N-acetyltransferase Research Articles

NAT2 knockdown inhibits the development of colorectal cancer and its clinical significance.

This study aims to explore the correlation between N-acetyltransferase 2 (NAT2) expression in colorectal cancer (CRC) tissues and the progression and prognosis of CRC. Through in vitro and in vivo experiments, the biological functions of NAT2 in the occurrence and development of CRC were explored. Immunohistochemical (IHC) staining, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot were used to detect the difference of NAT2 expression in CRC tissues and normal tissues. The role of NAT2 in the cell proliferation, apoptosis, migration, invasion, and tumorigenesis and development of CRC was analyzed by cell counting kit-8 (CCK-8), colony formation, flow cytometry, transwell cell invasion, wound-healing assays and construction of nude mouse xenograft model. The correlation between the expression level of NAT2, and the overall survival and clinicopathological characteristics of CRC patients were statistically analyzed to preliminarily determine the clinical significance of NAT2 in the diagnosis and prognosis of CRC. The expression level of NAT2 was notably upregulated in CRC. NAT2 knockdown inhibited the proliferation, migration, invasion and in vivo tumor formation of CRC cells, and promoted cell apoptosis. High NAT2 expression was associated with TNM stage, differentiation degree, tumor size, distant metastasis, lymph node metastasis and poor prognosis in CRC patients. This study showed that the expression level of NAT2 in CRC tissues was increased and closely related to the metastasis and prognosis of CRC. In addition, NAT2 can be used as a new prognostic biomarker and therapeutic target for CRC.

Identification of Serotonin as a Predictive Marker for Breast Cancer Patients.

PurposeCumulative evidence has demonstrated that breast cancer was the most commonly diagnosed cancer in women. Despite growing evidence for a link between serotonin and tumorigenesis, research on the expression of serotoninergic systems in the human breast cancer cell and tissue has only rarely been reported.MethodsFirst, immunofluorescence staining, ELISA and Western blotting were used to detect serotonin and melatoninergic systems in various breast cancer cell types. Then, serotonin expression was evaluated in the cultures of TPBC cell line BT-474 and TNBC cell line MDA-MB-231 using immunofluorescence assay. To further explore the diagnostic role of serotonin in breast cancer, serotonin expression was conducted in the TPBC and TNBC tumor sections by immunostaining analysis.ResultsOur results suggested that both human breast cancer cells and human breast epithelial cell line could synthesize serotonin and melatonin. Unlike melatonin, serotonin levels varied significantly between human breast cancer and breast epithelial cell line (p<0.01). In addition, serotonin N-acetyltransferase (NAT) and acetylserotonin methyltransferase (ASMT), the key enzymes in the pathway of melatonin synthesis from serotonin, were also detectable. In agreement with these findings of human breast cancer cell and human breast epithelial cell line, serotonin expression was also much higher in triple-negative (PR−, ER−, HER-2−) breast cancer (TNBC) and triple-positive breast cancer (TPBC) compared to para-carcinoma tissues (PCTs).ConclusionHere, we provided evidence that the human breast cancer cell (MCF-7, Bcap-37) and human breast epithelial cell (MCF-10A) could synthesize intrinsic serotonin and melatonin, and serotonin expression was higher in the breast cancer tissue compared with PCT. The findings suggested that serotonin might be used as a predictive marker for breast cancer patients.

Sequence Conservation and Structural Modeling of an Arginyl‐tRNA Transferase 1 (ATE1)

Arginyl-tRNATransferase 1 (ATE1) is a eukaryotic enzyme that arginylates cellular proteins, is an essential regulator of eukaryotic homeostasis via its involvement in the N-degron pathway, and regulates essential physiological processes such as embryogenesis, aging, cell migration, muscle contraction, and stress. Despite its importance, the structure, mechanism of action, and regulation of ATE1 has yet to be elucidated. The objective of this work is to model the three-dimensional fold of an ATE1, and to determine essential conserved residues that are involved in substrate recognition and enzymatic arginylation. In this study, we have modeled the structure of Saccharomyces cerevisiae ATE1 (ScATE1) and identified a putative location of the GCN5-related N-acetyl transferase (GNAT) fold, which is structurally conserved amongst amino-acid transferases. Additionally, we have performed a large-scale sequence alignment across eukaryotic ATE1s to map conserved residues onto our three-dimensional model. Notably, we have identified a highly-conserved His residue that would be consistent with the recognition of a negatively charged substrate and is located in a structural homologous site in bacterial amino-acid transferases. We hypothesize that this highly conserved (>90%) His residue is essential for substrate recognition, and site-directed mutagenesis is underway to verify this hypothesis. These results provide the first structural insights into the enzyme active site and mechanism of substrate recognition for ScATE1, and the strong sequence conservation suggest a common mode of action across eukaryotic homologs.

