Introduction

Abstract Introduction Results Discussion References

HLE¹ is a serine protease located in the azurophilic granules of the polymorphonuclear leukocytes (PMN). This enzyme has been implicated in the extracellular degradation of structural proteins such as elastin and collagen. Consequently, inhibition of HLE has been targeted as a potential therapy in diseases such as emphysema (1) and cystic fibrosis (2).

To date, numerous reports have been published concerning the synthesis and evaluation of nonpeptidyl, low molecular weight inhibitors of HLE (3-12). The potency of some of these potential inhibitors was screened in vitro, and then promising compounds were assessed for oral activity in hamsters and mice that had been instilled with HLE (8-11). Subsequently, pharmacokinetic studies were performed to determine whether leading drug candidates were bioavailable after oral administration to rats and monkeys. An example of one such orally active inhibitor of HLE is N-[1(R)-(1,3-benzodioxol-5-yl)butyl]-3,3-diethyl-2(S)-[4-[(4-methyl-1-piperazinyl)carbonyl]phenoxy]-4-oxo-1-azetidinecarboxamide (L-694,458²). This report describes the in vitro and in vivo metabolism of this monocyclic -lactam inhibitor in Sprague-Dawley rats and rhesus monkeys. These studies were performed in conjunction with a disposition study of this compound (13) in the two species that were used to determine its toxicological profile.

Materials and Methods

Chemicals. N-[1(R)-(1,3-benzodioxol-5-yl)butyl]-3,3-diethyl-2(S)-[4-[(4-methyl-1-piperazinyl)carbonyl]phenoxy]-4-oxo-1-azetidinecarboxamide (L-694,458, fig. 1A) was synthesized at the Merck Research Laboratories in Rahway, NJ (14). The radiolabeled ([butyl-3,4-[³H]₂]) and pentadeuterated ([butyl-3,4-[²H]₂, piperazinyl-N-C[²H]₃]) forms of L-694,458, and other reference standards (L-742,390 and L-740,447) were prepared at Merck Research Laboratories as well. Radiolabeled L-694,458 was 98% pure, as determined by HPLC analysis. Water was distilled in-house and purified by a Picosystem Ultra unit (Hydro, Garfield, NJ). Methanol and acetonitrile (optima grade), ammonium acetate (HPLC grade), and trifluoroacetic acid (reagent grade) were obtained from Fisher (Springfield, NJ). Argon and nitrogen collision gases were obtained from JWS Technologies (Piscataway, NJ).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   (A) Product ion mass spectrum of parent compound (L-694,458). (B) Source-induced fragmentation mass spectrum of parent compound. Proposed structures of designated fragment ions shown in fig. 2.

In Vitro Incubations. Rat and monkey liver microsomes. Subcellular fractions from rat and monkey livers were prepared by differential centrifugation using established procedures (15). Liver microsomes from Sprague-Dawley rats and rhesus monkeys (2 mg protein/ml) were incubated with L-694,458 (<1 to 100 µM) at 37°C for up to 1 hr in a pH 7.4 phosphate buffer and in the presence of an NADPH-regenerating system (1 mM NADP, 10 units glucose-6-phosphate dehydrogenase, and 20 mM glucose-6-phosphate). The microsomal protein fraction was precipitated with acetonitrile and the mixture was centrifuged. The supernatant was removed and dried under nitrogen, and the dried extract was reconstituted in 1:1 acetonitrile:water prior to HPLC-UV (220 nm) or LC-MS analysis.

Primary rat hepatocyte culture. Hepatocytes were isolated from a male Sprague-Dawley rat and plated in a petri dish at 1.5 × 10⁶ cells/ml for 48 hr with 10 µM dexamethasone (16). Cell viability was determined using trypan blue exclusion and was found to be 93%. The medium was changed at 2, 24, and 48 hr. The substrate was dissolved in DMSO and mixed with culture medium to achieve a final concentration of 50 µM. The final concentration of DMSO did not exceed 1% (v/v). Three ml of medium containing the substrate was added to the plated hepatocytes and incubated for 48 hr at 37°C. At the end of the incubation, the cells were scraped from the plate with a cell scraper. The medium and cell mixture was removed and the plate was washed with 3 ml of methanol. The methanol wash was combined with the medium and cell mixture and the entire volume was dried under nitrogen. Acetonitrile (5 ml) was used to resuspend the parent-related material and the supernatant was transferred to a clean tube. The remaining solid material was resuspended in 2 ml water and extracted with 5 ml ethyl acetate. The ethyl acetate and acetonitrile extracts were combined, dried under nitrogen, and resuspended in 1:1 acetonitrile:water for LC-MS analysis.

