John W. Wilks

The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism

The tetracycline analogs minocycline and doxycycline are inhibitors of metalloproteinases (MMPs) and have been shown to inhibit angiogenesis in vivo. To further study the mechanism of action of these compounds we tested them in an in vitro model of angiogenesis: aortic sprouting in fibrin gels. Angiogenesis was quantitated in this system by a unique application of planar morphometry. Both compounds were found to potently inhibit angiogenesis in this model. To further characterize the activity of these compounds against MMPs, we determined the IC50s of both compounds against representatives of three classes of metalloproteinases: fibroblast collagenase, stromelysin, and gelatinase A. Doxycycline was found to inhibit collagenase, gelatinase A and stromelysin with IC50s of 452 μM, 56 μM and 32 μM, respectively. Minocycline was found to inhibit only stromelysin in the micromolar range with an IC50 of 290 μM. Since these results suggest that these compounds may not have been inhibiting in vitro angiogenesis by an MMP-dependent mechanism, we decided to test the effects of the potent MMP inhibitor BB-94. This compound failed to inhibit aortic sprouting in fibrin gels, thus strongly suggesting that both doxycycline and minocycline act by an MMP-independent mechanism. These results have implications for the mechanism of action of tetracycline analogs, particularly where they are being considered for the treatment of disorders of extracellular matrix degradation including periodontal disease, arthritis, and tumor angiogenesis.

Serum FSH, LH and testosterone in the male rhesus following prostaglandin injection

Adult male rhesus were treated with PGE2, PGF2 alpha or the 13,14-dihydro-15-keto metabolite of PGE2 in a randomized crossover design. Serum concentrations of FSH, LH and testosterone were determined and compared to the respective values in the same uninjected animals. No significant changes were noted in controls or following the metabolite injection. FSH increased gradually for 4 hours after metabolite treatment. In contrast, injection of PGF2 alpha was followed by an abrupt (within 15 minutes) increase in LH and testosterone. FSH increased gradually in 2 of 3 treated animals. Injection of PGE2 was followed by a similar abrupt increase in LH concentration. This was not always associated with a significant increase in testosterone or FSH. These results demonstrate that injections of PGE2 or PGF2 alpha can change serum gonadotropin and testosterone concentrations in male rhesus monkeys, and that the effects of these two prostaglandins are qualitatively different.

Biologic activity of human chorionic gonadotropin following reversed-phase high-performance liquid chromatography

Human chorionic gonadotropin (hCG) was analyzed by reversed-phase high-performance liquid chromatography (HPLC) using mobile phases previously described in the literature, as well as newly developed solvent systems. Fractions of hCG collected following reversed-phase HPLC were bioassayed by activation of adenylate cyclase to determine their biologic potencies. hCG retained only 10-60% of its biologic activity following reversed-phase HPLC, depending on the chromatographic conditions employed. A portion of the reduced biologic activity was attributed to dissociation of the alpha- and beta-subunits of hCG at the low pH of the mobile phases, since neutralization of the pH prior to lyophilization and bioassay increased the biologic potency of the chromatographed hormone. The remaining loss in biologic activity is presumably due to organic solvent denaturation.

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.

Effects of (15S)-15-methyl prostaglandin F2α methyl ester and estrogens upon the corpus luteum and conceptus of the rhesus monkey

The corpus luteum inhibiting activity of (15S)-15-methyl prostaglandin F2 alpha methyl ester (15M-PGF2 alpha) was determined when given in combination with estrogens. Administration of 15M-PGF2 alpha alone (3 injections of 500 microgram each) to monkeys concomitantly receiving hCG reduced serum progesterone concentrations to 50% of values observed in control animals. Ethinyl estradiol or mestranol alone did not inhibit the corpus luteum of the hCG-treated, nonpregnant monkey. Serum progesterone values for nonpregnant monkeys treated with 15M-PGF2 alpha plus estradiol-17 beta, ethinyl estradiol or mestranol did not differ statistically from those observed in monkeys treated with 15M-PGF2 alpha alone. The dose of 15M-PGF2 alpha (3 x 500 microgram) which only partially inhibited the corpus luteum of the hCG-treated, nonpregnant monkey promptly terminated gestation when given on Day 28 od fertile menstrual cycles. Pregnancy terminated in two of three treated monkeys when the dose of 15M-PGF2 alpha was reduced ten-fold (3 x 50 microgram). Mestranol alone reduced serum progesterone and estradiol-17 beta concentrations to one-half and one-third of pretreatment values, but did not interrupt pregnancy. Pregnancy terminated in only two of five monkeys when mestranol was administered with the low dose of 15M-PGF2 alpha. It is concluded that: 1) estrogens do not enhance the corpus luteum inhibiting of 15M-PGF2 alpha in the nonpregnant monkey; 2) the primary action of 15M-PGF2 alpha during early pregnancy is upon the conceptus and not the corpus luteum; 3) concomitant treatment with mestranol and 15M-PGF2 alpha offers no advantage of 15M-PGF2 alpha alone for the termination of early pregnancy; and 4) mestranol alone can impair the steroidogenic potential of the corpus luteum of early pregnancy, but is not sufficiently active to terminate gestation.