Biobank Scale Pharmacogenomics Informs the Genetic Underpinnings of Simvastatin Use.

Studying drug-metabolizing enzymes, encoded by pharmacogenes, may inform biological mechanisms underlying the diseases for which a medication is prescribed. Until recently, pharmacogenes could not be studied at biobank scale. In 7,649 unrelated African-ancestry (AFR) and 326,214 unrelated European-ancestry (EUR) participants from the UK Biobank, we associated pharmacogene haplotypes from 50 genes with 265 (EUR) and 17 (AFR) medication use phenotypes using generalized linear models. In EUR, N-acetyltransferase 2 (NAT2) metabolizer phenotype and activity score were associated with simvastatin use. The dose of NAT2*1 was associated with simvastatin use when compared with NAT2*5 (the most common haplotype). This association was robust to effects of low-density lipoprotein cholesterol (LDL-C) concentration (NAT2*1 odds ratio (OR)=1.07, 95% CI: 1.05-1.09, P=1.14×10-8 ) and polygenic risk for LDL-C concentration (NAT2*1 OR=1.09, 95% CI: 1.04-1.14, P=2.26×10-4 ). Interactive effects between NAT2*1 and simvastatin use on LDL-C concentration (OR = 0.957, 95% CI: 0.916-0.998, P=0.045) were replicated in the electronic Medical Records and Genomics Pharmacogenetic Sequencing Pilot (eMERGE-PGx) cohort (OR = 0.987, 95% CI: 0.976-0.998, P=0.029). We used biobank-scale data to uncover and replicate an association between NAT2 locus variation and better response to statin therapy. Testing NAT2 alleles may be useful for making clinical decisions regarding the potential benefit (e.g., absolute risk reduction) in LDL-C concentration prior to statin treatment.

Molecular docking approach to elucidate metabolic detoxification pathway of polycyclic aromatic hydrocarbons

This study assessed the molecular interactions of (±)-anti-and-syn- dibenzo[a,l]pyrene-11,12-diol-13,14-epoxide (DBPDE), 7,12- dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (DMBADE), N2- hydroxylated-PhIP(N2-OH-PhIP), (±)-anti-and-syn-benzo[a]pyrene-7,8-diol- 9,10-epoxide (BPDE) with various Glutathione S-transferase (GST) and N- acetyltransferase (NAT) isozymes. Our in-silico data revealed that GSTP1 (- 8.83 kcal/mol), showing more plausible binding as compared to GSTM1 (-8.74 kcal/mol) and GSTA1 (ΔG: -8.03 kcal/mol) against (-)-anti-DBPDE and (+)- syn-DBPDE. We also investigated the involvement of GST and NAT isozymes in the conjugation of DMBADE andN2-OH-PhIP as a control despite their preferred routes sulfonation and glucuronidation for detoxification. The findings exhibited feeble binding of different classes of GSTs with metabolites of DMBA and PhIP, as highlighted by their free energy of binding. The enzymatic activity of GSTM1 against the most potent diol-epoxide of benzo[a]pyrene (BP), (+)-anti-BPDE, and (+)-syn-BPDE followed by GSTP1 and GSTA1 has well documented. In addition, these findings provide new perspectives for most probable mechanistic details of the detoxification pathway of PAHs and xenobiotics useful in combination therapy for future ligand-based drug discovery and development.

Influence of N-acetyltransferase 2 gene polymorphisms on the in vitro metabolism of the epidermal growth factor receptor inhibitor rociletinib.

Rociletinib showed activity in T790M-positive non-small cell lung cancer patients. It undergoes amide hydrolysis to form M502, followed by N-acetylation to M544 or amide hydrolysis to M460. We identified the enzymes responsible for rociletinib metabolism, and investigated the relationship between M544 formation and N-acetyltransferase 2 (NAT2) polymorphisms. Rociletinib and metabolites were incubated with carboxylesterase (CES)1b, CES1c, CES2, NAT1, NAT2, arylacetamide deacetylase, inhibitors, pooled human liver microsomes (HLM) and cytosols (HLC). Cytosols (n =107) were genotyped for NAT2 polymorphisms (rs1041983 and rs1801280) and incubated with M502. Human hepatocytes from intermediate (NAT2*6/*12A) and slow (NAT2*5B/*5B) acetylators were incubated with 10μM rociletinib and metabolites for 24 hours. Metabolites were measured by high-performance liquid chromatography. M502 was formed from rociletinib and M544 by CES2 and HLM; M544 and N-acetyl-M460 were formed by NAT2 and HLC; M460 was not formed by CES or arylacetamide deacetylase. M502 formation by HLM was inhibited by bis-(4-nitrophenyl)phosphate and eserine (10μM). M544 formation in HLC was inhibited by 100 μM quercetin and was associated with NAT2 genotype (P <.0001). M460 formation in HLM was inhibited by eserine, and M460 was N-acetylated in HLC. Hepatocytes formed M502, M544 and M460. The intermediate acetylator showed higher production (range: 3.4-5.1-fold) of N-acetylated metabolites than the slow acetylator. Results indicate that NAT2 and CES2 are involved in rociletinib metabolism, and polymorphic NAT2 could alter drug exposure in patients. Slow NAT2 acetylators would have higher exposure to M502 and M460 and consequently, be at increased risk of experiencing hyperglycaemia and QTc prolongation.

