Pregnancy interception with a combination of prostaglandins: studies in monkeys.

Treatment with combinations of synthetic prostaglandins, one with an ovarian site of action and one with a uterine site of action, terminated pregnancy in all rhesus monkeys given the injection on day 28 of fertile menstrual cycles. Single prostaglandins, even at higher doses, interrupted pregnancy in only one-third of the monkeys. The most effective treatment, 5-oxa-17-phenyl-18,19,20-trinor prostaglandin F1 alpha methyl ester plus 9-deoxo-16,16-dimethyl-9-methylene prostaglandin E2, promptly intercepted early pregnancy after a single administration and without side effects.

Synthetic matrix metalloproteinase inhibitors and tissue inhibitor of metalloproteinase (TIMP)-2, but not TIMP-1, inhibit shedding of tumor necrosis factor-alpha receptors in a human colon adenocarcinoma (Colo 205) cell line.

The solubilization of plasma membrane receptors through proteolytic cleavage of the ligand binding domain at the cell surface is an important mechanism for regulating cytokine function and receptor signaling. The inhibition of the shedding of a variety of receptors by synthetic inhibitors of the matrix metalloproteinases (MMPs) implicates metalloproteinases in this regulatory event. We examined the effects of two naturally occurring tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, and several synthetic MMP inhibitors (MMPIs) on the shedding of both tumor necrosis factor alpha receptor type I (TNFalpha-RI; Mr 55,000) and TNFalpha-RII (Mr 75,000) by the Colo 205 human colon adenocarcinoma cell line. Culture of Colo 205 cells for 48 h resulted in the shedding of both TNFalpha-RI and TNFalpha-RII, as determined by ELISA. The shedding of TNFalpha receptors was not affected by TIMP-1 or protease inhibitors aprotinin, pepstatin, or leupeptin but was inhibited in a dose-dependent manner by the following synthetic MMPIs: batimastat and marimastat (BB-94 and BB-2516, respectively, British Biotech, Inc.); CT1418 (Celltech Therapeutics); CGS27023A (Novartis Pharmaceuticals); and RO31-9790 (Roche), with IC50s ranging from 3.2 to 38.0 microM. Similarly, TIMP-2 from two different sources reproducibly inhibited the shedding of both TNFalpha-RI and TNFalpha-RII in a dose-dependent manner (IC50 = 286 +/- 33 nM for TNFalpha-RI shedding and 462 +/- 52 nM for shedding of TNFalpha-RII). The inhibition of TNFalpha-RI shedding was confirmed in the SW626 human ovarian adenocarcinoma cell line. The synthetic MMPIs and TIMP-2, but not TIMP-1, also caused a dose-dependent increase in the number of TNFalpha receptors retained on the surface of Colo 205 cells, as determined by flow cytometry. Inhibition of TNFalpha receptor shedding with TIMP-2 occurs at molar concentrations 10-100 times less than those required with low molecular weight, synthetic MMPIs but at concentrations greater than those required to inhibit collagen degradation. Modulation of TNFalpha receptor shedding by TIMP-2 could have important implications for the pleiotropic effects of TNFalpha in both normal and malignant cells and for the pharmacological activity of synthetic MMPIs.

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity.

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.

Steroidogenic responsiveness of the monkey corpus luteum to exogenous chorionic gonadotropin.

Groups of female rhesus monkeys were given a 5-day regimen of im injections of hCG (30, 60, 90, 180, and 360 IU, respectively) beginning 2, 6, 10, or 14 days after the midcycle LH surge (day 0). Serum progesterone concentrations in the day 2 treatment group did not differ markedly from values observed in control monkeys throughout normal menstrual cycles. In contrast, monkeys receiving hCG beginning on days 6, 10 or 14 had immediate significant increases in serum progesterone in response to the first injection of hCG. Dramatic responses were seen in the day 6 and 10 groups (2.5- and 5-fold elevations in serum progesterone, respectively); progesterone values plateaued at about 13 ng/ml during the hCG treatment interval. Monkeys receiving hCG on day 14 had 4-fold elevations in serum progesterone, but concentrations did not exceed 6 ng/ml. Serum estradiol increased significantly after hCG to concentrations between 200-300 pg/ml in all treatment groups; peak values were seen at the time of or in the days immediately after the last hCG injection. Serum testosterone concentrations were not significantly altered by hCG administration at any stage of the luteal phase. hCG did not sustain serum steroid hormone concentrations in monkeys with the corpus luteum removed on day 10 of the luteal phase. A 10-day regimen of increasing hCG doses beginning on day 10 of the luteal phase mimicked the steroid hormone secretion patterns observed in control monkeys during early pregnancy. The data show that the qualitative and quantitative steroidogenic capacities of monkey corpora lutea after a gonadotropin challenge are profoundly affected by luteal age.

