The Effect of Gene Variants on Levonorgestrel Pharmacokinetics When Combined With Antiretroviral Therapy Containing Efavirenz or Nevirapine.

Reduced levonorgestrel concentrations from the levonorgestrel contraceptive implant was previously seen when given concomitantly with efavirenz. We sought to assess whether single nucleotide polymorphisms (SNPs) in genes involved in efavirenz and nevirapine metabolism were linked to these changes in levonorgestrel concentration. SNPs in CYP2B6, CYP2A6, NR1I2, and NR1I3 were analyzed. Associations of participant demographics and genotype with levonorgestrel pharmacokinetics were evaluated in HIV-positive women using the levonorgestrel implant plus efavirenz- or nevirapine-based antiretroviral therapy (ART), in comparison to ART-naïve women using multivariate linear regression. Efavirenz group: CYP2B6 516G>T was associated with lower levonorgestrel log10 Cmax and log10 AUC. CYP2B6 15582C>T was associated with lower log10 AUC. Nevirapine group: CYP2B6 516G>T was associated with higher log10 Cmax and lower log10 Cmin . Pharmacogenetic variations influenced subdermal levonorgestrel pharmacokinetics in HIV-positive women, indicating that the magnitude of the interaction with non-nucleoside reverse transcriptase inhibitors (NNRTIs) is influenced by host genetics.

Interactions between tenofovir and nevirapine in CD4+ T cells and monocyte-derived macrophages restrict their intracellular accumulation.

OBJECTIVES: There is no pharmacokinetic interaction between tenofovir and nevirapine, but a higher emergence rate of resistance mutations has been reported when these drugs are coadministered. We sought to examine if there is a potential intracellular interaction that may account for the emergence of resistant virus. METHODS: Primary CD4+ and CD14+ cells were isolated from healthy volunteer blood. Monocyte-derived macrophages were differentiated from CD14+ cells. Accumulation of radiolabelled tenofovir and nevirapine was then assessed in these cells. RESULTS: We show here that tenofovir and nevirapine immune cell intracellular concentrations are lower when coincubated in CD4+ cells and monocyte-derived macrophages, but not in CD14+ cells. CONCLUSIONS: These data indicate a potential intracellular drug-drug interaction between these drugs that warrants further investigation.

Cultured CD4T cells and primary human lymphocytes express hOATPs: intracellular accumulation of saquinavir and lopinavir.

BACKGROUND AND PURPOSE: Drug efflux tranporters (P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP)) limit the cellular uptake of human immunodeficiency virus protease inhibitors but the contribution of influx transporters in cells that (over)express P-gp or MRP is less clear. Here, we studied the expression of one influx transporter system, human organic anion-transporting polypeptide (hOATP), in some T-cell lines (CEM, CEM(VBL), CEM(E1000)) and in peripheral blood mononuclear cells (PBMCs) and examined the effects of manipulation of influx/efflux transporters on the uptake of saquinavir and lopinavir. EXPERIMENTAL APPROACH: The expression of hOATPs was studied by PCR. We used hOATP substrate or inhibitor (estrone-3-sulphate (E-3-S) or montelukast, respectively) and inhibitors of P-gp (XR9576) and MRP (MK571 and frusemide) to study functional interactions between influx and efflux transporters in the uptake of saquinavir and lopinavir. Lipophilicity of the drugs was measured by octanol/saline partition coefficient. KEY RESULTS: CEM cells, their variants and PBMCs express various hOATP isoforms, with OATP3A1 detected in all of the cells. MK571, XR9576 and frusemide increased the uptake of saquinavir and lopinavir. E-3-S and montelukast reduced the uptake of saquinavir and lopinavir in some, but not all, of the cells. Pretreatment of the cells with MK571, XR9576 or frusemide, followed by E-3-S co-incubation reduced the cellular accumulation of saquinavir and lopinavir. Lopinavir is much more lipophilic than saquinavir. CONCLUSIONS AND IMPLICATIONS: Human OATPs, MRP, P-gp and lipophilicity determine the cellular uptake and retention of saquinavir and lopinavir. These data may have important implications for drug-drug interactions, drug safety and efficacy.

Comparison of the induction profile for drug disposition proteins by typical nuclear receptor activators in human hepatic and intestinal cells.

