Association of NAT2 genetic polymorphism with the efficacy of Neurotropin® for the enhancement of aggrecan gene expression in nucleus pulposus cells: a pilot study

Background Intervertebral disc degeneration, one of the major causes of low-back pain, results from altered biosynthesis/turnover of extracellular matrix in the disc. Previously, we reported that the analgesic drug Neurotropin® (NTP) had an anabolic effect on glycosaminoglycan synthesis in cultured nucleus pulposus (NP) cells via the stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1. However, its effect on the aggrecan core protein was not significantly detected, because of the data variance. A microarray analysis suggested that the effect of NTP on aggrecan was correlated with N-acetyltransferase 2 (NAT2), a drug-metabolizing enzyme. Specific NAT2 alleles are known to correlate with rapid, intermediate, and slow acetylation activities and side effects of various drugs. We investigated the association between the efficacy of NTP on aggrecan expression and the NAT2 genotype in cell donors. Methods NP cells were isolated from intervertebral disc tissues donated by 31 Japanese patients (28–68 years) who underwent discectomy. NTP was added to the primary cell cultures and its effect on the aggrecan mRNA was analyzed using real-time quantitative PCR. To assess acetylator status, genotyping was performed based on the inferred NAT2 haplotypes of five common single-nucleotide polymorphisms using allele-specific PCR. Results The phenotype frequencies of NAT2 in the patients were 0%, 42.0%, and 58.0% for slow, intermediate, and rapid acetylators, respectively. The proportions of responders to NTP treatment (aggrecan upregulation, ≥ 1.1-fold) in the intermediate and rapid acetylators were 76.9% and 38.9%, respectively. The odds ratio of the comparison of the intermediate acetylator status between responders and nonresponders was 5.2 (95% CI 1.06–26.0, P = 0.036), and regarding the 19 male patients, this was 14.0 (95% CI 1.54–127.2, P = 0.012). In the 12 females, the effect was not correlated with NAT2 phenotype but seemed to become weaker along with aging. Conclusions An intermediate acetylator status significantly favored the efficacy of NTP treatment to enhance aggrecan production in NP cells. In males, this tendency was detected with higher significance. This study provides suggestive data of the association between NAT2 variants and the efficacy of NTP treatment. Given the small sample size, results should be further confirmed.

Genetic characterization of N-acetyltransferase 2 variants in acquired multidrug-resistant tuberculosis in Indonesia

Background: Owing to the high resistance rate of tuberculosis (TB) to isoniazid, which is metabolized by N-acetyltransferase 2 (NAT2), we investigated the associations between NAT2 variants and multidrug-resistant (MDR)-TB. Materials & methods: The acetylator status based on NAT2 haplotypes of 128 patients with MDR-TB in Indonesia were compared with our published data from patients with anti-TB drug-induced liver injury (AT-DILI), TB and the general population. Results: NAT2*4 was more frequent in the MDR-TB group than in the AT-DILI group, TB controls and general controls. NAT2*4/*4 was significantly more frequent in patients with MDR-TB than in those with AT-DILI. NAT2*5B/7B, *6A/6A and *7B/*7B were detected at lower frequencies in patients with AT-DILI. Rapid acetylators were significantly more frequent in patients with MDR-TB than in those with AT-DILI. Conclusion: These results provide an initial data for optimizing TB treatment in the Indonesian population, and suggest that NAT2 genotyping may help to select appropriate treatment by predicting TB-treatment effect.

NAT2 genetic polymorphism and age dependent efficacy of neurotropin® for the enhancement of aggrecan gene expression in nucleus pulposus cells

Background Intervertebral disc degeneration, one of the major causes of low-back pain, results from altered biosynthesis/turnover of extracellular matrix in the disc. Previously, we reported that the analgesic drug Neurotropin® (NTP) had an anabolic effect on glycosaminoglycan synthesis in cultured nucleus pulposus (NP) cells via the stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1. However, its effect on the aggrecan core protein was not significantly detected, because of the data variance. A microarray analysis suggested that the effect of NTP on aggrecan was correlated with N-acetyltransferase 2 (NAT2), a drug-metabolizing enzyme. Specific NAT2 alleles are known to correlate with rapid, intermediate, and slow acetylation activities and side effects of various drugs. We investigated the association between the efficacy of NTP on aggrecan expression and the NAT2 genotype in cell donors. Methods NP cells were isolated from intervertebral disc tissues donated by 31 Japanese patients (28–68 years) who underwent discectomy. NTP was added to the primary cell cultures and its effect on the aggrecan mRNA was analyzed using real-time quantitative PCR. To assess acetylator status, genotyping was performed based on the inferred NAT2 haplotypes of five common single-nucleotide polymorphisms using allele-specific PCR. Results The phenotype frequencies of NAT2 in the patients were 0%, 42.0%, and 58.0% for slow, intermediate, and rapid acetylators, respectively. The proportions of responders to NTP treatment (aggrecan upregulation, ≥1.1-fold) in the intermediate and rapid acetylators were 76.9% and 38.9%, respectively. The odds ratio of the comparison of the intermediate acetylator status between responders and nonresponders was 5.2 (95% CI, 1.06–26.0, P = 0.036), and regarding the 19 male patients, this was 14.0 (95% CI, 1.54–127.2, P = 0.012). In the females, the effect was not correlated with NAT2 phenotype, but was negatively correlated with age (r = − 0.773, P = 0.006). Conclusions An intermediate acetylator status significantly favored the efficacy of NTP treatment to enhance aggrecan production in NP cells. In males, this tendency was detected with higher significance. Sex and age were also implicated in NTP efficacy. This study provides evidence of the association between NAT2 variants and the efficacy of NTP treatment.

