Azathioprine therapy and adverse drug reactions in patients with inflammatory bowel disease: impact of thiopurine S-methyltransferase polymorphism

The efficacy of the immunosuppressants azathioprine and 6-mercaptopurine has been well established in the therapy of inflammatory bowel diseases (IBD). However, its use has been complicated by a high incidence of serious adverse drug reactions such as hematotoxicity, hepatotoxicity, pancreatitis and gastrointestinal disturbances. Whereas azathioprine-related pancytopenia has been clearly linked to thiopurine S-methyltransferase (TPMT) polymorphism limited data are available to explain gastrointestinal side effects. In a retrospective analysis of 93 adults with IBD and azathioprine therapy both phenotyping and genotyping was used to explore systematically the relationship between TPMT and azathioprine-related adverse reactions. At time of inclusion, 69 patients were still receiving azathioprine therapy and had never experienced side effects. Azathioprine had been withdrawn in 10 patients for non-medical reasons or lack of response and 14 patients (15%) had stopped medication or were on reduced dose due to severe azathioprine-related side effects. Nine of these 14 patients had developed gastrointestinal side effects (hepatotoxicity, n = 3; pancreatitis, n = 3; others, n = 3), but their normal red blood cell TPMT activities were in accordance to TPMT wild-type. TPMT deficiency in one patient had led to pancytopenia whereas only two of the remaining four patients with hematotoxicity displayed an intermediate phenotype of TPMT. This study demonstrates that azathioprine-related gastrointestinal side effects are independent of the TPMT polymorphism. Nevertheless pharmacogenetic testing for TPMT prior to commencing thiopurine therapy should become routine practice in order to avoid severe hematotoxicity in TPMT deficient patients and lowering the incidence of hematological side effects in individuals heterozygous for TPMT.     

Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver

The human cytochrome P450, CYP2B6, is involved in the metabolism of several therapeutically important drugs and environmental or abused toxicants. In this study, we present the first systematic investigation of genetic polymorphism in the CYP2B6 gene on chromosome 19. A specific direct sequencing strategy was developed based on CYP2B6 and CYP2B7 genomic sequence information and DNA from 35 subjects was completely analysed for mutations throughout all nine exons and their exon-intron boundaries. A total of nine novel point mutations were identified, of which five result in amino acid substitutions in exon 1 (C64T, Arg22Cys), exon 4 (G516T, Gln172His), exon 5 (C777A, Ser259Arg and A785G, Lys262Arg) and exon 9 (C1459T, Arg487Cys) and four are silent mutations (C78T, G216C, G714A and C732T). Polymerase chain reaction-restriction fragment length polymorphism tests were developed to detect each of the five nonsynonymous mutations in genomic DNA. By screening a population of 215 subjects the C64T, G516T, C777A, A785G and C1459T mutations were found at frequencies of 5.3%, 28.6%, 0.5%, 32.6% and 14.0%, respectively. Haplotype analysis revealed six different mutant alleles termed CYP2B6*2 (C64T), *3 (C777A), *4 (A785G), *5 (C1459T), *6 (G516T and A785G) and *7 (G516T, A785G and C1459T). By analysing a large number of human liver samples, significantly reduced CYP2B6 protein expression and S-mephenytoin N-demethylase activity were found in carriers of the C1459T (R487C) mutation (alleles *5 and *7). These data demonstrate that the extensive interindividual variability of CYP2B6 expression and function is not only due to regulatory phenomena, but also caused by a common genetic polymorphism.     

Comprehensive analysis of the genetic factors determining expression and function of hepatic CYP2D6

