In Vivo Cytochrome P450 3A Isoenzyme Activity and Pharmacokinetics of Imatinib in Relation to Therapeutic Outcome in Patients With Chronic Myeloid Leukemia.

BACKGROUND: Cytochrome P450 3A (CYP3A) isoenzyme metabolic activity varies between individuals and is therefore a possible candidate of influence on the therapeutic outcome of the tyrosine kinase inhibitor imatinib in patients with chronic myeloid leukemia (CML). The aim of this study was to investigate the influence of CYP3A metabolic activity on the plasma concentration and outcome of imatinib in patients with CML. METHODS: Forty-three patients with CML were phenotyped for CYP3A activity using quinine as a probe drug and evaluated for clinical response parameters. Plasma concentrations of imatinib and its main metabolite, CGP74588, were determined using liquid chromatography-mass spectrometry. RESULTS: Patients with optimal response to imatinib after 12 months of therapy did not differ in CYP3A activity compared to nonoptimal responders (quinine metabolic ratio of 14.69 and 14.70, respectively; P = 0.966). Neither the imatinib plasma concentration nor the CGP74588/imatinib ratio was significantly associated with CYP3A activity. CONCLUSIONS: The CYP3A activity does not influence imatinib plasma concentrations or the therapeutic outcome. These results indicate that although imatinib is metabolized by CYP3A enzymes, this activity is not the rate-limiting step in imatinib metabolism and excretion. Future studies should focus on other pharmacokinetic processes so as to identify the major contributor to patient variability in imatinib plasma concentrations.

Using Whole-Exome Sequencing to Identify Genetic Markers for Carboplatin and Gemcitabine-Induced Toxicities.

PURPOSE: Chemotherapies are associated with significant interindividual variability in therapeutic effect and adverse drug reactions. In lung cancer, the use of gemcitabine and carboplatin induces grade 3 or 4 myelosuppression in about a quarter of the patients, while an equal fraction of patients is basically unaffected in terms of myelosuppressive side effects. We therefore set out to identify genetic markers for gemcitabine/carboplatin-induced myelosuppression. EXPERIMENTAL DESIGN: We exome sequenced 32 patients that suffered extremely high neutropenia and thrombocytopenia (grade 3 or 4 after first chemotherapy cycle) or were virtually unaffected (grade 0 or 1). The genetic differences/polymorphism between the groups were compared using six different bioinformatics strategies: (i) whole-exome nonsynonymous single-nucleotide variants association analysis, (ii) deviation from Hardy-Weinberg equilibrium, (iii) analysis of genes selected by a priori biologic knowledge, (iv) analysis of genes selected from gene expression meta-analysis of toxicity datasets, (v) Ingenuity Pathway Analysis, and (vi) FunCoup network enrichment analysis. RESULTS: A total of 53 genetic variants that differed among these groups were validated in an additional 291 patients and were correlated to the patients' myelosuppression. In the validation, we identified rs1453542 in OR4D6 (P = 0.0008; OR, 5.2; 95% CI, 1.8-18) as a marker for gemcitabine/carboplatin-induced neutropenia and rs5925720 in DDX53 (P = 0.0015; OR, 0.36; 95% CI, 0.17-0.71) as a marker for thrombocytopenia. Patients homozygous for the minor allele of rs1453542 had a higher risk of neutropenia, and for rs5925720 the minor allele was associated with a lower risk for thrombocytopenia. CONCLUSIONS: We have identified two new genetic markers with the potential to predict myelosuppression induced by gemcitabine/carboplatin chemotherapy.

[The usefulness of pharmacogenetics for a more individualized treatment. The example thiopurines in inflammatory bowel disease and childhood leukemia]. / Nyttan av farmakogenetik för en mer individualiserad behandling - Exemplet tiopuriner vid inflammatorisk tarmsjukdom och barnleukemi.

