Clinical Pharmacogenetics Implementation Consortium Guideline (CPIC) for CYP2C9 and Nonsteroidal Anti-Inflammatory Drugs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used analgesics due to their lack of addictive potential. However, NSAIDs have the potential to cause serious gastrointestinal, renal, and cardiovascular adverse events. CYP2C9 polymorphisms influence metabolism and clearance of several drugs in this class, thereby affecting drug exposure and potentially safety. We summarize evidence from the published literature supporting these associations and provide therapeutic recommendations for NSAIDs based on CYP2C9 genotype (updates at www.cpicpgx.org).

Application of pharmacogenetics in clinical practice: problems and solutions.

This paper discusses difficulties of pharmacogenomic data integration into clinical practice. It emphasizes the need for developing simple and easy to use bioinformatics tools to help prescribers to rapidly access and use genetic data in clinical decision-making at the point of encounter.

An analysis of allele, genotype and phenotype frequencies, actionable pharmacogenomic (PGx) variants and phenoconversion in 5408 Australian patients genotyped for CYP2D6, CYP2C19, CYP2C9 and VKORC1 genes.

Common polymorphisms in the genes encoding CYP2D6, CYP2C19, CYP2C9 and VKORC1 enzymes have an important role in predicting the occurrence of adverse effects and the efficacy of substrate medications. Drug-induced changes to the enzyme's phenotype, a process called phenoconversion, comprise another important factor contributing to interindividual variability in drug response. To date, there is lack of data on the frequency of these common polymorphisms and phenoconversion in the pan-ethnic Australian population. The aim of this study was to (1) describe allele, genotype and phenotype frequencies for CYP2D6, CYP2C19, CYP2C9 and VKORC1 enzymes in the pan-ethnic Australian population and (2) evaluate the frequency of actionable pharmacogenomic (PGx) variants and phenoconversion. Frequencies were calculated using the records of 5408 Australian patients (obtained from myDNA's propriety database), who were consecutively tested with the DNAdose PGx test which included the CYP2D6, CYP2C19, CYP2C9 and VKORC1 genes. In 2509 patients with listed medications at the time of testing, phenoconversion frequencies were calculated for CYP2D6, CYP2C19 and CYP2C9 enzymes. Allele, genotype and phenotype frequencies in our Australian patients correlated with a Caucasian population. Approximately 96% of patients had at least one actionable PGx variant. A five-fold increase in the frequency of poor metabolisers (PMs) for CYP2D6 and CYP2C19 was predicted by phenoconversion. Our study results indicate a high frequency of actionable PGx variants in our Australian population. With the addition of drug-induced phenoconversion, our results provide further support for the utilisation of PGx testing in clinical practice as another tool assisting prescribers in the application of personalised medicine.

Complex Drug-Drug-Gene-Disease Interactions Involving Cytochromes P450: Systematic Review of Published Case Reports and Clinical Perspectives.

Drug pharmacokinetics (PK) is influenced by multiple intrinsic and extrinsic factors, among which concomitant medications are responsible for drug-drug interactions (DDIs) that may have a clinical relevance, resulting in adverse drug reactions or reduced efficacy. The addition of intrinsic factors affecting cytochromes P450 (CYPs) activity and/or expression, such as genetic polymorphisms and diseases, may potentiate the impact and clinical relevance of DDIs. In addition, greater variability in drug levels and exposures has been observed when such intrinsic factors are present in addition to concomitant medications perpetrating DDIs. This variability results in poor predictability of DDIs and potentially dramatic clinical consequences. The present review illustrates the issue of complex DDIs using systematically searched published case reports of DDIs involving genetic polymorphisms, renal impairment, cirrhosis, and/or inflammation. Current knowledge on the impact of each of these factors on drug exposure and DDIs is summarized and future perspectives for the management of such complex DDIs in clinical practice are discussed, including the use of advanced Computerized Physician Order Entry (CPOE) systems, the development of model-based dose optimization strategies, and the education of healthcare professionals with respect to personalized medicine.

