Case Study 10: A Case to Investigate Acetyl Transferase Kinetics.

Major routes of metabolism for marketed drugs are predominately driven by enzyme families such as cytochromes P450 and UDP-glucuronosyltransferases. Less studied conjugative enzymes, like N-acetyltransferases (NATs), are commonly associated with detoxification pathways. However, in the clinic, the high occurrence of NAT polymorphism that leads to slow and fast acetylator phenotypes in patient populations has been linked to toxicity for a multitude of drugs. A key example of this is the observed clinical toxicity in patients who exhibit the slow acetylator phenotype and were treated with isoniazid. Toxicity in patients has led to detailed characterization of the two NAT isoforms and their polymorphic genotypes. Investigation in recombinant enzymes, genotyped hepatocytes, and in vivo transgenic models coupled with acetylator status-driven clinical studies have helped understand the role of NATs in drug development, clinical study design and outcomes, and potential roles in human disease models. The selected case studies herein document NAT enzyme kinetics to explore substrate overlap from two human isoforms, preclinical species considerations, and clinical genotype population concerns.

Acetylator Status Among Newly Diagnosed and Recurrent Tuberculosis Patients from Kupang, Eastern Part of Indonesia.

PURPOSE: N-acetyltransferase-2 enzyme in the liver, encoded by NAT2 gene, plays a central role in metabolizing tuberculosis (TB) drug isoniazid (INH). Low compliance of patients toward six-month TB therapy and internal host factors, ie comorbid diseases, immune status, and genetic profiles, are factors leading to treatment failure and recurrence of pulmonary TB infection. This study aimed to explore the NAT2 acetylator status among newly diagnosed and recurrent pulmonary TB patients in eastern part of Indonesia. PATIENTS AND METHODS: Archived DNA of TB patients (n=124) and healthy controls (n=124) were sequenced, and NAT2 acetylator status was determined, then categorized as fast, intermediate, or slow acetylators. Pulmonary TB patients who had no previous TB treatment history were designated as newly diagnosed pulmonary TB, whereas patients with a history of TB treatment were designated as recurrent pulmonary TB. The demographic, clinical, and microbiological data between pulmonary TB groups were compared, and acetylator status was described among groups. RESULTS: Male was more significantly prevalent in the recurrent pulmonary TB group (p=0.025), and anemia was more prevalent in new pulmonary TB (p=0.003). The acetylator status in pulmonary TB patients compared to healthy controls were rapid (33.9% vs 48.1%), intermediate (57.8% vs 33.0%), and slow acetylators (8.3% vs 18.9%), respectively. Interestingly, the rapid and intermediate acetylator were significantly more prevalent in pulmonary TB patients than in healthy controls (p=0.023, OR=2.58 (1.12-5.97). Furthermore, no differences were found in acetylator status between new and recurrent pulmonary (p=0.776). CONCLUSION: Rapid and intermediate acetylators status predominated the pulmonary TB patients in Kupang, eastern part of Indonesia, postulating different genetic makeup in this area. As the pulmonary TB patients in Kupang exhibit more rapid acetylator phenotype, the acetylator status might be relevant to be checked before TB therapy for adjusting treatment dose to prevent drug resistances.

Pharmacokinetics and pharmacogenetics of anti-tubercular drugs: a tool for treatment optimization?

INTRODUCTION: WHO global strategy is to end tuberculosis epidemic by 2035. Pharmacokinetic and pharmacogenetic studies are increasingly performed and might confirm their potential role in optimizing treatment outcome in specific settings and populations. Insufficient drug exposure seems to be a relevant factor in tuberculosis outcome and for the risk of phenotypic resistance. Areas covered: This review discusses available pharmacokinetic and pharmacogenetic data of first and second-line antitubercular agents in relation to efficacy and toxicity. Pharmacodynamic implications of optimized drugs and new options regimens are reviewed. Moreover a specific session describes innovative investigations on drug penetration. Expert opinion: The optimal use of available antitubercular drugs is paramount for tuberculosis control and eradication. Whilst trials are still on-going, higher rifampicin doses should be reserved to treatment for tubercular meningitis. Therapeutic Drug Monitoring with limiting sampling strategies is advised in patients at risk of failure or with slow treatment response. Further studies are needed in order to provide definitive recommendations of pharmacogenetic-based individualization: however lower isoniazid doses in NAT2 slow acetylators and higher rifampicin doses in individuals with SLCO1B1 loss of function genes are promising strategies. Finally in order to inform tailored strategies we need more data on tissue drug penetration and pharmacological modelling.

