Defining the in Vivo Role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50-90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.     

Inhibitory effect of propylthiouracil-induced hypothyroidism in rat on oxidative drug metabolism

The effect of propylthiouracil (PTU) pretreatment on in vivo and in vitro oxidative drug metabolism was determined in the rat. Whereas pentobarbital sleeping time (PBST) and zoxazolamine paralysis time (ZZPT) were used as indices of in vivo drug metabolizing activity, biotransformation of aminopyrine and aniline by hepatic microsomal preparations were used as indices of in vitro drug metabolizing enzymes activities. PTU pretreatment significantly prolonged both PBST and ZZPT. Whereas PTU did not affect microsomal protein concentration or cytochrome P-450 content, it significantly decreased microsomal cytochrome c reductase and aniline hydroxylase activities. These changes in enzymatic activities were observed in microsomal preparations from either non-fasted or 24-hr fasted rats. Our results suggest that PTU-induced hypothyroidism modifies the metabolism and effectiveness or toxicity of concomitantly administered drugs.     

The microsomal ethanol oxidizing system: its role in ethanol and xenobiotic metabolism

After chronic ethanol consumption, the activity of the microsomal ethanol-oxidizing system (MEOS) increases and contributes to ethanol tolerance, as most conclusively shown in alcohol-dehydrogenase-negative deermice. In man and animals, there is an associated rise in microsomal cytochrome P-450, including a specific form (P-450IIEI) with high affinity for ethanol and for the activation of some drugs (i.e. acetaminophen), carcinogens (i.e. N-nitrosodimethylamine) and hepatotoxic agents (i.e. CCl4), thereby contributing to the susceptibility of alcoholics to xenobiotics, including industrial solvents. In addition, a benzoflavone-inducible liver cytochrome P-450 isoenzyme distinct but catalytically similar to cytochrome P-450IIE1 was purified which may play a significant role in drinkers who also are heavy smokers. Cross-induction of other microsomal enzymes is associated with enhanced metabolism of various drugs, resulting in drug tolerance. Catabolism of retinol was also found to be accelerated, in part through activation of newly discovered vitamin A depletion and possibly toxicity. Thus, elucidation of the microsomal metabolism of ethanol explains a number of complications that develop in alcoholics.     

Intestinal absorption and metabolism of chlorpheniramine enantiomers in rat

Chlorpheniramine (CPAM) is a chiral antihistaminic drug commercialized as a racemic mixture. The intestinal absorption and metabolism of CPAM have been investigated in rat using in vivo (oral and IV administration), in situ (intestinal loop model), and in vitro (everted sac model) experiments. Oral and IV administrations of 20 mg/kg of the racemic mixture show that the pharmacokinetics of CPAM are stereoselective, with higher AUCs for the (+)-S-enantiomer compared to its antipode. The monodesmethyl metabolite (DCPM) was quantifiable in blood and its pharmacokinetics are stereoselective after oral but not after IV administration. Experiments using intestinal loops and everted sacs showed that the absorption is not stereoselective and that in vivo stereoselective formation of DCPM is presumably due to stereoselective hepatic metabolism. Moreover, the in vitro and in situ absorption of CPAM are not modified by modulators of P-glycoprotein and cytochromes P450 (cyclosporin A, ketoconazole).     

