Development of label-free electrochemical impedance spectroscopy for the detection of HLA-B*15:02 and HLA-B*15:21 for the prevention of carbamazepine-induced Stevens-Johnson syndrome.

BACKGROUND: Carbamazepine (CBZ) is an FDA-approved anticonvulsant that is widely used to treat epilepsy, bipolar disorder, trigeminal neuralgia and chronic pain. Several studies have reported a strong association between HLA-B*15:02 and carbamazepine-induced Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN). However, the HLA-B75 serotype (HLA-B*15:02, HLA-B*15:08, HLA-B*15:11 and HLA-B*15:21) has been found in patients with carbamazepine-induced SJS/TEN. METHODS: This study aimed to develop label-free electrochemical impedance spectroscopy (EIS) for the detection of HLA-B*15:02 and HLA-B*15:21 after PCR-SSP amplification. A total of 208 DNA samples were tested. The impedance was measured and compared to standard gel electrophoresis. RESULTS: The developed label-free EIS identified HLA-B*15:02 and HLA-B*15:21 alleles with 100% sensitivity (95% CI: 86.773%-100.000%) and 95.05% specificity (95% CI: 90.821%-97.714%), comparable to commercial DMSc 15:02 detection kits. CONCLUSIONS: We successfully developed a novel PCR-SSP associated with signal impedance changes to detect the HLA-B*15:02 allele and HLA-B*15:21 without downstream amplicon size analysis that is suitable for screening individuals before indication of CBZ therapy.

Genetic variants associated with severe cutaneous adverse drug reactions induced by carbamazepine.

AIMS: Carbamazepine (CBZ) is one of the most common causative drugs of severe cutaneous adverse drug reactions (SCARs) including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) and drug reactions with eosinophilia and systemic symptoms. Although genetic polymorphisms of the human leucocyte antigens (HLA) are well recognized key elements for the susceptibility to CBZ-induced SCARs, some evidence suggest that polymorphisms of microsomal epoxide hydrolase 1 (EPHX1) may also contribute to the risk of these SCARs. This study investigated the association between the HLA and EPHX1 polymorphisms on CBZ-induced SCARs in large sample sizes and well-defined SCARs patients. METHODS: Ninety-one CBZ-induced SCARs Thai patients and 144 CBZ-tolerant patients were enrolled in the study. The genotypes of HLA-A, HLA-B and EPHX1 were determined. RESULTS: Only 2 HLA alleles including HLA-B*15:02 and HLA-A*24:07 were statistically significant association with CBZ-induced SJS/TEN. The highest risk was observed in patients with HLA-B*15:02 allele (OR = 44.33, 95% confidence interval = 20.24-97.09, corrected P-value = 6.80 × 10-29 ). Moreover, HLA-B75 serotypes were significantly associated with CBZ-induced SJS/TEN groups with an odds ratio of 81.00 (95% confidence interval = 32.39-202.56, corrected P-value = 3.84 × 10-34 ). There is no association between EPHX1 c.337 T > C polymorphism and all phenotypes of CBZ-induced SCARs. CONCLUSION: The HLA-B*15:02 allele is the strongest genetic marker for the prediction of SJS/TEN induced by CBZ in Thai population. Screening for other alleles in the HLA-B75 serotype increases sensitivity for prediction of a life-threatening SCARs caused by CBZ.

Whole genome sequencing identifies genetic variants associated with co-trimoxazole hypersensitivity in Asians.

BACKGROUND: Co-trimoxazole, a sulfonamide antibiotic, is used to treat a variety of infections worldwide, and it remains a common first-line medicine for prophylaxis against Pneumocystis jiroveci pneumonia. However, it can cause severe cutaneous adverse reaction (SCAR), including Stevens-Johnson syndrome, toxic epidermal necrolysis, and drug reaction with eosinophilia and systemic symptoms. The pathomechanism of co-trimoxazole-induced SCAR remains unclear. OBJECTIVE: We aimed to investigate the genetic predisposition of co-trimoxazole-induced SCAR. METHODS: We conducted a multicountry case-control association study that included 151 patients with of co-trimoxazole-induced SCAR and 4631 population controls from Taiwan, Thailand, and Malaysia, as well as 138 tolerant controls from Taiwan. Whole-genome sequencing was performed for the patients and population controls from Taiwan; it further validated the results from Thailand and Malaysia. RESULTS: The whole-genome sequencing study (43 case patients vs 507 controls) discovered that the single-nucleotide polymorphism rs41554616, which is located between the HLA-B and MICA loci, had the strongest association with co-trimoxazole-induced SCAR (P = 8.2 × 10-9; odds ratio [OR] = 7.7). There were weak associations of variants in co-trimoxazole-related metabolizing enzymes (CYP2D6, GSTP1, GCLC, N-acetyltransferase [NAT2], and CYP2C8). A replication study using HLA genotyping revealed that HLA-B∗13:01 was strongly associated with co-trimoxazole-induced SCAR (the combined sample comprised 91 case patients vs 2545 controls [P = 7.2 × 10-21; OR = 8.7]). A strong HLA association was also observed in the case patients from Thailand (P = 3.2 × 10-5; OR = 3.6) and Malaysia (P = .002; OR = 12.8), respectively. A meta-analysis and phenotype stratification study further indicated a strong association between HLA-B∗13:01 and co-trimoxazole-induced drug reaction with eosinophilia and systemic symptoms (P = 4.2 × 10-23; OR = 40.1). CONCLUSION: This study identified HLA-B∗13:01 as an important genetic factor associated with co-trimoxazole-induced SCAR in Asians.

