Drug Metabolism In Man Research Articles

Aloe Vera Juice: IC50 and Dual Mechanistic Inhibition of CYP3A4 and CYP2D6

The aim of this study was to evaluate the inhibitory potency (IC₅₀ values) of ethanol extracts of two commercially available aloe vera juice (AVJ) products, on CYP3A4 and CYP2D6 activities in vitro and to determine if such inhibitions could be mechanism-based. Recombinant human CYP3A4 and CYP2D6 enzymes were used and the activities were expressed by the metabolism of testosterone and dextromethorphan with ketoconazole and quinidine as positive inhibitor controls, respectively. The formed metabolites were quantified by validated HPLC techniques. Time- and NADPH- dependent inhibition assays were performed to evaluate a possible mechanism-based inhibition. One of the AVJ extracts showed about twice the inhibitory potency towards both CYP enzymes over the other with IC₅₀ values of 8.35 ± 0.72 and 12.5 ± 2.1 mg/mL for CYP3A4 and CYP2D6, respectively. The AVJ was found to exert both CYP mediated and non-CYP mediated inhibition of both CYP3A4 and CYP2D6. This dual mechanistic inhibition, however, seems to be governed by different mechanisms for CYP3A4 and CYP2D6. Estimated IC₅₀ inhibition values indicate no major interference of AVJ with drug metabolism in man, but the dual mechanistic inhibition of both enzymes might be of clinical significance.

Malnutrition and drug metabolism in man.

Journal Article Malnutrition and Drug Metabolism in Man Get access Nutrition Reviews, Volume 34, Issue 8, August 1976, Pages 237–239, https://doi.org/10.1111/j.1753-4887.1976.tb05777.x Published: 01 August 1976

A new in vitro approach for the simultaneous determination of phase I and phase II enzymatic activities of human hepatocyte preparations

Primary hepatocytes are still the best qualified in vitro system to anticipate drug metabolism in man. Recent advances in hepatocytes cryopreservation have notably increased their use not only for drug metabolism studies, but also for other applications such as cell transplantation. Evaluation of the drug-metabolizing competence of each hepatocytes preparation is needed. To date, the metabolic characterization of hepatocytes preparations relies on the assessment of phase I activities and the role of phase II enzymes receives little attention. A novel approach for the rapid assessment of the metabolic functionality of hepatocytes has been developed. A five-probe cocktail was used to simultaneously determine the enzymatic activities of major human phase I CYPs (CYP1A2, CYP2A6, CYP2C9, CYP2E1, CYP3A4), as well as two phase II enzymes: glucuronidase (UGTs) and sulfotransferase (SULT). Liquid chromatography/tandem mass spectrometry was used as the technique of choice for the determination of the enzymatic activities in a single run. Results showed that the method described herein permits a rapid assessment of the metabolic capabilities of human hepatocyte preparations as well as an estimation of the quality of freshly isolated or cryopreserved hepatocytes.

Model Systems Based on Experimental Animals for Studies on Drug Metabolism in Man: (Mini)Pig Cytochromes P450 3A29 and 2E1

Model Systems Based on Experimental Animals for Studies on Drug Metabolism in Man: (Mini)Pig Cytochromes P450 3A29 and 2E1

Human Hepatocytes in Primary Culture: The Choice to Investigate Drug Metabolism in Man

