Thiopurine Drugs Research Articles

A well-tolerated and rapidly acting thiopurine for IBD?

Thiopurine drugs continue to be a cornerstone of inflammatory bowel disease (IBD) treatment. Thiopurines are economical compared with many newer medical treatments for IBD, other chronic inflammatory diseases and leukaemia, although they are not without their shortcomings. These include a slow-onset therapeutic action and many adverse drug reactions. This feature article surveys published data, unpublished in vitro and in vivo experiments, as well as clinical experience, underpinning a rationale for bringing a novel thiopurine drug formulation to market. This formulation has a rapid action making it suitable for the induction and maintenance treatment of IBD and avoids most thiopurine-associated adverse reactions.

A design for external quality assessment for the analysis of thiopurine drugs: pitfalls and opportunities.

Abstract Background For the analysis of 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine ribonucleotides (6-MMPR), no external quality assessment scheme (EQAS) is currently available and no quality control samples can be made because of the absence of pure substances. An experimental design is tested to compare laboratory analytical results. Methods In this EQAS, participating laboratories were asked to select patient samples from their routine analysis and exchange these with a coupled laboratory. Because of large differences in results between laboratories, all standard operating procedures were reviewed, revealing that the origin of these differences could be in the method of hydrolysis and the preparation of calibrators. To investigate the contribution of the calibrators to these differences, one participating laboratory was asked to prepare a batch of calibrators to be shipped to the participating laboratories for analysis. Results Results for 6-TGN differed more between laboratories, compared with results for 6-MMPR. For 6-TGN and 6-MMPR 43% and 24% of the results, respectively, were out of the 80%–120% range. When correcting the results from the exchange of the patient samples with the results of the calibrators, the mean absolute difference for 6-TGN improved from 24.8% to 16.3% (p<0.001), while the results for 6-MMPR worsened from 17.3% to 20.0% (p=0.020). Conclusions This first EQAS for thiopurine drugs shows that there is a difference between laboratories in the analysis of 6-TGN, and to a lesser extent in the analysis of 6-MMPR. This difference for 6-TGN can partially be explained by the use of in-house-prepared calibrators that differ among the participants.

Electrochemical Sensor Based on Nanocomposite of Ionic Liquid Modified Graphene Oxide – Chitosan and its Application for Flow Injection Detection of Anticancer Thiopurine Drugs

AbstractElectrochemical activity of carbositall electrodes (CSEs) covered with polymeric films of Chitosan (Chit) containing different types of graphene oxide (GO) dispersed in a room temperature ionic liquid (IL) was comparatively investigated by using cyclic voltammetry of [Fe(CN)6]3−/4− as a model redox probe. It was recognized that the nanocomposite film with in situ electrochemically reduced GO (ERGO) provided an improved electrochemical activity, including increased peak current and electron‐transfer efficiency, compared to the initial GO nanosheets and chemically reduced GO (RGO). The novel electrochemical sensor ERGO‐IL‐Chit/CSE showed enhanced electrochemical response characteristics towards the antimetabolic thiopurines such as 6‐thioguanine, 6‐mercaptopurine and azathioprine which play a significant role in medicinal chemistry and cancer therapy. The diagnostic characteristics of the voltammetric signals for all investigated compounds in neutral aqueous media were established and the nature of the corresponding redox‐reactions was discussed. The developed sensor was incorporated into a flow injection amperometric system for the automatic dissolution testing of thiopurine anticancer drugs with a sampling rate of 60 h−1.

Variants in TPMT, ITPA, ABCC4 and ABCB1 Genes As Predictors of 6-mercaptopurine Induced Toxicity in Children with Acute Lymphoblastic Leukemia.

