Pharmacological developments in male contraception.

To date, the current methods of male contraception are limited to condoms, coitus interruptus and vasectomy, all of which are beset with difficulties. The condom is inconvenient, dulls sensation, and although somewhat effective against sexually transmitted disease, has an increased failure rate over time of usage. Coitus interruptus reduces the pleasurable aspects of intercourse and is plagued with a high failure rate. Vasectomy is virtually sterilisation. The current research into new forms of contraception is as diverse as the mechanisms controlling male fertility. The majority of effort has focused on antispermatogenic agents. Hormonal agents that suppress spermatogenesis appear nearest to final development and are primarily centred around various testosterone esters. These can be administered alone or in combination with progestogens. Another promising line of study centres on gonadotropin releasing hormone (GnRH) antagonism resulting in suppression of gonadotropins. Non-hormonal antispermatogenic agents include numerous phytochemicals, and testicular enzyme inhibitors. Post-testicular approaches to male contraception include agents that interfere with sperm metabolism, motility, maturation or transport. This review summarises recent clinical and animal studies on these compounds with emphasis on their mechanism of action, advantages and drawbacks.

Four flavonoid glycosides from Peganum harmala.

The aerial parts of Peganum harmala yielded four new flavonoids: acacetin 7-O-rhamnoside, 7-O-[6"-O-glucosyl-2"-O-(3'''-acetylrhamnosyl)glucoside and 7-O-(2'''-O-rhamnosyl-2"-O-glucosylglucoside), and the glycoflavone 2'''-O-rhamnosyl-2"-O-glucosylcytisoside.

Enantiomeric resolution by HPLC of axial chiral amides using amylose tris[(S)-1-phenylethylcarbamate].

The enantiomeric resolution of a series of N-arylamides was examined on amylose tris[(S)-1-phenylethylcarbamate] coated onto aminopropylated 7 microns silica with 500 A diameter pores and on naked silica 5 microns particle size with 500 A diameter pores. The enantiomeric resolution obtained for this series was excellent on both columns. The enantioselectivity of cellulose and amylose tris(3,5-dimethylphenylcarbamate) coated onto APS-Hypersil (120 A pore size, 5 microns particle size) was also investigated for this series of compounds.

Effects of two antiprogestins on early pregnancy in the long-tailed macaque (Macaca fascicularis).

The abortifacient effects of mifepristone and HRP 2000 were compared in gravid long-tailed macaques. Thirty-six animals were studied with treatment administered either by the oral (0.5 or 5.0 mg/kg; N = 5 per antiprogestin per dose) or intramuscular (i.m.) routes (0.5 mg/kg; N = 5 per antiprogestin) on gestational days (GD) 23-26; six vehicle controls were included. Blood samples were collected for assay of progesterone (P4) and each of the antiprogestins (pre-treatment, daily GD 23-28, every other day GD 30-40), and animals were monitored sonographically throughout gestation. Results of these studies indicated high rates of abortion with i.m. administration (3/5 mifepristone, 4/5 HRP 2000) and 5.0 mg/kg oral route (4/5, 2/5, respectively), with less effects noted at oral doses of 0.5 mg/kg (2/5, 0/5, respectively). No early abortions were observed in the control groups. Following daily i.m. treatment, peak levels of 8-16 ng/ml mifepristone were detected whereas 6-10 ng/ ml of HRP 2000 were noted (GD 26-27). No serum levels of mifepristone were detected following either of the oral doses whereas serum levels of 2-6 ng/ml HRP 2000 were noted with high dose oral administratation. Results of these studies suggest: (1) both antiprogestins are roughly comparable in terminating early pregnancy although HRP 2000 may be more efficacious when administered i.m. whereas mifepristone may be more effective when administered orally; (2) similar levels of biological activity are seen with the i.m. and high dose oral dosing regimens, with little or no activity with the oral low dose; and (3) infants resulting from surviving pregnancies were not affected by early gestation exposure.