Alteration of the risk of pre-oral cancer and cancer in North Indian population by NAT1 and NAT2 polymorphisms genotypes and haplotypes.

The risk of oral cancer is strongly related to consumption of tobacco, smoking and drinking alcohol. N-acetyl transferases 1,2 are phase II metabolic enzymes, metabolize aryl and heterocyclic amines which are present in tobacco. NAT2 slows acetylator phenotype and the genotype is related to reduced ability to detoxify these xenobiotic that are carcinogenic to tissues. The aim of our study to determine the risk of oralcancer as well as oral precancerous lesions in North Indian population with polymorphisms in these two N-acetyl transferases 1,2 genes. A total of 250 patients with pre oral cancer, oral cancer and 250 healthy volunteers were genotypes for the NAT1 and NAT2 gene polymorphisms. Genotypes were identified by PCR and RFLP. Genotype frequencies were evaluated by Chi-square test and risk of disease was estimated by Odds ratio (OR) with 95% confidence interval. Our results showed that individuals with CT and TT genotypes of NAT1 C > T polymorphism were significantly lower risk of oral diseases (p value = 0.02, OR = 0.60 and p value = 0.04, OR = 0.58, respectively). For NAT2 C > T polymorphism, the TT genotype significantly increased the risk of OSMF (Oral Sub mucous Fibrosis) and Leukoplakia (p value = 0.001, OR = 4.16; p value = 0.002, OR = 4.38, respectively). In contrary, the CC genotype for NAT2 T > C polymorphism increased the risk of OSMF (p value = 0.01, OR = 3.00, 95% CI = 1.31-6.86). Our study concludes that the NAT1 polymorphism shows protective association with oral diseases and NAT2 polymorphism and haplotypes also influence the susceptibility to oral diseases in North Indian population subjects.

Association of Nucleotide Variants of GRHL3, IRF6, NAT2, SDC2, BCL3, and PVRL1 Genes with Nonsyndromic Cleft Lip With/Without Cleft Palate in Multigenerational Families: A Retrospective Study.

Background:Several genes are associated with the etiology of cleft lip and palate (CLP) in different populations. Many nucleotide variants on genes such as GRHL3, IRF6, NAT2, SDC2, BCL3, and PVRL1 were reported in different populations, but not studied in multigenerational cases in the Indian population.Aim and Objective:The aim of this study is to evaluate whether nucleotide variants rs41268753, rs861020, rs1041983, rs1042381, rs2965169, and rs10790332 are involved in the etiology of nonsyndromic CLP (NSCLP) in multigenerational Indian families.Study Design:Retrospective genetic study.Materials and Methods:Based on inclusion and exclusion criteria, 20 multigenerational families with nonsyndromic cleft lip with or without cleft palate (NSCL/P) were selected. Blood samples from both affected and unaffected participants were collected as a source of genomic DNA. Six nucleotide variants on these genes were genotyped to test for the association with NSCL/P. Genotyping was performed with the MassArray method. Genotype distribution was used to calculate the Hardy–Weinberg equilibrium using PLINK, a whole-genome association analysis toolset. The allelic association was compared among cases and controls using Chi-square test as implemented in PLINK. P ≤ 0.05 indicates statistical differences between groups.Results:No significant associations were found between individual single-nucleotide polymorphisms and NSCL/P. The odds ratio was 1.531, 1.198, 0.8082, 1.418, 1, and 0.5929 for polymorphisms rs41268753, rs861020, rs1041983, rs1042381, rs2965169, and rs10790332, respectively.Conclusion:Our findings suggest that among the multigenerational families in our population, the high-risk nucleotide variants GRHL3 rs41268753, IRF6 rs861020, NAT2 rs1041983, SDC2 rs1042381, BCL3 rs2965169, and PVRL1 rs10790332 are not associated with increased risk of NSCL/P.

The expression pattern of N-acetyltransferase 1 in healthy human skin.

