The effect of 677C>T and 1298A>C MTHFR polymorphisms on sulfasalazine treatment outcome in rheumatoid arthritis.

Despite the availability of several new agents for the treatment of rheumatoid arthritis (RA), sulfasalazine remains the mainstay because of both cost and experience with its use. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and several polymorphisms have been described in the MTHFR gene. Of these, the 677C>T and 1298A>C polymorphisms have been associated with altered enzyme activity. To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and sulfasalazine efficacy for the treatment of RA, a total of 117 RA patients treated with sulfasalazine (1 g daily; duration of treatment 17 +/- 5 months) were analyzed. The 677C>T and 1298 A>C polymorphisms were detected using a PCR-RFLP method. RA was diagnosed according to the criteria of the American College of Rheumatology (ACR). The remission of RA symptoms was evaluated according to the ACR 20% response criteria. Allele and genotype frequencies were compared by the two-sided Fisher exact test. The frequency of remission was 47.2% and 44.6% in carriers of 677T and 1298C alleles, compared to 40.7% and 42.0% in carriers of 677C and 1298A alleles, respectively. These differences were statistically non-significant. When the multivariate analysis was additionally adjusted for patients' age, gender and RA duration, the association of the MTHFR 677T allele with increased frequency of remission was statistically significant. Although RA remission rate in carriers of the MTHFR 677T and 1298C alleles was more frequently observed, it does not seem that 677C>T and 1298A>C MTHFR polymorphisms have a major influence on treatment outcome in RA patients treated with sulfasalazine.

The effect of 677C>T and 1298A>C MTHFR polymorphisms on sulfasalazine treatment outcome in rheumatoid arthritis

Despite the availability of several new agents for the treatment of rheumatoid arthritis (RA), sulfasalazine remains the mainstay because of both cost and experience with its use. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and several polymorphisms have been described in the MTHFR gene. Of these, the 677C>T and 1298A>C polymorphisms have been associated with altered enzyme activity. To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and sulfasalazine efficacy for the treatment of RA, a total of 117 RA patients treated with sulfasalazine (1 g daily; duration of treatment 17 ± 5 months) were analyzed. The 677C>T and 1298 A>C polymorphisms were detected using a PCR-RFLP method. RA was diagnosed according to the criteria of the American College of Rheumatology (ACR). The remission of RA symptoms was evaluated according to the ACR 20% response criteria. Allele and genotype frequencies were compared by the two-sided Fisher exact test. The frequency of remission was 47.2% and 44.6% in carriers of 677T and 1298C alleles, compared to 40.7% and 42.0% in carriers of 677C and 1298A alleles, respectively. These differences were statistically non-significant. When the multivariate analysis was additionally adjusted for patients’ age, gender and RA duration, the association of the MTHFR 677T allele with increased frequency of remission was statistically significant. Although RA remission rate in carriers of the MTHFR 677T and 1298C alleles was more frequently observed, it does not seem that 677C>T and 1298A>C MTHFR polymorphisms have a major influence on treatment outcome in RA patients treated with sulfasalazine.

Reduced folate carrier-1 80G>A polymorphism affects methotrexate treatment outcome in rheumatoid arthritis.