BACKGROUND AND PURPOSE: Certain nuclear receptors (NRs) such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR) mediate induction of some cytochrome P450 enzymes and ABC transporters but conflicting reports exist. The purpose of this study was to assess the reasons for these discrepancies and use a standardized approach to compare activators of NRs. EXPERIMENTAL APPROACH: Dexamethasone, pregnenolone 16alpha-carbonitrile, rifampicin, phenobarbital and chenodeoxycholic acid were incubated with HepG2, Caco-2 and cryopreserved human hepatocytes prior to analysis of mRNA and protein for CYP2B6, CYP3A4, CYP3A5, ABCB1, ABCC1, ABCC2, PXR, CAR and FXR. KEY RESULTS: Dexamethasone significantly up-regulated PXR, CYP3A4 and ABCB1 expression in HepG2 and Caco-2 cells. As a result, including dexamethasone as a media supplement masked the induction of these genes by pregnenolone 16alpha-carbonitrile, which may explain discrepancies between previous reports. In the absence of dexamethasone, significant activator-dependent induction was observed in all cell types. Significant correlations were observed between the fold increase in mRNA and in protein, which were, for most instances, logarithmic. Changes in mRNA expression were greater in cell lines than primary cells but for most transcripts correlations were observed between fold increases in HepG2 and hepatocytes. CONCLUSIONS AND IMPLICATIONS: Clearly, no in vitro system can imitate the physiology of a hepatocyte or intestinal cell within an intact organ in vivo, but these data explain some of the discrepancies reported between laboratories and have important implications for study design.

A simplified approach to determining P-glycoprotein expression in peripheral blood mononuclear cell subsets.

P-glycoprotein (P-gp), encoded by the MDR-1 (multidrug resistance) gene mediates the cellular efflux of several therapeutic agents with the potential of treatment failure. The differential expression of P-gp in many localised tissues and cells of the hematopoietic system implies diverse physiological and pharmacological roles. The exact function of P-gp involved in multidrug resistance remains unclear owing to the numerous discrepancies between different laboratories. The ability to characterise accurately P-gp expression has important clinical implications. However, a complete consensus recommendation regarding methods of P-gp detection has been difficult to reach. With the advancement in immune technology and new commercially available antibodies, we describe a simplified direct immunofluorescent assay capable of detecting surface P-gp expression in peripheral blood mononuclear cells (PBMCs) and subpopulations of lymphocytes in vivo by dual colour flow cytometry. Results were expressed as mean increase in fluorescence (MI) compared to isotypically matched controls. Using this assay, differential basal P-gp expression was found to exist in the following significant hierarchy CD56+ (MI=0.684+/-0.273; n=15)>CD8+ (MI=0.312+/-0.117; n=15)>CD4+ (MI=0.194+/-0.086; n=15). This method is rapid and reproducible and has potential use for in vitro and in vivo application.

Determination of P-gp and MRP1 expression and function in peripheral blood mononuclear cells in vivo.

P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) mediate the efflux of many therapeutic agents and have been implicated in the treatment failure of many infectious diseases and cancers. The ability to characterise the expression and function of these transporters in vivo is important when assessing the pharmacological activity of drugs. We investigated some of the problems involved in screening the multidrug resistance status of individuals using flow cytometry. Expression of P-gp and MRP1 on the surface of lymphocytes isolated from blood samples (30 ml) was determined by indirect immunofluorescence. Functional ability was assessed by measuring the efflux of specific fluorescent dyes. Results were expressed as a mean fold increase in fluorescence from the isotype control (expression) and a change in fluorescence compared to the load (function). Using these assays, we determined the expression of P-gp to be 2.01+/-0.40, n=30 and MRP1 to be 1.46+/-0.23, n=25. Functional ability was 6.98+/-4.97, n=25 for P-gp and 1.55+/-0.25, n=25 for MRP1. The dye efflux studies were associated with a lack of specificity and a number of methodological difficulties. There was no correlation between the expression and function of P-gp (r=0.338; p=0.10) or MRP1 (r=0.283; p=0.17). Therefore, we considered determination of P-gp and MRP1 expression to be a more reproducible and accurate approach to clinical investigation into the role of multidrug resistance.

Zidovudine phosphorylation determined sequentially over 12 months in human immunodeficiency virus-infected patients with or without previous exposure to antiretroviral agents.

