A comparison of the metabolism of the abortifacient compounds from Ponderosa pine needles in conditioned versus naive cattle.

Isocupressic acid (ICA) is the abortifacient compound in ponderosa pine (Pinus ponderosa L.) needles, which can cause late-term abortions in cattle (Bos taurus). However, cattle rapidly metabolize ICA to agathic acid (AGA) and subsequent metabolites. When pine needles are dosed orally to cattle, no ICA is detected in their serum, whereas AGA is readily detected. Recent research has demonstrated that AGA is also an abortifacient compound in cattle. The observation has been made that when cattle are dosed with labdane acids for an extended time, the concentration of AGA in serum increases for 1 to 2 d but then decreases to baseline after 5 to 6 d even though they are still being dosed twice daily. Therefore, in this study we investigated whether cattle conditioned to pine needles metabolize ICA, and its metabolites, faster than naïve cattle. Agathic acid was readily detected in the serum of naïve cattle fed ponderosa pine needles, whereas very little AGA was detected in the serum of cattle conditioned to pine needles. We also compared the metabolism of ICA in vitro using rumen cultures from pine-needle-conditioned and naïve cattle. In the rumen cultures from conditioned cattle, AGA concentrations were dramatically less than rumen cultures from naïve cattle. Thus, an adaptation occurs to cattle conditioned to pine needles such that the metabolism AGA by the rumen microflora is altered.

Implication of agathic acid from Utah juniper bark as an abortifacient compound in cattle.

Freshly ground Utah juniper [Juniperus osteosperma (Torr.) Little] bark was given via gavage at a dosage of 2.3 kg per cow twice daily to three pregnant cows starting on day 255 of gestation. All three cows aborted the calves after 4, 5 and 6 days of treatment. A fourth cow was dosed with Utah juniper needles and this cow calved early on day 268 of gestation with complications consistent with pine needle abortion. Chemical analysis of Juniperus osteosperma bark identified the major diterpene acid as the labdane acid known as agathic acid. Agathic acid was measured in the bark at a concentration of 1.5% (dry weight basis). Analysis of sera samples obtained from treated cows found detectable quantities of agathic acid, dihydroagathic acid and tetrahydroagathic acid, which are known serum metabolites of the abortifacient compound isocupressic acid. Based on the high incidence of induced abortion and detection of known metabolites in affected animals, the labdane acid known as agathic acid is considered to be an abortifacient compound in late-term pregnant cattle.

[Metabolism-based interaction of diphenytriazol and flavone compounds].

OBJECTIVE: To observe the metabolism-based interaction of diphenytriazol and flavone compounds. METHODS: Flavone compounds kaempferol, isoharmnten and Elsholtzia blanda benth extract were chosen as the substrate of glucuronidation in the phase II metabolism. The metabolism was investigated in different rat liver microsome incubates pretreated with beta-naphthoflavone (BNF), diphenytriazol and tea oil (control). The concentrations of residual substrate were determined by HPLC. Quercetin and kaempferol were coincubated with diphenytriazol in control microsome to evaluate the inhibition for phase I metabolism. The concentration of diphenytriazol was determined by HPLC. RESULT: The phase II metabolic activity of kaempferol, isoharmnten and Elsholtzia blanda benth extract in diphenytriazol-treated microsome was more potent than that in BNF-treated microsome (P<0.01). The phase I metabolism of diphenytriazol was markedly inhibited by quercetin and kaempferol, with the inhibition constants (Ki) (12.41 +/-0.26)microg . ml(-1) and (7.97 +/-0.08)microg . ml(-1), respectively. CONCLUSION: Diphenytriazol demonstrates metabolism-based interaction with flavone compounds in vitro.

Synthesis and biological activity of a novel, highly potent progesterone receptor antagonist.

Herein we describe the chemical synthesis and pharmacological characterization of a novel, highly potent progesterone receptor (PR) antagonist, ZK 230211. The introduction of a 17alpha-pentafluorethyl side chain in the D-ring of the steroid skeleton allowed the combination of high antiprogestagenic activity with little or no other endocrinological effects. In contrast to many other antiprogestins, ZK 230211 did not convert to an agonist in the presence of protein kinase A (PKA) activators and showed high antiprogestagenic activity on both PR isoforms PR-A and PR-B. This high antiprogestagenic activity could also be demonstrated in several in vivo models. Furthermore, this compound displayed only marginal antiglucocorticoid effects. In tumor models ZK 230211 exhibited strong antiproliferative action. The pharmacological properties of ZK 230211 may prove useful in the treatment of endometriosis, leiomyomas, breast cancer, and in hormone replacement therapy.

