LIBRARY OF MONOCLONAL ANTIBODIES
Inhibitory specific monoclonal antibodies (MAbs) are newly developed reagents for the precise identification and quantitation of the role of each human cytochrome P450 isoform for the metabolism of drugs or non-drug xenobiotics such as carcinogens, mutagens or environmental toxins. Drug metabolism in humans is catalysed by twelve major microsomal P450 enzymes which are heterogeneously distributed in liver and other tissues, and have different substrate and product specificities.
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Figure 1 is a summary of the wide applications of the new isoform specific inhibitory and immunoblotting MAbs, their mode of production and a summary description of their use for determining the metabolic contribution of the target P450 to the metabolism of a substrate in human liver microsomes. MAbs specific to individual P450 isoforms both identify and quantitate the metabolic role of the target P450 isoform(s) in human liver microsomes (HLM) by their inhibition of the of drug or other xenobiotic metabolism in vitro. The MAb system identifies drugs that are metabolized by only a single P450 or a single metabolic pathway. The drugs so identified can then be used as in vivo markers to determine the P450 phenotype of a patient. The MAbs also identify two or more drugs metabolized by a common P450 which may lead to toxic effects as a result of drug-drug interaction. The MAbs also identify drugs metabolized by polymorphic P450s such as P450 2D6. The potential toxicity of the drug in the polymorphically defective individual can be assessed by determining the amount of the drug normally metabolized by the defective polymorphic P450 relative to the amount of its metabolism by other non-defective P450s. Drugs metabolized by several P450s may negate the toxicity resulting from a polymorphically deficient P450. The MAbs determine inter-individual variation in the role amount and of each P450 isoform in different HLM samples by immunoblotting. The specificity of the MAbs may also make them useful for immunohistochemical tissue and cell localization. They can be used for mapping P450 isoforms in different human tissues. The MAbs being specific and highly inhibitory can be used as standards determining the potency and specificity of commonly used chemical inhibitors. The MAbs can be useful tools for defining the role of specific P450 isoforms in a variety of P450 dependent reactions such as mutagen activation, environmental toxicant formation, carcinogen activation, the formation of DNA and protein adducts and immunotoxicity. Further, the MAbs can be useful for determining factors such as age, sex, hormonal states, that may influence P450 activity. A major and important factor governing the clinical use of a drug is likely to require knowledge of the P450 specific metabolism of the drug.
Table 1 shows the immunoglobulin (Ig) subtype and the inhibitory effects of the MAbs on the enzyme activity of cDNA expressed P450 isoforms and the substrates used for the inhibition studies. All the MAbs are specific to a single target P450 isoform or a single 2C9 allele except for MAb 1-68-11 which is a potent inhibitor of all of the 2C human isoform P450s, 2C8, 2C9, 2C18 and 2C19. The single and the combinatorial use of the MAbs can "reaction phenotype", i.e. determine the metabolic contribution and interindividual variation of a P450 isoform for the metabolism of a drug or non-drug xenobiotic in human liver microsomes. The inhibitory activity was determined with the model substrates shown in Table 1. The MAb system offers large potential for studies of cytochrome P450 function useful in Drug Discovery and for the reduction of adverse drug reactions (ADR).
Table 2 shows that MAb 292-2-3 inhibits only a single allele, 2C9*2 of the three 2C9 alleles. MAb 293-2-3 inhibits only the 2C9*2 allele which differs form the wild type 2C9*1 and 2C9*3 by only a single amino acid of the 450 amino acid 2C9 proteins and thus demonstrating the extraordinary specificity potential of an MAb. The MAb 293-2-3 can also be used to define the extent of expression of each allele in an HLM sample from a 2C9 heterozygote individual and is a paradigm for the use of allele specific MAbs for determining the amount of expression of different alleles in heterozygotes.
Table 3 shows the Immunoblotting activity of MAbs to eight human liver P450 isoforms. Some of the MAbs are also inhibitory to the target P450 (see Table 1). Many of the MAbs exhibit both inhibitory and immunoblotting activity.