Cytochrome P450 Structure, Function and Clinical Significance: A Review.

BACKGROUND: The cytochrome P450 (CYP) enzymes are membrane-bound hemoproteins that play a pivotal role in the detoxification of xenobiotics, cellular metabolism and homeostasis. Induction or inhibition of CYP enzymes is a major mechanism that underlies drug-drug interactions. CYP enzymes can be transcriptionally activated by various xenobiotics and endogenous substrates through receptor-dependent mechanisms. CYP enzyme inhibition is a principal mechanism for metabolism- based drug-drug interactions. Many chemotherapeutic drugs can cause drug interactions due to their ability to either inhibit or induce the CYP enzyme system. Predictions based on in silico analyses followed by validation have identified several microRNAs that regulate CYPs. Genetic polymorphisms and epigenetic changes in CYP genes may be responsible for inter-individual and interethnic variations in disease susceptibility and the therapeutic efficacy of drugs. OBJECTIVE: The present review is a comprehensive compilation of cytochrome P450 structure, function, pharmacogenetics, pharmacoepigenetics and clinical significance. CONCLUSION: Knowledge about the substrates, inducers, and inhibitors of CYP isoforms, as well as the polymorphisms of CYP enzymes may be used as an aid by clinicians to determine therapeutic strategy, and treatment doses for drugs that are metabolized by CYP gene products.

Pharmacogenetic landscape of DPYD variants in south Asian populations by integration of genome-scale data.

AIM: Adverse drug reactions to 5-Fluorouracil(5-FU) is frequent and largely attributable to genetic variations in the DPYD gene, a rate limiting enzyme that clears 5-FU. The study aims at understanding the pharmacogenetic landscape of DPYD variants in south Asian populations. MATERIALS & METHODS: Systematic analysis of population scale genome wide datasets of over 3000 south Asians was performed. Independent evaluation was performed in a small cohort of patients. RESULTS: Our analysis revealed significant differences in the the allelic distribution of variants in different ethnicities. CONCLUSIONS: This is the first and largest genetic map the DPYD variants associated with adverse drug reaction to 5-FU in south Asian population. Our study highlights ethnic differences in allelic frequencies.

Species differences in drug glucuronidation: Humanized UDP-glucuronosyltransferase 1 mice and their application for predicting drug glucuronidation and drug-induced toxicity in humans.

More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an increase or decrease in blood drug concentration. To avoid drug-drug interactions and adverse drug reactions in individuals, therefore, it is important to understand whether UGTs are involved in metabolism of drugs and drug candidates. While most of glucuronides are inactive metabolites, acyl-glucuronides that are formed from compounds with a carboxylic acid group can be highly toxic. Animals such as mice and rats are widely used to predict drug metabolism and drug-induced toxicity in humans. However, there are marked species differences in the expression and function of drug-metabolizing enzymes including UGTs. To overcome the species differences, mice in which certain drug-metabolizing enzymes are humanized have been recently developed. Humanized UGT1 (hUGT1) mice were created in 2010 by crossing Ugt1-null mice with human UGT1 transgenic mice in a C57BL/6 background. hUGT1 mice can be promising tools to predict human drug glucuronidation and acyl-glucuronide-associated toxicity. In this review article, studies of drug metabolism and toxicity in the hUGT1 mice are summarized. We further discuss research and strategic directions to advance the understanding of drug glucuronidation in humans.

Protective effects of melatonin on long-term administration of fluoxetine in rats.

