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.

NAT2 Gene Polymorphisms and Plasma Isoniazid Concentration in Vietnamese Tuberculosis Patients

Isoniazid (INH) is one of the most common drugs for tuberculosis (TB) treatment and INH acetylation catalyzed by non-inducible hepatic enzyme arylamine N-acetyltransferase type 2 (NAT2). The isoniazid acetylation rates, which depend on NAT2 genotypes, is constant in an individual but can changes between patients. Phenotypic groups can be classified based on the genotype: slow, intermediate, and rapid acetylators. This study was performed to identify the relation between NAT2 gene polymorphisms and plasma INH concentrations among the different genotypes of Vietnamese tuberculosis patients. Blood samples of 136 adult TB patients treated with INH were collected and genotyped for NAT2 gene polymorphisms using Sanger sequencing. Two-hour post-dosing INH plasma concentrations were determined by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Among the 136 patients genotyped, there were 43 (31.62 %), 58 (42.65 %), and 35 (25.74 %) of slow, intermediate, and rapid acetylation phenotypes, with two-hour post dosing INH plasma concentrations of 3.4, 2.7, and 2.2 μg/ml, respectively. The differences in INH concentrations among the three genotypes were significant (P < 0.05). Genotyping of TB patients from Vietnam for NAT2 gene polymorphism revealed that 48 percent of the study population comprised slow acetylators. Two-hour INH levels were significantly different among CC and TT homozygous genotypes of NAT2(C282T), as 2.7 μg/ml and 3.9 μg/ml, respectively. This suggests that NAT2(C282T) could play a role in INH metabolism in TB patients. Methods: Blood samples of 136 adult TB patients treated with INH were collected and genotyped for NAT2 gene polymorphisms using Sanger sequencing. Two-hour post-dosing INH plasma concentrations were determined by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Results: Among the 136 patients genotyped, there were 43 (31.62%), 58 (42.65%) and 35 (25.74%) of slow, intermediate and rapid acetylation phenotypes with two-hour post dosing INH plasma concentration of 3.4, 2.7 and 2.2 μg/ml, respectively. The differences in INH concentrations among the three genotypes were significant (P<0.05). Conclusions: Genotyping of TB patients from Vietnam for NAT2 gene polymorphism revealed that 48 per cent of the study population comprised slow acetylators. Two-hour INH levels were significantly different among slow and rapid acetylators.

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.

Decreased Bioavailability of Rifampin and Other Antituberculosis Drugs in Patients with Advanced Human Immunodeficiency Virus Disease

ABSTRACT We evaluated the effects of human immunodeficiency virus (HIV) disease on pharmacokinetics of antituberculosis medications by measuring concentrations of isoniazid and rifampin in blood and of pyrazinamide and ethambutol in urine. Peak concentration and exposure were reduced for rifampin, and rapid acetylators of isoniazid had lower drug levels. HIV and HIV-tuberculosis patients who have diarrhea and cryptosporidial infection exhibit decreased bioavailability of antituberculosis drugs.