Metabolism of 13-cis-retinoic acid by a rat liver 9000g supernatant preparation

The in vitro metabolites formed on incubation of 13-cis-retinoic acid (13-cis-RA, isotretinoin) with a 9000g rat liver supernatant system were isolated by HPLC and identified by their mass and NMR spectra. The major metabolic pathway was hydroxylation at C4 to give 4-hydroxy-13-cis-RA, which was rapidly oxidized to 4-oxo-13-cis-RA, the major isolated metabolite. Further metabolism of this 4-oxo metabolite led to two novel compounds, 2-hydroxy-4-oxo-13-cis-RA and 3-hydroxy-4-oxo-13-cis-RA. In addition, small amounts of 13-cis-RA and 4-oxo-13-cis-RA were enzymatically converted to their all-trans isomers. Support for these pathways was obtained by the metabolism of reference samples of 4-hydroxy-13-cis-RA, 4-oxo-13-cis-RA, all-trans-RA, and 4-oxo-all-trans-RA. The predominant formation of 4-oxo metabolites of 13-cis-RA in this in vitro rat system and the results from previously reported in vivo metabolism studies suggest that oxidation at C4 is a major metabolic pathway of 13-cis-RA in both rats and humans.     

The pathways of propranolol metabolism in dog and rat liver 10,000g supernatant fractions

By use of an XAD-2 sample purification procedure, the quantitative patterns of the in vitro metabolism of propranolol and four of its metabolites have been examined by a GC/MS/DATA procedure. Supernatant fractions (10,000g) from both dog and rat livers have been used to degrade propranolol, 4-hydroxypropranolol, N-deisopropylpropranolol, propranolol glycol, and alpha-naphthoxylactic acid. There were both qualitative and quantitative differences in dog and rat metabolism; rat 10,000g supernatant fraction produced more hydroxylation than that of the dog. alpha-Naphthol is not produced from propranolol directly, but rather from alpha-naphthoxylactic acid with an intermediate step requiring NAD+ as a cofactor. We observed no degradation of 4-hydroxypropranolol from the 10,000g supernatant preparations. Thus, 1,4-naphthalenediol is more likely a product of 4-hydroxypropranolol glycol.     

Synthesis and identification of 3-(4-hydroxy-1-naphthoxy)lactic acid as a metabolite of propranolol in the rat, in man, and in the rat liver 9000 g supernatant fraction

The formation of 3-(4-hydroxy-1-naphthoxy)lactic acid (4-HO-NLA) from propranolol was investigated. Authentic 4-HO-NLA was synthesized from 4-methoxy-1-naphthol using methods previously used for preparation of naphthoxylactic acid (NLA). Cleavage of the 4-methyl ether was accomplished using iodotrimethylsilane in the presence of cyclopentene. After ip administration of propranolol-3,3-d2 (P-3,3-d2) (20 mg/kg) to rats, 4-HO-NLA-d2 was identified by GC-MS as a urinary metabolite. After administering pseudoracemic propranolol (S-P-d0/R-P-3,3-d2) ip to rats (20 mg/kg), parent ions of 4-HO-NLA-d0 and -d2 as methyl ester-trifluoroacetyl derivatives were monitored by GC-MS. 4-HO-NLA arose stereoselectively from S-propranolol (S/R ratio 2.6). After administering approximately equimolar quantities of 4-HO-P and P-3,3-d2 (10 mg/kg each, ip), only 4-HO-NLA-d2 arising from metabolism of P-3,3-d2 was observed by GC-MS indicating that 4-HO-P is probably not an important metabolic intermediate in vivo. In vitro experiments in the presence of the rat liver supernatant fraction performed with P-3,3-d2, NLA, and 4-HO-P showed that only NLA led to 4-HO-NLA. Incubation of NLA also produced two other hydroxylated NLA regioisomers. Incubation of 4-methoxypropanolol, a more lipophilic congener of 4-HO-P, produced no lactic acid metabolite in the presence of the rat liver supernatant fraction, indicating that poor lipophilicity is not the only deterrent to N-dealkylation of the side chain of 4-HO-P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reliability of human cryopreserved hepatocytes and liver microsomes as in vitro systems to predict metabolic clearance

