James C. Cornette

Return of ovulatory cyclicity following an intramuscular injection of medroxyprogesterone acetate (Provera)

Abstract This study was carried out to determine the relationship between peripheral serum concentrations of medroxyprogesterone acetate (Provera) and progesterone following an intramuscular injection of 150 mg of depomedroxyprogesterone acetate. Medroxyprogesterone acetate concentrations were maximal at 5–20 days post-injection (10–25 ng/ml), then decreased to 5–10 ng/ml by 30 days post-injection. In general, the concentrations decreased gradually, with little fluctuation, during the remainder of the study (260 days). Medroxyprogesterone acetate concentrations were 5.1, 0.8 and

Recombinant human interleukin-1α and recombinant human interleukin-1β stimulate cartilage matrix degradation and inhibit glycosaminoglycan synthesis

Recombinant human interleukin-1α (rhIL-1α) and recombinant human interleukin 1β (rhIL-1β) stimulated the time- and concentration-dependent release of glycosaminoglycan (GAG) from bovine nasal cartilage expiants. Maximum GAG release occurred during six to eight days of cartilage exposure to either species of rhIL-1; and rhIL-1α was consistently more potent than rhIL-1β. In addition to inducing cartilage matrix resorption, rhIL-1α and rhIL-1β also inhibited the incorporation of [35SO4]sulfate into cartilage, which is a reflection of the suppression of GAG synthesis. IL-1 had no capacity to stimulate GAG relase from or inhibit GAG synthesis by dead cartilage. Cycloheximide, an inhibitor of protein synthesis, and 1, 10-phenanthroline, a metalloproteinase inhibitor, suppressed rhIL-1-stimulated cartilage matrix resorption. Polyclonal antisera to rhIL-1α and rhIL-1β specifically neutralized the respective cytokines.

Renin activity determination using human plasma as a substrate

A method for human renin activity determination using human plasma as the substrate is described. Angiotensin I is generated by incubating renin with human plasma, which is available along with the commercial radioimmunoassay kit for angiotensin I. The method obviates the need to isolate and purify the substrate, human angiotensinogen, from human plasma. In addition, the assay is highly renin specific, sensitive, and convenient to use for the routine determination of active human renin during its isolation and purification from tissue extracts or from genetically engineered bacterial and nonbacterial expression systems.

Steroid-induced urogenital tract changes and urine retention in laboratory rodents.

Since previous literature suggested that estrogen-treated male mice are models for human benign prostatic hypertrophy, a series of studies was designed to examine urine retention and urogenital tract changes in rodents given chronic estradiol-17 beta (E) and dihydrotestosterone (DHT) treatments. In Study 1, intact and castrate male mice received E, DHT or E plus DHT for four weeks via subcutaneous Silastic capsules. Bladder urine volume increased in the groups given E and this effect was not altered by castration, DHT or removal of E capsules two weeks before necropsy. Estrogen treatment also increased mortality. In Study 2, intact male, intact female, adrenalectomized (Adx) male and sham Adx male mice received 16 weeks of steroid treatments. Bladder urine volume increased in all E treated groups regardless of sex or Adx. Hydronephrosis, hydroureter and increased mortality were found in the E treated mice of both sexes. Estrogen induced epithelial changes and edema of the prostate, vas deferens and the utriculus prostaticus. In further studies male rats, hamsters and guinea pigs were given several different dosages of E but no evidence of urine retention or increased mortality was found. Taken together these studies suggest that E-induced urine retention is unique to mice. Although urine retention and hydronephrosis found in the mice were similar to those in humans with BPH, the lesion that results in the urine obstruction is not similar.