Aprotinin antagonizes the binding of human chorionic gonadotropin to its receptor.

Aprotinin caused a dose-dependent inhibition of [125I]hCG binding to its receptor in plasma membranes prepared from luteinized rat ovaries. Displacement of [125I]hCG from its receptor by aprotinin was temperature dependent, with an ID50 of 300 microM at 4 C and an ID50 of 3700 microM at 22 C. Equilibrium binding data showed a decrease in the Ka for hCG in the presence of increasing concentrations of aprotinin; aprotinin had no effect on the number of binding sites. The rate of association of hCG with its receptor was markedly reduced in the presence of aprotinin, but aprotinin had little effect on the rate of dissociation. Control experiments established that aprotinin did not inhibit the binding of [125I]hCG to its receptor due to its lectin properties, an ionic effect or some irreversible impairment of the receptor or hormone. Aprotinin did not inhibit hCG binding when the receptor was solubilized out of ovarian membranes with Triton X-100. Aprotinin inhibited the hCG-mediated activation of adenylate cyclase in rat ovarian plasma membranes in a dose-dependent manner. Similarly, aprotinin antagonized the hCG-stimulated biosynthesis of progesterone by dispersed rat luteal cells. The maximal agonistic activity of hCG was attenuated in these two bioassays. The data are compatible with a hypothesis that aprotinin sterically hinders the entry of hCG into its receptor site by binding to a protease in the plasma membrane that is closely associated with the hCG receptor. This proposal is consistent with the emerging concept that integral membrane proteases are modulators of receptor function.

Periovulatory endocrine events in the stumptailed monkey (macaca arctoides).

Blood samples were obtained frequently from stumptailed monkeys (Macaca arctoides) at midcycle and assayed for FSH, LH, 17 beta-estradiol, and progesterone. Periovulatory endocrine events were generally similar to those which have been described in rhesus monkeys and women. Although the midcycle patterns of estradiol in individual monkeys appeared quite varied, careful evaluation indicated the following general characteristics: 1) a plateau or transient decline in estradiol 30-44 h before the LH peak, 2) a sharp rise in serum estradiol before the initiation of the LH rise, 3) a diminution in serum concentrations or a decline in the rate of estradiol increase at the initiation of the LH rise, and 4) an elevation in estradiol at the time of the highest serum LH value. The first elevation in serum progesterone occurred 0-12 h before the LH peak and after the initiation of the LH surge. An unusual finding was a second midcycle rise in serum FSH, which occurred 2-3 days after the LH peak in 10 of 13 menstrual cycles. The biological significance of this second FSH rise in stumptailed monkeys is not known with certainty, but available evidence suggests that it enhances postovulatory progesterone and estrogen syntheses. Two methods are presented for the RIA of serum FSH in the stumptailed macaque.

A new radioimmunoassay for follicle-stimulating hormone in macaques: ovulatory menstrual cycles.

A sensitive and specific radioimmunoassay system for macaque follicle-stimulating hormone (mFSH) was developed utilizing an antiserum (H-31) prepared in a rabbit against purified ovine FSH as the immunogen. Sera from castrated female, adult male, and juvenile rhesus monkeys, as well as urinary extracts from castrated rhesus and bonnet monkeys, were used to demonstrate parallelism with a standard of partially purified monkey pituitary gonadotropins (LER-M-907-D). An extract of baboon pituitary tissue also showed parallelism with the reference standard. A highly purified pituitary extract (WP-X-105-28), containing approximately 75% macaque luteinizing hormone (mLH) and 1% mFSH, was used to demonstrate the specificity of this mFSH assay system. Sera and urinary extracts obtained from hypophysectomized monkeys did not show cross-reactivity in the assay. Macaque chorionic gonadotropin (mCG) did not produce an inhibition curve in the assay, as determined from serum samples and urinary extracts collected from pregnant monkeys at the time of peak mCG secretion. Serum concentrations of mFSH were suppressed in ovariectomized monkeys by the administration of ethinyl estradiol for 3 days, but returned to near pretreatment values by 96 h after the last estradiol administration. The determination of serum mFSH concentrations in daily blood samples obtained from 20 rhesus monkeys throughout ovulatory menstrual cycles revealed a pattern similar to that previously reported for the rhesus monkey and the woman. The peak value of serum mFSH during the menstrual cycle coincided with the midcycle surge of mLH in each case. The gonadotropin peaks were preceded by increasing serum concentrations of estradiol and followed by rises in the serum concentrations of progesterone. The follicular phase of the menstrual cycle was characterized by continuously decreasing serum concentrations of mFSH, reaching a preovulatory nadir 48 h prior to the midcycle mFSH and mLH surges. Serum mFSH concentrations following the midcycle gonadotropin surges decreased progressively as serum progesterone concentrations increased and reached a plateau, and then increased during the last week of the menstrual cycle as corpus luteum function was waning. We have prepared a large pool of antiserum for distribution under the aegis of the Contraceptive Development Branch of the Center for Population Research, the National Institute of Child Health and Human Development.

