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A SIMPLE SEQUENTIAL INCUBATION METHOD FOR DECONVOLUTING THE COMPLICATED SEQUENTIAL METABOLISM OF CAPRAVIRINE IN HUMANS

Department of Pharmacokinetics, Dynamics & Metabolism (H.-Z.B., P.K., P.Z., W.F.P., E.Y.W.), Pfizer Global Research and Development, San Diego, California

(Received May 4, 2005; accepted June 30, 2005)

	Abstract

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Capravirine, a non-nucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus type 1, undergoes extensive oxygenations to numerous sequential metabolites in humans. Because several possible oxygenation pathways may be involved in the formation and/or sequential metabolism of a single metabolite, it is very difficult or even impossible to determine the definitive pathways and their relative contributions to the overall metabolism of capravirine using conventional approaches. For this reason, a human liver microsome-based "sequential incubation" method has been developed to deconvolute the complicated sequential metabolism of capravirine. In brief, the method includes three fundamental steps: 1) 30-min primary incubation of [¹⁴C]capravirine, 2) isolation of ¹⁴C metabolites from the primary incubate, and 3) 30-min sequential incubation of each isolated ¹⁴C metabolite supplemented with an ongoing (30 min) microsomal incubation with nonlabeled capravirine. Based on the extent of both the disappearance of the isolated precursor ¹⁴C metabolites and the formation of sequential ¹⁴C metabolites, definitive oxygenation pathways of capravirine were assigned. In addition, the percentage contribution of a precursor metabolite to the formation of each of its sequential metabolites (called sequential contribution) and the percentage contribution of a sequential metabolite formed from each of its precursor metabolites (called precursor contribution) were determined. An advantage of this system is that the sequential metabolism of each isolated ¹⁴C metabolite can be monitored selectively by radioactivity in the presence of all relevant metabolic components (i.e., nonlabeled parent and its other metabolites). This methodology should be applicable to mechanistic studies of other compounds involving complicated sequential metabolic reactions when radiolabeled materials are available.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Definitive metabolic scheme for oxygenations of capravirine in human liver microsomes. Arrows indicate definitive pathways, and an arrow with a cross-mark represents a possible pathway proposed but not observed. The percentage at the end of an arrow indicates the sequential contribution, whereas the percentage at the head of an arrow indicates the precursor contribution. No data shown at the end or head of an arrow indicate that the sequential or precursor contribution is 100% (or a single pathway responsible for the sequential metabolism or formation of a metabolite).

	Materials and Methods

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Microsomal Metabolism. [¹⁴C]Capravirine (2 µM) was incubated for 0 to 60 min at 37°C in an incubation system comprised of 100 mM potassium phosphate buffer, pH 7.4, 0.2 mg/ml human liver microsomes, and 1 mM ß-nicotinamide adenine dinucleotide phosphate-reduced tetrasodium salt (NADPH) in a final volume of 1 ml. After a 5-min preincubation, reactions were initiated by the addition of NADPH and terminated by the addition of 2 ml of ice-cold acetonitrile. Samples were vortexed and then centrifuged for 5 min. The supernatant from each incubation tube was transferred into an appropriate polypropylene tube for evaporation to dryness under N₂ at 40°C. The dried residues were reconstituted in 110 µl of 30:70 (v/v) methanol/20 mM ammonium acetate, pH 4, and an aliquot (100 µl) of each reconstituted solution was injected into an HPLC-MS-RAM system, as described below, for analysis.

Sequential Incubation. [¹⁴C]Capravirine (2 µM) was initially incubated with human liver microsomes (0.2 mg/ml) for 30 min at 37°C (called primary incubation; two 1-ml replicates). All other incubation conditions and sample preparation procedures were the same as described above. The reconstituted solution (total 1 ml) pooled from the two primary incubation tubes was injected at a volume of 0.9 ml for the isolation of ¹⁴C metabolites using the HPLC method as described below. Each ¹⁴C metabolite of interest was collected using a Gilson FC 204 fraction collector (Gilson Medical Electronics, Middleton, WI). The fraction collected for each ¹⁴C metabolite (with the exception that [¹⁴C]C4, [¹⁴C]C18, and [¹⁴C]C22 were pooled to generate a ¹⁴C metabolite mixture) was split equally into two incubation tubes that were evaporated to dryness under N₂ at 40°C.

