The effect of gossypol on fast axonal transport and microtubule assembly.

Gossypol at micromolar concentrations (2 microM) was found to inhibit axonal transport and a microsomal ATPase activity in the frog sciatic nerve, although axonal microtubules and the neuronal content of AMP, ADP and ATP were not affected. At slightly higher concentrations (30-40 microM), gossypol also inhibited microtubule assembly and neuronal energy metabolism. Gossypol accumulated in the nerve and the results indicate that gossypol may act as a potent neurotoxin.

PIP: The effect of gossypol on axonal transport, the translocation of substances along microtubules in neurites, was investigated in an experiment on sciatic nerves and 8th and 9th dorsal ganglia dissected from frogs. This effect was defined as the ratio between the radioactivity in front of the ligature in the experimental and the control nerve. When gossypol was present in the nerve compartment, axonal transport decreased from 52% at 2 mcgM to 13% at 30 mcgM. On the other hand, when the drug was present only in the ganglionic compartment, the inhibition was less marked, and at 30 mcgM axonal transport was still 70% of control. Since gossypol is lipid-soluble, its action may be related to its accumulation in myelin, which is more abundant in the nerve compartment than the ganglion. Support for this hypothesis was provided by the finding that, when frog nerves were incubated for 17 hours in Ringer solution containing 30 mcgM of gossypol, the concentration of gossypol in the nerve was 100-times higher than in the surrounding Ringer solution. Nerves incubated for 17 hours with 2 mcgM of gossypol did not show any significant changes in their content of ATP, ADP, or AMP. Moreover, the axonal morphology and the density of the microtubules were normal after treatment of the nerve with gossypol concentrations at or below 10 mcgM. At concentrations of 30-40 mcgM, gossypol did inhibit microtubule assembly and neuronal energy metabolism. The finding that gossypol accumulates in the nerve suggests a need for attention to the possible neurotoxicity of this male contraceptive.

Gossypol prevents activation of purified proenzyme of human seminal plasma acidic proteinase.

Gossypol inhibits the potential activity of the proenzyme form of human seminal plasma acidic proteinase, but has no effect on the active enzyme under the conditions tested. Inhibition of proenzyme is rapid and pH-dependent: 50% inhibition can be observed at gossypol concentrations of approx. 1.5 X 10(-5) M. SDS-polyacrylamide gel electrophoresis indicates that treatment of proenzyme with gossypol prevents the formation of active enzyme that normally occurs on acidification. Determination of free amino groups with 1-fluoro-2,4-dinitrobenzene suggests that gossypol reacts with 7.8 out of the 11.0 lysine residues in proenzyme: such a reaction could interfere with the activation process.

PIP: Studies of the effect of gossypol on the purified proenzyme of human seminal plasma acidic proteinase suggest that, while there is no effect on the active enzyme, the potential activity of the proenzyme form is inhibited. When the purified proenzyme was incubated at pH 8.0 with 0.25 mM of gossypol, 70% of potential activity was abolished within 2.5 minutes; moreover, this inhibitory activity was progressive, to the extent that only 5% of the original activity remained 90 minutes after incubation. When the concentrations of gossypol were varied, 50% inhibition of the potential activity of the proenzyme was observed at concentrations of approximately 15 mcgM. The reaction between gossypol and proenzyme was further found to be pH-dependent; a pH of at least 6.5, optimally 8.0, was required for activity inhibition. To determine whether treatment with gossypol altered protein structure, the active enzyme and proenzyme were preincubated with gossypol and subjected to SDS-polyacrylamide gel electrophoresis. Gossypol treatment decreased the potential activity of the proenzyme by 97%, but failed to affect the active enzyme. Studies on the amino acids affected suggested that gossypol protected 7.8 of 11 lysine residues in proenzyme from reaction with FDNB, presumably by formation of Schiff's bases. The physiological significance of the gossypol inhibition of seminal plasma acidic proteinase proenzyme remains unclear. Since the acidic proteinase requires acid for activation, it is unlikely that the enzyme plays a role in fertilization. It is more probable that seminal plasma acidic proteinase disposes of the remains of the ejaculate after intercourse, at a point when the vaginal pH has become acidic again. Thus, inhibition by gossypol could interfere with this "cleaning up" process.

Aromatization of norethindrone to ethinyl estradiol by human placental microsomes.

The interaction of 19-norethindrone [4-estren-17 alpha-ethinyl-17 beta-ol,3-one (NET)] with human placental microsomes was investigated using enzymatic and spectral techniques. The incubation of [6,7-3H]norethindrone with human placental microsomes, NADPH, and molecular oxygen resulted in the production of ethinyl estradiol [1,3,5-(10)estratrien-17 alpha-ethinyl-3,17 beta-diol (EE)]. The reaction was linear with respect to time and protein concentration. Androstenedione inhibited the enzymatic aromatization of NET to EE. The product was identified by thin layer chromatography, recrystallization to constant specific activity, and derivative formation. No acid or base was used in any step of product identification. To ensure that spontaneous aromatization of metabolites of NET did not contribute to our results, representative samples were treated with sodium borohydride before processing. Sodium borohydride reduces the 4-en-3-one grouping of the A-ring, thereby preventing chemical aromatization. Sodium borohydride treatment did not reduce our observed yields of EE from NET. The addition of NET to a preparation of solubilized, partially purified placental microsomal cytochrome P-450 yielded a type I cytochrome P-450 binding spectrum. The apparent spectral dissociation constant for NET binding to cytochrome P-450 was 28 microM. These results suggest that NET is enzymatically aromatized to EE by human placental microsomes.

Desogestrel- and levonorgestrel-containing oral contraceptives have different effects on urinary excretion of prostacyclin metabolites and serum high density lipoproteins.

