Concordance between tramadol and dextromethorphan parent/metabolite ratios: the influence of CYP2D6 and non-CYP2D6 pathways on biotransformation.

Cytochrome P4502D6 (CYP2D6) activity has been shown to be a determinant of both the pharmacokinetics and pharmacodynamics of tramadol in adults. This study evaluated the association between CYP2D6 activity, as determined by dextromethorphan (DM) urinary metabolite ratio, and tramadol biotransformation in 13 children (7-16 years). CYP2D6 genotype was determined by XL-PCR and PCR/RFLP. Phenotype was assessed by HPLC quantitation of DM and its metabolites from a 12- to 24-hour urine collection following a single oral dose of DM. There was only a modest correlation between tramadol/M1 (metabolite 1) plasma concentration or AUC and the DM/dextrorphan (DX) urinary molar ratio in the study cohort; however, when subjects were segregated based on the number of functional CYP2D6 alleles, a much stronger relationship was observed for subjects with two functional alleles, with essentially no relationship evident in those individuals with one functional allele. Further evaluation of these data suggested that the CYP2D6-mediated metabolite (M1) is formed to a lesser extent, and the formation of the non-CYP2D6 product (M2) is more pronounced in subjects with one versus two functional alleles. Thus, the number of functional CYP2D6 alleles and the availability of alternative cytochromes P450 capable of metabolizing tramadol may explain the poor association between DM, a well-characterized CYP2D6 probe, and tramadol in a population of CYP2D6 extensive metabolizers.

Montelukast dose selection in children ages 2 to 5 years: comparison of population pharmacokinetics between children and adults.

Montelukast, a leukotriene receptor antagonist, has demonstrated efficacy and tolerability in the treatment of asthma in patients age 6 years and older. The purpose of this open, one-period, multicenter population pharmacokinetic study was to identify a chewable tablet (CT) dose of montelukast for administration to children ages 2 to 5 years with asthma, yielding a single-dose pharmacokinetic profile (area under the plasma concentration-time curve [AUC]) comparable to that of the 10 mg film-coated tablet (FCT) dose in adults. Because patient numbers were small and the volume of blood that could be collected from individual 2- to 5-year-old patients was limited, a population pharmacokinetic approach was used to estimate population AUC (AUCpop). The 4 mg CT dose of montelukast was well tolerated and yielded an AUCpop (2721 ng.h/mL) similar to that of the adult AUCpop (2595 ng.h/mL) observed after a 10 mg FCT dose. These results support the selection of a 4 mg once-daily CT dose of montelukast for future efficacy and safety studies in children ages 2 to 5 years with asthma.

Montelukast dose selection in 6- to 14-year-olds: comparison of single-dose pharmacokinetics in children and adults.

Montelukast, an oral leukotriene-receptor antagonist, has demonstrated efficacy and tolerability for the treatment of chronic asthma in adults. A once-daily 10 mg dose (film-coated tablet) was selected as the optimal adult dose based on dose-ranging studies. Asthma is a similar disease and is treated with the same medications in children and adults. These observations suggested that a dose of montelukast in children providing overall drug exposure (i.e., montelukast plasma concentrations) similar to that of the 10 mg film-coated tablet dose in adults would be efficacious, well tolerated, and obviate the need for separate dose-ranging studies in children. Therefore, the dose of montelukast for 6- to 14-year-old children was selected by identifying the chewable tablet dose of montelukast yielding a single-dose area under the plasma concentration-time curve (AUC) comparable to that achieved with the adult 10 mg film-coated tablet dose. Based on this approach, which included dose normalization of data from several pediatric pharmacokinetic studies, a 5 mg chewable tablet dose of montelukast was selected for use in clinical efficacy studies in 6- to 14-year-old children with asthma.

Deficiency of cytosolic arylamine N-acetylation in the domestic cat and wild felids caused by the presence of a single NAT1-like gene.