We sought to determine whether the intracellular activation of zidovudine (ZDV) varied over time and with previous antiretroviral exposure in human immunodeficiency virus-infected individuals and to examine whether there is an association between virological responses and intracellular phosphorylation. A total of 23 patients (12 treatment naïve, 11 previously treated with ZDV) who commenced ZDV as part of dual nucleoside therapy were prospectively monitored for 12 months or until withdrawal from the study. No association was observed between virological responses at 2 weeks and 3 months and ZDV phosphorylation. The mean intracellular concentrations of ZDV mono-, di-, and triphosphates did not change significantly over time or with previous ZDV exposure. The rate of formation of total ZDV phosphates was increased in patients with CD4 counts <100 cells/mm(3). Previous reports from in vitro cell culture experiments or cross-sectional cohort studies suggesting alterations of ZDV phosphorylation over time are not confirmed by this longitudinal study.

The intracellular activation of lamivudine (3TC) and determination of 2'-deoxycytidine-5'-triphosphate (dCTP) pools in the presence and absence of various drugs in HepG2 cells.

AIMS: Lamivudine (3TC, 2'-deoxy-3'-thiacytidine) requires intracellular metabolism to its active 5'-triphosphate, 3TC-5'-triphosphate (3TCTP), to inhibit the replication of hepatitis B virus (HBV). We have investigated the activation of 3TC, in the presence and absence of a range of compounds, in HepG2 cells. The intracellular levels of the endogenous competitor of 3TCTP, 2'-deoxycytidine-5'-triphosphate (dCTP), were also determined and 3TCTP/dCTP ratios calculated. METHODS: The effects of a number of compounds on 3TC (3H; 1 microM) phosphorylation were investigated by radiometric h.p.l.c. dCTP levels were determined using a template primer extension assay. 3TCTP/dCTP ratios were calculated from these results. RESULTS: The phosphorylation of 3TC was significantly increased in the presence of either hydroxyurea (HU), methotrexate (MTX), or fludarabine (FLU). For example, at 100 microM HU, control 3TCTP levels were increased to 361% of control, whereas at 100 microM FLU, control 3TCTP levels were increased to 155%. dCTP pools were significantly reduced in the presence of HU and FLU, at 100 microM concentrations only. However, for all the above three compounds investigated, the ratio of 3TCTP/dCTP was favourably enhanced (e.g. at 1 microM MTX, 255% of control). Neither ganciclovir (GCV), lobucavir (LCV), penciclovir (PCV), adefovir dipivoxil (ADV), nor foscarnet (FOS) had any significant effects on 3TC phosphorylation or dCTP pools. CONCLUSIONS: These results suggest that the activity of 3TC may be potentiated when combined with one of the modulators studied. The lack of an interaction between 3TC and the other anti-HBV agents is reassuring. These in vitro studies can be used as an initial screen to examine potential interactions at the phosphorylation level.

The rabbit study: ritonavir and saquinavir in combination in saquinavir-experienced and previously untreated patients.

Thirteen protease inhibitor-naive patients with HIV-1 infection, and 12 patients with a median of 58 months prior treatment with saquinavir (SQV) monotherapy, were treated with SQV (400 mg twice daily) and ritonavir (RIT, 500 mg twice daily) in a study designed to assess the effect of prior treatment with SQV monotherapy on the antiretroviral activity of RIT-SQV combination therapy. Median baseline viral load and CD4+ cell counts were 155,000 and 262,000 copies/ml and 333 and 225 cells/mm3 in the naive and experienced groups, respectively. Mean viral load changes at 24 weeks were -1.63 and -0.27 log copies/ml in the naive and SQV-experienced groups, respectively (intent-to-treat analysis). Baseline genotype by point mutation assay and sequencing in the SQV-experienced group was highly predictive of virological response. Eight of 11 SQV-experienced patients had evidence of phenotypic resistance to RIT at baseline, despite previous treatment with SQV only. There was strong correlation between phenotypic resistance to RIT and the presence of the L90M mutation. We conclude that prolonged prior treatment with saquinavir monotherapy may produce cross-resistance to ritonavir and reduce the subsequent response to ritonavir-saquinavir in combination. In this study, both phenotypic resistance to ritonavir and presence of the L90M mutation predicted the viral load response to ritonavir-saquinavir.