The Thai reference exome (T-REx) variant database.

To maximize the potential of genomics in medicine, it is essential to establish databases of genomic variants for ethno-geographic groups that can be used for filtering and prioritizing candidate pathogenic variants. Populations with non-European ancestry are poorly represented among current genomic variant databases. Here, we report the first high-density survey of genomic variants for the Thai population, the Thai Reference Exome (T-REx) variant database. T-REx comprises exome sequencing data of 1092 unrelated Thai individuals. The targeted exome regions common among four capture platforms cover 30.04 Mbp on autosomes and chromosome X. 345 681 short variants (18.27% of which are novel) and 34 907 copy number variations were found. Principal component analysis on 38 469 single nucleotide variants present worldwide showed that the Thai population is most genetically similar to East and Southeast Asian populations. Moreover, unsupervised clustering revealed six Thai subpopulations consistent with the evidence of gene flow from neighboring populations. The prevalence of common pathogenic variants in T-REx was investigated in detail, which revealed subpopulation-specific patterns, in particular variants associated with erythrocyte disorders such as the HbE variant in HBB and the Viangchan variant in G6PD. T-REx serves as a pivotal addition to the current databases for genomic medicine.

Cost-effectiveness analysis of HLA-B*58: 01 genetic testing before initiation of allopurinol therapy to prevent allopurinol-induced Stevens-Johnson syndrome/toxic epidermal necrolysis in a Malaysian population.

OBJECTIVE: Studies found a strong association between allopurinol-induced Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and the HLA-B*58:01 allele. HLA-B*58:01 screening-guided therapy may mitigate the risk of allopurinol-induced SJS/TEN. This study aimed to evaluate the cost-effectiveness of HLA-B*58:01 screening before allopurinol therapy initiation compared with the current practice of no screening for Malaysian patients with chronic gout in whom a hypouricemic agent is indicated. METHODS: This cost-effectiveness analysis adopted a societal perspective with a lifetime horizon. A decision tree model coupled with Markov models were developed to estimate the costs and outcomes, represented by quality-adjusted life years (QALYs) gained, of three treatment strategies: (a) current practice (allopurinol initiation without HLA-B*58:01 screening); (b) HLA-B*58:01 screening before allopurinol initiation; and (c) alternative treatment (probenecid) without HLA-B*58:01 screening. The model was populated with data from literature review, meta-analysis, and published government documents. Cost values were adjusted for the year 2016, with costs and health outcomes discounted at 3% per annum. A series of sensitivity analysis including probabilistic sensitivity analysis were carried out to determine the robustness of the findings. RESULTS: Both HLA-B*58:01 screening and probenecid prescribing were dominated by current practice. Compared with current practice, HLA-B*58:01 screening resulted in 0.252 QALYs loss per patient at an additional cost of USD 322, whereas probenecid prescribing resulted in 1.928 QALYs loss per patient at an additional cost of USD 2203. One SJS/TEN case would be avoided for every 556 patients screened. At the cost-effectiveness threshold of USD 8695 per QALY, the probability of current practice being the best choice is 99.9%, in contrast with 0.1 and 0% in HLA-B*58:01 screening and probenecid prescribing, respectively. This is because of the low incidence of allopurinol-induced SJS/TEN in Malaysia and the lower efficacy of probenecid compared with allopurinol in gout control. CONCLUSION: This analysis showed that HLA-B*58:01 genetic testing before allopurinol initiation is unlikely to be a cost-effective intervention in Malaysia.

Risk factors of allopurinol-induced severe cutaneous adverse reactions in a Thai population.