Thiopurines are chemotherapeutic drugs used for treatment of inflammatory bowel diseases and childhood leukemia. Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurines. Individuals lacking TPMT are at increased risk for severe side effects when treated with conventional doses of thiopurines. A research group at the division of drug research at Linköping University is studying thiopurine pharmacogenetics. Since the year 2000, the lab has determined the TPMT status in over 12000 individuals, as an aid to decide thiopurine doses before starting treatment. New knowledge of how genetic factors influence thiopurine treatment effect are anticipated to improve the possibilities for individualization of thiopurine therapy.

Novel assay to improve therapeutic drug monitoring of thiopurines in inflammatory bowel disease.

BACKGROUND AND AIMS: The thiopurines are widely used in the treatment of inflammatory bowel disease, but are limited by poor dose-effect relationship. The objective was to assess the ability of a novel assay, determining the mono-, di-, and triphosphates, of thioguanine as well as methylthioinosine as individual metabolites in erythrocytes, to predict clinical outcome compared to a routine assay, determining metabolites as sums. METHODS: Samples from 79 patients with Crohn's disease or ulcerative colitis treated with azathioprine or mercaptopurine were analysed by both assays. Clinical status was determined by the Harvey-Bradshaw and Walmsley indices. The genotypes of thiopurine methyltransferase (TPMT) and inosine triphosphatase were determined. RESULTS: TPMT wild-type patients with thioguanine nucleotide (TGN) levels below the cut-off level were more likely to have active disease when TGN was measured by the novel assay (p=0.02), and when thioguanosine triphosphate (TGTP) was measured separately (p=0.01). When TGN was measured by the routine assay the correlation was not evident (p=0.12). Neither TGN levels nor TGTP correlated to disease activity in TPMT deficient patients. Patients with methyl thioinosine nucleotide (meTIN) levels above 1500 pmol/8×10^8 RBCs were more likely to have active disease (p=0.07). We observed good correlations between the mono-, di-, and triphosphates and their respective sums (R(2)>0.88). CONCLUSIONS: The novel TGN assay was better in predicting clinical outcome compared to the routine assay, while determination of TGTP had no clinical advantage and TGTP ratio was not correlated to disease activity.

Gene expression and thiopurine metabolite profiling in inflammatory bowel disease - novel clues to drug targets and disease mechanisms?

BACKGROUND AND AIMS: Thiopurines are effective to induce and maintain remission in inflammatory bowel disease (IBD). The methyl thioinosine monophosphate (meTIMP)/6-thioguanine nucleotide (6-TGN) concentration ratio has been associated with drug efficacy. Here we explored the molecular basis of differences in metabolite profiles and in relation to disease activity. METHODS: Transcriptional profiles in blood samples from an exploratory IBD-patient cohort (n = 21) with a normal thiopurine S-methyltransferase phenotype and meTIMP/6-TGN ratios >20, 10.0-14.0 and ≤4, respectively, were assessed by hybridization to microarrays. Results were further evaluated with RT qPCR in an expanded patient cohort (n = 54). Additionally, 30 purine/thiopurine related genes were analysed separately. RESULTS: Among 17 genes identified by microarray-screening, there were none with a known relationship to pathways of purines/thiopurines. For nine of them a correlation between expression level and the concentration of meTIMP, 6-TGN and/or the meTIMP/6-TGN ratio was confirmed in the expanded cohort. Nine of the purine/thiopurine related genes were identified in the expanded cohort to correlate with meTIMP, 6-TGN and/or the meTIMP/6-TGN ratio. However, only small differences in gene expression levels were noticed over the three different metabolite profiles. The expression levels of four genes identified by microarray screening (PLCB2, HVCN1, CTSS, and DEF8) and one purine/thiopurine related gene (NME6) correlated significantly with the clinical activity of Crohn's disease. Additionally, 16 of the genes from the expanded patient cohort interacted in networks with candidate IBD susceptibility genes. CONCLUSIONS: Seventeen of the 18 genes which correlated with thiopurine metabolite levels also correlated with disease activity or participated in networks with candidate IBD susceptibility genes involved in processes such as purine metabolism, cytokine signaling, and functioning of invariant natural killer T cells, T cells and B cells. Therefore, we conclude that the identified genes to a large extent are related to drug targets and disease mechanisms of IBD.

