Suggesting a testing strategy for possible endocrine effects of drug metabolites.

Most pharmaceuticals are extensively metabolized by organisms, which results in internal exposure to mixtures of parent compounds and various metabolites. Many of these metabolites are considered non-toxic, but some metabolites retain toxic properties of the parent compound or elicit other undesirable outcomes. Unfortunately, the effects of metabolites are often not considered when endocrine activities of chemicals are evaluated in vitro. In this study two approaches, an "effect-based" and a "compound-by-compound" testing design, were used to determine the effects of metabolites of the antidepressant sertraline on aromatase enzyme activity. In the "effect-based" approach, a mixture of sertraline metabolites, produced by liver microsomes, inhibited aromatase, but was less potent than sertraline. In the "compound-by-compound" testing design, three specific metabolites were evaluated individually and in mixtures. Though two N-desmethylated metabolites were more potent aromatase inhibitors than sertraline, hydroxyl ketone sertraline did not inhibit the enzyme and mixtures of these metabolites and sertraline were less potent than predicted from a concentration addition model. Our findings highlight the importance of considering aromatase inhibition, and potentially other biological activities, of pharmaceutical metabolites produced by liver microsome preparations and then comparing such observations to studies of specific metabolites available for testing in pure form. Subsequently, a five step integrated strategy for screening of the potential endocrine effects of drugs and their metabolites are proposed.

Inhibition of human aromatase complex (CYP19) by antiepileptic drugs.

Antiepileptic drugs and epilepsy are often associated with sexual disorder in women such as hyperandrogenism, menstrual disorders and ovarian cysts. In children, until puberty, a hormone imbalance may influence many aspects of development, e.g. growth and sexual maturation. The aromatase complex is the enzyme system that converts androgens to estrogens and consequently an inhibition may induce a hormone imbalance. Twelve antiepileptic drugs, used in mono or polytherapy for the treatment of children, were tested for their ability to inhibit aromatase (CYP19) with commercially available microsomes from transfected insect cells using dibenzylfluorescein as substrate. The drugs inhibiting CYP19 were: lamotrigine, oxcarbazepine, tiagabine, phenobarbital, phenytoin, ethosuximide, and valproate. The inhibitory effects (50% reduction in activity compared to enzymes without inhibitor present) were in the range of 1.4-49.7 mM. Carbamazepine, gabapentin, primidone, topiramate and vigabatrin showed no inhibition. Additionally, binary drug combinations were tested to investigate if combination therapy could potentiate the aromatase inhibition. Additive inhibition was seen in combination experiments with valproate and phenobarbital. When adding carbamazepine to a range of valproate concentrations no additional inhibition was seen. The data for some of the AEDs show that side effects on steroid synthesis in humans due to inhibition of aromatase should be considered.

1-Methyl-3-phenyl-1,2,4-triazinium-5-olate: a new zwitterion with cytotoxic activity against human cancer cell-lines.

One of 6 newly synthesized triazinium zwitterions (JR-1--JR-6) was shown to induce 51Cr-release from leukemic (HL60 and CEM) and solid tumor (MM170 and HeLa) cell-lines. Leukemic cells were more sensitive to this compound than solid tumors as demonstrated by dose-response and time-course studies. Other experiments showed that JR-6 (1-methyl-3-phenyl-1,2,4-triazinium-5-olate) significantly suppressed protein, RNA and DNA synthesis at tumoricidal concentrations.

Identification and synthesis of O-methylcatechol metabolites of phenobarbital and some N-alkyl derivatives.

5-Ethyl-5-(4-hydroxy-3-methoxyphenyl) barbituric acid was identified as a new, minor metabolite of phenobarbital in man. The identity of this O-methylcatechol metabolite was confirmed by an unequivocal chemical synthesis, and by GC-MS studies. Mephobarbital and the 1,3-dimethyl, 1-ethyl, and 1,3-diethyl analogues of phenobarbital yielded the corresponding N-alkylated O-methylcatechol metabolites, all of which were confirmed by synthesis. The N-alkyl barbiturates each gave additionally at least one O-methylcatechol metabolite in which N-dealkylation had occurred. These metabolites accounted for approximately 1-5% of the orally administered dose in man.

Rapid liquid-chromatographic determination of metformin in plasma and urine.

We describe an analysis for metformin in plasma and urine by use of cation-exchange "high-performance" liquid chromatography. The assay requires only 0.5 mL of sample and involves pretreatment with trichloroacetic acid containing an internal standard (1-propylbiguanide), followed by centrifugation and injection into the chromatograph. The column eluent is monitored at 230 nm. Metformin and 1-propylbiguanide give retention times of 8 and 10 min, respectively. Within-day and between-day precision (CV) for the assay of plasma containing 2 mg (1.2 X 10(-5) mol) of metformin hydrochloride per liter was 3.6 and 3.9%, respectively. Similar precision was obtained for urine. The limit of detection is about 0.1 mg (6.0 X 10(-7) mol) of metformin hydrochloride per liter. Several commonly used drugs, including other oral hypoglycemic agents, do not interfere with this method.

Inhibition of neutrophil locomotion, natural killer cell cytotoxicity and lymphocyte transformation by 1-methyl-3-phenyl-1,2,4-triazinium-5-olate, a novel triazinium zwitterion.

We studied a novel triazinium zwitterion compound for its effects on neutrophil locomotion and deoxyglucose uptake, Natural Killer (NK) cell cytotoxicity and mitogen-induced lymphocyte transformation. The results show significant inhibition of neutrophil locomotion at concentrations of 10 micrograms/ml or greater; by contrast, there was no significant effect on neutrophil deoxyglucose uptake. Significant suppression of NK cell cytotoxicity occurred at similar concentrations in a dose-dependent fashion. Marked suppression of mitogen-induced lymphocyte transformation was also observed for all three mitogens used in the assays. This effect was dose-dependent, reversible by washing and still evident even when it was added 37 h after the initiation of cultures. These results suggest that 1-methyl-3-phenyl-1,2,4-triazinium-5-olate may have application as an anti-inflammatory and immunosuppressive agent.

Effect of the novel triazinium zwitterion 1-methyl-3-phenyl-1,2,4-triazinium-5-olate on immunological responses and experimental brucellosis in mice.

The immunopharmacology of a novel triazinium zwitterion, designated JR-6, was studied in mice. Experiments show dose-response inhibition of antibody and delayed-type hypersensitivity (DTH) responses to sheep red blood cell antigens. Suppression of DTH was confirmed using trinitrochlorobenzene as a contact-sensitizing antigen. Using a model of experimental brucellosis in mice, it was found that JR-6 caused suppression of specific antibody and DTH reaction, as well as spleen weight, but a statistically significant increase in viable counts of Brucella abortus was not observed. Extensive short-term toxicology studies showed reduction in lymphocyte and neutrophil counts, and slow weight gain of treated mice. However, organ histology, liver function tests and biochemical profiles were normal.

Identification of p-hydroxyprimidone as a minor metabolite of primidone in rat and man.

Urine specimens from rats and humans who received single doses of primidone (PRM) have been investigated by GC/MS procedures. In addition to the previously documented metabolites of PRM, a small chromatographic peak was encountered which had a mass spectrum suggesting a hydroxy-PRM derivative. Synthesis of p-hydroxy-PRM from PRM was effected; the para isomer was separated from unwanted isomers by preparative HPLC. The PMR spectrum of the synthetic compound proved the position of the hydroxy substituent to be para. This compound had identical GC retention time and an almost identical mass spectrum with that obtained in the urinary extracts. Para-hydroxy-PRM was therefore confirmed as a new, minor metabolite of PRM in rat and man.