Tacrolimus CYP3A Single-Nucleotide Polymorphisms and Preformed T- and B-Cell Alloimmune Memory Improve Current Pretransplant Rejection-Risk Stratification in Kidney Transplantation.

Achieving fast immunosuppression blood exposure after kidney transplantation is key to abrogating both preformed and de novo anti-donor humoral and cellular alloresponses. However, while tacrolimus (TAC) is the cornerstone immunosuppressant inhibiting adaptive alloimmunity, its blood exposure is directly impacted by different single-nucleotide polymorphisms (SNPs) in CYP3A TAC-metabolizing enzymes. Here, we investigated how functional TAC-CYP3A genetic variants (CYP3A4*22/CYP3A5*3) influence the main baseline clinical and immunological risk factors of biopsy-proven acute rejection (BPAR) by means of preformed donor-specific antibodies (DSAs) and donor-specific alloreactive T cells (DSTs) in a large European cohort of 447 kidney transplants receiving TAC-based immunosuppression. A total of 70 (15.7%) patients developed BPAR. Preformed DSAs and DSTs were observed in 12 (2.7%) and 227 (50.8%) patients, respectively. According to the different CYP3A4*22 and CYP3A5*3 functional allele variants, we found 4 differential new clusters impacting fasting TAC exposure after transplantation; 7 (1.6%) were classified as high metabolizers 1 (HM1), 71 (15.9%) as HM2, 324 (72.5%) as intermediate (IM), and 45 (10.1%) as poor metabolizers (PM1). HM1/2 showed significantly lower TAC trough levels and higher dose requirements than IM and PM (p < 0.001) and more frequently showed TAC underexposure (<5 ng/ml). Multivariate Cox regression analyses revealed that CYP3A HM1 and IM pharmacogenetic phenotypes (hazard ratio (HR) 12.566, 95% CI 1.99-79.36, p = 0.007, and HR 4.532, 95% CI 1.10-18.60, p = 0.036, respectively), preformed DSTs (HR 3.482, 95% CI 1.99-6.08, p < 0.001), DSAs (HR 4.421, 95% CI 1.63-11.98, p = 0.003), and delayed graft function (DGF) (HR 2.023, 95% CI 1.22-3.36, p = 0.006) independently predicted BPAR. Notably, a significant interaction between T-cell depletion and TAC underexposure was observed, showing a reduction of the BPAR risk (HR 0.264, 95% CI 0.08-0.92, p = 0.037). Such variables except for DSAs displayed a higher predictive risk for the development of T cell-mediated rejection (TCMR). Refinement of pretransplant monitoring by incorporating TAC CYP3A SNPs with preformed DSAs as well as DSTs may improve current rejection-risk stratification and help induction treatment decision-making.

Influence of IL-18 and IL-10 Polymorphisms on Tacrolimus Elimination in Chinese Lung Transplant Patients.

Aims. The influence of interleukin-10 (IL-10) and interleukin-18 (IL-18) polymorphisms on tacrolimus pharmacokinetics had been described in liver and kidney transplantation. The expression of cytokines varied in different kinds of transplantation. The influence of IL-10 and IL-18 genetic polymorphisms on the pharmacokinetic parameters of tacrolimus remains unclear in lung transplantation. Methods. 51 lung transplant patients at Shanghai Pulmonary Hospital were included. IL-18 polymorphisms (rs5744247 and rs1946518), IL-10 polymorphisms (rs1800896, rs1800872, and rs3021097), and CYP3A5 rs776746 were genotyped. Dose-adjusted trough blood concentrations (C/D ratio, mg/kg body weight) in lung transplant patients during the first 4 postoperative weeks were calculated. Results. IL-18 rs5744247 allele C and rs1946518 allele A were associated with fast tacrolimus metabolism. Combined analysis showed that the numbers of low IL-18 mRNA expression alleles had positive correlation with tacrolimus C/D ratios in lung transplant recipients. The influence of IL-18 polymorphisms on tacrolimus C/D ratios was observed in CYP3A5 expresser recipients, but not in CYP3A5 nonexpresser recipients. No clinical significance of tacrolimus C/D ratios difference of IL-10 polymorphisms was found in our data. Conclusions. IL-18 polymorphisms may influence tacrolimus elimination in lung transplantation patients.

Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry.

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.

Pregnane X receptor is required for IFN-α-mediated CYP3A29 expression in pigs.

Pregnane X receptor (PXR) has been identified as a central mediator for coordinate responses to xenobiotic and drug metabolism, and is the major transcriptional regulator of cytochrome P-450 (CYP). Interferon (IFN)-α is known to induce antiviral mechanisms and exert immune regulatory capacity in various cell types. Here, we used primary porcine hepatocytes and a cultured hepatocyte cell line to identify the metabolic role of PXR in IFN-α-mediated CYP3A29 expression. We found that IFN-α could activate PXR in both time- and dose-dependent manners in pigs. Activation of PXR significantly increased CYP3A29 mRNA and protein expression. Meanwhile, the expression of CYP3A29 induced by IFN-α occurred after the increase of PXR expression in porcine hepatocytes. In addition, the IFN-α-induced CYP3A29 expression was blocked by PXR knockdown. The PXR-overexpressed cells (transfected with porcine PXR) increased CYP3A29 mRNA and protein expression. Furthermore, in animal experiments, we found that IFN-α increased both CYP3A29 mRNA and protein levels. Collectively, our results suggest that PXR plays an important role in IFN-α-mediated CYP3A29 expression in porcine hepatocytes.

Detection of anti-isoniazid and anti-cytochrome P450 antibodies in patients with isoniazid-induced liver failure.

UNLABELLED: Isoniazid (INH)-induced hepatotoxicity remains one of the most common causes of drug-induced idiosyncratic liver injury and liver failure. This form of liver injury is not believed to be immune-mediated because it is not usually associated with fever or rash, does not recur more rapidly on rechallenge, and previous studies have failed to identify anti-INH antibodies (Abs). In this study, we found Abs present in sera of 15 of 19 cases of INH-induced liver failure. Anti-INH Abs were present in 8 sera; 11 had anti-cytochrome P450 (CYP)2E1 Abs, 14 had Abs against CYP2E1 modified by INH, 14 had anti-CYP3A4 antibodies, and 10 had anti-CYP2C9 Abs. INH was found to form covalent adducts with CYP2E1, CYP3A4, and CYP2C9. None of these Abs were detected in sera from INH-treated controls without significant liver injury. The presence of a range of antidrug and autoAbs has been observed in other drug-induced liver injury that is presumed to be immune mediated. CONCLUSION: These data provide strong evidence that INH induces an immune response that causes INH-induced liver injury.

Extra-hepatic cancer represses hepatic drug metabolism via interleukin (IL)-6 signalling.

PURPOSE: In many cancer patients, the malignancy causes reduced hepatic drug clearance leading to potentially serious complications from the use of anticancer drugs. The mechanisms underlying this phenomenon are poorly understood. We aimed to identify tumor-associated inflammatory pathways that alter drug response and enhance chemotherapy-associated toxicity. METHODS: We studied inflammatory pathways involved in extra-hepatic tumor mediated repression of CYP3A, a major hepatic drug metabolizing cytochrome P450 subfamily, using a murine Engelbreth-Holm-Swarm sarcoma model. Studies in IL-6 knockout mice determined the source of elevated IL-6 in tumor-bearing animals and monoclonal antibodies against IL-6 were used to intervene in this inflammatory pathway. RESULTS: Our studies confirm elevated plasma IL-6 levels and reveal activation of Jak/Stat and Mapk signalling pathways and acute phase proteins in livers of tumor-bearing mice. Circulating IL-6 was predominantly produced by the tumor xenograft, rather than being host derived. Anti IL-6 antibody intervention partially reversed tumor-mediated inflammation and Cyp3a gene repression. CONCLUSIONS: IL-6 is an important player in cancer-related repression of CYP3A-mediated drug metabolism and activation of the acute phase response. Targeting IL-6 in cancer patients may prove an effective approach to alleviating cancer-related phenomena, such as adverse drug-related outcomes commonly associated with cancer chemotherapy.

Stimulation of human monocytic THP-1 cells by metabolic activation of hepatotoxic drugs.