Monitoring of thiopurine metabolites - a high-performance liquid chromatography method for clinical use.

A high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBCs were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials. Ten patient samples were analysed to demonstrate clinical application and to establish pilot ranges for all analytes. The method measured thioguanosine mono-(TGMP), di-(TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine). LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8 × 108 RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 4 °C for 8 h after sampling.

Impact of ABCB1 variants on neutrophil depression: a pharmacogenomic study of paclitaxel in 92 women with ovarian cancer.

The standard treatment for ovarian cancer in advanced stages is post-surgery treatment with taxane-platin chemotherapy. Despite an initial high response rate, most patients eventually relapse. The dose-limiting toxicities of paclitaxel are neutropenia and neuropathy, but the inter-individual variability is large. The aim of this prospective study was to investigate the impact of genetic variants in key drug metabolizing/transporter genes on toxicity and compliance. CYP2C8*3 and three ABCB1 polymorphisms were chosen for primary analysis, and a host of other candidate genes was explored in 92 prospectively recruited Scandinavian Caucasian women with primary ovarian cancer who were treated with paclitaxel and carboplatin. A single investigator assessed the clinical toxicity in 97% of the patients. Patients carrying variant alleles of ABCB1 C3435T experienced more pronounced neutrophil decrease (63%, 72% and 80% for 3435CC, CT and TT, respectively; p-value 0.03). A similar association was found for G2677T/A, p-value 0.02. For C1236T, there was a trend with p-value 0.06. No statistically significant correlations were found for paclitaxel compliance and sensory neuropathy in the primary analysis. Variants in the drug transporter ABCB1 gene are possibly associated with the neutrophil suppressing effect of paclitaxel in patients with ovarian cancer. This finding has implications for the understanding of bone marrow suppression and future tailored chemotherapy.

Impact of CYP3A5*3 and CYP2C8-HapC on paclitaxel/carboplatin-induced myelosuppression in patients with ovarian cancer.

The influence of genetic variants on paclitaxel-induced toxicity is of considerable interest for reducing adverse drug reactions. Recently, the genetic variants CYP2C8*3, CYP2C8-HapC, and CYP3A5*3 were associated with paclitaxel-induced neurotoxicity. We, therefore, investigated the impact of CYP2C8-HapC and CYP3A5*3 on paclitaxel/carboplatin-induced myelosuppression and neurotoxicity. Thirty-three patients from a prospective pharmacokinetics study were genotyped using pyrosequencing. Patients with variant alleles of CYP2C8-HapC were found to have significantly lower nadir values of both leukocytes and neutrophils (p < 0.05) than patients with the wild-type genotype. CYP3A5*3/*1 patients were shown to have borderline, significantly lower nadir values of leukocytes (p = 0.07) than *3/*3 patients. Combining the two genotypes resulted in a significant correlation with both leukopenia and neutropenia (p = 0.01). No effect of these genetic variants on neurotoxicity could be shown in this rather small study, but their importance for paclitaxel-induced toxicity could be confirmed.

The role of inosine-5'-monophosphate dehydrogenase in thiopurine metabolism in patients with inflammatory bowel disease.

