- Email: [email protected]
Polymer-enzyme inhibitor conjugates: influence of the molecular mass on the inhibition of membrane-bound aminopeptidase N activity
J. DRUG DEL. SCI. TECH., 14 (6) 495-498 2004
Polymer-enzyme inhibitor conjugates: influence of the molecular mass on the inhibition of membrane-bound aminopeptidase N activity V.M. Leitner1, A. Bernkop-Schnürch2* Institute of Pharmaceutical Technology and Biopharmaceutics, Centre of Pharmacy, University of Vienna, Althanstr. 14, 1090 Vienna, Austria 2 Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University Innsbruck, Innrain 52, Josef Möller Haus, 6020 Innsbruck, Austria *Correspondence: [email protected] 1
Conjugates of mucoadhesive polymers and protease inhibitors have been developed to protect incorporated (poly)peptides from enzymatic attack by luminally secreted or membrane-bound proteases. Within the present study, the influence of the molecular mass of such polymer conjugates on the inhibition of membrane-bound enzyme activity was evaluated. The aminopeptidase N inhibitor bacitracin (Bac) was covalently immobilised on the mucoadhesive polymer model poly(acrylic acid) with a molecular mass of 2 kDa (PAA2) and 3000 kDa (polycarbophil, PCP). The resulting conjugates PAA2-Bac and PCP-Bac exhibited comparable amounts of bacitracin (24.4 and 25.3% (m/m), respectively) and were used to perform inhibition studies on isolated and membrane-bound aminopeptidase N. The conjugates showed no differences in the inhibition of isolated aminopeptidase N, whereas PAA2-Bac exhibited a significantly higher inhibitory activity versus membrane-bound aminopeptidase than PCP-Bac. Due to its lower molecular mass, PAA2-Bac could more easily diffuse into the mucus layer to get in contact with the enzyme. Obtained results should provide helpful basic knowledge for the development of multifunctional polymers as auxiliary agents for the oral administration of peptide drugs. Key words: Mucoadhesion – Poly(acrylic acid) – Polymer-inhibitor conjugate – Bacitracin.
teases [e.g. 7, 8]. In order to substantiate our knowledge for the development of more effective polymer-inhibitor conjugates, it was the aim of the present study to evaluate the influence of the molecular mass of the conjugates on their efficacy to inhibit the most abundant brush border membrane bound enzyme aminopeptidase N. As mucoadhesive polymer model, poly(acrylic acid) with an average molecular mass of 2 kDa (PAA2) and 3000 kDa (polycarbophil, PCP) was chosen. Bacitracin (Bac), a cyclic dodecapeptide which has been reported to inhibit membranebound aminopeptidase N [9] was covalently linked to these polymers as illustrated in Figure 1. Both poly(acrylic acid)-
The combination of mucoadhesive polymers with enzyme inhibitors as means of delivering therapeutically active peptides and proteins via mucous membranes has received increasing attention in recent years. Mucoadhesive polymers, on the one hand, are supposed to ensure a prolonged and intimate contact of the delivery system with the absorbing membrane leading to improved drug absorption [1, 2] and a reduced presystemic degradation of the drug by shortening the way between the delivery system and the absorbing membrane. Enzyme inhibitors, on the other hand, are able to protect therapeutic (poly)peptides from enzymatic degradation with luminally secreted and/or membrane-bound proteases. Although it has been demonstrated that co-administered protease inhibitors are very efficient in improving the oral bioavailability of therapeutic (poly)peptides [e.g. 3, 4], their use remains questionable as they may cause side effects, such as an unintended disturbance of protein digestion, pancreatic hypersecretion due to a luminal feedback regulation as well as systemic toxic side effects [5]. To overcome these problems, the immobilisation of protease inhibitors to an unabsorbable drug carrier matrix such as mucoadhesive polymers seems to be a promising strategy combining the favourable properties of both excipients [6]. As the immobilised inhibitor remains concentrated on the drug carrier matrix, disturbing dilution effects as well as inhibitor absorption can be excluded leading to an enhanced protective effect whereby a reduced share of this auxiliary agent in the dosage form may be sufficient. To date, various polymer-inhibitor conjugates have been generated protecting incorporated (poly)peptides from enzymatic attack by luminally secreted or membrane-bound pro-
COOH
O
HN
S
H3C
N
O
CH3 His
D-Asp
D-Phe Ile
Asn ε
D-Orn
Leu D-Glu
Lys .α Ile
Figure 1 - Presumable chemical substructure of poly(acrylic acid)-bacitracin A conjugates. Bacitracin A is the major component of commercial bacitracin, which is a mixture of at least nine bacitracins. 495
J. DRUG DEL. SCI. TECH., 14 (6) 495-498 2004
Polymer-enzyme inhibitor conjugates: influence of the molecular mass on the inhibition of membrane-bound aminopeptidase N activity V.M. Leitner, A. Bernkop-Schnürch
bacitracin conjugates (PAA2-Bac, PCP-Bac) exhibited comparable amounts of immobilised bacitracin. The inhibitory effect of the conjugates was determined on isolated aminopeptidase N in comparison to an equal amount of unbound bacitracin. Furthermore aminopeptidase N inhibition studies were performed on porcine intestinal and bovine nasal mucosa to study the influence of the molecular size of the conjugates on their enzyme inhibitory properties.
