The impact of cholesterol and its metabolites on drug metabolism

INTRODUCTION: Global prevalence of Western-type diet has increased in the last decades resulting in occurrence of certain chronic diseases. This type of diet is also linked to high-cholesterol intake and increase in blood cholesterol. Many of the molecular mechanisms of dealing with increased levels of cholesterol and its metabolites have been elucidated in animal models and humans. It is also evident that cholesterol metabolism is closely connected to drug metabolism. Cholesterol/bile acids and drugs share many transporters, enzymes and regulatory proteins which are key points in the crosstalk. AREAS COVERED: This review presents an overview of the effect of cholesterol and its metabolites on drug metabolism with special emphasis on species-specific differences. The article focuses on the role of nuclear receptors farnesoid X receptor, vitamin D receptor and liver X receptor in the regulation of drug metabolism genes and the role of cholesterol biosynthesis intermediates, oxysterols and bile acids in the induction of drug metabolism through pregnane X receptor. EXPERT OPINION: Studies show that the regulation of drug metabolism by sterols is multileveled. Many species-dependent differences were observed which hinder the transfer of findings from model animals to humans. As of now, there is little evidence available for cholesterol impact on drug metabolism in vivo in humans. There is also the need to confirm the results obtained in animal models and in vitro analyses in human cells but this is very difficult given the current lack of tools.     

Liver function assessment by drug metabolism

Liver function can be assessed by administering an exogenous substance to quantify changes in hepatic blood flow, uptake, biotransformation, and excretion. Characterization of drug half-life, clearance, and product formation rates are possible methods for measuring hepatic efficiency. Allopurinol and caffeine have been used to measure metabolite formation followed by renal elimination of both parent substance and metabolite. Sorbitol, a substance with high intrinsic clearance, can reflect liver blood flow, while trimethadione, a low-extraction drug, has been used to measure liver enzyme capacity. Metabolites from lidocaine, methacetin, and aminopyrine have been measured in serum, urine, and breath tests. Salivary clearance measurements of caffeine and antipyrine are reported as suitable for routine use. Genetic diversity of isoenzymes and the many metabolic processes used by hepatocytes make it extremely difficult to quantify functional changes with one substance. Combinations of model substrates have been suggested to assess the many hepatic processes.     

Therapeutic drug monitoring of racemic venlafaxine and its main metabolites in an everyday clinical setting

When Efexor (venlafaxine) became available in Sweden, a therapeutic drug monitoring (TDM) service was developed in the authors' laboratory. This analytical service was available to all physicians in the country. From March 1996, to November 1997, 797 serum concentration analyses of venlafaxine (VEN) and its main metabolites, O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV), and N,O-didesmethylvenlafaxine (DDV) were requested. These samples, each of which was accompanied by clinical information on a specially designed request form, represented 635 inpatients or outpatients, comprising all ages, treated in a naturalistic setting. The first sample per patient, drawn as a trough value in steady state and with documented concomitant medication, was further evaluated pharmacokinetically (n = 187). The doses prescribed were from 37.5 mg/d to 412.5 mg/d. There was a wide interindividual variability of serum concentrations on each dose level, and the mean coefficient of variation of the dose-corrected concentrations (C/D) was 166% for C/D VEN, 60% for C/D ODV, 151% for C/D NDV, and 59% for C/D DDV. The corresponding CV for the ratio ODV/VEN was 110%. However, within patients over time, the C/D VEN and ODV/VEN variation was low, indicating stability in individual metabolizing capacity. Patients over 65 years of age had significantly higher concentrations of C/D VEN and C/D ODV than the younger patients. Women had higher C/D NDV and C/D DDV, and a higher NDV/VEN ratio than men, and smokers showed lower C/D ODV and C/D DDV than nonsmokers. A number of polycombinations of drugs were assessed for interaction screening, and a trend for lowered ODV/VEN ratio was found, predominantly with concomitant medication with CNS-active drug(s) known to inhibit CYP2D6.     

Inhibition of drug metabolism by hydroxylated metabolites: cross-inhibition and specificity.