Prostacyclin synthesis is stimulated in vitro by high density lipoproteins (HDL), which themselves are differently affected by desogestrel (DG)- and levonorgestrel (LN)- containing oral contraceptives. In this study we measured the urinary excretion of the metabolites of prostacyclin [6-keto-prostaglandin F 1 alpha(6-keto) and 2,3-dinor-6-keto-prostaglandin F1 alpha (dinor)] and of thromboxane A2 [thromboxane B2 (TxB2)] as well as serum HDL- and HDL2 cholesterol concentrations before and during DG and LN administration alone or in combination with ethinyl estradiol (EE) in 26 women. Before the trial, urinary dinor excretion correlated with serum total HDL cholesterol (r = 0.499; P less than 0.01) and HDL2 cholesterol levels (r = 0.668; P less than 0.001; n = 26). Administration of DG (150 micrograms/day; 14 women) or LN (150 micrograms/day; 12 women) for 2 weeks caused no changes in the excretion of these prostanoids, but LN administration decreased serum HDL cholesterol levels. After that, the women underwent a monophasic regimen of 150 micrograms DG or LN plus 30 micrograms EE for 3 months and thereafter polyphasic regimens of the same steroids for a further 3 months. The DG-containing pills increased urinary dinor excretion by 25-40%, but caused no changes in 6-keto and TxB2 excretion, as measured on days 19-21 of the cycles. LN-containing pills reduced urinary 6-keto excretion by 22% at the end of polyphasic treatment, but caused no changes in dinor and TxB2 output. DG plus EE, but not LN plus EE, increased serum total HDL and HDL2 cholesterol concentrations by a maximum of 25%. Thus, a DG plus EE combination may stimulate PGI2 synthesis by increasing the levels of HDL/HDL2. Theoretically, this stimulation protects against occlusive vascular disorders.

PIP: The effects of desogestrel or levonorgestrel alone and in combination with ethinyl estradiol on the urinary excretion of metabolites of antiaggregatory prostacyclin (PGI2) and thromboxane A2 (TxA2) and on serum high density lipoprotein (HDL) and HDL2 cholesterol concentrations were investigated in 26 women. Baseline urinary 6-keto, dinor, and TxB2 excretion and serum HDL or HDL2 cholesterol concentrations did not differ between study groups. Administration of desogestrel and levonorgestrel alone for 2 weeks caused no changes in PG excretion, but it reduced serum HDL and HDL2 cholesterol concentrations. The desogestrel-estradiol combination was accompanied by 40% and 25% rises in urinary dinor excretion and 20% and 15% rises in urinary 6-keto and dinor excretion at the end of the monophasic and polyphasic regimens, respectively, but no significant changes in urinary TxB2 excretion. The levonorgestrel-estradiol combination produced a 22% decrease in urinary 6-keto excretion during polyphasic treatment, but led to no change in the excretion of the other prostanoids. Both monophasic and polyphasic levonorgestrel and estradiol administration lowered serum HDL and HDL2 cholesterol levels, while monophasic desogestrel plus estradiol increased serum HDL2. The mean relative changes in serum HDL2 cholesterol and urinary dinor excretion were parallel in users of desogestrel plus estradiol, suggesting that a serum HDL cholesterol increase induced by this regimen could be related to increased vascular PGI2 production. Although the mechanism that causes increases in PGI2 and HDL during desogestrel-estradiol administration remains unknown, such a combination has the potential to reduce the risk of occlusive-thrombotic vascular disorders--currently the most serious side effect of oral contraceptive use.

The effect of a desogestrel-containing oral contraceptive on glucose tolerance and leptin concentrations in hyperandrogenic women.

Ovarian hyperandrogenism can be associated with insulin resistance, hyperinsulinemia, glucose intolerance, and obesity. High levels of the lipostatic hormone, leptin, have also been reported in this condition. The purpose of the present study was to examine the effect of an oral contraceptive (OC) of low androgenicity containing desogestrel on glucose tolerance in hyperandrogenic women and the impact of changes in androgenic/estrogenic status on leptin concentrations. Sixteen nondiabetic hyperandrogenic women, aged 29 +/- 1 yr with a body mass index (BMI) of 36.8 +/- 1.8 kg/m2, underwent an oral glucose tolerance test before and after 6 months of therapy with the OC. Free testosterone decreased and sex hormone-binding globulin increased after therapy (P < 0.001). Glucose tolerance deteriorated significantly, and two women developed diabetes. Body weight, BMI, and leptin did not change significantly. Leptin correlated with BMI before (r = 0.56; P = 0.02) and after (r = 0.51; P = 0.04) treatment, but not with glucose, insulin, total and free testosterone, or sex hormone-binding globulin before or after treatment. In conclusion, 1) glucose tolerance should be monitored in hyperandrogenic women using OC, even those of low androgenicity; and 2) changes in androgenic/estrogenic status had no effect on the leptin concentration, suggesting that its sexual dimorphism is not related to sex steroids.

PIP: Ovarian hyperandrogenism can be associated with insulin resistance, hyperinsulinemia, and glucose intolerance--all of which, in turn, have been linked to high levels of the lipostatic hormone, leptin. This study investigated the effect of an oral contraceptive (OC) containing a progestin of low androgenicity on glucose tolerance and insulinemia in hyperandrogenic women and the impact of changes in androgenic/estrogenic status on plasma leptin levels. 16 nondiabetic hyperandrogenic US women (mean age, 29 years) with a mean body mass index of 36.8 kg/sq. m underwent oral glucose tolerance testing before and after 6 months of treatment with an OC containing 30 mcg of ethinyl estradiol and 150 mcg of desogestrel. Treatment was associated with significant decreases in free testosterone and increased sex hormone-binding globulin (p 0.001). Glucose tolerance deteriorated moderately but significantly. After 6 months of treatment, 5 women had normal glucose tolerance, 9 had impaired glucose tolerance, and 2 developed non-insulin-dependent diabetes mellitus. There were no significant changes in serum insulin concentrations, body weight, body mass index, or leptin, but leptin levels were highly correlated with body mass index both before and after treatment. The data suggest that the sexual dimorphism of leptin is not caused by differences in sex hormones. Even when OCs containing low androgenic progestins are prescribed, women at high risk for diabetes should receive regular glucose tolerance tests.