Drug-induced liver injury (DILI) is thought to be involved in the participation of drugs that either directly affect the cell viability or elicit an immune response. However, there is limited information about the immune responses induced by drugs, including those drugs that are metabolically activated. In this study, we constructed an in vitro assay system to assess the involvement of immune-related factors induced by metabolic activation of drugs. To investigate whether CYP3A4-mediated metabolism of 10 hepatotoxic drugs is associated with immune-related responses, human monocytic leukemia THP-1 cells were co-incubated with CYP3A4 Supersomes. Cluster of differentiation (CD) 86 and CD54 expression levels on THP-1 cells were upregulated by treatment with albendazole and amiodarone (AMD), respectively, in the presence of CYP3A4. Additionally, N-desethylamiodarone (DEA), a major metabolite of AMD, upregulated the CD54 expression of THP-1 cells with CYP3A4. The release of interleukin (IL)-8 and tumor necrosis factor (TNF) α from THP-1 cells was significantly increased by the treatment of AMD or DEA with CYP3A4. Similarly, IL-8 and TNFα were also upregulated by the treatment of AMD and DEA with human liver microsomes, but were inhibited by adding ketoconazole to the cell culture. In this study, we first report that albendazole, AMD and DEA activate immune reaction when metabolically activated.

Immunochemical detection of cytochrome P450 enzymes in liver microsomes of 27 cynomolgus monkeys.

The cynomolgus monkey is widely used as a primate model in preclinical studies because of its evolutionary closeness to humans. Despite their importance in drug metabolism, the content of each cytochrome P450 (P450) enzyme has not been systematically determined in cynomolgus monkey livers. In this study, liver microsomes of 27 cynomolgus monkeys were analyzed by immunoblotting using selective P450 antibodies. The specificity of each antibody was confirmed by analyzing the cross-reactivity against 19 CYP1-3 subfamily enzymes using recombinant proteins. CYP2A, CYP2B6, CYP2C9/19, CYP2C76, CYP2D, CYP2E, CYP3A4, and CYP3A5 were detected in all 27 animals. In contrast, CYP1A, CYP1D, and CYP2J were below detectable levels in all liver samples. The average content of each P450 showed that among the P450s analyzed CYP3A (3A4 and 3A5) was the most abundant (40% of total immunoquantified P450), followed by CYP2A (25%), CYP2C (14%), CYP2B6 (13%), CYP2E1 (11%), and CYP2D (3%). No apparent sex differences were found for any P450. Interanimal variations ranged from 2.6-fold (CYP3A) to 11-fold (CYP2C9/19), and most P450s (CYP2A, CYP2D, CYP2E, CYP3A4, and CYP3A5) varied 3- to 4-fold. To examine the correlations of P450 content with enzyme activities, metabolic assays were performed in 27 cynomolgus monkey livers using 7-ethoxyresorufin, coumarin, pentoxyresorufin, flurbiprofen, bufuralol, dextromethorphan, and midazolam. CYP2D and CYP3A4 contents were significantly correlated with typical reactions of human CYP2D (bufuralol 1'-hydroxylation and dextromethorphan O-deethylation) and CYP3A (midazolam 1'-hydroxylation and 4-hydroxylation). The results presented in this study provide useful information for drug metabolism studies using cynomolgus monkeys.

Identification of cytochrome P450 enzymes responsible for metabolism of cannabidiol by human liver microsomes.

AIMS: Cannabidiol (CBD), one of the major constituents in marijuana, has been shown to be extensively metabolized by experimental animals and humans. However, human hepatic enzymes responsible for the CBD metabolism remain to be elucidated. In this study, we examined in vitro metabolism of CBD with human liver microsomes (HLMs) to clarify cytochrome P450 (CYP) isoforms involved in the CBD oxidations. MAIN METHODS: Oxidations of CBD in HLMs and recombinant human CYP enzymes were analyzed by gas chromatography/mass spectrometry. KEY FINDINGS: CBD was metabolized by pooled HLMs to eight monohydroxylated metabolites (6α-OH-, 6ß-OH-, 7-OH-, 1″-OH-, 2″-OH-, 3″-OH-, 4″-OH-, and 5″-OH-CBDs). Among these metabolites, 6α-OH-, 6ß-OH-, 7-OH-, and 4″-OH-CBDs were the major ones as estimated from the relative abundance of m/z 478, which was a predominant fragment ion of trimethylsilyl derivatives of the metabolites. Seven of 14 recombinant human CYP enzymes examined (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) were capable of metabolizing CBD. The correlations between CYP isoform-specific activities and CBD oxidative activities in 16 individual HLMs indicated that 6ß-OH- and 4″-OH-CBDs were mainly formed by CYP3A4, which was supported by inhibition studies using ketoconazole and an anti-CYP3A4 antibody. The correlation and inhibition studies also showed that CBD 6α-hydroxylation was mainly catalyzed by CYP3A4 and CYP2C19, whereas CBD 7-hydroxylation was predominantly catalyzed by CYP2C19. SIGNIFICANCE: This study indicated that CBD was extensively metabolized by HLMs. These results suggest that CYP3A4 and CYP2C19 may be major isoforms responsible for 6α-, 6ß-, 7-, and/or 4″-hydroxylations of CBD in HLMs.