SummaryBackgroundAcute lymphoblastic leukemia is the most common childhood malignancy. Optimal use of anti leukemic drugs has led to less toxicity and adverse reactions, and a higher survival rate. Thiopurine drugs, including 6-mercaptopurine, are mostly used as antileukemic medications in the maintenance phase of treatment for children with acute lymphoblastic leukemia. For those patients, TPMT genotype- tailored 6-mercaptopurine therapy is already implemented in the treatment protocols. We investigated the role of TPMT, ITPA, ABCC4 and ABCB1 genetic variants as predictors of outcome and 6-mercaptopurine induced toxicity during the maintenance phase of treatment in pediatric acute lymphoblastic leukemia.MethodsSixty-eight children with acute lymphoblastic leukemia were enrolled in this study. Patients have been treated according to ALL IC-BFM 2002 or ALL IC-BFM 2009 protocols. Toxicity and adverse events have been monitored via surrogate markers (off-therapy weeks, episodes of leu - ko penia and average 6-mercaptopurine dose) and a prob- abilistic model was employed to predict overall 6-mercaptopurine related toxicity.ResultsWe confirmed that patients with acute lymphoblastic leukemia that carry inactive TPMT allele(s) require 6- mercaptopurine dose reduction. ITPA and ABCC4 genetic variants failed to show an association with 6-mercapto - purine induced toxicity during the maintenance phase. Carriers of ABCB1 variant allele experienced greater hepatotoxicity. The probabilistic model Neural net which considered all the analysed genetic variants was assessed to be the best prediction model. It was able to discriminate ALL patients with good and poor 6-mercaptopurin tolerance in 71% of cases (AUC=0.71).ConclusionsThis study contributes to the design of a panel of pharmacogenetic markers for predicting thiopurineinduced toxicity in pediatric ALL.

Three Faces of Mercaptopurine Cytotoxicity In Vitro: Methylation, Nucleotide Homeostasis, and Deoxythioguanosine in DNA.

Mercaptopurine (MP) is a cytotoxic thiopurine important for the treatment of cancer and autoimmune diseases. MP and other thiopurine drugs undergo extensive intracellular metabolism, but the mechanisms of action are poorly characterized. In particular, it is unknown how different metabolites contribute to cytotoxicity and incorporation of thiopurine bases into DNA. The aim of this study was to ask whether cytotoxicity results from the incorporation of thioguanosine nucleotides into DNA, an alternative thiopurine metabolite, or a combination of factors. Therefore, we measured the cytotoxicity, metabolism, and incorporation of thioguanosine into DNA in response to MP or MP metabolites. Thiopurine metabolites varied in cytotoxicity, with methyl-thioinosine-mono-phosphate and thioguanosine-tri-phosphate the most toxic, and the methyl-thioguanosine nucleotides the least. We show, using liquid chromatography-tandem mass spectrometry, how different metabolites may perturb biochemical pathways, particularly disrupting guanosine nucleotide homeostasis, that may contribute to the mechanism of action of thiopurines. Although there was no correlation between metabolite cytotoxicity and the levels of 6-methylthioinosine-mono-phosphate or thioguanosine incorporation into DNA as individual factors, a combined analysis suggested that these factors together had a major influence on cytotoxicity. This study emphasizes the importance of enzymes of nucleotide homeostasis, methylation, and demethylation in thiopurine effects. These results will facilitate the development of dynamic biochemical models of thiopurine biochemistry that will improve our understanding of mechanisms of action in relevant target tissues.

Vanda v. West-Ward Pharmaceuticals: Good News for the Patent Eligibility of Diagnostics and Personalized Medicine, with Some Important Caveats

In Mayo v. Prometheus, decided in 2011, the U.S. Supreme Court invalidated patent claims directed towards diagnostic methods useful in the optimization of drug dosage for the needs of an individual patient, i.e., an example of personalized medicine, based on the Court’s determination that the claims were directed towards a patent ineligible law of nature. Notably, the claims in Mayo did not recite a step of applying the information generated by the test, e.g., a step of administering the drug to a patient at the optimized dosage. Some, including this author, have speculated that inclusion of such a step might have saved Prometheus’ claims. In 2018 the U.S. Court of Appeals of the Federal Circuit issued a decision in Vanda Pharmaceuticals v. West-Ward Pharmaceuticals International Limited wherein a divided panel held that inclusion of a step requiring action based on the result of a diagnostic test rendered the claims patent eligible. This is good news for innovators in diagnostics and personalized medicine, but there are some important caveats. While the Vanda majority makes a compelling argument that the claims at issue are patent eligible under Mayo, a well-reasoned dissent comes to the opposite conclusion. So it is not clear whether Vanda represents the last word on the subject, given that other judges on the Federal Circuit might agree with the dissent. Another important caveat is that the inclusion of an action step likely renders the claim difficult, if not impossible, to enforce, depending upon the nature of the alleged infringement. In Vanda, the court’s finding of infringement hinged upon the fact that the infringer, a generic drug company seeking FDA approval to market a generic version of the patent owner’s branded drug, was required by FDA law to include directions on the product label essentially advising doctors to perform the claimed method. Absent this fairly direct linkage between a mandated drug label and the claimed method, the issue of divided infringement will in many cases limit the enforce-ability of diagnostic method claims that recite an action step. In particular, if Prometheus had positively recited an action step in its claims the company would not have been able to establish infringement by a competing diagnostic testing company like Mayo.

