DMD Simcyp

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

Drug Metabolism and Disposition Fast Forward
First published on June 23, 2008; DOI: 10.1124/dmd.108.021444

0090-9556/08/3609-1896-1902$20.00
DMD 36:1896-1902, 2008

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dmd.108.021444v1
36/9/1896    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sharma, S.
Right arrow Articles by Venkataramanan, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sharma, S.
Right arrow Articles by Venkataramanan, R.

Identification of Enzymes Involved in the Metabolism of 17{alpha}-Hydroxyprogesterone Caproate: An Effective Agent for Prevention of Preterm Birth

Shringi Sharma, Junhai Ou, Stephen Strom, Don Mattison, Steve Caritis, and Raman Venkataramanan

Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania (S.Sh., J.O., R.V.); Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (S.St., R.V.); Center for Research for Mothers and Children, Obstetric-Fetal Pharmacology Research Units (OPRU) Network, National Institute of Child Health and Human Development, Bethesda, Maryland (D.M.); and Department of Obstetrics and Gynecology and Reproductive Sciences, Magee Women's Hospital, Pittsburgh, Pennsylvania (S.C.)

(Received March 13, 2008; Accepted June 19, 2008)


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Preterm delivery, that is delivery before 37 completed weeks of gestation, is the major determinant of neonatal morbidity and mortality. Until recently, no effective therapies for prevention of preterm birth existed. In a recent multicentered trial, 17{alpha}-hydroxyprogesterone caproate (17-OHPC) reduced the rate of preterm birth by 33% in a group of high-risk women. Limited pharmacologic data exist for this drug. The recommended dose is empiric; the metabolic pathways are not well defined especially in pregnant women; and the fetal exposure has not been quantified. To define the metabolic pathways of 17-OHPC we used human liver microsomes (HLMs), fresh human hepatocytes (FHHs), and expressed enzymes. HLMs in the presence of NADPH generated three metabolites, whereas two major metabolites were observed with FHHs. Metabolism of 17-OHPC was significantly inhibited by the CYP3A4 inhibitors ketoconazole and troleandomycin in HLM and FHH. Metabolism of 17-OHPC was significantly greater in FHH treated with the CYP3A inducers, rifampin and phenobarbital. Furthermore, studies with expressed enzymes showed that 17-OHPC is metabolized exclusively by CYP3A4 and CYP3A5. The caproic acid ester was intact in the major metabolites generated, indicating that 17-OHPC is not converted to the primary progesterone metabolite, 17{alpha}-hydroxyprogesterone. In summary, this study shows that 17-OHPC is metabolized by CYP3A. Because CYP3A is involved in the oxidative metabolism of numerous commonly used drugs, 17-OHPC may be involved in clinically relevant metabolic drug interactions with coadministered CYP3A inhibitors or inducers.

Preterm birth is the major determinant of neonatal morbidity and mortality (Paneth, 1995Go; Mattison et al., 2001Go). None of the numerous interventions to reduce the rate of preterm birth including labor-inhibiting drugs or strategies such as home uterine monitoring have proven effective. Recent evidence from the Maternal–Fetal Medicine Units Network showed that weekly injections of 250 mg of 17{alpha}-hydroxyprogesterone caproate (17-OHPC) reduced the rate of preterm birth in high-risk women by 33% (Meis et al., 2003Go). The dose used in this study was not based on the pharmacologic properties of 17-OHPC but rather on empiric doses used in clinical trials in the 1960s and 1970s. 17-OHPC (Fig. 1A) is a synthetic hormone produced by the esterification with caproic acid at the 17 carbon position of the metabolite of the natural female sex hormone progesterone (Fig. 1C). In animal studies, 17-OHPC produces a longer lasting and more robust progestational effect on the endometrium than progesterone (Wu and Allen, 1959Go).


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 1. Chemical structures of (A) product name: 17-OHPC (17{alpha}-hydroxy-4-pregnene-3, 20-dione hexanoate), (B) product name: 17{alpha}-hydroxyprogesterone (17{alpha}-hydroxy-4-pregnene-3, 20-dione), and (C) product name: progesterone (4-pregnene-3, 20-dione).

 
The mechanism of action of 17-OHPC is unknown, but the progestational effect is likely related to its concentration. We have recently reported plasma concentrations in pregnant women with twins treated with 17-OHPC (Caritis and Venkataramanan, 2007Go), but limited data exist on how this drug is metabolized and whether the drug or its metabolites reach the fetal circulation. The observation that only one third of cases benefit from treatment with 17-OHPC suggests that variability in 17-OHPC metabolism or drug interactions may also contribute to the observed variability in clinical response after a fixed dosing regimen. The purpose of the present study was to identify the various enzymes [cytochromes P450 (P450s) and flavin-containing monooxygenase (FMO) enzymes] involved in 17-OHPC metabolism and to characterize the drug's metabolites.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. 17{alpha}-OHPC (molecular mass 428.6) was a gift from Diosynth Inc., (Chicago, IL). The radioactive isotope of 17{alpha}-hydroxy-[1,2,6,7-3H]progesterone [1-14C]caproate was custom-synthesized by RTI International (Research Triangle Park, NC). Quinidine, sulfaphenazole, coumarin, ketoconazole, methimazole, {alpha}-naphthoflavone, testosterone, 6β-hydroxytestosterone, and NADPH were purchased from Sigma-Aldrich (St. Louis, MO). Methanol was purchased from Fisher Scientific (Fair Lawn, NJ). Microsomes derived from baculovirus-infected insect cells were purchased from BD-Gentest (Woburn, MA).