Background N‐acetyltransferase 1 (NAT1) is an enzyme expressed among others in keratinocytes in human skin. NAT1 is important in the biotransformation of aromatic amines, an important example being p‐phenylenediamine (PPD), a hair dye molecule. Unoxidized PPD penetrates the skin and is N‐acetylated by NAT1.ObjectivesTo investigate in detail the expression pattern of NAT1 in human skin.Materials and MethodsCryosections obtained from healthy human skin were stained for NAT1 and expression patterns were observed. NAT1 double stainings were performed with antibodies against different cellular organelles to determine expression patterns.ResultA speckled, granular expression of NAT1 was seen predominantly in the stratum basale. NAT1 was expressed in a cytoplasmic pattern, perinuclear, and in the nucleus. No co‐localisation was seen with the selected cellular organelles. Local differences in NAT1 expression patterns were observed between donors and between different biopsies obtained from the same donor.ConclusionsNAT1 is expressed predominantly in the stratum basale and can be found in the cytoplasm, nucleus, and perinuclear in human skin. Further studies should be performed to investigate expression of NAT1 in a larger sample size.

Functional Characterization of the Effects of N-acetyltransferase 2 Alleles on N-acetylation of Eight Drugs and Worldwide Distribution of Substrate-Specific Diversity.

Variability in the enzymatic activity of N-acetyltransferase 2 (NAT2) is an important contributor to interindividual differences in drug responses. However, there is little information on functional differences in N-acetylation activities according to NAT2 phenotypes, i.e., rapid, intermediate, slow, and ultra-slow acetylators, between different substrate drugs. Here, we estimated NAT2 genotypes in 990 Japanese individuals and compared the frequencies of different genotypes with those of different populations. We then calculated in vitro kinetic parameters of four NAT2 alleles (NAT2∗4, ∗5, ∗6, and ∗7) for N-acetylation of aminoglutethimide, diaminodiphenyl sulfone, hydralazine, isoniazid, phenelzine, procaineamide, sulfamethazine (SMZ), and sulfapyrizine. NAT2∗5, ∗6, and ∗7 exhibited significantly reduced N-acetylation activities with lower Vmax and CLint values of all drugs when compared with NAT2∗4. Hierarchical clustering analysis revealed that 10 NAT2 genotypes were categorized into three or four clusters. According to the results of in vitro metabolic experiments using SMZ as a substrate, the frequencies of ultra-slow acetylators were calculated to be 29.05–54.27% in Europeans, Africans, and South East Asians, whereas Japanese and East Asian populations showed lower frequencies (4.75 and 11.11%, respectively). Our findings will be helpful for prediction of responses to drugs primarily metabolized by NAT2.

Study of the Effect of Polymorphic Markers of the NAT2 Gene on the Risk of Adverse Drug Reactions in Patients with Pulmonary Tuberculosis Who Received Isoniazid and Rifampicin

Tuberculosis remains one of the most dangerous and widespread infectious diseases. More than 20 medicinal products are currently available for the treatment of tuberculosis. One of the most serious adverse drug reactions (ADRs) associated with anti-tuberculosis medicines is hepatotoxicity.The aim of the study was to assess the effect of polymorphic markers of the NAT2 gene on the ADR risk in patients with pulmonary tuberculosis who received isoniazid and rifampicin.Materials and methods. The study included 67 patients with different forms of pulmonary tuberculosis who received combination therapy with isoniazid and rifampicin. Single nucleotide polymorphisms (SNPs) of the NAT2 gene were determined by real-time PCR. Statistical processing was performed using SPSS Statistics 20.0.Results: Six SNPs were identified in the NAT2 gene. Based on these SNPs the following phenotypes were determined by the rate of NAT2 acetylation: fast acetylators—6 subjects, intermediate acetylators—24 subjects, and slow acetylators—37 subjects. The study assessed the relationship between the acetylator phenotype and the development of ADRs during combination therapy with isoniazid and rifampicin. Slow acetylators had a significantly greater increase in total bilirubin level (p=0.011) compared to intermediate acetylators. Loss of appetite was more often observed in fast acetylators than in intermediate acetylators (p=0.021).Conclusions. The obtained data suggest interrelation between the slow type of NAT2 acetylation and the risk of ADRs in patients undergoing pulmonary tuberculosis chemotherapy with isoniazid and rifampicin. Out of all the ADRs registered in the study, the fast acetylators were more likely to have loss of appetite, however, the expansion of the study population is needed to verify this observation. The studied polymorphisms have an impact on the development of ADRs in patients undergoing pulmonary tuberculosis chemotherapy with isoniazid and rifampicin and may be used to predict the safety profile of pharmacotherapy in this group of patients.

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

BackgroundIntervertebral 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.MethodsNP 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.ResultsThe 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.ConclusionsAn 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.

