Association of slow N-acetyltransferase 2 profile and anti-TB drug-induced hepatotoxicity in patients from Southern Brazil

Purpose  To determine the frequency of N-acetyltransferase 2 (NAT2) polymorphisms, the NAT2 acetylation profile and its relation to the incidence of gastrointestinal adverse drug reactions (ADRs), anti-tuberculosis (TB) drug-induced hepatotoxicity, and the clinical risk factors for hepatotoxicity in a population from Brazil. Methods  Two hundred and fifty-four Brazilian TB patients using isoniazid (INH), rifampicin (RMP), and pirazinamide (PZA) were tested in a prospective cohort study. NAT2 genotyping was performed by direct PCR sequencing. The association between gastrointestinal ADRs/hepatotoxicity and the NAT2 profile genotype was evaluated by univariate analysis and multiple logistic regression. Results  Of the 254 patients analyzed, 69 (27.2%) were slow acetylators and 185 (72.8%) were fast acetylators. Sixty-five (25.6%) patients were human immunodeficiency virus (HIV)-positive. Thirty-three (13%) and 14 (5.5%) patients developed gastrointestinal ADR and hepatotoxicity, respectively. Of the 14 hepatotoxicity patients, nine (64.3%) were slow acetylators and five (35.7%) were fast acetylators. Sex, age, presence of hepatitis C virus, alcohol abuse, and baseline aminotransferases were not found to be risk factors for hepatotoxicity. However, logistic regression analysis revealed that slow acetylator status and the presence of HIV (p < 0.05) were independent risk factors for hepatotoxicity. Conclusions  Our findings show that HIV-positive patients that have the slow acetylation profile are significantly associated with a higher risk of developing hepatotoxicity due to anti-TB drugs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The influence of dose and <Emphasis Type="Italic">N</Emphasis>-acetyltransferase-2 (NAT2) genotype and phenotype on the pharmacokinetics and pharmacodynamics of isoniazid

Objective This study evaluated the pharmacokinetics of isoniazid (INH) associated with optimal early bactericidal activity (EBA), defined as 90% of the maximum EBA (EBA₉₀) and the influence of N-acetyltransferase-2 (NAT2) subtype on the ability of pulmonary tuberculosis (PTB) patients to reach the identified pharmacokinetic values after INH doses ranging from 0.2 to 10–12 mg/kg body weight. Methods INH serum concentrations and NAT2 subtype were determined during four studies of PTB patients in three of whom the EBA of INH was determined. The relationship of EBA to area under the curve (AUC) and 2-h serum concentrations was examined by exponential regression and fitted curves estimated the and 2-h serum concentrations at which EBA₉₀ was reached. Results EBA₉₀ was reached at an of 10.52 μg/ml per hour and 2-h serum concentrations of 2.19 μg/ml. An of 10.52 μg/ml per hour was reached by all 66 patients receiving a 10–12 mg/kg INH dose and all 21 receiving 6 mg/kg, except 1 of 10 (10%) homozygous fast (FF) acetylators; however, at 5 mg/kg, 4 of 12 (33%) FF and 26 of 27 (96%) heterozygous fast (FS), but all 21 homozygous slow (SS) acetylators did so; and 1 of 3 (33%) FF, 2 of 6 (33%) FS, but all 4 SS acetylators at dose 3 mg/kg. An INH 2-h serum concentration of 2.19 μg/ml was reached by all 66 patients receiving 10–12 mg/kg and all 21 receiving 6 mg/kg, except for 2 (20%) FF acetylators at a dose of 5 mg/kg; however, only 3 (25%) of 12 FF acetylators, but 26 (96%) of 27 FS acetylators, and all 21 SS acetylators reached this concentration; and at a dose of 3 mg/kg, 1 (33%) of 3 FF acetylators, 2 (33%) of 6 FF, but all 4 SS acetylators. Conclusions At a 6 mg/kg dose, all except a minority of FF NAT2 acetylators, achieve an INH and 2-h INH serum concentrations associated with EBA₉₀, as did all 4 SS acetylators receiving 3 mg/kg. Any dose reduction below 6 mg/kg body weight will tend to disadvantage a significant proportion of faster acetylators, but, conversely, SS acetylators require only a 3 mg/kg dose to achieve a satisfactory exposure to INH.     

