Metabolism of megestrol acetate and related progesterone analogues by liver preparations in vitro.
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ABSTRACT:
SUBMITTER: Cooke BA
PROVIDER: S-EPMC1264745 | biostudies-other | 1965 Dec
REPOSITORIES: biostudies-other
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Project description:1. The isolation and partial purification of 11beta-hydroxy steroid dehydrogenase from rat and guinea-pig liver microsomes has been achieved by conventional methods. 2. The efficiency of different 11-oxygenated steroids as substrates has been examined. The relative efficiencies confirm in the main the stereochemical theory of the enzyme-coenzyme-substrate complex that was proposed earlier on the basis of studies in vivo. Delta(4)-3-Ketones and 5alpha-hydrogen steroids are readily metabolized by the enzyme. 5beta-Hydrogen steroids and Delta(4)-3-ketones with certain large alpha-substituents are metabolized to a limited extent or not at all. Halogen substitution in the 9alpha-position enhances the rate of reduction of 11-ketones but blocks the oxidation of the related 11beta-ols. 3. 9alpha-Fluorocortisol is a competitive inhibitor of the oxidation of cortisol, but 9alpha-fluorocortisone is reduced at five to ten times the initial velocity of cortisone. 4. 11beta-Hydroxy steroid dehydrogenase activity has been found in liver microsomes of rat, guinea pig, rabbit and calf. 5. Relative substrate efficiencies and K(m) values are similar in whole (debris-free) homogenates, washed microsomes and acetone-dried powders of washed microsomes. 6. A variety of conditions have been examined for the observation of 11beta-hydroxy steroid dehydrogenase activity. NADP(H) is an efficient and NAD(H) a very poor coenzyme for the reaction.
| S-EPMC1198650 | biostudies-other
Project description:1. Sex and species differences during the metabolism of [4-(14)C]progesterone by liver homogenates from rat, rabbit, guinea pig and hamster have been investigated. 2. Liver homogenate from male rat formed ;water-soluble' metabolites faster and in significantly larger amounts than did liver homogenate from female rat. About 65-70% of the added progesterone was conjugated as glucuronide by liver homogenate from male rat and about 45-50% by that from female rat. Liver homogenate from male rat also formed glucuronides faster than did liver homogenate from female rat. Sulphate formation was low (8-16%) in liver homogenates from both male and female rats. 3. Hamster-liver homogenate did not show any sex difference in the rate of formation of ;water-soluble' metabolites, but a sex difference was observed in the amount of free steroids recovered at low tissue:steroid ratios. Liver homogenate from female hamster formed glucuronides faster and in significantly larger amounts than did liver homogenate from male hamster, the reverse of what was found in rat liver. 4. Liver homogenates from male and female rabbits and guinea pigs formed ;water-soluble' metabolites that were almost entirely glucuronides. 5. Neither rabbit liver nor guinea-pig liver showed any significant sex difference in the rate or amount of formation of total ;water-soluble' metabolites or glucuronides, but guinea-pig liver was considerably less active than rabbit liver. 6. Glucuronides were quantitatively the major type of conjugate formed by the liver homogenates from both sexes of all species except the male hamster.
| S-EPMC1206918 | biostudies-other
Project description:Arnica tincture is a herbal medicinal preparation with anti-inflammatory activity which is used traditionally for the topical treatment of blunt injuries as well as rheumatic muscle and joint complaints. Its main bioactive constituents are sesquiterpene lactones (STLs) of the helenalin and 11α,13-dihydrohelenalin types. Besides the mentioned activity, the tincture and its isolated STLs have antileishmanial activity. In a recent in vivo study, a treatment with Arnica tincture cured cutaneous Leishmaniasis (CL) in a golden hamster model. CL is a neglected tropical disease affecting more than two million people every year, for which new treatments are urgently needed. In order to use Arnica tincture on open CL lesions of human patients, it is important to know how the constituents are metabolized. Therefore, in vitro metabolism experiments with liver microsomes of different species (rat, pig and human) were performed with the Arnica STLs helenalin acetate and 11α,13-dihydrohelenalin acetate. Phase I and phase II metabolism experiments were performed, as well as a combination of both. Glutathione conjugation plays a major role in the metabolism of these STLs, as could be expected based on previous reports on their reactivity. Besides glutathione conjugates, several other metabolites were formed, e.g., water conjugates and hydroxides. Our results show for the first time a detailed picture of the metabolism of Arnica STLs. The fast and extensive formation of glutathione conjugates makes it unlikely that low absorbed levels of these compounds, as expected after dermal absorption from Arnica tincture, could be of toxicological concern.