Establishing the minimum effective dose and additive effects of depot progestin in suppression of human spermatogenesis by a testosterone depot.

Hormonally induced azoospermia induced by weekly im injections of testosterone enanthate provides effective and reversible male contraception, but more practical regimens are needed. Given our previous findings that six 200-mg pellets implanted subdermally produced more stable, physiological T levels and reduced the delivered T dose by more than 50% while maintaining equally effective suppression of sperm output with fewer metabolic side-effects than weekly 200-mg testosterone enanthate injections, we sought in this study to determine 1) whether further dose-sparing could be achieved by lower testosterone doses while maintaining efficacy and 2) the efficacy of adding a depot progestin to a suboptimally suppressive depot testosterone dose as a model depot progestin/androgen combination male contraceptive. Healthy volunteers were randomized into groups (n = 10) who received either of two lower T doses (two or four 200-mg T pellets) or four 200-mg T pellets plus a single im injection of 300 mg depot medroxyprogesterone acetate (DMPA). Two T pellets (400 mg, 3 mg/day) had a negligible effect on sperm output. Four T pellets (800 mg, 6 mg/day) suppressed sperm output between the second to fourth postimplant months; output returned to normal by the seventh postimplant month, although only 4 of 10 men became azoospermic or severely oligozoospermic (< 3 mol/L/mL). The addition of a depot progestin markedly increased the extent, but not the rate, of sperm output suppression, with 9 of 10 becoming azoospermic and 10 of 10 becoming severely oligozoospermic. There were no serious adverse effects during the study. Plasma total and free testosterone levels remained within the eugonadal range at all times with each treatment. Plasma epitestosterone was suppressed by all 3 regimens, consistent with a dose-dependent inhibition of endogenous Leydig cell steroidogenesis. Plasma LH and FSH measured by a two-site immunoassay were suppressed in a dose-dependent fashion by T and further suppressed by the addition of DMPA. Sex hormone-binding globulin levels were decreased by DMPA, but not by either T dose. Prostate-specific antigen and lipids (total, low or high density lipoprotein cholesterol, and triglycerides) were not significantly changed in any group. Thus, a depot testosterone preparation with zero order release must be delivered at between 6-9 mg/day to provide optimal (but not uniform) efficacy at inducing azoospermia. The addition of a single depot dose of a progestin to a suboptimal testosterone dose (6 mg/day) markedly enhances the extent, but not the rate, of spermatogenic suppression, with negligible biochemical androgenic side-effects. These findings provide a basis for the use of a progestin/androgen combination depot for hormonal male contraception.

PIP: Clinical research conducted in Australia suggests that a progestin-androgen combination depot has potential for hormonal male contraception. The authors' previous research had indicated that 6 200-mg testosterone enanthate pellets implanted subdermally produced substantial reductions over injections in the delivered testosterone dose while maintaining equally effective suppression of spermatogenesis with few metabolic side effects. The present study sought to determine whether lower testosterone doses would maintain efficiency and to assess the efficacy of adding a depot progestin to a suboptimally suppressive depot testosterone dose (6 mg/day). 10 volunteers received either 2 or 4 200-mg testosterone pellets or 4 200-mg pellets plus a single intramuscular injection of 300-mg depot medroxyprogesterone acetate (DMPA). The testosterone implants alone achieved inadequate suppression of spermatogenesis for a male contraceptive; 400 mg of testosterone (3 mg/day) had a negligible effect on sperm output, while 800 mg (6 mg/day) produced azoospermia or severe oligozoospermia in only 4 of 10 men. However, the addition of DMPA markedly increased the extent, but not the rate, of sperm output suppression: azoospermia was achieved in 9 men and oligozoospermia in all 10 subjects, and sperm suppression persisted for 3 months. Epitestosterone concentrations, used as a marker of Leydig cell steroidogenesis, were decreased in a time- and dose-dependent manner, reaching castrate levels in the combined group. Plasma luteinizing hormone and follicle-stimulating hormone levels were suppressed in a dose-dependent fashion by testosterone and further suppressed by the addition of DMPA. Sex hormone-binding globulin levels were decreased by DMPA, but not by either testosterone dose. Prostate-specific antigens and lipids were not significantly altered by any regimen. There were no discontinuations or reports of side effects.

Effect of oral contraceptives on triazolam, temazepam, alprazolam, and lorazepam kinetics.

The effects of low-dose estrogen oral contraceptives (OC) on the elimination of the oxidized benzodiazepines triazolam (TRZ) and alprazolam (ALP) and the conjugated benzodiazepines temazepam (TMZ) and lorazepam (LOR) were studied in two parallel crossover studies of 20 women each. Women taking OC steroids containing low doses of estrogen and women matched for age, weight, and cigarette smoking received single oral doses of TRZ (0.5 mg) and TMZ (30 mg) or ALP (1 mg) and LOR (2 mg). Kinetics were determined as plasma concentrations during 48 hr after dosing. OCs inhibited the metabolism of ALP: The AUC increased and the elimination rate constant was greater in users of OCs. For TRZ, which has an intermediate extraction ratio, the AUC was increased by OCs but not significantly so. In contrast, OCs decreased the AUC for TMZ and the elimination rate constants for LOR and TMZ. The AUC of LOR was not affected by OCs. Low-dose estrogen OCs may therefore inhibit the metabolism of some oxidized benzodiazepines and accelerate the metabolism of some conjugated benzodiazepines.