Different types of hepatic tissue, including whole or split livers from organ donors or waste liver from therapeutic liver resections, are used to prepare human hepatocyte cultures. Characteristics of liver samples from different origins (gender, age, healthy/pathological status, xenobiotic treatment) as sources of human hepatocytes are key factors which notably determine viability and functionality of hepatocytes. The characterisation of the CYP system can be assessed in terms of activity (using specific substrates/inhibitors), protein (antibody analysis) and molecular biology-based mRNA amplification techniques (PCR technology and DNA microarrays). It could reasonably be considered that human hepatocytes reflect the heterogeneity of CYP expression in human liver and is a suitable model for drug metabolism studies. Several key issues need to be addressed at the early stages of drug development to better select drug candidates (metabolic profile and rate, identification of CYPs involved, drug-drug interactions due to enzyme induction/inhibition). The metabolic stability and metabolite profile of new chemicals can be easily investigated by incubating the drugs with fully competent metabolic models like hepatocyte suspensions or 24 h-cultured hepatocytes. CYP inhibitory effects are usually screened in recombinant CYP enzymes or microsomes, however, the actual concentration of substrate and inhibitor available to the CYP enzyme depends on processes missing in subcellular models (transport mechanisms, cytosolic enzymes, binding to intracellular proteins). Since intact cells more closely reflect the environment to which drugs are exposed in the liver, cultured hepatocytes constitute a more predictive model for drug-drug interactions. Screening of CYP inducers cannot be done in microsomes as it requires a cellular system fully capable of expressing CYP genes. Primary hepatocytes are still the unique in vitro model for global examination of inductive potential of drugs (monitored as increases in mRNA content or activity).

Minipig as a model for drug metabolism in man: Comparison of in vitro and in vivo metabolism of propafenone

To prove the suitability of minipigs as experimental animal in modeling of the drug metabolism and pharmacokine-tics in man, propafenone metabolism in vitro at the microsomal level as well as propafenone pharmacokinetics in the minipig was studied. The results were compared with those obtained for humans. It can be concluded that whereas the microsomal in vitro system of minipig may be a good model for drug metabolism in the man, the pharmacokinetics in the whole organism is more complex reflecting differences in substrate specificities of many enzymatic and transport systems. In this particular case, it has been documented that the glucuronidation of propafenone principal metabolite (5-hydroxypropafenone) is more efficient in the minipig.

Drug-metabolizing activity of human and rat liver, lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo Vmax in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.

Modelling human cytochrome P450-substrate interactions.

The cytochromes P450 (CYP) are extremely important and ubiquitous enzymes, being present in virtually all species studied to date and across the five biological kingdoms where they are associated with the phase I oxidative metabolism of a large number of structurally diverse chemicals, both exogenous and endogenous (Ortiz de Montellano 1995; Ioannides 1996; Lewis 1996, 2001; Rendic and DiCarlo 1997). Of the 1,200 or more P450s sequenced thus far, it has been established that a relatively small number (~10) of the human hepatic enzymes metabolize over 90% of the known drugs in current clinical use, with the CYP2C, CYP2D and CYP3A subfamilies constituting the major catalysts of drug metabolism in man (Rendic and DiCarlo 1997).

Minipig cytochrome P450 3A, 2A and 2C enzymes have similar properties to human analogs

BackgroundThe search for an optimal experimental model in pharmacology is recently focused on (mini)pigs as they seem not only to be an alternative source of cells and tissues for xenotherapy but also an alternative species for studies on drug metabolism in man due to similarities between (mini) pig and human drug metabolizing systems. The purpose of this work is to characterize minipig liver microsomal cytochromes P450 (CYPs) by comparing their N-terminal sequences with corresponding human orthologs.ResultsThe microsomal CYPs exhibit similar activities to their human orthologous enzymes (CYP3A4, nifedipine oxidation; 2A6, coumarin 7-hydroxylation; 2D6, bufuralol 1'-hydroxylation; 2E1, p-nitrophenol hydroxylation; and 2C9, tolbutamide hydroxylation). Specific minipig CYP (2A, 2C and 3A) enzymes were partially purified and proteins identified by immunostaining (using antibodies against the respective human CYPs) were used for N-terminal amino acid sequencing. From comparisons, it can be concluded that the sequence of the first 20 amino acids at the N-terminus of minipig CYP2A is highly similar to human CYP2A6 (70% identity). The N-terminal sequence of CYP2C shared about 50% similarity with human 2C9. The results on the minipig liver microsomal CYP3A yielded identical data with those obtained for amino acid sequences of the pig CYP3A29 showing 60% identity with human CYP3A4.ConclusionsThus, our results further support the view that minipigs may serve as model animals in pharmacological/toxicological studies with substrates of human CYP enzymes, namely, of the CYP3A and CYP2A forms.