ITPA Activity in Children Treated by Azathioprine: Relationship to the Occurrence of Adverse Drug Reactions and Inflammatory Response.

Azathioprine (AZA), a thiopurine drug, is widely used in the treatment of children with immunological diseases such as inflammatory bowel disease (IBD) and autoimmune hepatitis (AIH); however, interindividual variability in the occurrence of adverse drug reactions (ADRs) and drug response is observed. This study investigated (i) the relationships between inosine triphosphate pyrophosphatase (ITPA) activity, an enzyme involved in thiopurine metabolism, and the occurrence of ADRs in children with immunological disease on AZA therapy, and (ii) the relationship between ITPA activity and the inflammatory activity observed in children with IBD. ITPA and TPMT activities were determined in 106 children with immunological disease on AZA therapy. Markers of hepatotoxicity, myelotoxicity, pancreatitis and inflammation as well as clinical information were retrospectively collected during regular medical visits. No significant association was found between ITPA activity and hepatotoxicity or clinical ADRs such as cutaneous reactions, arthralgia, flulike symptoms and gastrointestinal disorders. Concerning myelotoxicity, a significant relation was observed between ITPA activity and RBC mean corpuscular volume (MCV; p=0.003). This observation may be related to the significant relationship found between high ITPA activity and the increase in γ-globulin level reflecting inflammation (p=0.005). In our study, ITPA activity was not associated with occurrence of ADRs, but a relationship between high ITPA activity and γ-globulin, a marker of inflammation, was found in children with IBD. Therefore, measurement of ITPA activity may help to identify children with IBD predisposed to residual inflammation on AZA therapy. Further prospective studies are needed to confirm this result.

Analytical and clinical validation of an LC-MS/MS method to measure thiopurine S-methyltransferase activity by quantifying d3-6-MMP

BackgroundIdentification of patients with thiopurine S-methyltransferase (TPMT) deficiency prior to thiopurine drug therapy has become routine clinical practice worldwide. To measure TPMT activity, traditional radiochemical assays have been replaced by chromatographic methods. MethodInspired by the increasing number of isotope labelled sources that may be of benefit for the TPMT assay, a new LC-MS/MS method for TPMT activity was developed and validated. Isotope labelled d3-S-adenosyl-l-methionine (d3-SAM) was selected for the enzymatic methylation of mercaptopurine during sample incubation; d3-6-methylmercaptopurine (d3-6-MMP) with d2-2, 8-hypoxanthine as the internal standard was quantified to ascertain individual TPMT activity. ResultsThe validation of the analytical part of this method showed good linearity (coefficient of determination 0.9999 in the range of 1–500 ng/mL) with the intra-and inter-day impression CV% between 7.6% and 9.1% and 3.7% and 9.2%, respectively. Recovery ranged from 94.9% to 112.3%. The specificity of the enzymatic reaction was validated by using 108 clinical check samples. After compared with traditional radiochemical assay and genotype results, all homozygous and heterozygous deficiency clinical checks fitted into the nominal groups, inter-batch and intra–batch impression CV% were between 2.3% and 9.7%. ConclusionWith the inclusion of isotope labelled substrate, interfering non-enzymatic methylation no longer results in potential false assignment of abnormal patients. Furthermore, the method can be applied to patients who have already been prescribed thiopurine drugs. This new LC-MS/MS is therefore a favourable clinical routine application to test TPMT activity, as it shows excellent performance in identifying patients with TPMT deficiency.