Human Liver Microsomes. Human liver samples were obtained from the Hepatocyte Transplantation Laboratory at the University of Pittsburgh. Donors of human liver tissue had no history of liver disease, but the liver was not used for transplantation.

Preparation of Human Liver Microsomes. Liver pieces were dissected and kept in cold saline on ice. Liver microsomes were prepared by a standard differential centrifugation procedure with minor modifications (Court and Greenblatt, 1997Go; Nelson et al., 2001Go). Briefly, liver pieces were homogenized with 3 volumes of a homogenization buffer (50 mM Tris-HCl buffer, 1.0% KCl, and 1 mM EDTA, pH 7.4) using an electrical homogenizer (Polytron, Brinkmann Instruments, Westbury, NY). The crude homogenate was centrifuged (Optima XL-100K ultracentrifuge, Beckman Instruments, Palo Alto, CA) at 10,000g for 20 min at 4°C. The supernatant was further centrifuged at 105,000g for 65 min at 4°C to sediment the microsomes. The microsomes were reconstituted using a manual homogenizer (Wheaton, Millville, NJ) in twice their weight of Tris-HCl buffer (50 mM Tris-HCl buffer, pH 7.4) containing 20% glycerol. Aliquots (1.0 ml) were immediately kept in storage at –80°C until used. The protein content was determined by Lowry's method (Lowry et al., 1951Go) using bovine serum albumin (Fluka, Buchs, Switzerland, 98% pure) as a standard.

Microsomal Incubations. Optimal conditions for the evaluation of the metabolism of 17-OHPC were selected by varying the time of incubation (0–180 min) and the microsomal protein concentrations (0–2 mg/ml). Different concentrations of 17-OHPC (0–200 µg/ml in methanol, final concentration 1%) were incubated with human liver microsomes (HLMs) (0.5 mg/ml, optimum protein concentration) and MgCl2 (10 mM) in 0.1 mM phosphate buffer, pH 7.4. The final volume was allowed to equilibrate in a shaking water bath for 5 min at 37°C. The reaction was initiated with the addition of NADPH (1 mM). In additional experiments the incubations were also carried out in the absence of NADPH. After 60 min of incubation (optimum incubation time), the reaction was stopped by immediately adding equal volume of cold methanol. The mixture was centrifuged at 700 relative centrifugal force for 20 min, and supernatant was injected into the high-performance liquid chromatography (HPLC).

Incubations of [3H], [14C]17-OHPC were also performed with HLMs. Radiolabeled 17-OHPC was incubated with three individual human liver microsomal preparations for 10 min using the abovementioned method, and the samples were analyzed directly by HPLC connected to a radioactivity detector.

Chemical Inhibition Studies. Various P450 inhibitors were used in the study to identify P450s that might be involved in the metabolism of 17-OHPC. Human liver microsomal preparations (n = 3) were used for the studies with the incubations being performed in triplicate. Experiments were done at different 17-OHPC concentrations (0–200 µg/ml expressed as 0–467 µM) spanning the concentrations seen clinically to identify the type of inhibition keeping the inhibitor concentration constant. P450 isoform selective chemical inhibitors were used at the following concentration: CYP1A2 ({alpha}-naphthoflavone, 10 µM), CYP2A6 (coumarin, 20 µM), CYP2C9 (sulfaphenazole, 5 µM), CYP2D6 (quinidine, 5 µM), CYP3A (ketoconazole, 1.0 µM), and FMO3 (methimazole, 200 µM). Additionally, inhibition studies were also carried out by incubating 17-OHPC (25 µM) with different concentrations of ketoconazole (0.01–10 µM) to determine the IC50 for the reaction. The formula used to determine the IC50 for microsomal incubations involving 17-OHPC and ketoconazole involves estimating percentage inhibition, which was calculated as follows: % Inhibition = [(17-OHPCwithout inhibitor – 17-OHPCwith inhibitor)/(17-OHPCwithout inhibitor)] · 100, where 17-OHPCwithout inhibitor is the amount of 17-OHPC metabolized in the absence of ketoconazole relative to the total amount of 17-OHPC. 17-OHPCwith inhibitor is the amount of 17-OHPC metabolized in the presence of ketoconazole relative to the total amount of 17-OHPC. Subsequently, IC50 values, or the inhibition concentration resulting in 50% inhibition of 17-OHPC metabolism, were determined from a plot of the percentage inhibition versus the logarithm of ketoconazole concentration.