Effect of protease inhibitors on nucleoside analogue phosphorylation in vitro.

AIMS: Combination antiretroviral therapy for human immunodeficiency virus (HIV) infection now involves both nucleoside analogues and protease inhibitors. Since intracellular phosphorylation is essential for the activity of all the nucleoside analogues this study was designed to investigate interactions with protease inhibitors at the intracellular level which may alter antiviral efficacy. METHODS: PHA-stimulated PBMCs (3 x 10[6] cell/plate) and U937 cells (4 x 10[6] cells/plate) were incubated with either radiolabelled zidovudine (ZDV), stavudine (d4T), zalcitabine (ddC), lamivudine (3TC) or didanosine (ddI) in the presence and absence of the protease inhibitors, indinavir, ritonavir, and saquinavir (0.1-10 microM) for 24 h. Cells were extracted overnight prior to analysis by radiometric h.p.l.c. Intracellular phosphates were standardised to pmol per million cells. RESULTS: None of the three protease inhibitors tested had any significant effect on the intracellular phosphorylation of the five nucleoside analogues. It is particularly important to focus on the active triphosphate anabolites and data for control vs ritonavir (10 microM) incubations in U937 cells were as follows: ZDVTP, 0.19 +/- 0.02 vs 0.21 +/- 0.2 pmol/10(6) cells (mean +/- s.d.; n = 5); d4TTP, 0.30 +/- 0.13 vs 0.27 +/- 0.26; 3TCTP, 0.32 +/- 0.12 vs 0.26 +/- 0.19; ddCTP, 0.07 +/- 0.04 vs 0.06 +/- 0.02, ddATP, 0.014 +/- 0.003 vs 0.018 +/- 0.006 pmol/10(6) cells. CONCLUSIONS: The protease inhibitors, indinavir, ritonavir and saquinavir have no effect on the enzymes responsible for phosphorylation. Combining protease inhibitors and nucleoside analogues should not lead to any intracellular interactions in vivo.

Lamivudine (3TC) phosphorylation and drug interactions in vitro.

Lamivudine (2'-deoxy-3'-thiacytidine; 3TC) is a dideoxynucleoside analogue that inhibits the replication of human immunodeficiency virus (HIV). We are currently investigating the intracellular metabolism of 3TC to its active triphosphate (3TCTP) in peripheral blood mononuclear cells (PBMC) and a monocytic cell line (U937). Optimal phosphorylation of 3TC was achieved after incubation for 24 hr, with 3TC diphosphate (3TCDP) the predominant metabolite formed, in both cell types investigated. Further studies in PBMCs followed preincubation with the mitogen phytohaemagglutinin (PHA) for 72 hr. This enabled greater detection of phosphates, compared to resting cells. A 3TC concentration of 1 microM was chosen for future interaction studies, allowing good detection of 3TC and phosphates on radiochromatograms whilst being similar to the plasma level found in clinical studies (i.e. 3 microM). With a shift in treatment to combination therapy, it is essential that potential interactions between nucleoside analogues are investigated at the phosphorylation level, as this could affect antiviral activity. Both deoxycytidine (dC) and 2',3'-dideoxycytidine (ddC) significantly inhibited 3TC phosphorylation (e.g. at dC 100 microM, no 3TCTP was detected in PBMCs; P < 0.001, whereas 66% of control 3TCTP production was observed in U937 cells; P < 0.01). Zidovudine (ZDV) caused a small but significant reduction of 3TC phosphate production in both PBMCs and U937 cells. However, this may be due to toxicity or an effect on endogenous dCTP pools. Neither 2',3'-dideoxyinosine (ddI) or 2',3'-didehydro-2',3'-dideoxythymidine (d4T) significantly inhibited 3TC phosphorylation. These results suggest it would be better to coadminister two nucleoside analogues with different activation pathways.

Effects of drugs on 2',3'-dideoxy-2',3'-didehydrothymidine phosphorylation in vitro.