BACKGROUND: Allopurinol is one of the most common causes of severe cutaneous adverse drug reactions (SCARs) including drug reactions with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN). This study identified the risk factors associated with the development of allopurinol-induced SCARs in a Thai population. PATIENTS AND METHODS: Eighty-six allopurinol-induced SCARs (i.e. 19 DRESS and 67 SJS/TEN) and 182 allopurinol-tolerant patients were enrolled in the study. The HLA-B*58:01 allele was determined. Clinical and medicinal data were collected. RESULTS: Results from multivariate analysis showed that only the HLA-B*58:01 and female sex were identified as risk factors of allopurinol-induced SCARs in this Thai population. Patients who carried the HLA-B*58:01 allele were at a higher risk of allopurinol-induced DRESS [odds ratio (OR)=149.2, 95% confidence interval (CI)=24.0-∞, P<1.00×10]. Similar results were observed in allopurinol-induced SJS/TEN (OR=175.0, 95% CI=44.3-690.9, P=1.69×10). The risk of allopurinol-induced SCARs in women was higher than that in men (OR=4.6, 95% CI=1.4-15.6, P=1.44×10). The overall mortality rate of allopurinol-induced SCARs was 11.39% and a higher mortality rate was observed in elderly women. CONCLUSION: Among the risk factors identified, the HLA-B*58:01 allele had the greatest impact on the development of both phenotypes of allopurinol-induced SCARs in this studied Thai population. In case HLA-B*58:01 genotyping cannot be accessed, close monitoring of allopurinol usage, especially in elderly women with impaired renal function, is necessary to reduce the mortality rate of these life-threatening SCARs.

Dapsone-induced severe cutaneous adverse drug reactions are strongly linked with HLA-B*13: 01 allele in the Thai population.

OBJECTIVES: A previous publication in Chinese leprosy patients showed that the HLA-B*13:01 allele is a strong genetic marker for dapsone-induced drug hypersensitivity reactions, however there are no data describing whether HLA-B*13:01 is a valid marker for prediction of dapsone-induced drug hypersensitivity reactions in other ethnicities or nonleprosy patients. The aim of this study is to investigate whether there is an association between HLA genotypes and dapsone-induced severe cutaneous adverse reactions (SCARs) in Thai nonleprosy patients. PATIENTS AND METHODS: HLA-B genotypes of 15 patients with dapsone-induced SCARs (11 drug reaction with eosinophilia and systemic symptoms, 4 Stevens-Johnson syndrome/toxic epidermal necrolysis), 29 control patients, and 986 subjects from the general Thai population were determined by the reverse PCR sequence-specific oligonucleotides probe. RESULTS: The HLA-B*13:01 allele was significantly associated with dapsone-induced SCARs compared with dapsone-tolerant controls (odds ratio: 54.00, 95% confidence interval: 7.96-366.16, P=0.0001) and the general population (odds ratio: 26.11, 95% confidence interval: 7.27-93.75, P=0.0001). In addition, HLA-B*13:01 associated with dapsone-induced SJS-TEN (OR: 40.50, 95% confidence interval: 2.78-591.01, P=0.0070) and DRESS (OR: 60.75, 95% confidence interval: 7.44-496.18, P=0.0001). CONCLUSION: This study demonstrated an association between HLA-B*13:01 and dapsone-induced SCARs including Stevens-Johnson syndrome/toxic epidermal necrolysis and drug reaction with eosinophilia and systemic symptoms in nonleprosy patients. Moreover, these results suggest that the HLA-B*13:01 allele may be a useful genetic marker for prediction of dapsone-induced SCARs in Thai and Han-Chinese populations.

The impact of non-genetic and genetic factors on a stable warfarin dose in Thai patients.

PURPOSE: The aim of this study was to investigate the contributions of non-genetic and genetic factors on the variability of stable warfarin doses in Thai patients. METHODS: A total of 250 Thai patients with stable warfarin doses were enrolled in the study. Demographics and clinical data, e.g., age, body mass index, indications for warfarin and concomitant medications, were documented. Four single nucleotide polymorphisms in the VKORC1 - 1639G > A, CYP2C9*3, CYP4F2 rs2108622, and UGT1A1 rs887829 genes were detected from gDNA using TaqMan allelic discrimination assays. RESULTS: The patients with variant genotypes of VKORC1 - 1639G > A required significantly lower warfarin stable weekly doses (SWDs) than those with wild-type genotype (p < 0.001). Similarly, the patients with CYP2C9*3 variant allele required significantly lower warfarin SWDs than those with homozygous wild-type (p = 0.006). In contrast, there were no significant differences in the SWDs between the patients who carried variant alleles of CYP4F2 rs2108622 and UGT1A1 rs887829 as compared to wild-type allele carriers. Multivariate analysis, however, showed that CYP4F2 rs2108622 TT genotype accounted for a modest part of warfarin dose variability (1.2%). In contrast, VKORC1 - 1639G > A, CYP2C9*3, CYP4F2 rs2108622 genotypes and non-genetic factors accounted for 51.3% of dose variability. CONCLUSIONS: VKORC1 - 1639G > A, CYP2C9*3, and CYP4F2 rs2108622 polymorphisms together with age, body mass index, antiplatelet drug use, amiodarone use, and current smoker status explained 51.3% of individual variability in stable warfarin doses. In contrast, the UGT1A1 rs887829 polymorphism did not contribute to dose variability.