Inhibition of drug metabolism was studied in adult male Sprague-Dawley rats. A hydroxylated metabolite of phenylbutazone (oxyphenbutazone) inhibited the elimination of phenytoin, which is metabolized by oxidative pathways. The biotransformation of a relatively polar and only slightly plasma protein-bound drug, antipyrine, was subject to product inhibition by a hydroxylated metabolite, 4-hydroxyantipyrine. Neither oxyphenbutazone nor 4-hydroxyantipyrine measurably affected the elimination kinetics or metabolic fate of a drug (sulfanilamide) that is not metabolized by oxidative pathways.     

The pharmacokinetics of codeine and its metabolites in Blacks with sickle cell disease

Purpose  We conducted a prospective, open-label study in 54 adult subjects with sickle cell disease to determine the relationship between morphine concentrations, cytochrome P450 (CYP) 2D6 genotype, and clinical outcomes. Methods  A blood sample was obtained for genotyping and serial blood samples were drawn to measure codeine and its metabolites in the plasma before and after oral codeine sulfate 30mg. Codeine and its metabolites were measured by liquid chromatography-tandem mass spectrometry (LC-MS). CYP2D6 genetic testing included four single nucleotide polymorphisms (SNP) indicative of three variant alleles: *17 (1023T); *29 (1659A, 3183A); and *41 (2988A) alleles. Results  Thirty subjects (group I) had a mean (standard deviation) maximal morphine concentration of 2.0 (1.0) ng/ml. Morphine was not measurable in the remaining 24 subjects (group II). Nine (30%) subjects in group I and 11 (46%) subjects in group II carried a variant *17, *29, or *41 allele (p = 0.23); one (3%) subject in group I and 5 (21%) subjects in group II were homozygous for *17 or *29 allele (p = 0.07). Emergency room visits (group I 1.5 ± 1.8 vs. group II 2.1 ± 4.3, p = NS) did not differ based on metabolic status, but more hospital admissions (0.9 ± 1.4 vs. 2.2 ± 4.1, p = 0.05) were documented in patients with no measurable morphine concentrations. Conclusions  We conclude that Blacks with sickle cell disease without measurable plasma morphine levels after a single dose of codeine were not more likely to be a carrier of a single variant allele commonly associated with reduced CYP2D6 metabolic capacity; however, homozygosity for a variant CYP2D6 allele may result in reduced metabolic capacity. Furthermore, it appears that subjects without measurable morphine concentrations were more likely to be admitted to the hospital for an acute pain crisis. This study was supported in part by the Illinois Department of Public Health awarded to the Sickle Cell Center, Janssen Medical Affairs, LLC (FEN-EMR4007) and a Clinical Translational Science Award from the Center for Clinical Translational Science at the University of Illinois awarded to Dr. Stacy S. Shord and the General Clinical Research Center at the University of Illinois Medical Center at Chicago (NIH grant M01-RR-13987).     

Safety assessment of drug metabolites: Characterization of chemically stable metabolites

Drug molecules are typically subjected to a variety of biotransformation reactions, and the metabolites formed through these reactions must be considered when conducting safety testing programs for new chemical entities. Metabolites that are chemically stable sometimes have pharmacological activity profiles similar to those of the parent compound but rarely have potent activity against off-target receptors that is unique relative to the parent profile. This fact argues for the thorough testing of drug metabolites for their pharmacological activity. It also argues for a significantly lower need for the thorough characterization and quantitation of stable metabolites not thought to substantially contribute to the pharmacodynamic effect. Given the tremendous resource requirements involved in the thorough characterization of drug metabolites, a more flexible, tiered approach to stable metabolite characterization would seem to provide the best utilization of resources while still allowing a complete evaluation of the toxicological profile of a new drug.     