BACKGROUND: There is a large interindividual variability in thiopurine metabolism. High concentrations of methylthioinosine-5'-monophosphate (meTIMP) and low concentrations of 6-thioguanine nucleotides (6-TGNs) have been associated with a lower response rate and an increased risk of adverse events. In this study, the role of inosine-5'-monophosphate dehydrogenase (IMPDH) for differences in metabolite patterns of thiopurines was investigated. METHODS: IMPDH activity and thiopurine metabolite concentrations were determined in patients with inflammatory bowel disease and a normal thiopurine methyltransferase (TPMT) phenotype and meTIMP/6-TGN concentration ratio > 20 (n = 26), in patients with a metabolite ratio ≤ 20 (n = 21), in a subgroup with a metabolite ratio <4 (n = 6), and in 10 patients with reduced TPMT activity. In vitro studies were conducted on human embryonic kidney cells (HEK293) with genetically engineered IMPDH and TPMT activities. RESULTS: Patients with metabolite ratios >20 had lower IMPDH activity than those with ratios ≤ 20 (P < 0.001). Metabolite ratios >20 were only observed in patients with normal TPMT activity. Downregulation of IMPDH activity in HEK293 cells was associated with an increase in the concentration of meTIMP (fold change: 17 up to 93, P < 0.001) but, unexpectedly, also of 6-thioguanosine monophosphate (fold change: 2.6 up to 5.0, P < 0.001). CONCLUSIONS: These data question the general view of IMPDH as the rate-limiting enzyme in the phosphorylation of thiopurines. Investigations of other mechanisms are needed to more fully explain the various metabolite patterns and outcomes in patients under treatment.

Drug therapy of cancer.

Cancer chemotherapy was introduced at the same time as antibacterial chemotherapy but has not been nearly such a success. However, there is a growing optimism in oncology today due to the introduction of several more or less target-specific drugs as complements to the conventional cytotoxic drugs introduced half a century ago. The success in the treatment of chronic myelogenous leukemia by imatinib, inhibiting the bcr-abl-activated tyrosine kinase and thereby interrupting the signal transduction pathways that lead to leukemic transformation with impressive survival benefit, has paved the way for this new optimism. Another success story is the introduction of trastuzumab in breast cancers overexpressing the HER-2 receptor. In contrast, there has been little progress in other malignancies such as metastatic malignant melanoma, although very recently, clinical trials with new targeted drugs have shown increased survival. All major pharmaceutical companies now have ambitious development programs in the cancer area. However, the high costs of the novel drugs cause economic distress in the health care system in many countries leading to an intense debate on the cost-effectiveness of these drugs in relation to other health care activities.

Retrospective study of the impact of pharmacogenetic variants on paclitaxel toxicity and survival in patients with ovarian cancer.

PURPOSE: Paclitaxel has a broad spectrum of anti-tumor activity and is useful in the treatment of ovarian, breast, and lung cancer. Paclitaxel is metabolized in the liver by CYP2C8 and CYP3A4 and transported by P-glycoprotein. The dose-limiting toxicities are neuropathy and neutropenia, but the interindividual variability in toxicity and also survival is large. The main purpose of this study was to investigate the impact of genetic variants in CYP2C8 and ABCB1 on toxicity and survival. METHODS: The 182 patients previously treated for ovarian cancer with carboplatin and paclitaxel in either the AGO-OVAR-9 or the NSGO-OC9804 trial in Denmark or Sweden were eligible for this study. Genotyping was carried out on formalin-fixed tissue. The patients' toxicity profiles and survival data were derived from retrospective data. CYP2C8*3, ABCB1 C1236T, G2677T/A, and C3435T were chosen a priori for primary analysis; a host of other variants were entered into an exploratory analysis. RESULTS: Clinical data and tissue were available from a total of 119 patients. Twenty-two single nucleotide polymorphisms (SNPs) in 10 genes were determined. Toxicity registration was available from 710 treatment cycles. In the primary analysis, no statistically significant correlation was found between CYP2C8*3, ABCB1 C1236T, G2677T/A, and C3435T and neutropenia, sensoric neuropathy, and overall survival. CONCLUSION: CYP2C8*3 and the ABCB1 SNPs C1236T, G2677T/A, and C3435T were not statistically significantly correlated to overall survival, sensoric neuropathy, and neutropenia in 119 patients treated for ovarian cancer with paclitaxel/carboplatin.