P731 The impact of thiopurine drugs on the natural history and surgical outcome of ulcerative colitis: A cohort study

Thiopurines are used as maintenance therapy in ulcerative colitis, but whether these drugs influence the natural history of the disease is unknown. We aimed to assess the effect of thiopurines in terms of colectomy, hospital admission, progression in disease extent and anti-TNF therapy within 10 years. Patients diagnosed with ulcerative colitis within the Örebro University Hospital catchment area, during 1963–2010 (n = 1007), who initiated thiopurines (n = 253) were included. To overcome the risk of confounding by indication, we used a novel approach and compared patients who stopped treatment within 12 months because of an adverse reaction (n = 76) with patients who continued therapy or discontinued due to other reasons (n = 177). Long-term outcomes were assessed by Cox-regression analyses with adjustments for confounding factors including sex, age, hospital admission at diagnosis, disease extent, and previous medication at first thiopurine exposure. No significant difference in baseline characteristics was observed between the two groups. The cumulative probability of colectomy within 10 years was 19.5% in tolerant patients compared with 29.0% in intolerant, (adjusted HR: 0.43; 95% confidence interval (CI) 0.24 to 0.79; Figure 1). The probability of hospital admission was 34.0 % in tolerant vs. 56.2% in intolerant patients (adjusted HR: 0.38; 95% CI 0.25 to 0.59). The risk for progression in disease extent was 20.4% in tolerant patients compared with 48.8% in intolerant, (adjusted HR: 0.44; 95% CI 0.20 to 0.99). Within 10 years, 16.1% of tolerant and 27.5% of intolerant patients received anti-TNF therapy, (adjusted HR: 0.43; 95% CI 0.24 to 0.79). Figure 1 Based on the novel approach of comparing patients tolerant and intolerant to thiopurines, we, for the first time, reveal that thiopurines have a profound beneficial impact of the natural history and long-term colectomy rates of ulcerative colitis.

Addition of Allopurinol for Altering Thiopurine Metabolism to Optimize Therapy in Patients with Inflammatory Bowel Disease.

Thiopurine drugs, including azathioprine and 6-mercaptopurine, are used commonly in patients with inflammatory bowel disease for maintenance of remission. Although generally well tolerated, adverse effects lead to discontinuation in a significant minority of patients. Pharmacogenomic studies have suggested that metabolic breakdown of azathioprine in an individual is genetically determined. Coupled with the fact that certain thiopurine metabolites, notably 6-thioguanine nucleotide and 6-methylmercaptopurine, are associated with antiinflammatory effects and adverse effects, respectively, some investigators have examined intentionally shunting the metabolism of azathioprine toward increasing 6-thioguanine nucleotide levels by using low doses of the xanthine oxidoreductase inhibitor allopurinol to improve efficacy and decrease toxicity of azathioprine in patients with inflammatory bowel disease. We performed a search of the MEDLINE and Embase databases for basic and clinical research reports of this modality. Pertinent articles were retrieved, reviewed, and assessed by the authors. Case series, cohort studies, and one randomized trial have investigated adding allopurinol to azathioprine therapy in patients with inflammatory bowel disease. Most reports primarily examined metabolite levels in these patients. In general, the literature suggests that this modality was successful at significantly increasing 6-thioguanine nucleotide levels while decreasing 6-methylmercaptopurine levels. Several small reports have suggested that patients with increased 6-thioguanine nucleotide levels had improved symptoms or symptom remission. Adverse effects and discontinuation rates remained similar or were improved in patients who were taking a thiopurine and started allopurinol. In conclusion, the addition of allopurinol may be an option for optimizing thiopurine metabolite production in select patients with low 6-thioguanine nucleotide levels. Appropriate care and monitoring of these patients are mandatory to prevent neutropenia or other adverse effects.

Thiopurine Drugs Repositioned as Tyrosinase Inhibitors.