NAT2 genotype guided regimen reduces isoniazid-induced liver injury and early treatment failure in the 6-month four-drug standard treatment of tuberculosis: A randomized controlled trial for pharmacogenetics-based therapy

Objective

This study is a pharmacogenetic clinical trial designed to clarify whether the N-acetyltransferase 2 gene (NAT2) genotype-guided dosing of isoniazid improves the tolerability and efficacy of the 6-month four-drug standard regimen for newly diagnosed pulmonary tuberculosis.

Methods

In a multicenter, parallel, randomized, and controlled trial with a PROBE design, patients were assigned to either conventional standard treatment (STD-treatment: approx. 5 mg/kg of isoniazid for all) or NAT2 genotype-guided treatment (PGx-treatment: approx. 7.5 mg/kg for patients homozygous for NAT2*4: rapid acetylators; 5 mg/kg, patients heterozygous for NAT2*4: intermediate acetylators; 2.5 mg/kg, patients without NAT2*4: slow acetylators). The primary outcome included incidences of 1) isoniazid-related liver injury (INH-DILI) during the first 8 weeks of therapy, and 2) early treatment failure as indicated by a persistent positive culture or no improvement in chest radiographs at the8th week.

Results

One hundred and seventy-two Japanese patients (slow acetylators, 9.3 %; rapid acetylators, 53.5 %) were enrolled in this trial. In the intention-to-treat (ITT) analysis, INH-DILI occurred in 78 % of the slow acetylators in the STD-treatment, while none of the slow acetylators in the PGx-treatment experienced either INH-DILI or early treatment failure. Among the rapid acetylators, early treatment failure was observed with a significantly lower incidence rate in the PGx-treatment than in the STD-treatment (15.0 % vs. 38 %). Thus, the NAT2 genotype-guided regimen resulted in much lower incidences of unfavorable events, INH-DILI or early treatment failure, than the conventional standard regimen.

Conclusion

Our results clearly indicate a great potential of the NAT2 genotype-guided dosing stratification of isoniazid in chemotherapy for tuberculosis.     

Arylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase 2 slow acetylator polymorphisms in unrelated Iranian individuals

Objective To determine the frequency of mutations at the polymorphic gene coding for arylamine N-acetyltransferase 2 (NAT2, EC 2.3.1.5) and NAT2 genotypes associated with slow acetylation in healthy Iranian individuals.Methods The polymorphisms in the NAT2 gene from 88 unrelated healthy subjects (48 men/40 women) from the general Tehran population were discriminated using polymerase chain reaction (PCR) with allele-specific primers (341 C>T) and PCR-restriction fragment length polymorphism analysis (481 C>T, 590 G>A, and 857 G>A).Results Frequencies of the studied polymorphisms showed the most common alleles to be NAT2*4 (0.43) and NAT2*5, 481 C>T (0.32), followed by NAT2*6 (0.19) and NAT2*7 (0.06), previously referred to as WT, M1, M2, and M3, respectively. The most prevalent genotypes were NAT2*4/*5 [(31.8%; 95% confidence interval (CI): 29–34%] and *4/*4 (18.2%; 95% CI: 16–21%). When grouped according to the expected phenotypical effects, the resulting genotypes revealed the significant prevalence of the subjects with slow (32.9%) and intermediate (48.9%) acetylation status compared with wild-type rapid (18.2%) acetylators (P<0.01).Conclusions The overall allele pattern and acetylator status distribution in Iranians displayed the considerable prevalence of slow acetylators over rapid acetylators, similar to those of Caucasians except for a minor difference observed in the frequency of the NAT2*7 allele. Nucleic acid testing for common NAT2 mutations might be a potentially useful tool for an accurate phenotype interpretation and identification of Iranian individuals at risk.     