Species differences in intestinal metabolic activities of cytochrome P450 isoforms between cynomolgus monkeys and humans.

The oral bioavailability of some drugs is markedly lower in cynomolgus monkeys than in humans. One of the reasons for the low bioavailability in cynomolgus monkeys may be the higher metabolic activity of intestinal CYP3A; however, the species differences in intestinal metabolic activities of other CYP isoforms between cynomolgus monkeys and humans are not well known. In the present study, we investigated the intrinsic clearance (CL(int)) values in pooled intestinal microsomes from cynomolgus monkeys and humans using 25 substrates of human CYP1A2, CYP2J2, CYP2C, and CYP2D6. As in humans, intestinal CL(int) values of human CYP1A2 and CYP2D6 substrates in cynomolgus monkeys were low. On the other hand, intestinal CL(int) values of human CYP2J2 and CYP2C substrates in cynomolgus monkeys were greatly higher than those in humans. Using immunoinhibitory antibodies and chemical inhibitors, we showed that the higher intestinal CL(int) values of the human CYP2J2 and CYP2C substrates in cynomolgus monkeys might be caused by monkey CYP4F and CYP2C subfamily members, respectively. In conclusion, there is a possibility that the greatly higher metabolic activity of CYP2C and CYP4F in cynomolgus monkey intestine is one of the causes of the species difference of intestinal first-pass metabolism between cynomolgus monkeys and humans.

Testosterone hydroxylation in bovine liver: enzyme kinetic and inhibition study.

In order to sort out the involvement of cytochrome P450 (CYP) 3A and possibly CYP2B in testosterone hydroxylation in cattle, enzyme kinetic and inhibition studies were performed. Most relevant kinetic constants (Km and Vmax) for 6beta-, 16beta- and 2beta-testosterone hydroxylase (OHT) activities were determined and accounted for 93.4 +/- 13.8, 36.4 +/- 6.1 and 110.8 +/- 15.2 muM, respectively, for Km and 0.558 +/- 0.03, 0.280 +/- 0.013, and 0.338 +/- 0.017 nmol min-1 mg-1 protein, respectively, for Vmax. Eadie-Hofstee plot analysis pointed out how these enzymatic activities in cattle follow a monophasic kinetic pattern. Preliminary inhibition studies conducted with the CYP3A inhibitor ketoconazole and the CYP2B inhibitors orphenadrine and 9-ethynylphenanthrene seemed to suggest the major involvement of CYP3A in testosterone hydroxylation in cattle. Immuno-inhibition studies with an anti-peptide antibody against bovine CYP3A4 confirmed the predominant role of CYP3A in testosterone hydroxylation in bovine liver, proving the usefulness of anti-peptide antibodies in defining the contribution of specific P450 isoforms in drug metabolism in veterinary species.

Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite.

The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically active metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the active metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the active metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel active metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.

Effects of epitope sequence tandem repeat and proline incorporation on polyclonal antibody production against cytochrome 1A2 and 3A4.

We describe a method for producing polyclonal antibodies against peptide antigen cytochrome P450 1A2 and 3A4 using a tandem repeat of the epitope region and incorporation of proline residue between the repeated sequences. An ELISA assay revealed more efficient generation of polyclonal antibodies to tandem repeat peptide antigens than mono-epitope peptides. The incorporation of proline residues further stimulated antibody production.

The combination of chemical and antibody inhibitors for superior P450 3A inhibition in reaction phenotyping studies.