Release, excretion, tissue uptake and biological effectiveness of estradiol from silastic devices implanted in rats

Abstract 3 H-Estradiol was incorporated into a silicone rubber polymer (Dow Corning Medical Grade Silastic) and formed into solid tubes of 4.7 mm diameter. These were implanted subcutaneously in rats. Estradiol release rates, excretion, tissue uptake and biological effectiveness were assessed. The amount of drug released from the implants was determined by analyses of the residual radioactivity. Determination of this activity in implants removed after varying intervals in vivo support the impression of sustained release over extended periods. Fecal excretion accounted for 79–85% of the radioactivity eliminated following subcutaneous treatment with 3 H-estradiol contained either in the silastic implant or in a cottonseed oil injection. The cumulative isotope excretion pattern approached linearity as the implant content was increased from 15 to 135 μg of estradiol. Implants containing 3.7 μg of estradiol altered normal reproductive cycles for 2–3 weeks. Duration of effectiveness increased with dosage; 100 to 200 μg of estradiol were effective for periods in excess of 200 days. Radioactivity was determined in blood, uterus, liver and muscle at 1 and at 10 days of treatment with comparable efficacious doses of 3 H-estradiol administered by oral, subcutaneous injection or subcutaneous implant routes. “Estradiol” content of the blood and tissues was substantially less for both subcutaneous routes relative to oral treatment; the implant provided lower more uniform levels than those following injection. By eliminating the daily fluctuation of drug in receptor tissues, implants may provide a preferred method of treatment relative to regimes requiring frequent administration by oral or injected routes.

Development of a sensitive activity assay for high-volume evaluation of human renin inhibitory peptides in rat serum : results with U-71,038

A sensitive activity assay for high volume evaluation of human renin inhibitory peptides (RIPs) in rat sera (range 2–80 ng/ml) was developed based on the low affinity of RIPs to rat renin and their high affinity to human renin. The utility of this activity assay was tested by measuring concentrations of a human RIP, U-71,038 (BOC-Pro-Phe-N-MeHis-Leu Ψ [CHOHCH2]Val-Ile-Amp), in rat sera, determined by the activity assay, by a sensitive radioimmunoassay (RIA), and by tracking tritiated drug. Rats were given radiolabeled drug as an intravenous bolus, and blood samples were collected at various times after dosing. The serum level of U-71,038 equivalents was determined by the three techniques. Whole blood was also counted for total radioactivity to evaluate the potential for U-71,038 incorporation into red blood cells. Results from the three serum assays indicate good agreement between the calculated U-71,038 equivalents for the 30 min and 1 hr collection times. The 2 and 4 hr collection times show excellent agreement for the activity assay and RIA; [3H]-U-71,038 determinations gave substantially higher values. Serum levels for U-71,038 determined 30 min after dosing averaged less than 300 ng equivalents/ml suggesting that less than 1% of the administered dose was in the systemic circulation at that time. Thus, U-71,038 was rapidly cleared. At the 4 hr collection time, the level of U-71,038 equivalents, as determined by activity assay and RIA, was ten times the in vitro IC50 for the renin inhibitory activity of U-71,038. Analysis of whole blood levels of 3[H]-U-71,038 indicated little or no incorporation of drug related material into red blood cells. In addition to predicting pharmacological response, the activity assay can be used to quantify human RIPs in rat serum when biotransformation is absent.

A primate bioassay for the determination of renin inhibitory peptides in serum.

The study of renin inhibitory peptides (RIPs) in rodents and primates requires the establishment of a simple, high volume method for determining the concentration of RIPs in serum after intravenous or oral dosing. The human renin inhibition assay useful for rodents is not directly applicable to primates due to inherent production of angiotensin I from the primate serum angiotensinogen and added recombinant human renin. Therefore, a novel approach to analyze the serum concentrations of RIPs in primates is described based on in vitro studies with monkey serum. The procedure involves the inactivation of monkey angiotensinogen and monkey renin by thermal denaturation prior to analysis. Application of this assay was demonstrated by analyzing serum samples from an in vivo study in monkeys using ditekiren (U-71,038), a renin inhibitory peptide, and by validation of the assay and results using a tritium-based radioimmunoassay (RIA) for ditekiren. The minimum detectable limit of ditekiren for both the RIA and the bioassay for primates was 10ng/ml serum. The reported bioassay should be of value for monitoring serum levels of thermostable RIPs from pharmacokinetic, bioavailability, and pharmacodynamic studies in primates as well as in humans.