Unified Pharmacogenetics-Based Parent–Metabolite Pharmacokinetic Model Incorporating Acetylation Polymorphism for Talampanel in Humans

The N-acetylation of the noncompetitive AMPA antagonist talampanel (TLP) represents a route of varying significance in various species. For a detailed analysis in humans, plasma concentrations of TLP and its N-acetyl metabolite (NAc–TLP) were measured for up to 48 h after administration of a single oral dose of 75 mg in 28 healthy volunteers following genotyping for the N-acetyltansferase NAT2 isozymes (alleles NAT2*4, *5, *6, and *7). Unified parent–metabolite pharmacokinetic (PK) models that allowed three different rates of acetylation were used to simultaneously fit plasma levels for both the parent drug and its metabolite following genotype-based classification as slow, intermediate, or fast acetylator. A perfect correspondence was found between the phenotype inferred from genotyping and the phenotype determined by using plasma metabolite-to-parent molar ratios indicating that this route of metabolism is indeed mediated by NAT2. Linear parent-metabolite PK models (first-order input, first-order elimination through two parallel routes one of which is through a metabolite with polymorphic rate of formation) gave adequate and sufficiently consistent fit. Parameters obtained suggest that for TLP in humans, N-acetylation represents only about 1/4th of the total elimination even in true (*4/*4 homozygous) fast acetylators, acetylation is about 8–12 times faster in fast and 3–6 times faster in intermediate acetylators than in slow acetylators, and the N-acetyl metabolite is eliminated faster than the parent drug. Such PK models can provide quantitative estimates of relative in vivo metabolism rates for routes catalyzed by functionally polymorphic enzymes.     

Arylamine N-acetyltransferase 2 genotype-dependent N-acetylation of isoniazid in cryopreserved human hepatocytes

Cryopreserved human hepatocytes were used to investigate the role of arylamine N-acetyltransferase 2 (NAT2; EC 2.3.1.5) polymorphism on the N-acetylation of isoniazid (INH). NAT2 genotype was determined by Taqman allelic discrimination assay and INH N-acetylation was measured by high performance liquid chromatography. INH N-acetylation rates in vitro exhibited a robust and highly significant (P<0.005) NAT2 phenotype-dependent metabolism. N-acetylation rates in situ were INH concentration- and time-dependent. Following incubation for 24 h with 12.5 or 100 µmol/L INH, acetyl-INH concentrations varied significantly (P = 0.0023 and P = 0.0002) across cryopreserved human hepatocytes samples from rapid, intermediate, and slow acetylators, respectively. The clear association between NAT2 genotype and phenotype supports use of NAT2 genotype to guide INH dosing strategies in the treatment and prevention of tuberculosis.     

DNA Adduct Levels and Intestinal Lesions in Congenic Rapid and Slow Acetylator Syrian Hamsters Administered the Food Mutagens 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) or 2‐Amino‐3‐methylimidazo[4,5‐f]quinoline (IQ

Epidemiological studies indicate that rapid acetylators with a high intake of well‐done red meat have an increased risk of colorectal cancer. Arylamine N‐acetyltransferase enzymes (E.C. 2.3.1.5) activate carcinogenic heterocyclic amines found in the crust of fried meat via O‐acetylation of their N‐hydroxylamines to reactive intermediates that bind covalently to DNA and produce mutations. Syrian hamsters as well as humans express two N‐acetyltransferase isozymes (NAT1 and NAT2) which differ in substrate specificity and genetic control. Nucleic acid substitutions in the NAT2 gene segregate individuals into rapid, intermediate and slow acetylator phenotypes. In the present paper, we examined the role of the polymorphic NAT2 acetylator genotype in carcinogenesis induced by the food mutagens 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) or 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ) by comparing Syrian hamster lines congenic at the NAT2 locus. No differences were found between rapid and slow acetylator congenic hamsters in levels of intestinal PhIP‐DNA adducts. In contrast to previous studies in rats, no carcinogen‐related induction of the preneoplastic lesions aberrant crypt foci or tumors was found in the intestines of rapid and slow acetylator congenic Syrian hamsters administered PhIP or IQ.     