Population Pharmacokinetic Analyses of Isoniazid in Tuberculosis Treatment: a Systematic Review

The aims of this systematic review are to provide knowledge concerning population pharmacokinetics of isoniazid (INH) and to identify factors influencing INH pharmacokinetic variability. Pubmed and Embase databases were systematically searched from inception to July, 2017. Relevant articles from reference lists were also included. All population pharmacokinetic studies of INH written in English, conducted in human (either healthy subjects or pulmonary tuberculosis patients) were included in this review. Ten studies were included in this review. Most studies characterized a two-compartment model with first-order kinetics for INH with a transit-compartment model for absorption suggested. Frequently reported significant predictors for INH clearance is NAT2 acetylator types (slow/intermediate/fast), while weight is a significant covariate for INH volume of distribution (both central and peripheral). In children, enzyme maturation had a profound affect on INH clearance.&#x0D; Keywords:&#x0D; Population pharmacokinetics, Isoniazid.&#x0D; References&#x0D; [1] World Health Organization, Global Tuberculosis Report 2019. https://apps.who.int/iris/bitstream/handle/10665/329368/9789241565714-eng.pdf (accessed 18 December 2019).[2] United Nations, Transforming our world: The 2030 agenda for sustainable development, New York, USA, 2015.[3] K. Takayama, L. Wang, H.L. David, Effect of isoniazid on the in vivo mycolic acid synthesis, cell growth, and viability of Mycobacterium tuberculosis, Antimicrob Agents Chemother 2.1 (1972) 29-35. https://doi.org/10.1128/aac.2.1.29 [4] A. Jindani, V.R. Aber, E. A. Edwards, D. A. Mitchison, The early bactericidal activity of drugs in patients with pulmonary tuberculosis. Am Rev Respir Dis 121(6) (1980) 939-49. https://doi.org/10.1164/arrd.1980.121.6.939 [5] P.R. Donald, The influence of human N-acetyltransferase genotype on the early bactericidal activity of isoniazid. Clin Infect Dis 39(10) (2004) 1425-30. https://doi.org/10.1086/424999 [6] D.A. Mitchison, Basic mechanisms of chemotherapy, Chest 76(6 Suppl) (1979) 771-81. https://doi.org/10.1378/chest.76.6_supplement.771 [7] H. McIlleron et al., Determinants of rifampin, isoniazid, pyrazinamide, and ethambutol pharmacokinetics in a cohort of tuberculosis patients, Antimicrob Agents Chemother 50(4) (2006) 1170-7. https://doi.org/10.1128/aac.50.4.1170-1177.2006 [8] S. Chideya et al., Isoniazid, rifampin, ethambutol, and pyrazinamide pharmacokinetics and treatment outcomes among a predominantly HIV-infected cohort of adults with tuberculosis from Botswana, Clin Infect Dis 48(12) (2009) 1685-94. https://doi.org/10.1086/599040 [9] N. Singh et al., Study of NAT2 gene polymorphisms in an Indian population: association with plasma isoniazid concentration in a cohort of tuberculosis patients. Mol Diagn Ther 13(1) (2009) 49-58. https://doi.org/10.1007/bf03256314 [10] N. Buchanan, C. Eyberg, M.D. Davis, Isoniazid pharmacokinetics in kwashiorkor. S Afr Med J 56(8) (1979) 299-300.[11] U.S. Food and Drug Administration (1999), "Guidance for Industry. Populationpharmacokinetics",Retrieved from http://www.fda.gov/downloads/Drugs/.../Guidances/UCM072137.pdf[12] D. R Mould, R. N. Upton, Basic concepts in population modeling, simulation, and model‐based drug development, CPT: pharmacometrics &amp; systems pharmacology 1(9) (2012) 1-14. https://doi.org/10.1038/psp.2012.4 [13] P. Denti et al., Pharmacokinetics of isoniazid, pyrazinamide, and ethambutol in newly diagnosed pulmonary TB patients in Tanzania, PLoS ONE 10(10) (2015), e0141002. https://doi.org/10.1371/journal.pone.0141002 [14] B. Guiastrennec et al., Suboptimal Antituberculosis Drug Concentrations and Outcomes in Small and HIV-Coinfected Children in India: Recommendations for Dose Modifications, Clin Pharmacol Ther 104(4) (2017), 733-741. https://doi.org/10.1002/cpt.987 [15] M. Kinzig-Schippers et al., Should we use N-acetyltransferase type 2 genotyping to personalize isoniazid doses?, Antimicrobial Agents and Chemotherapy 49(5) (2005), 1733-1738. https://doi.org/10.1128/aac.49.5.1733-1738.2005 [16] J.J. Kiser et al., Isoniazid pharmacokinetics, pharmacodynamics, and dosing in South African infants, Therapeutic Drug Monitoring 34(4) (2012) 446-451. https://doi.org/10.1097/ftd.0b013e31825c4bc3 [17] L. Lalande, Population modeling and simulation study of the pharmacokinetics and antituberculosis pharmacodynamics of isoniazid in lungs, Antimicrobial Agents and Chemotherapy 59(9) (2015) 5181-5189. https://doi.org/10.1128/aac.00462-15 [18] C. Magis-Escurra et al., Population pharmacokinetics and limited sampling strategy for first-line tuberculosis drugs and moxifloxacin, International Journal of Antimicrobial Agents 44(3) (2014) 229-234. https://doi.org/10.1016/j.ijantimicag.2014.04.019 [19] C.A. Peloquin et al., Population pharmacokinetic modeling of isoniazid, rifampin, and pyrazinamide, Antimicrobial Agents and Chemotherapy 41(12) (1997) 2670-2679. https://doi.org/10.1128/aac.41.12.2670 [20] K.Y. Seng et al., Population pharmacokinetic analysis of isoniazid, acetylisoniazid, and isonicotinic acid in healthy volunteers, Antimicrobial Agents and Chemotherapy 59(11) (2015) 6791-6799. https://doi.org/10.1128/aac.01244-15 [21] J.J. Wilkins et al., Variability in the population pharmacokinetics of isoniazid in South African tuberculosis patients, British Journal of Clinical Pharmacology 72(1) (2011) 51-62. https://doi.org/10.1111/j.1365-2125.2011.03940.x [22] S.P. Zvada et al., Population pharmacokinetics of rifampicin, pyrazinamide and isoniazid in children with tuberculosis: In silico evaluation of currently recommended doses, Journal of Antimicrobial Chemotherapy 69(5) (2014) 1339-1349. https://doi.org/10.1093/jac/dkt524 [23] World Health Organization, Guidance for national tuberculosis programmes on the management of tuberculosis in children (No. WHO/HTM/TB/2014.03). World Health Organization, 2014.[24] World Health Organization, &amp; Stop TB Initiative (World Health Organization), Treatment of tuberculosis: guidelines. World Health Organization, 2010.[25] J.S. Starke, S.M, Tuberculosis in: James D. Cherry, Ralph D. Feigin (Eds.), Textbook of Pediatric Infectious Diseases., Saunders: Philadelphia, 1998 pp. 1196-1238. [26] J.G. Pasipanodya, S. Srivastava, T. Gumbo, Meta-analysis of clinical studies supports the pharmacokinetic variability hypothesis for acquired drug resistance and failure of antituberculosis therapy, Clinical Infectious Diseases 55(2) (2012) 169-177. https://doi.org/10.1093/cid/cis353&#x0D;