Table 4 shows the monoclonal antibody based analysis of the metabolism of 13 drugs and the endobiotic testosterone. Testosterone conversion to the 6-Bhydroxy metabolite was primarily metabolized by P450 3A4. P450 3A4 catalyzed 73-90% of the metabolism in eight samples of HLM. Bufuralol is commonly used as an in vivo marker for phenotyping the presence of 2D6 activity MAb analysis showed a very high contribution of 2D6 to bufuralol metabolism. In sixteen HLM samples 2D6 based bufuralol metabolism was greater than 70%. However in three samples the 2D6 contribution was low and the 2C family contributed 11-56%. Further analysis with MAbs specific to different isoforms of the 2C family showed that 2C9 and 2C19 contribute significantly to bufuralol metabolism in addition to 1A2 and thus 2D6 cannot be used as a strict marker for in vivo phenotyping 2D6. Dextromethorphan has also been used commonly as a marker for in vivo 2D6 activity. In most of the 16 HLM 2D6 contributed a major part, of metabolism as much as 90%. However in three HLM, 2C9 contributed 38-45% of dextromethorpham metabolism. Thus dextromethorpham also is not definitively useful as a marker for phenotyping 2D6. In 16 HLM phenacetin metabolism is catalyzed primarily by P450 1A2 (64-84%). However, in 8 samples 2A6, 2C9 and 2C19 contributed low levels (0-17) of enzyme activity. In 16 HLM, chlorzoxazone metabolism was catalyzed from 32-60% by P450 2E1. The contribution of other P450s to chlorzoxazone metabolism was not determined. In all of 13 HLM, coumarin conversion to 7-hydroxy was catalyzed by 2A6 by greater than 90%. Thus, 7-hydroxy coumarin formation was dependent entirely on 2A6 and coumarin conversion to 7-OH is an excellent marker for phenotyping 2A6. 7-ethoxycoumarin conversion to 7-OH coumarin was primarily dependent on 2E1 (16-60%) but was also metabolized to a lesser degree by 2B6, 2A6 and 1A2 ranging from 6-48%. Diazepam (DZ) hydroxylation to temazepam (TMZ) by was entirely catalyzed by 3A4 (75-84%). In contrast conversion of diazepam to nordiazepam (NDZ) by n-demethylation was dependent on several P450s, 3A4, 2C8, and 2C9. Thus the metabolic routing of diazepam by hydroxylation is quite different than its n-demethylation. Diazepam hydroxylation to TMZ is entirely dependent on the single P450 3A4 and thus can be used for phenotyping 3A4 in vivo. DZ conversion to NDZ was dependent on from P450s, 2B6, 2C8, 2C9 and 3A4.
Diclofenac metabolism was almost entirely catalyzed by 2C9 (83-88%) but in some HLM a small contribution of 2C19 (7-8%) was detected, in 5-6 HLM imiprimine metabolism to 2-OH and desipramine was catalyzed variably, by 1A2, 2C family, 2D6 and 3A4. Mephenytoin metabolism is almost entirely metabolized by 2C19 and has been used as a phenotyping marker for 2C19. However, Table 4 indicates that 2C9 can also catalyze mephenytoin metabolism from 10-22% in 7 HLM. Taxol 6-hydroxylation was metabolized entirely by 2C8, whereas taxol 3-hydroxylation was almost entirely catalyzed by 3A4. Tolbutamide 6-hydroxylation was catalyzed primarily by 2C9 but also by 2C8 and 2C9 which contributed 3-8%. Metabolism of the xenobiotic carcinogen aflatoxin was entirely by 3A4 (not shown).
The MAb based analysis identify current drugs and new chemical entities (NCE's) that are metabolized by a single P450 or multiple P450s. As a consequence, the MAb system can determine which drugs or NCEs are metabolized by polymorphic P450s which when absent or deficient can cause toxicity. The MAb system identifies drugs or NCEs metabolized by a polymorphic P450 which are also metabolized by other non polymorphic P450s. In those cases, the metabolic deficiency caused by the defective polymorphic P450 can be compensated by other P450s and thereby reduce the potential detrimental effect of the deficient polymorphic P450.