Comparative pharmacodynamic effects of two clopidogrel formulations under steady-state conditions in healthy Thai volunteers
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OBJECTIVE: Clopidogrel is a commonly used antiplatelet aggregation agent. Compared with the reference clopidogrel product, most commercially available generic clopidogrel products contain different crystalline forms of clopidogrel. This study was aimed to compare the pharmacodynamics of a commonly used generic clopidogrel product in Thailand with the reference clopidogrel product under steady state conditions. METHODS: A multiple-dose, randomized 2-way crossover study was conducted in 32 healthy male Thai volunteers. The subjects were assigned to receive 75 mg once daily of the test or the reference product for 7 days with a 2-week wash out period. Blood samples were collected on days 1, 5, 6, and 7 prior to drug administration and at 1, 2, 3, 4, 8, 12, and 24 hours after the last dose administered. The antiplatelet aggregation effects of clopidogrel were determined by using two different ex-vivo platelet aggregation tests including the whole blood impedance assay (WBA) and the VerifyNow® P2Y12 assay. Both pharmacodynamic parameters, the maximal antiplatelet effect (Emax) and the areas under the antiplatelet effect-time curve (AUEC0-24h), were calculated. RESULTS: Neither the mean values of Emax (90.70 ± 15.15 vs. 89.50 ± 10.71% inhibition) nor of AUEC0-24h (1,892.84 ± 657.22 vs. 1,853.58 ± 673.95% inhibition × h) under steady-state conditions obtained using the WBA method of these two clopidogrel products were significantly different. The results obtained using the VerifyNow® P2Y12 assay were consistent with those of the WBA assay. CONCLUSION: This study clearly demonstrated that ex-vivo antiplatelet aggregation effect under steady-state conditions of the test product was not significantly different from the reference product.
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Associations between HLA class I and cytochrome P450 2C9 genetic polymorphisms and phenytoin-related severe cutaneous adverse reactions in a Thai population.

BACKGROUND: Phenytoin is one of the most common causative drugs of several types of severe cutaneous adverse reactions (SCAR) such as Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug reactions with eosinophilia and systemic symptoms (DRESS). Genetic polymorphisms of the human leukocyte antigens (HLA) and cytochromes P450 (CYP) have been proposed as key elements for the susceptibility to phenytoin-related SCAR in certain ethnicities. This study investigated the associations between the genetic polymorphisms of HLA class I and CYP2C9 and phenytoin-related SCAR in a Thai population. MATERIALS AND METHODS: Sixty phenytoin-related SCAR (i.e. 39 SJS/TEN and 21 DRESS) and 92 phenytoin-tolerant patients were enrolled in the study. The genotypes of HLA class I and CYP2C9 were determined. RESULTS: Six HLA alleles including HLA-A*33:03, HLA-B*38:02, HLA-B*51:01, HLA-B*56:02, HLA-B*58:01, and HLA-C*14:02 were significantly associated with phenytoin-related SJS/TEN, whereas only the HLA-B*51:01 was significantly associated with phenytoin-related DRESS. The odds ratios of phenytoin-related SJS/TEN in the patients who carried one of these alleles ranged from 4- to 10-fold. The frequencies of patients who carried the HLA-B*15:02 in the SJS/TEN (12.82%) or the DRESS (9.52%) groups were not significantly different from that of the controls (14.13%). The higher risk of phenytoin-related SJS/TEN was observed in the patients with CYP2C9*3 (odds ratio=4.30, 95% confidence interval=1.41-13.09, P<0.05). CONCLUSION: Neither SJS/TEN nor DRESS caused by phenytoin was significantly associated with the HLA-B*15:02. The CYP2C9*3 variant was significantly associated with phenytoin-related SJS/TEN, but not DRESS. Certain alleles of HLA, particularly HLA-B*56:02, were significantly associated with phenytoin-related SCAR in the study population.

Candidate HLA genes for prediction of co-trimoxazole-induced severe cutaneous reactions.