PIP: Because benzodiazepines and oral contraceptives (OCs) are among the most widely prescribed drugs and have a potential for interaction, 2 parallel crossover studies were conducted to determine the effects of OCs on the elimination of the oxidized benzodiazepines triazolam (TRZ) and alprozolam (ALP) and the conjugated benzodiazepines temazepam (TMZ) and lorazepam (LOR). 20 healthy women taking OCs containing does of under 35 mcg of ethinyl estradiol for 3 months or more and 20 women matched for age, weight, and cigarette smoking received single oral doses of TRZ (.5 mg) and TMZ (30 mg) or ALP (1 mg) and LOR (2 mg). Treatments were seperated by 28 days to control for effects of menstural cycle on drug metabolism. Kinetics were determined as plasma concentrations during 48 hours after dosing. The data indicated that OCs differentially affect the elimination of the benzodiazepines studied. OCs inhibited the metabolism of ALP: the area under the curve (AUC) increased and the elimination rate constant was greater in users of OCs. For TRZ, which has an intermediate extraction ratio, the AUC was increased by OCs but not significantly so. OCs decreased the AUC for TMZ and the elimination rate constants for LOR and TMZ. The AUC of LOR was not affected by OCs. It was concluded that low-dose estrogen OCs may inhibit the metabolism of some conjugated benzodiazepines and accelerate the metabolism of some conjugated benzodiazepines, but the clinical implications are unclear. The relationship between plasma concentration and effect has not been determined for most benzodiazepines, but the results suggest that the time required to achieve steady-state concentrations would be longer and that there would be higher steady state concentrations of ALP in OC users. Because both TMZ and LOR are eliminated more rapidly by OC users, women taking OCs should achieve steady-state concentrations more rapidly than nonusers. Because TMZ clearance is increased by OCs, mean plasma concentration may be decreased. The usual 30 mg dose of TMZ may therefore be less effective as a hypnotic in OC users.

The effects of rifampin and rifabutin on the pharmacokinetics and pharmacodynamics of a combination oral contraceptive.

BACKGROUND: Rifampin (INN, rifampicin), a CYP34A inducer, results in significant interactions when coadministered with combination oral contraceptives that contain norethindrone (INN, norethisterone) and ethinyl estradiol (INN, ethinylestradiol). Little is known about the effects of rifabutin, a related rifamycin. OBJECTIVES AND METHODS: The relative effects of rifampin and rifabutin on the pharmacokinetics and pharmacodynamics of ethinyl estradiol and norethindrone were evaluated in a prospective, randomized, double-blinded crossover study in 12 premenopausal women who were on a stable oral contraceptive regimen that contained 35 microg ethinyl estradiol/1 mg norethindrone. Subjects were randomized to receive 14 days of rifampin or rifabutin from days 7 through 21 of their menstrual cycle. After a 1-month washout period (only the oral contraceptives were taken), subjects were crossed over to the other rifamycin. RESULTS: Rifampin significantly decreased the mean area under the plasma concentration-time curve from time 0 to 24 hours [AUC(0-24)] of ethinyl estradiol and the mean AUC(0-24) of norethindrone. Rifabutin significantly decreased the mean AUC(0-24) of ethinyl estradiol and the mean AUC(0-24) of norethindrone. The effect of rifampin was significantly greater than rifabutin on each AUC(0-24). Despite these changes, subjects did not ovulate (as determined by progesterone concentrations) during the cycle in which either rifamycin was administered. Levels of mean follicle-stimulating hormone increased 69% after rifampin. CONCLUSION: In this study, rifampin (600 mg daily) was a more significant inducer of ethinyl estradiol and norethindrone clearance than rifabutin (300 mg daily), but neither agent reversed the suppression of ovulation caused by oral contraceptives. The carefully monitored oral contraceptive administration and the limited exposure to rifamycins may restrict the application of this study to clinical situations.

PIP: The relative effects of rifampin and rifabutin (a related rifamycin) on the pharmacokinetics and pharmacodynamics of ethinyl estradiol (EE) and norethindrone were evaluated in a prospective, randomized, double-blinded crossover study in 12 premenopausal women who were on a stable oral contraceptive regimen that contained 35 mcg EE and 1 mg norethindrone. Subjects were randomized to receive 14 days of rifampin or rifabutin from days 7 through 21 of their menstrual cycle. After a 1-month washout period (only the oral contraceptives were taken), subjects were crossed over to the other rifamycin. Findings showed that rifampin significantly decreased the mean area under the plasma concentration-time curve from time 0 to 24 hours [AUC (0-24)] of EE and the mean AUC (0-24) of norethindrone. Rifabutin significantly decreased the mean AUC (0-24) of EE and the mean AUC (0-24) of norethindrone. The effect of rifampin was significantly greater than rifabutin on each AUC (0-24). Despite these changes, subjects did not ovulate (as determined by progesterone concentrations) during the cycle in which either rifamycin was administered. Levels of mean follicle-stimulating hormone increased 69% after rifampin. This study suggests that rifampin (600 mg daily) was a more important inducer of EE and norethindrone clearance than rifabutin, but none of these agents were able to reverse the suppression of ovulation done by oral contraceptives.

The effect of olestra on systemic levels of oral contraceptives.