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Project description:Adult male rat liver and kidney preparations were incubated with (2-hydroxyoestradiol-1-yl)[(35)S]glutathione. The glutamic acid and glycine residues were removed by enzymes present in the kidney microsomal fraction; the liver preparations had no effect. The resulting 2-hydroxyoestradiol-cysteine conjugate was acetylated at the alpha-amino group by both liver and kidney homogenates fortified with acetyl-coenzyme A, but not significantly in the absence of this coenzyme, or by liver or kidney slices. These results suggest that an oestrogen-glutathione conjugate, if formed in vivo, would be converted into the corresponding mercapturic acid before excretion.
| S-EPMC1185515 | biostudies-other
Project description:1. There is limited knowledge regarding the metabolism of megestrol acetate (MA), as it was approved by FDA in 1971, prior to the availability of modern tools for identifying specific drug-metabolizing enzymes. We determined the cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) that metabolize MA, identified oxidative metabolites and determined pharmacologic activity at the progesterone, androgen and glucocorticoid receptors (PR, AR and GR, respectively). 2. Oxidative metabolites were produced using human liver microsomes (HLMs), and isolated for mass spectral (MS) and nuclear magnetic resonance (NMR) analyses. We screened recombinant P450s using MA at 62??M (HLM Km for metabolite 1; M1) and 28??M (HLM Km for metabolite 2; M2). UGT isoforms were simultaneously incubated with UDPGA, nicotinamide adenine dinucleotide phosphate (NADPH), CYP3A4 and MA. Metabolites were evaluated for pharmacologic activity on the PR, AR and GR. CYP3A4 and CYP3A5 are responsible for oxidative metabolism of 62??M MA. 3. At 28??M substrate concentration, CYP3A4 was the only contributing enzyme. Mass spectral and NMR data suggest metabolism of MA to two alcohols. After oxidation, MA is converted into two secondary glucuronides by UGT2B17 among other UGTs. MA, M1 and M2 had significant pharmacologic activity on the PR while only MA showed activity on the AR and GR.
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Project description:Herbal supplements are increasingly used in psychiatric practice. Our epidemiological study has identified several herbal preparations associated with adverse outcomes of antipsychotic therapy. In this study, we evaluated the in vitro effects of four herbal preparations-Radix Rehmanniae (RR), Fructus Schisandrae (FS), Radix Bupleuri (RB) and Fructus Gardeniae (FG)-on cytochrome P450s (CYPs) involved in the metabolism of clozapine in human liver microsomes (HLMs) and recombinant human cytochrome P450 enzymes (rCYPs). N-desmethylclozapine and clozapine N-oxide, two major metabolites of clozapine, were measured using high-performance liquid chromatography (HPLC). FG, RR and RB showed negligible inhibitory effects in both in vitro systems, with estimated half-maximal inhibitory concentrations (IC50) and apparent inhibitory constant values (Ki) greater than 1 mg/mL (raw material), suggesting that minimal metabolic interaction occurs when these preparations are used concomitantly with clozapine. The FS extract affected CYP activity with varying potency; its effect on CYP 3A4-catalyzed clozapine oxidation was relatively strong (Ki: 0.11 mg/mL). Overall, the weak-to-moderate inhibitory effect of FS on in vitro clozapine metabolism indicated its potential role in herb-drug interaction in practice.
| S-EPMC6273925 | biostudies-literature
Project description:Megestrol acetate is metabolized by homogenates of rat adrenal glands to three more polar metabolites Y(2), Y(3) and Y(4). Their structures were investigated by microchemical reactions, paper chromatography, and ultraviolet and infrared spectroscopy. Metabolite Y(3) was identified as 17alpha-acetoxy-11beta-hydroxy-6-methyl-pregna-4,6-diene-3,20-dione, its oxidation product being identical with authentic 17alpha-acetoxy-6-methylpregna-4,6-diene-3,11,20-trione. It is suggested that metabolite Y(2) is 17,18-dihydroxy-6-methylpregna-4,6-diene-3,20-dione and that it also exists in the form of an alpha-ketol formed between the 18-hydroxyl and 20-oxo groups. Metabolite Y(4) is probably a tautomeric form of metabolite Y(2) as they were partially interconverted on chromatography and also have similar properties.