Induction of fetal hemoglobin and ABCB1 gene expression in 9-ß-D-arabinofuranosylguanine-resistant MOLT-4 cells.

PURPOSE: To characterize resistance mechanisms to the nucleoside analog 9-ß-D-arabinofuranosylguanine (AraG) in the T-cell acute lymphoblastic leukemia cell line MOLT-4 and its AraG-resistant variant. METHODS: A gene expression microarray analysis was performed, as well as gene expression and enzyme activity measurements of key enzymes in the activation of AraG. Cytotoxicity of AraG and cross-resistance to other compounds were evaluated using a standard cytotoxicity assay. RESULTS: Gene expression microarray analysis revealed that fetal hemoglobin genes and the multidrug resistance ABCB1 gene, encoding the drug efflux pump P-gp, were the most highly upregulated genes in the resistant cells, while genes traditionally associated with nucleoside analog resistance were not. Fetal hemoglobin and ABCB1 induction can be due to global DNA hypomethylation. This phenomenon was studied using AraG during a period of 4 weeks in MOLT-4 cells and the lung adenocarcinoma cell line A549, leading to up-regulation of hemoglobin gamma and ABCB1 as well as DNA hypomethylation. Inhibiting P-gp in the AraG-resistant MOLT-4 cells led to decreased proliferation, reduced hemoglobin expression, and highly induced ABCB1 expression. CONCLUSIONS: We show that AraG can cause hypomethylation of DNA and induce the expression of the fetal hemoglobin gamma gene and the ABCB1 gene. We speculate that the induction of ABCB1/P-gp may occur in order to help with excretion of hemoglobin degradation products that would otherwise be toxic to the cells, and we present data supporting our theory that P-gp may be linked to the induction of hemoglobin.

Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene.

BACKGROUND: The activity of the human enzyme thiopurine methyltransferase (TPMT) varies greatly between individuals because of genetic polymorphism. TPMT is involved in the detoxification and activation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathioprine. These drugs are used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disease. A total of 29 sequence variants have been identified so far in the TPMT gene. However, most of these variants are rare and not fully characterized. METHODS AND RESULTS: In this study, we describe the identification and characterization of a novel TPMT sequence variant, originally found in a Swedish man of Italian origin. Sequencing of the variable number tandem repeats region of the TPMT promoter and exons III-X revealed a T-to-C transition at nucleotide 611, causing an amino acid substitution from isoleucine to threonine at amino acid 204, positioned in an α-helix, approximately 16 Å from the active site. This new variant was found in the patient and in his son. Both had intermediate enzyme activity (8.1 U/ml packed red blood cells and 8.8 U/ml packed red blood cells, respectively) and neither carried other variants in the coding region of the gene. To be able to study this variant in more detail, the TPMT*28 variant was expressed in Escherichia coli, and an in-vitro characterization of the variant revealed that the protein was destabilized and showed a stronger tendency towards degradation at 37°C than the wild-type protein. The individuals carrying the TPMT*28 variant had less TPMT protein and lower TPMT activity in both red and white blood cells compared with a wild-type control. CONCLUSIONS: We present a detailed in-vivo and in-vitro characterization of a novel TPMT sequence variant (TPMT*28) causing decreased TPMT activity. Individuals carrying TPMT*28 might have an increased risk for developing severe side effects if treated with conventional doses of thiopurines.

The association of reduced folate carrier 80G>A polymorphism to outcome in childhood acute lymphoblastic leukemia interacts with chromosome 21 copy number.

