Using in vitro receptor activity studies of synthetic cannabinoids to support the risk assessment of new psychoactive substances - A Swedish strategy to protect public health from harm.

In the past 15 years, close to 1000 of new psychoactive substances (NPS) have been reported in Europe and globally. At the time of identification, data on safety, toxicity and carcinogenic potential of many NPS are not available or very limited. To work more efficiently, a strategy and collaboration between the Public Health Agency of Sweden (PHAS) and the National Board of Forensic Medicine was established involving in vitro receptor activity assays to demonstrate neurological activity of NPS. This report summarizes the first results on the synthetic cannabinoid receptor agonists (SCRAs), and subsequent actions taken by PHAS. A total of 18 potential SCRAs were selected by PHAS for in vitro pharmacological characterization. 17 compounds could be acquired and investigated for their activity on the human cannabinoid-1 (CB1) receptors expressed together with the AequoScreen system in CHO-K1 cells. Dose-response curves were established using eight different concentrations in triplicates at three occasions with JWH-018 as reference. For the MDMB-4en-PINACA, MMB-022, ACHMINACA, ADB-BUTINACA, 5F-CUMYL-PeGACLONE, 5C-AKB48, NM-2201, 5F-CUMYL-PINACA, JWH-022, 5Cl-AB-PINACA, MPhP-2201, 5F-AKB57 the half maximal effective concentration values ranged from 2.2 nM (5F-CUMYL-PINACA) to 171 nM (MMB-022). EG-018 and 3,5-AB-CHMFUPPYCA were none-active. The results contributed to 14 of these compounds being scheduled as narcotics in Sweden. In conclusion, many of the emerging SCRAs are potent activators of the CB1 receptor in vitro, although some lack activity or are partial agonists. The new strategy proved useful when data on psychoactive effects of the SCRAs under investigation were not available or limited.

Pharmacogenetic studies of thiopurine methyltransferase genotype-phenotype concordance and effect of methotrexate on thiopurine metabolism.

The discovery and implementation of thiopurine methyltransferase (TPMT) pharmacogenetics has been a success story and has reduced the suffering from serious adverse reactions during thiopurine treatment of childhood leukaemia and inflammatory bowel disease. This MiniReview summarizes four studies included in Dr Zimdahl Kahlin's doctoral thesis as well as the current knowledge on this field of research. The genotype-phenotype concordance of TPMT in a cohort of 12 663 individuals with clinically analysed TPMT status is described. Notwithstanding the high concordance, the benefits of combined genotyping and phenotyping for TPMT status determination are discussed. The results from the large cohort also demonstrate that the factors of gender and age affect TPMT enzyme activity. In addition, characterization of four previously undescribed TPMT alleles (TPMT*41, TPMT*42, TPMT*43 and TPMT*44) shows that a defective TPMT enzyme could be caused by several different mechanisms. Moreover, the folate analogue methotrexate (MTX), used in combination with thiopurines during maintenance therapy of childhood leukaemia, affects the metabolism of thiopurines and interacts with TPMT, not only by binding and inhibiting the enzyme activity but also by regulation of its gene expression.

The influence of ABCG2 polymorphism on erlotinib efflux in the K562 cell line.

Single nucleotide polymorphisms (SNPs) in the gene for multidrug resistance protein ABCG2, an erlotinib transporter, is a possible contributor to the interindividual variation observed in erlotinib pharmacokinetics and toxicity. Therefore, the aim was to study erlotinib efflux by ABCG2 wild-type (wt) and ABCG2 polymorphic variants in the K562 cell line. The chronic myeloid leukemia K562 cell line, neither expressing EGFR nor ABCG2, was transduced with vectors containing the ABCG2 wt, the SNPs: 34 G > A and 421 C > A, or with empty vector (K562/ve). ABCG2-expressing cells were enriched using magnetic sorting and the expression was verified using flow cytometry. Intracellular erlotinib concentrations were analyzed by LC-MS/MS after incubation with 1 µmol/L erlotinib for 60 minutes. All recombinant cell lines were confirmed carriers of the vector and expressed ABCG2. Differences in intracellular erlotinib concentrations were observed between K562/ve and K562 ABCG2 wt and between K562/ve and K562 ABCG2 34G > A (both P ≤ .001, one-way ANOVA with Tukey HSD post hoc test), indicating that the cell lines carrying ABCG2 wt and ABCG2 34G > A actively transports erlotinib out of the cells. The ABCG2 34G > A cell line had a higher transport capacity compared with ABCG2 wt after adjusting for ABCG2 expression (P = .024, t test). No differences were observed between K562/ve and K562 ABCG2 421 C > A. Genetic polymorphism in the ABCG2 gene has an influence on the transport of erlotinib which can contribute to the observed variation in erlotinib pharmacokinetics and toxicity.