Human hepatic cell cultures: in vitro and in vivo drug metabolism

Drug metabolism is the major determinant of drug clearance, and the factor most frequently responsible for inter-individual differences in drug pharmacokinetics. The expression of drug metabolising enzymes shows significant interspecies differences, and variability among human individuals (polymorphic or inducible enzymes) makes the accurate prediction of the metabolism of a new compound in humans difficult. Several key issues need to be addressed at the early stages of drug development to improve drug candidate selection: a) how fast the compound will be metabolised; b) what metabolites will be formed (metabolic profile); c) which enzymes are involved and to what extent; and d) whether drug metabolism will be affected directly (drug-drug interactions) or indirectly (enzyme induction) by the administered compound. Drug metabolism studies are routinely performed in laboratory animals, but they are not sufficiently accurate to predict the metabolic profiles of drugs in humans. Many of these issues can now be addressed by the use of relevant human in vitro models, which speed up the selection of new candidate drugs. Human hepatocytes are the closest in vitro model to the human liver, and they are the only model which can produce a metabolic profile of a drug which is very similar to that found in vivo. However, the use of human hepatocytes is restricted, because limited access to suitable tissue samples prevents their use in high throughput screening systems. The pharmaceutical industry has made great efforts to develop fast and reliable in vitro models to overcome these drawbacks. Comparative studies on liver microsomes and cells from animal species, including humans, are very useful for demonstrating species differences in the metabolic profile of given drug candidates, and are of great value in the judicious and justifiable selection of animal species for later pharmacokinetic and toxicological studies. Cytochrome P450 (CYP)-engineered cells (or microsomes from CYP-engineered cells, for example, Supersomes) have made the identification of the CYPs involved in the metabolism of a drug candidate more straightforward and much easier. However, the screening of compounds acting as potential CYP inducers can only be conducted in cellular systems fully capable of transcribing and translating CYP genes.     

Failure of Corynebacterium parvum to inhibit antipyrine metabolism in man

Summary In animals, Corynebacterium parvum lowers the rate of drug metabolism and enhances the pharmacologic effect of drugs requiring hepatic microsomal enzyme activity for elimination. A pilot study was conducted to assess this drug interaction in patients given clinical protocol doses of C. parvum. In individual patients, C. parvum did not reduce microsomal drug metabolism as measured by antipyrine half-life. Conversely, antipyrine elimination appeared to be enhanced in 10 of 14 patients. Results from this small heterogenous patient group are not definitive, and further studies are needed to determine the clinical significance of the effects of nonspecific immunotherapy on drug metabolism.     

Hepatocytes--the choice to investigate drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. However, only a minor part of phenotypic variability in man is attributable to gene polymorphisms, thus making the definition of a normal liver complex. At present, the use of human in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Cultured human hepatocytes are considered to be the closest model to human liver. However, the fact that hepatocytes are located in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism variability observed in vitro actually reflect that of the liver in vivo? By comparing the metabolism of a model compound both in vitro and in vivo in the same individual, a good correlation between the in vitro and in vivo relative abundance of oxidized metabolites and the hydrolysis of the compound was observed. Thus, it is reasonable to consider that the variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of the P450 expression in human liver.     

Environmental factors in drug metabolism.

Although various kinds of environmental factors may alter the activity of cytochrome P-450 enzymes in liver micromes, their effects on the pharmacokinetics of drugs and other foreign compounds in living animals may not be as great as might be predicted from assays of these enzymes in vitro. Indeed, the effects will depend on the relative importance of excretory and metabolic mechanisms in the elimination of the drug, the relative importance of various metabolic reactions in different tissues, the extraction ratio of the drug by the liver, and in some instances on the route of administration of the drug. Moreover, the effect of the various environmental factors on the pharmacologic and the toxicologic actions of the drug will depend on whether these actions are caused by the parent foreign compounds or by one or more of their metabolites. It may also be important that the environmental factors may alter not only relative activiteis of the cytochrome P-450 in liver microsomes but also the activities of other drug-metabolizing enzymes and that the relative effects of the environmental factors of these enzymes may differ depending on the animal species or the animal strain. Indeed, a given factor may increase the pharmacologic effects of a drug metabolite in one animal species but decrease it in another. For these reasons, it frequently is not possible to predict the effects of environmental factors on drug action in living animals solely from in vitro rates of metabolism of model substrates.     