Drugs commonly administered to patients infected with the human immunodeficiency virus (HIV) have been studied for their propensity to alter the intracellular phosphorylation of the anti-HIV nucleoside analog stavudine (2',3'-dideoxy-2',3'-didehydrothymidine; d4T) in peripheral blood mononuclear cells (PBMCs) and U937 cells in vitro. PBMCs isolated from the blood of healthy volunteers were stimulated by the mitogen phytohemagglutinin (10 microg/ml) for 72 h. Stimulated PBMCs (3 x 10(6) cells/plate) were then incubated with [3H]d4T (0.65 microCi; 3 microM) and either acyclovir, dapsone, ddC, ddI, fluconazole, foscarnet, ganciclovir, itraconazole, lobucavir, ranitidine, ribavirin, rifampin, sorivudine, sulfamethoxazole, trimethoprim, lamivudine (3TC), zidovudine, or thymidine (30 and 300 microM) for 24 h. Doxorubicin and drugs showing some evidence of inhibition were also studied at 0.3 and 3 microM. Cells were extracted overnight with 60% methanol prior to analysis by radiometric high-performance liquid chromatography. Additional data for nine of the drugs were obtained by incubation with [3H]d4T in U937 cells for 24 h. The effect of d4T (0.2 to 20 microM) on zidovudine (0.65 microCi; 0.018 microCi) phosphorylation was also studied. Zidovudine significantly reduced d4T total phosphates in PBMCs and U937 cells (in PBMCs to 33% [P < 0.001] and 17% [P < 0.001] of that in control cells at 3 and 30 microM, respectively). A small reduction in zidovudine phosphorylation was seen with d4T but only at d4T:zidovudine ratios of 100 and 1,000. Of the other compounds screened, only thymidine, ribavirin, and doxorubicin produced inhibition of d4T phosphorylation in both PBMCs and U937 cells. However, doxorubicin was cytotoxic at 3 microM. The decrease in d4T phosphorylation in the presence of ribavirin is consistent with previous findings with zidovudine. Although ddC significantly inhibited the phosphorylation of d4T in PBMCs, this was not seen in U937 cells, and it is probable that the findings in PBMCs are related to mitochondrial toxicity [based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cytotoxicity assay]. The only drugs screened which may interfere with d4T phosphorylation at clinically relevant concentrations were zidovudine, ribavirin, and doxorubicin.

In vitro screening of nucleoside analog combinations for potential use in anti-HIV therapy.

With the results from the Delta and ACTG 175 clinical trials clearly showing an increased benefit of two drugs over monotherapy, combination nucleoside analog therapy looks set to play a major role in the battle against HIV. It is therefore essential that suitable combinations of drugs are used in clinical trials. We investigated the intracellular activation of zidovudine (ZDV), zalcitabine (ddC), and lamivudine (3TC) in MOLT-4 cells in two- and three-drug combinations at clinically achieved concentrations. The phosphorylation of ZDV and 3TC to their active triphosphate anabolites was not affected by the presence of the other drugs studied. However, the phosphorylation of ddC was significantly inhibited when incubated with 3TC, resulting in levels of ddC triphosphate (ddC-TP) less than 50% of control values. This can be explained by the requirement of both nucleoside analogs for the enzyme deoxycytidine kinase to carry out the initial step in their phosphorylation pathways, and by the comparatively low plasma concentrations of ddC achieved in vivo. These results suggest that regimens containing nucleoside analogs should be designed taking into account potential interactions affecting phosphorylation.

Drug interactions with zidovudine phosphorylation in vitro.

We have investigated the effect of a range of drugs (some commonly coadministered with zidovudine [ZDV] to human immunodeficiency virus-positive patients) on intracellular phosphorylation of ZDV by stimulated peripheral blood mononuclear cells, Molt 4 cells, and U937 cells in vitro. Of the drugs tested (azoles, antiviral agents, antibiotics, and anticancer agents), only doxorubicin and ribavirin caused inhibition of anabolite formation as measured by high-performance liquid chromatography. This in vitro approach may provide important leads to potential interactions at the phosphorylation level in patients with human immunodeficiency virus disease. It is reassuring that so many commonly administered drugs do not alter ZDV phosphorylation.

The metabolism of zidovudine by human liver microsomes in vitro: formation of 3'-amino-3'-deoxythymidine.

