zn 2+ increased with higher pH values. Carbomer showed a slightly higher Zn 2+ binding capacity than poly-

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H.L. Lueflen et al. / European Journal o f Pharmaceutical Sciences 4 (1996) 117-128 100

100

80

80

60

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O E

2O

20

0

0 0

50

100

150

200

0

250

60

120

180

240

incubation time |mini

incubation time [min]

Fig. 4. Degradation of the substrate L-leucine p-nitroanilide (LNA) by cytosolic leucine aminopeptidase in presence of polycarbophil and carbomer. +, control; &, 0.1% (w/v) carbomer; A, 0.1% (w/v) polycarbophil; e , 0.5% (w/v) carbomer; ©, 0.5% (w/v) polycarbophil. (mean-+ SD, N =

100

3).

8O

~

80

4.5. Rheological studies

~

40

To investigate whether inhibition of trypsin activity was caused by a possible immobilisation effect due to the viscous properties of the polymers, rheological measurements were performed. No relationship between trypsin inhibition and the viscosity of the different polymer preparations could be observed (Table 1). In principle, the viscosity values were ranging between 1 and 10 mPas. Methylcellulose in a concentration of 1% (w/v) displayed a viscosity of 30 mPas, but was not able to decrease trypsin activity.

20

I

0

50

100

150

200

250

incubation time [min I

Fig. 3. (a) Degradation of the substrate hippuryl-L-phenylalanine (HPA) by carboxypeptidase A in presence of polycarbophil. +, control; 0, 0.005% (w/v) polycarbophil; &, 0.01% (w/v) polycarbophil; 0, 0.025% (w/v) polycarbophil (mean -+ SD, N = 3). (b) Degradation of the substrate hippuryl-L-phenylalanine (HPA) by carboxypeptidase A in presence of carbomer. +, control; 0, 0.005% (w/v) carbomer; &, 0.01% (w/v) carbomer; Q, 0.025% (w/v) car.bomer (mean -+ SD, N = 3).

carbophil. Under the conditions of the degradation experiments at pH 6.7, 500 to 600 mg Zn 2+ was bound per gram polymer.

4.4. Calcium binding by the polymers The capacity of polycarbophil and carbomer to bind C a 2+ was strongly pH-dependent (Fig. 6). Binding of Ca 2÷ to the polymers increased between pH 4 and 6. At higher pH values, however, binding reached a plateau of approximately 240 mg Ca 2÷/g polymer.

5. Discussion

The present study showed that the poly(acrylate) derivatives polycarbophil and carbomer are potent inhibitors of the proteolytic enzymes trypsin, a-chymotrypsin, carboxypeptidase A and cytosolic leucine aminopeptidase, whereas the enzyme activities of microsomal leucine aminopeptidase and pyroglutamyl aminopeptidase are not affected by these polymers. The inhibitory properties of poly(acrylates) on intestinal proteases was firstly reported by Hutton et al. (1990). They found a strong reduction of albumin degradation by a mixture of proteases in the presence of carbomer 934P. Inhibition was

H.L. Lue[3en et al. / European Journal of Pharmaceutical Sciences 4 (1996) 117-128

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10 o E E

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N 0

0

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0

200

300

400

500

3

4

5

6

7

8

9

pH

incubation time [min]

Fig. 6. Binding capacity of 1 g polycarbophil or carbomer towards either calcium or zinc ions at different pH values. Calcium binding curves: A polycarbophil; I , carbomer. Zinc binding curves: + , polycarbophil; Q, carbomer (mean -+ SD, N = 3).

100

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500

incubation time [min]

Fig. 5. (a) Degradation of the substrate N-acetyl-L-tyrosine ethylester by a-chymotrypsin in presence of 0.5% (w/v) carbomer. Effect of Ca 2÷ ions on a-chymotrypsin inhibition was studied by adding 40 mmol/l Ca 2+ at different time points. + , control without polymer; O, control without polymer and with 40 m m o l / l Ca2+; ~, Ca 2+ was added just before a-chymotrypsin incubation; A, Ca 2+ was added after 10 min incubation; B; Ca 2÷ was added after 240 min incubation. (mean-+ SD, N = 3). (b) Degradation of the substrate hippuryl-L-phenylalanine (HPA) by carboxypeptidase A in presence of 0.1% (w/v) carbomer. Effect of Zn 2+ on carboxypeptidase A inhibition was studied by adding 10 m m o l / l Zn 2+ at different time points. + , control without polymer; O, control without polymer and with 10 m m o l / l Zn2+; &, Zn 2+ was added after 10 rain incubation; B , Zn 2+ was added after 240 min incubation. (mean-+ SD, U=3).

pH dependent, whereby the inhibitory effect of the polymer was strong at pH 4.5 and 7.5, but minor at pH 11. At pH 7.5 VmaX decreased and k m increased, indicating that inhibition could not be ascribed to either classical competitive or noncompetitive interactions.