The effect of olestra, a nonabsorbable, noncaloric fat replacement, on the absorption and efficacy of a highly lipophilic oral contraceptive was investigated in a double-blind, placebo-controlled crossover study with 28 women. Subjects consumed 18 gm/day olestra for 28 days while taking an oral contraceptive containing 300 micrograms of norgestrel and 30 micrograms ethinyl estradiol (Lo/Ovral-28). Blood taken on days 12 to 14 of the treatment cycles was analyzed for ethinyl estradiol and norgestrel. There was no statistically significant difference in time to attain maximum concentration, maximum concentration, or area under the concentration-time curve between the olestra and placebo treatments for either drug component. Measurements of serum progesterone indicated that olestra ingestion did not reduce efficacy as indicated by ovulation. The data show that ingestion of 18 gm/day olestra did not affect the absorption or efficacy of the highly lipophilic oral contraceptive.

Gabapentin does not interact with a contraceptive regimen of norethindrone acetate and ethinyl estradiol.

Anticonvulsants that induce hepatic metabolism increase clearance of oral contraceptive hormones and thereby cause contraceptive failure. Gabapentin is not metabolized in humans and has little liability for causing metabolic-based drug-drug interactions. In healthy women receiving 2.5 mg norethindrone acetate and 50 microg ethinyl estradiol daily for three consecutive menstrual cycles, concurrent gabapentin administration did not alter the steady-state pharmacokinetics of either hormone. Thus, gabapentin is unlikely to cause contraceptive failure.

PIP: Anticonvulsants that induce hepatic metabolism increase the clearance of synthetic estrogens and progestogens used in oral contraceptives (OCs), thereby potentiating contraceptive failure. In contrast, the anticonvulsant drug gabapentin is not metabolized in humans and has little liability for metabolic-based drug interactions. The present study sought to confirm whether concurrent administration of gabapentin would alter the pharmacokinetics of norethindrone acetate (2.5 mg) and ethinyl estradiol (50 mcg) in healthy US women. A total of 13 women were enrolled for three menstrual cycles each. Pharmacokinetic values did not change appreciably as a result of the addition of gabapentin. The rate and extent of absorption of both hormones were unaffected by the anticonvulsant. Gabapentin plasma concentration time profiles and pharmacokinetic values from this study were similar to historical values after administration of gabapentin alone. The observed lack of interaction between gabapentin and norethindrone acetate or ethinyl estradiol is consistent with the fact that gabapentin is not metabolized, is not an inducer or inhibitor of hepatic drug metabolizing enzymes, is absorbed via a specific transport system for amino acids, and is not bound to plasma proteins. Anticonvulsant drugs that do not interact with OCs should be considered for the treatment of epileptic women of childbearing age who are using this method of fertility control.

Immunosuppression with medroxyprogesterone acetate.

PIP: The immunosuppressive effects of MPA (medroxyprogesterone acetate) were tested in clinical studies with laboratory dogs and rabbits. The laboratory procedures employed in the studies are explained and results are tabulated and graphed. 2 microscopic photographs illustrate the observed effects. MPA significantly prolonged the survival of dogs with renal allografts. MPA was shown to enhance the immunosuppressive activity of azathioprine in dogs with renal allografts. In rabbits, MPA extended survival of rabbit skin allografts and suppressed the primary humoral antibody response. The influence of MPA on circulating leukocytes and on the histology of lymph nodes, spleen, thymus, liver, and kidney was also assessed. MPA is probably not as potent an immunosuppressive agent as azathioprine or corticosteroids. It is, however, useful in humans due to its low toxicity. It is possible that high local progesterone levels in the fetus and placenta during pregnancy help to prevent fetal rejection.^ieng

Effect of oestrogen dose on whole blood platelet activation in women taking new low dose oral contraceptives.

Oral contraceptive use is known to cause changes in the haemostatic system. These changes are thought to be related to oestrogen dose and to provide a possible link between the increased risk of thromboembolic disease known to occur in women taking oestrogen containing oral contraceptives. This study measured whole blood platelet activation, serially, in women taking oral contraceptives containing 20 micrograms and 30 micrograms ethinyloestradiol combined with desogestrel. Increased levels of ADP and arachidonic acid induced aggregation were observed in women taking the 30 micrograms ethinyloestradiol combination. Platelet release of beta-thromboglobulin (beta TG) was also significantly increased. Increased collagen induced aggregation was observed but this failed to reach statistical significance for the individual treatment groups. In women taking the 20 micrograms ethinyloestradiol combination, a significant increase was only observed when platelets were stimulated with arachidonic acid. Platelet factor 4 (PF4) levels were unchanged in both groups. Significantly higher levels of beta TG were observed in women taking the 30 micrograms ethinyloestradiol combination compared with women taking the 20 micrograms ethinyloestradiol combination. These results show that oral contraceptive use is associated with platelet activation. Women taking the 20 micrograms ethinyloestradiol combination show less changes in platelet activation than women taking the 30 micrograms ethinyloestradiol combination. This lower dose pill may therefore be particularly suitable for high risk women wishing to use oral contraception.

PIP: To evaluate the effects of low-dose oral contraceptives (OCs) on platelet function, hematologic measures were compared in 45 Irish women taking OCs containing 20 or 30 mcg of ethinyl estradiol as well as 150 mcg of desogestrel. Serum samples were collected before treatment and at 6, 14, and 22 weeks after OC use commenced. ADP induced whole blood platelet aggregation was significantly increased in users of OCs containing 30 mcg of ethinyl estradiol, reaching a maximum at 22 weeks, but not in users of the low-dose OC. A significant increase in collagen induced aggregation was observed when both groups of OC users were combined, but not when either was tabulated separately. Both groups showed significant increases in arachidonic acid induced aggregation. Platelet count, hematocrit, and platelet factor 4 levels were unaffected. Increased levels of beta-thromboglobulin were observed at 6, 14, and 22 weeks in the 30 mcg group; there was no significant change in the 20 mcg group. Since the low-dose 20 mcg ethinyl estradiol OC produced fewer changes in platelet activation, its use is recommended for women with risk factors for thromboembolic disease.

Effects of a long-acting progestin on reproductive function in female mice.