The degree and consequence of tissue injury are highly regarded during long-term exposure to selective antidepressant fluoxetine. Melatonin has been shown to palliate different lesions by scavenging free radicals, but its role in the reduction of the fluoxetine-induced injuries has been little known. Thirty-six mature male Wistar rats were randomly assigned into control and experimental groups. The experimental rats were included as following; 24mg/kg/bw fluoxetine for 4 weeks; 1mg/kg/bw melatonin for 4 weeks; fluoxetine+1-week melatonin, fluoxetine+2-week melatonin and fluoxetine+4-week melatonin. In the current experiment, we investigated weight gain, hematological and biochemical parameters, pathological injuries and oxidative status. We noted the positive effect of melatonin in weight loss of fluoxetine-treated rats (p<0.05). The significant reduction of superoxide dismutase, glutathione peroxidase, catalase activities in blood, liver, and kidneys and changes in serum total antioxidant capacity caused by fluoxetine were reversed by melatonin (p<0.05). Melatonin reduced the increased lipid peroxidation and transaminase activity in rats received fluoxetine (p<0.05). We also showed the potency of fluoxetine in inducing leukopenia, thrombocytopenia and hypochromic and macrocytic anemia which was blunted by melatonin. Both RBCs and platelets indices were also corrected. Rats received melatonin in combination with fluoxetine showed a reduction in the severity of degeneration and inflammatory changes in different tissues, brain, heart, liver, lungs, testes and kidneys as compared to the fluoxetine group. Therefore, melatonin fundamentally reversed the side effects of fluoxetine in the rat model which is comparable to human medicine.

Health regulatory communications of well-established safety-related pharmacogenomics associations in six developed countries: an evaluation of alignment.

Recommendations on genetic testing are typically conveyed by drug regulatory authorities through drug labels, which are legal requirements for market authorization of drugs. We conducted a cross-sectional study of drug labels focusing on three crucial aspects of regulatory pharmacogenomics communications: (i) intent; (ii) interpretation in the local context; and (iii) implications of the genetic information. Labels of drugs associated with well-established safety-related genetic markers for adverse drug reactions across six developed countries of United States, Canada, United Kingdom, Australia, New Zealand and Singapore were reviewed. We found differing medical advice for genotype-positive HLA-B*15:02, HLA-A*31:01, UGT1A1*28 and CYP2D6 ultra-rapid metabolisers in breastfeeding women. This raises questions on implications to clinical practice between these countries. Varying ways of presenting at-risk population and allele frequencies also raises question in incorporating such information in drug labels. An international guidance addressing these crucial aspects of regulatory pharmacogenomic communications in drug labels is long overdue.

Validation of a microdose probe drug cocktail for clinical drug interaction assessments for drug transporters and CYP3A.

A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions.

Managing the Risk of CYP3A Induction in Drug Development: A Strategic Approach.

Induction of cytochrome P450 (P450) can impact the efficacy and safety of drug molecules upon multiple dosing with coadministered drugs. This strategy is focused on CYP3A since the majority of clinically relevant cases of P450 induction are related to these enzymes. However, the in vitro evaluation of induction is applicable to other P450 enzymes; however, the in vivo relevance cannot be assessed because the scarcity of relevant clinical data. In the preclinical phase, compounds are screened using pregnane X receptor reporter gene assay, and if necessary structure-activity relationships (SAR) are developed. When projects progress toward the clinical phase, induction studies in a hepatocyte-derived model using HepaRG cells will generate enough robust data to assess the compound's induction liability in vivo. The sensitive CYP3A biomarker 4ß-hydroxycholesterol is built into the early clinical phase I studies for all candidates since rare cases of in vivo induction have been found without any induction alerts from the currently used in vitro methods. Using this model, the AstraZeneca induction strategy integrates in vitro assays and in vivo studies to make a comprehensive assessment of the induction potential of new chemical entities. Convincing data that support the validity of both the in vitro models and the use of the biomarker can be found in the scientific literature. However, regulatory authorities recommend the use of primary human hepatocytes and do not advise the use of sensitive biomarkers. Therefore, primary human hepatocytes and midazolam studies will be conducted during the clinical program as required for regulatory submission.