Influence of acetylator phenotype on the pharmacokinetics of a new vasodilator antihypertensive,endralazine

Summary Five and 10 mg single oral doses of a new vasodilator antihypertensive, endralazine (E) were given on separate occasions to 17 normal male volunteers (8 slow, 7 heterozygous fast and 2 homozygous fast acetylators). The homozygous fast acetylators were excluded from statistical comparisons. Only small differences were observed in the pharmacokinetics of E between the phenotypes and there was no evidence of non-linearity at the 2 dose levels studied. Terminal half-lives ranged from 2.59 to 7.14 h with a mean of 4.30±1.08 h for the 5 mg dose and 4.25±1.09 h for the 10 mg dose. There was no significant difference in half-lives between slow and heterozygous fast acetylators. The mean area under the plasma level-time curve (AUC ₀ ) was 18.2% lower (p<0.05) in the heterozygous fast acetylators than in the slow acetylators following the 5 mg dose and 11.0% lower (p>0.05) following the 10 mg dose. Extremely rapid absorption of the drug precluded accurate estimation of absorption rates. The AUC ₀ of the acetylation metabolite (methyltriazoloendralazine) was small compared to that of E although higher in the heterozygous fast acetylators than in the slow acetylators (p<0.01).     

4-Aminobiphenyl Downregulation of NAT2 Acetylator Genotype-Dependent N- and O-acetylation of Aromatic and Heterocyclic Amine Carcinogens in Primary Mammary Epithelial Cell Cultures from Rapid and Slow Acetylator Rats

Aromatic and heterocyclic amine carcinogens present in the dietand in cigarette smoke induce breast tumors in rats. N-acetyltransferase1 (NAT1) and N-acetyltransferase 2 (NAT2) enzymes have importantroles in their metabolic activation and deactivation. Humanepidemiological studies suggest that genetic polymorphisms inNAT1 and/or NAT2 modify breast cancer risk in women exposedto these carcinogens. p-Aminobenzoic acid (selective for ratNAT2) and sulfamethazine (SMZ; selective for rat NAT1) N-acetyltransferasecatalytic activities were both expressed in primary culturesof rat mammary epithelial cells. PABA, 2-aminofluorene, and4-aminobiphenyl N-acetyltransferase and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxalineO-acetyltransferase activities were two- to threefold higherin mammary epithelial cell cultures from rapid than slow acetylatorrats. In contrast, SMZ (a rat NAT1-selective substrate) N-acetyltransferaseactivity did not differ between rapid and slow acetylators.Rat mammary cells cultured in the medium supplemented 24 h with10µM ABP showed downregulation in the N-and O-acetylationof all substrates tested except for the NAT1-selective substrateSMZ. This downregulation was comparable in rapid and slow NAT2acetylators. These studies clearly show NAT2 acetylator genotype–dependentN- and O-acetylation of aromatic and heterocyclic amine carcinogensin rat mammary epithelial cell cultures to be subject to downregulationby the arylamine carcinogen ABP.     

Determination of NAT2 acetylation status in the Greenlandic population

N-acetyltransferase 2 (NAT2) is a well-studied phase II xenobiotic metabolizing enzyme relevant in drug metabolism and cancerogenesis. NAT2 activity is largely determined by genetic polymorphisms in the coding region of the corresponding gene. We investigated NAT2 acetylation status in 1556 individuals from Greenland based on four different single nucleotide polymorphism (SNP) panels and the tagging SNP rs1495741. There was good concordance between the NAT2 status inferred by the different SNP combinations. Overall, the fraction of slow acetylators was low with 17.5 % and varied depending on the degree of Inuit ancestry; in individuals with <50 % Inuit ancestry, we observed more than 25 % slow acetylators reflecting European ancestry. Greenland has a high incidence of tuberculosis, and individual dosing of isoniazid according to NAT2 status has been shown to improve treatment and reduce side effects. Our findings could be a first step in pharmacogenetics-based tuberculosis therapy in Greenland.     