The duration of activity of a long-acting progestin, medroxyprogesterone acetate, was compared using three tests for progestational activity: the induction of stromal mitosis in the endometrium, implantation of blastocysts and inhibition of ovulation. The duration of activity was similar in each test and was longer when higher doses were given.

PIP: Three experiments were conducted to evaluate the duration of activity of a long-acting progestin, medroxyprogesterone acetate: 1) induction of stromal mitosis in the endometrium; 2) implantation of blastocysts; and 3) inhibition of ovulation. In the 1st experiment, randomly bred albino mice were ovariectomized and injected with MPA 1 week later. They were injected at various intervals with 20 ng estradiol-17B in 0.05 ml arachis oil and killed 24 hours later. In the 2nd experiment, male and female mice were mated and the day of finding a vaginal plug was assigned day 1 of pregnancy. On day 4, both ovaries were removed and MPA was injected to maintain the state in which implantation can be initiated by injection of estradiol-17B. Successful initiation is shown by implantation swellings 72 hours later; absence of implantation suggests that the progestin is no longer present at an effective level. In the 3rd experiment, female mice were given MPA before they were mated with male mice. Ovulation suppresson was determined by the difference in time before parturition between MPA-treated mice and untreated mice. The results of all experiments were similar. Effective levels of hormone were observed for 6 days in most animals and maintained for up to 8 days in some animals. Some animals exhibited sufficient progestational activity to allow blastocyst implantation 12 days after injection, while stromal mitosis could not be induced after 8 days. It appears that the state of epithelium, rather than the stromal cells' ability to undergo hormone-induced proliferation, allows implantation as shown by the epithelial mitosis which was still suppressed at 10 days. 1 mg MPA induced progestin activity similar to the physiological state of early pregnancy. Mating was inhibited by higher doses of progestin for unduly long periods.

Plasma concentrations of vasopressin and a prostaglandin F2 alpha metabolite in women with primary dysmenorrhoea before and during treatment with a combined oral contraceptive.

Oral contraceptives reduce menstrual pain but the interaction with vasopressin and prostaglandin F2 alpha, two uterine stimulants related to the condition, is unknown. Ten women with a history of moderate to severe dysmenorrhoea were studied. Repeated blood samples were taken during a first menstrual cycle without treatment, during the first 21 days of a second cycle when they received an oral contraceptive (150 micrograms levonorgestrel and 30 micrograms ethynyloestradiol) and on the first or second day of the bleeding following hormonal withdrawal. Measurements were made of plasma concentrations of arginine vasopressin, 15-keto-13,14-dihydroprostaglandin F2 alpha, oestradiol-17 beta, progesterone, ethynyloestradiol, levonorgestrel, FSH, LH and prolactin, and serum osmolality was measured. Seven of the women rated their discomfort as moderate to severe on the first two menstruations, but as none or light at the withdrawal bleeding; with the rating scale for degree of pain that was used, this decrease in pain was significant (P less than 0.001). The plasma concentration of vasopressin in these seven women showed significant variation, with the highest concentrations being obtained at the beginning of the two painful menstruations (3.76 +/- 0.76 and 1.75 +/- 0.30 (S.E.M.) pmol/l) and at ovulation in the control cycle (1.91 +/- 0.58 pmol/l). During treatment the concentrations were consistently low, except on the first day of withdrawal bleeding (2.33 +/- 0.35 pmol/l). The concentrations of the prostaglandin F2 alpha metabolite showed less variation, but again the values at withdrawal bleeding (271 +/- 39 pmol/l) were not different from those obtained over the painful menstruations (255 +/- 24 and 217 +/- 25 pmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

PIP: To learn more about the beneficial effect of combined oral contraceptives (OCs) on symptoms in primary dysmenorrhea, plasma levels of vasopressin and a prostaglandin F2-alpha metabolite in dysmenorrheic women were investigated before and during treatment with a gestagen-dominated OC. The 10 subjects were administered an OC containing 150 mcg of levonorgestrel and 30 mcg of ethinyl estradiol for 21 days. The 7 women with dysmenorrheic symptoms at the time of blood sampling during the 1st menstruation graded their pain as averaging 2.1 (moderate to severe) + or - 0.3; during the 2nd menstruation, the average value was 2.9 (severe) + or - 0.1, indicating a significant increase in pain at the start of the withdrawal bleeding. Vasopressin concentrations in samples obtained on days 1-3 of the control cycle were significantly higher than those on days 6-8, 20-22, and 24-26 of the control cycle and days 1-2 of the next menstruation. Thus, the highest concentrations were obtained at the beginning of the 2 painful menstruations and at ovulation in the control cycle. During treatment, vasopressin concentrations were consistently low, except on the 1st day of withdrawal bleeding. The concentrations of the prostaglandin F2-alpha metabolite showed less variation, again, values at withdrawal bleeding were not different from those obtained during painful menstruation. Plasma concentrations of ovarian and adenohypophysial hormones, as well as osmolality, were normal throughout. Thus, the present study provided no evidence that there is a reduced release of vasopressin and/or prostaglandin F2-alpha capable of accounting for the beneficial effect of OCs on dysmenorrhea. It is possible, however, that a difference in ovarian hormone concentrations is more pronounced in uterine tissue than in plasma.

Influence of nutritional status on pharmacokinetics of contraceptive progestogens.

