No abstract

Isolated rat liver cells were found to catalyze the cytochrome P-450 dependent, oxidative metabolism of alprenolol [l-(2-allylphenoxy)-3-isopropylaminopropanol] at a rate similar to that obtained with the isolated microsomal fraction in the presence of a NADPH-generating system. Alprenolol was rapidly taken up into the liver cells and the apparent Michaelis constant was similar to that obtained with liver microsomes. Further, inhibitors of drug metabolism such as SKF 525-A and metyrapone showed similar inhibitory patterns in the cellular and microsomal systems. With liver cells isolated from control rats, NADPH generation from endogenous substrates was sufficient to support optimal alprenolol metabolism, whereas the addition of glucose or lactate stimulated this rate in liver cells isolated from starved, phenobarbital-treated rats. Inhibitors of mitochondria1 respiration such as rotenone, antimycin A and KCN and the uncoupler carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone inhibited cellular alprenolol metabolism, presumably by lowering the ATP/ADP ratio thereby interfering with NADPH generation via the malic enzyme system; this effect was more pronounced in liver cells from starved rats. Furthermore, in analogy with previous findings, the rate of gluconeogenesis from lactate was inhibited during alprenolol metabolism; this effect in turn was most pronounced in liver cells from phenobarbital-treated rats. The findings indicate that under certain conditions, the level of cytoplasmic NADPH may become rate-limiting for drug metabolism linked to cytochrome P-450 and support the previous assumption that in fed rats, generation of cytoplasmic NADPH is mainly via the pentose phosphate shunt whereas the malic enzyme system becomes more important for this purpose during starvation.

The effect of ethanol on N-demethylation of aminopyrine in rat liver slices and in the microsomal fraction and on microsomal hydroxylation of pentobarbital and aniline was studied. With liver slices N-demethylation of aminopyrine was stimulated by 35-40% at low ethanol concentrations (2mm), whereas no stimulation occurred at high concentrations (100mm). With the liver microsomal fraction, an inhibitory effect was observed only at high ethanol concentrations (100mm). This was also observed with the other drugs studied. In agreement with these results, only at a high concentration did ethanol interfere with the binding of drug substrates to cytochrome P-450. Further, as previously reported, ethanol produced a reverse type I spectral change when added to the liver microsomal fraction. Evidence that this spectral change is due to removal of substrate, endogenously bound to cytochrome P-450, is reported. A dual effect of ethanol is assumed to explain the present findings; in liver slices, at a low ethanol concentration, the enhanced rate of drug oxidation is the result of an increased NADH concentration, whereas the inhibitory effect observed with the microsomal fraction at high ethanol concentration is due to the interference by ethanol with the binding of drug substrates to cytochrome P-450.

The highly lipid soluble β‐receptor blocking drug alprenolol interacts with high affinity with rat liver microsomal cytochrome P‐450, is rapidly metabolized in the liver and exhibits a marked liver “first pass elimination” (FPE) in the rat. It thus has a low oral bioavailability in this species. In order to investigate the possible role of the cytochrome P‐450 system in the FPE we studied the influence of the three P‐450 inhibitors SKF 525‐A, imipramine and metyrapone and of phenobarbital treatment on the disposition kinetics of alprenolol in a series of experimental models. Alprenolol rapidly gave rise to a type I spectral change on addition to intact liver cells, indicating a rapid hepatic uptake. The maximal magnitude of this spectrum increased about twofold after phenobarbital treatment of rats in both microsomes and isolated liver cells. Imipramine, SKF 525‐A and metyrapone partly displaced 3H‐al‐prenolol from non‐metabolizing partly purified cytochrome P‐450 and liver cell preparations (20°). Imipramine and SKF 525‐A were about equally effective in this respect whereas metyrapone was much less potent. At 37° the metabolism of alprenolol was rapid and of about similar activity (per nmoles of cytochrome) in liver microsomes, isolated liver cells and in the perfused liver (at a high dose). The Km‐value was similar in microsomes and in isolated liver cells. A similar metabolic inhibitory pattern was found in microsomes and isolated liver cells. SKF 525‐A was the most efficient inhibitor followed by imipramine and then metyrapone. The same inhibitory pattern was found for the hepatic extraction of alprenolol. Moreover, the hepatic extraction of alprenolol was dose dependent. Imipramine in a high dose increased the area under the blood concentration curve by a factor of ten after oral administration of alprenolol in the conscious rat. The above findings suggest that cytochrome P‐450 is, at least partly, responsible for the degree of hepatic extraction and metabolism (FPE) of alprenolol. This view was also supported by the findings that the perfused liver showed an increased capacity for the extraction and metabolism of alprenolol after phenobarbital treatment. The cytochrome P‐450 system may influence the hepatic extraction of alprenolol in rat liver by providing an intracellular “high affinity binding pool” for the unchanged drug. The subsequent metabolic step seems to be important since it “unloads” P‐450 so that it can bind new drug molecules.

Metabolism of ethanol and alprenolol was studied in isolated rat liver cells. At low ethanol concentrations, where the rate of ethanol metabolism seemed to be limited by the rate of re-oxidation of NADH, ethanol metabolism was markedly stimulated by the addition of alprenolol, propranolol or hexobarbital. At higher concentrations of ethanol no stimulation by added alprenolol was observed. Alprenolol-stimulated ethanol metabolism was more sensitive to low concentrations of the ADH inhibitor 4-CHs-pyrazole, than unstimulated ethanol metabolism. Alprenolol metabolism in isolated liver microsomes and isolated liver cells was inhibited by addition of ethanol; however, at very low ethanol concentrations a slight stimulation of alprenolol metabolism was observed in isolated liver cells. The increased lactatelpyruvate ratio caused by ethanol metabolism in isolated liver cells was decreased by the addition of alprenolol. The present findings indicate that during simultaneous metabolism of ethanol and alprenolol, at low concentrations of ethanol, electrons can be diverted from the greatly reduced NADH/NAD+ couple by the microsomal monooxygenase reaction. Key-words:Ethanol metabolismdrug metabolismp-receptor blocking drugalprenololcytochrome P-450. Abbreviations: ADH Alcohol dehydrogenase (EC 1.1.1.1) NAD+ Nicotinamide adenine dinucleotide, oxidized form NADH Nicotinamide adenine dinucleotide, reduced form NADP+ Nicotinamide adenine dinucleotide phosphate, oxidized form NADPH Nicotinamide adenine dinucleotide phosphate, reduced formThe synergistic toxic interaction between ethanol and drugs has been a long established phenomenon of considerable importance in forensic medicine, both regarding drunken-driving offences (BURGER 1961; D~ENICKE &SIGMUND 1964). and lethal poisoning by combination of ethanol and hypnotic drugs