EFFECT OF ANGIOTENSINOGEN CONCENTRATION ON RENIN ACTIVITY FOLLOWING NEPHRECTOMY AND ADRENALECTOMY : IMPLICATIONS IN THE ACTIVATION OF PLASMA PRORENIN

We have studied the effects of bilateral nephrectomy and adrenalectomy on angiotensinogen concentration, plasma renin activity, and total plasma renin activity obtained after trypsin activation in rats and compared with controls. Our results show that the plasma angiotensinogen concentration of nephrectomized (NX) rats is 5-fold higher than that of adrenalectomized (AX) rats. On the other hand, plasma angiotensinogen concentration of AX rats was about 2.5-fold lower than that of control rats. While NX rat plasma possess no renin activity, its mixing (1:1, v/v) with AX plasma results in 2-fold increase in renin activity over that observed for AX plasma alone. These results suggest that the apparent increase in renin activity upon mixing these two plasmas is at least partly due to an overall increase in angiotensinogen concentration in the mixture. To show that it is not due to activation of NX plasma prorenin by a convertase from AX plasma, prorenin-free NX rat plasma was prepared by using an anti-renin immunoaffinity column. When this prorenin-free NX plasma was mixed (1:1, v/v) with AX, again a 2-fold increase in renin activity was observed which is attributed to the overall increase in plasma angiotensinogen in the mixture. It is concluded that rat plasma prorenin is probably not activated within the circulation by a prorenin convertase from the rat kidney.

Preparation and Application of Polyclonal Antibodies to Recombinant Human Interleukin-1α and Interleukin-1β

We report the preparation and application of polyclonal antisera to the analysis and quantitation of human interleukin-1 alpha (IL-1 alpha) and human interleukin-1 beta (IL-1 beta). The anti IL-1 alpha antibodies specifically react with the alpha form of IL-1 and do not cross react (less than 0.1%) with the beta form of IL-1 and vice versa. Data reported here demonstrate that detection of human IL-1 alpha or beta by a radioimmunoassay technique is sensitive enough to measure picogram levels of these lymphokines. The practical application of using these highly specific antisera for radioimmunoassays was established by measuring exogenously added IL-1 alpha or IL-1 beta to human plasma. Potential benefits of these reagents and the radioimmunoassay procedures described herein are discussed in relation to the biological assays which cannot distinguish between human IL-1 alpha and human IL-1 beta.

RETURN OF OVULATORY CYCLICITY FOLLOWING AN INTRAMUSCULAR INJECTION OF MEDROXYPROGESTERONE ACETATE (PROVERA

In order to determine the relationship between peripheral serum concentrations of medroxyprogesterone acetate (Provera) and progesterone following an intramuscular injection of 150 mg of depo-medroxyprogesterone acetate nonpregnant nonlactating healthy women between ages 19 and 35 and with a history of regular menstrual cycles and no history of pregnancy or use of oral contraceptives or other hormone therapy within the previous 3 months were selected for study. Following the injections at 5-20 days post-injection concentrations ranged from 10-25 ng/ml and then decreased to 5-10 ng/ml by 30 days post-injection. The concentration decreased gradually with little change during the remainder of the study. 1 exception was the increased concentration found at 63 and 70 days post-injecton in a subject. The minimal detectable concentration of medroxyprogenterone acetate was 0.5 ng/ml. All samples were above this level in all 3 subjects prior to 185 days post-injection. In 1 of the 3 patients levels were below this amount in some of the samples between days 185 and 228 but detectable levels were present in all 3 subjects wihtin 30 days of the initial progesterone increase. The study shows that 150 mg of intramuscularly administered medroxyprogesterone acetate (Provera) can suppress ovulation for 200 days.