Inhibition of the monkey corpus luteum with 15-methyl prostaglandins

The corpus luteum inhibiting properties of eighteen 15-methyl prostaglandin analogs were determined in the rhesus monkey during concomitant stimulation of the corpus luteum with chorionic gonadotropin. The methyl ester of (15S)-15-methyl PGF2 alpha (15M-PGF2 alpha, 12.5 mg/monkey) lowered serum progesterone to 12% of pretreatment values within 24 hours, however progesterone returned to normal limits within 48 hours. Elongation of the top side-chain by two carbons (2a,2b-dihomo-15M-PGF2 alpha methyl ester, 13 mg/monkey), substitution of a hydroxymethyl group at carbon 1 (2-decarboxy-2-hydroxymethyl-15M-PGF2 alpha, 12 mg/monkey), or the formation of the carbon 1 amide (15M-PGF2 alpha amide, 12.5 mg/monkey) improved the inhibitory activity of 15M-PGF2 alpha; serum progesterone for these 3 analogs was depressed to 15-30% of pretreatment levels within 24 hours, and did not return to control values. Luteal function was not inhibited (12 or more mg/monkey) when the 15-methyl group was placed in the R configuration, the top side chain was shortened by two carbons, an amino group was substituted for carbon 1, the 5-oxa modification was added, or the 1,9-lactone was formed. Some other modifications of 15M-PGF2 alpha were also inactive, although not all were tested at equivalent doses: 2,2-difluoro; 4,5-cis-didehydro; 9,11-dideoxy-9 alpha, 11 alpha-dichloro; 11-deoxy; 17-phenyl; 1,15-lactone; and the p-benzamidophenyl ester of 2a,2b-dihomo-15M-PGF2 alpha. (15S)-15-Methyl PGE2 methyl ester (1 mg/monkey) depressed serum progesterone concentrations to 42% of pretreatment values within 24 hours; 2a,2b-dihomo-11-deoxy-(15S)-15-methyl PGE2 methyl ester was inactive (5 mg/monkey). A corpus luteum inhibiting action of certain 15-methyl prostaglandins can be demonstrated in the rhesus monkey.

A procedure for evaluating luteolytic agents in primates.

A method of human fertility control based on the regulation of corpus luteum function has been the objective of a continuing research effort by reproductive biologists. The vast array of physiologic control mechanisms governing the corpus luteum among the mammalian species presents a formidable obstacle in extrapolating concepts developed in animal research for clinical application in women. As an example the uterus clearly regulates the secretory activity and life span of the corpus luteum in infraprimate animals, but a role for the uterus in altering luteal function in primates has not been established. The importance of this biological distinction is illustrated by the successful application of prostaglandin F2α (a physiologic luteolytic substance of uterine origin) to the regulation of estrous cycles in domestic animals (1), and the disappointing outcome of a clinical evaluation of this prostaglandin for control of the human corpus luteum (2).

Luteolytic action of 15-keto prostaglandin F2α in the rhesus monkey

The naturally-occurring metabolite of prostaglandin F2alpha, 15-keto prostaglandin F2alpha (15-keto PGF2alpha), elicited rapid and sustained declines in serum progesterone concentrations when administered to rhesus monkeys beginning on day 22 of normal menstrual cycles. Evidence for luteolysis of a more convincing nature was obtained in studies where a single dose of 15-keto PGF2alpha was given on day 20 of ovulatory menstrual cycles in which intramuscular injections of hCG were also given on days 18-20; serum progesterone concentrations fell precipitously in monkeys within 24 hours following intramuscular administration of 15-keto PGF2alpha. However, corpus luteum function was impaired in only 4 of 11 early pregnant monkeys when 15-keto PGF2alpha was administered on days 30 and 31 from the last menses, a time when the ovary is essential for the maintenance of pregnancy. Gestation failed in 2 additional monkeys 32 and 60 days after treatment with 15-keto PGF2alpha, but progressed in an apparently normal manner in the remaining 5 animals. Two pregnant monkeys treated with 15-keto PGF2alpha on day 42 from the last menstrual period, a time when the ovary is no longer required for gestation, continued their pregnancies uneventfully. Corpus luteum function was not impaired in 9 control monkeys which received injections of vehicle or hCG at appropriate times during the menstrual cycle or pregnancy.

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.

Prostaglandins and in vitro ovarian progestin biosynthesis

Abstract Prostaglandin F2α (PGF2α) did not alter the in vitro biosynthesis of progesterone by slices of luteinized rat ovaries when used in concentrations from 1 to 10,000 ng/ml of incubation medium; likewise, PGF2α did not affect the incorporation of acetate-1-14C into progestins. PGF1α, 15-keto PGF2α, and PGE1 did not alter the biosynthesis of progesterone by luteinized rat ovaries; PGE2 inhibited the production of progesterone when used at a concentration of 10 μg/ml, but not at lower doses. PGF2α in combination with luteinizing hormone (LH) enhanced the metabolism of progesterone to 20α-hydroxypregn-4-en-3-one in luteinized rat ovaries. Interestingly, PGF2α, at a high concentration of 10 μg/ml, did stimulate progesterone biosynthesis by slices of ovarian tissue from immature rats hormonally primed to simulate “pseudopregnancy,” suggesting a steroidogenic action of prostaglandins on the ovarian follicular or interstitial cell. PGF2α (10 μg/ml) did not stimulate the in vitro biosynthesis of progesterone or 20α-hydroxypregn-4-en-3-one by slices of rabbit corpora lutea or rabbit ovarian interstitial tissue. It is concluded that prostaglandins do not stimulate progestin biosynthesis in rat luteal tissue.