Polymorphisms of NAT2 in relation to sulphasalazine-induced agranulocytosis

Agranulocytosis is a rare, but serious adverse reaction to sulphasalazine. The polymorphic enzyme N-acetyltransferase 2 (NAT2) plays an important role in the metabolism of sulphasalazine. This study was conducted to analyse whether the risk of sulphasalazine-induced agranulocytosis is increased in slow acetylators. Patients were treated for inflammatory disease, mostly joint disease, with a mean dose of 2 g sulphasalazine daily. Thirty-nine patients reacted with agranulocytosis, while 75 patients had been treated for a minimum of 3 months without haematological side-effects. A population-based control panel of 448 individuals was used for comparison. All subjects were genotyped for NAT2 by polymerase chain reaction followed by restriction enzyme digestion. The six most common allelic variants were analysed: NAT2*4, NAT2*5A, NAT2*5B, NAT2*5C, NAT2*6 and NAT2*7. The proportion of slow acetylators was significantly higher in patients with sulphasalazine-induced agranulocytosis (69%) and population-based controls (64%) compared to patients who tolerated sulphasalazine (45%); odds ratio 2.71 [95% confidence interval (CI) 1.20; 6.15], P = 0.015, and odds ratio 2.17 (95% CI 1.32; 3.56), P = 0.002, respectively. Patients who developed agranulocytosis did not differ from population-based control subjects in the frequency of slow acetylators; odds ratio 1.25 (95% CI 0.62; 2.53), P = 0.535. The risk of agranulocytosis did not appear to be increased in slow acetylators, provided that the difference compared with sulphasalazine-treated control subjects was not due to a predominance of fast acetylators among patients with inflammatory joint disease. Instead, selection bias was suspected since more slow acetylators may have discontinued sulphasalazine therapy because of drug-intolerance.     

N-acetyltransferase 2 (Nat2) polymorphism in the sand rat Psammomys obesus

Human arylamine N-acetyltransferase 1 (NAT1) and its homologue in rodents (Nat2) are polymorphic xenobiotic metabolizing enzymes and also seem to play a role in endogenous metabolism. NAT1 and Nat2 polymorphism was associated to cancers under xenobiotic procarcinogens metabolism as well as under endogenous substrate metabolism. This study investigated the p-aminobenzoic acid (PABA) -Nat2 catalytic activity and its polymorphism in liver homogenates of adult sand rats Psammomys obesus Cretzschmar, 1828. These Saharian sand rats develop high incidence of spontaneous cancers under standard laboratory diet. The average value of PABA-Nat2 specific activity tested in nine sand rats was significant (2.96?±?2.16 nmoles/min/mg). The N-acetylation exhibited a bimodal distribution. There was a significant difference (p?<?0.01) between PABA-Nat2 activity in the fast acetylators group (4.10?±?1.67 nmol/min/mg) and slow acetylators group (0.7?±?0.27 nmol/min/mg). The percentage of the fast acetylator group was 66.66%. These results support the presence of Nat2 polymorphism in the liver of the strain sand rats Psammomys obesus. This strain is useful for investigating the role of Nat2 polymorphisms in susceptibility to cancers related to arylamine carcinogen exposures as well as to endogenous substrate metabolism.     

NAT1 genotypic and phenotypic contribution to urinary bladder cancer risk: a systematic review and meta-analysis

