Thiopurine methyltransferase activity in children with acute myeloid leukemia.

Activity of the enzyme thiopurine methyltransferase (TPMT) determines the anti-leukemic effect of thiopurines used in the chemotherapy of acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). TPMT status and its effects on treatment outcome have been studied extensively in ALL and autoimmune disorders, but few data is available on TPMT in AML. The present study assessed the genetic polymorphisms and activity of TPMT in children with AML at different treatment stages, and compared the results with those obtained for children with ALL. The study included 33 children with AML (0.7-19.7 years) treated with 6-thioguanine (6-TG) according to the AML-BFM 2004 Protocol. Blood samples were collected at diagnosis, during and following maintenance chemotherapy from 8, 10 and 17 patients with AML (the assay was performed at two time points in 2 patients), respectively. Blood samples from 105 children with ALL were obtained at diagnosis, during the maintenance chemotherapy and following the cessation of the chemotherapy from 16, 55 and 34 children, respectively. The activity of TPMT in red blood cells lysates was measured using an enzymatic reaction based on the conversion of 6-mercaptopurine into 6-methylmercaptopurine, involving S-adenozyl-L-methionine as the methyl group donor. TPMT mutations were determined using a polymerase chain reaction/restriction fragment length polymorphism method. Median TPMT activity at diagnosis, during maintenance chemotherapy and following chemotherapy was 43.1, 47,3 and 41.7 nmol 6-mMP g-1 Hb h-1, respectively. All patients with AML exhibited the homozygous TPMT*1/*1 genotype, with the exception of 1, who was a heterozygote with the TPMT*1/*3C genotype and demonstrated a TPMT activity level at diagnosis of 42.5 nmol 6-mMP g-1 Hb h-1. At each chemotherapy stage, the median TPMT activities in children with AML were significantly increased compared with the median TPMT activities in children with ALL. The preliminary results suggest that the TPMT activity in AML may be increased compared with that in ALL. Comprehensive studies on the association between thiopurine metabolism and treatment outcome in AML are required, with regard to the cytogenetic and molecular factors currently used for AML risk stratification.

Development of a Limited Sampling Strategy for the Estimation of Exposure to High-Dose Etoposide After Intravenous Infusion in Pediatric Patients.

BACKGROUND: Etoposide (VP-16), a podophyllotoxin derivative, is used in conditioning regimens before allogeneic hematopoietic stem cell transplantation in children with acute lymphoblastic leukemia. The aim of this study was to develop a limited sampling strategy (LSS) suitable for the prediction of exposure to VP-16 defined as area under time-concentration curve (AUC). METHODS: The study included 28 pediatric patients with acute lymphoblastic leukemia, who were administered a 4-hour infusion of 60 mg/kg VP-16. VP-16 concentrations were determined in samples collected 4-124 hours after the beginning of infusion. On obtaining the pharmacokinetic (PK) profiles, a population PK model was developed in NONMEM (ICON Development Solutions, Hanover, MD) with first-order conditional estimation with interaction algorithm. LSSs were chosen by means of a multivariate regression analysis and cross-validated with a leave-one-out approach. Predictive performance of LSSs was assessed by calculating relative prediction error (PE), mean PE, mean absolute PE, and root mean squared PE for model-predicted and observed AUC. RESULTS: VP-16 PKs was best described by a 2-compartment first-order model, and a large variability in the PK parameters was observed. A 3-sample strategy allowed the estimation of VP-16 with highest accuracy and precision (mean relative PE = 0.18%, 95% confidence interval, 1.73%-2.09%; mean absolute relative PE = 3.47%, 95% confidence interval, 2.28%-4.66%; root mean squared PE = 4.43%). The final equation was AUC = 6.85 × C6 h + 3.88 × C12 h + 46.11 × C28 h + 282.0 (adjusted R = 0.9540). CONCLUSIONS: In conclusion, developed LSS allows accurate and precise estimation of VP-16 AUC and might be useful for therapeutic drug monitoring.

Clinical and In Vitro Studies on Impact of High-Dose Etoposide Pharmacokinetics Prior Allogeneic Hematopoietic Stem Cell Transplantation for Childhood Acute Lymphoblastic Leukemia on the Risk of Post-Transplant Leukemia Relapse.