The reduced folate carrier (RFC) is involved in the transport of methotrexate (MTX) across the cell membrane. The RFC gene (SLC19A1) is located on chromosome 21, and we hypothesized that the RFC80 G>A polymorphism would affect outcome and toxicity in childhood leukemia and that this could interact with chromosome 21 copy number in the leukemic clone. A total of 500 children with acute lymphoblastic leukemia treated according to the common Nordic treatment protocols were included, and we found that the RFC AA variant was associated with a 50% better chance of staying in remission compared with GG or GA variants (P = .046). Increased copy numbers of chromosome 21 appear to improve outcome also in children with GA or GG variant. In a subset of 182 children receiving 608 high-dose MTX courses, we observed higher degree of bone marrow toxicity in patients with the RFC AA variant compared with GA/GG variants (platelet 73 vs 99/105 x 10(9)/L, P = .004, hemoglobin 5.6 vs 5.9/6.0 mmol/L, P = .004) and a higher degree of liver toxicity in patients with RFC GG variant (alanine aminotransferase 167 vs 127/124 U/L, P = .05). In conclusion, the RFC 80G>A polymorphism interacts with chromosome 21 copy numbers and affects both efficacy and toxicity of MTX.

Characterisation and utility of thiopurine methyltransferase and thiopurine metabolite measurements in autoimmune hepatitis.

BACKGROUND & AIMS: Corticosteroids alone or in conjunction with azathioprine (AZA) is the standard treatment in autoimmune hepatitis (AiH). Individual variations in thiopurine (TP) metabolism may affect both drug efficacy and toxicity. Our aim was to investigate the utility of thiopurine methyltransferase (TPMT) as well as thioguanine nucleotide (TGN) and methylthioinosine monophosphate (meTIMP) metabolite measurements with regard to clinical outcome. METHODS: Two hundred thirty-eight patients with AiH were included in this cross-sectional study. TPMT status was assessed in all patients, while TGN and meTIMP were measured in patients with ongoing TP medication. Clinical outcome was evaluated by liver tests and the ability to withdraw steroids. RESULTS: TPMT genotyping (n=229) revealed 207 (90.4%) wild-type and 22 heterozygous patients. One hundred forty-three patients had ongoing TP therapy with AZA (n=134) or mercaptopurine (MP; n=9); response was judged as complete response (CR) in 113 patients and partial response (PR) in 30 patients. Both TP dose (1.64 vs 1.19 mg/kg; p=0.012) and TPMT activity (14.3 vs 13.5; p=0.05) were higher in PR, resulting in similar TGN levels (PR: 121 pmol/8 x 10(8) red blood cells [RBC]; CR: 113 pmol/8 x 10(8) RBC; p=0.33) but higher meTIMP levels in PR (1350 vs 400 pmol/8 x 10(8) RBC; p=0.004). Patients able to withdraw steroids or who were using 5 mg prednisolone daily were treated with lower TP doses than patients on higher steroid doses (1.15 vs 1.18 vs 1.82 mg/kg; p<0.001). CONCLUSIONS: TP metabolite measurements are of clinical value in AiH patients who do not respond to standard TP treatment and for the identification of a shifted metabolism, which may demand an alternative treatment strategy.

Monitoring of thiopurine metabolites in patients with inflammatory bowel disease-what is actually measured?

Azathioprine and 6-mercaptopurine are often used in the treatment of patients with inflammatory bowel disease (IBD). They are prodrugs and undergo a complex metabolism to active and inactive metabolites. Thiopurine treatment is monitored in many laboratories by measuring metabolite concentrations in erythrocytes (red blood cells). The metabolites of interest are not measured directly but as hydrolysis products, which can be produced from several metabolites. The aim of this study was to examine which metabolites are actually measured during routine monitoring. Samples from 18 patients treated with a thiopurine were analyzed by a typical routine high-performance liquid chromatography method for therapeutic drug monitoring and by a newly developed specific method measuring thioguanosine monophosphate (TGMP), thioguanosine diphosphate (TGDP), and thioguanosine triphosphate (TGTP), as well as methylthioinosine monophosphate (meTIMP), and the results were compared. 6-Thioguanine nucleotide (TGN) values detected by the routine method were 69% (range 40%-90%) of the sum of TGMP, TGDP, and TGTP measured by the specific method. TGTP and TGDP contributed 85% (range 78%-90%) and 14% (range 10%-21%) of the TGN total, respectively. Thioguanosine was not found in any patient sample. The concentration of meTIMP obtained by the routine method was 548% of the value obtained by the specific method (range 340%-718%). The difference in TGN measurements between the routine and specific methods can be explained by low hydrolysis efficiency in the routine method, although the most likely explanation for the difference in meTIMP values is that not yet identified metabolites are codetermined in the routine high-performance liquid chromatography method. Concentrations reported as TGN during therapeutic drug monitoring of thiopurine metabolites consist of TGDP and TGTP with a minor contribution of the TGMP. Concentrations reported as meTIMP or methyl mercaptopurine consist in part of meTIMP, but other not yet identified metabolites are codetermined.

