Heyo K. Kroemer

Identification of P450 enzymes involved in metabolism of verapamil in humans

SummaryThe calcium channel blocker verapamil[2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] is widely used in the treatment of hypertension, angina pectoris and cardiac arrythmias. The drug undergoes extensive and variable hepatic metabolism in man with the major metabolic steps comprising formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile] and norverapamil [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoxtanitrile]. The enzymes involved in metabolism of verapamil have not been characterized so far. Identification of these enzymes would enable estimation of both interindividual variability in verapamil metabolism introduced by the respective pathway and potential for metabolic interactions. We therefore characterized the enzymes involved in formation of D-617 and norverapamil.The maximum rate of formation of D-617 and norverapamil was determined in the microsomal fraction of 21 human livers which had been previously characterized for the individual expression of various P450 enzymes (CYP1A2, CYP2C, CYP2D6, CYP2E1 and CYP3A3/4) by means of Western blotting. Specific antibodies directed against CYP3A were used to inhibit formation of D-617 and norverapamil. Finally, formation of both metabolites was investigated in microsomes obtained from yeast cells which were genetically engineered for stable expression of human P450.Formation of D-617 was correlated with the expression of CYP3A (r=0.85; P<0.001) and CYP1A2 (r=0.57; P<0.01) in the microsomal fraction of 21 human livers after incubation with racemic verapamil. Formation of norverapamil was correlated with the expression of CYP3A (r=0.58; P<0.01) and CYP1A2 (r=0.5; P<0.05) in the same preparations after incubation with racemic verapamil. Antibodies against CYP3A reduced maximum rate of formation of D-617 (to 37.1±11% and 40.6±6.801o of control after incubation with S- and R-verapamil, respectively) and norverapamil (to 38.2±4.5% and 29.2±5.5% of control after incubation with S- and R-verapamil, respectively). Both D-617 and norverapamil were formed by stable expressed CYP3A4 (16.6 pmol/mg protein/min and 22.6 pmol/mg protein/min, respectively). In summary, formation of D-617 and norverapamil is catalyzed mainly by CYP3A4. D-617 is also formed by CYP1A2. Veraparnil therefore has the potential to interact with other drugs which are substrates or inducers of CYP3A and CYP1A2.

Glucuronidation of drugs : a re-evaluation of the pharmacological significance of the conjugates and modulating factors

SummaryGlucuronides of drugs are considered to be generally inactive and rapidly eliminated. Therefore, these metabolites are often not taken into account in evaluating drug effects. The present review describes examples of both direct and indirect contributions of glucuronides to net drug effects. Multiple lines of evidence indicate that morphine-6-glucuronide has analgesic activity. This compound has a high affinity to the μ-receptor, is capable of penetrating the blood/brain barrier and is a potent analgesic after administration to patients. Indirect activity of glucuronides may consist of a systemic cycle in which an active parent compound is derived from the glucuronide by enzymatic action. Such systemic cycling has been demonstrated for clofibric acid. In addition, some acyl glucuronides are subject to intramolecular rearrangement and the resulting metabolites are resistant to β-glucuronidase. Covalent protein binding of glucuronides by different mechanisms may contribute to drug toxicity and immune responses. If glucuronides are accepted as potential modifiers of net drug action it is important to determine what factors modulate disposition of these compounds. Therefore, the later section of this review describes glucuronidation under different pathophysiological conditions. Examples for alterations of the rate and/or extent of glucuronidation by concurrent disease processes, age and coadministration of other drugs are provided.

“It's the genes, stupid” Molecular bases and clinical consequences of genetic cytochrome P450 2D6 polymorphism

In this review we highlight the information available on the genetic polymorphism of cytochrome P4502D6 expression in man. An absent function of this enzyme is observed in 7-10 percent of the Caucasian population which are referred to as Poor metabolizers as opposed to the remainder of the population (Extensive Metabolizers). More than 30 widely used drugs have been identified as substrates for CYP2D6. Disposition and action of these compounds depend on the individual phenotype. Both the molecular bases of the variable enzyme activity and the consequences for drug therapy are outlined. While mutations on the DNA level have been investigated in great detail larger scale clinical trials are lacking and information on therapeutic consequences of CYP2D6 mediated polymorphic drug oxidation is restricted to case reports. Besides these implications for drug metabolism several lines of evidence indicate that CYP2D6 could be involved in biotransformation of endogenous compounds.