Luteal phase defects in the rhesus monkey: the significance of serum FSH:LH ratios.

Luteal phases of an abbreviated duration or suboptimal progesterone secretion were identified in the rhesus monkey. Characterization of menstrual cycles with short luteal phases (8 days) in 5 monkeys revealed the following deviations from normal: a. a failure of serum FSH to decline progressively from an early follicular phase high to a preovulatory nadir; b. a commonly inadequate, or absent, preovulatory FSH surge; c. subnormal serum LH concentrations during both the follicular and luteal portions of the menstrual cycle; d. ratios of serum FSH:LH which did not progressively decrease during the follicular phase; and e. serum estradiol concentrations below normal values during the luteal portion of the menstrual cycle. One monkey had abnormally low serum concentrations of progesterone, although the luteal phase was 19 days in duration. Serum gonadotropin and estradiol patterns in this latter monkey were identical to those observed in monkeys with short luteal phases. It is concluded that inappropriate ratios of serum FSH:LH during the follicular phase of the menstrual cycle result in impaired corpus leteum function. The data document the usefulness of the nonhuman primate for investigating those luteal phase defects which also occur in the woman.

Synthesis and biological activity of prostaglandin lactones.

Most of the primary prostaglandins and several biologically important prostaglandin analogues were converted to 1,9-, 1,11- or 1,15-lactones, in order to investigate the biological profiles of these internal esters and to assess their potential as prodrugs for the corresponding open-chain hydroxy acids. In each case, the key lactonization step was done using Corey's "double activation" procedure (cyclization of omega-hydroxy-2-pyridinethiol esters). In general, the 1,9-lactones exhibited less than 1% of the biological activity of the parent hydroxy acids in the standard prostaglandin test systems. The 1,11- and 1,15-lactones, on the other hand, were essentially equal to the parent hydroxy acids as antifertility agents (a 4-day assay which would allow time for in vivo enzymatic lactone hydrolysis). The 1,11- and 1,15-lactones exhibited very low activity in acute or in vitro screens (e.g., rat blood pressure and gerbil colon stimulation), assays which more closely reflect the intrinsic activity of the lactones themselves. These results are consistent with the observed relative ease of enzymatic hydrolysis of the prostaglandin lactones (1,15 greater than or equal to 1,11 much greater than 1,9). Several of the lactones whose parent hydroxy acids are resistant to metabolic inactivation (e.g., 15-methyl, 16-phenoxy, and 17-phenyl) exhibited potent abortifacient activity in the hamster. These lactones, with greatly diminished activity in the blood pressure and smooth muscle assays (indicators of potential side effects), represent a therapeutically useful class of antifertility agents.

Synthesis of a series of stromelysin-selective thiadiazole urea matrix metalloproteinase inhibitors.

The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metalloproteinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads to include an alpha-amino acid side chain with variable terminal amide substituents provided a series of ureas which were moderately effective stromelysin inhibitors, with Ki's between 0.3 and 1.0 microM. The most effective analogues utilized an L-phenylalanine as the amino acid component. In particular, unsubstituted 46 had a Ki of 710 nM, while the p-fluoro analogue 52 displayed increased potency (100 nM). Stromelysin inhibition was further improved using a pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually provided for enhanced activity, with 71 inhibiting gelatinase with a Ki of 770 nM. The combination of this heterocycle with a p-fluorophenylalanine substituent provided the only analogue, 69, with collagenase activity (13 microM). The SAR for analogues described within this series can be rationalized through consideration of the X-ray structure recently attained for70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of stromelysin. Careful selection of the amide substituent can also provide for analogues with modest gelatinase inhibition.

Inhibition of the primate ovarian cycle by a porcine follicular fluid protein(s).