BACKGROUND: Co-trimoxazole is a sulfonamide-containing antibiotic that is effective in the treatment of several infections and for prophylaxis of Pneumocystis jiroveci pneumonia. This drug has been reported as a common culprit drug for the Stevens-Johnson syndrome (SJS) and for toxic epidermal necrolysis (TEN). Human leukocyte antigens (HLAs) play a key role in the immunopathogenesis of severe cutaneous reactions induced by several drugs. This study investigated the association between the HLA class I and HLA-DRB1 polymorphisms and co-trimoxazole-induced SJS/TEN in a Thai population. METHODS: Forty-three patients with co-trimoxazole-induced SJS/TEN and 91 co-trimoxazole-tolerant patients were enrolled in the study. HLA class I and HLA-DRB1 were genotyped using the reverse sequence-specific oligonucleotide probe method. RESULTS: The frequencies of three alleles of HLA, namely HLA-B*15:02, HLA-C*06:02, and HLA-C*08:01, were significantly higher in the co-trimoxazole-induced SJS/TEN group compared with controls. The risks for co-trimoxazole-induced SJS/TEN in patients with the HLA-B*15:02, HLA-C*06:02, or HLA-C*08:01 allele were about 3-11-fold higher when compared with those who did not carry one of these alleles. Individuals who carried the HLA-B*15:02-C*08:01 haplotype had a 14-fold higher risk for co-trimoxazole-induced SJS/TEN. CONCLUSION: Evidence of associations between co-trimoxazole-induced SJS/TEN and HLA alleles including HLA-B*15:02, HLA-C*06:02, and HLA-C*08:01 were found in the study population. These findings may suggest that apart from the HLA molecules, other molecules involved in the molecular pathogenesis of these severe cutaneous adverse drug reactions may play an important role in the susceptibility of individuals to SJS/TEN caused by co-trimoxazole.

PGE2 signaling and its biosynthesis-related enzymes in cholangiocarcinoma progression.

Prostaglandin E2 (PGE2) involves in progression of various chronic inflammation-related cancers including cholangiocarcinoma (CCA). This study aimed to determine the role of PGE2 signaling, its biosynthesis-related enzymes in a clinical prognosis, and their targeted inhibition in CCA progression. The immunohistochemical staining of cyclooxygenase (COX)-1, COX-2, mPGES-1, EP1, and EP4 was examined in CCA tissues, and their expressions were compared with clinicopathological parameters. The effect of PGE2 on levels of its signaling molecules was examined in CCA cell lines using proteome profiler array. The suppression of mPGES-1 using a small-molecule inhibitor (CAY10526) and small interfering RNA (siRNA) was determined for growth and migration ability in CCA cells. The results indicated that strong expressions of COX-1, COX-2, mPGES-1, EP1, and EP4 were found in CCA tissues as 87.5, 47.5, 52.5, 55, and 80 % of frequencies, respectively. High mPGES-1 expression was significantly correlated with tumor stages III-IV (p = 0.001), lymph node metastasis (p = 0.004), shorter survival (p = 0.009), and prognostic indicator of CCA patients (HR = 2.512, p = 0.041). Expressions of COX-1, COX-2, and EP receptors did not correlate with data tested from patients. PGE2 markedly enhanced protein levels of integrinα6, VE-cadherin, Jagged1, and Notch3, and CAY10526 suppressed those protein levels as well as PGE2 production in CCA cells. CAY10526 and siRNA mPGES-1 markedly suppressed mPGES-1 protein levels, growth, and migration abilities of CCA cell lines. In conclusion, PGE2 signaling strongly promotes CCA progression. Therefore, inhibition of PGE2 synthesis by suppression of its biosynthesis-related enzymes could be useful for prevention and treatment of CCA.

Pharmacogenomics of drug-induced hypersensitivity reactions: challenges, opportunities and clinical implementation.

Drug hypersensitivity reactions affect many patients leading to a variety of clinical manifestations, mainly the cutaneous adverse reactions ranging from milder skin reactions to severe cutaneous adverse reactions (SCARs). Hypersensitivity reactions are unpredictable and are thought to have an underlying genetic etiology, as suggested by case reports. With the scientific knowledge of pharmacogenomics and the evidence based on the genomic testing, it is possible to identify genetic predisposing factors for these serious adverse reactions and personalize drug therapy. The most significant genetic associations have been identified in the major histocompatibility complex (MHC) genes encoded for human leukocyte antigens (HLA) alleles. Drugs associated with hypersensitivity reactions with strong genetic predisposing factors include abacavir, nevirapine, carbamazepine, and allopurinol. In this review, strong genetic associations of drug-induced SCARs are highlighted so as to improve drug safety and help to select optimal drugs for individual patients. Further investigation, however, is essential for the characterization of other genes involved in the hypersensitivity reactions with the use of several genetic strategies and technologies.