Population Pharmacokinetic Analysis of Isoniazid among Pulmonary Tuberculosis Patients from China

ABSTRACT The blood concentration of isoniazid (INH) is evidently affected by polymorphisms in N-acetyltransferase 2 (NAT2), an enzyme that is primarily responsible for the trimodal (i.e., fast, intermediate, and slow) INH elimination. The pharmacokinetic (PK) variability, driven largely by NAT2 activity, creates a challenge for the deployment of a uniform INH dosage in tuberculosis (TB) patients. Although acetylator-specific INH dosing has long been suggested, well-recognized dosages according to acetylator status remain elusive. In this study, 175 blood samples were collected from 89 pulmonary TB patients within 0.5 to 6 h after morning INH administration. According to their NAT2 genotypes, 32 (36.0%), 38 (42.7%), and 19 (21.3%) were fast, intermediate, and slow acetylators, respectively. The plasma INH concentration was detected by liquid chromatography-tandem mass spectrometry. Population pharmacokinetic (PPK) analysis was conducted using NONMEM and R software. A two-compartment model with first-order absorption and elimination well described the PK parameters of isoniazid. Body weight and acetylator status significantly affected the INH clearance rate. The dosage simulation targeting three indicators, including the well-recognized efficacy-safety indicator maximum concentration in serum (Cmax; 3 to 6 μg/ml), the reported area under the concentration-time curve from 0 h to infinity (AUC0–∞; ≥10.52 μg·h/ml), and the 2-h INH serum concentrations (≥2.19 μg/ml), was associated with the strongest early bactericidal activity. The optimal dosages targeting the different indicators varied from 700 to 900 mg/day, 500 to 600 mg/day, and 300 mg/day for the rapid, intermediate, and slow acetylators, respectively. Furthermore, a PPK model for isoniazid among Chinese tuberculosis patients was established for the first time and suggested doses of approximately 800 mg/day, 500 mg/day, and 300 mg/day for fast, intermediate, and slow acetylators, respectively, after a trade-off between efficacy and the occurrence of side effects.

Genotype-Guided Hydralazine Therapy

<b><i>Background:</i></b> Despite its approval in 1953, hydralazine hydrochloride continues to be used in the management of resistant hypertension, a condition frequently managed by nephrologists and other clinicians. Hydralazine hydrochloride undergoes metabolism by the N-acetyltransferase 2 (NAT2) enzyme. NAT2 is highly polymorphic as approximately 50% of the general population are slow acetylators. In this review, we first evaluate the link between NAT2 genotype and phenotype. We then assess the evidence available for genotype-guided therapy of hydralazine, specifically addressing associations of NAT2 acetylator status with hydralazine pharmacokinetics, antihypertensive efficacy, and toxicity. <b><i>Summary:</i></b> There is a critical need to use hydralazine in some patients with resistant hypertension. Available evidence supports a significant link between genotype and NAT2 enzyme activity as 29 studies were identified with an overall concordance between genotype and phenotype of 92%. The literature also supports an association between acetylator status and hydralazine concentration, as fourteen of fifteen identified studies revealed significant relationships with a consistent direction of effect. Although fewer studies are available to directly link acetylator status with hydralazine antihypertensive efficacy, the evidence from this smaller set of studies is significant in 7 of 9 studies identified. Finally, 5 studies were identified which support the association of acetylator status with hydralazine-induced lupus. Clinicians should maintain vigilance when prescribing maximum doses of hydralazine. <b><i>Key Messages:</i></b> NAT2 slow acetylator status predicts increased hydralazine levels, which may lead to increased efficacy and adverse effects. Caution should be exercised in slow acetylators with total daily hydralazine doses of 200 mg or more. Fast acetylators are at risk for inefficacy at lower doses of hydralazine. With appropriate guidance on the usage of <i>NAT2</i> genotype, clinicians can adopt a personalized approach to hydralazine dosing and prescription, enabling more efficient and safe treatment of resistant hypertension.