Drug repositioning is the application of the existing drugs to new uses and has the potential to reduce the time and cost required for the typical drug discovery process. In this study, we repositioned thiopurine drugs used for the treatment of acute leukaemia as new tyrosinase inhibitors. Tyrosinase catalyses two successive oxidations in melanin biosynthesis: the conversions of tyrosine to dihydroxyphenylalanine (DOPA) and DOPA to dopaquinone. Continuous efforts are underway to discover small molecule inhibitors of tyrosinase for therapeutic and cosmetic purposes. Structure-based virtual screening predicted inhibitor candidates from the US Food and Drug Administration (FDA)-approved drugs. Enzyme assays confirmed the thiopurine leukaemia drug, thioguanine, as a tyrosinase inhibitor with the inhibitory constant of 52 μM. Two other thiopurine drugs, mercaptopurine and azathioprine, were also evaluated for their tyrosinase inhibition; mercaptopurine caused stronger inhibition than thioguanine did, whereas azathioprine was a poor inhibitor. The inhibitory constant of mercaptopurine (16 μM) was comparable to that of the well-known inhibitor kojic acid (13 μM). The cell-based assay using B16F10 melanoma cells confirmed that the compounds inhibit mammalian tyrosinase. Particularly, 50 μM thioguanine reduced the melanin content by 57%, without apparent cytotoxicity. Cheminformatics showed that the thiopurine drugs shared little chemical similarity with the known tyrosinase inhibitors.

Validation of a high-performance liquid chromatography method for thiopurine S-methyltransferase activity in whole blood using 6-mercaptopurine as substrate.

Variation in metabolism, toxicity and therapeutic efficacy of thiopurine drugs is largely influenced by genetic polymorphisms in the thiopurine S-methyltransferase (TPMT) gene. Determination of TPMT activity is routinely performed in patients to adjust drug therapy. We further optimized a previously established high-performance liquid chromatography (HPLC) method by measuring TPMT activity in whole blood instead of isolated erythrocytes, which is based on conversion of 6-mercaptopurine to 6-methylmercaptopurine using S-adenosyl-methionine as methyl donor. The simplified TPMT whole-blood method showed similar or better analytical and diagnostic performance compared with the former erythrocyte assay. The whole-blood method was linear for TPMT activities between 0 and 40 nmol/(mL·h) with a quantification limit of 0.1 nmol/(mL·h). Within-day imprecision and between-day imprecision were ≤5.1% and ≤8.5%, respectively. The optimized method determining TPMT activity in whole blood (y) showed agreement with the former method determining TPMT activity in erythrocytes (x) (n=45, y=1.218+0.882x; p>0.05). Phenotype-genotype concordance (n=300) of the whole-blood method was better when TPMT activity was expressed per volume of whole blood (specificity 92.2%), whereas correction for hematocrit resulted in lower genotype concordance (specificity 86.9%). A new cutoff for the whole-blood method to distinguish normal from reduced TPMT activity was determined at ≤6.7 nmol/(mL·h). This optimized TPMT phenotyping assay from whole blood using 6-MP as substrate is suitable for research and routine clinical analysis.

Capillary Electrophoresis Hyphenated with Mass Spectrometry for Determination of Inflammatory Bowel Disease Drugs in Clinical Urine Samples.

Azathioprine is the main thiopurine drug used in the treatment of immune-based inflammations of gastrointestinal tract. For the purpose of therapy control and optimization, effective and reliable analytical methods for a rapid drug monitoring in biological fluids are essential. Here, we developed a separation method based on the capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) for the simultaneous determination of azathioprine and its selected metabolites (6-thioguanine, 6-mercaptopurine, and 6-methylmercaptopurine) as well as other co-medicated drugs (mesalazine, prednisone, and allopurinol). The optimized CE-MS/MS conditions provided a very efficient and stable system for the separation and sensitive detection of these drugs in human urine matrices. The developed method was successfully applied for the assay of the targeted drugs and their selected metabolites in urine samples collected from patients suffering from inflammatory bowel disease (IBD) and receiving azathioprine therapy. The developed CE-MS/MS method, due to its reliability, short analysis time, production of complex clinical profiles, and favorable performance parameters, evaluated according to FDA guidelines for bioanalytical method validation, is proposed for routine clinical laboratories to optimize thiopurine therapy, estimate enzymatic activity, and control patient compliance with medication and co-medication.