NAT2 polymorphisms as a cause of metamizole-induced agranulocytosis.

Metamizole is a widely prescribed NSAID with excellent analgesic and antipyretic properties. Although very effective, it is banned in some countries because of the risk for severe agranulocytosis. We here describe three patients with metamizole-associated agranulocytosis. Patient #1 suffered from agranulocytosis and tonsillitis followed by severe sepsis by Streptococcus pneumoniae and Epstein-Barr virus reactivation. Her dizygotic twin sister (patient #2) also suffered from agranulocytosis after a surgical intervention. Patient #3 initially had a tonsillitis and also developed neutropenia after metamizole intake. For all patients, pharmacogenetic diagnostic for the genes CYP2C9, CYP2C19 and NAT2, which are involved in metamizole metabolism and degradation of toxic metabolites, was initiated. Pharmacogenetic analysis revealed NAT2 slow acetylator phenotype in all three patients. Additionally, patient #2 is an intermediate metabolizer for CYP2C19 and patient #3 is a poor metabolizer for CYP2C9. Impairment of these enzymes causes a reduced degradation of toxic metabolites, for example, 4-methylaminoantipyrine (4-MAA) or 4-aminoantipyrine. The metabolite 4-MAA can complex with hemin, which is an early breakdown product during hemolysis. Hemolysis is often observed during invasive infections or after surgical procedures. It is known that the 4-MAA/hemin complex can induce cytotoxicity in the bone marrow and interrupt granulocyte maturation. In conclusion, metamizole-induced agranulocytosis most likely was a consequence of the underlying genetical predisposition, that is, polymorphisms in the genes NAT2, CYP2C9 and CYP2C19. Hemolysis may have increased the toxicity of metamizole metabolites.

AtreMorine Treatment Regulates DNA Methylation in Neurodegenerative Disorders: Epigenetic and Pharmacogenetic Studies