Pharmacogenetic studies of Paclitaxel in the treatment of ovarian cancer.

The purpose of this study was to evaluate the role of sequence variants in the CYP2C8, ABCB1 and CYP3A4 genes and the CYP3A4 phenotype for the pharmacokinetics and toxicity of paclitaxel in ovarian cancer patients. Thirty-eight patients were treated with paclitaxel and carboplatin. The genotypes of CYP2C8*1B, *1C, *2, *3, *4, *5, *6, *7, *8 and P404A, ABCB1 G2677T/A and C3435T, as well as CYP3A4*1B, were determined by pyrosequencing. Phenotyping of CYP3A4 was performed in vivo with quinine as a probe. The patients were monitored for toxicity and 23 patients underwent a more extensive neurotoxicity evaluation. Patients heterozygous for G/A in position 2677 in ABCB1 had a significantly higher clearance of paclitaxel than most other ABCB1 variants. A lower clearance of paclitaxel was found for patients heterozygous for CYP2C8*3 when stratified according to the ABCB1 G2677T/A genotype. In addition, the CYP3A4 enzyme activity in vivo affected which metabolic pathway was dominant in each patient, but not the total clearance of paclitaxel. The exposure to paclitaxel correlated to the degree of neurotoxicity. Our findings suggest that interindividual variability in paclitaxel pharmacokinetics might be predicted by ABCB1 and CYP2C8 genotypes and provide useful information for individualized chemotherapy.

IMPDH activity in thiopurine-treated patients with inflammatory bowel disease - relation to TPMT activity and metabolite concentrations.

AIMS: Azathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and occurrence of adverse events. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the role of inosine-5'-monophosphate dehydrogenase (IMPDH), which is a key enzyme in the de novo synthesis of guanine nucleotides and also strategically positioned in the metabolic pathway of thiopurines. METHODS: IMPDH was measured in 100 healthy blood donors. IMPDH, TPMT and metabolite concentrations were studied in 50 patients with IBD on stable thiopurine therapy. IMPDH activity was measured in peripheral blood mononuclear cells. TPMT activity, 6-methylthioinosine 5'-monophosphate (meTIMP) and 6-thioguanine nucleotide (6-TGN) concentrations were measured in red blood cells, which is the current practice in clinical monitoring of thiopurines. Enzyme activities were related to metabolite concentrations and clinical characteristics. RESULTS: A wide range of IMPDH activity was observed both in healthy blood donors (median 13.1, range 4.7-24.2 nmol mg(-1) protein h(-1)) and IBD patients (median 14.0, range 7.0-21.7). There was a negative correlation between IMPDH activity and dose-normalized meTIMP concentrations (r(s) = -0.31, P = 0.03), but no evident correlation to 6-TGN concentration or the meTIMP/6-TGN ratio. There were no significant correlations between TPMT activity and metabolite concentrations. CONCLUSION: Even though the meTIMP concentrations correlated inversely to the IMPDH activity, the role of IMPDH in balancing the formation of methylated and phosphorylated metabolites was not evident. Taken together, the results give cause to question established opinions about thiopurine metabolism.