Complete mitochondrial genomes of patients from Thailand with cardiovascular diseases.

Several previous studies have reported that both variation and haplogroups of mitochondrial (mt) DNA were associated with various kinds of diseases, including cardiovascular diseases, in different populations, but such studies have not been carried out in Thailand. Here, we sequenced complete mtDNA genomes from 82 patients diagnosed with three types of cardiovascular disease, i.e., Hypertrophic Cardiomyopathy (HCM) (n = 26), Long Q-T Syndrome (LQTS) (n = 7) and Brugada Syndrome (BrS) (n = 49) and compared these with 750 previously published mitogenome sequences from interviewed normal individuals as a control group. Both patient and control groups are from the same geographic region of northeastern Thailand. We found 9, 2, and 5 novel mutations that were not both damaging and deleterious in HCM, LQTS, and BrS patients, respectively. Haplogroup R9c was significantly associated with HCM (P = 0.0032; OR = 62.42; 95%CI = 6.892-903.4) while haplogroup M12b was significantly associated with LQTS (P = 0.0039; OR = 32.93; 95% CI = 5.784-199.6). None of the haplogroups was found to be significantly associated with BrS. A significantly higher density of mtDNA variants in the rRNA genes was found in patients with HCM and BrS (P < 0.001) than in those with LQTS or the control group. Effects of detected SNPs in either protein coding or tRNA genes of all the mitogenome sequences were also predicted. Interestingly, three SNPs in two tRNA genes (MT-TA m.5618T>C and m.5631G>A heteroplasmic variants in two BrS patients and MT-TQ m.4392C>T novel homoplasmic variant in a HCM patient) were predicted to alter tRNA secondary structure, possibly leading to abnormal tRNA function.

Comparative bioavailability of two moxifloxacin tablet products after single dose administration under fasting conditions in a balanced, randomized and cross-over study in healthy volunteers.

Moxifloxacin, a 4th generation of fluoroquinolones, is a broad spectrum antibacterial agent against respiratory tract pathogens, including Gram-positive and Gram-negative bacteria, anaerobic bacteria and atypical respiratory tract pathogens. In order to evaluate the efficacy and safety of generic moxifloxacin products, the bioequivalence of these generic products with an approved reference formulation should be demonstrated. Thus, the aim of this study was to compare the rate and extent of absorption of a new generic film coated moxifloxacin tablet product (Rapiflox®, Atlantic Laboratories Corporation Ltd., Bangkok, Thailand) with that of a reference product (Avelox®, Bayer Health Care AG, Leverkusen, Germany) when given as a single dose. A crossover study was performed in 20 healthy Thai volunteers. The subjects received either a 400 mg tablet of the reference or test product after overnight fasting. Blood samples were collected at pre-dose (0 hour) and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 34 hours post-dose. Moxifloxacin plasma level was measured by a validated HPLC method with fluorescence detector. Pharmacokinetic parameters were calculated using a non-compartmental model. The geometric mean of maximum concentration (Cmax) of the test product was 4,069.64 ng/ml, with median time to achieve maximum concentration (tmax) at 2 hours (range 0.25 - 6.00 hours), while the geometric mean Cmax and median tmax of the reference product was 4,211.98 ng/ml and 2.00 hours (range 0.25 - 8.00 hours). Furthermore, the geometric means of AUC0-tlast and AUC0-∞ for the test product were 49,731.66 and 51,865.89 ng×h/ml while those of the reference product were 51,927.97 and 54,455.93 ng×h/ml. The geometric mean of the ratios of Test/Reference for the logtransformed Cmax, AUC0-tlast and AUC0-∞ of moxifloxacin and their 90% CIs were 96.62% (83.21 - 112.19%), 95.77% (87.07 - 105.34%) and 95.24 (86.52 - 104.85%), respectively. Therefore, it can be concluded that these two moxifloxacin tablet products were bioequivalent in healthy Thai volunteers under fasting condition.

Cost-effectiveness analysis of HLA-B*13:01 screening for the prevention of co-trimoxazole-induced severe cutaneous adverse reactions among HIV-infected patients in Thailand.