The characterization of the enzymatic step(s) involved in the reduction of 3'-azido-3'-deoxythymidine (zidovudine)(ZDV) to 3'-amino-3'-deoxythymidine (AMT) was pursued. AMT formation by human liver microsomes was NADPH dependent, enhanced under anaerobic conditions, and increased by flavin adenine dinucleotide (FAD) and FMN. Carbon monoxide inhibited AMT formation by up to 80%. The effect of theophylline (CYP1A substrate), tolbutamide (CYP2C substrate), chlorzoxazone, thiobenzamide, p-nitrophenol, mercaptoethanol, isoniazid (CYP2E substrates), cortisol (CYP3A substrate), ketoconazole, itraconazole, fluconazole, cimetidine, micronazole (CYP inhibitors), methimazole (flavin-containing mono-oxygenase inhibitor), chloramphenicol (undergoes nitroreduction), allopurinol (xanthine oxidase inhibitor) and dicoumarol (DT-diaphorase inhibitor) on AMT formation were studied to see if the reduction reaction was mediated by a particular isozyme. The greatest inhibition was observed with ketoconazole (concentration producing 50% inhibition = 78.0 microM). At this concentration ketoconazole acted as a non-selective inhibitor of several CYP isozymes. Overall, these data suggested that ZDV reduction was probably mediated by both cytochrome P450 isozymes and NADPH-cytochrome P450 reductase. Formation of AMT, as measured by intrinsic clearance (Clint), was significantly increased in microsomes from rats pre-treated with phenobarbitone, dexamethasone and clofibrate (inducers of CYP2B, CYP3A and CYP4A, respectively). Pre-treatment of rats with beta-naphthoflavone and ethanol (CYP1A and CYP2E1 inducers, respectively) had no effect on AMT formation.

Zidovudine pharmacokinetics in zidovudine-induced bone marrow toxicity.

1. The major adverse effect of zidovudine (ZDV) is haematological toxicity which results in anaemia and granulocytopenia. The aim of the present study was to investigate if HIV-positive patients developing erythroid aplasia/hypoplasia are exposed to higher plasma concentrations of ZDV owing to impaired hepatic metabolism to the major metabolite, 3'-azido-3'-deoxy-5'-beta-D-glucopyranuronosylthymidine (GZDV). 2. Twelve HIV-positive male patients were studied, six having developed bone marrow aplasia/hypoplasia within the first 6 months of ZDV therapy. Each of the patients exhibiting toxicity were matched for age, weight, risk factors for HIV infection and disease stage with patients who had no evidence of early bone marrow toxicity. 3. ZDV was administered orally in doses of 3-10 mg kg-1 and blood samples taken at intervals to 6 h. Urine was collected over the whole 6 h period. ZDV and GZDV were assayed by h.p.l.c. 4. There were no significant differences in the pharmacokinetic parameters between the two groups of patients. For patients with early bone marrow toxicity the elimination half-life of ZDV was 1.10 +/- 0.16 h with an oral clearance of 2752 +/- 1031 ml min-1 compared with values of 1.06 +/- 0.18 h and 2843 +/- 730 ml min-1 seen in the control group. Similarly there was no significant difference in the pharmacokinetics of GZDV or the urinary ratio of GZDV to ZDV. 5. Therefore, despite the fact that ZDV toxicity to haematopoietic progenitor cells has been previously shown to be dose related, there was no indication from this study that it is directly related to plasma concentrations of ZDV.

Metabolism of the oral contraceptive steroids ethynylestradiol, norgestimate and 3-ketodesogestrel by a human endometrial cancer cell line (HEC-1A) and endometrial tissue in vitro.