The folate antagonist methotrexate (MTX) is a drug currently used in the treatment of rheumatoid arthritis (RA). MTX enters the cells through the reduced folate carrier (RFC-1) and is activated to polyglutamates. Previous studies have shown that RFC-1 expression may influence the efficacy of therapy with MTX. The studies suggest that G80A polymorphism in RFC-1 is associated with altered folate/antifolate levels and the subjects carrying homozygous mutant 80AA genotype tend to have higher plasma folate and MTX concentrations and higher erythrocyte polyglutamate levels compared with those with the wild type or heterozygous genotype. It is possible that this polymorphism might influence MTX treatment outcome in patients with RA. In the present study, we examined the association between RFC-1 G80A polymorphism and treatment outcome in patients with RA administered MTX. The study was carried out on 174 patients diagnosed with RA treated with MTX (7.5-15.0 mg weekly) plus low doses of methylprednisone. The RFC-1 80G>A polymorphism (resulting in a histidine-to-arginine substitution at codon 27 of RFC-1) was detected using a polymerase chain reaction-restriction fragment length polymorphism method. The probability of remission of RA symptoms was 3.32-fold higher in carriers of 80AA genotype as compared with patients with 80GG genotype (P=0.021, OR=3.32, 95% CI: 1.26-8.79). The frequency of A allele among MTX responders was 62.1, compared to 47.8% in a group of poor MTX responders (P=0.013, OR=1.78, 95% CI: 1.13-2.81). Moreover, the increase of aminotransferase activity was noted more frequently in carriers of 80AA genotype. The present data suggest that evaluation of RFC-1 gene 80G>A polymorphism may be a useful tool to optimize MTX therapy in patients with RA.

Lack of association between arylamine N-acetyltransferase 2 (NAT2) polymorphism and systemic sclerosis.

OBJECTIVES: It has been shown that exposure to some environmental toxins may induce scleroderma-like illness in predisposed individuals, but the etiopathogenesis of the idiopathic form of systemic sclerosis (SSc) remains obscure. The genetic background of this illness has been confirmed in multiple studies. We investigated whether patients with SSc differ from healthy subjects with regard to the enzymatic activity of polymorphic N-acetyltransferase 2 (NAT2). METHODS: The study was carried out in 39 patients with SSc; 15 fulfilled the criteria of diffuse SSc (dSSc) and 24 of limited SSc (lSSc); an ethnically matched control group consisted of 100 healthy volunteers. Acetylation phenotype was estimated using the isoniazid as a model drug. The most common mutations in the Caucasian population at positions 481T, 803G, 590A and 857A on the NAT2 gene were determined using the polymerase chain reaction-restriction fragment length polymorphism method with deoxyribonucleic acid (DNA) extracted from peripheral blood. RESULTS: In the group of patients with SSc, the frequency of fast acetylator genotypes was 38.5% (95% CI 23.4-55.4), while that for the genotypes coding slow acetylator status was 51.3% (95% CI 34.8-67.6). There was a strong correlation between NAT2 phenotype and NAT2 genotype with a concordance of 97%. We did not observe a preponderance of slow acetylators among patients with SSc and in two subsets of SSc. With the sample size analyzed in the present study, there is a 90% probability of detecting significant differences in distribution of slow, fast, and intermediate phenotypes between patients with SSc and controls, there is a difference of at least 30.3, 28.7 and 21.9% in the distribution of these phenotypes in the general population, respectively. CONCLUSION: Acetylator status does not seem to be the significant factor in the development of SSc in patients with both subsets of this autoimmune disease, but further studies are required to confirm this conclusion.

[Evaluation of methotrexate levels in biological fluids released from bone cement filling]. / Ocena stezenia metotreksatu w plynach biologicznych uwalnianych z plomby cementowej.

The authors present an analysis of methotrexate concentration in blood, urine and drainage liquid (both deep and superficial), as well as the results of biochemical analysis of blood and urine in a population of 42 patients treated surgically because of pathologic fractures of long bones. In 8 of the 42 patients chemotherapy was applied 14 days before the surgical procedure. Lithic lesions in the long bones were filled with cement with 2 g of methotrexate and further stabilisation was achieved by applying metal implants. The highest methotrexate concentrations were noted in deep drainage fluid and in blood. Methotrexate concentration reached its peak in body fluids 12-24 hours after the procedure, and fell to a minimum 48-72 hours post surgery. The lowest level of the drug compared to all body fluids was found to be in the blood. Only after 72 hours did it exceed the minimum required level of 0.05 umol/l. All biochemical markers in blood and urine except AspAT were found within normal range in all cases 72 hours post surgery. After 24-72 hours post-op a decrease in the number morphotic elements of blood was noted, particularly lymphocytes and thrombocytes.