3. Determination of the bacitracin content of the conjugates
The bacitracin content (%) was determined by quantifying the amount of covalently bound bacitracin within the polymer photometrically (Lambda 16; Perkin-Elmer, Vienna, Austria). PAA2-Bac, PCP-Bac and unmodified control polymers were hydrated in demineralised water to obtain a concentration of 0.125% (m/v) and the absorbance at 255 nm was determined (n = 3). As reference, 0.125% (m/v) solutions of PAA2 and PCP were used, respectively. The amount of immobilised bacitracin on the polymers was calculated by using a standard curve obtained by the measurement of a series of reference solutions containing increasing amounts of bacitracin.
I. MATERIALS AND METHODS 1. Materials
Polycarbophil with an average molecular mass of 3000 kDa (PCP) was kindly provided by Noveon (Raubling, Germany). Poly(acrylic acid) with an average molecular mass of 2 kDa (PAA2), microsomal aminopeptidase (aminopeptidase N; EC 3.4.11.2.), L-leucine-p-nitroanilide, bacitracin and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) were purchased from Sigma (St. Louis, MO, United States). All other chemicals were of reagent grade and obtained from Sigma (St. Louis, MO, United States).
4. Inhibition studies with isolated aminopeptidase N
L-leucine-p-nitroanilide was used as enzyme substrate for degradation studies with aminopeptidase N. The PAA2-Bac and PCP-Bac conjugates were dissolved in TBS (50 mM Tris-HCl buffer containing 2.9% NaCl, pH 7.5) in a concentration of 0.5% (m/v) and aliquots of 100 µl were transferred into wells of a microtitration plate. Aminopeptidase N (50 µl of 10 µg/ml TBS) was added and the mixtures were incubated for 1 h at room temperature. Thereafter, 50 µl of the substrate solution (0.5 mg of L-leucine-p-nitroanilide/ml TBS) was added and the increase in absorbance was immediately recorded at 405 nm and room temperature using a microtitration plate reader (Anthos reader 2001, Salzburg, Austria). TBS and unbound bacitracin dissolved in TBS in a concentration of 0.124% (m/v) (equivalent to the bacitracin content of 0.5% (m/v) conjugate) were used as controls. Concentrations of the hydrolyzed substrate were calculated by interpolation from a standard curve obtained from the analysis of solutions containing increasing amounts of p-nitroaniline [9].
2. Synthesis of PAA2-Bac and PCP-Bac conjugates
The available amino-groups of bacitracin were covalently bound to the carboxyl groups of PAA2 and PCP via a condensation reaction mediated by EDAC, as described previously [9]. The poly(acrylates) (150 mg) were hydrated in 30 ml of demineralised water and the pH was adjusted to 6 with 1 M NaOH. EDAC was added in a final concentration of 50 mM and mixtures were stirred for 15 min at room temperature in order to activate the carboxylic acid groups of the polymers. Then, 75 mg of bacitracin were added and the reaction was allowed to proceed for 3 h with stirring at room temperature. The resulting polymer-bacitracin conjugates were purified by dialysing against 5 mM NaOH containing 1% (m/v) NaCl five times each for 12 h at 10°C and then exhaustively against demineralised water. Control polymers were prepared and purified in the same way as described for the conjugates; however, EDAC was omitted during the coupling reaction (Table I). The polymer-bacitracin conjugates and controls were lyophilised by drying frozen aqueous polymer solutions at - 30°C and 0.01 mbar (Christ beta 1-8K; Osterode am Harz, Germany) and stored at 4°C.
5. Inhibition studies with membrane-bound aminopeptidase N
The inhibitory effect of the conjugates was tested on intact bovine nasal mucosa and porcine intestinal mucosa, as described previously [10]. In brief, a plastic cylinder with an internal surface area of 1.77 cm2 was placed vertically on top of the mucosal side of the intestinal tissue and clamped. PAA2-Bac and PCP-Bac conjugates were prepared in TBS in a final concentration of 0.5% (m/v). These solutions were equilibrated at 37°C, added into the cylinder and incubated for 60 min. After adding 1 ml of L-leucine-p-nitroanilide (2 mM, 37°C) samples of 300 µl were withdrawn at predetermined time points. To stop the reaction 20 µl of 20% (v/v) TFA was added and samples were centrifuged at 20 000 g, at 4°C for 5 min. A 200-µl sample of the supernatant was transferred to a microtitre plate and the absorbance was measured at 405 nm. TBS and 0.124% (m/v) of unbound bacitracin (corresponding to the bacitracin content of 0.5% (m/v) conjugate) served as controls.