The purpose of this study was to determine the molecular basis for a relative deficiency in the cat of cytosolic arylamine N-acetyltransferase (NAT), an enzyme family that is important in the metabolism of xenobiotics and that normally consists of at least two related enzymes, NAT1 and NAT2. N-acetyltransferase in feline liver showed high affinity (mean Km = 2.1 microM) for p-aminobenzoic acid, an NAT1 selective substrate in humans and rabbits, but showed a very poor affinity (mean Km > 10 mM) for sulfamethazine, an NAT2 selective substrate in humans and rabbits. Immunoreactive N-acetyltransferase was detected in feline liver, bladder and colon using an NAT1-specific antipeptide antibody, but was not detected in any tissues using an NAT2-specific antibody. Southern blot analysis of genomic DNA demonstrated a single band in domestic cats using each of six restriction digests; single bands were also found on Southern blot analysis of six wild felids. The deduced amino acid sequence of the central portion of feline N-acetyltransferase, obtained by polymerase chain reaction amplification in both domestic cats and seven wild felids (lion, tiger, lynx, snow leopard, bobcat, Asian leopard cat and cheetah), contained three residues, Phe125, Arg127, and Tyr129, which determine NAT1-like substrate specificity in humans. These results support the conclusion that cytosolic arylamine N-acetylation activity is low in the cat because of the presence of a single N-acetyltransferase that has substrate specificity, immunogenicity and sequence characteristics similar to human NAT1, and that the unusual presence of only a single N-acetyltransferase gene appears to be a family wide trait shared by other felids.

Patients with delayed-onset sulfonamide hypersensitivity reactions have antibodies recognizing endoplasmic reticulum luminal proteins.

Sulfonamide antimicrobials cause a delayed-onset, hypersensitivity-type syndrome characterized by fever, skin rash and multiorgan toxicity occurring 7 to 14 days after initiation of therapy. The pathogenesis is believed to be immune-mediated. We investigated whether patients with delayed-onset sulfonamide hypersensitivity reactions had antibodies recognizing hapten-microsomal protein conjugates and/or native microsomal proteins. By immunoblotting using rat liver as a source of microsomal protein, 17 of 21 patients had antibodies recognizing one or more of three native endoplasmic reticulum proteins of 55 kDa (14 of 21 patients), 80 kDa (4 of 21 patients) or 96 kDa (3 of 21 patients) in size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No control subjects (n = 11) and only 1 of 18 patients with adverse events not consistent with sulfonamide hypersensitivity reactions had antibodies against these microsomal proteins under the conditions used. Only 1 patient had antibodies that recognized the sulfonamide hapten, sulfamethoxazole. The 55-kDa protein was identified as protein disulfide isomerase. The 80-kDa protein was identified as grp78. The 96-kDa protein was not identified. Delayed-onset sulfonamide hypersensitivity reactions are therefore primarily associated with antibodies recognizing specific protein epitopes and not anti-drug antibodies.

Cytosolic arylamine N-acetyltransferase (NAT) deficiency in the dog and other canids due to an absence of NAT genes.

The purpose of this study was to determine the molecular basis in the dog for an unusual and absolute deficiency in the activity of cytosolic N-acetyltransferase (NAT), an enzyme important for the metabolism of arylamine and hydrazine compounds. NAT activity towards two NAT substrates, p-aminobenzoic acid and sulfamethazine, was undetectable in dog liver cytosol, despite substrate concentrations ranging from 10 microM to 4 mM and a wide range of incubation times. Similarly, no protein immunoreactive to NAT antibody was evident on western blot analysis of canine liver cytosol. Southern blot analysis of genomic DNA from a total of twenty-five purebred and mixed bred dogs, and eight wild canids, probed with a full-length human NAT2 cDNA, suggested an absence of NAT sequences in all canids. Polymerase chain reaction amplification of genomic DNA using degenerate primers designed to mammalian NAT1 and NAT2 consensus sequences generated products of the expected size in human, mouse, rabbit, and cat DNA, but no NAT products in any dog or wild canids. These results support the conclusion that cytosolic NAT deficiency in the domestic dog is due to a complete absence of NAT genes, and that this defect is shared by other canids.

