New antiprogestins with partial agonist activity: potential selective progesterone receptor modulators (SPRMs) and probes for receptor- and coregulator-induced changes in progesterone receptor induction properties.

A pharmacologically relevant property of steroid hormone-regulated gene induction is the partial agonist activity of antisteroid complexes. We now report that dexamethasone-mesylate (Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of the glucocorticoid-selective steroid dexamethasone (Dex), are two new antiprogestins with significant amounts of agonist activity with both the A and B isoforms of progesterone receptor (PR), for different progesterone-responsive elements, and in several cell lines. These compounds continue to display activity under conditions where another partial antiprogestin (RTI-020) is inactive. These new antiprogestins were used to determine whether the partial agonist activity of PR complexes can be modified by changing concentrations of receptor or coregulator, as we have recently demonstrated for glucocorticoid receptors (GRs). Because GR and coregulator concentrations simultaneously altered the position of the physiologically relevant dose-response curve, and associated EC(50), of GR-agonist complexes, we also examined this phenomenon with PR. We find that elevated PR or transcriptional intermediary factor 2 (TIF2) concentrations increase the partial agonist activity of Dex-Mes and Dex-Ox, and the EC(50) of agonists, independently of changes in total gene transactivation. Furthermore, the corepressors SMRT (silencing mediator for retinoid and thyroid receptors) and NCoR (nuclear receptor corepressor) each suppresses gene induction but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces the EC(50) and increases the partial agonist activity of antiprogestins. These comparable responses of GR and PR suggest that variations in receptor and coregulator concentrations may be a general mechanism for altering the induction properties of other steroid receptors. Finally, the magnitude of coregulator effects on PR induction properties are often not identical for agonists and the new antagonists, suggesting subtle mechanistic differences. These properties of Dex-Mes and Dex-Ox, plus the sensitivity of their activity to cellular differences in PR and coregulator concentrations, make these steroids potential new SPRMs (selective progesterone receptor modulators) that should prove useful as probes of PR induction properties.

Steroid-induced conformational changes of rat glucocorticoid receptor cause altered trypsin cleavage of the putative helix 6 in the ligand binding domain.

Steroid-induced changes in receptor protein conformation constitute a logical means of translating the variations in steroid structures into the observed array of whole cell biological activities. One conformational change in the rat glucocorticoid receptor (GR) can be readily discerned by following the ability of trypsin digestion to afford a 16-kDa fragment. This fragment is seen after proteolysis of steroid-free receptors but disappears in digests of either glucocorticoid- or antiglucocorticoid-bound receptors. The location of this cleavage site has now been located unambiguously as R651, in helix 6 of the ligand binding domain, by a combination of point mutagenesis, arginine specific protease digestion, and radiochemical sequencing. This 16-kDa species, corresponding to amino acids 652-795, was non-covalently associated with another, approximately 17-kDa species that was determined to be amino acids 518-651 after a comparison of co-immunoprecipitated fragments from wild type and two chimeric receptors. These assignments revise our earlier report of amino acids 537-673 being the 16-kDa fragment and suggest that sequences of the entire ligand binding domain are required for high affinity and specificity binding. This was supported by the observation that trypsin digestion of the steroid-free R651A mutant GR gave rise to the 30-kDa meroreceptor (amino acids 518-795), which displayed wild type affinity. This 30-kDa species is thus the smallest non-associated fragment of GR possessing wild type steroid binding affinity. This suggests that other GR regions do not influence steroid binding affinity. The above results are reminiscent of those observed for the estrogen receptor. However, unlike the estrogen receptor or the more closely related progesterone receptor, the precise proteolytic cleavage points of both the steroid-free and -bound GR fall within regions that are predicted, on the basis of X-ray crystal structures of related receptors, to be alpha-helical and resistant to proteolysis. Thus, the tertiary structure of the GR ligand binding domain may be distinctly different from that of estrogen and progesterone receptors.

Fetal and newborn death associated with maternal cocaine use.

Data collected from 25 cases of fetal or newborn death associated with maternal cocaine use are reported. The average week of gestation at which fetal death occurred was week 30. Abruptio placentae was observed in 7 cases and placental infarct was found in 4 cases. The average fetal blood cocaine and benzoylecgonine levels were 0.26 and 1.73 micrograms/mL. The average maternal levels were 0.14 and 1.80 micrograms/mL, respectively.

A 5-year stability study of common illicit drugs in blood.

The present study was designed to determine the stability of common illicit drugs in stored blood at various time intervals for a period of up to 5 years. The drugs of interest were cocaine and benzoylecgonine, methamphetamine and amphetamine, nonconjugated morphine and codeine, and phencyclidine (PCP). All specimens were from live individuals and were collected in gray-top Vacutainer tubes containing sodium fluoride and potassium oxalate; the tubes were stored at ambient temperature. The results of the study showed that cocaine and benzoylecgonine have poor stability and require quantitative confirmation within a reasonable time period for reliable interpretation. Methamphetamine and PCP were both fairly stable and had a high probability of confirmation upon reanalysis. The stability of nonconjugated morphine showed wide variation throughout the study. Initially, the morphine concentration decreased, then increased at the 3-year interval, and finally decreased at the 4- and 5-year intervals. The significance of the analytical findings are discussed in this report.

