Inhibition of hepatic microsomal lipid peroxidation by drug substrates without drug metabolism

Experiments were performed to study the mechanism of action of drug substrates on lipid peroxidation in rat hepatic microsomes. Addition of the drug substrates, aniline, β-diethylaminoethyl diphenylpropylacetate (SKF-525A), aminopyrine, benzo[a]pyrene or ethylmorphine, to hepatic microsomes causes almost complete inhibition of NADPH-induced (enzymatic) lipid peroxidation. These substrates also produce similar inhibition of ascorbate-induced (non-enzymatic) lipid peroxidation in microsomes in which drug-metabolizing enzymes were inactivated by heat treatment. The substrate concentrations producing half-maximal inhibition ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ are also similar for NADPH- and ascorbate-induced lipid peroxidation. Addition of metyrapone, an inhibitor of drug metabolism, has no effect on either the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values or on the maximal substrate inhibition of NADPH-induced lipid peroxidation. All five drug substrates also inhibit Fe²⁺-stimulated oxidation of linoleic acid. These results demonstrate that inhibition of lipid peroxidation in hepatic microsomes by drug substrates is independent of drug metabolism and is probably due to the antioxidant properties of the substrates.     

The effects of cyclosporin A (CsA) on hepatic microsomal drug metabolism in the rat

The effects of cyclosporin A (CsA), a powerful immunosuppressant, on the hepatic microsomal mixed function oxidase (MFO) system was studied in male rats. Difference spectroscopy studies indicated that CsA binds to cytochrome P-450 producing a type I spectral change. To investigate potential interactions with the MFO system, CsA was administered orally at doses of either 25 mg/kg or 50 mg/kg once daily for 9 days. Various metabolic parameters were examined, including: levels of microsomal protein, cytochrome P-450, and cytochrome b5, NADPH-cytochrome c reductase activity, N-demethylation of ethylmorphine (ETM), and p-hydroxylation of aniline (ANL). Rats treated with 50 mg/kg showed a 25% or greater decrease over controls in all parameters examined except microsomal protein and cytochrome b5 levels. Rats treated with 25 mg/kg showed a 28% or greater decrease in all parameters except microsomal protein, cytochrome b5, and cytochrome P-450. Kinetic studies of ETM-N-demethylase and ANL-hydroxylase activities were conducted either with microsomes prepared from CsA-treated animals (50 mg/kg/day for 5 days) or with pooled microsomes prepared from untreated animals to which CsA was added directly. Enzyme reaction velocities were measured and apparent KM and apparent Vmax were determined. Studies with CsA-treated animals revealed a 57% decrease in both KM and Vmax for ETM-N-demethylase, and a 46% decrease in KM and a 32% decrease in Vmax for ANL-hydroxylase. Studies involving direct addition of CsA to microsomes at final concentrations of 0.01 mM and 0.10 mM revealed no significant changes in apparent KM or Vmax for either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of hepatic microsomal drug metabolism by the calcium channel blockers diltiazem and verapamil

Diltiazem and verapamil were found to be inhibitors of the cytochrome P-450-dependent biotransformation of drugs. Diltiazem and verapamil competitively inhibited the N-demethylation of aminopyrine in hepatic microsomes with Ki values of 100 and 140 microM respectively. Both diltiazem and verapamil were N-demethylated themselves by hepatic microsomes with Km values of 62 and 145 microM respectively. Both drugs also interacted directly with cytochrome P-450 as measured by difference spectra. Diltiazem caused a type I spectral change and verapamil caused a reverse type I spectral change. No metabolic intermediate complexes could be demonstrated for either drug. Inhibition also occurred in vivo as both drugs could prolong pentobarbital-induced sleeping times in mice at doses comparable to those used in man. These results suggest that diltiazem and verapamil may have the potential to cause drug interactions involving inhibition of drug biotransformation.     

Inhibition of hepatic microsomal drug metabolism by the steroid hydroxylase inhibitor SU-10'603

SU-10'603 is a pyridine derivative that has been widely used as a steroid 17-hydroxylase inhibitor. Studies were done to compare the effects of SU-10'603 with those of the structurally related compound, metyrapone, on hepatic microsomal drug metabolism in vitro in rats and guinea pigs. In rat liver microsomes, SU-10'603 produced a concentration-dependent (0.01 to 1.0 mM) inhibition of ethylmorphine demethylation, aniline hydroxylation, and benzo[a]pyrene hydroxylation. A concentration of 0.1 to 0.2 mM decreased the metabolism of all three substrates by approximately 50%. SU-10'603 was a more potent inhibitor of ethylmorphine metabolism than metyrapone, and its relative potency was even greater with respect to aniline and benzo[a]pyrene metabolism. Similar results were obtained with guinea pig liver microsomes. SU-10'603 and metyrapone produced type II spectral changes in hepatic microsomes, but the apparent affinity of SU-10'603 for cytochrome(s) P-450 was greater than that of metyrapone. Both compounds inhibited the binding of type I substrates to microsomal cytochromes P-450; SU-10'603 was the more potent inhibitor. The results indicate that SU-10'603 is a potent inhibitor of hepatic microsomal monooxygenases whose mechanism of action is similar to that of metyrapone.     

