Recovery of Rat Nasal Mucosa from the Effects of Aminopeptidase Inhibitors

Recovery of Rat Nasal Mucosa from the Effects of Aminopeptidase Inhibitors MUNIRA. HUSSAIN,CHRISTOPHER A. KOVAL, ASHOK 6. SHENVI, AND BRUCEJ. AUNGST' Received June 6, 1989, from DuPont Company, Medical Products and Central Research and Development Departments, Experimental Station, Accepted for publication August 8, 1989. Wilmington, DE 79880-0400. Abstract 0Aminopeptidase inhibitors may be useful for improving the systemic bioavailability of peptide drugs administered nasally or by other

routes. Preferably,their effects would be rapidly reversible. The recovery of peptide hydrolytic activity after exposure of the rat nasal cavity to various aminopeptidase inhibitors was evaluated. Leucine enkephalin (0.1 mM) was used as a model peptide which is predominantly metabolized by aminopeptidases nasally. All experiments involved in situ perfusion of the rat nasal cavity with leucine enkephalin and the inhibitor for 90 min, followed by a washout with saline, and finally a second experimental phase of perfusion with leucine enkephalin but no inhibitor. Boroleucine (0.1 pM) was a potent inhibitor of leucine enkephalin metabolism, and, after its removal, the leucine enkephalin metabolism rate returned to control levels. Much higher concentrations of bestatin (0.1 mM) and puromycin (1 mM) did not inhibit leucine enkephalin metabolism as much as did boroleucine. Furthermore, when these inhibitors were washed out, the rates of leucine enkephalin disappearance rebounded to higher than control levels. With bestatin this could have been partially due to membrane disruption, as evidenced by significantly increased protein concentrations in the perfusates. However, protein release was much lower than that caused by sodium glycocholate, a nasal membrane permeation enhancer. In considering the use of peptidase inhibitors as pharmaceutical adjuvants for peptide drugs, the aminoboronic acid derivatives, including boroleucine,have the advantages of efficacy at very low concentrations and reversibility of effects.

Nasal administration is an alternative to injecting peptide drugs, and several peptide and protein drug candidates have been in clinical trials in dosage forms for nasal administration. Nasal bioavailability of these compounds is generally much greater than oral bioavailability because the proteolytic digestive enzymes of the gut are circumvented. However, the nasal bioavailability of peptides is usually
used. Although this method does not provide data on systemic absorption, it enables study of the interactions of nasal mucosal enzymes, peptide substrates, and metabolic inhibitors, and this has important implications for systemic absorption.

Experimental Section Boroleucine [3-methyl-1-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane2-yl)-l-butamine, trifluoroacetic acid salt] was prepared as described previously.1 Leucine enkephalin (Tyr-Gly-Gly-Phe-Leu), destyrosyl-leucine enkephalin (Gly-Gly-Phe-Leu), bestatin, and puromycin were obtained from Sigma Chemical Company. Leucine enkephalin metabolism in a solution perfused through the rat nasal cavity was characterized. The methods for perfusion of the rat nasal cavity were as described previously.z33Lewis rats weighing ~ 3 0 g0 were maintained under pentobarbital anesthesia throughout the study; rats were usually lying on a heating blanket. A cannula was inserted via the esophagus to abut the posterior part of the nasal cavity. Flow of the perfusate through the nasal cavity was from the cannula, through the nasal cavity, and out the nares. The effluent was collected with a funnel into a reservoir maintained at 37 "C. The reservoir solution was recirculated and was the site of sample removal. The initial volume of perfusate solution was 10 mL, and 0.1-mL samples were taken. The flow rate was 1.5 mL/min. The perfusion solution was 0.1 M phosphate buffer, pH 7.4,and initially contained leucine enkephalin a t a concentration of 60 pg/mL (0.1 mM). Boroleucine, bestatin, or puromycin were also added at various concentrations in some experiments. There were two experimental phases for each rat. In phase I, the perfusate contained leucine enkephalin and the aminopeptidase inhibitor (boroleucine, bestatin, puromycin, or control with no inhibitor). After phase I, the nasal cavity was perfused for 30 min with 50 mL of saline, which was not recirculated. Then, in phase 11, the perfusate contained only leucine enkephalin, without the aminopeptidase inhibitor. "he samples were immediately diluted with two volumes of 0.1 M citric acid to quench further metabolism. Leucine enkephalin and des-tyrosyl-leucine enkephalin concentrations were then determined by HPLC. The mobile phase was 0.1 M monobasic sodium phosphate:phosphoric acidacetonitrile (916:5:275) and the flow rate was 1.7 mL/min. An octylsilane column (DuPont) was used. Detection was by UV absorbance at 210 nm. Typical retention times for leucine enkephalin and des-tyrosyl-leucine enkephalin were 7.3 and 4.8 min, respectively. Experiments were performed in separate rats as described above, but without leucine enkephalin in the perfusion solution. The effects of the aminopeptidase inhibitors on protein release from the nasal cavity were evaluated. A perfusion solution containing 1%sodium glycocholate (Sigma) was also evaluated in this experiment. Protein concentrations in the perfusion solution were determined using a commercially available kit (BCA Protein Assay Reagent; Pierce). The aminopeptidase inhibitors and sodium glycocholate did not interfere in the protein determinations.