Erlotinib treatment induces cytochrome P450 3A activity in non-small cell lung cancer patients.

AIMS: Erlotinib is a tyrosine kinase inhibitor used in the treatment of non-small cell lung cancer highly metabolized by the cytochrome P450 (CYP) 3A. Hence, CYP3A4 activity might be a useful predictor of erlotinib pharmacokinetics in personalized medicine. The effect of erlotinib on CYP3A activity was therefore studied in non-small cell lung cancer patients. METHODS: The study included 32 patients scheduled for erlotinib monotherapy. CYP3A activity was assessed using quinine as a probe before and during erlotinib treatment. Plasma from blood samples drawn 16 hours post quinine administration were analysed using HPLC with fluorescence detection to determine the quinine/3-OH-quinine ratio. RESULTS: Matched samples, available from 13 patients, showed an induction of CYP3A activity (P = 0.003, Wilcoxon's signed rank test) after 2 months of treatment. The quinine/3-OH-quinine ratio decreased from 20.2 (± 13.4) at baseline to 11.0 (± 4.34). Single-point samples, available from 19 patients, supported the decrease in ratio (P = 0.007, Mann-Whitney U-test). Generally, females had a higher CYP3A activity both at baseline and after two months of treatment. Statistical analysis by gender also showed significant increase in CYP3A activity (males, n = 10, P = 0.001, and females, n = 22, P = 0.001). CONCLUSIONS: An induction of CYP3A activity was observed after 2 months of erlotinib treatment which was also seen when subdividing based on gender. It could be important to take this into consideration for patients co-administering other CYP3A-metabolizing drugs during erlotinib treatment and also makes it difficult to use baseline CYP3A activity to predict erlotinib pharmacokinetics.

Simple and cost-effective liquid chromatography-mass spectrometry method to measure dabrafenib quantitatively and six metabolites semi-quantitatively in human plasma.

Dabrafenib is an inhibitor of BRAF V600E used for treating metastatic melanoma but a majority of patients experience adverse effects. Methods to measure the levels of dabrafenib and major metabolites during treatment are needed to allow development of individualized dosing strategies to reduce the burden of such adverse events. In this study, an LC-MS/MS method capable of measuring dabrafenib quantitatively and six metabolites semi-quantitatively is presented. The method is fully validated with regard to dabrafenib in human plasma in the range 5-5000 ng/mL. The analytes were separated on a C18 column after protein precipitation and detected in positive electrospray ionization mode using a Xevo TQ triple quadrupole mass spectrometer. As no commercial reference standards are available, the calibration curve of dabrafenib was used for semi-quantification of dabrafenib metabolites. Compared to earlier methods the presented method represents a simpler and more cost-effective approach suitable for clinical studies. Graphical abstract Combined multi reaction monitoring transitions of dabrafenib and metabolites in a typical case sample.

Combination treatment with 6-mercaptopurine and allopurinol in HepG2 and HEK293 cells - Effects on gene expression levels and thiopurine metabolism.

Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP+oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. AP's effects in relation to thiopurine metabolism are complex. The network-identified susceptibility genes represented biological processes mainly associated with purine nucleotide biosynthetic processes, lymphocyte proliferation, NF-KB activation, JAK-STAT signaling, and apoptotic signaling at oxidative stress.

Determination of loss of consciousness: a comparison of clinical assessment, bispectral index and electroencephalogram: An observational study.

BACKGROUND: Computer-processed algorithms of encephalographic signals are widely used to assess the depth of anaesthesia. However, data indicate that the bispectral index (BIS), a processed electroencephalography monitoring system, may not be reliable for assessing the depth of anaesthesia. OBJECTIVE: The aim of this study was to evaluate the ability of the BIS monitoring system to assess changes in the level of unconsciousness, specifically during the transition from consciousness to unconsciousness, in patients undergoing total intravenous anaesthesia with propofol. We compared BIS with the electroencephalogram (EEG), and clinical loss of consciousness (LOC) defined as loss of verbal commands and eyelash reflex. DESIGN: This was an observational cohort study. SETTING: University Hospital Linköping, University Hospital Örebro, Finspång Hospital and Kalmar Hospital, Sweden from October 2011 to April 2013. PATIENTS: A total of 35 ASA I patients aged 18 to 49 years were recruited. INTERVENTIONS: The patients underwent total intravenous anaesthesia with propofol and remifentanil for elective day-case surgery. Changes in clinical levels of consciousness were assessed by BIS and compared with assessment of stage 3 neurophysiological activity using the EEG. The plasma concentrations of propofol were measured at clinical LOC and 20 and 30 min after LOC. MAIN OUTCOME MEASURES: The primary outcome was measurement of BIS, EEG and clinical LOC. RESULTS: The median BIS value at clinical LOC was 38 (IQR 30 to 43), and the BIS values varied greatly between patients. There was no correlation between BIS values and EEG stages at clinical LOC (r = -0.1, P = 0.064). Propofol concentration reached a steady state within 20 min. CONCLUSION: There was no statistically significant correlation between BIS and EEG at clinical LOC. BIS monitoring may not be a reliable method for determining LOC. CLINICAL TRIALS REGISTRY: This trial was not registered because registration was not mandatory at the time of the trial.