In the case of mechanism-based inhibitor-like troleandomycin (100 µM), preincubation was done for 30 min at 37°C before adding the substrate (17-OHPC, 25 µM). In inhibition experiments with ketoconazole (1, 10 µM), the substrate was coincubated with inhibitor.

Expressed Enzyme Microsomal Incubations. The incubations (n = 3) were carried similarly to the method described for HLMs. To evaluate the involvement of P450 isoforms in 17-OHPC metabolism, 20 pmol of each expressed enzyme tested was incubated for 60 min with 17-OHPC, and the samples were analyzed using HPLC-UV.

Preparation of Human Hepatocytes. Hepatocytes were prepared by a three-step collagenase perfusion technique (Strom et al., 1996Go). Hepatocytes were plated on Falcon six-well culture plates (1.5 x 106 cells/well; Falcon; BD Biosciences Discovery Labware, Bedford, MA) previously coated with rat tail collagen in hepatocyte maintenance medium (HMM) supplemented with 0.1 µM insulin, 0.1 µM dexamethasone, 0.05% streptomycin, 0.05% penicillin, 0.05% amphotericin B, and 10% bovine calf serum. After allowing the cells to attach for 4 to 6 h, medium was replaced with serum-free medium containing all the supplements described above. Cells were maintained in culture at 37°C in an atmosphere containing 5% CO2 and 95% air. After 24 h in culture, unattached cells were removed by gentle agitation, and the medium was changed. The medium was changed every 24 h, and the hepatocytes were maintained in culture for the experiment.

Incubations with Fresh Human Hepatocytes. Briefly, hepatocytes were maintained in culture in the presence of the chemical under study or vehicle control (dimethyl sulfoxide 0.1% or MeOH 0.1%). On the day of the experiment, cells were washed with HMM devoid of insulin, dexamethasone, antibiotics, and antifungal drugs. Drug stocks were prepared in methanol at 1000-fold incubation concentration (100 mM). Ten microliters of this 100 mM stock was added to a vial containing 10 ml of HMM. Reactions were started by incubating six-well cell culture plates containing human hepatocytes (1.5 million cells/well) with the drug in HMM solutions for 60 min. At the end of that time, 1 ml of medium was sampled and stored at –80°C analysis. The remaining media were aspirated, and the cells were harvested in phosphate buffer (0.1 M, pH 7.4) and stored at –80°C for protein determination.

For acute inhibition experiments (n = 4), human hepatocytes were coincubated with 17-OHPC (25 µM) in the presence and absence of inhibitors (troleandomycin and ketoconazole). The samples were incubated for 30 min and collected as described above.

The induction experiments were initiated 24 h after plating the cells. The hepatocytes (n = 3) were incubated with the inducers (rifampin, phenobarbital, and clotrimazole) for 4 days before adding HMM/17-OHPC (50 µM) to estimate the effect of CYP3A induction on 17-OHPC metabolism.

Correlation Studies. Testosterone 6β-hydroxylation was used as the marker for CYP3A activity (Chiba et al., 1996Go). Formation rates of 17-OHPC metabolite (M2) were measured using microsomes (n = 7) and fresh human hepatocytes (FHHs) (n = 7) at a substrate concentration of 100 µM. These rates were correlated with 6β-hydroxytestosterone formation activity to assess the involvement of CYP3A isoforms.

Analytical Procedure. HPLC-UV. Analysis of the unmetabolized drug and the potential metabolites obtained from HLM-based incubations was performed using a HPLC system equipped with UV detection. The HPLC system comprised an autosampler (712 WISP, Waters, Milford, MA) and solvent delivery system (Waters 501) attached to a UV detector (Waters 486). Chromatography was performed with a 4.6 x 250-mm, 100Å, 5-µm Symmetry C18 (Waters) column. Isocratic elution was performed with a mobile phase of 90% (v/v) methanol in water at a flow rate of 0.8 ml/min, column temperature of 25°C, and eluent monitored at 242 nm. The intraday and interday variation expressed as coefficient of variation did not exceed 10% in any of the assays. The concentration of 17-OHPC in samples was quantitated by comparing the peak areas in samples with a standard curve of the pure drug. The metabolites were quantitated by expressing them in terms of 17-OHPC equivalents.

The concentration of 6β-hydroxytestosterone in the medium was measured by HPLC-UV method as previously described by Kostrubsky et al. (1999Go). The HPLC system comprised a LiChrospher 100 RP-18 column (4.6 x 250 mm, 5 µm, Merck, Darmstadt, Germany). 6β-Hydroxytestosterone was eluted with a mobile phase of methanol/water (60:40, v/v) at a flow rate of 1.2 ml/min, and the eluents were monitored at 242 nm. The concentration of the metabolite was quantitated by comparing the peak areas in samples with a standard curve containing known amount of the metabolite.