PIP: In response to recent studies from India suggesting that malnutrition, as assessed by anthropometric indexes, affects metabolism of oral progestogens, this study administered a mini-pill containing .35 mg of norethindrone (NET) and combination pills containing 250 or 150 mcg of d-norgestrel (d-NG) and either 50 or 30 mcg ethinyl estradiol as a single dose for fasting women of high and low income. Blood samples were collected for up to 24 hours for NET and 80 hours for the combination pills. Pharmacokinetics were evaluated by a least-squares method. Anthropometric measurements were also made. Peak NET levels occurred within 1-2 hours; half-life of plasma NET was shorter among low income, malnourished women compared with high income, well-nourished women. A direct correlation between weight/height and half-life of the drug suggests that malnutrition enhanation rate and reduces NET's half-life. Peak levels for d-NG also were reached between 1 and 2 hours after dosing. In well-nourished women, the decline in plasma d-NG was tri-exponential; malnourished women showed a biphasic curve with a neglible alpha-phase. Therefore, the lower the nutrition status, the faster the plasma clearance of these 2 orally administered compounds. Studies inn rabbits designed to elucidate this connection showed a significant elevation in specific activities of liver microsomal glucuronyl transferase and cytochrome-p450 in undernourished compared with control animals. There was also an increase in the amount (but not affinity) of uterine progesterone receptors in undernourished animals. Another study of a small group of Thai and Indian women showed positive correlation between anthropometric indexes and post peak plasma NET levels; however, an obesity study of Thai women found no such correlation.^ieng

A review of the endometrial changes in Norplant users.

PIP: Prolonged exposure to levonorgestrel associated with use of Norplant contraceptive implants leads, in many acceptors, to menstrual irregularities. Endometrial biopsies collected from a group of Norplant acceptors with persistent or prolonged breakthrough bleeding indicated this side effect is associated with proliferative-type endometrium. The number of both proliferative and secretory endometrium was markedly reduced. The stroma showed edema, sloughing, and inflammatory infiltration. Despite chronic levonorgestrel exposure, only 31% of stroma showed poorly developed decidualization. There was no association between endometrial findings and levonorgestrel levels. Hysteroscopy revealed markedly engorged superficial vessels in long-term Norplant users. It is hypothesized that progestin-related bleeding abnormalities may be due to the changes in the vascular system (e.g., damaged vessels) and hemostatic factors. Confirmation of this hypothesis requires detection of the presence of hemostatic plugs in patients with and without abnormal bleeding, study of the perivascular expression of tissue factor, measurement of estrogen and progesterone receptors at bleeding and non-bleeding sites, and identification of factors that increase angiogenesis and the fragility of uterine vessels.^ieng

Hormonal and nonhormonal factors affecting sex hormone-binding globulin levels in blood.

PIP: Researchers in Utrecht, the Netherlands have studied the effects of different factors, such as oral contraceptives (OCs), on sex hormone binding globulin (SHBG) levels in blood. The SHBG levels in women who continuously used OCs consisting only of .05 mg of ethinyl estradiol (EE2) rose as high as 260% + or - 25% of those in women not using OCs. Further, mean SHBG levels of women using combination OCs of EE2 and levonorgestrel were 10-60% higher than women not using OCs. SHBG levels were significantly higher than the use of a sequential OC containing decreasing amounts of EE2 and increasing amounts of levonorgestrel than those cause by use of a continuous combined OC with .03 mg and .15 mg respectively. As the dosage of EE2 increased in combination OCs with 2.5 mg lynestrenol, the SHBG increased from 20% (.05 mg EE2) to 150% (.75 mg EE2). SHBG levels after taking EE2 and cyproterone acetate increased significantly more (240%) than levels after EE2 and desogestrel (170%), or after EE2 and gestoden (140%) [p.001]. SHBG levels of women who took OCs containing only .03 mg of levonorgestrel daily decreased 35% (p.01). These levels fell by 30% in women who received 150 mg of medroxyprogesterone acetate intramuscularly every 3 months (p.001). SHBG concentrations increased when estrogens were taken orally for noncontraceptive purposes, but they did not change when they were administered percutaneously. As body weight increased the SHBG levels decreased despite hormonal status or sex. Further, the lower the fat content of one's diet the higher the SHBG levels and vice versa. SHBG levels are higher in males with flaccid lungs than they are in males with healthy lungs.^ieng

A risk-benefit assessment of the levonorgestrel-releasing intrauterine system.

The levonorgestrel-releasing intrauterine system (LNG-IUS), has been developed by Leiras Pharmaceuticals, Turku, Finland. It is a new systemic hormonal contraceptive that releases levonorgestrel 20 micrograms every 24 hours. The device provides fertility control comparable with that of female sterilisation, complete reversibility and convenience, and has an excellent tolerability record. The low dosage of levonorgestrel released by its unique delivery system ensures minimal hormone-related systemic adverse effects, which tend to be in the category of 'nuisance' rather than hazardous, and gradually diminish after the first few months of use. In some respects, the contraceptive characteristics of the LNG-IUS have over-shadowed a substantial range of noncontraceptive beneficial effects that are rarely seen with inert or copper-releasing intrauterine contraceptive devices (IUDs), and have important and positive gynaecological and public health implications. This applies particularly to the profound reduction in duration and quantity of menstrual bleeding, and alleviation of dysmenorrhoea, which are associated with the use of the device. Recent studies have shown that the LNG-IUS is effective in preventing endometrial proliferation associated with oral or transdermal estradiol therapy, and in inducing regression of endometrial hyperplasia. Further research is required to determine whether it has a role in regulating the growth of uterine fibroids, and preventing pelvic inflammatory disease. The unique unwanted noncontraceptive effects of the system, including possible development of functional ovarian cysts, and the relationship between menstrual bleeding pattern and ovarian function, also require better understanding, in order to offer appropriate patient counselling and maximise acceptability and continuation of use of the method.