Table I - Concentrations of reagents used for the preparation of PCPBac and PAA2-Bac conjugates and resulting bacitracin content (means ± SD, n = 3). Polymer
Polymer conc. % (m/v)
Bacitracin % (m/v)
EDAC
Amount of coupled bacitracin % (m/m)
PAA2-Bac PAA2-control
0.5 0.5
0.25 0.25
50 mM -
24.4 ± 0.5 -
PCP-Bac PCP-control
0.5 0.5
0.25 0.25
50 mM -
25.3 ± 1.5 -
6. Statistical data analysis
Statistical data analyses were performed using the Student t test with p < 0.05 as the minimal level of significance. Calculations were done using the software Xlstat version 5.1 v1. 496
J. DRUG DEL. SCI. TECH., 14 (6) 495-498 2004
Polymer-enzyme inhibitor conjugates: influence of the molecular mass on the inhibition of membrane-bound aminopeptidase N activity V.M. Leitner, A. Bernkop-Schnürch
II. RESULTS AND DISCUSSION 1. Characterisation of the PAA2-Bac and PCP-Bac conjugates
140 120
The presumable chemical substructure of the poly(acrylic acid)-bacitracin A conjugates is shown in Figure 1. The lyophilised polymers appeared white and of fibrous structure and were quickly swellable in water and buffer solutions. The amount of bacitracin coupled to the polymers was photometrically determined. Purified polymers prepared in the same way, but omitting EDAC during the coupling reaction served as negative controls. As shown in Table I, the PAA2-Bac and PCP-Bac conjugates exhibited comparable amounts of bacitracin. The amount of bacitracin within the negative controls was negligible, demonstrating the efficiency of the purification method.
p-nitroaniline [µmol/l]
100 80 60 40 20 0
2. Influence of PAA2-Bac and PCP-Bac conjugates on the activity of isolated aminopeptidase N
0
Figure 2 shows the effect of PAA2-Bac, PCP-Bac and unbound bacitracin on the activity of isolated aminopeptidase N. For a positive control, bound and unbound bacitracin was omitted during the enzymatic reaction. Due to the covalent attachment of bacitracin both conjugates inhibited aminopeptidase N activity, but no significant difference in the inhibitory effect of the conjugates could be detected. Therefore, differences in the molecular mass of the conjugates do not influence their inhibitory activity towards isolated aminopeptidase N. The inhibitory activity of unbound bacitracin cannot be directly compared with that of the conjugated, as the chemical modification may lead to changes in binding affinity and/or an altered diffusion behaviour due to immobilization.
2
4
6
8
10
time [min] Figure 2 - Inhibition of isolated aminopeptidase N (2.5 µg/ml TBS) by 0.5% (m/v) PAA2-Bac (O), 0.5% (m/v) PCP-Bac (◆) and 0.124% (m/v) unbound bacitracin (■) in comparison to control buffer (X). Pre-incubation time 1 h; substrate: 0.125 mg of L-leucine-p-nitroanilide/ml TBS; each point represents the mean ± SD of three experiments.
450 400
p-nitroaniline [µmol/l]
350
3. Influence of the molecular mass of PAA2-Bac and PCP-Bac conjugates on the activity of membrane-bound aminopeptidase N
Aminopeptidase N inhibition studies were performed on intact intestinal and nasal mucosa. As depicted in Figure 3, both PAA-bac conjugates were able to inhibit membranebound aminopeptidase N, however, the PAA2-Bac conjugate showed a significantly stronger inhibitory effect than the high molecular mass conjugate PCP-Bac. This result cannot be explained by a difference in the enzyme inhibitory efficacy of the two conjugates as both of them exhibited similar inhibitory activities versus isolated aminopeptidase N (Figure 2). As the PAA2-Bac molecule is approximately 1500-fold smaller than PCP-Bac, its stronger inhibitory effect on membrane-bound aminopeptidase N may be the result of a higher amount of PAA2-Bac conjugate that was able to diffuse through the mucus layer to get in direct contact with the enzyme. The mucus layer covering the gastrointestinal epithelia was reported to represent a barrier for the direct interaction between membrane-bound enzymes and enzyme inhibitors immobilised in a mucoadhesive polymer [5]. Therefore, the question whether a direct contact between bacitracin and aminopeptidase N is necessary for enzyme inhibition seems to be relevant for the interpretation of the results. Mäkinen [11] was able to provide evidence for a direct inhibitory effect of bacitracin on aminopeptidase N. For instance, this property of bacitracin to bind proteases allows the use of the antibiotic for protease
300 250 200 150 100 50 0 0
10
20
30 time [min]
40
50
60
Figure 3 - Inhibitory effect of 0.5% (m/v) PAA2-Bac (O), 0.5% (m/v) PCP-Bac (◆) and 0.124% (m/v) unbound bacitracin (■) toward membrane-bound aminopeptidase N from intestinal mucosa in comparison to control buffer (X). Pre-incubation time 1 h; substrate: 0.125 mg of L-leucine-p-nitroanilide/ml TBS; each point represents the mean ± SD of three experiments.