In vitro metabolism of indiplon and an assessment of its drug interaction potential

This study was designed to study the in vitro metabolism of indiplon, a novel hypnotic agent, and to assess its potential to cause drug interactions. In incubations with pooled human liver microsomes, indiplon was converted to two major, pharmacologically inactive metabolites, N-desmethyl-indiplon and N-desacetyl-indiplon. The N-deacetylation reaction did not require NADPH, and appeared to be catalyzed by organophosphate-sensitive microsomal carboxylesterases. The N-demethylation of indiplon was catalyzed by CYP3A4/5 based on the following observations: (1) the sample-to-sample variation in N-demethylation of indiplon ([S]?=?100?µM) in a bank of human liver microsomes was strongly correlated with testosterone 6β-hydroxylase (CYP3A4/5) activity (r²?=?0.98), but not with any other CYP enzyme; (2) recombinant CYP1A1, CYP1A2, CYP3A4, CYP3A5 and CYP3A7 had the ability to catalyze this reaction; (3) the N-demethylation of indiplon was inhibited by CYP3A4/5 inhibitors (ketoconazole and troleandomycin), but not by a CYP1A2 inhibitor (furafylline). In pooled human liver microsomes, indiplon exhibited a weak capacity to inhibit CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4/5 and carboxylesterase (p-nitrophenylacetate hydrolysis) activities (IC50?≥?20?µM). Clinical data available on indiplon support the conclusions of this paper that the in vitro metabolism of indiplon is catalyzed by multiple enzymes, and indiplon is a weak inhibitor of human CYP enzymes.     

In vitro metabolism of indiplon and an assessment of its drug interaction potential

This study was designed to study the in vitro metabolism of indiplon, a novel hypnotic agent, and to assess its potential to cause drug interactions. In incubations with pooled human liver microsomes, indiplon was converted to two major, pharmacologically inactive metabolites, N-desmethyl-indiplon and N-desacetyl-indiplon. The N-deacetylation reaction did not require NADPH, and appeared to be catalyzed by organophosphate-sensitive microsomal carboxylesterases. The N-demethylation of indiplon was catalyzed by CYP3A4/5 based on the following observations: (1) the sample-to-sample variation in N-demethylation of indiplon ([S] = 100 microM) in a bank of human liver microsomes was strongly correlated with testosterone 6beta-hydroxylase (CYP3A4/5) activity (r(2) = 0.98), but not with any other CYP enzyme; (2) recombinant CYP1A1, CYP1A2, CYP3A4, CYP3A5 and CYP3A7 had the ability to catalyze this reaction; (3) the N-demethylation of indiplon was inhibited by CYP3A4/5 inhibitors (ketoconazole and troleandomycin), but not by a CYP1A2 inhibitor (furafylline). In pooled human liver microsomes, indiplon exhibited a weak capacity to inhibit CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4/5 and carboxylesterase (p-nitrophenylacetate hydrolysis) activities (IC50 >/= 20 microM). Clinical data available on indiplon support the conclusions of this paper that the in vitro metabolism of indiplon is catalyzed by multiple enzymes, and indiplon is a weak inhibitor of human CYP enzymes.     

Inhibition of theophylline metabolism by suplatast and its metabolites in rats

The inhibitory effect of suplatast (ST), an anti-allergic drug, on theophylline (TP) metabolism was investigated in rats in vivo and in vitro. Intravenous injection of aminophylline (AP) at 10 mg/kg of TP equivalent was performed with or without pretreatment by oral administration of 100 mg/kg of ST 2.5 h prior to AP. In the ST-pretreated group, plasma concentration (Cp), the area under Cp-time profile (AUC) and urinary excretion of TP increased significantly, and urinary excretion of TP metabolites, 1,3-dimethyluric acid (DMU) and 1-methyluric acid (1MU) decreased significantly. Metabolic clearance of DMU (CL(DMU)) and that of 1MU (CL(1MU)) were remarkably suppressed by ST pretreatment, however, renal clearance (CLr) of TP did not change. To compare the inhibitory effect of ST on TP metabolism with that of its main metabolite (M1) in vivo, a concomitant intravenous injection of AP (10 mg/kg of TP equivalent) with ST or M1 (40 mg/kg of ST equivalent) was carried out. In the M1 group, Cp and AUC of TP increased significantly, and the total body clearance of TP decreased significantly. In contrast, ST did not induce these changes. Then, the inhibitory effect of ST and M1 on TP metabolism in vitro was evaluated using rat-liver microsomes. ST and M1 suppressed DMU formation in a competitively inhibitory manner, and their equilibrium dissociation constants (Ki) were 822 and 731 microM, respectively. In conclusion, inhibition of TP metabolism by ST was demonstrated in vivo and in vitro, and the involvement of M1 and/or other metabolites in this drug interaction was suggested.     