Species differences in metabolism of ketotifen in rat, rabbit and man: demonstration of similar pathways in vivo and in cultured hepatocytes

In vitro drug metabolism by cultured rat, rabbit and human adult hepatocytes has been studied, using ketotifen (ZADITEN) as a model substrate because it is biotransformed in vivo by various metabolic pathways in man and animals. The major in vivo pathways were demonstrated in vitro, namely oxidation in rat hepatocytes, oxidation, glucuronidation and sulfation in rabbit hepatocytes, reduction and glucuronidation in human hepatocytes. Human hepatocytes were the most stable in culture, displaying ketotifen biotransformation for at least one week. These results clearly demonstrated that cultured hepatocytes retain their in vivo specific drug metabolizing activities, including inter-species polymorphism, for a few days. Therefore, pure hepatocyte cultures represent a useful system suitable for drug metabolism studies.     

Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.     

Evaluation of surfactants as solubilizing agents in microsomal metabolism reactions with lipophilic substrates

Solubilizing agents are routinely added when investigating the biotransformation of lipophilic substrates using hepatic microsomes. For highly lipophilic compounds, the concentration of solvent or surfactant necessary for dissolution can be detrimental to enzyme activity. This study evaluates the effect of 12 surfactants on microsomal metabolism and the ability of the same surfactants to improve the aqueous solubility of the pentabrominated diphenyl ether BDE-100, a lipophilic environmental contaminant previously found to be recalcitrant to in vitro metabolism. Of the surfactants investigated, Cremophor EL and Tween 80 displayed the best combination of increased BDE-100 solubility and minimal inhibition of microsomal metabolism. However, a comparison of the in vitro metabolism products of BDE-100 in the presence of the two surfactants revealed varying amounts of metabolites depending on the surfactant used.     

Toxicity and in vitro metabolism of t-permethrin in eastern subterranean termite (Isoptera: Rhinotermitidae)

Toxicity and metabolism of t-permethrin were evaluated in two colonies (UF and ARS) of the eastern subterranean termite, Reticulitermes flavipes (Kollar), collected in Gainesville, FL. The UF colony (LC50 = 1.86 micrograms per vial) was approximately twofold more tolerant of t-permethrin than the ARS colony (LC50 = 0.89 microgram per vial) at the LC50. The synergists piperonyl butoxide and S,S,S-tributylphosphorotrithioate increased t-permethrin toxicity four- and threefold (at the LC50) in the UF and ARS colonies, respectively. Despite these differences in t-permethrin susceptibility, microsomal oxidase activities toward surrogate substrate (aldrin epoxidase, and methoxyresorufin O-demethylase), cytochrome P450 content, and microsomal esterase activity toward alpha-naphthyl acetate did not differ significantly between the colonies. Moreover, no significant differences in qualitative and quantitative metabolism of [14C]t-permethrin were observed between the UF and ARS colonies for three enzyme sources (microsomal oxidase, microsomal esterase, and cytosolic esterase). Based on in vitro metabolism assays, the major detoxification route of t-permethrin in the UF and ARS termite colonies appears to be hydrolysis catalyzed by microsomal esterases.     

Cytochromes P450 and experimental models of drug metabolism

For the development of new drugs, evaluation of drug-drug interactions with already known compounds, as well as for better understanding of metabolism pathways of various toxicants and pollutants, we studied the drug metabolism mediated by cytochromes P450. The experimental approach is based on animal drug-metabolising systems. From the ethical as well as rational reasons, the selection of an appropriate system is crucial. Here, it is necessary to decide on the basis of expected CYP system involved. For CYP1A-mediated pathways, all the commonly used experimental models are appropriate except probably the dog. On the contrary, the dog seems to be suitable for modelling of processes depending on the CYP2D. With CYP2C, which is possibly the most large and complicated subfamily, the systems based on monkey ( Maccacus rhesus ) may be a good representative. The CYP3A seems to be well modelled by pig or minipig CYP3A29. Detailed studies on activities with individual isolated CYP forms are needed to understand in full all aspects of inter-species differences and variations.     