Results and Discussion Leucine enkephalin solutions were perfused through the nasal cavities of anesthetized rats. The studies were done in two phases. During phase I, the perfusion solutions contained leucine enkephalin along with an aminopeptidase inhibitor. The control group was perfused with leucine enkephalin oO22-3549/90/05oO-O398$0 7 .OO/O 0 1990, American Pharmaceutical Association

alone. A perfusion rinse with 50 mL of saline followed. Then, in phase 11, all groups were perfused with buffer containing leucine enkephalin as in phase I, but with no inhibitor. The concentrations of leucine enkephalin in the recirculated perfusion solution decayed with time, and des-tyrosyl-leucine enkephalin, the product of aminopeptidase metabolism, appeared (data not reported). Figure 1 shows the leucine enkephalin concentration versus time profiles for the control group, with no inhibitor, and for rats for which the perfusion solution also contained boroleucine. In all groups of rats, the decay of leucine enkephalin was apparently zero order, a t least within the first 60 min. Decay rate constants were calculated as the slopes of individual concentration versus time profiles, using 0-60-min time points or 0-90-min time points in the presence of boroleucine. Disappearance from the perfusion solution was apparently not due to absorption because when metabolism was inhibited, the leucine enkephalin concentration hardly decreased. This has also been shown in previous studies employing similar experimental methods with leucine enkephalin.2~3The perfusion protocol submits a large volume of solution to a proportionally small area of membrane for a discontinuous period of time, and this may account for the absence of absorption. In control rats, there was little difference in leucine enkephalin disappearance rates between phases I and 11, both of which were in the absence of an aminopeptidase inhibitor. Boroleucine (0.1 pM)effectively prevented leucine enkephalin degradation. After the nasal cavity was rinsed with saline, the leucine enkephalin disappearance rates returned to levels similar to control rats (Figure 11, indicating that the inhibition of aminopeptidase activity was reversible. The other aminopeptidase inhibitors, bestatin and puromycin, were both much less potent than boroleucine in preventing leucine enkephalin degradation. Rate constants for all groups are summarized in Table I. The effects of bestatin and puromycin were concentration dependent, but in the presence of 0.1 mM bestatin or 1 d puromycin, leucine enkephalin still was metabolized more rapidly than in the presence of 0.1 pM boroleucine. After perfusion with lower concentrations of bestatin (0.01 mM) and puromycin (0.1 mM) in phase I, the leucine enkephalin disappearance rates returned to control levels in phase 11. However, when 10-fold higher inhibitor I

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Table CLeucine Enkephaiin Disappearance Rate Constants (mean f SEM) in Perfusates of the Rat Nasal Cavity In the Presence of Various Aminopeptidase Inhibitors (Phase I) and after Washout of the inhibitors (Phase ii) Inhibitor

Control (no inhibitor) Boroleucine (0.1pM) Bestatin (0.01 mM) Bestatin (0.1mM) Puromycin (0.1mM) Puromycin (1 mM)

Disappearance Rate Constant (4, pg/mUmin Phase I Phase II 0.45 2 0.05 0.006a.b

0.0342 0.21 2 0.11 -r0.32 0.14 2

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Figure 1-Leucine enkephalin concentration versus time profiles in solutions perfused through the nasal cavity of control rats (0). in the presence of the aminopeptidase inhibitor, boroleucine (0, phase I), and after washout of the boroleucine (phase Ii). Data are expressed as mean + SEM of four rats.

Figure 2-Leucine enkephalin disappearance rate constants in solutions perfused through the rat nasal cavity in the presence of aminopeptidase inhibitors (phase I, shaded bars) and after washout of the inhibitors (phase II, open bars). Journal of Pharmaceutical Sciences I 399 Vol. 79, No. 5, May 1990

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Figure &Protein concentrations in rat nasal cavity perfusates contain1 pM boroleucine (O), 0.1 mM bestatin (A), 1 mM puromycin (0),or 1% sodium glycocholate (V).The additives were present during phase I, which was followed by a saline rinse, and no additives were present in phase 11.

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400 I Journal of Pharmaceutical Sciences Vol. 79, No. 5, May 1990

trations in rats perfused with inhibitors were not different from controls, except for the phase I1 value for rats perfused with 0.1 mM bestatin. Bestatin may therefore have some membrane disruption effect a t concentrations required for inhibition of aminopeptidases. This membrane disruption effect is relatively small, however, in comparison with that seen with sodium glycocholate. Sodium glycocholate and other bile salts have been used in numerous studies to promote nasal peptide and protein absorption by increasing the permeability of the membrane. The rebound in aminopeptidase hydrolysis of leucine enkephalin after puromycin perfusion was apparently not related to a greater release of protein from the mucosa. This study has shown that boroleucine can be used to prevent leucine enkephalin metabolism by the nasal mucosa and that its effect is reversed when the inhibitor is removed. The same cannot be said for the other aminopeptidase inhibitors studied, bestatin and puromycin. Although bestatin and puromycin did inhibit leucine enkephalin metabolism, higher concentrations would be required to approach the level of inhibition seen with boroleucine. After exposure to these concentrations the nasal mucosa did not return to the normal level of aminopeptidase activity. The mechanisms for the rebound of aminopeptidase activity are not known, but may involve membrane disruption.

References and Notes 1. Shenvi, A. B.Biochemistry 1986,25,12861291. 2. Hussain, M.A.; Shenvi, A. B.; Rowe, S. M.; Shefter, E. Pharm. Res. 1989,6,186189. 3. Hussain, A.; Faraj, J.; Aramaki, Y.; Truelove, J. E. Biochem. Biophys. Res. Commun. 1985,133,923-928.