purification by means of bacitracin-affinity chromatography [12]. Furthermore, bacitracin seems to exhibit metal-complexing capabilities as it was shown to inhibit metalloproteases [13]. According to this, polymer/bacitracin conjugates can be regarded as polymer/inhibitor conjugates of mixed mechanism of inhibition. The isolated aminopeptidase N hydrolysed 2.8 nmol substrate/min, whereas membrane bound aminopeptidase N showed a turnover of 13.2 nmol substrate/min, which represents a 4.7-fold higher activity. However, the activity of the isolated and membrane bound enzymes cannot be directly compared 497
J. DRUG DEL. SCI. TECH., 14 (6) 495-498 2004
Polymer-enzyme inhibitor conjugates: influence of the molecular mass on the inhibition of membrane-bound aminopeptidase N activity V.M. Leitner, A. Bernkop-Schnürch
with each other due to likely differences in binding constants and/or diffusion behaviour. Unbound bacitracin showed the strongest inhibitory effect on isolated as well as on membrane-bound aminopeptidase N. This may be attributed to an easier accessibility of the unbound inhibitor for the protease or to a loss in activity of the immobilized bacitracin due to the coupling process. Since unbound bacitracin can pass through the membrane, an additional dilution effect might be expected under in vivo conditions, leading to a comparatively weaker inhibitory effect in comparison to the immobilized inhibitor [9]. No differences in the inhibitory effects of the two PAA-Bac conjugates could be found in aminopeptidase N inhibition studies on intact nasal mucosa (data not shown). This finding can be explained by differences in the thickness of the mucus layers. The mucus layer of intestine and colon and was reported to vary between 50 and 450 µm, whereas the respiratory part of the nasal cavity is only 0.5-2 µm thick [14]. The comparatively thin mucus layer of the nasal cavity seems therefore not to represent a real diffusion barrier for the PAA-Bac conjugates. As both conjugates can easily get in contact with the membrane-bound aminopeptidase N, they show a similar inhibitory effect. The results of this study are in good agreement with a recent interpenetration study performed with fluorescent-labelled poly(acrylic acid) of increasing molecular mass (2-3000 kDa). It showed that even PCP could penetrate the mucus gel layer of intestinal mucosa in significant quantities, whereby the amount of interpenetrated polymer increased with decreasing molecular mass of the polymer [15].
3.
4.
5.
6. 7.
8.
9. 10.
11. 12. 13.
*
* *
14.
PAA2-Bac showed a significantly stronger inhibitory effect on membrane-bound aminopeptidase N than PCP-Bac, whereas no differences in the inhibition of isolated aminopeptidase N could be found. Due to its low molecular mass, PAA2-Bac could more easily diffuse into the mucus layer to get in contact with the membrane-bound enzyme. These results provide helpful basic information for the design of non-invasive (poly)peptide delivery systems via mucosal membranes based on multifunctional polymers.
15.
ACKNOWLEDGEMENTS The authors wish to thank Mr. Ströbl and co-workers from the slaughterhouse Totzenbach for the supply of bovine nasal and porcine intestinal mucosa. This work was supported by Grant No. P15373-MOB from the Fonds zur Förderung der wissenschaftlichen Forschung (FWF) to A. Bernkop-Schnürch.
REFERENCES 1. 2.