Background: Neurodegenerative disorders are one of the major health problems in Western countries. Genetic and epigenetic mechanisms play crucial roles in the origin and progression of these disorders. DNA methylation is the most widely studied epigenetic mark and is an important regulator of gene expression. Objective: Little is known about the influence of bioactive dietary components on epigenetic mechanisms in neurodegenerative diseases. In this study, we investigated the effects of E-PodoFavalin-15999 (AtreMorine®), a bioproduct with potent neuroprotective and dopamine enhancing capabilities, on DNA methylation patterns in Alzheimer’s (AD) and Parkinson’s Disease (PD). We also aimed to assess, in patients with PD, the effects that genetic variation across candidate pharmacogenes may have on dopamine synthesis and release in response to treatment with AtreMorine. Methods: We analyzed global DNA methylation and de novo DNA methyltransferase (DNMT) expression in a transgenic (3xTg) mouse model of AD, and further examined global DNA methylation in blood samples from patients with PD. Results: AtreMorine treatment increased global DNA methylation in 3xTg mice and in patients with Parkinson´s disease, and produced high DNMT3a expression in AD mice. We observed varied responses to AtreMorine across the following pharmacogenetic genophenotypes analyzed, cytochrome P450 oxidases (CYP2D6, CYP2C19, CYP2C9, CYP3A4, CYP3A5, CYP1A2), human arylamine N-acetyltransferase 2 (NAT2), the vitamin K epoxide reductase complex subunit 1 (VKORC1), ATP-binding cassette subfamily B member 1 (ABCB1), and solute carrier organic anion transporter family member 1B1 (SLCOB1). Conclusion: Our results suggest that AtreMorine regulates DNA methylation in neurodegenerative disorders and may constitute a new therapeutic option for the treatment of these pathologies.

A case report of isoniazid adverse drug reaction in a pediatric patient with defective NAT2 gene

Abstract Rationale: Adverse drug reactions (ADRs) represent a major health problem worldwide, with high morbidity and mortality rates, ranging from 0.5% to 16.8% in hospitalized children. One of the most effective and widely used drugs for anti-tuberculosis treatment is Isoniazid (INH). INH is a pro-drug and following oxidative activation, forms an adduct with NAD+ that inhibits NADH-dependent targets of Mycobacterium tuberculosis. Although this regimen is effective in treating active tuberculosis, it is associated with many ADRs considered as the main complication during therapy Patient concerns: The patient, a 14-year-old girl, presented with nodal tuberculosis exhibiting mobile laterocervical and angle-mandibular lymphadenopathy Diagnoses: INH plasmatic concentration levels above the therapeutic range and a custom NGS gene panel including NAT2 gene allowed the identification of the NAT2∗5C/∗6B slow metabolizer haplotype Interventions: The Patient was hospitalized and initially treated with quadruple antituberculosis therapy. After treatment, she was not able to continue the anti-tuberculosis treatment owing to ADR including overwhelming vomiting, nausea, worsening of clinical conditions, and hypertransaminasemia. Outcomes: Isoniazid and Rifampicin dosage was halved and then, because of the further increase of transaminases, anti-tuberculosis therapy was suspended. Clinical conditions and hematic parameters rapidly improved after suspension of anti-tuberculosis drugs Lessons: We report a 14-year-old girl with defective NAT2 that showed ADRs during anti-tuberculosis therapy. Combined strategy of therapeutic drug monitoring and pharmacogenetic analysis should be widely adopted during the routine clinical practice in order to optimize therapy and minimize ADRs

Development of a limited sampling strategy for the estimation of isoniazid exposure considering N-acetyltransferase 2 genotypes in Korean patients with tuberculosis

A limited sampling strategy (LSS) to estimate the exposure to isoniazid was developed considering N-acetyltransferase 2 (NAT2) genotypes in Korean patients with tuberculosis. The influence of the genotypes on the pharmacokinetics of isoniazid was also evaluated. A total of 33 participants participated in the study and received isoniazid 300 mg once daily. Evaluable participants consist of ten slow (SA), fourteen intermediate (IA) and six rapid acetylators (RA). As expected, isoniazid exposure was higher (mean AUC, 28.4 versus 7.6 mg*h/L) and systemic clearance lower (mean apparent clearance, 14.8 versus 50.6 L/h) in SAs than RAs. The formulas to estimate isoniazid exposure were constructed using one or more concentration-time points that correlate with the area under the concentration-time curve (AUC). The LSS using a formula of single concentration-time point at 4 h post dose (C4) is applicable for all acetylators to the therapeutic drug monitoring (TDM) of isoniazid in patients with tuberculosis when evaluated using the Deming regression and Bland-Altman plot (AUC = 1.53 + 10.03*C4, adjusted r2 = 0.95, p < 0.001). Considering that SAs are more prone to adverse effects, pre-dose NAT2 genotyping would be valuable for optimal isoniazid dosing in conjunction with TDM.