Thiopurine Therapy Risk Evaluation for Inflammatory Bowel Disease Patients Using TPMT Enzyme Expression Method

Introduction: Thiopurine drugs, what are widely used medications for the treatment of inflammatory bowel diseases (IBD), have remarcable adverse effects like myelosuppression, leading to life threatening complications (severe infection or bleeding). Thiopurine methyltransferase (TPMT) plays a significant role in the metabolism of thiopurine drugs. Low TPMT activity in body is associated with pathological thiopurine drug metabolisms, overproduction of cytotoxic metabolites and myelosuppression. Methods: A purpose of our study was to compare TPMT enzyme expression analysis in patients with ulcerative colitis (UC) and Crohn's disease (CD) who are using azathioprine therapy with patients who have not yet begun this therapy. A prospective pilot study included 20 patients (55% female n=11 and 45% male n=9) having an age ranging from 22 to 79 years. All participants where admitted to Pauls Stradins Clinical university hospital (Riga, Latvia) from January 2017 to May 2017. All included patients received standard treatment of 5-ASA, steroid or azathioprine; leucocyte level was no lower than 4 x 109/L. The expression of TPMT in each blood sample was determined using ELISA (MyBioSource, USA) method. Results: All patient was previously diagnosed with IBD and now was admitted to hospital because of IBD exacerbation. 70 % of patients (n=14) was diagnosed with UC, 30 (n=6) with CD. 75% (n=15) of patients had not previously received azathioprine. 15% (n=3) had received azathioprine therapy, but stopped using it because of negative side effects like dyspepsia, acute pancreatitis, rash or exacerbation symptoms. 10% (n=2) was still receiving azathioprine therapy. Patients TPMT expression was in the range of 1.4 to 50 U/mL. All respondents were divided into the three TPMT enzyme activity groups. Activity of TPMT was low (< 5.5 U/mL) in 10% (n=2), intermediate (5.6-15.5 U/mL) in 5% (n=1) and normal/high (>15.6 U/mL) ir 85% (n=17) of patients. Conclusion: The results of this study confirmed the important role of the TPMT enzyme activity in the therapeutic drug monitoring. 15% of examined patients had decreased TPMT activity. Further studies in combination with TPMT genotype analysis are necessary for additional evaluation of treatment risks.

Pharmacokinetic Effect of Anti-tumor Necrosis Factor-alpha Agents on Thiopurine Drug Metabolism in Inflammatory Bowel Diseases

Introduction: Thiopurines and anti-tumor necrosis factor-alpha (anti-TNF) drugs are commonly used in treatment of inflammatory bowel diseases (IBD). Studies have established that thiopurines, when used in combination with anti-TNF drugs, affect the metabolism of anti-TNF drugs, resulting in higher serum drug concentrations. However, the data on the effect of anti-TNF drugs on thiopurine metabolism is limited. Methods: All adult IBD patients followed at our IBD center who had thiopurine metabolite concentrations measured between 2000-2016 were eligible for inclusion if they had at least 1 level measured while on thiopurine monotherapy, and 1 level measured while on combination therapy with an anti-TNF and thiopurine drug with the same thiopurine drug dose. Thiopurine metabolite concentration, white blood cell (WBC) count, mean corpuscular volume (MCV) and serum alanine aminotransferase (ALT) levels were compared on monotherapy and combination therapy. Paired t-test and Wilcoxon rank sum test were used for analysis of variables with normal and non-normal distribution, respectively. Results: 30 patients met the inclusion criteria (Table 1). Mean 6TGN level (in pmol/8 x 108 RBCs) on monotherapy and combination therapy were 201 and 267 respectively (% increase 33%; 95% CI 30.1-105.8; p 0.0004). There was a corresponding decrease in mean WBC count (in x1000/mL) from 7.6 to 6.1 (% decrease 20%; 95% CI 0.49-2.85; p 0.01) (Figure 1). There was no significant difference in 6-MMP levels, MCV and ALT levels on monotherapy and combination therapy (Table 2). On subgroup analysis, patients with therapeutic 6TGN levels >230 (n=13) had a significant increase in 6TGN levels (301 vs 369; p 0.03), and a corresponding decrease in WBC count (8.0 vs 5.7; p 0.07) on combination therapy. One patient developed leukopenia (WBC 2.8) after starting infliximab with an increase in 6TGN levels from 425 to 516.Figure: Thiopurine metabolite concentration and WBC count on monotherapy and combination therapy.Table: Table. Baseline CharacteristicsTable: Table. Lab parameters on monotherapy and combination therapyConclusion: In patients with IBD on thiopurine monotherapy, the addition of an anti-TNF agent can lead to an increase in 6-TGN concentration, thereby increasing the risk of leukopenia. Hence, elective thiopurine dose reduction should be considered prior to starting an anti-TNF agent, particularly in patients with therapeutic 6TGN concentration on thiopurine monotherapy. Our findings also suggest that benefits of combination therapy may be, in part, due to increase in thiopurine metabolite concentration with addition of an anti-TNF agent.

