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.

Simultaneous determination of rivanol and mifepristone in human plasma by a HPLC-UV method with solid-phase extraction.

A HPLC method with UV detection was developed and validated for the simultaneous determination of rivanol and mifepristone in human plasma. Norethisterone was used as the internal standard. Separation was performed by a C18 reversed-phase column maintained at 20 degrees C. The mobile phase was a mixture of methanol-acetonitrile-0.05% sodium dodecylsulfonate in a 0.05 M phosphate buffer with the pH adjusted to 3.0 (30:30:40, v/v/v) at a flow rate of 0.8 ml/min. Dual wavelength mode was used, with mifepristone monitored at UV 302 nm, while rivanol and norethisterone at 272 nm. A reliable biological sample pre-treatment procedure by means of solid-phase extraction was used, which allowed to obtain good extraction efficiency (>93%) for both of the analytes and the internal standard. The calibration curves were both linear with the correlation coefficient r equal to 0.9999. For rivanol, the assay gave CV% values for precision always lower than 7.8% and mean accuracy values higher than 95.3%. As to mifepristone, precision was always lower than 10.1% and mean accuracy values were higher than 93.8%. The limit of detection for the assay of rivanol and mifepristone was 1.1 and 3 ng/ml, respectively. The method is simple, sensitive and accurate, and allow for simultaneous determination of nanogram levels of rivanol and mifepristone in human plasma. It could be applied to assess the plasma level of rivanol and mifepristone in women undergoing polypharmacy with the two drugs.

An HPLC method for the determination of ng mifepristone in human plasma.

An HPLC method was developed and validated for the determination of mifepristone in human plasma. C(18) solid-phase extraction cartridges were used to extract plasma samples. Separation was by C(18) column; mobile phase, methanol-acetonitrile-water (50:25:25, v/v/v); flow rate, 0.8 ml/min; UV detection at 302 nm. The calibration curve was linear in the concentration range of 10 ng/ml to 20 microg/ml (r=0.9991). Within- and between-day variability were acceptable. The limit of detection for the assay was 6 ng/ml. Plasma samples were stable for at least 7 days in the state of plasma or residue treated at -20 degrees C. The method was simple, sensitive and accurate, and allowed to determine ng mifepristone in human plasma. It could be applied to assess the plasma level of mifepristone in women receiving low oral doses of mifepristone.

Development of enzyme-linked immunosorbent assays for isocupressic acid and serum metabolites of isocupressic acid.

The consumption of ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), common juniper (Juniperus communis), and Monterey cypress (Cupressus macrocarpa) causes abortions in pregnant cattle. Recent studies have identified isocupressic acid (1) as the primary abortificient compound in these plants. In vitro and in vivo studies using rumen and blood have shown isocupressic acid (1) is rapidly metabolized to agathic acid (3), dihydroagathic acid (4), and tetrahydroagathic acid (5). Rapid and sensitive diagnostic techniques are needed to identify poisoned animals, to study toxicokinetics, and to elucidate the mechanism of isocupressic acid-induced abortion in cattle. In this study, four competitive inhibition enzyme-linked immunosorbent assays for isocupressic acid and its sera metabolites were developed using polyclonal antibodies. One assay is specific to 1, whereas the other three assays show cross-reactivity to 3-5 in addition to 1. The assay specific to 1 had a limit of detection of 44.1 pg. The other assays which demonstrated cross-reactivity to the isocupressic acid blood metabolites also had comparably low limits of detection. One assay was used to follow the absorption and elimination profile of isocupressic acid metabolites in both cow serum and urine after oral dosage of a cow with common juniper.

Plasma levels of antiprogestin RU 486 following oral administration to non-pregnant and early pregnant women.

RU 486 is a synthetic steroid which acts as an antiprogestin at the receptor level. The clinical usefulness of the compound for menstrual regulation and termination of early pregnancy is currently being evaluated. The aim of the present study was to determine the plasma levels of RU 486 following the oral administration of the compound to 42 pregnant and 10 non-pregnant women. The levels of RU 486 were measured by a radioimmunoassay method which uses chromatography on Sephadex LH 20 columns. The identity of the compound assayed as RU 486 was confirmed, but the presence of small amounts of two highly cross-reacting metabolites (monodemethyl and didemethyl RU 486) in the analyzed fractions could not be excluded. Following the ingestion of a single tablet containing 25 and 50 mg of the compound, a peak plasma value of approximately 3.5 to 4.0 mumol/l in both the pregnant and non-pregnant subjects was reached one to two hours later. The half-lives of elimination were about 20 hours in both the pregnant and the non-pregnant women. Following the repeated oral administration of 50, 100 or 200 mg of RU 486 daily for four days, maximum plasma levels of 2.9, 4.5 and 5.4 mumol/l, respectively, were found. Thus, the increase in plasma levels was not directly proportional to the increase in the dose. No accumulation of RU 486 in the plasma was found, even when the duration of treatment was prolonged to six days. The data partly explain the reported lack of relation between ingested dose and frequency of induced abortion and they may be useful for designing future studies on the use of compound to prevent implantation, induce menstruation or terminate an early pregnancy.

Plasma levels of 9-deoxo-16,16-dimethyl-9-methylene-PGE2 in connection with its development as an abortifacient.

Gaschromatographic-mass spectrometric quantitation of 9-deoxo-16,16-dimethyl-9-methylene-PGE2 in plasma samples obtained during constant intravenous infusion of the drug revealed that a plasma level of about 20 ng/ml was associated with high enough uterine contractility for induction of second trimester abortions. This level was therefore aimed for during the development of formulations and dose schedules for interruption of pregnancy with this drug. For the first time it was possible to induce second trimester abortions through oral administration of a prostaglandin analog, although the plasma levels were low giving a moderate success rate (about 50%) within 25 hours. Rectal administration of 20 mg of the drug at 6 hours intervals resulted in high enough plasma levels for second trimester abortions. Highly efficient dose schedules for interruption of early first trimester ("menses induction") and second trimester pregnancies through vaginal administration were developed. The frequency of side effects in the early first trimester were so low that "home treatment" was possible. Formulations suitable for 3, 6 or 12 hours preoperative dilatation of the cervix were also developed.