Variable expression and function of the cytochrome P4502D6 (CYP2D6) leads to distinct phenotypes termed ultrarapid (UM), extensive (EM), intermediate (IM) and poor metabolizer (PM). Whereas the PM phenotype is known to be caused by two null-alleles leading to absence of functional CYP2D6 protein, the large variability among individuals with functional alleles remained largely unexplained. In this study, we systematically investigated 76 liver biopsies from individuals with known sparteine metabolic ratios (MRS) for the relationships between CYP2D6 genotype, microsomal protein expression, bufuralol 1'-hydroxylase activity and in-vivo phenotype. Average CYP2D6 protein levels ranged from undetectable in PMs (MRS > 20) to 2.6 +/- 2.7 pmol/mg microsomal protein in IMs (1.2 < MRS< 20), 7.6 +/- 4.7 in EMs (0.2 < MRS < 1.2) and 23.8 +/- 7.7 in UMs (MRS < 0.2), respectively. Analysis with respect to genotype demonstrated gradually increased expression and function for individuals with no, one, two or three functional gene copies per genome. The recently discovered -1584 C/G promoter polymorphism was identified as another major factor for expression and function with the mutant [-1584G] promoter type being consistently associated with significantly higher expression than [-1584C]. To investigate functional differences between the detected variant protein forms CYP2D6.1, 2D6.2, 2D6.9 and 2D6.10, we expressed them recombinantly in insect cells. The most significant difference was a decrease in the relative P450 holoprotein content of all allelic forms, including the common functional variant 2D6.2, in comparison to 2D6.1, whereas only modest Km changes were observed. Taken together, these data provide further insight into the complex mechanisms that govern the highly variable expression and function of CYP2D6.     

Reduced Efficacy of Intermittent Preventive Treatment of Malaria in Malnourished Children

Intermittent preventive treatment in infants with sulfadoxine-pyrimethamine (IPTi-SP) reduces malaria episodes by 20 to 59% across Africa. This protective efficacy, however, may be affected by the high frequency of malnutrition in African infants. We analyzed the impact of malnutrition as defined by anthropometry on the incidence of malaria and on the protective efficacy of IPTi in a cohort of 1,200 children in northern Ghana, where malaria is hyperendemic. These children received IPTi-SP or placebo at 3, 9, and 15 months of age and were monitored until 24 months of age. Malnutrition was present in 32, 40, and 50% of children at ages 3, 9, and 15 months, respectively. It was associated with increased risks of severe anemia and death but not an increased risk of malaria. Although malaria slightly contributed to chronic malnutrition, IPTi did not substantially improve child growth. Importantly, the protective efficacies of IPTi in malnourished children were roughly half or even less of those observed in nonmalnourished children. In the first year of life, IPTi reduced the incidence of malaria to a significantly lesser extent in infants who received both doses in a malnourished condition (25%; 95% confidence interval [CI], −7 to 48%) compared to that of nonmalnourished children (46%; 95% CI, 30 to 58%; P = 0.049). Moreover, in contrast to nutritionally advantaged children, the rate of severe malaria appeared to be increased in malnourished children who took IPTi. IPTi might exhibit reduced efficacy in regions of abundant malnutrition. Concomitant nutrition programs may be needed in these places to achieve the desired impact.Intermittent preventive treatment in infants (IPTi) with sulfadoxine-pyrimethamine (SP) appears to be a promising tool of malaria control in young children. The initial IPTi trial in Tanzania reported a protective efficacy (PE) against uncomplicated malaria in infancy of 59% and some degree of protection persisting into the second year of life (28, 29). In subsequent studies at sites of differing endemicity in sub-Saharan Africa, protection in infancy was confirmed; however, the PEs were lower, at 20 to 33% (5, 12, 15, 17, 23). We have reported previously that in Tamale, northern Ghana, IPTi reduced the incidence of asymptomatic parasitemia, uncomplicated malaria, and severe anemia from 3 to 24 months of age by 29, 17, and 15%, respectively. These effects were greatest in the first year of life and less pronounced in the second (23).As in many regions of Africa, malnutrition is abundant in northern Ghana, reaching prevalences as high as 50% in preschool children, depending on seasonality and food availability (32 and http://www.who.int/nutgrowthdb/database/countries/nchs_reference/gha.pdf). Malnutrition causes relative immunosuppression, and repeated or chronic infections may contribute to poor nutritional status (27). However, the effect of malnutrition on malaria is less clear cut than would be expected: protein-energy malnutrition has been associated with greater malaria morbidity and mortality in some areas but not in others (4, 6, 8, 21, 24, 30). Moreover, the risk of antimalarial treatment failure appears to be increased in malnourished children (13, 14, 22, 39). Taken together, these findings suggest that malnutrition is one factor contributing to malaria-associated morbidity and that malaria control strategies without concomitant nutrition programs may not have the desired impact on childhood morbidity on a large scale (8). We hypothesized that malnutrition affects both malaria morbidity and IPTi efficacy. Alternatively, IPTi might improve children''s growth and nutritional status. We reanalyzed data from a cohort from northern Ghana (23) regarding the effect of malnutrition on the PE of IPTi, and here we report the results of this secondary analysis.     