Differences in cardiovascular toxicities associated with cigarette smoking and snuff use revealed using novel zebrafish models.

Tobacco use is strongly associated with cardiovascular disease and the only avoidable risk factor associated with development of aortic aneurysm. While smoking is the most common form of tobacco use, snuff and other oral tobacco products are gaining popularity, but research on potentially toxic effects of oral tobacco use has not kept pace with the increase in its use. Here, we demonstrate that cigarette smoke and snuff extracts are highly toxic to developing zebrafish embryos. Exposure to such extracts led to a palette of toxic effects including early embryonic mortality, developmental delay, cerebral hemorrhages, defects in lymphatics development and ventricular function, and aneurysm development. Both cigarette smoke and snuff were more toxic than pure nicotine, indicating that other compounds in these products are also associated with toxicity. While some toxicities were found following exposure to both types of tobacco product, other toxicities, including developmental delay and aneurysm development, were specifically observed in the snuff extract group, whereas cerebral hemorrhages were only found in the group exposed to cigarette smoke extract. These findings deepen our understanding of the pathogenic effects of cigarette smoking and snuff use on the cardiovascular system and illustrate the benefits of using zebrafish to study mechanisms involved in aneurysm development.

Novel rapid liquid chromatography tandem masspectrometry method for vemurafenib and metabolites in human plasma, including metabolite concentrations at steady state.

A novel, rapid and sensitive liquid chromatography tandem-mass spectrometry method for quantification of vemurafenib in human plasma, that also for the first time allows for metabolite semi-quantification, was developed and validated to support clinical trials and therapeutic drug monitoring. Vemurafenib was analysed by precipitation with methanol followed by a 1.9 min isocratic liquid chromatography tandem masspectrometry analysis using an Acquity BEH C18 column with methanol and formic acid using isotope labelled internal standards. Analytes were detected in multireaction monitoring mode on a Xevo TQ. Semi-quantification of vemurafenib metabolites was performed using the same analytical system and sample preparation with gradient elution. The vemurafenib method was successfully validated in the range 0.5-100 µg/mL according to international guidelines. The metabolite method was partially validated owing to the lack of commercially available reference materials. For the first time concentration levels at steady state for melanoma patients treated with vemurafenib is presented. The low abundance of vemurafenib metabolites suggests that they lack clinical significance. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Role of cytochrome P450 2C8*3 (CYP2C8*3) in paclitaxel metabolism and paclitaxel-induced neurotoxicity.

AIM: The CYP2C8*3 allele has been suggested as a risk factor for paclitaxel-induced neuropathy but the data hitherto published are conflicting. MATERIALS & METHODS: In total 435 patients were investigated with respect to maximum neuropathy grade and accumulated paclitaxel dose. The enzymatic properties of CYP2C8.3 variant were analyzed using heterologous mammalian HEK293 cell expression system. RESULTS: No significant association between CYP2C8*3 allele and neuropathy was found, although a trend was observed. The paclitaxel and amodiaquine metabolism by CYP2C8.3 were found similar to CYP2C8.1, whereas CYP2C8.3 was more efficient in the metabolism of rosiglitazone. CONCLUSION: These results indicate a difference in substrate specificity between CYP2C8.1 and CYP2C8.3; however, the CYP2C8*3 allele has no major impact on paclitaxel metabolism in vitro or of paclitaxel-induced neuropathy in vivo. Original submitted on 6 February 2015; revision submitted on 9 April 2015.

A validated liquid chromatography tandem mass spectrometry method for quantification of erlotinib, OSI-420 and didesmethyl erlotinib and semi-quantification of erlotinib metabolites in human plasma.