Thiopurine S-methyltransferase testing for averting drug toxicity: a meta-analysis of diagnostic test accuracy.

Thiopurine S-methyltransferase (TPMT) deficiency increases the risk of serious adverse events in persons receiving thiopurines. The objective was to synthesize reported sensitivity and specificity of TPMT phenotyping and genotyping using a latent class hierarchical summary receiver operating characteristic meta-analysis. In 27 studies, pooled sensitivity and specificity of phenotyping for deficient individuals was 75.9% (95% credible interval (CrI), 58.3-87.0%) and 98.9% (96.3-100%), respectively. For genotype tests evaluating TPMT*2 and TPMT*3, sensitivity and specificity was 90.4% (79.1-99.4%) and 100.0% (99.9-100%), respectively. For individuals with deficient or intermediate activity, phenotype sensitivity and specificity was 91.3% (86.4-95.5%) and 92.6% (86.5-96.6%), respectively. For genotype tests evaluating TPMT*2 and TPMT*3, sensitivity and specificity was 88.9% (81.6-97.5%) and 99.2% (98.4-99.9%), respectively. Genotyping has higher sensitivity as long as TPMT*2 and TPMT*3 are tested. Both approaches display high specificity. Latent class meta-analysis is a useful method for synthesizing diagnostic test performance data for clinical practice guidelines.The Pharmacogenomics Journal advance online publication, 24 May 2016; doi:10.1038/tpj.2016.37.

Pharmacokinetic drug interactions in liver disease: An update.

Inhibition and induction of drug-metabolizing enzymes are the most frequent and dangerous drug-drug interactions. They are an important cause of serious adverse events that have often resulted in early termination of drug development or withdrawal of drugs from the market. Management of such interactions by dose adjustment in clinical practice is extremely difficult because of the wide interindividual variability in their magnitude. This review examines the genetic, physiological, and environmental factors responsible for this variability, focusing on an important but so far neglected cause of variability, liver functional status. Clinical studies have shown that liver disease causes a reduction in the magnitude of interactions due to enzyme inhibition, which is proportional to the degree of liver function impairment. The effect of liver dysfunction varies quantitatively according to the nature, reversible or irreversible, of the inhibitory interaction. The magnitude of reversible inhibition is more drastically reduced and virtually vanishes in patients with advanced hepatocellular insufficiency. Two mechanisms, in order of importance, are responsible for this reduction: decreased hepatic uptake of the inhibitory drug and reduced enzyme expression. The extent of irreversible inhibitory interactions is only partially reduced, as it is only influenced by the decreased expression of the inhibited enzyme. Thus, for appropriate clinical management of inhibitory drug interactions, both the liver functional status and the mechanism of inhibition must be taken into consideration. Although the inducibility of drug-metabolizing enzymes in liver disease has long been studied, very conflicting results have been obtained, mainly because of methodological differences. Taken together, the results of early animal and human studies indicated that enzyme induction is substantially preserved in compensated liver cirrhosis, whereas no definitive conclusion as to whether it is significantly reduced in the decompensated state of cirrhosis was provided. Since ethical constraints virtually preclude the possibility of performing methodologically rigorous investigations in patients with severe liver dysfunction, studies have recently been performed in animals rigorously stratified according to the severity of liver insufficiency. The results of these studies confirmed that enzyme induction is virtually unaffected in compensated cirrhosis and indicated that the susceptibility of enzyme induction to severe liver dysfunction depends on the type of nuclear receptor involved and also varies among enzyme isoforms under the transcriptional control of the same nuclear receptor. These findings make it clear that no general conclusion can be reached from the study of any particular enzyme and partly explain the conflicting results obtained by previous studies. Since no general guidelines can be provided for the management of drug interactions resulting from enzyme induction, both the effects and the plasma concentration of the induced drug should be strictly monitored. The findings discussed in this review have important methodological implications as they indicate that, contrary to current guidelines, the magnitude of metabolic drug-drug interactions in patients with liver disease cannot be inferred from studies in healthy subjects.