Genotype of N-acetyltransferase 2 (NAT2) polymorphism in children with immunoglobulin E-mediated food allergy.

We investigated whether patients with immunoglobulin E-mediated food allergy differed from healthy individuals with regard to genotype of the polymorphic enzyme N -acetyltransferase 2 (NAT2). The genetic polymorphism of acetylation can alter the toxic and therapeutic response to certain xenobiotics and may be also a factor that influences the susceptibility toward certain partly chemically induced diseases. We compared 136 children with immunoglobulin E-mediated food allergy with 123 healthy children. The NAT2 alleles (*4, *5, *6, and *7 ) were determined by polymerase chain reaction-restriction fragment length polymorphism methods with DNA extracted from peripheral blood. A statistically significant increase in the proportion of homozygous slow acetylators (76.5%) was found among patients with food allergy compared with healthy subjects (53.7%; P < .001). There were no homozygous fast acetylators within this group of individuals with severe forms of food allergy. The risk of development of immunoglobulin E-mediated food allergy was almost 3-fold greater in slow acetylators than that in the healthy subjects (odds ratio, 2.8; 95% confidence interval, 1.6 to 4.9). We therefore concluded that the slow acetylation genotype may be an important factor of individual susceptibility to immunoglobulin E-mediated food allergy.

[Effect of therapeutic drug monitoring of amitriptyline and genotyping on efficacy and safety of depression therapy]. / Przydatnosc terapii monitorowanej stezeniem leku we krwi oraz badan farmakogenetycznych w ocenie skutecznosci i bezpieczenstwa leczenia depresji amitryptylina.

Modern pharmacotherapy is based on precise adjustment of a dosage schedule to individual requirements of patient. Therapeutic drug monitoring is a method that allows for a more effective treatment approach, especially in the case of a narrow therapeutic index of a drug. Tricyclic antidepressant drugs are characterised by narrow therapeutic index as well as relationship between serum drug concentration and side effects. It was demonstrated that interindividual variability of blood concentrations of tricyclic antidepressant drugs is related to genetic polymorphism of oxidating enzymes participating in metabolism of these drugs. The aim of the study was to estimate the impact of therapeutic drug monitoring of tricyclic antidepressant drugs as well as genotyping on efficacy and safety of endogenous depression therapy. The study included 9 patients with established diagnosis of endogenous depression. Blood serum concentrations of amitryptyline was measured by fluorescence polarisation immune assay (FPIA, Abbott system). Genotype of cytochrome P450 isoenzyme CYP2D6 was determined using PCR-RFLP method. It was demonstrated that monitoring therapy of tricyclic antidepressant drugs in combination with determination of the genotype seems to be more safe and effective. Monitoring therapy and genotyping may be less expensive than the costs of prolonged hospitalisation and risk of side effects.

CYP2D6 and GSTM1 genotypes in a Polish population.

OBJECTIVE: The aim of the present study was to investigate the distribution of the CYP2D6 and GSTM1 genotypes in a Polish population. METHODS: One hundred and forty-five unrelated healthy individuals from the western region of Poland were studied. The CYP2D6 genotype was analysed by means of polymerase chain reaction (PCR) amplification for the CYP2D6*3 and CYP2D6*4 alleles. The GSTM1 genotype was also analysed by means of a PCR assay to determine two genotypes: GSTM1-1 (positive) and GSTM1-0 (negative). RESULTS: Fourteen subjects (9.6%) were classified as poor metabolisers. The frequency of CYP2D6*4 and CYP2D6*3 was 23.1% and 2.1%, respectively. The frequency of GSTM1 nulled genotype in a Polish population came to 49%. CONCLUSION: The frequencies of poor metabolisers for CYP2D6 and GSTM1 nulled genotype among a Polish population were similar to those observed in other Caucasian populations.