PIP: The primary objective was to compare the relative potencies of mifepristone and another newly synthesized antiprogestin, HRP 2000 (17-alpha-acetoxy-11-beta-[4-N,N-dimethylaminophenyl]-pregna-4,9- diene-3,20-dione) in terminating early pregnancy in gravid long-tailed macaques. 36 animals were studied with treatment administered either by the oral (0.5 or 5.0 ng/kg; N = 5 per antiprogestin per dose) or intramuscular (im) routes (0.5 ng/kg; N = 5 per antiprogestin) on gestational days (GD) 23-26; 6 vehicle controls were included. Blood samples were collected for assay of progesterone (P4) and each of the antiprogestins (pre-treatment, daily GD 23-28, every other day GD 30-40), and the animals were monitored sonographically throughout gestation. Results of these studies indicated high rates of abortion with im administration (3/5 mifepristone, 4/5 HRP 2000) and the 5.0 mg/kg oral route (4/5, 2/5, respectively), with less effects noted at oral doses of 0.5 mg/kg (2/5, 0/5, respectively). No early abortions were observed in the control groups. Following daily im treatment, peak levels of 8-16 ng/ml mifepristone were detected, whereas 6-10 ng/ml of HRP 2000 were noted (GD 26-27). No serum levels of mifepristone were detected following either of the oral doses, whereas serum levels of 2-6 ng/ml HRP 2000 were noted with high-dose oral administration. Results of these studies suggest: 1) both antiprogestins are roughly comparable in terminating early pregnancy, although HRP 2000 may be more efficacious when administered im, whereas mifepristone may be more effective when administered orally; 2) similar levels of biological activity are seen with the im and high-dose oral dosing regimens, with little or no activity with the oral low dose; and 3) infants resulting from surviving pregnancies were not affected by early gestation exposure.

The contraceptive effects of etoprine on male mice and rats.

We had previously found that 2,4-diaminopyrimidines affected spermatogenesis, possibly through the inhibition of testicular dihydrofolate reductase (DHFR). The current study examined the effects of etoprine, a highly lipophilic 2,4-diaminopyrimidine that is also a potent DHFR inhibitor, on the fertility of male mice at various dosages (0.1-50 mg/kg/day) for 55 days and male rats at 5 mg/kg/day for 65 days. Two other substituted diaminopyrimidines were tested at dosages of 50 mg/kg/day for 55 days. Results of breeding trials along with assessment of various parameters indicative of male fertility were noted. We found that of the compounds tested, etoprine is a potent antifertility agent that causes complete infertility at doses of > or = 5 mg/kg/day in mice with a threshold of effectiveness occurring between 1 and 5 mg/kg/day. The antifertility action of etoprine may be related to its capacity to inhibit testicular DHFR and its high degree of lipophilicity.

Developing gossypol derivatives with enhanced antitumor activity.

Preclinical and clinical studies have pointed to the antitumor potential of the naturally occurring polyphenolic binaphthyl dialdehyde, gossypol, as well as its purified (-,+) enantiomers. To explore further the antitumor properties of this multifunctional agent, we synthesized several reactive derivatives including the (-,+) enantiomers of gossypolone and four different gossypol Schiff's bases (AR1, AR2, AR3, AR4). The biological activities of these new agents were screened by measuring their in vitro antiproliferative activity against malignant (MCF-7, MCF-7/adr) or immortalized (HBL-100) human breast epithelial cell lines. Racemic gossypolone showed relatively uniform antiproliferative activity against all of the breast epithelial cell lines with 3- to 5-fold less activity than (--)-gossypol against MCF-7 and MCF-7/adr cells. Of interest, the relative antitumor potency of purified gossypolone enantiomers was reverse that of gossypol enantiomers, since (+)-gossypolone showed up to 3-fold greater inhibition of MCF-7 culture growth than (--)-gossypolone. Of the Schiff's base derivatives only AR3 with its isopropyl amine substituent demonstrated cytotoxic activity comparable to that of (--)-gossypol; derivatives with ethyl, propyl, or butyl amine substituents (AR1, AR2, AR4) had little growth inhibitory activity at culture concentrations up to 25 microM. AR3 activity was greatest against HBL-100 and MCF-7 cells [MCF-7 IC50 values: AR3 = 0.9 microM, (--)-gossypol = 2.3 microM]; unlike (--)-gossypol, however, AR3 showed substantially reduced activity against the multidrug-resistant subline, MCF-7/adr. These structure-activity comparisons suggest that isolation of (-,+)-enantiomers of AR3 and additional chemical modifications including the synthesis of an isopropyl amine Schiff's base of gossypolone will likely yield a newer generation of gossypol analogues with enhanced anticancer potential.

Male antifertility compounds from Tripterygium wilfordii Hook f.

Extracts of the Chinese medicinal plant, Tripterygium wilfordii, cause reversible infertility in male animals. Sub-fractionation studies have now revealed that the plant extracts contain a number of compounds which are potent antifertility agents in male mammals, including the diterpenes triptolide and tripdiolide and an isomer of the latter. A triptolide, 12,13-chlorohydrin, which is a transformation product formed reversibly by interaction of triptolide with HCl, was also found to be active.