Liquid chromatography/tandem mass spectrometry. Samples obtained from FHH-based incubations were analyzed by a liquid chromatography/mass spectrometry (LC/MS) (Thermo Electron, San Jose, CA) system consisting of a Surveyor quaternary LC pump, a Surveyor autosampler, coupled to a triple quadrupole mass spectrometer (TSQ Quantum) and equipped with an atmospheric pressure ionization electrospray interface. Instrument control and data acquisition were performed with the Xcalibur software (Thermo Electron, 2.0). Tandem MS (MS/MS) conditions for the analytes were optimized by pump infusion of 17-OHPC stock solutions using the Quantum Tune Master software (Thermo Electron). The HPLC column used was a Symmetry C18 (150 x 2.1 mm, 3.5 µm, Waters) with an appropriate guard column (10 x 2.1 mm, Symmetry). The mobile phases used were A, water (0.1% formic acid) and B, acetonitrile (0.1% formic acid). The total run time was 35 min at a flow rate of 0.2 ml/min. A gradient profile was used starting from a mobile phase containing 10% solution B, increased linearly to 90% B over first 15 min, isocratic 90% B for 10 min, followed by returning to the initial condition of 10% B to achieve the baseline.

Radio-HPLC. Analytical separations were achieved using conditions similar to the abovementioned HPLC method. The metabolites were analyzed using a radiomatic model 525TR/FLO-ONE flow-through radioactivity detector (PerkinElmer Life Sciences, Boston, MA), and peak areas were integrated with Windows-based (Microsoft, Redmond, WA) FLO-ONE version 3.61.

Data Analysis. Data are expressed as the mean ± S.D. Student's t test was used to assess the significance of results. IC50 was calculated using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Metabolism of 17-OHPC by P450s in Human Liver Microsomal and FHH Preparations. Incubation of 17-OHPC with HLMs resulted in the generation of three (M1–M3HLM) main metabolites wherein (M2HLM) was the major metabolite. The formation of metabolites was observed to increase up to 60 min using a microsomal protein concentration of 0.5 mg/ml. Hence, the abovementioned conditions were used for all the incubations unless specified otherwise. The formation of metabolites (M1HLM and M3HLM) was low compared with M2HLM; thus, accurate data could not be obtained for these minor metabolites, and the article focuses on the major metabolite (M2HLM). Incubation of 17-OHPC with FHHs resulted in the generation of five metabolites (M1–M5HH). The major metabolite generated was M2HH, which was used to characterize 17-OHPC metabolism in FHHs. Figure 2 depicts the chromatograms obtained using LC-UV and MS for analysis. Figure 2, A and B, shows the metabolism of 17-OHPC in microsomal incubates in the presence/absence of NADPH. All the metabolites were formed in an NADPH-dependent manner only. Figure 2C shows the metabolism of 17-OHPC in FHHs incubated for 1 h.


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 2. Chromatograms illustrating the metabolism of 17-OHPC by human liver microsomal preparations. A, chromatogram (HPLC-UV) showing retention times and absorption peaks for metabolites 1, 2, 3 (M1HLM, M2HLM, M3HLM) and 17-OHPC having retention times of 4.84, 5.10, 5.94, and 8.55, respectively, after microsomal incubation in the presence of NADPH. B, chromatogram (HPLC-UV) depicting incubation of 17-OHPC with HLM in the absence of NADPH. C, chromatogram (LC/MS) showing absorption peaks for metabolites (M1HH, M2HH, M3–5HH) and 17-OHPC after incubation with FHHs for 60 min. The retention times for M1, M2, and 17-OHPC were observed to be 6.64, 7.99, and 21.71, respectively. D, chromatogram (LC/MS) showing absorption peak for 17-OHPC after incubation with FHHs for 1 min. No metabolite formation was detected. The retention time for 17-OHPC was 21.97.

 
Incubation of radiolabeled 17-OHPC with HLM generated one major and two minor metabolite peaks (data not shown). The recovery of radioactivity from the incubations was 85 to 97%. The major metabolite constituted approximately 60 to 65% of the metabolized 17-OHPC, and the two minor metabolites were estimated to be approximately 15%. The remaining 20% of the metabolite could not be accounted for because of limitations of the analytical method used. The two ([14C, 3H]) labels on the 17-OHPC molecule were observed to remain intact in the metabolites generated, confirming that the caproate side chain or the ring structure was not cleaved during metabolism in human livers.

Metabolic Profiles in HLMs and Hepatocytes. Metabolism of 17-OHPC was evaluated over time using HLMs and hepatocytes. The concentrations of unmetabolized 17-OHPC and the major metabolite (expressed in terms of 17-OHPC equivalent) were determined over a time period of 0 to 180 min incubation. Figure 3, A and B, shows that the concentration of 17-OHPC decreased in a time-dependent manner, and the concentration of the metabolite increased proportionately. Approximately 60% of the parent drug (17-OHPC) was metabolized within 60 min, and M2(HLM, HH) accounted for almost 50% of metabolized 17-OHPC.