N-acetyltransferases: pharmacogenetics and clinical consequences of polymorphic drug metabolism.

Since the discovery of polymorphic N-acetylation of drugs nearly 40 years ago, great progress has been made in understanding the molecular genetics of acetylation as well as the clinical consequences of being a rapid or slow acetylator. Inborn errors (several different alleles) at the NAT2 locus are responsible for the traditional acetylator polymorphism. Studies have revealed variant alleles at the NAT1 locus as well. The consequences of pharmacogenetic variation in these enzymes include (i) altered kinetics of specific drug substrates; (ii) drug-drug interactions resulting from altered kinetics; (iii) idiosyncratic adverse drug reactions. The latter have been extensively investigated for the arylamine-containing sulfonamide antimicrobial drugs. Individual differences in multiple metabolic pathways can increase the likelihood of covalent binding of reactive metabolites of the drugs to cell macromolecules with resultant cytotoxicity and immune response to neoantigens. This can result clinically in an idiosyncratic hypersensitivity reaction, manifested by fever, skin rash, and variable toxicity to organs including liver, bone marrow, kidney, lung, heart, and thyroid. Slow acetylation by NAT2 is a risk factor for such reactions to sulfonamides. Given the incidence of these severe adverse drug reactions (much less than 1/1000), slow acetylation cannot be the sole mechanism of predisposition in the population. Differences in rates of production of hydroxylamine metabolites of the drugs by cytochrome P450 (CYP2C9), myeloperoxidase, and thyroid, roxidase, along with an inherited abnormality in detoxification of the hydroxylamines are critically important in determining individual differences in adverse reaction risk. Both NATs, particularly NAT1, also can further metabolize hydroxylamine metabolites to N-acetoxy derivatives. Intensive investigation of patients with these rare adverse reactions using a variety of tools from in vitro cell toxicity assays through molecular genetic analysis will help elucidate mechanisms of predisposition and ultimately lead to diagnostic tools to characterize individual risk and prevent idiosyncratic drug toxicity.

Covalent binding of sulfamethoxazole reactive metabolites to human and rat liver subcellular fractions assessed by immunochemical detection.

Potentially serious idiosyncratic reactions associated with sulfamethoxazole (SMX) include systemic hypersensitivity reactions and hepatotoxicity. Covalent binding of SMX to proteins subsequent to its N-hydroxylation to form N4-hydroxysulfamethoxazole (SMX-HA) is thought to be involved in the pathogenesis of these reactions. A polyclonal antibody was elicited in rabbits against a SMX--keyhole limpet hemocyanin conjugate that recognized covalent protein adducts of SMX in microsomal protein and was used to characterize the covalent binding of SMX and its putative reactive metabolites to hepatic protein in vivo and in vitro. In vitro covalent binding of SMX to rat and human liver microsomal protein was NADPH-dependent, while binding of SMX-HA was not dependent on NADPH. SMX and SMX-HA produced similar patterns of covalent binding, with major protein targets in the region of 150, 100 (two bands), 70 (two bands), and 45-55 kDa. The pattern of covalent binding to human and rat liver microsomal protein was similar. Binding of SMX-HA was completely eliminated by GSH or by addition of cytosolic fractions and acetylcoenzyme A. The acetoxy metabolite of SMX also led to covalent binding, but it was primarily attributable to the formation of SMX-HA from acetoxySMX. In vivo exposure of rats to SMX did not result in detectable covalent binding by the methods employed. When rat liver slices were incubated with 2 mM SMX or 500 microM SMX-HA, no toxicity was observed and yet covalent binding of SMX-HA to 130, 100, 70, and 55 kDa proteins could be detected. These results confirm that covalent binding of SMX occurs via the formation of SMX-HA and that covalent binding of SMX-HA in vitro results from its conversion to the more reactive nitroso metabolite. Acetylation of SMX-HA protected against its covalent binding. Further studies are required to determine how this in vitro covalent binding relates to in vivo covalent binding in humans and to either direct or immune-mediated cytotoxicity in SMX idiosyncratic drug reactions.