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Figure 2 examines the potency and the specificity of ketoconazole, commonly used as an inhibitor of 3A4. MAb 3-29-9 is specific and highly inhibitory to 3A4. Phenanthrene metabolism is metabolized variably by each of the 12 expressed P450 isoforms and can be used to test specificity. Figure 2 examines the specificity of ketoconazole relative to MAb 3-29-9, a potent inhibitor of 3A4 in HLM. Figure 2 shows that at very low concentrations (2mm) ketoconazole is as potent and inhibitory of 3A4 as the MAb. However, at higher concentration (10-100Ám) ketoconazole inhibits phenanthrene metabolism (50-80%) far more than the MAb, indicating that ketoconazole is inhibiting P450s other than 3A4. Chemical inhibitors of P450 isoforms often lack the necessary specificity and potency. The MAbs, being completely specific and highly potent inhibitors, mostly by greater than 90%, can be used as standards for assessing the adequacy of chemical inhibitors.
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P450 1A2 and 2E1 have been reported as major catalysts of 7-ethoxycoumairn deethylation (ECOD) in HLMs. Figure 3 shows the ECOD of twelve cDNA-expressed human P450 isoforms. 1A1 exhibited the highest ECOD but its activity is likely irrelevant for ECOD in HLM since 1A1 is absent or present at extremely low levels in HLM. The ECOD metabolism of expressed P450 shows activity for 2B6 and 2E1and moderate activity for 1A2, 2B6 and 2E1is not observed with HLM analysis. Thus high activity of an expressed P450 isoform does not indicate a large role for it in metabolism in HLM.
The inhibitory effect on ECOD in four HLM by MAbs added singly or combinatorally are shown in Table 5 and indicates that the inhibitory effect of MAbs added singly or combinatorally is the same thus validating the use of the combinatorial assay system. The average difference between inhibition by MAbs added singly or combinatorally was 4.4% with a standard deviation of 2.2%. In this system, MAbs also detected a 2A6 and 2B6 contribution to ECOD in HLMs not previously reported. The combinatorial analyses successfully defines the role of a target P450 in the presence of other P450 isoforms competing for the same drug.
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Diazepam (DZ) is used worldwide as an anxiolytic/hypnotic drug. Human P450s metabolize DZ by N1-demethylation to NDZ and by C3-hydroxylation to TMZ. Figure 4 shows the enzyme activities of twelve cDNA-expressed human P450s for the metabolism of DZ. Expressed P450 2B6 exhibited very high enzyme activity for N1-demethylation to NDZ whereas P450 2C8, 2C9, 2C18, 2C19, and 3A4/5 all exhibited lower but significant activity. No activity was exhibited by the other P450s examined, 1A1, 1A2, 2A6, 2D6 and 2E1.
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With the combinatorial addition of MAbs to HLM, Figure 5A shows that the formation of TMZ was inhibited by more than 80% by anti-3A4/5 (MAb 3-29-9) while the addition of the MAb 1-68-11 to the 2C subfamily (MAb 1-68-11) did not produce any added inhibition and was not significantly involved in C3-hydroxylation. Figure 5B also shows that the combinatorial addition of MAb inhibited NDZ formation to different extents defining the role of each target P450 to DZ demethylation. In HL39, 3A4/5 contributed 45% of demethylation activity, the 2C family 12%, and 2B6 23%. With HL41, the contribution of 3A4/5 was 17%, 61% for the 2C subfamily, and 6% for 2B6. Among the different samples, the range of contribution of 3A4/5 was 14-45%, the 2C subfamily 12-16% and 2B6 6-23%. No inhibition was observed with the addition of MAbs to 1A1, 1A2, 2A6, 2D6 and 2E1 indicating that these P450s do not contribute to DZ demethylation.