NAT2 slow acetylator is associated with anti-tuberculosis drug-induced liver injury severity in indonesian population

Aim: We investigated the contribution of NAT2 variants and acetylator status to anti-tuberculosis drug-induced liver injury (AT-DILI) severity. Materials & methods: 100 patients with clinically severe AT-DILI and 210 non-AT-DILI controls were subjected to NAT2 genotyping by direct DNA sequencing. Results: NAT2 slow acetylator was significantly associated with AT-DILI risk (p = 2.7 × 10-7; odds ratio [95% CI] = 3.64 [2.21–6.00]). Subgroup analysis of NAT2 ultra-slow acetylator revealed a stronger association with AT-DILI risk (p = 4.3 × 10-6; odds ratio [95% CI] = 3.37 [2.00–5.68]). Subset analysis of NAT2 acetylator status and severity grade confirmed these results in AT-DILI patients with more severe disease whereas fast and intermediate acetylator phenotypes were associated with a decreased AT-DILI risk. Conclusion: We elucidated the role of NAT2 phenotypes in AT-DILI in Indonesian population, suggesting that NAT2 genotype and phenotype determination are important to reduce AT-DILI risk.

Effect of diabetes mellitus on TB drug concentrations in Tanzanian patients

Background Diabetes mellitus (DM) is associated with poor TB treatment outcome. Previous studies examining the effect of DM on TB drug concentrations yielded conflicting results. No studies have been conducted to date in an African population. Objectives To compare exposure to TB drugs in Tanzanian TB patients with and without DM. Patients and methods A prospective pharmacokinetic study was performed among 20 diabetic and 20 non-diabetic Tanzanian TB patients during the intensive phase of TB treatment. Plasma pharmacokinetic parameters of isoniazid, rifampicin, pyrazinamide and ethambutol were compared using an independent-sample t-test on log-transformed data. Multiple linear regression analysis was performed to assess the effects of DM, gender, age, weight, HIV status and acetylator status on exposure to TB drugs. Results A trend was shown for 25% lower total exposure (AUC0–24) to rifampicin among diabetics versus non-diabetics (29.9 versus 39.9 mg·h/L, P=0.052). The AUC0–24 and peak concentration (Cmax) of isoniazid were also lower in diabetic TB patients (5.4 versus 10.6 mg·h/L, P=0.015 and 1.6 versus 2.8 mg/L, P=0.013). Pyrazinamide AUC0–24 and Cmax values were non-significantly lower among diabetics (P=0.08 and 0.09). In multivariate analyses, DM remained an independent predictor of exposure to isoniazid and rifampicin, next to acetylator status for isoniazid. Conclusions There is a need for individualized dosing of isoniazid and rifampicin based on plasma concentration measurements (therapeutic drug monitoring) and for clinical trials on higher doses of these TB drugs in patients with TB and DM.

N-Acetyltransferase 2 (NAT2) Acetylator Status among Systemic Lupus Erythematosus Patients from A Tuberculosis Endemic Area in Bandung, Indonesia

BACKGROUND: Systemic lupus erythematosus (SLE) patients living in Indonesia are prone to tuberculosis (TB) infection, since this country ranks second globally for TB prevalence. Isoniazid, an anti-tuberculosis (TB) drug, is metabolized by enzyme N-acetyltransferase 2 (NAT2) that is encoded by NAT2 gene. NAT2 haplotype, referring as acetylator status, may predispose as genetic factor in SLE development or complicate SLE therapy. This study explored the NAT2 haplotypes and acetylator status among SLE patients living in a TB endemic area.METHODS: Genomic DNA of 260 registered SLE patients at The Rheumatology Clinic of Dr. Hasan Sadikin General Hospital, Bandung, Indonesia were isolated. NAT2 gene was amplified and sequenced, then NAT2 haplotypes and the acetylator status among SLE patients with or without TB history were determined and presented.RESULTS: Most of SLE patients registered were female (n=250; 96.2%). The median age of patients when SLE was diagnosed for the first time was 27 years old (8-69 years), with organ involvement predominantly in musculoskeletal (80.8%) and mucocutaneous (73.1%). TB history, mostly pulmonary TB, was present in 23.1% of SLE patients of whom TB was diagnosed before SLE (10.4%) or after SLE (10.7%) or both before and after SLE (2%). The acetylator status was mostly intermediate (61.5%) with the NAT2*4/*6B was the most prevalent haplotype (25.8%).CONCLUSION: There is a high number of intermediate and low acetylator status among SLE patients. Since these SLE patients live in TB endemic area, the NAT2 acetylator status determination among SLE patients before starting TB therapy may have clinical benefit to decrease a possible drug induced liver injury, and this warrants further study.KEYWORDS: NAT2, acetylator, systemic lupus erythematosus, tuberculosis