N-acetyltransferase 1 (NAT1), a polymorphic Phase II enzyme, plays an essential role in metabolizing heterocyclic and aromatic amines, which are implicated in urinary bladder cancer (BCa). This systematic review investigates a possible association between the different NAT1 genetic polymorphisms and BCa risk. Medline, PubMed, EMBASE, Scopus, Web of Science, OpenGrey, and BASE databases were searched to identify eligible studies. The random-effect model was used to calculate pooled effects estimates. Statistical heterogeneity was tested with Chi-square and I². Twenty case-control studies, including 5606 cases and 6620 controls, met the inclusion criteria. Pooled odds ratios (OR) analyses showed a statistically significant difference in NAT1*10 versus non-NAT1*10 acetylators in the total sample (OR: 0.87; 95% CI: 0.79–0.96) but was borderline among Caucasians (OR: 0.88 with 95% CI: 0.77–1.01). No statistically significant differences in BCa risk were found for: NAT1*10 versus NAT1*4 wild type (OR: 0.97; 95% CI: 0.78–1.19), NAT1 ‘Fast’ versus ‘Normal’ acetylators (OR: 1.03; 95% CI: 0.84–1.27), and NAT1 ‘Slow’ versus ‘Fast’ (OR: 2.32; 95% CI: 0.93–5.84) or ‘Slow’ versus ‘Normal’ acetylators (OR: 1.84; 95% CI: 0.92–3.68). When stratifying by smoking status, no statistically significant differences in BCa risk were found for NAT1*10 versus non-NAT1*10 acetylators among the different subgroups. Our study suggests a modest protective role for NAT1*10 and a possible risk contributory role for slow acetylation genotypes in BCa risk. Further research is recommended to confirm these associations.     

Acetylator phenotype and serum levels of sulfapyridine in patients with inflammatory bowel disease

Summary Twenty-eight outpatients receiving sulfasalazine for inflammatory bowel disease were monitored. Assessment of acetylator phenotype according to the percentage of acetylated sulfapyridine in serum provided a clear distinction between rapid and slow acetylators. In comparison, the percentage of acetylated sulfapyridine in saliva or urine was a less precise index of phenotype. Determination of saliva concentrations of sulfapyridine and N⁴-acetylsulfapyridine did not provide a reliable estimate of serum levels. Slow acetylators had significantly higher serum concentrations of sulfapyridine (21.9±14.0 [SD] µg/ml) than rapid acetylators (8.8±4.3 µg/ml) and had a higher incidence of toxicity (not statistically significant,p>0.05). Serum concentrations of sulfapyridine were significantly higher in patients with symptoms of toxicity (23.2±15.9 µg/ml) than those without (13.9±9.5 µg/ml) (p<0.05). However, serum concentrations of total sulfapyridine (sulfapyridine plus N⁴-acetylsulfapyridine) were not significantly different in patients with (32.9±21.2 µg/ml) or without (22.8±12.0 µg/ml) toxicity (p>0.05). For all patients serum concentrations of sulfapyridine (total sulfapyridine) ranged from 3.5 to 73.1 (5.7 to 95.1) µg/ml in patients with controlled disease and 6.3 to 38.0 (14.0 to 54.7) µg/ml in patients with active disease. A significant correlation between clinical status of disease and serum drug concentrations was only apparent for rapid acetylators (p<0.05). The daily sulfasalazine dosage (mg/kg of body weight, log value) and serum drug concentrations (log values) were highly correlated (p<0.05). For clinical evaluation of inflammatory bowel disease patients determination of serum sulfapyridine concentrations appears to be more important for monitoring toxicity than therapeutic efficacy of sulfasalazine. Assessment of acetylator status appears to be useful for predicting serum sulfapyridine levels in patients receiving sulfasalazine therapy.     

N-acetylation of the heterocyclic amine batracylin by human liver.