N-acetylation and hydroxylation polymorphisms in type II diabetics with microvascular disturbances.

The N-acetylation and hydroxylation (CYP2D6) genetic polymorphisms were assessed in 43 healthy subjects and in 84 type II (non-insulin-dependent) diabetics. The proportions of slow and fast acetylators as well as poor and extensive metabolisers in a group of diabetics suffering from microvascular disturbances (nephropathy, retinopathy and neuropathy) were compared with the control group and with diabetics without such complications. Sulphadimidine was used as a probe for polymorphic acetylation and debrisoquine for CYP2D6. Debrisoquine and its 4-OH metabolite were assayed by means of HPLC, and sulphadimidine using a modified Bratton-Marshall procedure. The frequency of the slow phenotype (63%) was significantly higher in diabetics with microvascular disturbances than in patients without diabetic complications (P < 0.005). In patients with type II diabetes (84), only the extensive phenotype of hydroxylation was observed.

Comparative pharmacokinetics of theophylline in rabbits and in humans with hyperlipidemia.

UNLABELLED: The study was carried out on male rabbits divided into two groups: a control and an experimental one, fed on a high-fat diet. Humans were also ascribed into two groups: control and those affected with primary, mixed form of hyperlipidemia. The animals and humans were given theophylline intravenously as a single dose. Blood was sampled after 5, 10, 15, 30 and 45 min and 1, 2, 4, 6, 8, 12 and 24 h following theophylline administration. FPIA method was used to determine blood serum concentrations of theophylline. Considerable alterations of theophylline pharmacokinetics in humans suffering from mixed form of hyperlipidemia were observed. Marked decrease in area under the concentration-time curve (AUC), diminished volume of distribution, increased total body clearance, and shortened elimination half-life were observed. On the contrary, in rabbits with alimentary induced lipid metabolism disturbances t1/2 of theophylline was practically unchanged and AUC only slightly increased. IN CONCLUSION: (1) hyperlipidemia affects the pharmacokinetics of theophylline in human beings, (2) rabbit model with dietetary induced lipid metabolic disturbances is not a suitable subject for estimation of pharmacokinetics of xanthine derivatives.

[Comparative pharmacokinetics of theophylline in hyperlipidemia in animals and humans]. / Porównawcza farmakokinetyka teofiliny w hiperlipidemii u zwierzat i u ludzi.

The incidence of hereditary hyperlipidemia amounts to about 8% and rises when secondary causes of lipid metabolism disturbances are taken into consideration. In contemporary literature there is paucity of data on the influence of hyperlipidemia on pharmacokinetics of drugs. That is especially important in the case of drugs characterized by a narrow therapeutic index, such as theophylline, which can be administered to patients affected by an altered lipid status. The investigation was aimed at evaluating the feasibility of an animal model of hyperlipidemia for pharmacokinetic studies of theophylline in humans suffering from lipid metabolism disturbances. The study was carried out on male rabbits divided into two groups: a control and an experimental one, fed on a high-fat diet. Humans were also ascribed to two groups: controls and those affected by primary, mixed-form of hyperlipidemia. The animals were given theophylline intravenously in a single dose of 12 mg/kg, whereas humans received intravenously injected theophylline in a single dose of 3.5 mg/kg. Blood was sampled after 5, 10, 15, 30, 45 minutes and 1, 2, 4, 6, 8, 12, 24 hours following theophylline administration. FPIA method was used to determine blood serum concentrations of theophylline (Tab. 1,3). The two-compartment open model for intravenous administration was applied for calculations. Considerable alterations of theophylline pharmacokinetics in humans suffering from mixed form of hyperlipidemia were observed (Tab. 4), whereas no marked changes were noted in animals with alimentary-induced hyperlipidemia (Tab. 2). In hyperlipidemic rabbits theophylline behaves as lipophilic agent, despite its poor penetration into the adipose tissue. A considerable decrease in area under the concentration-time curve of theophylline, increase in transfer constants as well as accelerated elimination of the drug were observed in humans with mixed form of hyperlipidemia. The behaviour of theophylline in hyperlipidemic rabbits was different from that in patients with mixed form of hyperlipidemia. Comparison of theophylline pharmacokinetics in hyperlipidemic animals and in human subjects revealed that an animal model of hyperlipidemia was inappropriate for studying the effect of lipid metabolism disturbances on pharmacokinetics of drugs as it is shown in the study on theophylline. This can be explained, to some extent, by different mechanisms of hyperlipidemia in rabbits and in humans. Hyperlipidemia in rabbits was induced by alimentary factors, while in humans lipid metabolism disturbances were of primary origin. Basing on the results of the present study it may be suggested that there are no general rules for anticipating the influence of hyperlipidemia on the pharmacokinetics of drugs in various organisms. Therefore, it is most likely that studies on the influence of hyperlipidemia on the pharmacokinetics of drugs should be performed for every particular drug separately.