Many proteases have bivalent cations as zinc and calcium as essential co-factors within their structure. The endoproteases trypsin and o~chymotrypsin were chosen as representatives of the group of Ca2+-containing serine proteases. The enzymes carboxypeptidase A, microsomal and cytosolic aminopeptidase belong to the group of Zn2+-dependent exopeptidases. A classification of the family of zinc metalloproteases has been recently described by Hooper (1994). Depletion of zinc reduces or totally inhibits their activity (Himmelhoch, 1969; Salvesen and Nagase, 1989; DiGregorio et al., 1988). Pyroglutamyl aminopeptidase was chosen as a member of the group of cysteine proteases (Armentrout and Doolittle, 1969). From Table 1 it is evident that from all the mucoadhesive polymers studied only the poly(acrylates) polycarbophil and carbomer were able to inhibit trypsin activity. This indicates that mucoadhesive properties are not primarily responsible for trypsin inhibition. Direct binding interactions between the enzyme structure, as a hydrophilic macromolecule, and the mucoadhesire polymer may occur, thereby leading to inactivation of protease activity. In our previous studies (LueBen et al., 1995), binding between proteins and both polycarbophil and carbomer could only be observed at pH 4, but not at pH 6.7 where the enzyme inhibition studies were

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performed. Another aspect for enzyme activity studies in viscous systems is the possibility of immobilization of both enzyme and substrate in the gel matrix of the different investigated polymers. The viscosities of the inhibiting polycarbophil and carbomer preparations, however, were quite low in comparison with the other polymers studied which were not able to inhibit trypsin activity (e.g. 1% (w/v) methylcellulose; Table 1). Only under the influence of 2% (w/v) methylcellulose less enzyme activity could be found. At this concentration, however, the viscosity was so high (about 300 mPas), that reduced trypsin activity is most probably due to hampered diffusion of both the enzyme and substrate. Our results revealed a non-linear metabolite versus time profile, thus a decrease of degradation activity of trypsin with time. In the case of 0.35% polycarbophil and 0.15% and 0.25% carbomer a time period of 20-30 min was required before complete loss of trypsin activity could be detected. This time-dependency indicates that trypsin inhibition is not due to a rapid enzymeinhibitor interaction, but to a more complex pattern of different kinetic parameters. It was recently reported that trypsin inhibition by poly(acrylates) could be ascribed to deprivation of Ca 2÷ ions out of the trypsin structure (Luef3en et al., 1995). The efficacy of the poly(acrylates) is underlined by the observation that 7.5% (w/v) of the chelating agent E D T A was not sufficient to inhibit trypsin activity at pH 6.7 (Table 1), demonstrating that the binding affinity of polycarbophil and carbomer to calcium is much higher than E D T A at this pH value. The second C a 2+ containing serine protease studied, a-chymotrypsin, shows high similarities in its tertiary structure with trypsin (Tsukada and Blow, 1985; Hedstrom et al., 1994). In comparison to the trypsin degradation studies, higher polymer concentrations were required to inhibit a-chymotrypsin in order to display a comparable reduction of enzyme activity. For example, 0.1% carbomer in the trypsin experiment showed an inhibitory profile comparable to 0.5% carbomer in the a-chymotrypsin study. As found for trypsin, a-chymotrypsin showed a non-linear reduction of its activity under the influence of the

poly(acrylates) which could be observed in the first 20 min after addition of the enzyme to the incubation medium. This indicates that some steps in the inhibition kinetics are slow, such as dissociation of Ca 2+ from the enzyme and denaturation processes (e.g. autodegradation). As also observed in the trypsin studies, there seems to be a narrow poly(acrylate) concentration range in which the enzymes lose their activities. Obvious is the polycarbophil concentration range between 0.25% and 0.35% for trypsin. The concentration of 0.3% appears to be a transitionary concentration, as indicated by the huge standard deviations. For c~-chymotrypsin 0.5% polycarbophil seems to serve as such a transitionary concentration, switching between inhibition and no inhibition. a-Chymotrypsin seems to be less sensitive to autodegradation than trypsin after deprivation of calcium ions from its structure. In contrast to reversibility studies with trypsin (Luegen et al., 1995), a-chymotrypsin activity could be partially recovered. This observation may be explained that, under deprivation of calcium ions from trypsin, basic amino acid groups are exerted and cleaved more easily by the enzyme itself, whereas this does not seem to be the case for ~-chymotrypsin which has a high preference for aromatic amino acid groups. Carboxypeptidase A, requiring Z n 2+ for its activity (Vallee et al., 1960) could also be inhibited by polycarbophil and carbomer at neutral pH values. In principle, the concentrations of the two polymers to achieve enzyme inhibition was quite comparable, but the concentrations required for inhibition were much lower compared to trypsin and a-chymotrypsin. Reversibility studies on carboxypeptidase A activity by the addition of zinc indicate a strong effect of this bivalent cation on the inhibitory properties of the poly(acrylates). Binding of zinc to the poly(acrylates) by depletion from the secondary structure of the enzyme may explain the time-dependent inactivation of carboxypeptidase A. The lower polymer concentrations required to display an inhibitory effect as compared to the degradation studies with trypsin and a-chymotrypsin may be ascribed to either a higher dissociation constant of the cation out of the enzyme structure