Molecular Genetic Studies of Intraoperative Ligamentum Flavum Bioptates of Patients with Spinal Canal Stenosis

Background. Ligamentum flavum is involved in the development of stenosis of the spinal canal and dural sac, but the mechanisms underlying degenerative-dystrophic changes have not yet been sufficiently studied. Aim. To study the expression of candidate genes potentially involved in connective tissue metabolism in intraoperative samples of Ligamentum flavum of patients with stenosis processes of spinal canal and duralsac on the lumbar spine. Materials and methods. During the surgical treatment, Ligamentum flavum biopsies were collected from 33 people with stenosis processes of spinal canal and dural sac on the lumbar spine (16 men, 17 women). RNA was isolated, then reverse transcription was performed. After that, real time PCR was performed (CFX96, Biorad) with the specific primers (RealTimePrimers.com). Results. The characteristics of the expression of candidate genes that are active in the Ligamentum flavum of the examined patient population are given: NAT1 and NAT2 acetyltransferase genes; genes that determine the intensity of local metabolism (Dio1, Dio2 and Dio3); estradiol receptor genes ESR1 and ESR2; genes encoding receptors for growth factors and parathyroid hormone. All Ligamentum flavum samples showed activity of the NAT, ESR 2, FGFR1, FGFR3, NA20, PTH1R, and PTH2R genes. In the vast majority of samples (93.9–97.0 %), the following genes were active: PDGFA, ESR1, CALCR, and PDGFB. In 21.2–39.4 % of samples, “silence” of the Dio1, Dio2, Dio3, NAT2 and GDF5 genes was detected, and only 39.4 % of samples revealed NAT1 transcripts. The highest heterogeneity of transcript content was observed for Dio1, NAT2, and Dio2. The most stable expression in Ligamentum flavum tissue and a narrow range of fluctuations were characterized by genes with a high level of activity AANAT, ESR2, NAA20 and genes with an average level of activity FGFR1, FGFR3, PTH1R and PTH2R. Conclusions . Ligamentum flavum is a promising little-studied substrate for molecular genetic research. The revealed features of gene expression provide new information about pathogenesis, and the new fundamental knowledge obtained can provide a basis for developing methods for preventing the development of epidural fibrosis.

Population pharmacokinetic model of isoniazid in patients with tuberculosis in Tunisia

AimsTo develop a pharmacokinetic model of isoniazid (INH) concentration taking into account demographic factors and genetic variables [N-acetyltransferase 2 (NAT2) genotype], and to propose an initial INH dosage that could maximize the probability of achieving the desired INH concentration. MethodsA retrospective analysis was undertaken of INH concentration data collected from patients with tuberculosis in Tunisia. ResultsIn total, 118 patients were included in this study. The one-compartment model [volume of distribution (V), elimination rate (Ke)] was found to have good predictive performance. Multi-variate analysis showed that NAT2 affected both V and Ke significantly, but age, gender and weight did not. Internal validation of the final model showed correlation of 0.95 between individual predicted INH concentration 3 h after drug intake (C3) and observed C3. External validation showed that percentage mean absolute prediction error and percentage root mean squared error were 9.11% (range 0.62–35.8%) and 11.6%, respectively. Monte-Carlo simulation showed that doses of at least 225 mg/24 h and at least 450 mg/24 h attained a therapeutic concentration in >80% of patients in the NAT2 slow acetylator group and the NAT2 rapid/intermediate acetylator group, respectively. ConclusionThe pharmacokinetic model allowed optimization of individual dosing regimens of INH in patients with tuberculosis in Tunisia. This tool may facilitate improved efficacy of INH and prevent its toxicity in this population.

Biochemical Characterization of Arylamine N-acetyltransferases From Vibrio vulnificus.

Vibrio vulnificus is a zoonotic bacterium that is capable of causing highly lethal diseases in humans; this pathogen is responsible for 95% of all seafood-related deaths in the United States. Arylamine N-acetyltransferases (NAT, E.C. 2.3.1.5) is a major family of xenobiotic-metabolizing enzymes that can biotransform aromatic amine chemicals. In this research, to evaluate the effect of NAT on acetyl group transformation in arylamine antibiotics, we first used sequence alignment to study the structure of V. vulnificus NAT [(VIBVN)NAT]. The nat gene encodes a protein of 260 amino acids, which has an approximate molecular mass of 30 kDa. Then we purified recombinant (VIBVN)NAT and determined the enzyme activity by PNPA and DTNB methods. The DTNB method indicates that this prokaryotic NAT has a particular substrate specificity towards aromatic substrates. However, (VIBVN)NAT lost most of its activity after treatment with high concentrations of urea and H2O2. In addition, we also explored the stability of the enzyme at different temperatures and pH values. In analyzing the influence of metal ions, the enzyme activity was significantly inhibited by Zn2+ and Cu2+. The kinetic parameters Km and Vmax were determined using hydralazine, isoniazid, 4-amino salicylic acid, and 4-chloro-3-methylaniline as substrates, and the Tm, Tagg and size distribution of (VIBVN)NAT were observed. In particular, a molecular docking study on the structure of (VIBVN)NAT was conducted to understand its biochemical traits. These results showed that (VIBVN)NAT could acetylate various aromatic amine substrates and contribute to arylamine antibiotic resistance in V. vulnificus.