Tamoxifen as an antifertility agent in the long-tailed macaque (Macaca fascicularis).

The potential use of tamoxifen, a nonsteroidal antiestrogen, as an antifertility agent was studied in the long-tailed macaque (Macaca fascicularis). Twenty-six cycling females were bred, then treated with a single oral dose of tamoxifen (5 mg/kg) (N = 13) or vehicle (N = 13) on day 4 post-ovulation. Serum progesterone (P4) and tamoxifen concentrations were evaluated on post-ovulation days 4, 8, 12, 16, and 18. No effects of treatment were observed on P4 concentrations or on the fertility rate--pregnancy was achieved in 4/13 controls (31%) and 6/13 treated females (46%). Analysis for serum tamoxifen concentrations in samples collected during the fertility and a supplemental pharmacokinetic study (N = 3; single oral dose of 10 mg/kg; urine and serum evaluated) failed to reveal any detectable tamoxifen levels. It was concluded that (1) absorption of tamoxifen may be negligible under the described treatment regimens or (2) tamoxifen metabolism/clearance occurs at a rapid rate.

Differential effects of the (+)- and (-)-gossypol enantiomers upon Entamoeba histolytica axenic cultures.

The in-vitro anti-amoebic effects of (+/-)-, (+)-, (-)-gossypol and emetine were tested against axenic trophozoites from five Entamoeba histolytica strains. The (-)-isomer was more active than the racemate and the (+)-isomer. These results indicate that the gossypol anti-amoebic activity is mainly due to its content of (-)-gossypol in all strains tested.

Entamoeba histolytica: inhibition of malic enzyme and alcohol dehydrogenase by (+/- )-, (+)-, and (-)- gossypol.

Gossypol, a natural racemic mixture with action on NADP- and NAD-oxidoreductases from diverse species, has been proposed as a possible antiamebic medication considering several of its pharmacological properties. In this study it was found that malic enzyme and alcohol dehydrogenase from Entamoeba histolytica are strongly inhibited by (+/-)-gossypol, and both (+)- and (-)- enantiomers. The inhibition was of the noncompetitive type among their respective substrates in all cases. The (+/-), (+)-, (-)-gossypol half-maximal inhibitory concentrations (IC50) for the malic enzyme were 3.71, 13.37 and 1.03 microM, and against the alcohol dehydrogenase 79.64, 124.43 and 42.56 microM, respectively. Therefore, the (-) enantiomer resulted 3.6 and 13.0 times more potent than the racemic mixture and (+)-gossypol, respectively, to inhibit the malic enzyme, and 1.9 times and 2.9 times more potent than the racemic mixture and (+)-gossypol, respectively, against the alcohol dehydrogenase. Accordingly, one possible mechanism of the antiamebic effect of gossypol could be the inhibition of vital NADP-dependent enzymes as those analyzed in this study.

Progesterone-releasing vaginal rings for use in postpartum contraception. I. In vitro release rates of progesterone from core-loaded rings.

Vaginal rings of Dow Corning 382 Silastic polymer, having identical outside dimensions, were fabricated to contain cores of different diameters loaded with 25% w/w progesterone. Elution of rings was carried out in continuously flowing baths of isotonic saline at 37 degrees C and quantities of progesterone released in 24 h periods measured for up to 128 days. Release of the steroid was shown to be a membrane diffusion-controlled process, modified by the development of a gradually increasing zone of depletion at the core surface. Rings of a suitable core diameter were selected to give initial release of 5 mg/24 h progesterone and sterile batches of these rings, prepared for WHO-sponsored clinical studies in post-partum, lactating women, were shown to give highly consistent and reproducible rates of in vitro drug delivery. A comparison was made with the in vitro release rates of rings containing a homogeneous dispersion of progesterone.

PIP: Chemists from London, England and a chemist from WHO in Geneva, Switzerland compared release rates of progesterone from vaginal rings with cores of different diameters (4, 5, 6, 6.7, and 7.24 mm). The manufacturer loaded each core with 25% w/w progesterone. The technique used to dissolve the progesterone from the silicone rubber core consisted of placing the rings in continuously flowing baths of isotonic saline at 37 degrees Celsius. The learned that a membrane diffusion controlled process, modified by the development of a gradually increasing zone of depletion at the core surface, did indeed release the progesterone. The used the UV absorption method to measure the amount of progesterone released in 24 hour periods for as much as 128 days. The vaginal ring with the 6 mm core released 3.6-5.5 mg progesterone/day in a 90 day period. The daily range of maximum and minimum values for each set of rings demonstrated good reproducibility. Progesterone release was inversely related to diffusion distance (between core surface and ring surface) for each day. Since, in their clinical trials in postpartum women, WHO wanted to use vaginal rings which initially released 5 mg progesterone/day and declines by about 0.5 mg/month under conditions of membrane limited diffusion as the depletion zone grew thicker, the study showed that the rings with a 6 mm core met the criteria. The chemists found that these rings and 4 sterile batches of these rings have highly consistent and reproducible rates in vitro drug delivery. They also compared the vaginal rings with a 6 mm core with rings with at homogeneous dispersion of progesterone throughout the polymer. The homogenous rings 1st released much progesterone then fell quickly from 10-20 mg/day during the 1st week to a gradual release of about 6 mg/day during the end of the 90 days.