Pine needle abortion in cattle: metabolism of isocupressic acid.

The rumen and hepatic metabolism of the cattle abortifacient compound isocupressic acid (ICA) was examined in vitro and in vivo. ICA was incubated for 56 h in bovine rumen inoculum and was found to be converted to three compounds identified as imbricatoloic acid, a structurally uncharacterized isomer of imbricatoloic acid, and dihydroagathic acid. In preparations of liver homogenates, ICA was found to be oxidized to agathic acid. No differences in ICA metabolites were detected in comparing the cow, sheep, pig, goat, guinea pig, and rat livers; however, guinea pig and rat liver homogenates were less efficient in converting ICA to agathic acid. ICA had been administered to cows orally and by intravenous infusion and induced abortions after either method of treatment. After intravenous infusion, agathic acid was identified as the major metabolite together with minor amounts of dihydroagathic acid. After oral administration, dihydroagathic acid was identified as the major metabolite with minor amounts of agathic acid, imbricatoloic acid, and a structurally uncharacterized metabolite tentatively identified as tetrahydroagathic acid.

A novel approach to human fertility control: contragestion by the anti-progesterone RU 486.

PIP: The introductory presentation for a session on antiprogestins summarizes the action of RU 486 relating to its use as a contragestive agent. RU 486, or mifepristone (Roussel-Uclaf), is a progestin analogue resembling norethindrone with a longer 17-alpha side chain and a 11-beta phenyl group that confers the progesterone antagonist activity. It binds the progesterone receptor with a half-life of about 20 hours. Since progesterone receptors are only located in a few tissues, notably the decidualized endometrium and the corpus luteum, its action can be targeted. RU 486 interrupts the cycle regardless of whether fertilization occurred. A single 600 mg dose arrests over 80% of pregnancies up to 41 days, then a decreasing proportion, down to 60% at 10 weeks, due to a greater chance of incomplete evacuation. There have been no recorded side effects in 4000 trails. The effect of prostaglandins is magnified when progesterone action is suppressed. 400 trials have confirmed that over 98% successful termination of pregnancy can be obtained if 600 mg RU 486 is followed by 1 mg synthetic prostaglandin 36 hours later. RU 486 has also tested successfully for delivery of dead fetuses, cervical dilation, a postcoital contraceptive, and as an antiglucocorticoid agent. It has been submitted to the French Ministry of Health, and will probably be approved for marketing in 1988. Clinical trials are underway in 15 countries. This drug is a useful contragestive agent, active in the beginning of the continuum of pregnancy, and may defuse the abortion issue.^ieng

Absorption and elimination of a prostaglandin F analog, fenprostalene, in lactating dairy cows.

Pharmacokinetic characteristics of the prostaglandin F2 alpha analog, fenprostalene, were studied in five lactating Holstein cows. Blood samples, milk, urine, and feces were collected for up to 7 d following a single subcutaneous injection of 1 mg of 13,14-hydrogen-3-fenprostalene in polyethylene glycol-400. The maximum concentration of tritium in plasma, observed 4 h after injection, equated to .17 ngeq/ml fenprostalene and declined with a fractional disappearance rate of .051 X h-1 to less than .04 ngeq/ml by 48 h. Likewise, milk contained .53 ngeq/ml fenprostalene at 4 h and the concentration declined with a fractional disappearance rate of .069 X h-1 to less than .03 ngeq/ml by 48 h. Milk was a very minor route of elimination of fenprostalene with only .46% of the injected dose recovered over a 7-d sampling. Recovery of tritium in urine accounted for 55% of the total dose and recovery in feces accounted for an additional 43%. Residues from fenprostalene at 7 d after injection were less than .1 ppb in all edible tissues. Differences in the molecular structure, formulation, and route of injection of fenprostalene resulted in a slower rate of absorption and elimination of this analog than previously reported for other prostaglandin products. Nonetheless, the percentage of the injected dose of fenprostalene secreted in milk was not increased appreciably, and no persistent tissue residues of fenprostalene were observed.