| S-EPMC1186760 | biostudies-other
Project description:Acetate kinase (ACK) (EC no: 2.7.2.1) interconverts acetyl-phosphate and acetate to either catabolize or synthesize acetyl-CoA dependent on the metabolic requirement. Among all ACK entries available in UniProt, we found that around 45% are multiple ACKs in some organisms including more than 300 species but surprisingly, little work has been done to clarify whether this has any significance. In an attempt to gain further insight we have studied the two ACKs (AckA1, AckA2) encoded by two neighboring genes conserved in Lactococcus lactis (L. lactis) by analyzing protein sequences, characterizing transcription structure, determining enzyme characteristics and effect on growth physiology. The results show that the two ACKs are most likely individually transcribed. AckA1 has a much higher turnover number and AckA2 has a much higher affinity for acetate in vitro. Consistently, growth experiments of mutant strains reveal that AckA1 has a higher capacity for acetate production which allows faster growth in an environment with high acetate concentration. Meanwhile, AckA2 is important for fast acetate-dependent growth at low concentration of acetate. The results demonstrate that the two ACKs have complementary physiological roles in L. lactis to maintain a robust acetate metabolism for fast growth at different extracellular acetate concentrations. The existence of ACK isozymes may reflect a common evolutionary strategy in bacteria in an environment with varying concentrations of acetate.
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Project description:Several medical plants belonging to the genera Passiflora, Viola, and Crataegus accumulate flavonoid C-glycosides, which likely contribute to their efficacy. Information regarding their phase I and II metabolism in the liver are lacking. Thus, in vitro liver metabolism of orientin, isoorientin, schaftoside, isoschaftoside, vitexin, and isovitexin, all of which accumulated in Passiflora incarnata L., was investigated by incubation in subcellular systems with human liver microsomes and human liver S9 fraction. All metabolite profiles were comprehensively characterized using HPLC-DAD and UHPLC-MS/MS analysis. Mono-glycosylic flavones of the luteolin-type orientin and isoorientin showed a broad range of mono-glucuronidated and mono-sulfated metabolites, whereas for mono-glycosylic flavones of the apigenin-type vitexin and isovitexin, only mono-glucuronidates could be detected. For di-glycosylic flavones of the apigenin-type schaftosid and isoschaftosid, no phase I or II metabolites were identified. The main metabolite of isoorientin was isolated using solid-phase extraction and prep. HPLC-DAD and identified as isoorientin-3'-O-α-glucuronide by NMR analysis. A second isolated glucuronide was assigned as isoorientin 4'-O-α-glucuronide. These findings indicate that vitexin and isovitexin are metabolized preferentially by uridine 5'-diphospho glucuronosyltransferases (UGTs) in the liver. As only orientin and isoorientin showed mono-sulfated and mono-glucuronidated metabolites, the dihydroxy group in 3',4'-position may be essential for additional sulfation by sulfotransferases (SULTs) in the liver. The diglycosylic flavones schaftoside and isoschaftoside are likely not accepted as substrates of the used liver enzymes under the chosen conditions.
| S-EPMC8587677 | biostudies-literature
Project description:1. [1-(14)C]Acetate undergoes metabolism when incubated aerobically at 37 degrees in the presence of rat brain-cortex slices, forming (14)CO(2) and (14)C-labelled amino acids (glutamate, glutamine, aspartate and relatively small quantities of gamma-aminobutyrate). In the absence of glucose the yield of (14)C-labelled aspartate exceeds that of (14)C-labelled glutamate and glutamine. The addition of glucose brings about a doubling of the rate of formation of (14)CO(2) and a greatly increased yield of (14)C-labelled glutamate or glutamine, whereas that of (14)C-labelled aspartate is diminished. 2. The addition of potassium chloride (100mm) to the incubation medium causes an increased rate of (14)CO(2) formation in the presence or absence of glucose and an increased rate of utilization of acetate. 3. The addition of 2,4-dinitrophenol (0.1mm) suppresses the rate of utilization of [1-(14)C]acetate. 4. The presence of ouabain (10mum) suppresses the rate of formation of (14)CO(2) from [1-(14)C]acetate and the rate of acetate utilization. Acetate conversion into carbon dioxide in the rat brain cortex is both Na(+)- and K(+)-dependent and controlled by operation of the active sodium-transport process. Only the Na(+)-stimulated rate is suppressed by ouabain. 5. Sodium fluoroacetate (1mm) decreases the rate of (14)CO(2) evolution from [1-(14)C]acetate in the presence of rat brain cortex without affecting the respiratory rate. The results are consistent with the conclusion that fluoroacetate competes with, or blocks, a transport carrier for acetate, so that in its presence only the passive diffusion rate of acetate takes place. 6. The presence of sodium propionate or sodium butyrate suppresses the utilization of [1-(14)C]acetate in rat brain cortex and leads to a concentration ratio (tissue/medium) of [1-(14)C]-acetate greater than unity. 7. The presence of NH(4) (+) diminishes acetate utilization, this being attributed to a diminished ATP concentration. Glycine is also inhibitory. It is concluded that acetate transport into the brain is carrier-mediated and dependent on the operation of the sodium pump.
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