ments, limits and novel approaches to bioadhesion. - Eur. J. Drug Metab. Pharmacokinet., 21, 139-148, 1996. LANGGUTH P., MERKLE H.P., AMIDON G. - Oral absorption of peptides-the effect of absorption side and enzyme inhibition on the systemic availability of metkephamid. - Pharm. Res., 11, 528-535, 1994. YAMAMOTO A., TANIGUCHI T., RIKYUN K., TSUJI T., FUJITA T., MURAKAMI M., MURANISHI S. - Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats. - Pharm. Res., 11, 1496-1500, 1994. BERNKOP-SCHNÜRCH A. - Polymer-inhibitor conjugates: a promising strategy to overcome the enzymatic barrier to perorally administered (poly)peptide drugs? - STP Pharma Sciences, 9, 78-87, 1999. BERNKOP-SCHNÜRCH A., DUNDALEK K. - Novel bioadhesive drug delivery system protecting (poly)peptides from gastric enzymatic degradation. - Int. J. Pharm., 138, 75-83, 1996. PLATE N.A., VALUEV I.L., SYTOV G.A., VALUEV L.I. - Mucoadhesive polymers with immobilized protease inhibitors for oral administration of protein drugs. - Biomaterials, 23, 1673-1677, 2002. LARIONOVA N.I., VARTANOV S.S., SOROKINA N.V., GLADYSHEVA I.P., VARFOLOMEYEV S.D. - Conjugation of the Bowman-Birk soybean proteinase inhibitor with hydroxyethylstarch. - Appl. Biochem. Biotechnol., 62, 175-182, 1997. BERNKOP-SCHNÜRCH A., MARSCHÜTZ M.K. - Development and in vivo evaluation of systems to protect peptide drugs from aminopeptidase N. - Pharm. Res., 14, 181-185, 1997. BERNKOP-SCHNÜRCH A., ZARTI H., WALKER G.F. - Thiolation of polycarbophil enhances its inhibition of soluble and intestinal brush border membrane bound aminopeptidase N. - J. Pharm. Sci., 90, 1907-1914, 2001 MÄKINEN K.K. - Inhibition by bacitracin of some hydrolytic enzymes. - Int. J. Protein Res., 4, 21-28, 1972 IBRAHIM-GRANET O., BERTRAND O. - Separation of proteases: old and new approaches. - J. Chromatogr. B Biomed. Appl., 684, 239-263, 1996. MING L.J. - Structure and function of «metalloantibiotics». - Med. Res. Rev., 23, 697-762, 2003. KHANVILKAR K., DONOVAN M.D., FLANAGAN D.R. - Drug transfer through mucus. - Adv. Drug Deliv. Rev., 48, 173-193, 2001. IMAM M.E., HORNOF M., VALENTA C., REZNICEK G., BERNKOP-SCHNÜRCH A. - Evidence for the interpenetration of mucoadhesive polymers into the mucus gel layer. - STP Pharma Sci., 13, 171-176, 2003.
DUCHENE D., PONCHEL G. - Principle and investigation of the bioadhesion mechanism of solid dosage forms. - Biomaterials, 13, 709-714, 1992. LEHR C.M. - From sticky stuff to sweet receptors - achieve-
MANUSCRIPT Received 2 February 2004, accepted for publication 20 April 2004.
498
Polymer-enzyme inhibitor conjugates: influence of the molecular mass on the inhibition of membrane-bound aminopeptidase N activity V.M. Leitner1, A. Bernkop-Schnürch2* Institute of Pharmaceutical Technology and Biopharmaceutics, Centre of Pharmacy, University of Vienna, Althanstr. 14, 1090 Vienna, Austria 2 Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University Innsbruck, Innrain 52, Josef Möller Haus, 6020 Innsbruck, Austria *Correspondence: [email protected] 1
Conjugates of mucoadhesive polymers and protease inhibitors have been developed to protect incorporated (poly)peptides from enzymatic attack by luminally secreted or membrane-bound proteases. Within the present study, the influence of the molecular mass of such polymer conjugates on the inhibition of membrane-bound enzyme activity was evaluated. The aminopeptidase N inhibitor bacitracin (Bac) was covalently immobilised on the mucoadhesive polymer model poly(acrylic acid) with a molecular mass of 2 kDa (PAA2) and 3000 kDa (polycarbophil, PCP). The resulting conjugates PAA2-Bac and PCP-Bac exhibited comparable amounts of bacitracin (24.4 and 25.3% (m/m), respectively) and were used to perform inhibition studies on isolated and membrane-bound aminopeptidase N. The conjugates showed no differences in the inhibition of isolated aminopeptidase N, whereas PAA2-Bac exhibited a significantly higher inhibitory activity versus membrane-bound aminopeptidase than PCP-Bac. Due to its lower molecular mass, PAA2-Bac could more easily diffuse into the mucus layer to get in contact with the enzyme. Obtained results should provide helpful basic knowledge for the development of multifunctional polymers as auxiliary agents for the oral administration of peptide drugs. Key words: Mucoadhesion – Poly(acrylic acid) – Polymer-inhibitor conjugate – Bacitracin.