Nonlabeled capravirine (2 µM) was incubated with human liver microsomes for 30 min (called supplemental incubation; as many 1-ml replicates as needed). All other incubation conditions were identical to those for the primary incubation. At 30 min, 1 ml of ongoing supplemental incubation (not terminated) mixture was quickly transferred into each of the two aforementioned dried tubes containing the isolated ¹⁴C metabolite(s). After vortexing (5 s), one tube was terminated immediately by the addition of 2 ml of ice-cold acetonitrile (control). The other tube was incubated for an additional 30 min at 37°C (called sequential incubation) and then terminated by the addition of 2 ml of ice-cold acetonitrile. The same sequential incubation procedure was executed for each isolated ¹⁴C metabolite of interest. All subsequent sample preparation procedures were the same as described above, except that dried residues were reconstituted in 1 ml of 30:70 (v/v) methanol/20 mM ammonium acetate, pH 4. An aliquot (0.9 ml) of each reconstituted solution was injected into the HPLC-MS-RAM system for analysis.

Metabolite Profiling. Metabolite profiling was performed on an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA) coupled with an IN/US model 3 ß-RAM radiodetector (IN/US Systems, Inc., Tampa, FL), an ARC Model C StopFlow system (AIM Research Company, Newark, DE), and a Thermo Electron LCQ-Deca ion-trap mass spectrometer (Thermo Electron Corporation, Waltham, MA). An Agilent 1100 autosampler used in the study was upgraded with a 900-µl injection loop and pump head. Thus, a maximal volume of 900 µl could be singly injected. Separation was achieved using a Phenomenex Aqua C18 column (150 x 4.6 mm, 5 µ) (Phenomenex, Torrance, CA) at a flow rate of 1.0 ml/min. The effluent was split to allow 20% to the mass spectrometer via the supplied electrospray ionization source and 80% to mix with either ULTIMA FLO-M scintillation cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA) at 2.4 ml/min or ARC StopFlow AD scintillation cocktail (AIM Research) at 1.2 ml/min and then flow through the radiodetector. A mobile phase gradient of (A) 20 mM ammonium acetate, pH 4, and (B) methanol was programmed as follows: initiated with 100% A for 10 min, changed to 60% A from 10 to 30 min, changed to 55% A from 30 to 35 min, held at 55% A from 35 to 60 min, changed to 40% A from 60 to 70 min, changed to 10% A from 70 to 80 min, held at 10% A from 80 to 90 min, changed to 100% A from 90 to 92 min, and held at 100% A from 92 to 100 min for column equilibration. All of the above gradient changes were linear. Major operating parameters for the ion-trap electrospray ionization-MS method were as follows: positive ion mode with a spray voltage of 4.5 kV, capillary temperature of 200°C, sheath gas flow rate of 70 (arbitrary), and an auxiliary gas flow rate of 20 (arbitrary). LAURA 3, version 3.0 (IN/US Systems), ARC data system, version 2.4 (AIM Research Company), and Xcalibur V1.4 (Thermo Electron Corporation) were used to control the ß-RAM detector, the StopFlow system, and the liquid chromatography-MS system, respectively, for data acquisition and processing. The metabolite profiling of sequential incubations of the isolated ¹⁴C metabolites was performed using the StopFlow system-controlled radiodetector, where time intervals were preset for stop-flow radiochemical detection in terms of known metabolite information. Outside the preset intervals, no radiochemical detection was executed. The ARC StopFlow-controlled radiodetector is 10- to 20-fold more sensitive than the conventional flow-through detection method (Nassar et al., 2003). However, it takes a much longer time to finish a run because the system stops the flow for radioactivity counting during most of the run time (stop for 60 s per every 8 s in this study).

View larger version (13K): [in this window] [in a new window]   FIG. 2. A representative radiochromatogram generated from a 30-min primary incubation of [14C]capravirine (2 µM) in human liver microsomes (0.2 mg/ml). The radioactivity was detected using the regular (nonstop) radiodetector.