A total of 110 drugs, selected to cover a range of physicochemical and pharmacokinetic properties, were used to explore standard approaches to the prediction of in vivo metabolic clearance using drug-depletion profiles from human liver microsomes (HLMs) and cyropreserved hepatocytes. A total of 41 drugs (37% of the compounds tested) showed measurable depletion rates using HLMs (depletion by 20% or more over the time course). The most reliable correlations in terms of bias (average fold error (AFE) = 2.32) and precision (root mean square error (RMSE) = 3501) were observed by comparing in vivo intrinsic clearance (CL(int)), calculated using the parallel-tube model and incorporating the fraction unbound in blood, with in vitro CL(int) adjusted for microsomal binding. For these reference drugs, 29% of predictions were within two-fold of the observed values and 66% were within five-fold. Compared with HLMs, clearance predictions with cryopreserved hepatocytes (57 drugs) were of similar precision (RMSE = 3608) but showed more bias (AFE = 5.21) with 18% of predictions within two-fold of the observed values and 46% within five-fold. However, with a broad complement of drug-metabolizing enzymes, hepatocytes catalysed measurable CL(int) values for a greater proportion (52%) of the reference compounds and were particularly proficient at defining metabolic rates for drugs with predominantly phase 2 metabolic routes.     

Reliability of human cryopreserved hepatocytes and liver microsomes as in vitro systems to predict metabolic clearance

A total of 110 drugs, selected to cover a range of physicochemical and pharmacokinetic properties, were used to explore standard approaches to the prediction of in vivo metabolic clearance using drug-depletion profiles from human liver microsomes (HLMs) and cyropreserved hepatocytes. A total of 41 drugs (37% of the compounds tested) showed measurable depletion rates using HLMs (depletion by 20% or more over the time course). The most reliable correlations in terms of bias (average fold error (AFE) = 2.32) and precision (root mean square error (RMSE) = 3501) were observed by comparing in vivo intrinsic clearance (CL_int), calculated using the parallel-tube model and incorporating the fraction unbound in blood, with in vitro CL_int adjusted for microsomal binding. For these reference drugs, 29% of predictions were within two-fold of the observed values and 66% were within five-fold. Compared with HLMs, clearance predictions with cryopreserved hepatocytes (57 drugs) were of similar precision (RMSE = 3608) but showed more bias (AFE = 5.21) with 18% of predictions within two-fold of the observed values and 46% within five-fold. However, with a broad complement of drug-metabolizing enzymes, hepatocytes catalysed measurable CL_int values for a greater proportion (52%) of the reference compounds and were particularly proficient at defining metabolic rates for drugs with predominantly phase 2 metabolic routes.     

Isolated rat hepatocytes as a model system for screening chelators for use in cadmium intoxication

The utility of isolated rat hepatocytes as a model system for screening potential chelators in treatment of Cd intoxication was studied. The ability of the chelators diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 2,3-dimercaptosuccinic acid (DMSA), diethyldithiocarbamic acid (DDC), d,1-penicillamine (PEN), nitrilotriacetic acid (NTA), and 2,3-dimercaptopropanol (BAL) to decrease the cellular concentration of Cd was correlated with the previously reported effectiveness of these agents in the treatment of Cd intoxication in vivo. The results of cellular studies with either control or metallothionein-induced hepatocytes were compared to the in vivo effect of the chelators administered before or after the induction of metallothionein, respectively. The effects of DTPA, EDTA, DDC, and BAL in the hepatocyte model screening system correlated well with their reported in vivo effects. The results with NTA, PEN, and DMSA were not correlated as well but were explained by the relative differences between in vivo doses versus in vitro concentrations of the respective chelators. Therefore, the proposed model for screening potential chelators for use in cadmium intoxication appears to be a system which may prove to be an economical and rapid method to facilitate the search for efficacious chelators.     

Isolated perfused liver technology for studying metabolic and toxicological problems

The isolated perfused liver system is a versatile model for investigating the effects and mechanisms of action of hepatotoxins and the metabolism of endogenous and exogenous compounds. The interpretation of metabolic data and apparent toxic events is dependent upon the viability and reproducibility of the model. In this study, a new approach has been undertaken to assess the viability of isolated liver preparations. This has involved the continuous monitoring of multistage processes namely, the synthesis and secretion of radiolabelled proteins, glycoproteins and lipoproteins on the one hand and the uptake of macromolecules by receptor-mediated endocytosis on the other. The consistency of these complex integrated processes from one liver to another and in particular the steady-state rate of production of radiolabelled macromolecules over 6h perfusion periods suggests that this model can be used with confidence for metabolic/toxicological investigations. The selectivity of the responses to chemical challenge(s) shows that this system can be exploited for (a) screening potential hepatotoxins; (b) identifying areas of metabolism which are affected and (c) advancing basic knowledge of liver biochemistry.     