Ginkgo biloba extract inhibits the development of experimental atherosclerosis in rabbits.

Forty male mongrel rabbits were divided into 4 equal groups: (1) controls, (2) animals receiving a high-fat diet (HFD) containing cholesterol and coconut oil, (3) HFD + standardized. Ginkgo biloba extract (GB), and (4) HFD + rutin (as a well known substance it was used for comparison of pure single flavonol diglycoside activity with total extract). The experiment lasted 12 weeks. The most pronounced reduction in lipid metabolism disturbances and in the severity of plaque formation occurred after the GB had been administered. Lipid content in serum, liver homogenate and liver microsomes was depressed. HDL-cholesterol concentration was increased. Diminution of the malondialdehyde level in plasma was accompanied by an increase in the concentration of ascorbate free radicals in the liver in vitro. Testosterone concentrations in serum were normalized whilst the cytochrome P-450 content in liver microsomes was increased. Surface area of the lipid deposits at 12 weeks measured planimetrically averaged 85.5 % in HFD-fed animals vs 54.7 % in GB-treated rabbits. Quite distinct superiority of GB in antiatherosclerotic activity, as compared with rutin, was shown. These findings suggest that GB, in addition to lowering serum lipid level and possessing antioxidant properties in rabbits on an experimental diet, also affects metabolic processes in the liver and may modify lipid deposition in major arteries.

[Pharmacokinetics of acetaminophen in individuals occupationally exposed to polyvinyl chloride modified with plasticizers]. / Farmakokinetyka n-acetylo-p-aminofenolu u osób narazonych zawodowo na dzialanie polichlorku winylu modyfikowanego plastyfikatorami.

The aim of this study was to evaluate the health state of a random group of subjects occupationally exposed to polyvinyl chloride. The emphasis was put on the hepatic functional tests in comparison with acetaminophen pharmacokinetics. Forty two males were assigned to two groups: the control group free from occupational exposure and the group exposed to polyvinyl chloride. The latter one was divided into two subgroups according to the length of exposure. The following examinations were carried out in all subjects: subjective and physical examinations, basic laboratory and functional hepatic tests, radioisotope examinations of the liver and spleen, and acetaminophen pharmacokinetics was analysed as well. Acetaminophen was administered intragastrically in a single dose of 1.0 g. Blood was collected within 24 hrs after drug administration. The method of Routh et al. was used for determination of paracetamol concentration. A two-compartment open model for extravascular administration was used for calculation of pharmacokinetic parameters. The results suggest that polyvinyl chloride leads to an accelerated acetaminophen biotransformation, regardless of the length of occupational exposure. There was no correlation between changes in paracetamol metabolism and standard functional hepatic tests. The acetaminophen test can be useful in detecting alterations in the activity of hepatic microsomal enzymes, a symptom of early occupational exposure to chemical compounds with potential hepatoxic properties.