PIP: The levonorgestrel-releasing intrauterine system (LNG-IUS) provides fertility control comparable to female sterilization, convenience, and complete reversibility. This method appears to combine the benefits of oral contraception and the IUD, while avoiding most of their side effects. The low level of LNG released (20 mcg every 24 hours) minimizes the systemic adverse effects associated with hormonal contraception. Unlike inert or copper IUDs, the LNG-IUS is associated with a profound reduction in the duration and quantity of menstrual bleeding and alleviates dysmenorrhea. Moreover, there is evidence that the LNG-IUS prevents the endometrial proliferation associated with estradiol therapy and induces regression of endometrial hyperplasia; its potential for regulating the growth of uterine fibroids and preventing pelvic inflammatory disease remains undetermined. Although large multicenter studies have not detected differences in cervical cytology or breast cancer incidence between copper IUD and LNG-IUS users, long-term epidemiological studies are needed to confirm this finding. Fundal positioning of the LNG-IUS is essential to ensure uniform exposure of the endometrium to the progestogen, prevent expulsion, and maximize efficacy. A promising future use for the LNG-IUS is in protecting the endometrium during postmenopausal hormone replacement therapy. Overall, the research suggests that the LNG-IUS comes close to meeting many of the requirements of an ideal contraceptive.

Clinical significance of the androgenicity of progestins in hormonal therapy in women.

Optimal efficacy has been achieved in both oral contraception and postmenopausal replacement therapy. The current challenge is to minimize the side effects and metabolic impact of the administered hormones in both oral contraceptives and hormone replacement agents. When the dose of estrogen in oral contraceptives was reduced the risk of thromboembolism decreased, but the androgenic side effects of the progestin became increasingly apparent. The addition of progestins to hormone replacement therapy reduces the risk of endometrial cancer associated with unopposed estrogen, but their androgenicity offsets the favorable effects of estrogen on lipid metabolism. Androgens not only cause troublesome clinical side effects but also induce changes in blood levels of lipoproteins that have been associated with an increased risk of atherogenesis and coronary heart disease, as well as alterations in glucose and insulin levels. Both the side effects and the adverse effects on lipoprotein and glucose metabolism can be reduced by the use of less androgenic progestins.

PIP: In order to offset the undesirable clinical effects of progestins in oral contraceptives (OCs) or in hormone replacement therapy, effort has been made to reduce the amount of progestin used and to use progestins with lower androgenicity. It is pointed out that the androgenicity is related to the structural relationship between progestins and 19-nortestosterone. Based on the relative binding affinities (RBAs) for rat prostatic androgen receptors and for sex hormone binding globulin (SHBG), it has been noted that levonorgestrel, which is the active isomer of norgestrel, has twice the androgenicity of norethisterone. There have been research results which confirm OCs with progestins with reduced androgenicity; research shows norethindrone in Ortho-Novum 7/7/7 and levonorgestrel in Triphasil both minimize the effect on lipid metabolism. Another study shows only those more androgenic progestins reduce HDL. The newest low dose progestins in OCs are norgestimate, desogestrel, and gestodene. The action of progestins is on lipid and carbohydrate metabolism.

Sequential regimen of the antiprogesterone RU486 and synthetic progestin for contraception.

OBJECTIVE: To examine the effect of sequential use of the antiprogesterone RU486 and synthetic progestin on ovarian function of healthy women. DESIGN: Healthy women were given a sequential antiprogesterone-progestin treatment. Blood samples were taken twice a week during one control cycle and one to three treatment cycles; prospective analysis. SETTING: The outpatient clinic of the Helsinki City Maternity Hospital, Helsinki, Finland, and Steroid Research Laboratory, Department of Medical Chemistry, University of Helsinki, Helsinki, Finland. PATIENTS: Eleven healthy women, volunteers, 20 to 34 years of age. INTERVENTIONS: A dose of 25 mg/d of RU486 was given during cycle days 1 to 21, and synthetic progestin (5 mg of norethisterone to six and 5 mg of medroxyprogesterone acetate to five women) during cycle days 22 to 31. MAIN OUTCOME MEASURES: Serum P, E2, FSH, and LH were measured from serum samples. RESULTS: In 20 of the 24 treatment cycles analyzed the serum concentrations of P were anovulatory. In the remaining 4 cycles, P levels rose above 3 ng/mL, suggestive of ovulation. Folliculogenesis was not completely inhibited, but serum E2 profiles were subnormal and delayed. Bleeding control was satisfactory. CONCLUSIONS: Antiprogesterone RU486 hampers or delays follicular development, suggesting a possible use as an estrogen-free oral contraceptive. However, the synthetic progestins used in this regimen induced serum P rises in some cycles. The synthetic progestin provides the cycle control, but its possible effect on the reliability of the method remains to be evaluated.

PIP: To assess the effectiveness of an estrogen-free oral contraceptive (OC) involving the sequential administration of an antiprogesterone and synthetic progestin, 11 healthy Finnish women 20-34 years of age were enrolled in a prospective investigation for 1-3 cycles. 25 mg of RU-486 was administered for the first 21 days of the cycle, followed by 5 mg of norethisterone or medroxyprogesterone acetate for the next 10 days. It was hypothesized that prolonged RU-486 administration would prevent the follicle from surviving for ovulation. During the RU-486 phase, P increases above 3 ng/ml (suggestive of ovulation) were recorded in only four of the 24 treatment cycles analyzed. Mean serum estradiol concentrations were 147 pg/ml in ovulatory women and 72 pg/ml in anovulatory women, indicating that follicle development was subnormal and delayed, but not completely inhibited. Baseline secretion of luteinizing hormone (LH) increased during RU-486 administration, while an LH surge greater than 100% over baseline was recorded in the majority of cycles during progestin administration. No significant changes occurred in follicle-stimulating hormone levels. Menstrual bleeding began 1-5 days after progestin discontinuation; women who took norethisterone experienced no breakthrough bleeding or spotting during treatment. It is concluded that the main antiovulatory effect of RU-486 is attributable to its antiprogestational effect on the preovulatory P rise.