In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames+MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames+MNvit--benzyl acetate, toluene, morphine and thiabendazole--and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames+MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames+MNvit.

A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins.

The cDNA of the mineralocorticoid receptor (MR) was cloned 30 years ago, in 1987. At that time, spirolactone, the first generation of synthetic steroid-based MR antagonists (MRAs), which was identified in preclinical in vivo models, had already been in clinical use for 30 years. Subsequent decades of research and development by Searle & Co., Ciba-Geigy, Roussel Uclaf and Schering AG toward identifying a second generation of much more specific steroidal MRAs were all based on the initial 17-spirolactone construct. The salient example is eplerenone, first described in 1987, coincidentally with the cloning of MR cDNA. Its launch on the market in 2003 paralleled intensive drug discovery programs for a new generation of non-steroidal MRAs. Now, 30 years after the cDNA cloning of MR and 60 years of clinical use of steroidal MRAs, novel non-steroidal MRAs such as apararenone, esaxerenone and finerenone are in late-stage clinical trials in patients with heart failure, chronic kidney disease (CKD), hypertension and liver disease. Finerenone has already been studied in over 2000 patients with heart failure plus chronic kidney disease and/or diabetes, and in patients with diabetic kidney disease, in five phase II clinical trials. Here, we reflect on the history of the various generations of MRAs and review characteristics of the most important steroidal and non-steroidal MRAs.

/pdf/30-years-of-the-mineralocorticoid-receptor-mineralocorticoid-2qfdanqgu9.pdf

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Mineralocorticoid receptor antagonists: 60 years of research and development.

This survey is a compendium of genotoxicity and carcinogenicity information of 838 marketed drugs, whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time. Of these 838 drugs, 366 (43.7%) do not have retrievable genotoxicity or carcinogenicity data. The remaining 472 (56.3%) have at least one genotoxicity or carcinogenicity test result. Of the 449 drugs with at least one genotoxicity test result, 183 (40.8%) have at least one positive finding. Of the 338 drugs with at least one carcinogenicity test result, 160 (47.3%) have at least one positive result. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, of the 315 drugs which have both genotoxicity and carcinogenicity data 116 (36.8%) are neither genotoxic nor carcinogenic, 50 (15.9%) are non-carcinogens which test positive in at least one genotoxicity assay, 75 (23.8%) are carcinogenic in at least one sex of mice or rats but test negative in genotoxicity assays, and 74 (23.5%) are both genotoxic and carcinogenic. Only 208 (24.8%) of the 838 drugs considered have all data required by current guidelines for testing of pharmaceuticals. However, it should be noted that a large fraction of the drugs considered were developed and marketed prior to the present regulatory climate. Although the laws do not require re-testing based on revised standards, in the absence of epidemiological studies excluding a carcinogenic risk to humans, a re-evalutation would be appropriate.

Update on genotoxicity and carcinogenicity testing of 472 marketed pharmaceuticals.

Publisher Summary The vast majority of drugs or other foreign chemicals entering the mammalian organism are chemically altered. Many of these changes are effected in a biphasic process—phase I transformations consist of oxidation, reduction, and hydrolysis reactions, while phase II transformations involve conjugative reactions (synthetic reactions) between the drug or its metabolite and common biochemicals found in the body. Cytochrome P450 systems from mammals have been widely studied because of their well known involvement in many types of oxidative drug transformations, such as aromatic and aliphatic hydroxylations. The most striking similarities in microbial and mammalian metabolic systems lie in their respective cytochrome P450 hemoprotein monooxygenases. Most oxidative phase I reactions in mammals are catalyzed by cytochrome P450-linked monooxygenases located primarily in the liver. The routine screening of a carefully selected group of 10-40 microorganisms may be used to quickly assess the probability of producing drug metabolites.

Microbial models for drug metabolism.

Studies on the destruction of adrenal and testicular cytochrome P-450 by spironolactone. Requirement for the 7alpha-thio group and evidence for the loss of the heme and apoproteins of cytochrome P-450.

