Objective
Plasma concentrations of 17-alpha-hydroxyprogesterone caproate (17-OHPC) vary substantially in pregnant patients who receive an identical dose. Endogenous steroid hormones may alter 17-OHPC metabolism, which contributes to this large variability.
Study Design
Pooled human liver microsomes were incubated with 17-OHPC alone or in combination with progesterone, hydroxyprogesterone, estrone, estradiol, or estriol. High-performance liquid chromatography with ultraviolet detection was used to quantify 17-OHPC.
Results
Under the conditions that were studied, 17-OHPC metabolism was inhibited by 37% by a combination of endogenous steroid hormones. Progesterone alone significantly inhibited 17-OHPC metabolism by 28% ( P < .001).
Conclusion
17-OHPC metabolism is inhibited significantly by endogenous steroids and, in particular, progesterone. This effect may account for some of the large variation in plasma 17-OHPC concentrations that is seen in pregnant patients who receive a fixed dose of medication.
A synthetic progestin, 17-alpha-hydroxyprogesterone caproate (17-OHPC), is prescribed commonly for the prevention of recurrent, spontaneous preterm birth. The largest trial to investigate the clinical use of 17-OHPC for the reduction of recurrent, spontaneous preterm birth showed a 34% reduction in the incidence of recurrent preterm birth. However, two-thirds of patients who were treated with the standard dose of 17-OHPC in this trial still delivered preterm. Plasma concentrations of 17-OHPC vary substantially in pregnant women who are treated with the standard dose of 17-OHPC. Because both 17-OHPC and endogenous steroid hormones are metabolized by the cytochrome P450 3A4 enzyme (CYP3A4), the variability in plasma concentration may, in part, be due to a drug-endogenous steroid interaction at the level of metabolism by CYP3A4. Any alteration in 17-OHPC metabolism may affect plasma concentrations and ultimately impact the efficacy of 17-OHPC. Thus, the objective of this study was to examine the effect of endogenous steroid hormones on 17-OHPC metabolism in vitro with the use of human liver microsomes (HLM) that expressed the CYP3A4 enzyme. This study primarily evaluated the loss of 17-OHPC in the presence and absence of various concentrations of several steroid hormones.
Materials and Methods
Chemicals
The 17-OHPC was purchased from the United States Pharmacopeia (Rockville, MD). Progesterone, 17-hydroxyprogesterone, estrone, 17-beta-estradiol, estriol, testosterone, 6β-hydroxytestosterone, and β-nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO). All of the substrates and hormones were dissolved in dimethyl sulfoxide.
HLMs
Human liver samples were provided by the laboratory of Steve Strom, PhD, University of Pittsburgh, as part of the Liver Tissue and Cell Distribution System, #N01-DK-7-0004/HHSN267200700004C, under protocol 0411122. Tissue was obtained with informed consent and consent for research from organ donor material that was not suitable for transplantation or from patients who underwent liver resections mainly for cancer treatment. Tissue is provided to Liver Tissue and Cell Distribution System investigators without identifiers. This study was reviewed by the institutional review board at the University of Pittsburgh and was deemed exempt from full board review (PR011120226).
Preparation of HLMs
HLMs were prepared according to methods previously described. Total protein concentration (20 mg/mL) was determined by Lowry’s method with bovine serum albumin as a standard. Pooled HLMs from 3 liver preparations were used in studies. CYP3A4 activity in pooled HLMs was characterized by quantification of the conversion of testosterone to 6β-hydroxytestosterone.
Microsomal incubations
Incubation mixtures contained phosphate buffer (50 mmol/L; pH 7.4), magnesium chloride (1 mmol/L), and pooled HLM (0.05 mg of protein/mL, optimal protein concentration). Incubations were initiated by the addition of NADPH (1 mmol/L) and were carried out in a final volume of 250 μL in a 37°C shaking water bath. Dimethyl sulfoxide was used as a solvent for 17-OHPC and did not exceed 1% of the final reaction volume in all cases. Preliminary studies were conducted to establish the optimal protein concentration of HLM (0-2 mg/mL), incubation time (0-120 min), and 17-OHPC concentration (0-328 μg/mL) to be used in the experimental incubations. Depletion of parent compound in HLM was linear with respect to incubation time and microsomal protein concentration over ranges that were relevant to this study. From the data that used various concentrations of 17-OHPC, we also calculated the Vm (maximum rate of metabolism) and Km (substrate concentration at which the metabolic velocity is half of Vm) for 17-OHPC. Concentrations of 17-OHPC that are below the Km value were selected for the evaluation of the interaction between various hormones and 17-OHPC. The metabolic reactions were stopped by the addition of 250 μL of methanol. All samples were processed in triplicate. The mixture was centrifuged, and 100 μL of the supernatant was injected onto high-performance liquid chromatography for the analysis of 17-OHPC. Control incubations with no cofactor, no protein, and/or no substrate were performed concurrently to validate CYP3A4-dependent metabolism of 17-OHPC.