Pharmacogenetics and blood dyscrasias.

Pharmacogenetic differences in the handling of and response to drugs can markedly alter the risk of severe idiosyncratic adverse drug reactions, including neutropenia, agranulocytosis and aplastic anaemia. Inherited deficiencies of drug metabolizing enzymes can shunt the metabolism of drugs to metabolites which are directly toxic (e.g. 6-mercaptopurine metabolism to 6-thioguanine nucleotides) or towards electrophilic metabolites which can kill cells and/or lead to a host immune response (e.g. sulphonamide metabolism to hydroxylamine metabolites). Defects in detoxification pathways (e.g. glutathione conjugation) similarly can predispose patients to adverse outcomes. The advent of molecular screening tools to define individual (rather than population) risk may lead to the use of clinical laboratory tests to identify/predict idiosyncratic adverse drug reactions.

Further investigations of the role of acetylation in sulphonamide hypersensitivity reactions.

Abstract Sulphonamide hypersensitivity reactions are believed to be mediated through reactive intermediates derived from oxidation of the paraamino group to form sulphonamide hydroxylamines. Sulphamethoxazole hydroxylamine (SMX-HA) can be acetylated by N-acetyltransferase (NAT) enzymes to form an acetoxy metabolite (acetoxySMX). In the current studies, acetoxySMX was found to be not toxic over the concentration range of 0 to 500 µM towards a human lymphoblastoid cell line (RPMI 1788) or a human hepatoma cell line (HepG2). Further, transient expression of NAT1 in COS-1 cells or stable transfection of NAT1 andNAT2 in HepG2 cells did not alter the toxicity of SMX-HA in vitro. The activity of NAT1 in isolated mononuclear leucocytes (a reflection of systemic NAT1 activity) determined with paraaminobenzoic acid as a substrate was not different between controls (n = 11) or patients with a known hypersensitivity reaction (n = 5) (4.1 ±1.2 nmol min(-1)mg(-1) vs 5.7 ± 1.4 nmol min(-1) mg(-1)). Thus, acetoxy SMX is unlikely to play a significant role in mediating SMX hypersensitivity reactions anda constitutive deficiency in NAT1 activity is not a common finding in patients susceptible to SMX hypersensitivity reactions.

In vitro formation, disposition and toxicity of N-acetoxy-sulfamethoxazole, a potential mediator of sulfamethoxazole toxicity.

Variation in the formation and disposition of the hydroxylamine of (SMX-HA) is thought to play an important role in the pathogenesis of sulfamethoxazole (SMX)-induced idiosyncratic adverse drug reactions. We hypothesized that, in analogy to carcinogenic arylamines, SMX-HA might be further converted to an electrophilic N-acetoxy metabolite which could play a role in mediating SMX toxicity. Accordingly, we chemically synthesized N-acetoxy-SMX, and examined the characteristics of its formation, metabolism, cytotoxicity and mutagenicity in human and bacterial test systems. The human arylamine N-acetyl-transferases, (NAT)1 and NAT2, were capable of converting SMX-HA to N-acetoxy-SMX. NAT1 and NAT2 possessed similar affinities for SMX-HA (apparent Km values of 650 and 520 microM, respectively), but the apparent maximal velocity of the NAT1-mediated acetylation was higher than that of NAT2. (1332 vs. 37 nmol/min/U of immunoreactive NAT protein). Human peripheral blood mononuclear cells 12,000 x g supernatant fractions converted N-acetoxy-SMX mainly back to SMX-HA, and also to a lesser extent to SMX, at clinically relevant concentrations. Similar pathways were observed in human hepatic cytosolic fractions. In a cytotoxicity assay, N-acetoxy-SMX was significantly more toxic to human peripheral blood mononuclear cells than SMX-HA (16.6 vs. 11.5% dead cells at a concentration of 300 microM). N-acetoxy-SMX was weakly mutagenic to the Salmonella typhimurium TA100 strain in the Ames test. These data suggest that the N-acetoxy metabolites of sulfonamides could potentially play a role in mediating sulfonamide idiosyncratic adverse drug reactions.