3H-labelled RU 38486: characterization of binding sites in human uterine cytosol.

The behaviour of the antifertilizing synthetic steroid RU 38486 towards human uterine progestin receptor was investigated. RU 38486 competed in the same order of magnitude as progesterone for the [3H]R 5020 binding site of progestin receptor, whereas R 5020 was unable to compete against [3H]RU 38486. This apparent contradiction could be explained by means of HPLC-chromatography. HPLC-chromatography with an anion exchange column (MonoQ, Pharmacia, Uppsala, Sweden) showed that [3H]RU 38486 forms at least two stable complexes with uterine cytosol, on one hand with serum albumin, which presents almost 90% of bound radioactivity, and on the other hand with the two native progestin receptor forms, corresponding to 4 S and 8 S receptor forms in sucrose density gradient analysis. Whether reduced binding of salt-activated RU 38486 receptor complexes to DNA-cellulose is due to reduced activation is still uncertain and remains to be further investigated.

Absorption, excretion and tissue residue in feedlot heifers injected with the synthetic prostaglandin, fenprostalene.

Preliminary studies on use of the synthetic prostaglandin, fenprostalene, as an abortifacient had indicated that maximum effectiveness was dependent upon slow delivery. Because both route of administration and formulation control delivery rates, the influences of intramuscular (im) vs subcutaneous (sc) injections, and aqueous acetate buffer (AAB) vs polyethylene glycol-400 (PEG) vehicles on the plasma concentration and urinary excretion of fenprostalene were compared. Feedlot heifers were administered 1 mg injections of [13,14-3H]-fenprostalene. Blood samples and total urine excretion were collected during the following 96 h. The maximum concentration of tritium in plasma occurred at 2 h for AAB-im (.90 ng eq/ml), PEG-im (.75 ng eq/ml) and AAB-sc (.64 ng eq/ml), and then declined throughout 24 h with t 1/2 values of 6.1, 9.4 and 9.2 h, respectively. The peak concentration from PEG-sc was lower (.37 ng eq/ml, P less than .05), observed later (4h, P less than .05) and declined more slowly following peak concentration (t 1/2 = 15.1 h, P less than .05). Consistent with delayed absorption, a smaller fraction (P less than .05) of the total radioactivity excreted in urine was recovered during the first 24 h after injection for PEG-sc (85%) than for PEG-im (95%), AAB-sc (97%) or AAB-im (99%). In a tissue distribution study, plasma, urine and fecal samples were collected and heifers were slaughtered at various times following sc injection of 1 mg of [3H] fenprostalene in PEG. Peak concentrations of tritium in plasma occurred between 4 and 8 h and declined with a t 1/2 of 15.2 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic pathways of the contragestational agent, 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole (DL 111-IT), in the rat.

The metabolic pathways of the non-hormonal anti-fertility agent 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole (DL 111-IT) were studied in rats given the 14C-labelled compound intramuscularly. The diaryltriazole, once absorbed, was metabolized rapidly by three phase I reactions: (a) hydroxylation at the C-4 of the methoxyphenyl ring, (b) hydroxylation at the alpha-C of the ethyl chain, and (c) demethylation of the methoxyl function. Seven free metabolites and three conjugates have been isolated and characterized by u.v., i.r., n.m.r. and mass spectroscopy. The products of the first step of metabolism of the diaryltriazole were tested for their pregnancy-terminating activity in the rat. They were only 5-9% as effective as the parent compound, indicating that the unchanged drug is the active molecule.

Biophysical studies on molecular mechanisms of abortifacient action of prostaglandins. V. CNDO/2 estimation of the relative affinities of the cations Na+, Mg2+ and Ca2+ to the carboxylic group.

The paper describes the study of relative affinities of the cations Na+, Mg2+ and Ca2+ to the carboxylic group, their electronic structure and optimum binding positions on the basis of the CNDO/2 technique, using the well known supramolecular approach. The cation for this purpose was made to approach the carboxylic group in various possible directions in and out of the plane C1O1AO1B and the final parameters were evaluated for the optimum planar as well as nonplanar geometries. With the exception of Ca2+ the nonplanar geometries were always favoured over the planar ones for binding at O1A and O1B, whereas for symmetric binding between O1A and O1B planar geometries were favoured for Mg2+ and Ca2+. The distance between the cation and oxygen was slightly larger for this geometry and Etot which followed the order Mg2+ less than Ca2+ less than Na+ had the lowest value.