The impact of etoposide (VP-16) plasma concentrations on the day of allogeneic hematopoietic stem cell transplantation (allo-HSCT) on leukemia-free survival in children with acute lymphoblastic leukemia (ALL) was studied. In addition, the in vitro effects of VP-16 on the lymphocytes proliferation, cytotoxic activity and on Th1/Th2 cytokine responses were assessed. In 31 children undergoing allo-HSCT, VP-16 plasma concentrations were determined up to 120 h after the infusion using the HPLC-UV method. For mentioned in vitro studies, VP-16 plasma concentrations observed on allo-HSCT day were used. In 84 % of children, VP-16 plasma concentrations (0.1-1.5 µg/mL) were quantifiable 72 h after the end of the drug infusion, i.e. when allo-HSCT should be performed. In 20 (65 %) children allo-HSCT was performed 4 days after the end of the drug infusion, and VP-16 was still detectable (0.1-0.9 µg/mL) in plasma of 12 (39 %) of them. Post-transplant ALL relapse occurred in four children, in all of them VP-16 was detectable in plasma (0.1-0.8 µg/mL) on allo-HSCT day, while there was no relapse in children with undetectable VP-16. In in vitro studies, VP-16 demonstrated impact on the proliferation activity of stimulated lymphocytes depending on its concentration and exposition time. The presence of VP-16 in plasma on allo-HSCT day may demonstrate an adverse effect on graft-versus-leukemia (GvL) reaction and increase the risk of post-transplant ALL relapse. Therefore, if 72 h after VP-16 administration its plasma concentration is still above 0.1 µg/mL then the postponement of transplantation for next 24 h should be considered to protect GvL effector cells from transplant material.

RISK FACTORS FOR ABDOMINAL OBESITY IN CHILDREN AND ADOLESCENTS FROM CRACOW, POLAND (1983-2000).

The aim of this study was to determine abdominal obesity risk factors in two successive cohorts of children and adolescents aged 4-18 from Cracow, Poland, examined during the years of political transformation. The influence of biological, socio-demographic and lifestyle factors on abdominal obesity was analysed by calculating odds ratios and 95% confidence intervals using logistic regression analysis. It was found that for girls obesity in both parents (OR=4.31; 95% CI 1.73-20.70) and high birth weight (OR=1.78; 95% CI 1.12-2.82) were significant risk factors for abdominal obesity in the 1983 cohort. In the 2000 cohort obesity in both parents for boys and girls (boys: OR=5.85; 95% CI 1.36-25.10; girls: OR=4.82; 95% CI 1.17-19.77), low level of parental education in girls (OR=2.06; 95% CI 1.15-3.69), having only one son (OR=1.96; 95% CI 1.36-3.40), parents' smoking habits in girls (OR=2.94; 95% CI 1.46-5.91) and lack of undertaking physical activity in sport clubs in boys (OR=6.11; 95% CI 1.46-25.47) were significant abdominal obesity risk factors. Higher number of hours of leisure time physical activity (OR=0.89; 95% CI 0.81-0.97) significantly lowered the risk of abdominal obesity in boys in the 2000 cohort. The greater differentiation of abdominal obesity risk factors in the 2000 cohort in comparison to the 1983 cohort may have resulted from the social and economic changes taking place in Poland at the end of the 20th century.

Effect of mycophenolate mofetil on hematological side effects incidence in renal transplant recipients.

Mycophenolate mofetil (MMF), an immunosuppressant administered after solid organ transplantation, is generally well tolerated; however, it frequently causes hematological toxicity. In this study, we aimed to assess the relation between the pharmacokinetic parameters of MMF metabolites (mycophenolic acid [MPA] and 7-O-MPA glucuronide [MPAG]) and the adverse effects on the hematopoietic system in renal transplant recipients. The four-h pharmacokinetic profiles of MPA and MPAG were determined using the HPLC method for MMF-treated patients (n = 61) among 106 renal transplant recipients (during the late post-transplant period) participating in the study. Anemia was more frequently observed in the study group compared with the control group (30.7% vs. 20.0%) and although the difference was insignificant, plasma iron concentrations were significantly higher in patients treated with MMF (32.9 ± 9.4 µmol/L vs. 28.7 ± 9.4 µmol/L; p = 0.032). Iron supplementation was more frequently applied to patients with anemia (48.2%) compared with patients with hemoglobin within the norm (20.3%; p = 0.005). As all MPAG pharmacokinetic parameters correlated negatively with hemoglobin and hematocrit, and MPAG pharmacokinetic parameters were higher in patients with anemia, MPAG may be the predicting factor of MMF side effects. In renal transplant recipients, especially with deteriorated renal function, extensive iron supplementation may be ineffective as anemia was associated with declined renal function and was not caused by low iron concentration.