Sequential contribution represents the percentage contribution of a precursor metabolite to the formation of each of its sequential metabolites when the precursor metabolite is metabolized to two or more sequential metabolites. For example, a precursor metabolite (PM) is metabolized to two sequential metabolites (SM), SM1 and SM2. The percentage contribution of PM to the formation of SM1 or SM2 during the sequential incubation course can be calculated as percentage contribution (PM-to-SM1) = [A_SM1/(A_SM1 + A_SM2)] x 100% and percentage contribution (PM-to-SM2) = [A_SM2/(A_SM1 + A_SM2)] x 100%, where A_SM1 and A_SM2 represent the radiochromatographic peak area (or radioactivity) of SM1 and SM2, respectively.

Precursor contribution represents the percentage contribution of a sequential metabolite formed from each of its precursor metabolites when the sequential metabolite is formed from two or more precursor metabolites. For example, an SM is formed from two precursor metabolites, PM1 and PM2. The percentage contribution of SM formed from PM1 or PM2 during the sequential incubation course can be calculated as percentage contribution (SM-from-PM1) = [A_SM-from-PM1/(A_SM-from-PM1 + A_SM-from-PM2)] x 100% and percentage contribution (SM-from-PM2) = [A_SM-from-PM2/(A_SM-from-PM1 + A_SM-from-PM2)] x 100%, where A_SM-from-PM1 and A_SM-from-PM2 represent the radiochromatographic peak area (or radioactivity) of SM formed from PM1 and PM2, respectively.

	Results

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Microsomal Metabolism of Capravirine. Capravirine was extensively oxygenated to a variety of primary and sequential metabolites in human liver microsomes (Fig. 2). Note that all of the metabolites observed in this in vitro study were identified in our previous study (Bu et al., 2004). In the current study, the identity of the in vitro metabolites was confirmed simply by the similarity in retention times and MS² ion-trap mass spectra between the in vitro and in vivo data. The primary metabolites (monooxygenated) were formed via sulfoxidation (metabolite C23), N-oxidation at the pyridinyl nitrogen atom (metabolite C26), and tertiary hydroxylation (metabolite C19) and primary hydroxylation (metabolite C20) at the isopropyl group. All sequential metabolites (di-, tri-, and tetra-oxygenated) were formed via combinations of the four types of monooxygenation reactions (Fig. 1).

After the incubation of [¹⁴C]capravirine in human liver microsomes, the oxygenation time courses of the parent and its metabolites are shown in Fig. 3. It should be noted that metabolites C19, C20, and C25b were present at trace/minor levels over the entire incubation time. In contrast, C23 and C26 represent the two most abundant metabolites of capravirine in human liver microsomes. All of the other metabolites have low-to-moderate abundances (Fig. 3).

View larger version (22K): [in this window] [in a new window]   FIG. 3. Time courses of parent disappearance and metabolite formation in primary incubations of [14C]capravirine (2 µM) in human liver microsomes (0.2 mg/ml). The radioactivity was detected using the regular (nonstop) radiodetector.

Sequential Incubation. The primary incubations of [¹⁴C]capravirine in human liver microsomes were conducted to generate ¹⁴C metabolites. The ¹⁴C metabolites with sufficient abundances were isolated for sequential incubations. Note that 70% of each ¹⁴C metabolite in the primary incubation was isolated for its sequential incubation. The isolated ¹⁴C metabolites from the primary incubations included C4, C9, C15, C18, C22, C23, and C26. The ¹⁴C metabolites C3 and C11 were not isolated, because they did not show any sequential metabolism. The ¹⁴C metabolites C19, C20, and C25b were not isolated directly from the primary incubations because of their low abundances (<1% of total radioactivity, Fig. 2). However, the ¹⁴C metabolite C25b, which might undergo sequential oxygenations, was isolated from a surrogate incubation of [¹⁴C]capravirine in dog CYP3A12 supersomes in which C25b was formed at a sufficient amount for isolation (data not shown). The supplemental incubations of nonlabeled capravirine in human liver microsomes were conducted to generate ongoing incubation mixtures for sequential incubations of the isolated ¹⁴C metabolites. When an isolated ¹⁴C metabolite was mixed with an ongoing supplemental incubation, the sequential incubation of the ¹⁴C metabolite was conducted under conditions that mimicked continued incubation of the supplemental mixture. Thus, the sequential incubation method has the ability to monitor selectively, by radioactivity, the disappearance of each isolated ¹⁴C metabolite and the appearance of its sequential ¹⁴C metabolite(s) in the presence of all relevant metabolic components (nonlabeled). It should be noted that the incubation time of 30 min for each of the primary, supplemental, and sequential incubations was selected based on the oxygenation time courses of capravirine and its metabolites (Fig. 3). The total radioactivity measured from the 30-min sequential incubations (Figs. 4, 5, 6, 7, 8, panels B) was 97 to 108% of that measured from the 0-min sequential incubations (Figs. 4, 5, 6, 7, 8, panels A). In addition, it was assumed that the concentrations of the added ¹⁴C metabolites would not cause significant changes in metabolic rates under the linear conditions (Fig. 3).