Studies found a strong association between HLA-B*13:01 allele and co-trimoxazole-induced severe cutaneous adverse reactions (SCARs). Genetic screening before initiation of co-trimoxazole may decrease the incidence of co-trimoxazole-induced SCARs. This study aims to evaluate the cost-effectiveness of HLA-B*13:01 screening before co-trimoxazole initiation in HIV-infected patients in Thailand. A combination of a decision tree model and a Markov model was used to estimate lifetime costs and outcomes of two strategies including 1) HLA-B*13:01 screening before co-trimoxazole initiation and 2) usual practice from a societal perspective. Alternative drugs are not considered because dapsone (the second-line drug) also presents a genetic risk. Input parameters were obtained from literature, government documents, and part of the TREAT Asia HIV Observational Database (TAHOD). One-way sensitivity analyses and probabilistic analyses were performed to determine robustness of the findings. HLA-B*13:01 screening resulted in 0.0061 quality-adjusted life years (QALYs) loss with an additional cost of 370 THB ($11.84). At the cost-effectiveness threshold of 160,000 THB ($5,112.85), the probability of the genetic screening strategy being cost-effective is 9.54%. This analysis demonstrated that HLA-B*13:01 allele screening before initiation of co-trimoxazole among HIV-infected patients is unlikely to be cost-effective in Thailand. Our findings will help policymakers make an evidence-informed decision making.

Association between HLA alleles and beta-lactam antibiotics-related severe cutaneous adverse reactions.

Introduction: Beta-lactam antibiotics are one of the most common causes of antibiotics-related severe cutaneous adverse reactions (SCARs) including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), drug reactions with eosinophilia and systemic symptoms (DRESS), and acute generalized exanthematous pustulosis (AGEP). Recent evidence demonstrated that the human leukocyte antigen (HLA) polymorphisms play important roles in the development of drug-related SCARs. This study aimed to extensively characterize the associations between HLA genetic polymorphisms and several phenotypes of SCARs related to beta-lactam antibiotics. Methods: Thirty-one Thai patients with beta-lactam antibiotics-related SCARs were enrolled in the study. A total of 183 unrelated native Thai subjects without any evidence of drug allergy were recruited as the control group. Genotyping of HLA class I and class II alleles was performed. Results: Six HLA alleles including HLA-A*01:01, HLA-B*50:01, HLA-C*06:02, HLA-DRB1*15:01, HLA-DQA1*03:01, and HLA-DQB1*03:02, were significantly associated with beta-lactam antibiotics-related SCARs. The highest risk of SCARs was observed in patients with the HLA-B*50:01 allele (OR = 12.6, 95% CI = 1.1-142.9, p = 0.042), followed by the HLA-DQB1*03:02 allele (OR = 5.8, 95% CI = 1.5-22.0, p = 0.012) and the HLA-C*06:02 allele (OR = 5.7, 95% CI = 1.6-19.9, p = 0.011). According to the phenotypes of SCARs related to beta-lactam antibiotics, the higher risk of SJS/TEN was observed in patients with HLA-A*03:02, HLA-B*46:02 (OR = 17.5, 95% CI = 1.5-201.6, p = 0.033), HLA-A*02:06, HLA-B*57:01 (OR = 9.5, 95% CI = 1.3-71.5, p = 0.028), HLA-DQB1*03:02 (OR = 7.5, 95% CI = 1.8-30.9, p = 0.008), or HLA-C*06:02 (OR = 4.9, 95% CI = 1.1-21.4, p = 0.008). While eight HLA alleles including HLA-A*02:05, HLA-A*02:11, HLA-B*37:01, HLA-B*38:01, HLA-B*50:01, HLA-C*06:02, HLA-C*03:09, and HLA-DRB1*15:01 were associated with AGEP, the highest risk of AGEP was observed in patients with the HLA-B*50:01 allele (OR = 60.7, 95% CI = 4.8-765.00, p = 0.005). Among the four HLA alleles associated with DRESS including HLA-C*04:06, HLA-DRB1*04:05, HLA-DRB1*11:01, and HLA-DQB1*04:01, the HLA-C*04:06 allele had the highest risk of beta-lactam antibiotics-related DRESS (OR = 60.0, 95% CI = 3.0-1202.1, p = 0.043). However, these associations did not achieve statistical significance after Bonferroni's correction. Apart from the HLA risk alleles, the HLA-A*02:07 allele appeared to be a protective factor against beta-lactam antibiotic-related SCARs (OR = 0.1, 95% CI = 0.0-0.5, p = 3.7 × 10-4, Pc = 0.012). Conclusion: This study demonstrated the candidate HLA alleles that are significantly associated with several phenotypes of beta-lactam antibiotics-related SCARs. However, whether the HLA alleles observed in this study can be used as valid genetic markers for SCARs related to beta-lactam antibiotics needs to be further explored in other ethnicities and larger cohort studies.

Effect of albumin on human liver microsomal and recombinant CYP1A2 activities: impact on in vitro-in vivo extrapolation of drug clearance.