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 3. Metabolism of 17-OHPC by human hepatocytes and HLMs (n = 3). A, incubation of 17-OHPC (150 µM) with fresh adult human hepatocytes generated five metabolites (M1–5HH). The major metabolite (M2HH, {blacksquare}) showed a time-dependent increase throughout the incubation that corresponded with a decrease in the amount of 17-OHPC ({blacktriangleup}). B, incubation of 17-OHPC (50 µM) with HLMs generated metabolites 1, 2, and 3. Metabolite 2 (M2HLM, {blacksquare}) was the major metabolite that depicted time-dependent increase in concentration corresponding to a decrease in 17-OHPC concentration ({blacktriangleup}). The amount of metabolite has been expressed in terms of 17-OHPC equivalents.

 
Identification of Human P450 Isoforms. Incubations with HLMs. 17-OHPC was incubated with microsomes for 60 min in the presence of various representative inhibitors of P450 isoforms. A similar reaction was performed in the absence of the inhibitors, and the rate of major metabolite (M2HLM) generated was compared between the two conditions to identify the isoforms responsible for 17-OHPC metabolism. As summarized in Fig. 4, {alpha}-naphthoflavone, sulfaphenazole, coumarin, quinidine, and methimazole did not inhibit the metabolism of 17-OHPC by the corresponding P450s. In contrast, the amount of M2HLM was significantly decreased in the presence of ketoconazole, indicating that the metabolism of 17-OHPC was markedly inhibited. The Vmax (0.29 ± 0.02 nmol/min/mg) and Km (77.94 ± 19.4 µM) values for M2HLM were observed to decrease significantly in the presence of ketoconazole (Vmax = 0.03 ± 0.002, Km = 15.38 ± 3.9). An IC50 (0.17 µM) value for the inhibition of 17-OHPC metabolism by ketoconazole in HLMs (n = 3) was also calculated (Fig. 5).


Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 4. Ketoconazole inhibits 17-OHPC metabolism. Of the various inhibitors evaluated, namely, coumarin (CYP2A6, {blacksquare}), {alpha}-naphthoflavone (CYP1A2, {blacktriangleup}), sulfaphenazole (CYP2C9, {blacksquare}), quinidine (CYP2D6, {square}), ketoconazole (CYP3A, {diamondsuit}), and methimazole (FMO3, bullet), only ketoconazole (1 µM) showed significant inhibition of 17-OHPC (0–200 µg/ml expressed as 0–467 µM) metabolism in HLMs (n = 3) when compared with control (no inhibitor, {Delta}). This indicated the role of CYP3A isoforms in metabolizing 17-OHPC and the potential involvement of CYP3A4. Approximately 80% inhibition of 17-OHPC was observed at 1 µM ketoconazole (*, p < 0.05). V (µmol/min/mg microsomal protein) denotes the major metabolite formation (M2HLM) expressed in terms of 17-OHPC equivalents.

 

Figure 5
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 5. Estimation of IC50. IC50 value for the ketoconazole-mediated inhibition of 17-OHPC (25 µM) metabolism was calculated to be 0.17 µM in HLMs (n = 3). Inhibition of 17-OHPC metabolism was evaluated by estimating the formation of M2HLM.

 
Incubations with baculovirus-expressed human P450 isoforms. Studies were also performed in baculovirus-infected insect cells expressing various P450s. The metabolizing activity of each CYP3A isoform for 17-OHPC was compared with control microsomes, which were devoid of any P450 activity. The results indicate the involvement of CYP3A4/5 in the metabolism of 17-OHPC and the formation of the major metabolite (M2HLM) was a result of CYP3A4/5 pathway (Table 1).


View this table:
[in this window]
[in a new window]

  		TABLE 1 Identification of P450s involved in the metabolism of 17-OHPC

Mean ± S.D. of triplicate in vitro preparations. Amount of metabolite is expressed in terms of HPC equivalent. Significant levels of metabolite were detected on incubation of 17-OHPC (100 µM) with expressed CYP3A4/5, whereas no metabolite could be detected for other P450s.

 

Incubations with human hepatocytes: inhibition studies. Results obtained from inhibition experiments performed in HLM were confirmed in fresh adult human hepatocytes (Fig. 6) using chemical inhibitors for CYP3A (ketoconazole and troleandomycin). Troleandomycin and ketoconazole inhibited 17-OHPC metabolism (M2HH formation) by 75 and 89%, respectively, indicating involvement of CYP3A4/5.


Figure 6
View larger version (50K):
[in this window]
[in a new window]

  		FIG. 6. CYP3A4/5 metabolizes 17-OHPC in fresh adult human hepatocytes. Ketoconazole (K, 1 and 10 µM) and troleandomycin (TAO, 100 µM) showed significant (p < 0.05) inhibition of 17-OHPC (25 µM) metabolism in FHHs (n = 4). This indicated the role of CYP3A isoforms in metabolizing 17-OHPC and the potential involvement of CYP3A4. Inhibition of 17-OHPC metabolism was evaluated by estimating the formation of M2HH.