Human endometrial cancer cells and human endometrial tissue have been extensively used to study steroid hormone action and metabolism. The natural estrogens estradial (E2) and estrone (E1) are known to be metabolized by both cells and tissue with the interconversion of the two steroids and the formation of sulphate conjugates. The aim of the present work was to see if the commonly used oral contraceptive steroids ethynylestradiol (EE2), norgestimate (Ngmate) and 3-ketodesogestrel (3-KDG) were metabolized by a human endometrial cancer cell line (HEC-1A) and human endometrial tissue in vitro. Metabolites were analysed by on-line radiometric HPLC. Endometrial tissue was obtained from women undergoing dilation and curettage or hysterectomy operations. In preliminary studies with endogenous estrogens, HEC-1A cells were able to interconvert E1 and E2; the equilibrium favouring the formation of E2. Normal endometrial tissue extensively converted E2 to E1, tumour tissue appeared to catalyse this reaction much less avidly. In addition sulphate conjugates were formed by normal tissue from some patients. Cell line and endometrial tissue was able to hydrolyse estrone 3-sulphate. With EE2 as substrate there was no evidence of phase I metabolism by cell line or tissue. However, conversion to the presumed 3-sulphate conjugate was observed following incubation with normal tissue from some women. Deacetylation of the progestogen Ngmate to norgestrel oxime (NgOx) was complete within 24 h. There was also some loss of the oxime moiety to give norgestrel (Ng) following incubation with HEC-1A cells. Metabolism of Ngmate was also complete within 24 h following incubation with endometrial tissue. There were both qualitative and quantitative differences in metabolite formation between tissue obtained from different women. In contrast, 3-KDG was relatively resistant to metabolism by cell line and tissue. The major metabolite formed by HEC-1A cells accounted for only 3.3 +/- 0.4% of total added radiolabelled steroid and co-chromatographed with 3 alpha-hydroxydesogestrel. Smaller amounts of other radiometabolites were formed. No phase I metabolites of 3-KDG were formed by normal endometrial tissue, however small amounts of radiometabolites appeared to be formed by malignant tissue. These studies have provided evidence to suggest that the oral contraceptives EE2, Ngmate and 3-KDG are metabolized in the human endometrium. Knowledge of the metabolism of these in target tissues such as the endometrium may be pertinent considering the possibility that metabolites may exert specific effects.

PIP: In England, pharmacologists and a biochemist at the University of Liverpool used an established human endometrial cancer cell line (HEC-1A) and human endometrial tissue in vitro to confirm that HEC-1A and tissue metabolize oral contraceptive (OC) steroids. They used on-line radiometric high-performance liquid chromatography to analyze metabolic activity. Surgeons obtained the endometrial tissue from women undergoing dilatation and curettage or hysterectomy at the Royal Liverpool University Hospital. Earlier research showed that HEC-1A cells interconvert estrone (E1) and 17 beta-estradiol (E2), with E2 predominating in the equilibrium. In this in vitro study, healthy endometrial tissue extensively changed E2 to E1, while malignant tissue caused this conversion to a much lesser extent. The healthy endometrial tissue of some patients formed sulphate conjugates. Both HEC-1A and endometrial tissue hydrolyzed E1 3-sulphate. They did not bring about phase I metabolism when ethinyl estradiol (EE2) was the substrate. Yet, incubation with healthy tissue from some women did lead to conversion of the presumed 3-sulphate conjugate. Incubation with HEC-1A cells completely removed the acetyl group from norgestimate, resulting in mainly norgestrel oxime (55.1% of metabolites) within 24 hours. It also resulted in some norgestrel (16.3%). Incubation with endometrial tissue also brought about complete metabolism of norgestimate within 24 hours. The tissue from different women brought about qualitative and quantitative differences. HEC-1A and endometrial tissue did not metabolize much of 3-ketodesogestrel (3-KDG). In fact, the major metabolite formed by HEC-1A was 3 alpha-hydroxydesogestrel, which made up 3.3% of total added radiolabeled steroid. Healthy endometrial tissue did not produce any phase I metabolites of 3-ketodesogestrel, while tumor tissue may have produced a small amount of radiometabolites. These findings indicate that the endometrium does metabolize the OC EE2, 3-KDG, and norgestimate.

Influence of gestodene and desogestrel as components of low-dose oral contraceptives on the pharmacokinetics of ethinyl estradiol (EE2), on serum CBG and on urinary cortisol and 6 beta-hydroxycortisol.