Steroid hormone-17-OHPC interaction
To evaluate the effect of various steroid hormones on 17-OHPC metabolism, we used fixed concentrations of 17-OHPC (2.5 μg/mL) that were incubated in the presence of a combination of steroid hormones (progesterone, 27.5 μg/mL; 17-hydroxyprogesterone, 1.5 μg/mL; estrone, 2 μg/mL; 17-beta-estradiol, 3.5 μg/mL; estriol, 11 μg/mL) or with individual steroid hormones that included progesterone, 17-hydroxyprogesterone, estrone, 17-beta-estradiol, and estriol at the same concentrations. Both the concentration of 17-OHPC and the endogenous steroid hormones that were used for this experiment were much higher than the concentrations that were observed during pregnancy (micrograms per milliliter vs nanograms per milliliter). These higher concentrations were necessary to ensure adequate substrate for the detection of metabolic interactions over a prolonged period of incubation in the HLM. To more closely mimic in vivo conditions, the ratio of 17-OHPC to endogenous steroid hormone concentration was kept physiologically proportional to the concentrations that were reported during pregnancy. Once significant interactions were identified with the use of the supraphysiologic concentrations of various hormones and 17-OHPC, we performed additional experiments with physiologic concentrations of progesterone and 17-OHPC. Specifically, for these studies in which physiologic concentrations were used, incubations were prepared as described earlier but with a final HLM concentration of 0.0025 mg of protein/mL, 17-OHPC concentration of 50 ng/mL, and progesterone concentrations of 183 and 550 ng/mL. All incubations were carried out for 60 minutes and were performed in duplicate. Reactions were stopped and processed as described earlier.
Additionally, we evaluated whether there is a concentration-dependent relationship between progesterone and the degree of inhibition of 17-OHPC. Various concentrations of progesterone (0 μg/mL, 1.72 μg/mL, 3.44 μg/mL, 6.88 μg/mL, 13.75 μg/mL, 27.5 μg/mL, 55 μg/mL, 110 μg/mL) were incubated with 17-OHPC (2.5 μg/mL). The incubation solution was otherwise prepared as described earlier. All incubations were carried out for 60 minutes and were performed in triplicate. Reactions were stopped and processed as described earlier.
High-performance liquid chromatography–ultraviolet analysis for 17-OHPC
The high-performance liquid chromatography system comprised a Waters 2695 Separations Module attached to Waters 2998 Photodiode Array Detector (Waters Corp, Millford, MA). Chromatographic separations were achieved on a 250 ×4.6 mm, 5 μm Waters Symmetry C18 column (Interlink Scientific Services Limited, Dartford, UK) with an isocratic elution that was monitored at 242 nm with a mobile phase (delivered at 1.0 mL/min) of 90% (vol/vol) methanol in water. The retention time of 17-OHPC was 5.9 minutes. The concentration of 17-OHPC in samples was quantitated according to its calibration curve. The intraday and interday variations that were expressed as coefficient of variation did not exceed 10% in any of the assays. The limit of detection was 5 ng, and the limit of quantitation was 20 ng on the column.
Data analysis
Data from the 60-minute incubation time point was analyzed by 1-way analysis of variance with a Dunnett’s correction for multiple comparisons with a control. Statistical analysis was performed with Stata software (version 11; StataCorp, College Station, TX).
Results
Incubation conditions for HLMs
Preliminary experiments were carried out to establish the optimal incubation conditions for the HLM system. Incubations were carried out both in the presence and absence of NADPH, which was the cofactor necessary for CYP3A4-mediated metabolism. Figure 1 shows that, in the absence of NADPH, 17-OHPC was not metabolized. With the addition of NADPH, the concentration of 17-OHPC decreased by 65.3%, which indicated robust metabolism within 60 minutes. This finding supports the fact that 17-OHPC was metabolized by cytochrome P450 enzymes in the HLM system. Additionally, in the absence of NADPH, there was consistent recovery of 17-OHPC from the incubation tubes, which indicated that the decrease that was observed in the presence of NADPH was secondary to metabolism and not spontaneous decomposition. Beyond 60 minutes, little metabolism occurred that likely was due to exhaustion of the NADPH. Thus, for the purposes of this experiment, 60 minutes was the time point that was selected for all data analysis.
Effects of endogenous steroid hormones on 17-OHPC metabolism
To investigate the effects of endogenous steroid hormones on 17-OHPC metabolism that was catalyzed by CYP3A4 in HLM, 17-OHPC was added to reaction mixtures with individual steroids or a combination of steroids. Approximately 59% of 17-OHPC was metabolized under control conditions with NADPH. When 17-OHPC was incubated with a combination of the endogenous steroid hormones, only 37.1% of 17-OHPC was metabolized, which was significantly less compared with control conditions ( P < .001). Also, in the presence of progesterone alone, only 42.1% of 17-OHPC was metabolized, which was significantly less when compared with control ( P < .001). Rates of 17-OHPC metabolism that were similar to control conditions were observed when 17-OHPC was incubated with 17-alpha-hydroxyprogesterone (58.4%), estrone (62.1%), estradiol (60.8%), and estriol (60.9%).
The same dataset was used to calculate the percent inhibition of 17-OHPC metabolism. Percent inhibition was calculated by subtraction of the percent metabolism in the presence of steroid hormone from the percent metabolism under control conditions (absence of steroid hormone). This difference was then divided by the percent metabolism under control conditions ( Figure 2 ) . Control conditions were considered to be zero inhibition and were used as the comparison for the other incubation conditions. When incubated with a combination of steroid hormones, 17-OHPC metabolism was significantly inhibited by 36.5% ( P < .001) relative to control conditions. Also, in the presence of progesterone, 17-OHPC metabolism was inhibited by 27.9%, which was significant when compared with control conditions ( P < .001). Relative to the control, the presence of 17-hydroxyprogesterone (2.9%), estrone (0%), 17-beta-estradiol (2.3%), or estriol (0.8%) did not result in significant inhibition of 17-OHPC metabolism.
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