The stereoselective metabolism of halofantrine to desbutylhalofantrine in the rat: evidence of tissue-specific enantioselectivity in microsomal metabolism

The pharmacokinetics of the antimalarial drug (+/-)-halofantrine are stereoselective in humans and rats. To better understand the stereoselective metabolism of the drug to its primary metabolite, desbutylhalofantrine (DHF), a series of in vitro and in vivo experiments were undertaken in the rat. Formation of (-)-DHF exceeded that of (+)-DHF in liver microsomes [(-):(+) ratio of intrinsic formation clearances = 1.4]. In contrast, in intestinal microsomes no significant stereoselectivity was noted in the formation of the DHF enantiomers. Intestinal microsomes were also less efficient at producing the DHF enantiomers than were liver microsomes. Based on kinetic analysis of the DHF formation, there appeared to be more than one enzyme involved in the biotransformation. (+/-)-Ketoconazole (KTZ) effectively inhibited the formation of both DHF enantiomers by both liver and intestinal microsomes, although the reduction was more marked in liver microsomes. Through a combination of the use of CYP antibodies and recombinant CYP isoenzymes, the involvement of CYP 2B1/2, 3A1, 3A2, 1A1, 2C11, 2C6, 2D1, and 2D2 were implicated in the metabolism of halofantrine to DHF. Of these, CYP3A1/2 and CYP2C11 appeared to be the primary isoenzymes involved, although CYP2C11 showed greater (+)-DHF than (-)-DHF formation, whereas for CYP3A1 it was similar to the isolated rat liver microsomes. In vivo, oral (+/-)-KTZ caused significant increases in plasma halofantrine and decreases in DHF enantiomer plasma concentrations.     

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.Key words: mAb IgG, neonatal Fc receptor (FcRn), pharmacokinetics, subcutaneous bioavailability, animal model, minipig     

The metabolism of benzimidazole anthelmintics

The benzimidazole carbamates are important broad-spectrum drugs for the control of helminth parasites in mammals. David Gottschall, Vassilios Theodorides and Richard Wang explain that the metabolism of these compounds depends heavily on the substituent present on carbon-5 of the benzimidazole nucleus and involves a wide variety of reactions. Work in vitro has shown that two major enzyme systems, the cytochrome P-450 family and the microsomal flavin monooxygenases are primarily responsible for these biotransformations. The parent compound is generally short-lived and its metabolites predominate in the tissues and excreta of treated animals. The metabolic pathways can be exploited therapeutically to overcome the problems of poor water solubility and adsorbtion of benzimidazoles by the development and use of more soluble prodrugs.     

Drug metabolism in Octodon degus: low inductive effect of phenobarbital

1. Differential effects of phenobarbital pre-treatment on liver microsomal drug metabolizing enzymes were registered in Octodon degus. 2. Glucuronidation reaction for morphine was decreased but that for p-nitrophenol was significantly increased. 3. Oxidative reactions such as naphthalene hydroxylation, morphine and aminopyrine N-demethylation were modestly increased. 4. In phenobarbital treated Octodon degus, testosterone metabolic pathways were decreased, not inducible or absent. 5. Spectral studies revealed two binding sites with different affinities for aniline in Octodon degus liver microsomes. 6. The poor phenobarbital induction on drug metabolism in Octodon degus may be a result of deficiency of androgen metabolic pathways associated to drug metabolizing enzymes.     

Some high-performance liquid-chromatographic studies of the metabolism of aflatoxins by rat liver microsomal preparations.