Batracylin (8-aminoisoindolo[1,2-b]quinazolin-12(10 H)-one; BAT) is a heterocyclic amine that exhibits antitumor activity in a number of in vivo and in vitro models. The acetyl product has been implicated in BAT toxicity in animals, cells, and bacteria. The ability of human N-acetyltransferase (NAT) to form this product was investigated. Nine human liver samples were analyzed for NAT1 and NAT2 genotypes. Seven of the samples possessed at least one NAT1*4 allele. Three samples contained one or more NAT2*4 allele and were classified as rapid acetylators. The remaining six had two alleles associated with the slow phenotype. NAT activities were evaluated with BAT, sulfamethazine (SMZ), a preferential substrate for human NAT2, and p-aminobenzoic acid, a substrate for NAT1. BAT activities in the nine donor samples ranged from 14.9 to 0.56 nmol/min/mg. The mean apparent K(m) values in rapid acetylators for BAT, SMZ, and p-aminobenzoic acid were 6.59 +/- 3.21, 278 +/- 69.4, and 31.2 +/- 12.5 microM, respectively. The apparent K(m) values for slow acetylators did not differ from the rapid acetylator phenotype. However, a significant difference in the apparent V(max) for BAT and SMZ was observed between rapid and slow acetylators. Comparing the apparent intrinsic clearance (V(max)/K(m)) for BAT and SMZ, a significant correlation (r(2) = 0.97, p <.001) was observed. These data demonstrate that BAT N-acetylation is similar to SMZ, and suggests that BAT is a preferential substrate for human NAT2. Thus, rapid acetylators would be more likely to develop toxicity when exposed to this drug.     

Re-investigation of the concordance of human NAT2 phenotypes and genotypes

A comparative study of N-acetyltransferase 2 (NAT2) genotyping and phenotyping (caffeine test method) was performed on 211 persons to elucidate apparent discrepancies in the assignment of NAT2*12 and NAT2*13 alleles which occur in the literature. The study used the standard procedures of genotyping (two PCR runs and application of seven restriction enzymes) and phenotyping (determination of the two caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (1X)), as documented in detail and validated by the Deutsche Forschungsgemeinschaft. The data were consistent with an AFMU/1X molar ratio of 0.85 as cut-off point (antimode) between phenotypically slow and rapid acetylators. Under this provision, several R/S allele combinations did not comply, either fully or partly, with their associated phenotypes. In particular, there was a wide phenotypic overlap of the alleged rapid allele combination groups (i) NAT2*12A/*5A; NAT2*12C/*5D; NAT2*4/*5B, (ii) NAT2*13/*6B; NAT2*4/*6A, and (iii) NAT2*13/*7A; NAT2*4/*7B. These groups obviously contained both phenotypically rapid and slow acetylators. If one assumes that the presence of one wild type allele NAT2*4 defines a rapid acetylator the assignment of the alleles NAT2*12A, NAT2*12C, and NAT*13 as determinants of a rapid acetylator phenotype must be questioned. This refers in particular to the nucleotide changes A⁸⁰³G (NAT2*12A, NAT2*12C) and C²⁸²T (NAT2*13). Based on discussions in the literature and the data presented here, there is accumulating evidence that current assignments of the NAT2*12 and NAT2*13 alleles as determinants of a rapid acetylator state should be reconsidered.     

Plasma kinetics of dipyrone metabolites in rapid and slow acetylators

Summary The pharmacokinetics of the dipyrone metabolites 4-methylaminoantipyrine (MAA), 4-aminoantipyrine (AA), 4-formylaminoantipyrine (FAA) and 4-acetylaminoantipyrine (AAA) were evaluated following the administration of a single oral 1.0 g dose of dipyrone to 23 healthy volunteers. Twelve were slow and 11 were rapid acetylators as previously determined by dapsone phenotyping. For MAA and FAA the mean peak plasma concentrations were 10.5±2.8 µg/ml and 2.1±0.8 µg/ml and the half-lives were 3.3±1.0 and 10.1±1.8 h, respectively. No significant difference was found between rapid and slow acetylators in MAA and FAA kinetics. For AA, the mean peak plasma concentrations were 2.7±0.6 and 1.6±0.7 µg/ml (p<0.01), the peak times 6.7±2.1 and 3.1±1.1 h (p<0.01) and the half-lives were 5.5±1.0 and 3.8±1.2 h in slow and rapid acetylators, respectively. For AAA, the mean peak plasma concentrations were 1.6±0.4 and 4.4±1.1 µg/ml (p<0.01) and the peak time 16.1±5.1 and 10.0±2.6 h (p<0.01) in slow and rapid acetylators, respectively. There was no difference in the elimination half-life between the two groups (10.6±2.2 h). Thus, it has been demonstrated that the AAA/AA ratio is an indicator of the acetylation phenotype, as it is closely correlated with that determined by dapsone (r=0.895, p<0.0005).     