Clinical Weighting of Drug-Drug Interactions in Hospitalized Elderly.

Adverse drug reactions impact on patient health, effectiveness of pharmacological therapy and increased health care costs. This investigation intended to detect the most critical drug-drug interactions in hospitalized elderly patients, weighting clinical risk. We conducted a cross-sectional study between January and April 2014; all patients 70 years or older, hospitalized for >24 hr and prescribed at least one medication were included in the study. Drug-drug interactions were estimated by combining Stockley's, Hansten and Tatro drug interactions. Drug-drug interactions were weighted using a risk-analysis method based on failure modes, effects and criticality analysis. We calculated a criticality index for each drug involved in the drug-drug interactions based on the severity of the interaction mechanism, the frequency the drug was involved in drug-drug interactions and the risk of drug-drug interactions in patients with impaired renal function. The average number of drugs consumed in the hospital was 6 ± 2.69, involving 160 active ingredients. The most frequent were as follows: Furosemide, followed by Enalapril. Of drug-drug interactions, 2% were classified as contraindicated, 14% advised against and 83% advised caution during the hospital stay. Thirty-four drug-drug interactions were assessed, of which 23 were pharmacodynamic drug-drug interactions and 12 were pharmacokinetic drug-drug interactions (1 was both). The clinical risk calculated for each drug-drug interaction included heparins + non-steroidal anti-inflammatory drugs (NSAIDs) or Digoxin + Calcium Gluconate, cases which are pharmacodynamic drug-drug interactions with agonist effect and clinical risk of bleeding, one of the most common clinical risks in the hospital. An index of clinical risk for drug-drug interactions can be calculated based on severity by the interaction mechanism, the frequency that the drug is involved in drug-drug interactions and the risk of drug-drug interactions in an elderly patient with impaired renal function.

Prospective DPYD genotyping to reduce the risk of fluoropyrimidine-induced severe toxicity: Ready for prime time.

5-Fluorouracil (5-FU) and capecitabine (CAP) are among the most frequently prescribed anticancer drugs. They are inactivated in the liver by the enzyme dihydropyrimidine dehydrogenase (DPD). Up to 5% of the population is DPD deficient and these patients have a significantly increased risk of severe and potentially lethal toxicity when treated with regular doses of 5-FU or CAP. DPD is encoded by the gene DPYD and variants in DPYD can lead to a decreased DPD activity. Although prospective DPYD genotyping is a valuable tool to identify patients with DPD deficiency, and thus those at risk for severe and potential life-threatening toxicity, prospective genotyping has not yet been implemented in daily clinical care. Our goal was to present the available evidence in favour of prospective genotyping, including discussion of unjustified worries on cost-effectiveness, and potential underdosing. We conclude that there is convincing evidence to implement prospective DPYD genotyping with an upfront dose adjustment in DPD deficient patients. Immediate benefit in patient care can be expected through decreasing toxicity, while maintaining efficacy.

Genotype-phenotype correlations in 5-fluorouracil metabolism: a candidate DPYD haplotype to improve toxicity prediction.