The metabolism of aflatoxin B1 in vitro was examined in rat liver microsomal preparations. 2. H.p.l.c. (high-performance liquid-chromatographic) systems were used. A silica column was used to separate non-polar metabolites. A system utilizing a reversed-phase column which separates both poar and non-polar metabolites was also developed. 3. The principal metabolites of aflatoxin B1 found were aflatoxin M1, aflatoxin Q1 and a compound which co-chromatographed with a degradation product of aflatoxin B1 2,3-dihydrodiol. 4. The time course of metabolism of aflatoxin B1 by microsomal preparations isolated from control and phenobarbitone-pretreated rats was examined. The rate and extent of metabolism was greater with microsomal preparations from the latter. The formation of aflatoxin Q1 was enhanced 4--5-fold by phenobarbitone pretreatment, whereas the production of aflatoxin M1 was only increased 1--2-fold. The formation of the degradation product of aflatoxin B1 2,3-dihydrodiol was increased 4--5-fold by the pretreatment with phenobarbitone. 5. The microsomal metabolism of aflatoxins M1, P1 and Q1 was examined. Aflatoxin M1 apparently underwent very limited microsomal metabolism to more polar compounds. Aflatoxin P1 was not metabolized. The situation with aflatoxin Q1 was complicated in that it was metabolized in the absence of NADPH to an unidentified metabolite. Aflatoxin B1 appeared as a metabolite of aflatoxin Q1 only when NADPH was present, and the formation of more polar metabolites was also then observed.     

Cimetidine and ranitidine: comparison of effects on hepatic drug metabolism.

Paired studies of hepatic microsomal function were conducted in eight subjects during treatment with two histamine H2 antagonists, cimetidine and ranitidine. Cimetidine but not ranitidine inhibited the metabolism of antipyrine (phenazone) and demethylation of aminopyrine (aminophenazone) as measured by breath 14CO2 production after intravenous injection of 14C-aminopyrine. These results suggest that the metabolic inhibitory actions on the liver may be separated from H2 antagonist effects, and that ranitidine has an advantage over cimetidine by not inhibiting microsomal drug oxidative function.     

Triacylglycerol metabolism in the phenobarbital-treated rat

1. Various aspects of triacylglycerol metabolism were compared in rats given phenobarbital at a dose of 100mg/kg body wt. per day by intraperitoneal injection; controls were injected with an equal volume of 0.15m-NaCl by the same route. Animals were killed after 5 days of treatment. 2. Rats injected with phenobarbital demonstrated increased liver weight, and increased microsomal protein per g of liver. Other evidence of microsomal enzyme induction was provided by increased activity of aminopyrine N-demethylase and cytochrome P-450 content. Increased hepatic activity of γ-glutamyltransferase (EC 2.3.2.2) occurred in male rats, but not in females, and was not accompanied by any detectable change in the activity of this enzyme in serum. 3. Phenobarbital treatment increased the hepatic content of triacylglycerol after 5 days in starved male and female rats, as well as in non-starved male rats; non-starved females were not tested in this regard. At 5 days after withdrawal of the drug, there was no difference in hepatic triacylglycerol content or in hepatic functions of microsomal enzyme induction between the treated and control rats. 4. After 5 days, phenobarbital increased the synthesis in vitro of glycerolipids in cell-free liver fractions fortified with optimal concentrations of substrates and co-substrates when results were expressed per whole liver. The drug caused a significant increment in the activity of hepatic diacylglycerol acyltransferase (EC 2.3.1.20), but did not affect the activity per liver of phosphatidate phosphohydrolase (EC 3.1.3.4) in cytosolic or washed microsomal fractions. A remarkable sex-dependent difference was observed for this latter enzyme. In female rats, the activity of the microsomal enzyme per liver was 10-fold greater than that of the cytosolic enzyme, whereas in males, the activities of phosphohydrolases per liver from both subcellular fractions were similar. 5. The phenobarbital-mediated increase in hepatic triacylglycerol content could not be explained by a decrease in the hepatic triacylglycerol secretion rate as measured by the Triton WR1339 technique. Since the hepatic triacylglycerol showed significant correlation with microsomal enzyme induction functions, with hepatic glycerolipid synthesis in vitro and with diacylglycerol acyltransferase activity, it is likely to be due to enhanced triacylglycerol synthesis consequent on hepatic microsomal enzyme induction. 6. In contrast with rabbits and guinea pigs, rats injected with phenobarbital showed a decrease in serum triacylglycerol concentration in the starved state; this decrease persisted for up to 5 days after drug administration stopped, and did not occur in non-starved animals. It seems to be independent of the microsomal enzyme-inducing properties of the drug, and may be due to the action of phenobarbital at an extrahepatic site.