teases [e.g. 7, 8]. In order to substantiate our knowledge for the development of more effective polymer-inhibitor conjugates, it was the aim of the present study to evaluate the influence of the molecular mass of the conjugates on their efficacy to inhibit the most abundant brush border membrane bound enzyme aminopeptidase N. As mucoadhesive polymer model, poly(acrylic acid) with an average molecular mass of 2 kDa (PAA2) and 3000 kDa (polycarbophil, PCP) was chosen. Bacitracin (Bac), a cyclic dodecapeptide which has been reported to inhibit membranebound aminopeptidase N [9] was covalently linked to these polymers as illustrated in Figure 1. Both poly(acrylic acid)-
The combination of mucoadhesive polymers with enzyme inhibitors as means of delivering therapeutically active peptides and proteins via mucous membranes has received increasing attention in recent years. Mucoadhesive polymers, on the one hand, are supposed to ensure a prolonged and intimate contact of the delivery system with the absorbing membrane leading to improved drug absorption [1, 2] and a reduced presystemic degradation of the drug by shortening the way between the delivery system and the absorbing membrane. Enzyme inhibitors, on the other hand, are able to protect therapeutic (poly)peptides from enzymatic degradation with luminally secreted and/or membrane-bound proteases. Although it has been demonstrated that co-administered protease inhibitors are very efficient in improving the oral bioavailability of therapeutic (poly)peptides [e.g. 3, 4], their use remains questionable as they may cause side effects, such as an unintended disturbance of protein digestion, pancreatic hypersecretion due to a luminal feedback regulation as well as systemic toxic side effects [5]. To overcome these problems, the immobilisation of protease inhibitors to an unabsorbable drug carrier matrix such as mucoadhesive polymers seems to be a promising strategy combining the favourable properties of both excipients [6]. As the immobilised inhibitor remains concentrated on the drug carrier matrix, disturbing dilution effects as well as inhibitor absorption can be excluded leading to an enhanced protective effect whereby a reduced share of this auxiliary agent in the dosage form may be sufficient. To date, various polymer-inhibitor conjugates have been generated protecting incorporated (poly)peptides from enzymatic attack by luminally secreted or membrane-bound pro-
COOH
O
HN
S
H3C
N
O
CH3 His
D-Asp
D-Phe Ile
Asn ε
D-Orn
Leu D-Glu
Lys .α Ile
Figure 1 - Presumable chemical substructure of poly(acrylic acid)-bacitracin A conjugates. Bacitracin A is the major component of commercial bacitracin, which is a mixture of at least nine bacitracins. 495
J. DRUG DEL. SCI. TECH., 14 (6) 495-498 2004
Polymer-enzyme inhibitor conjugates: influence of the molecular mass on the inhibition of membrane-bound aminopeptidase N activity V.M. Leitner, A. Bernkop-Schnürch
bacitracin conjugates (PAA2-Bac, PCP-Bac) exhibited comparable amounts of immobilised bacitracin. The inhibitory effect of the conjugates was determined on isolated aminopeptidase N in comparison to an equal amount of unbound bacitracin. Furthermore aminopeptidase N inhibition studies were performed on porcine intestinal and bovine nasal mucosa to study the influence of the molecular size of the conjugates on their enzyme inhibitory properties.
3. Determination of the bacitracin content of the conjugates
The bacitracin content (%) was determined by quantifying the amount of covalently bound bacitracin within the polymer photometrically (Lambda 16; Perkin-Elmer, Vienna, Austria). PAA2-Bac, PCP-Bac and unmodified control polymers were hydrated in demineralised water to obtain a concentration of 0.125% (m/v) and the absorbance at 255 nm was determined (n = 3). As reference, 0.125% (m/v) solutions of PAA2 and PCP were used, respectively. The amount of immobilised bacitracin on the polymers was calculated by using a standard curve obtained by the measurement of a series of reference solutions containing increasing amounts of bacitracin.
I. MATERIALS AND METHODS 1. Materials
Polycarbophil with an average molecular mass of 3000 kDa (PCP) was kindly provided by Noveon (Raubling, Germany). Poly(acrylic acid) with an average molecular mass of 2 kDa (PAA2), microsomal aminopeptidase (aminopeptidase N; EC 3.4.11.2.), L-leucine-p-nitroanilide, bacitracin and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) were purchased from Sigma (St. Louis, MO, United States). All other chemicals were of reagent grade and obtained from Sigma (St. Louis, MO, United States).
4. Inhibition studies with isolated aminopeptidase N
L-leucine-p-nitroanilide was used as enzyme substrate for degradation studies with aminopeptidase N. The PAA2-Bac and PCP-Bac conjugates were dissolved in TBS (50 mM Tris-HCl buffer containing 2.9% NaCl, pH 7.5) in a concentration of 0.5% (m/v) and aliquots of 100 µl were transferred into wells of a microtitration plate. Aminopeptidase N (50 µl of 10 µg/ml TBS) was added and the mixtures were incubated for 1 h at room temperature. Thereafter, 50 µl of the substrate solution (0.5 mg of L-leucine-p-nitroanilide/ml TBS) was added and the increase in absorbance was immediately recorded at 405 nm and room temperature using a microtitration plate reader (Anthos reader 2001, Salzburg, Austria). TBS and unbound bacitracin dissolved in TBS in a concentration of 0.124% (m/v) (equivalent to the bacitracin content of 0.5% (m/v) conjugate) were used as controls. Concentrations of the hydrolyzed substrate were calculated by interpolation from a standard curve obtained from the analysis of solutions containing increasing amounts of p-nitroaniline [9].
2. Synthesis of PAA2-Bac and PCP-Bac conjugates
The available amino-groups of bacitracin were covalently bound to the carboxyl groups of PAA2 and PCP via a condensation reaction mediated by EDAC, as described previously [9]. The poly(acrylates) (150 mg) were hydrated in 30 ml of demineralised water and the pH was adjusted to 6 with 1 M NaOH. EDAC was added in a final concentration of 50 mM and mixtures were stirred for 15 min at room temperature in order to activate the carboxylic acid groups of the polymers. Then, 75 mg of bacitracin were added and the reaction was allowed to proceed for 3 h with stirring at room temperature. The resulting polymer-bacitracin conjugates were purified by dialysing against 5 mM NaOH containing 1% (m/v) NaCl five times each for 12 h at 10°C and then exhaustively against demineralised water. Control polymers were prepared and purified in the same way as described for the conjugates; however, EDAC was omitted during the coupling reaction (Table I). The polymer-bacitracin conjugates and controls were lyophilised by drying frozen aqueous polymer solutions at - 30°C and 0.01 mbar (Christ beta 1-8K; Osterode am Harz, Germany) and stored at 4°C.