Mammalian drug metabolism.

Drugs and other chemicals that do not occur in mammalian systems are metabolized by a wide variety of enzymes. Reactions catalyzed by these enzymes have been classified into two general phases. Phase I reactions include oxidations, reductions, and hydrolyses, whereas Phase II reactions are broadly defined as conjugation reactions and include glucuronidation, sulfation, acylation, methylation, and conjugation with glutathione. The mechanisms of these biotransformations are outlined to demonstrate how both non-toxic and toxic metabolites are produced. The mammalian metabolism of acetaminophen, a widely used mild analgesic, and R-(+)-pulegone, the major constituent terpene of pennyroyal oil, will be discussed to illustrate specific features of mammalian drug metabolism.

DL 111, a new non-hormonal antifertility agent: contragestational and kinetic profile in baboons.

It was previously shown that 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4 triazole (DL 111) given parenterally in single or multiple doses during the early stage of embryonal development terminates pregnancy in the mouse, the hamster, the rat, the rabbit and the dog. In the present work, the studies have been extended to the baboon. In this sub-human primate, single and/or multiple intramuscular injections of the compound terminated pregnancy when given between day 34 and 54 of gestation. The effectiveness of DL 111 was greater when earlier in gestation and the optimal mode of treatment appears to be a multiple one. DL 111 appears to act by a direct action on the conceptus, with consequent suppression of the endocrine function of the placenta, progesterone withdrawal and abortion. In all the baboons that aborted, the menstrual cycles resumed within a reasonable length of time and subsequent cycles were regular. All the animals that did not abort have given birth to normal and health term infants. Fertility appears to be unimpaired and the progeny resulting form these pregnancies did not show any abnormalities. No significant drug-related side-effects or alterations in plasma enzymes or haematological parameters were observed. Pharmacokinetic and activity relationships strongly suggest that sustained exposure of the conceptus to the drug action is indispensable for optimizing the pregnancy-terminating effect.

Disposition of ORF 9326, a novel contragestational steroid, in animals.

The disposition of ORF 9326 [17BETA-acetoxy-2alpha-chloro-3(p-nitrophenoxy) imino-5-androstane], an O-aryl oxime of 2beta-chlorodihydrotestosterone acetate, was studied in rats, dogs, monkeys and rabbits. Intravenous administration of 3H-ORF 9326 dissolved in PEG-400 to rats, dogs and monkeys resulted in a rapid decline of radioactivity in blood followed by a terminal slope suggesting long retention of radioactivity. Apparent half lives of radioactivity in blood were calculated to be from 50--95 hours for the three species, which peak levels of radioactivity in whole blood occurring within 4--7 hours after administration of the compound. Tissue distribution studies in the rat and dog indicate that body fat is one of the major depot areas for the drug and/or its metabolites. The major route of excretion for ORF 9326 and/or its metabolites in dog and rat is biliary whereas in monkey and rabbit it appears to be renal. Greater than 90% of the radioactive compounds excreted in the urine of dogs and monkeys following intravenous administration of 3H-ORF 9326 appear to be in the form of conjugates.

Comparative bioavailability of a lipophilic steroid.

17beta-Acetoxy-2alpha-chloro-3-(p-nitrophenoxy)imino-5alpha-androstane (I) is a lipophilic steroid with postimplantive antifertility activity in laboratory animals. The bioavailability of micronized I from solutions and suspensions was compared in four groups of adult female Wistar rats. Each group received varying concentrations of micronized 3H-I (specific activity of 0.38--8.94 muCi/mg) in sesame oil by oral gavage. Samples of whole blood and urine collected following drug administration were assayed for radioactive content. Calculation of the mean area under the blood radioactivity versus time curve, when corrected for the quantity of drug administered, indicated that a substantially larger fraction of the dose was absorbed in the two instances where I was present only in solution. A linear relationship between the amount of I absorbed based on whole blood radioactivity and urinary excretion and the administered dose was found primarily for groups receiving the drug in solution. Preliminary results in humans indicate that 3H-I was absorbed to a much greater extent following oral administration of the drug in sesame oil than when admixed with lactose.