Rat intestinal homogenate. Intestines were obtained from six fed male Sprague-Dawley rats. Small intestines were isolated from below the duodenum to just above the cecum. Large intestines were isolated from below the cecum. The intestinal segments were sliced open longitudinally and emptied by rinsing the contents in ice cold sodium chloride solution (0.9%; pH 5.6). Homogenates were prepared by cutting up the intestines into 3- to 5-mm segments and dropping these segments into a hand-held glass homogenizer filled with 5 ml NaCl solution. After homogenization of the tissue, the soluble fraction with buffer was removed and kept on ice until ready for use.

In Vivo Samples. Male Sprague-Dawley rats were used in all experiments as described in the preceding paper (13). Rats weighing 300-400 g (3 to 5 per dose) were dosed with [³H]-labeled L-694,458 orally at 10 mg/kg and iv at 5 mg/kg. Intravenous dosing was via the tail vein or via a previously implanted catheter in the femoral vein for biliary excretion studies, and oral dosing was by gavage.

HPLC-UV and HPLC-Radiometric Analysis. Chromatographic separation of microsomal metabolites was performed isocratically at a flow rate of 1 ml/min using 40% acetonitrile in water with 10 mM ammonium acetate and 0.1% TFA on a DuPont Zorbax SB-CN column (4.6 × 250 mm). In experiments in which mass spectrometry was not used, the UV absorbance of the eluate was monitored at 220 nm and the radioactivity was monitored on-line with a radiometric detector.

LC-MS/MS Analysis. Characterization of metabolites was performed on a Sciex (Perkin Elmer, Concord, Ontario, Canada) Model API III Plus triple-quadrupole mass spectrometer equipped with a Sciex ionspray source operated at 5000 V. Positive ion detection was used in all cases. The collision gas was argon for CID experiments. The source-induced fragmentation (SIF) mass spectra were produced by setting the orifice voltage to 65 or 70 V, which induced the ionized molecules to fragment prior to entering the first quadrupole of the mass spectrometer.

NMR Analysis. NMR spectra were acquired in CD₃OD at 25°C on a Varian Unity 400 MHz spectrometer. Chemical shifts are on a ppm scale relative to the residual solvent CD₂H signal set at 3.30 ppm.

Metabolite Designations. The designation of the individual metabolites has been coded to specify the structure and the source of the biotransformation product. The initial letter R or M refers to the animal source: rat or monkey. The second letter M, H, P, or B refers to the tissue source or incubation medium: microsomes, hepatocytes, plasma, or bile, respectively. The number given to a metabolite corresponds to a specific chemical entity regardless of the source in which it was identified. For example, metabolite 1 is invariably the catechol, whether it is found in rat hepatocytes (RH1) or in monkey liver microsomes (MM1).

	Results

Abstract Introduction Results Discussion References

Mass Spectrometric Analysis. The [M+H]⁺ ion of parent compound appeared at m/z 565. The product ion mass spectrum and structure of L-694,458 are shown in fig. 1A. The SIF mass spectrum shown in fig. 1B was obtained under TIS conditions, and it is similar to the conventional CID product ion mass spectrum shown in fig. 1A. The SIF spectra can be extremely useful because they often obviate the need to isolate fractions or repeatedly inject a complex mixture of metabolites under conventional CID conditions to obtain structural information on the individual metabolites.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Proposed structures of fragment ions generated by collisionally induced dissociation of parent compound.

Identification of In Vitro Metabolites. Incubations with liver microsomes. HPLC-UV profiles obtained after the incubation of L-694,458 with liver microsomes from male Sprague-Dawley rats and male rhesus monkeys suggested that several metabolites had been generated (13). Subsequent on-line LC-MS analysis of these samples was used to characterize the metabolites. Fig. 3 shows EIC chromatograms obtained from LC-MS analysis of the monkey liver microsomal extract. Subsequent CID experiments were performed on each of the suspected metabolites to produce MS/MS spectra to aid in their structural characterization. Table 1 summarizes the [M+H]⁺ ion observed for each of these metabolites and the fragment ions observed in each of their product ion mass spectra.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Extracted ion current chromatograms of primary metabolites generated in incubations of parent compound with monkey liver microsomes. Incubation and HPLC separation were performed as described in Materials and Methods. The parent compound is designated as P.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   NMR spectra of parent compound (A) and metabolite 6 isolated from rat liver microsomes by HPLC fractionation (B).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Extracted ion current chromatograms of novel metabolites generated in incubations of parent compound with rat liver microsomes. The incubation was carried out as described in Materials and Methods.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Product ion mass spectra of metabolite 9 generated in incubations of L-694,458 (A) and [2H]-labeled L-694,458 (B) with rat liver microsomes.