A preliminary clinical study of gossypol in advanced human cancer.

A total of 34 patients with advanced cancer were given weekly or daily escalating doses of oral gossypol, a cottonseed-oil constituent showing evidence of antineoplastic activity in pre-clinical studies. No major adverse events occurred and there was no evidence of haematological or biochemical disturbance. As determined by dose escalation in 17 patients, the dose-limiting toxicity was emesis in 16 patients. There was no evidence of tumour regression in any of the 20 patients assessed for response. We conclude that gossypol is safe but unlikely to be clinically useful in patients with advanced cancer.

Further studies on the in vitro activity of gossypol as antiamebic agent.

Gossypol has an in vitro antiamebic effect, which is 11,39, and 980 times more potent than emetime, metronidazole and diiodohydroxyquinolein, respectively, on five Entamoeba histolytica axenic strains. The trophozoite NADP-dependent malic enzyme and alcohol dehydrogenase are inhibited by (+/-)-gossypol, in a noncompetitive manner with respect to the substrate. From the (-)- and (+)- enantiomers, which integrate the natural gossypol mixture, the first one is three and four times more active in inhibiting both the amebic culture growth and the mentioned oxidoreductases, respectively. The above results justify the analysis of gossypol as an antiamebic drug in experimentally infected animals.

Modulation of resistance to alkylating agents in cancer cell by gossypol enantiomers.

Several cell lines resistant to alkylating agents possess increased activity of glutathione-S-transferase (GST) drug detoxifying enzymes. Inhibition of certain enzymes of the glutathione redox system may affect cellular sensitivity to alkylators. We report that the (-.)enantiomer of gossypol is a potent and selective inhibitor of GST alpha and GST pi isozymes, and that in combination with buthionine sulfoximine (BSO), causes the enhanced modulation of alkylator resistance in two drug resistant cell lines with increased GST activity. The use of (-)gossypol alone had no effect on the 2-5-fold resistance of MCF-7 Adr and Walker resistant cells to chlorambucil, melphalan and BCNU. Cellular depletion of glutathione with BSO resulted in a 2-4-fold modulation of cell sensitivity to these alkylators. However, the combination of (-)gossypol with BSO resulted in a markedly greater modulation of alkylator sensitivity than with either inhibitor alone. Therefore, the complementary inhibition of glutathione and GST by BSO and (-)gossypol, respectively, produced a synergistic modulation of alkylator cytotoxicity in these drug resistant cell lines. The favorable clinical pharmacokinetics of (-)gossypol suggest its further evaluation for use in combination with BSO and alkylating agents in clinical trials.

Gossypol effects on endothelial cells and tumor blood flow.

Isomers (-, +) of the antitumor agent gossypol (G) were studied for their ability to reduce tumor ATP and blood flow in rats bearing subcutaneously implanted pancreatic tumors. A 50% reduction in tumor ATP/Pi within ih of a single injection of -G was associated with a 60% decline in tumor blood flow. To determine if these changes in tumor physiology could be due to a direct drug effect on tumor endothelium, G isomers were compared for their ability to alter protein (125I-BSA) permeability and metabolic (32P) labelling of cultured endothelial cells. Treatments for ih produced no endothelial cell leakage, but 24h exposures to either -G (5 microM) or +G (50 microM) produced complete permeability of the monolayers to 125I-BSA. In contrast, 0.5-I.Oh exposures to -G (4 microM) produced 2 to 3-fold increases in phosphorylated 27 kDa heat-shock protein, hsp-27. Hsp-27 phosphoprotein isoforms were differentially labelled following -G and +G exposures with the phosphorylation profile of -G appearing most similar to that of oxyradical producing agents known to induce hsp-27 and injure endothelial cells. We postulate that the tumor ischemic effects of -G are mediated by endothelial response to oxyradical production in a mechanism similar to that of tissue ischemia-reperfusion injury.