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MAb based combinatorial analyses of diazepam metabolism in HLMs with MAbs to 2B6, 3A4, 2C8, 2C9 and 2C19 is shown in Figure 5C. A previous report revealed the validity of the combinatorial system and in a previous application showed that 3A4 and 2B6 were the major contributors to diazepam N-demethylation to NDZ in HLMs. In Figure 5C the MAbs anti-2B6 and 3A4 activity from the HLMs so that the role of 2C8, 2C9, and 2C19 would be better assessed for NDZ formation in multiple HLM. In Figure 5C MAbs anti-3A4 and anti-2B6 were added together and the combined contribution of activity of 3A4 and 2B6 showed large interindividual variation among nine HLMs ranging from 20% in HLM 79-90% in HLM 74. The 2C9 contribution was also highly variable, greater than 30% in four HLMs (77,78,79, and 81) and showed a lesser contribution, from 5 to 25% in four other HLMs (74, 75, 76 and 80). 2C8 was found to significantly contribute to NDZ formation, ranging variably from 2 to 35%, and 2C19 does not contribute to NDZ formation (Figure 5C)
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Figures 6A & B show that in 6 of 8 HLM, bufuralol metabolism was largely catalyzed by 2D6, greater than 75%. In two samples, however, the 2D6 catalyzed metabolism was only 50%. Further analysis of these two HLM shown in Figure 6B indicate that 1A2, and 2C9 catalyzed 40-50% of bufuralol metabolism and 2C8 and 2C19 catalyzed 5-6% of bufuralol metabolism. Thus the combinatorial method is generally applicable to MAb analysis of metabolism and is especially useful for detection of metabolic activity contributed by P450s other than the major P450 catalysts of metabolism.
A variety of cDNA expression vectors such as vaccinia baculovirus, bacteria and yeast produce pure cytochromes P450s which can be characterized for substrate and product specificity. Analyses of the pure P450 isoforms does not adequately portray the P450s metabolic function in HLM. Different expression systems yield different amounts of P450 isoforms, account for interindividual P450 content and catalytic activity in human liver microsomes which vary as much as ten fold. Interindividual differences in metabolism and P450 protein content and further NADPH P450 oxidoreductase, cytochrome b5 and the membrane lipid environment may significantly influence the metabolic role of a P450 in human liver are not considered with the expressed P450s.
Polyclonal antibodies (PAbs) which have been used for assessing P450 metabolic function are heterogeneous reagents derived from a single immunization, commonly exhibit considerable cross-reactivity. Sequential or parallel immunizations with identical immunogens result in PAbs that consists of different heterogeneous populations of antibodies and are non-identical reagents which cannot be standard for universal use. In contrast hybridomas are stable, potentially immortal and produce an MAb that bid to specific epitopes on a protein.
There has been an undisciplined proliferation in the use of chemical inhibitors for reaction phenotyping. The chemical inhibitors, are aptly termed "selective" and not "specific" and commonly both isoform selectivity and inhibitory potency are undefined. Chemical inhibitors lacking defined character are generally not suitable for determining the metabolic role of a P450 isoforms in drug metabolism. The MAbs being specific and highly inhibitory can be used as standards for determining chemical inhibitor specificity and inhibitory potency.
Hepatic cytochrome P450 catalyzed metabolism is central to drug disposition and thus important to the clinical value of a drug. There are clinical implications of variable P450 activities for the metabolism of a variety of drugs including anti-arrhythmic antidepressants and neuroleptic drugs often these often display a high degree of inter-individual therapeutic efficacy and toxicity. Some of these differences may be due to genetic or environmental factors. The MAb system can determine interindividual variation and the effects of P450 inducers, inhibitors, as well as factors such as, ethnicity age, gender, developmental and nutritional states. The MAb system can identify drugs or metabolic pathways metabolized by a single P450 isoform which can be used in vivo, as a marker for phenotyping a patients P450 isoform profile. Defining a drug's P450 catalyzed metabolism, coupled with a knowledge of the P450 phenotype of the patient will be clinically useful for drug choice, dosage and the prediction of toxicity of the drug. The comprehensive library of MAbs will be of value in achieving a better understanding of P450 related therapeutic value, a reduction of adverse drug reaction (ADR) and in Drug Discovery.
Address for correspondence:H.V. Gelboin
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