A randomized controlled clinical trial was undertaken over a 6-month treatment period with two low-dose combined oral contraceptives (OC) to investigate whether the metabolism and elimination of ethinyl estradiol (EE2) is differently influenced by the two progestational components gestodene (G) and desogestrel (D), an issue which has been very controversial recently. The two formulations contained 30 micrograms EE2 each, together with either 75 micrograms G or 150 micrograms D. Of the 40 young women recruited for each formulation, 31 of each group were available for statistical evaluation. The pharmacokinetics of serum EE2 were studied on day 1, 10 and 21 of cycle 1, 3 and 6. There were no significant differences between the two groups in any cycle with respect to parameters measured. This was true for the distinct intracyclical rise in the mean EE2 serum levels from day 1 to day 10 and the smaller further increase between day 10 and day 21, with no change in this respect between the cycles studied. Respective changes were seen with regard to the area under the EE2 serum concentration curve up to 4 and 24 hours (AUC0-4 and AUC0-24), cmax and tmax of serum EE2. The estrogen-dependent corticoid-binding globulin (CBG) increased similarly in the two groups intracyclically and slightly also intercyclically at all times tested. Except for the first treatment cycle, urinary excretion of cortisol and 6 beta-hydroxycortisol displayed a tendency to lower values intracyclically as well as intercyclically, again with no differences between the two groups. Also, the 6 beta-hydroxycortisol-to-cortisol ratio was not different between the groups, showing a slight tendency to rise from about 4 at the beginning of the medication to around 5.5 at the end of the 6th treatment cycle in both groups. It is concluded that G and D as components of low-dose OCs exert comparable effects on the metabolism and elimination of EE2.

PIP: The question of whether the pharmacokinetics of ethinyl estradiol (EE2) is affected differently by the progestins in low-dose combined oral contraceptives containing gestodene or desogestrel was revisited. 80 randomly allocated women took 30 mcg EE2 and either 75 mcg gestodene or 150 mcg desogestrel for the first 21 days of each cycle for 6 months. Blood samples taken on days 1, 10, and 21 of the 1st, 3rd and 6th cycle, at frequent times for 24 hours after pill intake, were analyzed for EE2, corticosteroid binding globulin, cortisol and 6beta-hydroxycortisol. 31 women in each group completed the study. Minor side effects such as headache, breast tension, acne, and nausea occurred in each group; 1 subject dropped out because of headache, nausea, and hypermenorrhea and 1 because of a hematoma. No significant differences were seen in serum EE2 levels including the rise in mean EE2 on days 1-10, or the smaller rise between days 10-21, or the pharmacokinetic parameters Cmax, tmax, area under the curve (AUC) at 0-4 hours, or AUC at 0-24 hours. There was a maximal variation of 11% in intracyclical increases in serum EE2, but no change in intercyclical variations. There were also no significant differences between groups in the expected estrogen-induced increase in corticosteroid binding globulin. Urinary hydroxycortisol increased slightly over each cycle, somewhat more in the 1st cycle, and a bit more in the desogestrel cycles than in gestodene cycles, but not significantly. This study was contrasted in detail with the reports that prompted the controversy over pharmacokinetics of estradiol during intake of the involved combined pills. The import of the assays for cortisol metabolites is the fact that estradiol and cortisol are metabolized by the same liver cytochrome P450 isoenzyme.

Radioimmunological analysis of ethinylestradiol in human serum. Validation of the method and comparison with a gas chromatographic/mass spectrometric assay.

The majority of combination oral contraceptives contain ethinylestradiol (EE2, CAS 57-63-6) as estrogenic component at doses between 50 and 20 micrograms/unit. Since the concentrations of EE2 in the serum of women under oral contraceptive (OC) therapy are in the lower pg-range, highly sensitive and specific analytical methods are required. Radioimmunoassay (RIA) has been the method of choice, but evidence of specificity in the presence of the coadministered progestogens and their metabolites has not always been provided. The present study compares two radioimmunological methods, which use the same antiserum but different sample volumes and standard curves (extracted vs. non-extracted), with a newly developed gas chromatographic/mass spectrometric (GC/MS) method, in order to cross-validate the methods. For that purpose, 51 serum samples obtained from women who had been taking two different combination oral contraceptives were analysed independently by all three methods. The specificity of the antiserum was further examined by submitting ex vivo serum samples obtained from OC-users to a combination of high pressure liquid chromatography (HPLC) and RIA. The results obtained by the three methods were very similar and correlation coefficients (r) obtained from linear regression analysis were about 0.7. There was no interference from the coadministered progestins on the analysis of EE2 as carried out by the three methods. There were no metabolites in the extracts of ex vivo serum samples which showed cross-reactivity with the antiserum used. Modifications of the radioimmunoassay procedure did not markedly affect the results of EE2 determination. If properly validated, radioimmunoassay can be used as an alternative to GC/MS in pharmacokinetic studies.