Cardiovascular effects of an immunosuppressive agent cyclosporin A

Cyclosporin A (CsA) is now routinely used for transplantation of all solid organs, bone marrow transplantation, and for an increasing number of immunological diseases. However, treatment with CsA is an important iatrogenic cause of post-transplant hypertension, hyperlipidemia, and diabetes, which may contribute to the high cardiovascular morbidity in transplant recipients. Recently, the calcineurin inhibitor CsA has been employed in vivo and in vitro to examine the role of calcineurin in the signal transduction leading to cardiac hypertrophy. A cell culture study demonstrated the inhibitory effect of CsA on cytokine production by cardiac myxoma cells, the most common primary tumor of the heart. This review discusses recent data on the cardiovascular effects of CsA.     

Species-dependent hepatic metabolism of immunosuppressive agent tacrolimus (FK-506)

1. The current study aims to investigate species-related differences in the in-vitro hepatic metabolism of tacroliums using liver microsomes obtained from rat, hamster, guinea pig, rabbit, pig, dog, baboon and humans.

2. Tacrolimus metabolism was characterized using high-performance liquid chromatography- ultraviolet light (HPLC-UV) and two soft ionization mass spectrometric techniques; matrix-assisted lasers desorption/ionization (MALDI) and time-of-flight-secondary ion mass spectrometry (TOF-SIMS).

3. The extent of tacrolimus metabolism, when normalized to the cytochrome P-450 content, was in the order: rat < hamster < rabbit < pig < guinea pig < dog < human < baboon. Tacrolimus metabolism exhibited significant qualitative and quantitative differences between the animal species tested.

4. Desmethyl- (MI–MIII), didesmethyl- (MIV–MVI), monohydroxy- (MVII), dihydroxy- (MVIII), epoxide- (MIX), dihydrodiol- (MX), monodesmethyl and monohydroxy- (MXI–MXIII), and didesmethyl and monohydroxy- (MXIV–MXVI) tacroliums metabolites were identified in the species tested. MI–MX were identified in all the species tested; MXI–MXVI were identified in all species except rat, rabbit and guinea pig; and MXIV–MXVI were identified only in baboon.

5. The current investigation was unable to detect any phase II metabolites due to the limitations of the test system used.

6. The analytical methods were not able to differentiate optical and positional isomers of metabolites due to the nature of the analytical tools used, therefore groups of metabolites were identified based on their molecular weights and available information.

7. From the current in-vitro metabolism studies, the pattern of tacroliums metabolism in baboons closely resembled that in humans and thus it is ideal for studying tacroliums metabolism-related work of clinical relevance.

     

Serum cyclosporin levels, hepatic drug metabolism and renal tubulotoxicity

The present study was designed to examine inter-relationships between serum cyclosporin (CsA) levels, hepatic drug metabolising enzyme activity and CsA induced nephrotoxicity. CsA (25 mg/kg p.o.) was administered daily to male Sprague-Dawley rats: groups of animals were killed on days 0, 4, 7, 10 and 14 and thereafter at weekly intervals over the 7-week course of the experiment. Nephrotoxicity was evaluated by measuring tubular enzymuria and by light microscopy and serum CsA levels (parent drug plus certain metabolites) were determined by radioimmunoassay. The hepatic microsomal mono-oxygenase enzyme system was monitored by measurement of cytochrome P-450, aminopyrine N-demethylase and NADPH-cytochrome c reductase. Nephrotoxicity appeared within 4 days of starting treatment and continued for 4 weeks. Between weeks 4 and 6 there was a period of complete remission followed by the return of renal damage. Aminopyrine N-demethylase activity fell during the first 4 weeks. During the period of remission, however, N-demethylase activity rose to a point significantly higher than pretreatment values and serum CsA levels fell to their lowest concentration. With relapse, hepatic N-demethylase activity again fell below normal and serum drug levels rose to their pre-remission values. From the third week onward, changes in NADPH-cytochrome c reductase activity paralleled those in N-demethylase activity. The hepatic microsomal concentration of cytochrome P-450 did not, however, change significantly during the 7-week period of CsA treatment. Our results suggest that the spontaneous remission of CsA-induced nephrotoxicity is due to a reduction in circulating drug levels caused by increased hepatic CsA metabolism.     