5. Inhibition studies with membrane-bound aminopeptidase N
The inhibitory effect of the conjugates was tested on intact bovine nasal mucosa and porcine intestinal mucosa, as described previously [10]. In brief, a plastic cylinder with an internal surface area of 1.77 cm2 was placed vertically on top of the mucosal side of the intestinal tissue and clamped. PAA2-Bac and PCP-Bac conjugates were prepared in TBS in a final concentration of 0.5% (m/v). These solutions were equilibrated at 37°C, added into the cylinder and incubated for 60 min. After adding 1 ml of L-leucine-p-nitroanilide (2 mM, 37°C) samples of 300 µl were withdrawn at predetermined time points. To stop the reaction 20 µl of 20% (v/v) TFA was added and samples were centrifuged at 20 000 g, at 4°C for 5 min. A 200-µl sample of the supernatant was transferred to a microtitre plate and the absorbance was measured at 405 nm. TBS and 0.124% (m/v) of unbound bacitracin (corresponding to the bacitracin content of 0.5% (m/v) conjugate) served as controls.
Table I - Concentrations of reagents used for the preparation of PCPBac and PAA2-Bac conjugates and resulting bacitracin content (means ± SD, n = 3). Polymer
Polymer conc. % (m/v)
Bacitracin % (m/v)
EDAC
Amount of coupled bacitracin % (m/m)
PAA2-Bac PAA2-control
0.5 0.5
0.25 0.25
50 mM -
24.4 ± 0.5 -
PCP-Bac PCP-control
0.5 0.5
0.25 0.25
50 mM -
25.3 ± 1.5 -
6. Statistical data analysis
Statistical data analyses were performed using the Student t test with p < 0.05 as the minimal level of significance. Calculations were done using the software Xlstat version 5.1 v1. 496
J. DRUG DEL. SCI. TECH., 14 (6) 495-498 2004
Polymer-enzyme inhibitor conjugates: influence of the molecular mass on the inhibition of membrane-bound aminopeptidase N activity V.M. Leitner, A. Bernkop-Schnürch
II. RESULTS AND DISCUSSION 1. Characterisation of the PAA2-Bac and PCP-Bac conjugates
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The presumable chemical substructure of the poly(acrylic acid)-bacitracin A conjugates is shown in Figure 1. The lyophilised polymers appeared white and of fibrous structure and were quickly swellable in water and buffer solutions. The amount of bacitracin coupled to the polymers was photometrically determined. Purified polymers prepared in the same way, but omitting EDAC during the coupling reaction served as negative controls. As shown in Table I, the PAA2-Bac and PCP-Bac conjugates exhibited comparable amounts of bacitracin. The amount of bacitracin within the negative controls was negligible, demonstrating the efficiency of the purification method.
p-nitroaniline [µmol/l]
100 80 60 40 20 0
2. Influence of PAA2-Bac and PCP-Bac conjugates on the activity of isolated aminopeptidase N
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Figure 2 shows the effect of PAA2-Bac, PCP-Bac and unbound bacitracin on the activity of isolated aminopeptidase N. For a positive control, bound and unbound bacitracin was omitted during the enzymatic reaction. Due to the covalent attachment of bacitracin both conjugates inhibited aminopeptidase N activity, but no significant difference in the inhibitory effect of the conjugates could be detected. Therefore, differences in the molecular mass of the conjugates do not influence their inhibitory activity towards isolated aminopeptidase N. The inhibitory activity of unbound bacitracin cannot be directly compared with that of the conjugated, as the chemical modification may lead to changes in binding affinity and/or an altered diffusion behaviour due to immobilization.
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time [min] Figure 2 - Inhibition of isolated aminopeptidase N (2.5 µg/ml TBS) by 0.5% (m/v) PAA2-Bac (O), 0.5% (m/v) PCP-Bac (◆) and 0.124% (m/v) unbound bacitracin (■) in comparison to control buffer (X). Pre-incubation time 1 h; substrate: 0.125 mg of L-leucine-p-nitroanilide/ml TBS; each point represents the mean ± SD of three experiments.