Biochemical correlates of the antitumor and antimitochondrial properties of gossypol enantiomers.

Racemic gossypol has been shown to have antitumor properties that may be due to its ability to uncouple tumor mitochondria or to its inhibitory effects on a variety of nonmitochondrial enzymes. We have studied the antimitochondrial and enzyme-inhibiting properties of gossypol in human carcinoma cell lines of breast (MCF-7, T47-D), ovarian (OVCAR-3) colon (HCT-8), and pancreatic (MiaPaCa) origin by comparing the effects of its purified (+)- and (-)-enantiomers. (-)-Gossypol shows up to 10-fold greater antiproliferative activity than (+)-gossypol in the cancer cell lines and in normal hematopoietic stem cells grown in vitro, with IC50 values ranging from 1.5 to 4.0 microM for the cancer cells and from 10 to 20 microM for the human marrow stem cells. As well, multidrug-resistant MCF/Adr cells appear more resistant to (-)-gossypol than their parental cell line. Electron microscopy indicates that the earliest ultrastructural change in tumor cells exposed to a cytotoxic (10 microM) concentration of (-)-gossypol is the selective destruction of their mitochondria. Consistent with this observation, 31P magnetic resonance spectroscopy detects pronounced changes in tumor cell high energy phosphate metabolism within 24 hr of (-)-gossypol treatment, manifest by 1.6- to greater than 50-fold differential reductions in the intracellular ratios of ATP/Pi, relative to (+)-gossypol-treated cell lines; the magnitude of these antimitochondrial effects correlates with the antiproliferative activity of (-)-gossypol. Northern blot RNA analyses suggest that treatment with a 5-10 microM dose of (-)-gossypol induces a transient increase in the expression of heat shock gene products, particularly hsp-70 transcripts. The mean 5-fold increase in (-)-gossypol-induced hsp-70 mRNA appears coincident with a comparable heat-stimulated increase in transcript levels, as compared with control or (+)-gossypol-treated cells. The enzyme-inhibiting properties of gossypol enantiomers were compared in cell-free assays measuring glutathione-S-transferase-alpha, -mu, and pi activities, calmodulin stimulation of cyclic nucleotide phosphodiesterase, and protein kinase C activity. Both enantiomers are near equivalent antagonists of calmodulin stimulation and protein kinase C activity, exceeding the potency of known inhibitors such as phenothiazines by as much as 50-fold. In contrast, (-)-gossypol is a 3-fold more potent inhibitor of glutathione-S-transferase-alpha and -pi isozyme activity, resulting in IC50 values of 1.6 and 7.0 microM, respectively, for these two isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative cross-over pharmacokinetic study on two types of postcoital contraceptive tablets containing levonorgestrel.

A pharmaceutical and pharmacokinetic study was carried out on levonorgestrel tablets from two different sources (Hungarian- and Chinese-made). Both preparations contained 0.75 mg levonorgestrel and had been shown to have similar contraceptive efficacy and side effects when used for postcoital contraception. Absorption and bioavailability of the Hungarian-made tablets were greater as evidenced by higher serum concentrations of levonorgestrel, a greater area under the concentration-time curve during the first 24 hours, and a more marked suppressive effect on SHBG levels. These differences most probably reflect differences in their pharmaceutical formulation, in particular the extent of tablet dissolution and the degree of micronisation of levonorgestrel.

The effect of tamoxifen on the function and lifespan of the corpus luteum and on subsequent ovarian function.

The aim of the present study was to investigate the effects of tamoxifen on pituitary and luteal function and on the bleeding pattern when administered continuously in the secretory phase. The study included 16 women with regular menstrual cycles followed during one control, one treatment and one follow-up cycle. Each volunteer received 20 mg tamoxifen twice daily from cycle day 18 to menstruation in the treatment cycle. The luteal phase was slightly, but significantly prolonged during treatment, and FSH, progesterone, 17-hydroxyprogesterone, 20 alpha- dihydro progesterone, estrone, estrone sulphate and estradiol significantly elevated in comparison with corresponding data during the control cycle. The results indicate that estrogen may be of some importance for the regulation of the life span of the corpus luteum in the human. The significantly elevated levels of pregnanediol glucuronide and estrone glucuronide during the follow-up cycle are most likely a result of either a direct effect of remaining circulating tamoxifen levels on the ovary, or mediated through the increased release of FSH. If estrogens are of importance for the process of implantation, which has recently been suggested in sub-human primates, also in the human remains unclear. Studies on the effect of anti-estrogens on the endometrium during the secretory phase of the cycle are ongoing.