Pharmacological effects of CM6912 and its main metabolites

Pharmacodynamic effects of ethyl 7-chloro-2,3-dihydro-5-(2-fluorophenyl)-2-oxo-1H-1,4- benzodiazepine-3-carboxylate (CM6912), a new benzodiazepine derivative, and its main metabolites (CM6913 = M1, CM7116 = M2) on the peripheral systems were investigated in several species of animals. In pentobarbital-anesthetized rabbits, CM6912 and M2 (1 or 5 mg/kg, i.v.) had little effect on blood pressure, heart rate and ECG, but it slightly reduced the respiration rate. M1 decreased the heart rate without affecting respiration, blood pressure and ECG. In conscious rabbits, CM6912 and M2 (1 mg/kg, i.v.) did not affect respiration, blood pressure, heart rate and ECG, but M1 (1 mg/kg, i.v.) increased the heart rate. CM6912 (5 or 30 mg/kg), when administered orally, also increased heart rate. In pentobarbital-anesthetized dogs, CM6912 and its metabolites (5 mg/kg, i.v.) decreased respiration and heart rate without affecting blood pressure and ECG. CM 6912 (5 mg/kg, i.v.) did not affect cardiovascular responses to the carotid occlusion, vagus stimulation, and pre- and post-ganglionic stimulation of cardiac ganglion in anesthetized dogs. CM6912 and its metabolites affected neither the spontaneous contraction nor the heart rate of isolated rabbit atria. These compounds also had no action on isolated aortic strips from rabbits. CM6912 and its metabolites did not affect the muscle tone of isolated guinea pig intestine, and it had no effects on the contractile responses to acetylcholine, histamine, serotonin and barium chloride. In isolated rabbit intestine, CM6912 and M2 slightly reduced the amplitude of contraction, while M1 had no effect. CM6912 and its metabolites did not affect the spontaneous motility of isolated non-pregnant and pregnant rat uteri as well as in situ non-pregnant rat uterus and isolated guinea pig vas deferens, including the contractile response to adrenaline. CM6912 and M2 relaxed isolated guinea pig trachea strips only at high concentrations. CM6912 and its metabolites did not affect the contractile responses of isolated rat diaphragm to electrical stimulation of the phrenic nerve. CM6912 (2 or 10 mg/kg, p.o.) did not affect the rat renal and hepatic functions. CM6912 influenced neither blood coagulation in rabbits nor blood hemolysis in rats. CM6912 and its metabolites did not affect the pupil size and its light reflex, and they did not produce a local anesthesia and edema. The present results suggest that CM6912 and its main metabolites exert only slight effects on the peripheral systems in animals.     

Urinary elimination of nitrazepam and its main metabolites.

Nitrazepam and its main metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, free and conjugated, were determined in the human urine after a single oral dose of 5 mg. The determinations were performed by GLC method using 63Ni-EC-detector for unchanged nitrazepam and nitrogen selective detector for the metabolites. Unchanged nitrazepam was poorly eliminated through the kidneys (about 1% of the dose). The interindividual variation of total excreted urinary metabolites was large ranging between 848-4933 microgram (17-99% of the dose during 7 days). Of this amount conjugated metabolites made up 57%. The urinary half-lives of free and conjugated-7-aminonitrazepam were (mean and ranges) 44 (23-65) and 46 (25-69) hrs, and of 7-acetamidonitrazepam 12 (5-31) and 18 (5-46) hrs, respectively. The half-lives of the excreted amounts of the metabolites did not correlate with any pharmacokinetic parameter of unchanged nitrazepam in serum.     