[Pharmacokinetics of procainamide and its metabolite depending on acetylator phenotype]. / Farmakokinetyka prokainamidu i jego metabolitu w zaleznosci od fenotypu acetylacji.

Procainamide (Pa) and its active metabolite--N-acetylprocainamide (NAPA)--pharmacokinetics was studied in 12 healthy volunteers in relation to acetylation phenotype. Acetylation phenotype was determined with sulphadimidine test. Blood serum levels of PA and NAPA were determined 0.5; 1.0; 1.5; 2.0; 3.0; 4.0; 8.0; and 12 hours following a single oral dose of 500 mg. Blood levels of both PA and NAPA were assayed with immunofluorescence polarization technique (FPIA), using TDx apparatus manufactured by Abbott. Pharmacokinetic parameters were calculated with the aid of pharmacokinetics independent of the model principles. All results were analysed statistically (AWOA). It was found that PA and NAPA pharmacokinetics depends on acetylation phenotype. Blood serum PA levels were higher in slow acetylators during the whole follow-up whereas NAPA levels were lower. Blood serum PA levels in rapid acetylators were decreased while NAPA levels were increased. Acetylation phenotype determined in sulphadimidine test confirmed bimodal procainamide acetylation.

[Pharmacokinetics of phenazone in patients with fast and slow euthyroidism after methimazole treatment]. / Farmakokinetyka fenazonu u chorych szybko i wolno osiagajacych kliniczna eutyreoze pod wplywem leczenia metymazolem.

Hyperthyroidic patients treated with methimazole were retrospectively divided into two groups, depending on the period of time required for euthyreosis: these attaining euthyreosis up to 28 days of therapy (A) and these in whom thyroid gland functioning is normalized after a 35-day therapy with full dose of methimazole (B). The study aimed at investigating whether clinical euthyreosis is related to the activity of microsomal enzymes in the liver using phenazone elimination test for this purpose. Phenazone elimination test was performed just before the treatment and after 8 weeks of methimazole administration. A decrease in kel and Clt as well as an increase in AUC were noted. These changes were more pronounced in patients of group A than those of group B. It might be concluded that phenazone pharmacokinetics is different in patients quickly attaining euthyreosis. The difference is probably due to the difference in biotransformation of methimazole in the liver in which microsomal enzymes play some role.

[Acetylation phenotype in Graves' disease]. / Fenotyp acetylacji w chorobie Gravesa-Basedowa.

Acetylation phenotype was determined with sulfamidine technique in 25 patients with Graves-Basedow disease including 19 female and 6 male patients aged between 21 and 64 years. The control group consisted of 23 healthy volunteers (8 female and 15 male individuals aged between 28 and 57 years) from the Szczecin province . Acetylation phenotype was determined before and after therapy with thiamazole++. Prevalence of fast acetylators was noted in the patients with Graves-Basedow disease in comparison with healthy population. The administered treatment decreased the percentage of fast acetylators. Fast acetylators responded to the treatment earlier achieving clinical euthyreosis.

Effect of unilateral nephrectomy on the pharmacokinetics of rolitetracycline in rabbits.

An analysis of the single-dose pharmacokinetics of rolitetracycline in rabbits after unilateral nephrectomy is presented. Rolitetracycline was injected i.v. 3.5 mg/kg before operation and after 2 weeks, 3 months and 6 months from unilateral nephroctomy. The concentration of the antibiotic in plasma was estimated by the fluorimetric method of Kohn. The two-compartment open model was applied to calculate rolitetracycline concentration changes. The calculations of pharmacokinetic parameters were performed using the Hewlett-Packard 9830B computer and fitting the concentrations of the antibiotic in blood to the general biexponential equation. AUC of rolitetracycline was increased in operated animals by 130% after 6 months. The mean kel at this time decreased, while t0.5 beta was prolonged from 1.0 h to 3.0 h. It is assumed that the dosage regimen of rolitetracycline should be changed in subjects possessing one kidney.