ITPA Activity in Adults and Children Treated With or Without Azathioprine: Relationship Between TPMT Activity, Thiopurine Metabolites, and Co-medications.

The implication of inosine triphosphate pyrophosphatase (ITPA) on thiopurine drug response variability has been investigated but little data are available on its role on thiopurine metabolites. The ability of ITPA to modify the thiopurine metabolite levels is currently used to optimize azathioprine (AZA) therapy in relation to thiopurine S-methyltransferase (TPMT) activity, the aim of this study is to investigate ITPA phenotype in a large population and to evaluate the relation between ITPA and TPMT activities and thiopurine metabolites. ITPA activity was determined in 183 adults and 138 children with or without AZA therapy. 6-thioguanine nucleotides (6-TGN), 6-methylmercaptopurine nucleotides (6-MeMPN) levels, and ITPA as well as TPMT activities were measured in red blood cells. Using the Gaussian mixture model, distribution of ITPA activity was evaluated. Intraindividual variability and influence of age, sex, AZA treatment and associated co-medications on ITPA activity were also assessed. This retrospective study shows a quadrimodal distribution in ITPA activity. No influence of age, sex, AZA therapy, and co-medications was found. In adults, ITPA activity was not significantly associated with 6-TGN or 6-MeMPN concentrations, whereas a weak negative correlation was observed with 6-MeMPN levels in pediatric populations (rs = -0.261; P = 0.024). A weak positive correlation was observed between ITPA and TPMT activities in children (rs = 0.289; P = 0.001). ITPA activity was poorly influenced by nongenetic parameters and has no influence on 6-TGN and 6-MeMPN concentrations in adults and only a weak correlation with 6-MeMPN and TPMT activity in children. These results demonstrate that ITPA is not a rate-limiting enzyme in the formation of 6-TGN but suggest that a decrease in ITPA activity in children may be a risk factor for accumulation of 6-MeMPN in cells.

Thiopurine S-methyltransferase polymorphisms in acute lymphoblastic leukemia, inflammatory bowel disease and autoimmune disorders: influence on treatment response.

The thiopurine S-methyltransferase (TPMT) gene encodes for the TPMT enzyme that plays a crucial role in the metabolism of thiopurine drugs. Genetic polymorphisms in this gene can affect the activity of the TPMT enzyme and have been correlated with variability in response to treatment with thiopurines. Advances in the pharmacogenetics of TPMT allowed the development of dosing recommendations and treatment strategies to optimize and individualize prescribing thiopurine in an attempt to enhance treatment efficacy while minimizing toxicity. The influence of genetic polymorphisms in the TPMT gene on clinical outcome has been well-documented and replicated in many studies. In this review, we provide an overview of the evolution, results, conclusions and recommendations of selected studies that investigated the influence of TPMT pharmacogenetics on thiopurine treatment in acute lymphoblastic leukemia, inflammatory bowel disease and autoimmune disorders. We focus mainly on prospective studies that explored the impact of individualized TPMT-based dosing of thiopurines on clinical response. Together, these studies demonstrate the importance of preemptive TPMT genetic screening and subsequent dose adjustment in mitigating the toxicity associated with thiopurine treatment while maintaining treatment efficacy and favorable long-term outcomes. In addition, we briefly address the cost-effectiveness of this pharmacogenetics approach and its impact on clinical practice as well as the importance of recent breakthrough advances in sequencing and genotyping techniques in refining the TPMT genetic screening process.