Gas chromatography-mass spectrometry was used to identify metabolites of spironolactone in human blood and urine. In three healthy men about 20% of the radioactivity was excreted in the urine within 24 hr after an oral dose of [20-3H]spironolactone (200 mg + 200 muCi). About half of this radioactivity was extracted with chloroform at pH 3 and from this extract four stable metabolites were isolated by use of column and thin-layer chromatography. Two of these were the previously identified metabolites, canrenone (VII; 2.9% of dose) and the 6beta-hydroxy-sulfoxide (X; 1.8% of the dose). The remaining were the new metabolites, 15alpha-hydroxycanrenone (XI; 0.8% of dose) and the 6beta-hydroxy-thiomethyl derivatives (VI; 0.5% of dose). The principal water-soluble urinary metabolite was canrenoate ester glucuronide (XII; 4.5% of dose). In the 24- to 32-hr pooled serum, canrenone (VII) was the principal metabolite in the organic-extractable fraction; VI was present in appreciable amounts but X and XI were present at extremely low levels.

Spironolactone metabolism in man studied by gas chromatography-mass spectrometry.

The metabolic fates of potassium canrenoate (PC) and spironolactone (SP) were compared for the rat in vivo and in vitro. Approximately 18% of an in vivo dose of SP was metabolized to canrenone (CAN) and related compounds in the rat. In vitro, 20–30% of SP was dethioacetylated to CAN and its metabolites by rat liver 9000 g supernatant (S9). Thus, the major route of SP metabolism is via pathways that retain the sulfur moiety in the molecule. PC was metabolized by rat hepatic S9 to 6α, 7α- and 6β, 7β-epoxy-CAN. The β-epoxide was further metabolized to its 3α- and 3β-hydroxy derivatives as well as its glutathione (GSH) conjugate. Both 3α- and 3β-hydroxy-6β, 7β-epoxy-CAN were shown to be direct acting mutagens in the mouse lymphoma assay, whereas 6α, 7α- and 6β, 7β-epoxy-CAN were not. These mutagenic metabolites, their precursor epoxides and their GSH conjugates were not formed from SP under identical conditions. The above findings appear to be due to inhibition of metabolism of CAN formed from SP by SP and/or its S-containing metabolites, since the in vitro metabolism of PC by rat hepatic microsomes was appreciably reduced in the presence of SP. The hypothesized mechanism(s) for this inhibition is that SP and its S-containing metabolites specifically inhibit an isozyme of hepatic cytochrome P-450 or SP is a preferred substrate over PC/CAN for the metabolizing enzymes. Absence of the CAN epoxide pathway in the metabolism of SP provides a possible explanation for the observed differences in the toxicological profiles of the two compounds.

Difference in metabolic profile of potassium canrenoate and spironolactone in the rat: mutagenic metabolites unique to potassium canrenoate.

1. Several new hydroxylated and reduced metabolites of spironolactone in the urine and faeces of the rat, dog and monkey after 50 mg/kg oral administration of [22-14C]spironolactone were purified by preparative t.l.c. and identified by comparing their chromatographic (t.l.c. and g.l.c.) and mass spectral characteristics with those of synthetic samples.2. Metabolites were divided into those in which the sulphur of spironolactone was removed and those in which this sulphur was retained.3. Canrenone (CAN) was the primary metabolite in the first class. It was further metabolized by three main pathways: (1) opening of the y-lactone ring to canrenoic acid which was excreted as canrenoate ester glucuronide (2) hydroxy-lation to 15α-hydroxy-CAN, 15β-hydroxy-CAN and 21-hydroxy-CAN and (3) reduction to 6,7-dihydro-CAN, 4,5β,6,7-tetrahydro-CAN, 4,5α,6,7-tetra-hydro-CAN, 3β-hydroxy, 4,5α-tetrahydro-CAN, 3β-hydroxy,4,5β,6,7-hexahydro-CAN and 3α-hydroxy,4,5β,6,7-hexahydro-CAN.4. Metabolites in the second class were the...

Spironolactone: Diversity in Metabolic Pathways

Identification of carbamate derivatives formed during chloroform extraction of tricyclic antidepressants from urine.

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Spironolactone metabolism in man studied by gas chromatography-mass spectrometry.

Difference in metabolic profile of potassium canrenoate and spironolactone in the rat: mutagenic metabolites unique to potassium canrenoate.

Spironolactone: Diversity in Metabolic Pathways

Identification of carbamate derivatives formed during chloroform extraction of tricyclic antidepressants from urine.