The role of β-glucuronidase in drug disposition and drug targeting in humans

SummaryGlucuronides of drugs often accumulate during long term therapy. The hydrolysis of glucuronides can be catalysed by β-glucuronidase, an enzyme expressed in many tissues and body fluids in humans. The possible contribution of β-glucuronidase to drug disposition in humans has not been assessed in a systematic manner, but this enzyme may be able to release, locally or systemically, the active or inactive parent compound from drug glucuronides, thereby modifying the disposition and action of these drugs.Based on the information available on the localisation, expression and variability of β-glucuronidase, the concept of β-glucuronidase-mediated drug metabolism is outlined in this article using examples from the literature. Since some issues surrounding the β-glucuronidase-mediated deconjugation of drug glucuronides still need to be clarified in humans, additional data from animal models supporting this concept have been included. Moreover, as β-glucuronidase has already been proven to be useful in tumour specific bioactivation of glucuronide prodrugs of anticancer agents, we also focus on anticancer prodrug approaches utilising β-glucuronidase. This review summarises the role of β-glucuronidase in drug disposition and drug targeting in humans.

Cytochromes of the P450 2C subfamily are the major enzymes involved in the O-demethylation of verapamil in humans

The calcium channel blocker verapamil [2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] undergoes extensive biotransformation in man. We have previously demonstrated cytochrome P450 (CYP) 3A4 and 1A2 to be the enzymes responsible for verapamil N-dealkylation (formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile]), and verapamil N-demethylation (formation of norverapamil [2,8-bis(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile]), while there was no involvement of CYP3A4 and CYP1A2 in the third initial metabolic step of verapamil, which is verapamil O-demethylation. This pathway yields formation of D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and D-702 [2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)6-methyl-2-isopropyl-6-azaoctanitrile]. The enzymes catalyzing verapamil O-demethylation have not been characterized so far. We have therefore identified and characterized the enzymes involved in verapamil O-demethylation in humans by using the following in vitro approaches: (I) characterization of O-demethylation kinetics in the presence of the microsomal fraction of human liver, (II) inhibition of verapamil O-demethylation by specific antibodies and selective inhibitors and (111) investigation of metabolite formation in microsomes obtained from yeast strain Saccharomyces cerevisiae W(R), that was genetically engineered for stable expression of human CYP2C8, 2C9 and 2C18.In human liver microsomes (n=4), the intrinsic clearance (CLint), as derived from the ratio of Vmax/Km, was significantly higher for O-demethylation to D-703 compared to formation of D-702 following incubation with racemic verapamil (13.9±1.0 vs 2.4±0.6 ml*min-1*g-1 mean±SD; p<0.05), S-Verapamil (16.8±3.3 vs 2.2±1.2 ml* mini*g-1, p<0.05) and R-verapamil (12.1±2.9 vs 3.6 ±1.3 ml*min-1* g-1; p<0.05), thus indicating regioselectivity of verapamil O-demethylation process. The CLint of D-703 formation in human liver microsomes showed a modest but significant degree of stereo selectivity (p<0.05) with a S/R-ratio of 1.41±0.17. Anti-LKM2 (anti-liver/kidney microsome) autoantibodies (which inhibit CYP2C9 and 2C19) and sulfaphenazole (a specific CYP2C9 inhibitor) reduced the maximum rate of formation of D-703 by 81.5±4.5% and 45%, that of D-702 by 52.7±7.5% and 72.5%, respectively. Both D-703 and D-702 were formed by stably expressed CYP2C9 and CYP2C18, whereas incubation with CYP2C8 selectively yielded D-703.In conclusion, our results show that enzymes of the CYP2C subfamily are mainly involved in verapamil O-demethylation. Verapamil therefore has the potential to interact with other drugs which inhibit or induce these enzymes.