 

Incubations with human hepatocytes: induction studies. Induction studies were performed in FHHs to further confirm CYP3A to be the primary enzyme responsible for 17-OHPC metabolism. CYP3A inducers like rifampin, phenobarbital, and clotrimazole increased M2HH formation (Fig. 7). Rifampin showed the maximum (2.2-fold) induction in 17-OHPC metabolism. Phenobarbital showed 2.1-fold, whereas clotrimazole showed the least, 1.2-fold induction in human hepatocytes.


Figure 7
View larger version (33K):
[in this window]
[in a new window]

  		FIG. 7. CYP3A inducers increase 17-OHPC metabolism in fresh adult human hepatocytes. Incubation of FHHs (n = 3) with specific CYP3A4 inducers, namely, rifampin (p < 0.05), phenobarbital (p < 0.05), and clotrimazole (p = 0.054) increased the metabolism of 17-OHPC (50 µM) as compared with control (dimethyl sulfoxide). The estimation of induction was based on the generation of the major metabolite (M2HH) in hepatocytes.

 
Incubations with human hepatocytes: correlation studies. Rates of formation of 17-OHPC metabolite (M2) with liver microsomes and fresh adult human hepatocytes were measured at a substrate concentration of 100 µM. There was considerable interindividual variation in the values of metabolite generated (Fig. 8). Formation of M2HLM and M2HH correlated significantly with the testosterone 6β-hydroxylation activity (CYP3A4) in both microsomes (r = 0.89) and hepatocytes (r = 0.91), respectively.


Figure 8
View larger version (28K):
[in this window]
[in a new window]

  		FIG. 8. Correlation analysis of 17-OHPC metabolite M2 with CYP3A-dependent 6β-hydroxytestosterone formation. A, correlation analysis in FHHs; n = 7. B, correlation analysis in HLMs; n = 7. CYP3A activity was estimated by incubating 17-OHPC (100 µM) and testosterone (100 µM) in individual human liver microsomal preparations and FHH cultures. The experiment was carried out in triplicate in both cases.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
To the best of our knowledge, this is the first report identifying the human hepatic enzymes, which play a major role in the metabolic pathway of 17-OHPC. The metabolism of 17-OHPC was evaluated using HLMs, fresh human adult hepatocytes, and recombinant systems expressing P450s and FMOs. Incubation of 17-OHPC with heat-inactivated microsomes did not result in any loss of 17-OHPC or generation of any 17-OHPC metabolites. Significant metabolism of 17-OHPC, however, was seen in all three systems that were tested. Generation of metabolites and loss of parent drug confirmed this observation. No metabolites were observed in the absence of NADPH, thus confirming the metabolism to be P450-mediated. Incubation of 17-OHPC with HLMs under the conditions for direct generation of conjugates did not alter 17-OHPC concentrations or yield any 17-OHPC metabolites, ruling out a direct role of UDP glucuronosyltransferase-mediated pathway. However, studies evaluating the role of UDP glucuronosyltransferase as a sequential pathway in 17-OHPC metabolism are being carried out currently.

Multiple approaches were used to identify the P450 enzymes involved in the metabolism of 17-OHPC. Metabolism of medroxyprogesterone acetate, a potent progestogenic compound, has been reported in literature (Kobayashi et al., 2000Go) to be catalyzed mainly by CYP3A4. Because medroxyprogesterone acetate is structurally similar to 17-OHPC, it was expected that the metabolic pathways for both compounds would likely coincide. The results obtained confirmed this expectation. In HLMs, 17-OHPC was metabolized to one major and two minor metabolites. P450 inhibition experiments indicated CYP3A4/5 to form the main metabolic pathway. Ketoconazole (CYP3A inhibitor) at 1.0 µM inhibited 90% of 17-OHPC biotransformation in HLMs. These findings and the lack of effect of {alpha}-naphthoflavone, quinidine, coumarin, and sulfaphenazole (inhibitors for CYP1A2, CYP2D6, CYP2A6, and CYP2C9) suggest a major role for CYP3As in 17-OHPC metabolism.

In fresh human adult hepatocytes, five (M1–5HH) metabolites were observed on incubation of 17-OHPC with M2HH being the major metabolite. Ketoconazole and troleandomycin (a known CYP3A inhibitor) significantly inhibited 17-OHPC metabolism. Inducers of CYP3A, namely, rifampicin, phenobarbital, and clotrimazole, significantly increased 17-OHPC metabolism in comparison with control. Thus, the results in HLMs were successfully reproduced in FHHs. This showed CYP3A to play the major role in 17-OHPC metabolism in hepatocytes as well.