Thiopurine S-Methyltransferase as a Pharmacogenetic Biomarker: Significance of Testing and Review of Major Methods.

Background: Thiopurine S-methyltransferase (TPMT) enzyme metabolizes thiopurine drugs which are widely used in various disciplines as well as in leukemias. Individual enzyme activity varies depending on the genetic polymorphisms of TPMT gene located at chromosome 6. Up to 14% of popu-lation is known to have a decreased enzyme activity, and if treated with standard doses of thiopurines, these individuals are at a high risk of severe Adverse Drug Reactions (ADR) as myelosuppression, gas-trointestinal intolerance, pancreatitis and hypersensitivity. However, TPMT-deficient patients can suc-cessfully be treated with decreased thiopurine doses if enzyme status is identified by a prior testing. TPMT status identification is a pioneering experience in application of pharmacogenetic testing in clini-cal settings. 4 TPMT (*2, *3A, *3B, *3C) alleles are known to account for 80-95% of a decreased en-zyme activity, and therefore, identifying the presence of these alleles supported by phenotypic measure-ment of the enzyme activity can reveal patient’s TPMT status. Evaluation of the levels of thiopurine me-tabolites further supports the practice of appropriate dose adjustment by providing the efficient monitor-ing of drug cytotoxicity.Conclusion: We hereby review the thiopurine pharmacogenetics and the methods applied in common practice to evaluate patient’s TPMT status.

Branched-chain and dendritic lipids for nanoparticles

Lipid nanoparticles (LNPs) for drug-delivery applications are largely derived from natural lipids. Synthetic lipids, particularly those incorporating branched hydrocarbons and hyper-branched hydrocarbon architectures, may afford enhanced lipophilicity with enhanced fluidity and thereby lead to LNP stabilization. Hydrocarbon anchors based on serinol diesters were prepared from linear Cn (n = 14, 16, 18) and branched (n = 16) acids with Boc-protected serinol. These diesters were further dimerized on an iminodiacetamide backbone to provide eight branched-chain and dendritic lipid anchors. Derivatization of these core structures provided eight PEG-lipids and seven thiopurine linked lipid–drug conjugates. LNPs were prepared by microfluidic mixing from mixed lipids in ethanol diluted into aqueous media. The lipid–drug conjugates incorporated 5 mol% of a phosphocholine and 5 mol% of a commercial PEG-lipid to form LNPs with a thiopurine drug loading of 15 wt%. The PEG–lipids prepared were formulated at 1.5 mol% as a surface stabilizer to LNPs containing dsDNA lipoplexes. The stability of the LNPs was assessed under different storage conditions through monitoring of particle size. For both LNPs from lipid–thiopurine conjugates and the PEG-lipid systems, there is strong preliminary evidence that hydrocarbon branching results in LNP stabilization. Four of the lipid–drug conjugate formulations were stable to cell culture conditions (10% serum, 37 °C) and the toxicity of these LNPs was assessed in two cell lines relative to the free thiopurines in the medium. The observed toxicity is consistent with cellular uptake of the LNPs and reductive release of the cargo thiopurine within the cell.

Pharmacogenetics of anti-cancer drugs: State of the art and implementation – recommendations of the French National Network of Pharmacogenetics

Individualized treatment is of special importance in oncology because the drugs used for chemotherapy have a very narrow therapeutic index. Pharmacogenetics may contribute substantially to clinical routine for optimizing cancer treatment to limit toxic effects while maintaining efficacy. This review presents the usefulness of pharmacogenetic tests for some key applications: dihydropyrimidine dehydrogenase (DPYD) genotyping for fluoropyrimidine (5-fluorouracil, capecitabine), UDP glucuronosylstransferase (UGT1A1) for irinotecan and thiopurine S-methyltransferase (TPMT) for thiopurine drugs. Depending on the level of evidence, the French National Network of Pharmacogenetics (RNPGx) has issued three levels of recommendations for these pharmacogenetic tests: essential, advisable, and potentially useful. Other applications, for which the level of evidence is still discussed, will be evoked in the final section of this review.