Incubations with rat hepatocytes in culture. Selected EIC chromatograms obtained from a 48-hr incubation of L-694,458 in cultured rat hepatocytes are shown in Fig. 7. A component with [M+H]⁺ of 539 Da (RH10) that co-eluted with the parent compound was observed in this incubation, and this was believed to be the metabolite that had been observed previously in the incubation of L-694,458 with rat liver microsomes (RM10). Fig. 7 also shows that the N-desmethyl metabolite (RH5) was accompanied by a co-eluting metabolite (RH11) with [M+H]⁺ of 525 Da. These two components were unusual in that they were 26 and 40 Da lower in molecular weight than parent compound, which was not suggestive of any standard route of biotransformation. Subsequent CID analyses of RH10 yielded the product ion mass spectrum shown in fig. 8. Intact fragment ions i and j indicate that the 4-butyl-methylenedioxyphenyl portion of the molecule was not altered, and intact fragment ions e and g suggest that the -lactam ring was not cleaved (see fig. 2). Assignment of the fragment ions at m/z 164, 195, 277, 332, and 363 suggest that an ethylene group is missing from the piperazine ring, corresponding to a net loss of 26 Da from the molecule (table 1). The structural assignment of RH11 was established in a similar manner using the fragment ions listed in table 1. 

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Extracted ion current chromatograms of novel metabolites generated in incubations of parent compound with rat hepatocytes in culture. The incubation was carried out as described in Materials and Methods. SCO indicates signal carryover from another ion channel.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Product ion mass spectrum of metabolite 10 generated in cultured rat hepatocytes. Correlation of designated fragment ions to parent compound fragment ions shown in table 1.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   (A) Product ion mass spectrum of acetylated metabolite 10 after derivatization of an extract from cultured rat hepatocytes. (B) Source-induced fragmentation mass spectrum of acetylated metabolite 10 after derivatization of an extract from cultured rat hepatocytes. Derivatization was carried out using acetic anhydride in pyridine.

Incubations with rat intestinal homogenates. A 4-hr incubation of L-694,458 in homogenates of small and large intestine produced LC-MS profiles consisting mainly of the primary metabolites generated in liver microsomes of rats and monkeys (data not shown). These included the catechol, N-desmethyl, and N-oxide metabolites of parent compound. Minute amounts of the lactam-ring opened products (metabolites 7, 8, and 9) were observed in the ethyl acetate extract of the incubation of L-694,458 with homogenate of small intestine, but they were not detectable in the extract from homogenate of large intestine.

Identification of In Vivo Metabolites. The N-desmethyl and piperazine N-oxide metabolites were identified in plasma, lung, and liver obtained from rats dosed orally with [³H]-labeled L-694,458. The catechol resulting from O-dealkylation of the methylenedioxyphenyl group was observed in rat liver and lung, but was not detected in rat plasma. Two hydroxylated metabolites were also found in the plasma, lung, and liver samples. Plasma also contained the two metabolites that had resulted from -lactam ring opening (RP7 and RP8); however, it is not clear if these components were true biotransformation products because they were also observed as degradation products (D1 and D2) in the in vitro incubations at 37°C with plasma from untreated rats (13). The substituted urea derivative (metabolite 7) was also identified in the lung extract; the presence of this derivative in liver was not determined because of the limited mass range scanned during that experiment.

View larger version (13K): [in this window] [in a new window]   Fig. 10.   Radioactivity profile of bile (2-4 hr postdose) obtained from rats dosed orally with [3H]-labeled L-694,458. Acetonitrile extract was analyzed on a Zorbax phenyl column eluted at 1 ml/min with a 30-min gradient from 70% A (2 mM ammonium acetate with 0.2% TFA) to 80% B (acetonitrile with 0.2% TFA). Radioactivity was measured with an on-line radiometric detector.

View larger version (20K): [in this window] [in a new window]   Fig. 11.   (A) and (B): Extracted ion current chromatograms of selected metabolites observed in an ethyl acetate extract of bile obtained 0-2 hr postdose from rats dosed with [3H]-labeled parent compound. SCO indicates signal carryover from another ion channel.

View larger version (30K): [in this window] [in a new window]   Fig. 12.   Proposed structures of selected metabolites of parent compound observed in incubations with cultured rat hepatocytes and in a rat bile extract.