5-Fluorouracil is among the most widely used anticancer drug, but a fraction of treated patients develop severe toxicity, with potentially lethal injuries. The predictive power of the available pretreatment assays, used to identify patients at risk of severe toxicity, needs improvements. This study aimed to correlate a phenotypic marker of 5-fluorouracil metabolism (the individual degradation rate of 5-fluorouracil-5-FUDR) with 15 functional polymorphisms in the dihydropyrimidine dehydrogenase gene (DPYD). Single SNP (single-nucleotide polymorphism) analysis revealed that the SNPs rs1801160, rs1801265, rs2297595 and rs3918290 (splice site variant IVS14+1G>A) were significantly associated with a decreased value of 5-FUDR, and the rs3918290 causing the larger decrease. Multi-SNP analysis showed that a three-SNP haplotype (Hap7) involving rs1801160, rs1801265 and rs2297595 causes a marked decrease in 5-FUDR, comparable to that caused by the splice site variant rs3918290, which is the main pharmacogenetic marker associated with severe fluorouracil toxicity. The similar effect played by Hap7 and by the splice site variant rs3918290 upon individual 5-FUDR suggests that Hap7 could also represent a similar determinant of fluorouracil toxicity. Haplotype assessment could improve the predictive value of DPYD genetic markers aimed at the pre-emptive identification of patients at risk of severe 5-fluorouracil toxicity.The Pharmacogenomics Journal advance online publication, 28 July 2015; doi:10.1038/tpj.2015.56.

[Role of Human Orphan Esterases in Drug-induced Toxicity].

Esterases hydrolyze compounds containing ester, amide, and thioester bonds, causing prodrug activation or detoxification. Among esterases, carboxylesterases have been studied in depth due to their ability to hydrolyze a variety of drugs. However, there are several drugs for which the involved esterase(s) is unknown. We found that flutamide, phenacetin, rifamycins (rifampicin, rifabutin, and rifapentine), and indiplon are hydrolyzed by arylacetamide deacetylase (AADAC), which is highly expressed in human liver and gastrointestinal tissues. Flutamide hydrolysis is considered associated with hepatotoxicity. Phenacetin, a prodrug of acetaminophen, was withdrawn due to side effects such as methemoglobinemia and renal failure. It was demonstrated in vitro and in vivo using mice that AADAC is responsible for phenacetin hydrolysis, which leads to methemoglobinemia. In addition, it was shown that AADAC-mediated hydrolysis attenuates the cytotoxicity of rifamycins. Thus AADAC plays critical roles in drug-induced toxicity. Another orphan esterase, α/ß hydrolase domain containing 10 (ABHD10), was found responsible for deglucuronidation of acyl-glucuronides including mycophenolic acid acyl-glucuronide and probenecid acyl-glucuronide. Because acyl-glucuronides appear associated with toxicity, ABHD10 would function as a detoxification enzyme. The roles of orphan esterases are becoming increasingly understood. Further studies will facilitate our knowledge of the pharmacologic and toxicological significance of orphan esterases in drug therapy.

Computational predictions of the site of metabolism of cytochrome P450 2D6 substrates: comparative analysis, molecular docking, bioactivation and toxicological implications.

Cytochrome P450 2D6 (CYP2D6) is a polymorphic enzyme responsible for metabolizing approximately 25% of all drugs. CYP2D6 is highly expressed in the brain and plays a role as the major CYP in the metabolism of numerous brain-penetrant drugs, including antipsychotics and antidepressants. CYP2D6 activity and inhibition have been associated with numerous undesirable effects in patients, such as bioactivation, drug-associated suicidality and prolongation of the QTc interval. Several in silico tools have been developed in recent years to assist safety assessment scientists in predicting the structural identity of CYP2D6-derived metabolites. The first goal of this study was to perform a comparative evaluation on the ability of four commonly used in silico tools (MetaSite, StarDrop, SMARTCyp and RS-WebPredictor) to correctly predict the CYP2D6-derived site of metabolism (SOM) for 141 compounds, including 10 derived from the Genentech small molecule library. The second goal was to evaluate if a bioactivation prediction model, based on an indicator of chemical reactivity (ELUMO-EHOMO) and electrostatic potential, could correctly predict five representative compounds known to be bioactivated by CYP2D6. Such a model would be of great utility in safety assessment since unforeseen toxicities of CYP2D6 substrates may in part be due to bioactivation mechanisms. The third and final goal was to investigate whether molecular docking, using the crystal structure of human CYP2D6, had the potential to compliment or improve the results obtained from the four SOM in silico programs.