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p-nitroaniline [µmol/l]
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3. Influence of the molecular mass of PAA2-Bac and PCP-Bac conjugates on the activity of membrane-bound aminopeptidase N
Aminopeptidase N inhibition studies were performed on intact intestinal and nasal mucosa. As depicted in Figure 3, both PAA-bac conjugates were able to inhibit membranebound aminopeptidase N, however, the PAA2-Bac conjugate showed a significantly stronger inhibitory effect than the high molecular mass conjugate PCP-Bac. This result cannot be explained by a difference in the enzyme inhibitory efficacy of the two conjugates as both of them exhibited similar inhibitory activities versus isolated aminopeptidase N (Figure 2). As the PAA2-Bac molecule is approximately 1500-fold smaller than PCP-Bac, its stronger inhibitory effect on membrane-bound aminopeptidase N may be the result of a higher amount of PAA2-Bac conjugate that was able to diffuse through the mucus layer to get in direct contact with the enzyme. The mucus layer covering the gastrointestinal epithelia was reported to represent a barrier for the direct interaction between membrane-bound enzymes and enzyme inhibitors immobilised in a mucoadhesive polymer [5]. Therefore, the question whether a direct contact between bacitracin and aminopeptidase N is necessary for enzyme inhibition seems to be relevant for the interpretation of the results. Mäkinen [11] was able to provide evidence for a direct inhibitory effect of bacitracin on aminopeptidase N. For instance, this property of bacitracin to bind proteases allows the use of the antibiotic for protease
300 250 200 150 100 50 0 0
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30 time [min]
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Figure 3 - Inhibitory effect of 0.5% (m/v) PAA2-Bac (O), 0.5% (m/v) PCP-Bac (◆) and 0.124% (m/v) unbound bacitracin (■) toward membrane-bound aminopeptidase N from intestinal mucosa in comparison to control buffer (X). Pre-incubation time 1 h; substrate: 0.125 mg of L-leucine-p-nitroanilide/ml TBS; each point represents the mean ± SD of three experiments.
purification by means of bacitracin-affinity chromatography [12]. Furthermore, bacitracin seems to exhibit metal-complexing capabilities as it was shown to inhibit metalloproteases [13]. According to this, polymer/bacitracin conjugates can be regarded as polymer/inhibitor conjugates of mixed mechanism of inhibition. The isolated aminopeptidase N hydrolysed 2.8 nmol substrate/min, whereas membrane bound aminopeptidase N showed a turnover of 13.2 nmol substrate/min, which represents a 4.7-fold higher activity. However, the activity of the isolated and membrane bound enzymes cannot be directly compared 497
J. DRUG DEL. SCI. TECH., 14 (6) 495-498 2004
Polymer-enzyme inhibitor conjugates: influence of the molecular mass on the inhibition of membrane-bound aminopeptidase N activity V.M. Leitner, A. Bernkop-Schnürch
with each other due to likely differences in binding constants and/or diffusion behaviour. Unbound bacitracin showed the strongest inhibitory effect on isolated as well as on membrane-bound aminopeptidase N. This may be attributed to an easier accessibility of the unbound inhibitor for the protease or to a loss in activity of the immobilized bacitracin due to the coupling process. Since unbound bacitracin can pass through the membrane, an additional dilution effect might be expected under in vivo conditions, leading to a comparatively weaker inhibitory effect in comparison to the immobilized inhibitor [9]. No differences in the inhibitory effects of the two PAA-Bac conjugates could be found in aminopeptidase N inhibition studies on intact nasal mucosa (data not shown). This finding can be explained by differences in the thickness of the mucus layers. The mucus layer of intestine and colon and was reported to vary between 50 and 450 µm, whereas the respiratory part of the nasal cavity is only 0.5-2 µm thick [14]. The comparatively thin mucus layer of the nasal cavity seems therefore not to represent a real diffusion barrier for the PAA-Bac conjugates. As both conjugates can easily get in contact with the membrane-bound aminopeptidase N, they show a similar inhibitory effect. The results of this study are in good agreement with a recent interpenetration study performed with fluorescent-labelled poly(acrylic acid) of increasing molecular mass (2-3000 kDa). It showed that even PCP could penetrate the mucus gel layer of intestinal mucosa in significant quantities, whereby the amount of interpenetrated polymer increased with decreasing molecular mass of the polymer [15].
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PAA2-Bac showed a significantly stronger inhibitory effect on membrane-bound aminopeptidase N than PCP-Bac, whereas no differences in the inhibition of isolated aminopeptidase N could be found. Due to its low molecular mass, PAA2-Bac could more easily diffuse into the mucus layer to get in contact with the membrane-bound enzyme. These results provide helpful basic information for the design of non-invasive (poly)peptide delivery systems via mucosal membranes based on multifunctional polymers.
15.
ACKNOWLEDGEMENTS The authors wish to thank Mr. Ströbl and co-workers from the slaughterhouse Totzenbach for the supply of bovine nasal and porcine intestinal mucosa. This work was supported by Grant No. P15373-MOB from the Fonds zur Förderung der wissenschaftlichen Forschung (FWF) to A. Bernkop-Schnürch.
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MANUSCRIPT Received 2 February 2004, accepted for publication 20 April 2004.
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