A liquid chromatography tandem mass spectrometry method was developed and validated for quantification of erlotinib and its metabolites in human plasma. The method is suitable for therapeutic drug monitoring and pharmacokinetic studies. The substances were extracted using protein precipitation, separated on a BEH XBridge C18 column (100 ×2.1 mm, 1.7 µm) by gradient elution at 0.7 mL/min of acetonitrile and 5 mM ammonium acetate. The concentration was determined using a Waters Xevo triple quadrupole mass spectrometer in a multi reaction monitoring mode. The total run time was 7 min. Deuterated erlotinib and OSI-597 were used as internal standard for erlotinib and its metabolites, respectively. Erlotinib, OSI-420 and didesmethyl erlotinib were quantified in the concentration range 25-5000 ng/mL, 0.5-500 ng/mL and 0.15-10 ng/mL, respectively. Precision and accuracy was <14% except for OSI-420 at LLOQ (17%). Extraction recovery was above 89%, 99% and 89% for erlotinib, OSI-420 and didesmethyl erlotinib, respectively. The human liver microsomes generated 14 metabolites, three of them not previously reported. Twelve metabolites were measured semi-quantitatively and validated with respect to selectivity, precision and stability.

MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool.

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

Single-nucleotide polymorphisms of ABCG2 increase the efficacy of tyrosine kinase inhibitors in the K562 chronic myeloid leukemia cell line.

OBJECTIVE: The tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia are substrates for the efflux transport protein ATP-binding cassette subfamily G member 2 (ABCG2). Variations in ABCG2 activity might influence pharmacokinetics and therapeutic outcome of TKIs. The role of ABCG2 single-nucleotide polymorphisms (SNPs) in TKI treatment is not clear and functional in-vitro studies are lacking. The aim of this study was to investigate the consequences of ABCG2 SNPs for transport and efficacy of TKIs [imatinib, N-desmethyl imatinib (CGP74588), dasatinib, nilotinib, and bosutinib]. MATERIALS AND METHODS: ABCG2 SNPs 34G>A, 421C>A, 623T>C, 886G>C, 1574T>G, and 1582G>A were constructed from ABCG2 wild-type cDNA and transduced to K562 cells by retroviral gene transfer. Variant ABCG2 expression in cell membranes was evaluated and the effects of ABCG2 SNPs on transport and efficacy of TKIs were measured as the ability of ABCG2 variants to protect against TKI cytotoxicity. RESULTS: Wild-type ABCG2 had a protective effect against the cytotoxicity of all investigated compounds except bosutinib. It was found that ABCG2 expression provided better protection against CGP74588 than its parent compound, imatinib. ABCG2 421C>A, 623T>C, 886G>C, and 1574T>G reduced cell membrane expression of ABCG2 and the protective effect of ABCG2 against imatinib, CGP74588, dasatinib, and nilotinib cytotoxicity. CONCLUSION: These findings show that the ABCG2 SNPs 421C>A, 623T>C, 886G>C, and 1574T>G increase the efficacy of investigated TKIs, indicating a reduced transport function that might influence TKI pharmacokinetics in vivo. Furthermore, the active imatinib metabolite CGP74588 is influenced by ABCG2 expression to a greater extent than the parent compound.

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.

Increased sensitivity to thiopurines in methylthioadenosine phosphorylase-deleted cancers.

The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are used in the treatment of leukemia. Incorporation of deoxythioguanosine nucleotides (dG(s)) into the DNA of thiopurine-treated cells causes cell death, but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Because the growth of MTAP-deleted tumor cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP(-)) transfected to express MTAP constitutively (A549-MTAP(+)). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intracellularly to MeTIMP, was markedly higher in both cell lines under MTAP(-) conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dG(s) incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.

A validated and rapid high-performance liquid chromatography method for the quantification of conversion of radio-labelled sex steroids.

The 17ß-hydroxysteroid dehydrogenase enzymes modify the availability of potent sex steroids and have thus attracted interest in the study of several steroid-dependent pathologies including breast, endometrial and prostate cancers. An increased awareness of the importance of steroidogenic enzymes has brought forth a demand for efficient assays to study the effects of individual enzymes on steroid levels. Methods used for assessing steroid conversion are often laborious and frequently involve hazardous sample preparation steps. We developed and validated an optimised simple method for sample preparation of sex steroids using protein precipitation by the addition of zinc sulphate/sodium hydroxide. The interconversion of radio-labelled oestrogens and androgens was quantified using high-performance liquid chromatography separation of oestrone, oestradiol, androstenedione and testosterone followed by online radiometric flow scintillation analysis. The method, which can be applied for assessing, e.g., the efficacy of inhibitors of steroidogenic enzymes, was successfully used for evaluating oestrogenic interconversion in breast cancer cell lines MCF7 and T-47D.