	Discussion

Abstract Introduction Results Discussion References

The present study has demonstrated the identification and characterization of the in vitro metabolites of L-694,458 in male Sprague-Dawley rats and male rhesus monkeys. This characterization was invaluable in determining the structures of the in vivo metabolites observed in a sample as complex as bile obtained from orally dosed rats. The results have shown that the major in vitro metabolites are formed via four primary metabolic pathways: a) O-dealkylation of the methylenedioxyphenyl group, which results in the catechol derivative (metabolite 1); b) N-demethylation of the methylpiperazine group (metabolite 5); c) N-oxidation of the methylpiperazine nitrogen (metabolite 6), which was confirmed by NMR analysis; and d) cleavage of the -lactam ring, which results in three by-products: the 4-butyl-methylenedioxyphenyl-substituted urea derivative (metabolite 7), the vinyl-substituted urea derivative, which retains part of the -lactam ring (metabolite 8), and the C-4 leaving group, 4-methylpiperazinyl-carbonyl-p-phenol (metabolite 9). Based on HPLC analysis with UV and radiometric detection (13), the catechol was the major metabolite generated in monkey liver microsomes, while the N-oxide was the major metabolite produced in rat liver microsomes. The incubation of L-694,458 with rat intestinal homogenates produced profiles qualitatively similar to those in liver microsomes. Both the small and large intestinal homogenates generated metabolites 1, 5, and 6, while only the former was capable of producing minute amounts of metabolites 7, 8, and 9.

The incubation of L-694,458 with rat hepatocytes in culture produced many of the same metabolites identified in the microsomal incubations. A notable exception was the observation of metabolites 12 and 13, the two methoxy derivatives of the catechol (see table 1), which were not observed in incubations of L-694,458 with liver microsomes. This is because catechol-O-methyl transferase is a cytosolic enzyme (17), which should be present in the cultured hepatocyte system but not in the hepatic microsomes.

The hepatocyte culture system also produced substantial amounts of some unusual metabolites that do not possess an intact piperazine ring; instead, these biotransformation products appeared to contain an amido-ethylamine group. Acetylation was selected as a means of verifying the proposed ring fission product, which was expected to undergo N-acetylation readily, in contrast to the intact parent compound. Subsequent LC-MS/MS analysis of the acetylated derivatives in the hepatocyte extract indeed supported the assignment of metabolites 10 and 11 (see table 1).

While cleavage of the piperazine ring seemed to be an unusual metabolic pathway, there have been several precedents in the literature for this type of in vivo biotransformation. Studies have been made of the metabolism of several drugs containing a piperazine ring, including some phenothiazines (18-20), antihistamines (20, 21), an inotropic agent (22), and ketoconozole (23-25). In addition, investigation of the in vitro metabolism of the hypotensive agent clonidine, which contains a five-membered imidazolidine ring, has indicated that ring scission may proceed either via an epoxide-diol pathway or after two successive hydroxylations at adjacent carbons in the ring (26).

It is interesting that oxidative cleavage of the methylpiperazine ring occurred with and without retention of the piperazine N-methyl group; this phenomenon was noted previously by Breyer et al. as well (19). This observation suggests that further studies of the enzymes that catalyze methylpiperazine N-demethylation and piperazine ring scission are warranted.

Several metabolites of L-694,458 observed in the plasma or tissues obtained from orally dosed rats have been characterized herein, and most of the primary metabolites identified in the in vivo samples had been observed in vitro. For example, the -lactam ring-opened derivatives of L-694,458 (metabolites 7 and 8) were observed in the plasma of orally dosed rats, as well as in the in vitro incubations of L-694,458 with plasma from untreated rats (13). These results suggest that although these in vivo metabolites could have been generated in the liver, they also could have arisen from ex vivo degradation of circulating parent compound owing to the action of a type B esterase (13). Alternatively, the mechanism of formation of these metabolites in plasma may be pH-dependent, as evidenced by the incubation of several analogs of L-694,458 in basic media which produced the corresponding analogs of metabolites 7, 8, and 9 (27). These metabolites were also observed after incubation of related inhibitors with HLE in vitro (28). It should be noted that while analogs of metabolites 7, 8, and 9 have been generated as a consequence of the normal mechanism of HLE inhibition (27-30), it has not been established whether this same mechanism of inhibition has occurred in animals that have been dosed with L-694,458.

Rat bile yielded the most interesting and complex metabolite profile, which was comprised of at least 40 parent-related components. Many of these species were primary metabolites that had been observed in vitro, while several others were secondary metabolites that had not been seen before. The large number of metabolites circulating in bile is attributed to oxidation and subsequent cleavage of the piperazine ring of these primary and secondary metabolites. Detailed mass spectral analysis of the bile sample was useful in demonstrating that the large broadened peak in the radioprofile (fig. 10) corresponded to more than 20 parent-related components that eluted between 12 and 15 min (see figs. 11A and 11B).

In summary, incubation of L-694,458 with liver microsomes and cultured hepatocytes was used successfully to generate and characterize numerous primary and secondary metabolites by LC-MS/MS. The results from the in vitro studies were subsequently applied to the deconvolution of a very complex in vivo metabolite profile observed in bile obtained from orally dosed rats. Notably, a very unusual metabolic pathway resulting in numerous biliary metabolites was identified as cleavage of the piperazine ring, presumably owing to oxidation of adjacent methylenes in the ring. This pathway was interesting in that it occurred with and without molecular retention of the piperazine N-methyl group.