Plasma esterase activities in young and old patients undergoing open inguinal hernia repair

Previous research has shown substantial decrements in enzymes of drug metabolism (esterases) in older patients following fracture neck of femur and hip replacement surgery. The main aim of this study was to examine the effect of open inguinal hernia repair on the activities of four plasma esterases in old and young patients. Seventeen older patients (mean age±S.E.M. was 67.6±1.8) and 12 young patients (mean age±S.E.M. was 38±3.3) undergoing open hernia repair for clinical reasons were recruited. The activities of plasma benzoylcholinesterase, butyrylcholinesterase, acetylcholinesterase, and aspirin esterase were determined spectrophotometrically, before the operation, 1 day post operatively and 1 week later. There was no significant effect of hernia repair surgery on any of the four enzymes measured in young or old patients. Neither was there any significant difference in enzyme activity between young and old at any of the three time points. Hernia repair surgery is not associated with decrements in enzymes of drug metabolism in man. This contrasts with the previous findings in hip replacement surgery.

Modelling Human Cytochromes P450 for Evaluating Drug Metabolism: An Update

Cytochrome P450 (CYP) enzymes represent the major catalysts for the Phase 1 metabolism of drugs and other xenobiotics in Mammalia, including Homo sapiens. There is considerable current interest in evaluating and, consequently, predicting the metabolic fate of new chemical entities (NCEs) via modelling molecular interactions with P450 constructs, such that sites of metabolism, particular CYP involvement and binding affinities, can be estimated. This paper focuses on the principles for homology modelling of typical enzyme-substrate interactions within the putative active sites of major P450s associated with drug metabolism in man. It also represents an update on previously published work in this journal /1/.

Book Review: Advances in Drug Metabolism in Man

Book Review: Advances in Drug Metabolism in Man

Molecular basis for differences in susceptibility to toxicants: introduction

Many toxicants depend upon metabolism for their elimination and/or conversion to the toxic species. As a consequence, differences in metabolism can have a profound influence on interspecies and interindividual susceptibility to toxicity. Until relatively recently, the molecular basis for such differences were not known. However, over the past decade and a half, our understanding of the factors controlling the expression and regulation of the enzymes of drug metabolism has increased dramatically. Thus, the molecular basis of the common polymorphisms of drug metabolism in man, in CYP2D6 (debrisoquine 4-hydroxylase) and NAT2 (arylamine N-acetyltransferase) has been determined, and considerable progress has been made in elucidating the regulatory control of inducible enzymes such as CYP1A1 and CYP4A1. The number of drug metabolising enzymes subject to genetic polymorphism in man is not known, but rare genetic differences in enzymes such as epoxide hydrolase are increasingly being implicated in susceptibility to toxicants previously attributed to idiosyncrasy Indeed, the whole concept of idiosyncrasy in reactions to toxicants is being rendered obsolete by such advances. As information on the basis of differences in susceptibility accrues, the challenge is to apply this knowledge in identifying the “at risk” population. Although much progress has been made in this area, there are still many unanswered questions. What controls the tissue, and indeed cell, selective expression of some enzymes of drug metabolism? What is the molecular basis of species differences in specificity? And most importantly, what factors beyond metabolism are involved in determining susceptibility, and how do these interact with differences in metabolism?

Primary culture of cryopreserved adult human hepatocytes on homologous extracellular matrix and the influence of monocytic products on albumin synthesis