N-Alkyl barbiturates. A series of compounds for the study of metabolic structure-activity relationships

Many therapeutic agents are metabolised along multiple pathways, but up to now there have been few investigations addressing the question of which chemical features of drugs govern the participation in, and quantitative significance of, different biotransformation pathways. To assess the influence of variations of the chemical structure upon metabolism, a series of novel barbiturate analogues has been synthesized. The N1-monoalkylated and N1,N2-dialkylated phenobarbitones and 2-desoxyphenobarbitones have been synthesized via condensation of ethylphenylmalonic acid derivatives with different N-alkylated ureas or thioureas, and/or by base-catalyzed N-alkylation of different barbituric acids.     

普罗帕酮和利多卡因在大白鼠肝匀浆中合用时的代谢

验证Pro和Lid结构中N原子上的烷基受类似同功酶催化。采用肝匀浆上清液孵育法,观察到Pro和Lid合用后,原药浓度随其代谢的相互制约,可分别提高0.7和0.5倍,DGX生成减少2/3。提示:1)两药均由细胞色素P_(450)ⅢA_4同功酶催化,并呈饱和现象。2)由于DGX生成减少,可降低由此引起的毒性。3)两药合用有益于抗心律失常的治疗。     

The metabolism of thioamides by the supernatant fraction of rat liver homogenate

α-Phenyl-α-(2-pyridyl) thioacetamide (SC 15396; antigastrin) is metabolized by the supernatant fraction of rat liver homogenate to give two metabolites. The major metabolite has been identified as α-phenyl-α-(2-pyridyl)acetonitrile. Three other thioamides, analogues of SC 15396 also give the corresponding nitriles in these conditions.     

In vitro metabolic study of EGYT-3615 using rat liver microsomes

In vitro metabolism of EGYT-3615, a prospective new antidepressant was studied by using rat liver microsomes. The metabolite (M) found in earlier in vivo studies was unanimously found to be the product of a microsomal enzymatic process. The Michaelis-Menten constant of the reaction was calculated.     

Oxidation of barbiturates and the glucuronidation of 1-napthol in perfused rat liver and in microsomes

Summary An attempt has been made to relate quantitatively drug metabolism in perfused liver with that in microsomes. Therefore in both systems microsomal monooxygenase (hexobarbital as substrate) and UDP-glucuronyltransferase (1-naphthol as substrate) have been studied.The rate of hexobarbital oxidation in perfused liver was slightly higher than in microsomes incubated with an NADPH regenerating system, indicating that the generation of reducing equivalents is not rate-limiting in livers of normal rats. The rate of phenobarbital oxidation was only about 3% of the rate of hexobarbital oxidation in perfused liver.Microsomal UDP-glucuronyltransferase can be activated about 10-fold in vitro. The formation of naphthol glucuronide in perfused liver (determined from the appearance of glucuronide in liver tissue, perfusate and bile) corresponded well with UDP-glucuronyltransferase activity in non-activated microsomes when the intracellular UDP-glucuronate level was taken into account. This suggests that UDP-glucuronyltransferase is mostly latent within the cell.     

Hepatic pharmacokinetics of taurocholate in the normal and cholestatic rat liver

The disposition kinetics of [3H]taurocholate ([3H]TC) in perfused normal and cholestatic rat livers were studied using the multiple indicator dilution technique and several physiologically based pharmacokinetic models. The serum biochemistry levels, the outflow profiles and biliary recovery of [3H]TC were measured in three experimental groups: (i) control; (ii) 17 alpha-ethynylestradiol (EE)-treated (low dose); and (iii) EE-treated (high dose) rats. EE treatment caused cholestasis in a dose-dependent manner. A hepatobiliary TC transport model, which recognizes capillary mixing, active cellular uptake, and active efflux into bile and plasma described the disposition of [3H]TC in the normal and cholestatic livers better than the other pharmacokinetic models. An estimated five- and 18-fold decrease in biliary elimination rate constant, 1.7- and 2.7-fold increase in hepatocyte to plasma efflux rate constant, and 1.8- and 2.8-fold decrease in [3H]TC biliary recovery ratio was found in moderate and severe cholestasis, respectively, relative to normal. There were good correlations between the predicted and observed pharmacokinetic parameters of [3H]TC based on liver pathophysiology (e.g. serum bilirubin level and biliary excretion of [3H]TC). In conclusion, these results show that altered hepatic TC pharmacokinetics in cholestatic rat livers can be correlated with the relevant changes in liver pathophysiology in cholestasis.     