In vitro characterization of the human cytochrome P‐450 involved in polymorphic oxidation of propafenone

Propafenone is a new class 1 antiarrhythmic agent. The drug is extensively metabolized. 5‐Hydroxylation and N‐dealkylation constitute major metabolic pathways. Recently it has been demonstrated that the in vivo metabolism of propafenone is controlled by the debrisoquin/sparteine polymorphism. To elucidate which of the above metabolic reactions is catalyzed by cytochrome P‐450db1, the formation of 5‐hydroxypropafenone and N‐desalkylpropafenone was studied in the microsomal fraction of four human kidney donor livers previously characterized with regard to their ability to hydroxylate the β‐adrenergic antagonist bufuralol. The lhydroxylation of bufuralol is catalyzed by the P‐450db1 responsible for polymorphic debrisoquin/sparteine oxidation. The formation of 5‐hydroxypropafenone but not N‐desalkylpropafenone was closely related to bufuralol lhydroxylation. Incubation with LKM1 antibodies, which selectively recognize P‐450db1, inhibited 5‐hydroxypropafenone formation completely whereas N‐dealkylation was unimpaired. Propafenone was a strong competitive inhibitor of bufuralol lhydroxylation. Thus it can be concluded that 5‐hydroxypropafenone is formed by the cytochrome P‐450 isozyme involved in polymorphic bufuralol oxidation.

Use of probe drugs as predictors of drug metabolism in humans.

The pharmacokinetics of many drugs often vary considerably among individuals, largely because of variations in the expression of different cytochrome P‐450 (CYP) enzymes in the liver and other tissues. Relatively selective substrate probes in vivo have been discovered for several major CYP isoforms involved in oxidative drug metabolism. Regarding isoforms that show genetic polymorphism (CYP2C19 and CYP2D6), genotyping as well as phenotyping with appropriate probe drugs can be used to distinguish between “poor” and “extensive” metabolizers. Measurement of CYP2D6 activity, which is being performed increasingly by means of genotyping, has an established role in the individualization of the dosage of selected CYP2D6 substrates. However, the therapeutic implications of extremely high CYP2D6 activity in some patients (ultrarapid metabolizers) need more attention. The therapeutic consequences of CYP2C19 polymorphism are not as well characterized as those of CYP2D6 polymorphism, but are likely to be of little significance with most CYP2C19 substrates. Probe‐based assays are also available for measurement of in vivo activity of CYP1A2, CYP2E1 and CYP3A4; those will be discussed in detail in this review. These tests can be used, for example, to compare the activity of a specific isoform among patients and to characterize effects of such environmental factors as drugs and compounds in the diet on enzyme activity. However, it should be recognized that attempts to develop valid probe‐based assays of in vivo activity of specific, nonpolymorphic CYP isoforms have proved relatively difficult; for example, none of the several putative probes of CYP3A4, the most important drug‐metabolizing CYP isoform, is completely satisfactory. It is now clear that many diverse factors must be considered in the validation of these tests.