Thiopurine S-methyltransferase phenotype-genotype correlation in children with acute lymphoblastic leukemia.

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability mainly as a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. The aim of this study was to determine the TPMT genotype and phenotype (activity) and investigate the correlation between TPMT genotype and enzyme activity in 43 Polish children receiving 6-MP during maintenance therapy in course of acute lymphoblastic leukemia (ALL), in 16 children with ALL at diagnosis and 39 healthy controls. TPMT activity was measured in RBC by HPLC method. Patients were genotyped for TPMT *2, *3A and *3C variant allelesusing PCR-RFLP and allele-specific PCR methods. In the group of children with ALL during maintenance therapy, median TPMT activity (29.3 nmol 6-mMP g(-1) Hb h(-1)) was significantly higher compared to the group of children with ALL at diagnosis (20.6 nmol 6-mMP g(-1) Hb h(-1), p = 0.0028), as well as to the control group (22.8 nmol 6-mMP g(-1) Hb h(-1), p = 0.0002). Percentages of individuals heterozygous for TPMT variant allele in respective groups were: 9.3, 6.2 and 15.5% (p > 0.05). In all the study groups heterozygous patients manifested a significantly lower TPMT activity as compared to the wild type homozygotes (16.7 +/- 2.1 vs. 31.2 +/- 6.8 nmol 6-mMP g(-1) Hb h(-1), p = 0.002, in children during maintenance therapy, 11.9 +/- 2.7 vs. 24.6 +/- 9.5, p = 0.0003, in the combined group of children with ALL at diagnosis and controls). The results present that commencement of the thiopurine therapy caused an increase in the TPMT activity in RBCs by approximately 20%. All patients heterozygous for the TPMT variant allele revealed decreased TPMT activity compared to TPMT wild-type patients. Since decreased TPMT activity is associated with higher risk for toxicity after receiving standard doses of thiopurine drugs, pretreatment determination of TPMT status, with phenotypic or genetic assay, should be performed routinely, also in Poland.

[Anthropometric indexes of the state of nutrition and eating habits, and recreational physical activity of working physically men aged 20-60 of urban population]. / Rekreacyjna aktywnosc fizyczna a antropometryczne wskazniki stanu odzywienia i zachowania zywieniowe pracujacych fizycznie mezczyzn w wieku 20-60 lat z populacji wielkomiejskiej.

The aim of this studies was the comparison of somatic indexes and eating habits of working physically men who prefer different ways (active vs. passive) of spending their free time. The studies has been carried out on a group of 1271 people who work in HTS (steelworks) in Nowa Huta (one of Cracow's districts), including 523 men aged 20-40 (181 active and 342 non-active) and 748 men aged 40-60 (194 active and 554 non-active). Men referred to as active declared active spending of their free time and taking up recreational physical activity at lest twice a week. The presented research has not revealed statistically important differentiation of somatic parameters depending on preferred way of spending free time, or a connection between the physical activity level during free time and some eating habits indicating more rational choices, connected with the control of energy value of the diet, larger consumption of vegetables and fruit and smaller consumption of sweet products, and less frequently appearance of 'canine appetite' in the case of active men.

Pharmacokinetics of high-dose etoposide administered in combination with fractionated total-body irradiation as conditioning for allogeneic hematopoietic stem cell transplantation in children with acute lymphoblastic leukemia.

Etoposide (VP-16) is one of the most widely used antitumor agents in pediatric oncology as well as chemotherapeutic agents used in conditioning regimen prior to allo-HSCT for childhood ALL. This study included 21 children with ALL who underwent allo-HSCT after conditioning with FTBI and high-dose of VP-16 (60 mg/kg) given intravenously as single four-h infusion on day -3 (n=2) or day -4 (n=19) prior to allo-HSCT. Blood samples were collected at defined time intervals until 120 h elapsed from the end of infusion. VP-16 plasma concentrations were determined using validated HPLC method. Three-compartment model was assumed for assessing PK parameters of VP-16. The median value of VP-16 C(max) measured at the end of infusion was 188.0 µg/mL (range 148.0-407.0 µg/mL). Out of 21 studied children, VP-16 was still detectable in 17 patients 72 h (median concentration 0.31 µg/mL) and in eight patients 96 h (median concentration 0.31 µg/mL) after the end of infusion. VP-16 concentration 96 h after the end of infusion was positively correlated with VP-16 AUC and negatively correlated with VP-16 CL normalized to body weight.