Nipradilol, a new β-blocker with vasodilatory properties, in experimental portal hypertension: A comparative haemodynamic study with propranolol

The haemodynamic effects of nipradilol, a new non-selective β-adrenoreceptor blocker with vasodilating actions like nitroglycerin, were examined in rats with portal hypertension due to portal vein stenosis. Portal hypertensive rats were divided into five groups receiving infusion of placebo, 3 mg of propranolol, 300, 600 and 1200 μg of nipradilol. At its highest dose, nipradilol achieved a reduction of 34.4 ± 4.4% in heart rate which was similar to that in the propranolol group (36.5 ± 2.4%). Also for other systemic haemodynamic parameters, the nipradilol 1200 μg group exhibited changes not significantly different from those in the propranolol group; mean arterial pressure (- 13 vs - 14%), cardiac index (- 37 vs - 31%) and systemic vascular resistance (+ 29 vs+ 32%). In contrast to the similar changes in the systemic circulation, a 1200 μg dose of nipradilol lowered portal pressure significantly more than propranolol (- 4.3 ± 0.6 vs - 2.9 ± 0.2 mmHg, P≤ 0.05). Nipradilol then reduced portal blood flow by 22% (P≤ 0.05) without a significant change in portocollateral resistance. On the other hand, propranolol not only caused a reduction in portal blood flow of 30% (P≤ 0.01), but also an increase in portocollateral resistance of 21% (P≤ 0.05). The results suggest that nipradilol may ensure a more effective control of portal hypertension than propranolol, presumably via its venodilatory action on portocollateral vessels.     

The genetic polymorphism of debrisoquine/sparteine metabolism-molecular mechanisms

The genetic polymorphism of debrisoquine/sparteine metabolism is one of the best studied examples of a genetic variability in drug response. 5-10% of individuals in Caucasian populations are 'poor metabolizers' of debrisoquine, sparteine and over 20 other drugs. The discovery and the inheritance of deficient debrisoquine/sparteine metabolism are briefly described, followed by a detailed account of the studies leading to the characterization of the deficient reaction and the purification of cytochrome P-450IID1, the target enzyme of this polymorphism. It is demonstrated by immunological methods that deficient debrisoquine hydroxylation is due to the absence of P-450IID1 protein in the livers of poor metabolizers. The cloning and sequencing of the P-450IID1 cDNA and of IID1 related genes are summarized. The P-450IID1 cDNA has subsequently led to the discovery of aberrant splicing of P-450IID1 pre-mRNA as the cause of absent P-450IID1 protein. Finally, the identification of mutant alleles of the P-450IID1 gene (CYP 2D) by restriction fragment length polymorphisms in lymphocyte DNA of poor metabolizers is presented.     

Clonal expansion accounts for an excess of antimicrobial resistance in Staphylococcus aureus colonising HIV-positive individuals in Lagos, Nigeria

Nasal colonisation with Staphylococcus aureus is a risk factor for invasive infection in human immunodeficiency virus (HIV)-positive individuals. This study aimed to characterise colonising S. aureus from regions with a high HIV prevalence. Single nasal swabs were taken from a total of 374 HIV-positive and 370 healthy individuals. Overall, 202 S. aureus carriers were detected. Compared with healthy individuals, HIV-positive subjects were more likely to be S. aureus nasal carriers (33% vs. 21%; P=0.0001). Isolates from HIV-positive individuals were more often resistant to meticillin (16% vs. 8%; P=0.13), chloramphenicol (47% vs. 16%; P<0.0001), sulfamethoxazole/trimethoprim (SXT) (90% vs. 55%; P<0.0001) and ciprofloxacin (18% vs. 0%; P<0.0001). Strains belonging to the spa clonal complexes 3772/ST25 and 064/ST8 were significantly more often isolated from HIV-positive individuals and exhibited greater resistance to ciprofloxacin, SXT and chloramphenicol (spa-CC 3772) or to meticillin (spa-CC 064), respectively. Panton-Valentine leukocidin gene content was high overall and was equally distributed between isolates from HIV-positive and healthy individuals (33% vs. 30%). Genotypic characteristics of colonising isolates were similar to those reported to cause invasive infection in Nigeria. The HIV pandemic contributes to the evolution of antimicrobial resistance in S. aureus. Measures to contain antimicrobial resistance of S. aureus in Nigeria must target risk groups such as HIV-positive individuals.