Cultured human hepatocytes provide a useful method for studies of specific metabolic functions, such as plasma protein synthesis or drug metabolism in man and specific interaction of microorganisms with the human liver cell. In vitro studies using human hepatocytes are seriously hampered by the limited availability of viable tissue and the lack of suitable methods for preservation of hepatocytes. In addition, normal adult human hepatocytes, under classical culture conditions, do not proliferate in vitro. Recently, we have reported a method for long-term storage of human hepatocytes, using a cryopreservation technique. However, after thawing, the efficiency of cell seeding onto tissue culture plastic and, accordingly, the survival of hepatocytes in primary cultures were decreased as compared to freshly prepared and cultured hepatocytes. In the present study, we report the effects of extracellular matrix (ECM) obtained from normal liver of kidney donors on the attachment efficiency, survival, some metabolic functions and fine structure of human hepatocytes. It was found that the deleterious effect of deep-freeze storage on attachment efficiency and survival of hepatocytes could be significantly reduced by using tissue culture plastic precoated with human liver ECM. Hepatocytes survived for more than 4-6 weeks, without evidence of fibroblast overgrowth. Using this in vitro experimental system, we have also shown that these hepatocytes synthesize several liver-specific acute phase proteins, and monocytic products were able to decrease the hepatocytic synthesis of albumin while total protein synthesis remained unchanged. These results support our previous observations in cultured rodent hepatocytes, indicating the important role of monocytic products in the regulation of liver synthesis of albumin in inflammatory diseases in man.

The effect of cigarette smoking on drug metabolism in the liver and placenta: the use of cotinine in verifying smoking status.

Placental and hepatic xenobiotic-metabolising activities were studied in smokers and non-smokers, who were classified by anamnestic interview, plasma thiocyanate and plasma cotinine determinations. Plasma thiocyanate assay is inadequate in the classification of smokers and non-smokers. Plasma cotinine levels reflect more accurately the smoking status. The anamnestic smokers remained smokers and several self-declared non-smokers proved to be smokers. On the basis of plasma cotinine determination all real smokers had higher 7-ethoxyresorufin 0-deethylase (ERDE) activities measured either in placental microsomes or liver biopsy homogenates than non-smokers. Classification based on plasma cotinine levels showed a statistically significant (P less than 0.001) difference between smokers and non-smokers in liver homogenate ERDE activity. However, cotinine levels did not correlate with any of the xenobiotic-metabolising activities tested. An objective biochemical marker, such as cotinine determination seems to be necessary when evaluating the effect of smoking on drug metabolism in man.

Pharmacokinetic and pharmacodynamic consequences of stereoselective drug metabolism in man

The examples discussed demonstrate the importance of stereoselective drug metabolism and raise the question of whether the therapeutic use of racemic drugs is still justified. There is no straightforward answer to this question. If only quantitative differences in therapeutic activity exist and the less active enantiomer is not predominantly responsible for side effects, the therapeutic benefit gained by using the more active enantiomer is only marginal and does not justify the substantial increase in costs involved in manufacturing such a drug preparation. However, if stereoselectivity in therapeutic activity is pronounced and adverse drug reactions are caused mainly by the less active isomer then an isomeric pure drug preparation should be used.

Metabolism of 5-fluorouracil to an N-cholyl-2-fluoro-beta-alanine conjugate: previously unrecognized role for bile acids in drug conjugation.

Recently we demonstrated clinically significant levels of a previously unrecognized metabolite of the anticancer drug 5-fluorouracil (FUra) in bile of cancer patients. In the present study, reanalysis of bile from these patients demonstrated the presence of not one but two previously unrecognized metabolites. The major unrecognized metabolite was purified by reversed-phase HPLC, after which its molecular weight was determined by fast-atom-bombardment mass spectrometry to be 497. The similarity in HPLC retention times and molecular weights of this FUra derivative and the bile acids N-cholylglycine (Mr 465) and N-cholyltaurine (Mr 515), along with the structural similarity of the FUra catabolite 2-fluoro-beta-alanine and the amino acids glycine and taurine, led to the hypothesis that this metabolite could be a conjugate of 2-fluoro-beta-alanine and cholic acid. This hypothesis was tested and confirmed by hydrolyzing the purified metabolite by cholylglycine hydrolase after which: 2-fluoro-beta-alanine was demonstrated by using a sensitive HPLC technique capable of resolving all of the known putative FUra metabolites, and unconjugated cholic acid was identified by both GC and GC-MS. Additionally, chemically synthesized N-cholyl-2-fluoro-beta-alanine was shown to cochromatograph on HPLC and TLC with the purified biliary metabolite. In summary, this study demonstrates a unique, so far as we know, pathway of drug metabolism in man in which an amino acid drug metabolite is conjugated with cholic acid and eliminated into the bile. Furthermore, the finding that 2-fluoro-beta-alanine is conjugated to bile acids may provide some insight into the mechanism of cholestasis that is frequently observed after administration of fluoropyrimidine by hepatic arterial infusion.