Relative antioxidant capacities of propofol and its main metabolites

The antioxidant activity of propofol, a widely used anesthetic, has previously been demonstrated, but no study has focused on propofol metabolites although propofol undergoes extensive metabolism. In the present study, the antioxidant properties of propofol and its metabolites were studied by measuring malondialdehyde (MDA) produced from lipid peroxidation by microsomes triggered with several free radical generating systems. True MDA determination was performed using a specific high performance liquid chromatography technique. Gas chromatography–isotope ratio mass spectrometry methodology was also used to assess the antioxidant action in a homogeneous aqueous environment. Propofol, 2,6-di-isopropyl-1,4-quinol (1,4-quinol) metabolite and 3,5-di-tert-butyl-4-hydroxytoluene markedly inhibit lipid peroxidation at concentrations lower than 5 µM. The binding of the glucuroconjugated moiety to either one of two hydroxyl groups of 1,4-quinol lowers the radical scavenging activity. Propofol glucuronide did not exert any radical scavenging activity except when peroxidation was induced with tert-butylhydroperoxide. Our data demonstrate that propofol and its metabolites inhibit lipid peroxidation at concentrations similar to those measured in human plasma during anesthesia. Their antioxidant efficiency is influenced by several factors, including the type of radical initiator involved and the site of radical production.     

Simultaneous detection in urine of cocaine and its main metabolites

The simultaneous detection in urine of cocaine (CO), and the main biotransformation products, benzoylecgonine (BE) and ecgonine methyl ester (EME), is difficult due to their different physicochemical properties.

The method presented involves a bonded silica solid-phase extraction procedure that allows mixed ionic and apolar interactions with the analyte. After extraction the compounds are derivatized sequentially with ethyliodide to obtain the BE ethyl ester derivative, and with N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) to obtain the O-TMS derivatives. The derivatized compounds are then analysed by a capillary (methylphenylsilicone) gas chromatographic system equipped with a specific nitrogen—phosphorus detector.

The method is suitable for the confirmation and quantitation of CO and its main metabolites in urine. BE levels in urine samples (n = 20) measured by the described method and by an immunological technique were in close agreement (r = 0.999).     

Quantitation of phenobarbital and its main metabolites in human urine

Summary A method for the quantitative determination of phenobarbital and free and conjugatedp-hydroxyphenobarbital in urine samples is described. The method includes initial extraction, purification on a small chromatographic column and finally determination by gas chromatography. The barbituric acids are methylated by trimethylanilinium hydroxide which serves as a flash heater methylating agent. The conjugate ofp-hydroxyphenobarbital, which appears to be a glucuronide, is hydrolysed with hydrochloric acid.     

Lacidipine metabolism in rat and dog: identification and synthesis of main metabolites

1. The main metabolites of lacidipine were isolated from bile and plasma of rats and dogs following an oral dose of the ¹⁴C-labelled drug (10?mg/kg for rats: 2 and 1?mg/kg for dogs). They were identified by comparison of chromatographic and spectral data with authentic reference compounds synthesized ad hoc.

2. Five metabolites (I-V) were isolated and identified in dog bile by gradient?h.p.l.c. with u.v. detection and?h.p.l.c.-thermospray mass spectrometry. In all metabolites the heterocyclic ring has been oxidized to pyridine. Further biotransformation reactions involved hydroxylation of the methyl substituents and hydrolysis of the ethyl and t-butyl ester groups to produce carboxylic acids and a lactone. Some of these metabolites also occurred as glucuronide conjugates.

3. A metabolite retaining the intact dihydropyridine ring, the des-ethyl analogue of lacidipine (VI), was isolated from rat plasma where it accounted for 60% of the total circulating radioactivity up to 24?h after administration. To characterize this metabolite,?h.p.l.c. with photodiode array u.v. detection also was employed. This compound was detected in dog plasma, but there was no evidence of its presence in dog bile samples.

4. Profiles of circulating metabolites were qualitatively similar in rats and dogs. Identified metabolites accounted for the large majority of total radioactivity in all the analysed samples.