To further confirm our results, we conducted experiments to check the ability of expressed enzyme systems to catalyze the biotransformation of 17-OHPC. CYP3A isoforms were the only enzyme systems that metabolized 17-OHPC significantly. The enzyme activity for 17-OHPC metabolism was observed to be higher for CYP3A5 isoform than CYP3A4. The reason for the differential activity is not known at this time.

The CYP3A subfamily is known to be expressed most abundantly (i.e., from 10–60% of total P450s) in human liver and plays a pivotal role in the oxidative metabolism of many clinically important drugs. Among the CYP3A isoforms tested (i.e., CYP3A4 and CYP3A5), CYP3A4 is the major isoform in adult humans. CYP3A5 is polymorphically expressed in approximately 10 to 20% of the adult livers (Wrighton et al., 1990Go). Overall, we can predict that CYP3A4 would be the major P450 isoform responsible for the hepatic metabolism of 17-OHPC in the majority of adult patients given 17-OHPC. Given that CYP3A5 has higher activity for 17-OHPC, genetic polymorphism in CYP3A5 may have a significant role in 17-OHPC metabolism and pharmacokinetics, but this remains to be evaluated. It has been proposed that the prolonged and more potent action of 17-OHPC over progesterone involves the cleavage of 17-OHPC molecule to 17{alpha}-hydroxyprogesterone (Fig. 1B) and release of free caproic acid. It was also suggested that caproic acid could affect genomic pathways and hence have an effect on progesterone signaling pathways (Attardi et al., 2007Go). However, results of radio-HPLC–based method confirmed that the structure of 17-OHPC remained intact during metabolism by human enzymes. Our observation does not support the hypothesis that 17-OHPC is a prodrug that gets metabolized to progesterone or hydroxy progesterone and thus prevents preterm labor. Furthermore, LC/MS-based analysis of the major metabolite (M2HLM, m/z = 445) generated from incubations in HLMs indicated toward a possible monohydroxylation or an oxidation mechanism, although di- and tri-hydroxy metabolites (data not shown) were also observed in HLMs. Identity of the major metabolite (M2HH) generated from human hepatocytes needs to be elucidated, although preliminary data based on retention time and LC/MS data analysis indicate M2HH (m/z = 445) to be similar to M2HLM. Similar results have recently been reported by Yan et al. (2008Go). The study, performed in HLMs, reported the lack of 17-OHPC cleavage to release caproate ester based on radio-HPLC experiment. Furthermore, generation of mono-, di-, and tri-hydroxylated metabolites was also observed. Our study reports the production of three main metabolites from human liver microsomal incubation, whereas the Yan study reported the formation of 21 metabolites. This can be attributed to our study being based on a simpler, isocratic LC method with UV detection as compared with a gradient LC method with MS (single quadrupole) detection used by the previous study. However, M2HLM (m/z = 445.0) is the major metabolite (>60%) reported by both studies, and this was the primary focus of this publication.

Our study shows that the metabolism of 17-OHPC is predominantly mediated by CYP3A isoforms, mainly CYP3A4. Given that the activity of CYP3A enzyme is known to vary between subjects, one would expect large variation in the pharmacokinetics of 17-OHPC in pregnant subjects. In women pregnant with twins we noted a large variation in concentrations despite a fixed dosing regimen (Caritis and Venkataramanan, 2007Go). CYP3A4 plays a major role in the metabolism of various drugs because of its abundance in the liver and its broad substrate specificity. Numerous clinically important drugs are known as substrates of CYP3A4 (Rendic and Di Carlo, 1997Go). Thus, further in vitro and clinical studies are required to assess 17-OHPC–associated clinically relevant metabolic drug interaction with any coadministered CYP3A4 substrates/inhibitors.

Pregnancy is a dynamic state of the human body that is characterized by significant variations in the physiology and metabolism. Changes in the metabolizing activity of P450s, especially CYP3A4, have been reported in literature (Tracy et al., 2005Go). Activity of CYP3A4 has been shown to increase significantly in all the trimesters in humans. Furthermore, the expression level of CYP3A isoforms varies from individual to individual. Thus, we expect significant interindividual fluctuations in 17-OHPC plasma concentrations in pregnant patients over time. In clinical studies, 17-OHPC is administered as a fixed dose regimen of 250 mg weekly. The therapy was observed to be effective in 33% of the patients in the study of Meis et al. (2003Go). It is possible that the low success rate of 17-OHPC may be attributable to the significant variation in CYP3A-mediated metabolism of 17-OHPC in these patients. Based on the abovementioned facts, it may be necessary to investigate various dosing regimens and individualize the therapy with 17-OHPC. Monitoring 17-OHPC plasma levels and adjustment of dose accordingly may be needed to improve therapeutic outcomes.


   Acknowledgments
 
We thank Brianne Raccor for help with the radio-HPLC and acknowledge the overall support and contribution of all members of the Obstetrical-Fetal Pharmacology Research Network (OPRU).


   Footnotes
 
This work is supported in part by National Institute of Child Health and Human Development Grant HD-047905-2.

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

doi:10.1124/dmd.108.021444.