Pharmacokinetics of the Experimental Non‐Nucleosidic DNA Methyl Transferase Inhibitor N‐Phthalyl‐l‐Tryptophan (RG 108) in Rats

DNA methyl transferase (DNMT) inhibitors can re‐establish the expression of tumour suppressor genes in malignant diseases, but might also be useful in other diseases. Inhibitors in clinical use are nucleosidic cytotoxic agents that need to be integrated into the DNA of dividing cells. Here, we assessed the in vivo kinetics of a non‐nucleosidic inhibitor that is potentially free of cytotoxic effects and does not require cell division. The non‐specific DNMT inhibitor N‐phthalyl‐l ‐tryptophan (RG 108) was injected subcutaneously in rats. Blood was drawn 0, 0.5, 1, 2, 4, 6, 8 and 24 hr after injection and RG 108 in plasma was measured by high‐performance liquid chromatography coupled to mass spectrometry. Trough levels and area under the curve (AUC) were significantly higher with multiple‐dose administration and cytochrome inhibition. In this group, time to maximal plasma concentration (t_max, mean ± S.D.) was 37.5 ± 15 min., terminal plasma half‐life was approximately 3.7 h (60% CI: 2.1–15.6 h), maximal plasma concentration (C_max) was 61.3 ± 7.6 μM, and AUC was 200 ± 54 μmol·h/l. RG 108 peak levels were not influenced by cytochrome inhibition or multiple‐dose administration regimens. Maximal tissue levels (C_max in μmol/kg) were 6.9 ± 6.7, 1.6 ± 0.4 and 3.4 ± 1.1 in liver, skeletal and heart muscle, respectively. We conclude that despite its high lipophilicity, RG 108 can be used for in vivo experiments, appears safe and yields plasma and tissue levels in the range of the described 50% inhibitory concentration of around 1 to 5 μM. RG 108 can therefore be a useful tool for in vivo DNMT inhibition.     

The influence of exercise and dehydration on the urine concentrations of salbutamol after inhaled administration of 1600 µg salbutamol as a single dose in relation to doping analysis

The present study investigated the influence of exercise and dehydration on the urine concentrations of salbutamol after inhalation of that maximal permitted (1600 µg) on the 2015 World Anti‐Doping Agency (WADA) prohibited list. Thirteen healthy males participated in the study. Urine concentrations of salbutamol were measured during three conditions: exercise (EX), exercise+dehydration (EXD), and rest (R). Exercise consisted of 75 min cycling at 60% of VO_2max and a 20‐km time‐trial. Fluid intake was 2300, 270, and 1100 mL during EX, EXD, and R, respectively. Urine samples of salbutamol were collected 0–24 h after drug administration. Adjustment of urine concentrations of salbutamol to a specific gravity (USG) of 1.020 g/mL was compared with no adjustment. The 2015 WADA decision limit (1200 ng/mL) for salbutamol was exceeded in 23, 31, and 10% of the urine samples during EX, EXD, and R, respectively, when unadjusted for USG. When adjusted for USG, the corresponding percentages fell to 21, 15, and 8%. During EXD, mean urine concentrations of salbutamol exceeded (1325±599 ng/mL) the decision limit 4 h after administration when unadjusted for USG. Serum salbutamol C_max was lower (P<0.01) for R(3.0±0.7 ng/mL) than EX(3.8±0.8 ng/mL) and EXD(3.6±0.8 ng/mL). AUC was lower for R (14.1±2.8 ng/mL·?h) than EX (16.9±2.9 ng/mL·?h)(P<0.01) and EXD (16.1±3.2 ng/mL·?h)(P<0.05). In conclusion, exercise and dehydration affect urine concentrations of salbutamol and increase the risk of Adverse Analytical Findings in samples collected after inhalation of that maximal permitted (1600 µg) for salbutamol. This should be taken into account when evaluating doping cases of salbutamol. Copyright © 2015 John Wiley & Sons, Ltd.