H.L. Lueflen et al. / European Journal of Pharmaceutical Sciences 4 (1996) 117-128

or to a higher binding affinity of the poly(acrylates) to zinc in comparison with calcium. Cytosolic leucine aminopeptidase was the second zinc dependent protease which could be inhibited by the poly(acrylates), but the required polymer concentrations to achieve inhibition were higher compared to carboxypeptidase A. This may be due to a lower dissociation constant between Zn 2+ and cytosolic leucine aminopeptidase than between Zn 2+ and carboxypeptidase A. In contrast, microsomal leucine aminopeptidase, also a zinc metallo enzyme, could not be inhibited by polycarbophil and carbomer, suggesting that the binding ability of the polymers toward Zn 2+ is not high enough to deplete cations from the enzyme structure. These two aminopeptidases represent two different Zn 2+dependent aminopeptidases. Microsomal leucine aminopeptidase binds only one ZnZ+-ion per subunit (Himmelhoch, 1969; DiGregorio, 1988), whereas cytosolic leucine aminopeptidase binds two bivalent cations (Van Wart and Lin, 1981; Taylor, 1993). In the commercially available form, in which MgC12 is added to the stock suspension, the metalloenzyme complex can be described as [(LAP)ZnxMgy ] (Allen et al., 1983). For the non-magnesium containing enzyme a tenfold lower kcat was reported (Van Wart and Lin, 1981). Thus the reduced but not completely inhibited enzyme activity may also be due to binding of the more easily dissociating Mg 2+ cation than Zn 2÷ to the poly(acrylate), resulting in a still measurable amount of enzyme activity. Pyroglutamyl aminopeptidase belongs to the family of cysteine exoproteases (Amentrout and Doolittle, 1969). The enzyme does not contain any bivalent cations, but exhibits an increased activity in presence of chelating agents such as E D T A (Szewczuk and Mulczyk, 1969). Accordingly, polycarbophil and carbomer rather increased than inhibited the activity of pyroglutamyl aminopeptidase. This enzyme is of pharmaceutical importance, since it is involved in the intestinal degradation of luteinizing hormone-releasing hormone and its analogous such as the peptide drug buserelin (Sandow, 1989). The poly(acrylates) studied showed a pronounced binding ability for the bivalent cations calcium and zinc. The binding capacity of these

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polymers increased with higher pH values. An explanation for this phenomenon may be that at higher pH values the carboxylic groups of poly(acrylates) dissociate, forming a polyanionic polymer which tends to salt out in the presence of cations. Estimating that the molecular mass of one acrylic acid m o n o m e r [ C 3 H 4 0 2 ] in the polymer chain is 72 g, it can be calculated that one gram of polymer consists of 13.9 mmol of carboxylic groups. At neutral pH, where all carboxylic groups are mostly dissociated, a range of 500 to 600 mg Z n 2+ bound to 1 gram of polymer could be found, which means a molar ratio (Zn2+: carboxylic group) of 1:1.8 to 1:1.4. For calcium a binding capacity of approximately 250 mg Ca R+ per gram of polymer was measured, which correlates to a molar ratio of 1:2.3 (Ca2+: carboxylic group). This suggests that two carboxylic groups bind one calcium cation to form a neutralized charge complex. Consequently, this Ca,[acrylate]2 . complex reduces the hydrophilicity of the polymer, resulting in a non-soluble precipitate.

Acknowledgements The authors wish to thank the German National Scholarship Foundation (Bonn, Germany) and Lohmann Therapie Systeme (Neuwied, Germany) for financial support and Prof. Dr. B.W. Miiller (Kiel, Germany) and his co-workers for allowing us to perform rheological measurements in their Institute.

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