View larger version (13K): [in this window] [in a new window]   FIG. 4. Representative metabolite profiles after sequential incubation of the isolated metabolite [14C]C23 in human liver microsomes (0.2 mg/ml) for 0 (A) and 30 min (B). The radioactivity was detected using the StopFlow system-controlled radiodetector with the preset stop-flow detection intervals. The flow was stopped for radiochemical detection for 60 s per every 8 s. Outside the preset intervals, no radiochemical detection was performed.

View larger version (9K): [in this window] [in a new window]   FIG. 5. Representative metabolite profiles after sequential incubation of the isolated metabolite [14C]C26 in human liver microsomes (0.2 mg/ml) for 0 (A) and 30 min (B). The StopFlow radiochemical detection was the same as that described in Fig. 4.

View larger version (11K): [in this window] [in a new window]   FIG. 6. Representative metabolite profiles after sequential incubation of the isolated metabolite [14C]C9 in human liver microsomes (0.2 mg/ml) for 0 (A) and 30 min (B). The StopFlow radiochemical detection was the same as that described in Fig. 4.

View larger version (10K): [in this window] [in a new window]   FIG. 7. Representative metabolite profiles after sequential incubation of the isolated metabolite [14C]C15 in human liver microsomes (0.2 mg/ml) for 0 (A) and 30 min (B). The StopFlow radiochemical detection was the same as that described in Fig. 4.

View larger version (11K): [in this window] [in a new window]   FIG. 8. Representative metabolite profiles after sequential coincubation of the three isolated metabolites [14C]C4, [14C]C18, and [14C]C22 in human liver microsomes (0.2 mg/ml) for 0 (A) and 30 min (B). The StopFlow radiochemical detection was the same as that described in Fig. 4.

Metabolite [¹⁴C]C9 underwent extensive sequential biotransformation with a metabolite profile similar to that of [¹⁴C]capravirine for all applicable metabolites (Fig. 6 versus 2). This suggested that C9 represents an important di-oxygenated metabolite responsible for sequential tri- and tetra-oxygenations of capravirine in humans. In contrast, metabolite [¹⁴C]C15 only underwent trace-level sequential metabolism to C4 (Fig. 7), suggesting a negligible role of C15 in sequential oxygenations of capravirine in humans. In addition, the sequential incubation of metabolite [¹⁴C]C25b indicated that C25b was sequentially oxygenated to C22 only (data not shown).

In theory, each of the three tri-oxygenated metabolites, [¹⁴C]C4, [¹⁴C]C18, and [¹⁴C]C22, may be further oxygenated to the tetra-oxygenated metabolite C11 only. Therefore, the relative contributions of the three metabolites to the formation of C11 can be estimated based on the extent of the disappearance of [¹⁴C]C4, [¹⁴C]C18, and [¹⁴C]C22. All three ¹⁴C metabolites were coincubated to evaluate their sequential metabolism (Fig. 8). After the 30-min sequential coincubation, both [¹⁴C]C4 and [¹⁴C]C22 did not show any disappearance, whereas [¹⁴C]C18 completely disappeared (Fig. 8), suggesting that only C18 undergoes further oxygenation to C4.