Liver enlargement and modification of hepatic microsomal drug metabolism in rats by pyrethrum

Oral administration of pyrethrum to male rats at 200 mg/kg for 23 days resulted in liver enlargement with decreases in hepatic DNA concentrations. While total lipid concentrations were increased significantly, the increases did not account for the enlargement. Protein concentrations of whole liver homogenates, 9000 g supernatants and the 105,000 g pellet were not different from controls. No significant changes occurred in hepatic water concentrations. Significant decreases in hexobarbital-induced hypnosis without concomitant changes in barbital-induced hypnosis suggested a pyrethrum-caused alteration in hepatic drug metabolism. The activities of hepatic microsomal enzymes responsible for EPN detoxification, p-nitroanisole demethylation and hexobarbital oxidation were increased to 150, 173 and 241% of control, respectively. No significant increases were noted in the demethylation of aminopyrine or oxidation of l-tryptophan. Increases in liver weight, the detoxification of EPN and demethylation of p-nitroanisole were found to be dose-related. Small increases in enzyme activities were observed at the lowest dose used of 85 mg/kg/day for 15 days. At a daily dose of 500 mg/kg/day, liver weights and enzyme activities were increased up to 17 days of treatment but returned to control level within 7 days after cessation of treatment. NADPH cytochrome c reductase activity and P-450 concentration were also increased. At high dose levels, pyrethrum will cause induction of microsomal enzymes.     

Inhibition of hepatic microsomal drug-metabolizing enzymes by imperatorin

The effect of imperatorin on hepatic microsomal mixed function oxidases (MFO) was investigated. On acute treatment, imperatorin (30 mg/kg, i.p.) caused a significant reduction in activities of hepatic aminopyrine N-demethylase, hexobarbital hydroxylase and aniline hydroxylase as well as cytochrome p-450 content in rats and mice. Kinetic studies on rat liver enzymes revealed that imperatorin appeared to be a competitive inhibitor of aminopyrine N-demethylase (Ki, 0.007 mM), whereas a non-competitive inhibitor of hexobarbital hydroxylase (Ki, 0.0148mM). Imperatorin also inhibited non-competitively aniline metabolism (Ki, 0.2mM). Imperatorin binds to phenobarbital-induced cytochrome p-450 to give a typical type 1 binding spectrum (max. 388nm, min 422nm). Multiple administrations of imperatorin (30 mg/kg, i.p. daily for 7 days) to mice shortened markedly the duration of hexobarbital narcosis and increased activities of hepatic aminopyrine N-demethylase and hexobarbital hydroxylase and the level of cytochrome p-450 whereas aniline hydroxylase activity was unaffected.     

Inhibition of microsomal drug metabolism by mitochondria and cytochrome c oxidation of extramitochondrial NADPH

Microsomal aniline p-hydroxylase and aminopyrine N-demethylase activities were inhibited by mitochondria. The magnitude of the inhibition increased in parallel with the amount of added mitochondria. The inhibition was reverted by 0.2 mM KCN. Marked inhibition of these microsomal enzyme activities was observed also in the presence of cytochrome c and low amounts of mitochondria causing negligible inhibition in themselves. The inhibition increased with the concentration of cytochrome c and it was reverted by KCN. Microsome-free mitochondria did not oxidize NADPH even in the presence of cytochrome c, although NADH oxidation has been demonstrated under these circumstances [Sottocasa et al., J. cell Biol. 32, 415, (1967)]. However, completion of the system by addition of microsomes resulted in the oxidation of NADPH, which was inhibited by KCN. These findings may indicate the cooperation of the microsomal and mitochondrial compartments in the regulation of drug metabolism.     

Inhibition of some rat hepatic microsomal enzymes by ethoxyquin

Administration of single intragastric doses of ethoxyquin (500 mg/kg) to rats dramatically increased the hexobarbitone sleeping time. Conversely, dietary administration of ethoxyquin (0.5% w/w) for 14 days led to a 50 per cent decrease in the hexobarbitone sleeping time. Single doses of ethoxyquin had little effect on hepatic mixed function oxidase activity. Ethoxyquin was shown to be a potent competitive inhibitor of rat hepatic microsomal biphenyl 4-hydroxylase and ethylmorphine N-demethylase in vitro (K_i = 4.3 × 10^?6 M and 6.0 × 10^?6 M respectively) and displayed a type 1 binding spectrum with cytochrome P-450 with a very high affinity (K_s = 1.06 × 10^?5 M). Ethoxyquin had no inhibitory effect on the activity of glucose-6-phosphatase in vitro.