Clinical Pharmacokinetic Considerations in the Use of Plasma Expanders

SummaryThis review deals with the pharmacokinetics of dextrans and hydroxyethylstarch, the most commonly used plasma expanders. The complex composition of these colloidal agents (broad range of molecular weight distribution in vitro and in vivo, ) confounds their specific assay and meaningful pharmacokinetic analysis. In addition, the time-dependent decline of plasma concentrations of the plasma expanders is at least biphasic, and in some clinical studies the time period for plasma concentration monitoring has been inadequate to characterise the terminal elimination phase.According to their average molecular weight, dextrans can be differentiated into dextran 1, dextran 40, dextran 60 and dextran 70. Metabolism of dextrans by dextranases and extrarenal excretion account for only 2 to 10% of the overall drug loss from the body. Persistence of dextrans in the systemic circulation and elimination by the renal route are dependent on the size of dextrans and their molecular weight distribution. Dextran species with a molecular weight below 15,000 daltons are filtered unrestricted, and consequently the elimination half-life of dextran 1 is relatively short (2 hours) and that of dextran 40 (10 hours) or dextran 60 (42 hours) much longer. In patients with renal insufficiency elimination is impaired in parallel to the reduction in glomerular filtration rate, and smaller doses are advisable in these patients. Dosage reduction might be also indicated if multiple infusions of dextrans are used, since dextran 40 accumulates considerably during long term use (particularly the fractions with higher molecular weights). As only about 50 to 70% of a single dose could be recovered within 48 hours in the urine, the remainder of the dose is probably stored somewhere in the body.n Disposition of hydroxyethylstarch is dependent on 2 major factors. As with dextrans, the molecular weight distribution affects the rate of renal elimination. In addition, the degree of substitution with hydroxyethyl groups mainly determines the metabolism of hydroxyethylstarch by α-amylase, and thus the overall elimination rate. A higher molecular weight range (e.g. hydroxyethylstarch 450,000 vs 200,000) and a more extensive degree of substitution (e.g. 0.7 vs 0.5) result in a slower elimination, as can be seen by comparing the half-life values of hydroxyethylstarch 450/0.7 (48 days) and hydroxyethylstarch 200/0.5 (20 days). Since only 40 to 65% of an infused dose could be recovered in the urine in humans, the remainder of the dose may be stored in the body. Animal experiments suggest that certain fractions of hydroxyethylstarch might be stored in some tissues. However, during multiple infusions with hydroxyethylstarch 200/0.5 for 10 days no accumulation was observed in the plasma of patients.In conclusion, the disposition and pharmacological effects of plasma expanders are related to time-dependent changes in the molecular weight distribution of the plasma concentration decline. Unfortunately, the analytical assays applied in most studies were not able to differentiate the complex mixture of the infused colloids.

Expression of cytochrome P 450 3A enzymes in human lung: a combined RT-PCR and immunohistochemical analysis of normal tissue and lung tumours.

We have previously demonstrated expression of cytochrome P 450 3A (CYP3A) protein in pulmonary carcinomas and surrounding normal tissue, using immunohistochemistry. These results suggested that different CYP3A enzymes may be expressed in normal and tumour tissue. Therefore, the aim of the present study was to identify specific CYP3A enzymes expressed in normal human lung and lung tumours. Both normal lung tissue and tumour tissue from eight patients was analyzed for CYP3A4, CYP3A5 and CYP3A7 mRNA using a specific RT-PCR (reverse transcriptase-polymerase chain reaction) method. Identical samples were subjected to immunohistochemical analysis of CYP3A protein. CYP3A5 was the major enzyme of the CYP3A subfamily present at the mRNA level in both normal human lung and lung tumours. CYP3A5 mRNA was detected in normal lung tissue in all eight cases and in tumour tissue in four cases. CYP3A7 mRNA was detected in five cases in normal tissue and in one tumour. Notably, no CYP3A4 mRNA was found in any of the samples. Immunohistochemical staining for CYP3A protein was found in normal lung tissue in each case. Interestingly, all pulmonary carcinomas showed immunostaining for CYP3A, while mRNA for CYP3A enzymes was found in only four cases. In summary, our study indicates a specific expression pattern of the members of the CYP3A subfamily in normal human lung and lung tumours. These findings have potential clinical significance, since it has been recently shown that CYP3A5 catalyzes the activation of the anticancer pro-drugs cyclophosphamide and ifosfamide. Thus, local activation of these agents may take place in pulmonary carcinomas and surrounding normal tissues.

IMMUNOHISTOCHEMICAL LOCALIZATION OF CYTOCHROME P450 3A IN HUMAN PULMONARY CARCINOMAS AND NORMAL BRONCHIAL TISSUE

The cytochrome P450 (CYP) enzymes metabolize drugs and other xenobiotics in liver and also in some extrahepatic tissues. We have studied the expression and localization of CYP3A in primary lung tumours and normal lung tissue from the same patients. Thirtytwo patients undergoing partial or total lung resection for therapy of primary pulmonary carcinoma were included in this study. Immunohistochemical staining for CYP3A was performed with a modification of the ABC technique. Eight of the 32 cases of primary pulmonary carcinoma showed expression of CYP3A. In 12 of the 32 cases of normal tissue, the seromucous glands were positive for CYP3A. The bronchial epithelium was positive for CYP3A in 11 cases. We observed no correlation between CYP3A expression in tumour tissue and that in seromucous glands or bronchial epithelium. We conclude that CYP3A is present in both normal and cancerous lung tissue. Our findings suggest, however, no co-expression of CYP3A in lung cancer.