The metabolic effects of diuron in the rat liver

A systematic study on the effects of diuron on the hepatic metabolism was conducted with emphasis on parameters linked to energy metabolism. The experimental system was the isolated perfused rat liver. The results demonstrate that diuron inhibited biosynthesis (gluconeogenesis) and ammonia detoxification, which are dependent of ATP generated within the mitochondria. Conversely, it stimulated glycolysis and fructolysis, which are compensatory phenomena for an inhibited mitochondrial ATP generation. Furthermore, diuron diminished the cellular ATP content under conditions where the mitochondrial respiratory chain was the only source of this compound. Besides the lack of circulating glucose due to gluconeogenesis inhibition, one can expect metabolic acidosis due to excess lactate production, impairment of ammonia detoxification and cell damage due to a deficient maintenance of its homeostasis. Some of the general signs of toxicity that were observed in diuron-treated rats can be attributed, partly at least, to the effects of the herbicide on energy metabolism.     

Genetic control of responsiveness of rat liver supernatant aldehyde dehydrogenase to phenobarbital and 3-methylcholanthrene

The responsiveness of the hepatic supernatant NAD⁺-dependent aldehyde dehydrogenase with a high Km value (high Km-AldDH) to phenobarbital (PB) and 3-methylcholanthrene (3-MC) treatment was studied in male rats of three strains; Wistar, Long-Evans, and Sprague-Dawley.A remarkable strain difference in the response of the enzyme to PB or 3-MC was observed. In rats of the Wistar strain the enzyme activity remained unchanged (non-responsive) in all rats after treatment with PB while it increased (responsive) 5- to 19-fold in all rats after treatment with 3-MC. The enzyme activity increased 8- to 20-fold and 2- to 8-fold respectively after treatment with PB and 3-MC in all rats of the Long-Evans strain. In rats of the Sprague-Dawley strain the enzyme activity remained unchanged in half of all the rats treated with PB or 3-MC and increased 2- to 7-fold over the basal level in half of the treated rats. The non-responsive rats to PB were all responsive to 3-MC treatment while the responsive rats to PB were responsive in 65% and non-responsive in 35% to 3-MC treatment.     

Kinetic studies on the deethylation of ethoxybenzamide: A comparative study with isolated hepatocytes and liver microsomes of rat

Kinetic parameters (K_m and V_max) of ethoxybenzamide deethylation in isolated rat hepatocytes and liver microsomes were compared. Adjustment of cofactors in microsomal deethylation, such as NADPH and Mg²⁺, to give optimum conditions, and appropriate correction of the apparent kinetic parameters for nonspecific binding and microsomal yield resulted in good agreement among the kinetic parameters of isolated hepatocytes [$\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 0.0863 μmole · min}{}^{\mathrm{?1}}\text{· (g liver)}{}^{\mathrm{?1}}$ and K_m = 0.459 mM] and microsomes [V_max = 0.124 μmoles · min^?1 · (gliver)^?1 and K_m = 0.378 mM].     

Effect of spin traps in isolated rat hepatocytes and liver microsomes

Spin traps are increasingly employed in the detection of free radicals in biological systems, including liver microsomes and isolated hepatocytes. Two spin traps phenyl-t-butyl nitrone (PBN) and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) have been tested for their effects on hepatocyte viability and mixed-function oxidase activity. High concentration of PBN but not of 4-POBN proved to moderately affect liver cell integrity, without interfering with intracellular ATP or cytochrome P-450 content. PBN also decreased hepatocyte GSH content, probably as the result of its metabolism to benzaldehyde. The two spin traps were found to inhibit aminopyrine demethylase and ethoxycoumarin deethylase activity in hepatocytes and microsomes. At low concentrations (1-5 mM) PBN enhanced aniline hydroxylase while high concentrations of the spin trap inhibited this activity. The inhibition of the monooxygenase system was not caused by damage of microsomal enzymes, but rather by competition with other substrates for the binding to the haemoprotein. The effects of spin traps on mixed function oxidase systems should be taken into account when evaluating the results of spin trapping experiments.