Monkeys received twice daily intramuscular injections of 3 mg of purified porcine follicular fluid protein(s) for the first 14.5 days of the menstrual cycle. Two of five treated monkeys had anovulatory menstrual cycles. Three monkeys had cycles characterized by long follicular phases, low follicular and luteal phase serum estradiol concentrations, and subnormal luteal progesterone production. Serum gonadotropin concentrations were not affected by the follicular fluid protein(s). The data demonstrate in the nonhuman primate that porcine follicular fluid contains a protein(s) that acts at the ovarian level to inhibit gonadotropin action.

Arrest of folliculogenesis and inhibition of ovulation in the monkey following weekly administration of progestins.

Studies were undertaken in the rhesus monkey to determine whether development of a dominant ovarian follicle could be repeatedly arrested by the administration of a progestin on day 7 of the menstrual cycle, and then every 7 days thereafter regardless of menstrual bleeding history. Progesterone (7.5 mg), norethisterone (1.5 mg), and 17 alpha-ethinyl-17 beta-methoxy-7 alpha-methyl-4-estren-3-one (1.0 or 1.5 mg) effectively inhibited ovulation when injected intramuscularly once a week for 8 weeks. Orally administered STS 557 (17 alpha-cyanomethyl-17 beta-hydroxy-4,9-estradien-3-one, 1.0 mg) also inhibited ovulation. Two structurally related steroids (17 beta-methoxy-4-estren-3-one, 1.0 mg; and 17 beta-methoxy-7 alpha-methyl-4-estren-3-one, 1.5 mg) did not inhibit ovulation when given intramuscularly at the indicated doses. Although weekly administration of certain progestins effectively arrested follicular development and inhibited ovulation in the primate, the treatment was accompanied by disturbances in menstrual bleeding patterns.

Steroid binding specificity of the hamster uterine progesterone receptor.

The relative binding affinities (RBA) of 51 steroids were determined for the uterine progesterone receptor of the proestrous hamster. The receptor demonstrated a high specificity for progesterone; most structural modifications to the progesterone molecule resulted in a substantial reduction in binding affinity. Only six steroids had relative binding affinities similar to progesterone (RBA=100): 17 alpha-ethinyl-17 beta-methoxy-4-androsten-3-one (RBA=85); 6 alpha-fluoro-4-pregnene-3,20-dione (RBA=94); 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (RBA=96); 19-nor-4-pregnene-3,20-dione (RBA=110); 21-fluoro-4-pregnene-3,20-dione (RBA=119); and 17 alpha-ethinyl-17 beta-methoxy-4-estren-3-one (RBA=123).

The effect of oxytocin on the corpus luteum of the monkey.

The effect of a pharmacologic dose of synthetic oxytocin on corpus luteum function was evaluated in rhesus monkeys during normal menstrual cycles, or during menstrual cycles in which the corpus luteum was concomitantly stimulated by injections of human chorionic gonadotropin (hCG). Oxytocin administered by intramuscular injection at a total dose of 4.5 milligrams (2250 I.U.) on Day +6 of the normal luteal phase (Day 0 is the day of the midcycle LH surge) did not change the concentrations of progesterone in the peripheral serum of monkeys or alter the duration of the luteal phase. The same dose of oxytocin, administered to monkeys on Day 22 of menstrual cycles in which hCG was also given on Days 20-22, caused a small, but statistically significant, reduction in serum progesterone values. The results indicate that oxytocin does not alter luteal life span or markedly change blood progesterone concentrations in primates.

Inhibition of preovulatory gonadotropin secretion in the rhesus monkey by [(

We report the first example of a complete inhibition of preovulatory gonadotropin secretion resulting from administration of a luteinizing hormone releasing hormone antagonist during a spontaneous menstrual cycle. The antagonist, [(

Visualization of histamine binding to nuclei.

The binding of histamine to cultured microvascular endothelial cells and glycol methacrylate embedded ovarian tissue sections has been localized using fluorescein-albumin-histamine conjugate. Histamine conjugate was bound to the plasma membranes and nuclei of luteal, endothelial, and ovarian stromal cells. An apparent increase in the binding of histamine to nuclei was observed in the presence of cimetidine but the plasma membrane staining was still evident. Unlike cimetidine, pyrilamine completely inhibited the binding of histamine to the plasma membrane. Instead, in the presence of pyrilamine, histamine bound exclusively to the nuclei of endothelial, germinal epithelial, granulosa, and stromal cells. However, the nuclei of terminally differentiated luteal cells and oocytes were not labeled. The functional significance of these nuclear histamine binding sites remains to be determined.