[Level of education comparing to eating behaviours and anthropometrical indicators of nutritional status among men of Cracovian population]. / Poziom wyksztalcenia a zachowania zywieniowe i antropometryczne wskazniki stanu odzywienia mezczyzn z populacji krakowskiej.

The purpose of this study was to estimate of educating level effect as one indicator of social status on eating behaviours and anthropometrical parameters of nutritional status in professionally active men aged 20-60 at city environment. The research was conducted into 1320 workers of Tadeusz Sendzimir's Steelworks in Cracov. The research tool was the author's questionnaire which included questions about meal consumption regularity and frequency of consuming selected groups of foodstuffs. The indicators of nutritional status were fixed on the base of anthropometrical measurements, whereas the body content was estimated by method of bioimpendation with the use of electronic scales TBF-300P. Differentiation of some eating behaviours depending on the level of education was proved; but one cannot definitely estimate the relation of these parameters, as the higher educated people aged 40-60 years old more frequently declare two meal style of eating and more often consume confectionery than the lower educated; in turn vocationally educated men aged 20-40 more often declare consuming fast food products. Statistically considerable differentiation in some anthropometrical indicators of nutritional status depending of the level of education among men aged 40-60 was also proved. Men of vocational education are characterized by the highest value of WHR indicator but at the same time lower value of the 4 skin-fatty folds sum than higher educated people.

Thiopurine S-methyltransferase phenotype-genotype correlation in hemodialyzed patients.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme, catalyzing S-methylation of thiopurine drugs. TPMT exhibits autosomal codominant polymorphism. Patients carrying a variant genotype have low TPMT activity, and produce elevated levels of 6-thioguanine nucleotides (6-TGN) in their red blood cells (RBC). 6-TGN accumulation may result in azathioprine (AZA)-induced bone marrow myelosuppression in the course of treatment with the drug in a standard dosage regimen in patients following renal transplantation. In the current study, TPMT activity (phenotype) and genotype were determined in dialyzed patients, qualified for renal transplantation. TPMT activity was measured in RBC after dialysis by HPLC method. Patients were genotyped for TPMT *2, *3A and *3C variant alleles using PCR-RFLP and allele-specific PCR methods. TPMT activity ranged between 12.2 and 45.5 nmol 6-mMP/g Hb/h (median value 30.6). A significant correlation between TPMTphenotype and genotype was noted: the heterozygous patients (11.5%) demonstrated significantly lower mean TPMT activity as compared to the wild homozygotes (17 +/- 3.6 vs. 32.4 +/- 4.8 nmol 6-mMP/g Hb/h, p < 0.0003). No overlap in TPMT activity values between the group of heterozygous (range 12.2-20.6) and wild-type homozygous patients (range 22.7-45.5) was noted. TPMT activity, established after hemodialysis and TPMT genotyping results seem to be convergent in dialyzed patients, so both methods can be used for the identification of patients with lower TPMT activity. Such tests could be helpful in AZA dose individualization, and thus in reducing the risk of myelosuppression during AZA therapy following renal transplantation.

Modeling of purine derivatives transport across cell membranes based on their partition coefficient determination and quantum chemical calculations.

Mercaptopurine (6-MP), thioguanine (6-TG), and azathioprine (AZA) are purine antimetabolites introduced as anticancer or immunosuppressive drugs decades ago. Methylated AZA, called MAZA, is among the investigational drugs. The present study compares MAZA to the widely recognized drugs AZA, 6-MP, and 6-TG with respect to the ability of being transported across cell membranes. The obtained octanol/water phases partition coefficients and results of quantum chemical calculations predict the following sequence of hydrophobicity: MAZA > AZA > 6-TG > 6-MP.

Comparative kinetics of azathioprine and metazathioprine mercaptolysis in presence of physiological thiols.

Kinetic parameters of mercaptolysis of azathioprine (AZA) and metazathioprine (MAZA) to 6-mercaptopurine in phosphate buffer, pH 7.4, under the influence of physiological thiols (glutathione and cysteine) at 25 degrees, 30 degrees and 37 degrees C were determined and compared. It comes out that the mercaptolysis of MAZA is significantly faster under the influence of both mentioned thiols if compared to that reaction of AZA. Furthermore, the mercaptolysis of MAZA and AZA proceeded significantly faster under the influence of cysteine than on the glutathione heterolysis.