Fetal and infant deaths associated with maternal methamphetamine abuse.

Eight cases of fetal and infant death related to maternal methamphetamine abuse are presented. The mean fetal blood concentration of methamphetamine was 0.36 microgram/mL (range, 0.03-1.20 micrograms/mL), and the mean concentration of amphetamine was 0.05 microgram/mL (range, 0-0.08 microgram/mL). Both maternal and fetal blood methamphetamine concentrations were obtained in two cases. The maternal and fetal methamphetamine concentrations for these two cases were 0.21 and 0.40 microgram/mL and 0.18 and 1.20 micrograms/mL, respectively. The cause of death for each case, as listed by the pathologist, is also discussed.

Postmortem tissue methamphetamine concentrations following selegiline administration.

Selegiline is a relatively new antiparkinson's drug whose metabolites include methamphetamine and amphetamine. A 72-year-old female with apparently suicidal intentions was found dead at her residence. The cause of death was listed as coronary heart disease with a history of polypharmacy. The tissue methamphetamine and amphetamine concentrations resulting from selegiline administration are presented in this report.

Two fatal cases of venlafaxine poisoning.

Venlafaxine is a phenethylamine derivative that has recently been approved for use in the treatment of depression. It is chemically unrelated to tricyclic, tetracyclic, or other available antidepressant agents. Anticholinergic, hypotensive, hypertensive, and cardiotoxic side effects are rare. Two fatal cases encountered at separate laboratories are discussed, both involve high levels of venlafaxine. Concentrations of the drug in peripheral blood, heart blood, urine, vitreous humor, and liver are reported. Descriptions of extraction and gas chromatographic methods for confirmation and quantitation are included.

Zolpidem tissue concentrations in a multiple drug related death involving Ambien.

Zolpidem (Ambien), a relatively new nonbenzodiazepine sedative-hypnotic, was involved in the death of a 39-year-old obese male who was being treated for depression and insomnia. The identification and quantitation procedures of zolpidem in postmortem tissues included dual-column gas chromatography (GC) with nitrogen-phosphorus detection and GC-mass spectrometry. Zolpidem was present at concentrations of 2.91, 1.40, and 2.13 microg/mL in the heart blood, peripheral blood, and urine, respectively. The liver had zolpidem present at a concentration of 4.74 microg/g, and the gastric contents had a total of 172 mg zolpidem. Additional drugs present included hydrocodone and morphine (nonconjugated) at 0.16 and 0.04 microg/mL, respectively. The cause of death was determined to be multiple drug intoxication. This report describes the analytical techniques and significance of the zolpidem findings.

Clozapine tissue concentrations following an apparent suicidal overdose of Clozaril.

Clozapine is a tricyclic dibenzodiazepine derivative that is classified as an "atypical" antipsychotic drug. A 25-year-old male was brought to a hospital emergency room following the ingestion of an estimated 20 100-mg tablets of clozapine. After several hours in the hospital, the patient died. The cause of death was listed as acute clozapine intoxication. It was also noted upon autopsy that the patient had an unusual eosinophilic myocarditis. The toxicological and pathological findings are presented in this report.

Pennyroyal toxicity: measurement of toxic metabolite levels in two cases and review of the literature.

BACKGROUND: Pennyroyal is a widely available herb that has long been used as an abortifacient despite its potentially lethal hepatotoxic effects. However, quantitative data for pennyroyal constituents and their metabolites in humans have not been previously reported. OBJECTIVES: To quantify pennyroyal metabolites in human overdose, to correlate these findings with clinical variables, and to place these findings in the context of previously reported cases of pennyroyal toxicity. DESIGN: Clinical case series of pennyroyal ingestions; quantification of pennyroyal metabolites by gas chromatography and mass spectrometry; qualitative detection of protein-bound adducts of the metabolites of pennyroyal constituents in human liver by Western blot assay; and review of the literature based on a search of MEDLINE, Index Medicus, and the reference citations of all available publications. RESULTS: We report four cases of pennyroyal ingestion. One patient died, one received N-acetylcysteine, and two ingested minimally toxic amounts of pennyroyal and were not treated with N-acetylcysteine. In the fatal case, postmortem examination of a serum sample, which had been obtained 72 hours after the acute ingestion, identified 18 ng of pulegone per mL and 1 ng of menthofuran per mL. In a serum sample from the patient treated with N-acetylcysteine, which had been obtained 10 hours after ingestion, the menthofuran level was 40 ng/mL. Review of 18 previous case reports of pennyroyal ingestion documented moderate to severe toxicity in patients who had been exposed to at least 10 mL of pennyroyal oil. CONCLUSION: Pennyroyal continues to be an herbal toxin of public health importance. Data on human metabolites may provide new insights into the toxic mechanisms and treatment of pennyroyal poisoning, including the potential role of N-acetylcysteine. Better understanding of the toxicity of pennyroyal may also lead to stricter control of and more restricted access to the herb.