Cytochrome P450 (P450) reaction phenotyping is a key process toward accurately determining the contribution of different P450s to the metabolism of new chemical entities. The significance of P450s to drug disposition has led to the identification of selective chemical and antibody inhibitors for individual P450 enzymes. Despite these advances, the maximal inhibition attainable is limited by the use of inhibitor concentrations that maintain selectivity for the individual P450s. Thus, most commercially available inhibitors produce a maximal inhibition of approximately 80%. Herein, the combination of chemical plus antibody inhibitors is explored to find whether P450 3A could be selectively and completely (>99%) inhibited by using both inhibitors simultaneously.

Characterization of the physiological turnover of native and inactivated cytochromes P450 3A in cultured rat hepatocytes: a role for the cytosolic AAA ATPase p97?

Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno- and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in Saccharomyces cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal (MG-132 and MG-262) and ALD [NH4Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase with immunoprecipitation analyses revealed a basal 52% 35S-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH4Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were cross-linked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-cross-linking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.

Cytochrome P450 3A-dependent metabolism of a potent and selective gamma-aminobutyric acid Aalpha2/3 receptor agonist in vitro: involvement of cytochrome P450 3A5 displaying biphasic kinetics.

In vitro metabolism studies were conducted to determine the human cytochrome P450 enzyme(s) involved in the biotransformation of 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3b]pyridazine (TPA023), a selective agonist of human gamma-aminobutyric acid(A) receptor alpha2 and alpha3 subunits. Incubation of TPA023 with NADPH-fortified human liver microsomes resulted in the formation of t-butyl hydroxy TPA023, N-desethyl TPA023, and three minor metabolites. Both t-butyl hydroxylation and N-deethylation reactions were greatly inhibited (>85%) in the presence of CYP3A-selective inhibitory antibodies and chemical inhibitors, indicating that members of the CYP3A subfamily play an important role in TPA023 metabolism. Eadie-Hofstee plots of t-butyl hydroxylation and N-deethylation in pooled CYP3A5-rich human liver microsomes revealed a low K(m) (3.4 and 4.5 microM, respectively) and a high K(m) (12.7 and 40.0 microM, respectively) component. For both metabolites, the high K(m) component was not observed with a pool of microsomal preparations containing minimal levels of CYP3A5. Preincubation of liver microsomes with mifepristone (selectivity for CYP3A4 > CYP3A5) greatly inhibited both t-butyl hydroxylation and N-deethylation (>75%); however, the residual activities were significantly higher in the pooled CYP3A5-rich liver microsomes (p < 0.0005). In addition, elevated levels of residual t-butyl hydroxylase and N-deethylase activities were observed in the presence of both CYP3A5-rich and CYP3A5-deficient preparations when the substrate concentration increased from 4 to 40 microM. In agreement, metabolite formation catalyzed by recombinant CYP3A5 was described by a biphasic model. It is concluded that CYP3A4 plays a major role in TPA023 metabolism, and CYP3A5 may also contribute at higher concentrations of the compound.

Generation of specific antibodies and their use to characterize sex differences in four rat P450 3A enzymes following vehicle and pregnenolone 16alpha-carbonitrile treatment.

The purpose of this study was to identify isozyme-specific antibodies and use them to determine the expression levels of four P450 3A enzymes in the livers of vehicle- and pregnenolone 16alpha-carbonitrile (PCN)-treated rats of both sexes, since previous work on mRNA levels has shown considerable sexual dimorphism. Using Western blot analysis with four isozyme-specific antibodies, we show that P450 3A1, 3A2, and 3A9 were expressed in vehicle-treated adult female rats at very low levels whereas P450 3A18 was not detected. PCN treatment of females strongly induced the expression of P450 3A1 in the livers with protein product increases of 214-, 3-, and 5-fold for P450 3A1, 3A2, and 3A9, respectively, and P450 3A18 was induced to 3.7 pmol/mg protein. In contrast, all four P450 3As were detected in livers of vehicle-treated males, in the order of 3A2 >> 3A18 > 3A9 approximately = 3A1. The protein product increases induced by PCN treatment of male rats were 92-, 3-, 6-, and 16-fold for P450 3A1, 3A2, 3A9, and 3A18, respectively.