CYP2E1 polymorphism, acetylator profiles and drug-induced liver injury incidence of Indonesian tuberculosis patients.

OBJECTIVE: A polymorphism of CYP2E1 may be directly associated with the development of INH hepatotoxicity. We conducted this study to evaluate the association between polymorphisms of CYP2E1, Isoniazid (INH) concentration and the acetylator status of INH in cases of Indonesian tuberculosis patients with drug-induced liver disease (DILI). METHODS: We conducted our study with a cohort design consisting of 55 Indonesian adult tuberculosis (TB) patients. Acetylating phenotypes were studied in using the metabolic ratio of plasma AcHZ/HZ. DILI was defined using CTCAV version 4.0. The allelic and genotypic frequency distributions of CYP2E1 rs 3813867 were studied using the polymerase chain reaction - amplification refractory mutation system (ARMS) methodology. RESULTS: Patients with an INH concentration of more than 7µg/mL showed a higher risk of developing DILI when compared with patients who showed a therapeutic range of 3-6µg/mL INH (OR: 1.3, 95% CI: 0.2-8.2). Slow acetylators had a higher incidence of DILI when compared with rapid acetylators (OR: 4.6, 95% CI: 1.3-15.9). Meanwhile, subjects with GC had a higher risk of DILI incidence (OR: 4.3, 95% CI: 0.8-24.4). CONCLUSION: Our study shows that polymorphisms of CYP2E1 and slow acetylator may have role in the DILI incidence.

Allelic frequencies of single nucleotide polymorphisms (SNPs) in the N-acetyltransferase 2 (NAT2) gene of Filipinos

OBJECTIVES:The present study aims to determine the frequency of occurrence of NAT2*4, NAT2*5A, NAT2*6B, NAT2*7A and NAT2*14A alleles by PCR-RFLP among Filipino volunteers. These alleles correspond to substitutions in the following sites: C341T, G590A, G857A and G191A, respectively, of the NAT2 gene. The presence of specific SNP combination was also used to deduce acetylation status and estimate genotype frequency and describe them in comparison with other populations based on literature. METHODS: Genomic DNA from peripheral blood lymphocytes from 129 healthy Filipino volunteers was used to amplify the NAT2 gene segment. The RFLP analysis was done by restricting the expected PCR product with Kpn1, Taq1, BamH1 and Msp1/Al1, respectively, to detect the 4 alleles: NAT2*4, NAT2*5A, NAT2*6B, NAT2*7A and NAT2*14A. RESULTS:The calculated allelic frequencies in Hardy-Weinberg equilibrium of NAT2*5A (C481T), NAT2*6B (G590A), NAT2*7A (G857A) and NAT2*14A (G191A) were 0.058, 0.097, 0.182 and 0.046, respectively. NAT2*4 had an allele frequency of 0.617. Nine genotypes were determined: NAT2*4/*4, NAT2*4/*5A, NAT2*4/*6B, NAT2*4/*7A, NAT2*4/*14A, NAT2*5A/*7A, NAT2*6B/*7A, NAT2*6B/*14A and NAT2*7A/*14A. From these genotypes, acetylator phenotypes were deduced. A trimodal pattern of distribution was established: rapid, intermediate and slow acetylators with the following percentages, 47.3%, 41.1 % and 11.6%. Among the slow acetylator SNPs determined, NAT2*7A was found as the most frequent allele and NAT2*14A was found as the least frequent allele. CONCLUSION:The study showed the mutation profile and observed genotypic similarities and differences of Filipinos with other Asian populations and Americans and other Caucasians based on literature. The results also suggest a trimodal pattern of distribution of acetylators and lesser number of slow acetylators among Filipino populations, a characteristic similar to other Asian populations but significantly different from Americans and other Caucasians. The occurrence of NAT2*7A and NAT2*14A can be further sequenced to verify the observed genotype.