ABBREVIATIONS: 17-OHPC, 17{alpha}-hydroxyprogesterone caproate; P450, cytochrome P450; FMO, flavin-containing monooxygenase; HLM, human liver microsome; HPLC, high-performance liquid chromatography; HMM, hepatocyte maintenance medium; FHH, fresh human hepatocyte; LC/MS, liquid chromatography/mass spectrometry; MS/MS, tandem mass spectrometry.

Address correspondence to: Raman Venkataramanan, 731 Salk Hall, 3501 Terrace Street, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261. E-mail: rv{at}pitt.edu


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 


Attardi BJ, Zeleznik A, Simhan H, Chiao JP, Mattison DR, and Caritis SN (2007) Comparison of progesterone and glucocorticoid receptor binding and stimulation of gene expression by progesterone, 17-alpha hydroxyprogesterone caproate, and related progestins. Am J Obstet Gynecol 197: 599.e591–e597.

Caritis S and Venkataramanan R (2007) Pharmacokinetics of 17-alpha hydroxyprogesterone caproate in women with twin gestation. Reprod Sci Suppl 14: 294.

Chiba M, Hensleigh M, Nishime JA, Balani SK, and Lin JH (1996) Role of cytochrome P450 3A4 in human metabolism of MK-639, a potent human immunodeficiency virus protease inhibitor. Drug Metab Dispos 24: 307–314.[Abstract]

Court MH and Greenblatt DJ (1997) Molecular basis for deficient acetaminophen glucuronidation in cats. An interspecies comparison of enzyme kinetics in liver microsomes. Biochem Pharmacol 53: 1041–1047.[CrossRef][Medline]

Kobayashi K, Mimura N, Fujii H, Minami H, Sasaki Y, Shimada N, and Chiba K (2000) Role of human cytochrome P450 3A4 in metabolism of medroxyprogesterone acetate. Clin Cancer Res 6: 3297–3303.[Abstract/Free Full Text]

Kostrubsky VE, Ramachandran V, Venkataramanan R, Dorko K, Esplen JE, Zhang S, Sinclair JF, Wrighton SA, and Strom SC (1999) The use of human hepatocyte cultures to study the induction of cytochrome P-450. Drug Metab Dispos 27: 887–894.[Abstract/Free Full Text]

Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275.[Free Full Text]

Mattison DR, Damus K, Fiore E, Petrini J, and Alter C (2001) Preterm delivery: a public health perspective. Paediatr Perinat Epidemiol 15(Suppl 2): 7–16.[CrossRef][Medline]

Meis PJ, Klebanoff M, Thom E, Dombrowski MP, Sibai B, Moawad AH, Spong CY, Hauth JC, Miodovnik M, Varner MW, et al. (2003) Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate. N Engl J Med 348: 2379–2385.[Abstract/Free Full Text]

Nelson AC, Huang W, and Moody DE (2001) Variables in human liver microsome preparation: impact on the kinetics of l-alpha-acetylmethadol (LAAM) n-demethylation and dextromethorphan O-demethylation. Drug Metab Dispos 29: 319–325.[Abstract/Free Full Text]

Paneth NS (1995) The problem of low birth weight. Future Child 5: 19–34.[CrossRef][Medline]

Rendic S and Di Carlo FJ (1997) Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab Rev 29: 413–580.[Medline]

Strom SC, Pisarov LA, Dorko K, Thompson MT, Schuetz JD, and Schuetz EG (1996) Use of human hepatocytes to study P450 gene induction. Methods Enzymol 272: 388–401.[Medline]

Tracy TS, Venkataramanan R, Glover DD, Caritis S, for the National Institute for Child Health and Human Development Network of Maternal-Fetal-Medicine Units (2005) Temporal changes in drug metabolism (CYP1A2, CYP2D6 and CYP3A activity) during pregnancy. Am J Obstet Gynecol 192: 633–639.[CrossRef][Medline]

Wrighton SA, Brian WR, Sari MA, Iwasaki M, Guengerich FP, Raucy JL, Molowa DT, and Vandenbranden M (1990) Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3). Mol Pharmacol 38: 207–213.[Abstract]

Wu DH and Allen WM (1959) Maintenance of pregnancy in castrated rabbits by 17-alpha-hydroxy-progesterone caproate and by progesterone. Fertil Steril 10: 439–460.[Medline]

Yan R, Nanovskaya TN, Zharikova OL, Mattison DR, Hankins GD, and Ahmed MS (2008) Metabolism of 17alpha-hydroxyprogesterone caproate by hepatic and placental microsomes of human and baboons. Biochem Pharmacol 75: 1848–1857.[CrossRef][Medline]



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dmd.108.021444v1
36/9/1896    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sharma, S.
Right arrow Articles by Venkataramanan, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sharma, S.
Right arrow Articles by Venkataramanan, R.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
All ASPET Journals Molecular Pharmacology Pharmacological Reviews
Molecular Interventions Drug Metabolism and Disposition