Long-chain unsaturated fatty acids inhibit several cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes involved in drug metabolism, including CYP2C8, CYP2C9, UGT1A9, UGT2B4, and UGT2B7. Bovine serum albumin (BSA) enhances these cytochrome P450 and UGT activities by sequestering fatty acids that are released from membranes, especially with human liver microsomes (HLM) as the enzyme source. Here, we report the effects of BSA on CYP1A2-catalyzed phenacetin (PHEN) O-deethylation and lidocaine (LID) N-deethylation using HLM and Escherichia coli-expressed recombinant human CYP1A2 (rCYP1A2) as the enzyme sources. BSA (2% w/v) reduced (p < 0.05) the K(m) values of the high-affinity components of human liver microsomal PHEN and LID deethylation by approximately 70%, without affecting V(max). The K(m) (or S(50)) values for PHEN and LID deethylation by rCYP1A2 were reduced to a similar extent. A fatty acid mixture, comprising 3 µM concentrations each of oleic acid and linoleic acid plus 1.5 µM arachidonic acid, doubled the K(m) value for PHEN O-deethylation by rCYP1A2. Inhibition was reversed by the addition of BSA. K(i) values for the individual fatty acids ranged from 4.7 to 16.7 µM. Single-point in vitro-in vivo extrapolation (IV-IVE) based on the human liver microsomal kinetic parameters obtained in the presence, but not absence, of BSA predicted in vivo hepatic clearances of PHEN O-deethylation and LID N-deethylation that were comparable to values reported in humans, although in vivo intrinsic clearances were underpredicted. Prediction of the in vivo clearances of the CYP1A2 substrates observed here represents an improvement on other experimental systems used for IV-IVE.

Effects of Phyllanthus amarus on the pharmacokinetics of midazolam and cytochrome P450 activities in rats.

Phyllanthus amarus, a commonly used medicinal herb, was investigated for possible herb-drug interactions. The effect on CYP3A-mediated drug metabolism in rats after single dose administration of P. amarus extract was investigated using midazolam (MDZ) as a probe substrate. The effect of multiple dose administration of P. amarus extract on activity and expression of various CYP isoforms were studied. Oral administration of P. amarus extract (800 mg/kg) 1 h before oral MDZ increased the C(max) and AUC(0--∞) of MDZ by 3.9- and 9.6-fold and decreased the clearance by 12%, but did not alter the pharmacokinetics of intravenous MDZ. Daily administration of P. amarus extract (200 or 800 mg/kg/day) for 15 days in rats increased the activity and expression of CYP3A and CYP2B1/2. In contrast, the activities and expressions of CYP1A, CYP2C and CYP2E1 were not significantly changed. The dual effects of P. amarus extract on CYP enzymes were demonstrated. Single dose administration of the extract increased oral bioavailability of MDZ through inhibition of intestinal CYP3A whereas repeated administration of the extract slightly induced hepatic CYP3A and CYP2B1/2 in rats, which suggested that herb-drug interactions by P. amarus may potentially occur via CYP3A and 2B.

HLA-B*1502 strongly predicts carbamazepine-induced Stevens-Johnson syndrome and toxic epidermal necrolysis in Thai patients with neuropathic pain.

BACKGROUND: Carbamazepine (CBZ) is one of the standard pharmacological treatments for neuropathic pain. However, its serious adverse drug reactions include Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Recently, HLA-B*1502 allele was implicated as a genetic marker of CBZ-induced SJS/TEN in some Asian epilepsy populations. METHODS: This is a case control study to describe the clinical characteristics of SJS/TEN in Thai patients with neuropathic pain who were treated with CBZ, and to determine the association of HLA-B*1502 in these patients, comparing with those who exposed to CBZ for at least 6 months without any cutaneous reactions. RESULTS: Thirty-four SJS/TEN patients and 40 control patients were included in this study. Mean age of SJS/TEN patients was 47 years. SJS/TEN was developed in 10.8 ± 1.4 days after initiation of CBZ. HLA-B*1502 allele was found in 32 of 34 SJS/TEN patients (94.1%) but it was found only in 7 of 40 control patients (17.5%). The association was very strong with an odds ratio of 75.4. Sensitivity and specificity of this HLA-B*1502 genotype test were 94.1% and 82.5%, respectively, while the positive predictive value and negative predictive value were 1.43% and 99.98%, respectively. Positive and negative likelihood ratios were 5.37 and 0.07, respectively. CONCLUSIONS: HLA-B*1502 is a strong genetic marker for CBZ-induced SJS/TEN in Thai patients with neuropathic pain. The screening for this marker should be performed prior to initiation of CBZ treatment to assess the risk of this serious side effect.