Caffeine consumption and vegetarian diets affect D-glucaric acid excretion in man

The urinary excretion of D-glucaric acid (DGA) has been used as a nonspecific measure of the induction of hepatic enzymes associated with drug metabolism in man. A survey of 124 nonmedicated men (18–56 years of age), who kept a 5-day food, beverage, and drug intake record and collected their total urinary output for the last three days of the five, was conducted to assess the relationship between DGA excretion and various dietary factors. Eighteen nonmedicated healthy women collected the same data, but started on the eighth day from the commencement of menstruation. DGA was determined by an enzymatic assay and recorded as umoles D-glucaro-1,4-lactone/g creatinine. A positive association was found between total DGA excreted and the use of marijuana, caffeine, and tobacco products, the heaviest users excreting higher levels of urinary DGA than the moderate or low users of the same substance (P<0.05). Analysis of variance of mean DGA excretion also revealed differences (P<0.05) between females (17.0±3.7) and males (14.3±5.2); male vegetarians (17.4±5.5) and nonvegetarians (13.9±5.1); and female vegetarians (19.8±4.6) and nonvegetarians (16.2±3.1). Ethanol consumption did not relate to increased DGA excretion in either sex. Multiple regression analysis revealed that vegetarianism and caffeine consumption were the two strongest predictors of DGA excretion accounting for 32 percent of the total variance (P<0.005), while ethanol and marijuana consumption affected DGA the least. These results indicate that dietary and environmental factors may cause important changes in DGA excretion, and these changes may identify dietary inducers of hepatic enzymes associated with drug, xenobiotic and carcinogen biotransformations in humans.

High-performance liquid chromatographic assays for bufuralol 1′-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver

Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency of oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for the in vitro metabolism of (+)- and (−)-bufuralol, debrisoquine, and dextromethorphan in human liver microsomes and reconstituted purified cytochrome P-450 isozymes. These assays combine nonextractive sample preparation by precipitation of protein with perchloric acid with reversed-phase inorganic ion-pair HPLC and fluorescence detection. The minimal detectable levels of the major metabolites formed are 1′-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 ng/ml; and dextrorphan, 0.1 ng/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 μg of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)- and (−)-bufuralol 1′-hydroxylation and dextromethorphan O-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (<1 m m) studied. These data, in combination with those obtained by purified human cytochrome P-450 isozymes indicate the involvement of the same enzyme in the metabolism of all three substrates investigated. However, additional and distinct activities contribute to the metabolism of (+)- and (−)-bufuralol and dextromethorphan.

Genetics of drug transformation

Biotransformations of drugs are controlled or strongly affected by genetic factors. During the past few years several genetic deficiencies of drug-metabolizing reactions catalyzed by members of the family of cytochrome P-450 were observed. Choice of the appropriate drug to study and attention to urinary metabolites have been the essential ingredients for the recent discovery of genetic deficiencies of drug metabolism in man which include recessive deficiency of debrisoquine/sparteine metabolism and of mephenytoin metabolism. The clinical significance of these defects is discussed. Ethanol after metabolism to acetaldehyde is further metabolized to acetic acid by aldehyde dehydrogenase. Numerous isozymes of aldehyde dehydrogenase exist, one of which possesses a high affinity for acetaldehyde. Approximately 40% of the Oriental population lack this high affinity isozyme so that in these individuals who may have symptoms of flushing and other unpleasant effects the acetaldehyde formed is destroyed only at high plasma concentrations.