Based on the above sequential incubation results of the isolated ¹⁴C metabolites, the definitive oxygenation pathways were assigned and the relative contributions of the pathways during the sequential incubation course were estimated for the extensive metabolism of capravirine in human liver microsomes (Fig. 1). In general, it was straight-forward to perform the pathway assignments and the estimations of relative contributions using the methods described under Materials and Methods. One exception was the difficulty in estimating the sequential contributions of C23 to its direct sequential metabolites, C9, C15, and C25b, because of their further sequential oxygenations. After the sequential incubation of [¹⁴C]C23 in human liver microsomes, the sum of the abundances of C22 and C25b represented the sequential contribution of C23 to C25b, the sum of the abundance of C15 and 10% C4 was the sequential contribution of C23 to C15, and the sum of the abundances of C9, C3, C18, and C11 and 90% C4 accounted for the sequential contribution of C23 to C9. Thus, the sequential contributions of C23 to C25b, C15, and C9 during the sequential incubation course were calculated to be 17, 31, and 52%, respectively, by the normalization of the three individual sums (Fig. 1). Likewise, the percentage contributions of capravirine to the formation of the four monooxygenated metabolites, C19, C20, C23, and C26, were estimated based on the time courses of the parent disappearance and metabolite formation in the incubations of [¹⁴C]capravirine in human liver microsomes (Figs. 1 and 3).

	Discussion

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For a drug undergoing extensive sequential metabolism via numerous metabolic pathways, it is very difficult or even impossible to identify definitive pathways and determine the relative contribution of each pathway to the total metabolism of the drug using conventional approaches such as performing individual incubations of synthetic or isolated metabolites (radiolabeled or nonlabeled) in liver microsomes and hepatocytes. Based on in vitro studies using synthetic nonlabeled metabolites, possible tentative pathways can be proposed but relative contributions of these pathways cannot be determined. In practice, it is very expensive, time-consuming, or even infeasible to synthesize all metabolites of a compound. Alternatively, radiolabeled metabolites of a compound can be isolated from in vitro incubations and/or in vivo samples after dosing of the radiolabeled compound. By conducting individual incubations of the isolated radiolabeled metabolites, possible metabolic pathways can be proposed and sequential contributions of these pathways can be determined. Because the above sequential metabolism studies of the individual metabolites are usually conducted both at concentrations irrelevant to their abundances in primary incubations and in the absence of other metabolic components (the parent and its other metabolites), the data obtained through the conventional in vitro approaches can only be tentative or may be irrelevant to the observations from the primary incubations.

Ideally, a sequential incubation of a precursor metabolite should be performed in an incubation medium containing the same metabolic components as in the incubation medium of the parent drug where all relevant parent-metabolite/metabolite-metabolite interactions are maintained. Allowing sequential metabolism to occur in the natural incubation milieu ensures the relevance of the data obtained. The sequential incubation methodology developed in the present study is composed of three steps: 1) 30-min primary incubation of [¹⁴C]capravirine, 2) isolation of ¹⁴C metabolites of interest from the incubate, and 3) 30-min sequential incubation of each isolated ¹⁴C metabolite supplemented with an ongoing (30 min) microsomal incubation with nonlabeled capravirine. The strategically designed three-step process ensures that all sequential liver microsomal incubations of the isolated ¹⁴C metabolites are conducted under the conditions that mimic the intact incubation of the parent drug.

This strategy for the mechanistic study of the sequential metabolism of capravirine in humans shows a number of advantages over the conventional approaches. These are summarized as follows: 1) no need for the synthesis of metabolite standards, 2) efficient isolation of all radiolabeled metabolites of interest from only two 1-ml microsomal incubations with minimal cost and time, 3) achieving definitive information on all metabolic pathways and the relative contribution of each pathway to the total metabolism of the parent, and 4) a readily usable method for the characterization of sequential biotransformation reactions of other chemical/biochemical compounds and drugs when radiolabeled materials are available.

	Acknowledgments

 
We thank Drs. Deepak Dalvie, James Ferrero, Wei-Zhu Zhong, and Geoffrey Peng for reviews of the manuscript.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.005413.

ABBREVIATIONS: AG1549, 2-carbamoyloxymethyl-5-(3,5-dichlorophenyl)thiol-4-isopropyl-1-(4-pyridyl)methyl-1H-imidazole; NADPH, ß-nicotinamide adenine dinucleotide phosphate-reduced tetrasodium salt; PM, precursor metabolite; SM, sequential metabolite; HPLC, high-performance liquid chromatography; MS, ion-trap mass spectrometry; RAM, radioactivity monitoring.

Address correspondence to: Dr. Hai-Zhi Bu, Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, San Diego, CA 92121. E-mail: haizhi.bu{at}pfizer.com

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.005413v1 33/10/1438    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bu, H.-Z. Articles by Wu, E. Y. Search for Related Content PubMed PubMed Citation Articles by Bu, H.-Z. Articles by Wu, E. Y.