N4-hydroxylation of sulfamethoxazole by cytochrome P450 of the cytochrome P4502C subfamily and reduction of sulfamethoxazole hydroxylamine in human and rat hepatic microsomes.

The N4-hydroxylation of sulfamethoxazole (SMX) to its hydroxylamine (SMX-HA) metabolite is the first step in the formation of reactive metabolites responsible for mediating hypersensitivity reactions associated with this compound. In rat hepatic microsomes, the NADPH-dependent oxidation of SMX to SMX-HA was increased 3-fold by pretreatment of rats with phenobarbital. Other cytochrome P450 (CYP) inducers were ineffective. The constitutive and induced SMX N-hydroxylation activities were inhibited by tolbutamide, and induction of SMX-HA activity paralleled the induction of progesterone 21-hydroxylase activity, a marker for CYP2C6. SMX N-hydroxylation in phenobarbital-treated rat hepatic microsomes was inhibited 70% by anti-CYP2C6 antisera. Thus, the N4-hydroxylation of SMX by rat hepatic microsomes was mediated by members of the CYP2C subfamily, probably CYP2C6. In a panel of human microsomes, SMX-HA formation correlated with tolbutamide hydroxylase activity (r = 0.75; p = 0.01); CYP2C9 content (r = 0.79; p < 0.01) and was inhibited 70% by 500 microM tolbutamide and 90% by 100 microM sulfaphenazole. Recombinant CYP2C9 catalyzed the N-hydroxylation of SMX. SMX-HA formation in human hepatic microsomes was therefore mediated predominantly by CYP2C9. CYP-mediated reduction of SMX-HA to SMX was markedly induced in dexamethasone and phenobarbital-treated rat hepatic microsomes, and was attributed to CYP3A and CYP2B forms. In uninduced rat and human hepatic microsomes, SMX-HA reduction was mediated predominantly by an NADH-dependent microsomal hydroxylamine reductase under aerobic conditions. Under anaerobic conditions, troleandomycin at > or = 1 microM inhibited the reduction of SMX-HA in human hepatic microsomes by 45%, whereas sulfaphenazole had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced differential diagnosis of anticonvulsant hypersensitivity reactions by an integrated Bayesian and biochemical approach.

OBJECTIVE: The differential diagnosis of hypersensitivity reactions associated with anticonvulsants requires accuracy because of the many implications for patient management. We tested an integrated Bayesian and biochemical diagnostic approach. METHODS: The patients were analyzed clinically by two tests. One test, the Bayesian Adverse Reaction Diagnostic Instrument (BARDI), calculates the posterior probability of a drug being the cause based on epidemiologic and case data. The other, the lymphocyte toxicity assay, is an in vitro rechallenge that determines the percentage of cell death attributable to a drug's toxic metabolites. The setting for the study was an adverse drug reaction clinic at Sunnybrook Health Science Centre and the Hospital for Sick Children, Toronto, Ontario, Canada. Fifty-one patients who had hypersensitivity reactions after receiving aromatic anticonvulsants were tested. Four of these patients had more than one reaction reported, with different anticonvulsants generating 56 distinct events. RESULTS: Compared to the lymphocyte toxicity assay, BARDI had 94% sensitivity, 93% accuracy, and 50% specificity. When lymphocyte toxicity assay data were incorporated into BARDI, agreement rose from 93% to 100%. BARDI also identified which drug was a more likely cause for 11 patients receiving multiple anticonvulsants. CONCLUSION: These findings show that BARDI and the lymphocyte toxicity assay have high concordance and, when used in an integrated approach, these tests can improve the diagnostic accuracy and enhance the management of patients with hypersensitivity reactions.