Pharmacokinetics of Mycophenolate Mofetil Metabolites in Older Patients on the Seventh Day After Renal Transplantation.

BACKGROUND: Currently, immunosuppression schemes are age-independent; however, physiological changes may alter drugs' pharmacokinetics in the older population. We compared mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) pharmacokinetics among patients aged <60 and >60 years on the seventh day after renal transplantation. METHODS: We included 7 and 10 renal transplant recipients, aged >60 and <60 years, respectively, treated with mycophenolate mofetil. MPA and MPAG concentrations were determined using the high-performance liquid chromatography method with ultraviolet detection (HPLC-UV). Noncompartmental pharmacokinetic analysis was performed. RESULTS: In patients aged >60 years, mean MPA and MPAG concentrations before the next dose and ratio of MPAG area under the concentration-time curve (AUC0-12) to MPA AUC0-12 were higher by 1.6-fold, 1.4-fold, and 1.9-fold, respectively. Other MPAG concentrations appeared to be slightly higher (1.2- to 1.5-fold) in older patients. MPA apparent clearance was similar in both groups, whereas volume of distribution at steady state was slightly higher (1.6-fold) in patients aged >60 years. The variability of most MPA and some MPAG pharmacokinetics was greater in patients aged >60 years. The MPA AUC0-12 target was achieved in 40% and 14% of patients aged <60 and >60 years, respectively. The highest MPAG concentrations and AUC0-12 were observed for patients with the lowest glomerular filtration rate. CONCLUSIONS: Higher variability of MPA and MPAG pharmacokinetic parameters, MPA AUC0-12 above the reference range, higher values of MPAG pharmacokinetics in patients with lower glomerular filtration rates, as well as lower proportion of patients achieving MPA targets all indicate the need for therapeutic drug monitoring in renal transplant recipients aged >60 years and to verify target MPA AUC0-12 for this population.

Pharmacokinetics of Enteric-Coated Mycophenolate Sodium Metabolites in Patients Over 60 Years Old Within the First Year After Renal Transplantation.

OBJECTIVE: It is still unclear whether mycophenolic acid (MPA) doses should be adjusted for older patients. Therefore, we compared the pharmacokinetics of MPA, mycophenolic acid glucuronide (MPAG), and free MPA (fMPA) between older and younger renal transplant recipients. METHODS: We included 12 patients <60 years and 6 patients >60 years within the first year after renal transplantation, who were receiving enteric-coated mycophenolate sodium, tacrolimus, and steroids. Blood samples were collected up to 12 hours after drug administration. RESULTS: MPA and fMPA pharmacokinetics were similar for patients <60 and >60 years; however, the MPA area under the concentration-time curve from 0 to 12 hours (AUC0-12) was 1.2-fold lower in the older patients. MPAG pharmacokinetics were more than 1.5-fold higher in patients >60 years, which might be related to deteriorated renal function in older people. Moreover, the mean (MPAG AUC0-12)/(MPA AUC0-12) ratio was more than 2-fold higher in patients >60 years. The second maximal MPA concentration was more frequently observed in patients <60 years, although all patients received tacrolimus. The percentage of patients with MPA concentration before the next drug dose (Ctrough) and AUC0-12 within and below target was the same in both groups. All patients >60 years had MPA AUC0-12 >30 µg·h/mL within 22 to 114 days after transplantation. CONCLUSIONS: MPA therapeutic monitoring should be recommended in enteric-coated mycophenolate sodium--treated patients >60 years because MPA AUC0-12 exceeded the recommended value in half of the studied patients.

The extent of overweight index in children and adolescents from Cracow, Poland (1971-2000).

Obesity indices describe the percentage of overweight and obese children in a given population but they do not show the extent to which the norms have been exceeded. The aim of this work was to determine the extent of overweight index (EOW), suggested by Jolliffe (2004a,b), by examining children and adolescents from Cracow in order to obtain information on overweight and obesity prevalence and on the amounts by which the BMI age- and sex-specific norms are exceeded, emphasising usefulness of EOW in population studies. The study material comprises three randomly selected groups, representative for Cracow: (1) measured in 1971 which includes 4090 individuals of both sexes and aged 7-19 years, (2) measured in 1983 with 6542 individuals aged 3-19 years and (3) measured in 2000 with 4524 boys and girls aged 3-19 years. The EOW index of overweight is a mean relative deviation from BMI threshold, assuming that for values lower than the threshold ones, the deviation amounts to zero. The EOW index values in boys increased from 0.9 in 1971 up to 2.2 in 2000 i.e. by 144%, indicating an increase of both prevalence of overweight and obesity and an increase of the amount by which the limits of overweight are exceeded. In girls the index also increased, though less dramatically, from 0.7 in 1971 to 1.5 in 2000 i.e. by 114%. An analysis of the results showed that the increasing prevalence of overweight and obesity in children and adolescents is accompanied by an increase of the amount by which the BMI threshold values are exceeded.

Antioxidant capacity in renal transplant patients.

The aim of the study was to analyse the relation between total antioxidant capacity and immunosuppressive therapies, renal function and hematocrit in kidney transplant patients. The study included 46 adult patients during the maintenance period (>1 year) following renal transplantation, treated with different combinations of immunosuppressive agents--most commonly with cyclosporine (n = 23) or tacrolimus (n = 15). The total antioxidant capacity (TAOC) of plasma was measured using Trolox-equivalent antioxidant capacity (TEAC) assay. Patients treated with cyclosporine had significantly greater TAOC compared with those treated with tacrolimus (1.16 +/- 0.46 mmol/L vs. 0.80 +/- 0.37 mmol/L, p = 0.018, respectively). There was a significantly negative correlation between TAOC and plasma creatinine (rs = -0.551, p = 0.033) and a positive correlation between TAOC and creatinine clearance or hematocrit in patients treated with tacrolimus but not with cyclosporine (r = 0.525, p = 0.045 or rs = 0.629, p = 0.012, respectively). Immunosuppressive therapy with cyclosporine was associated with higher TAOC. Anemia can be an independent risk factor for an increase of oxidative stress. Although subject numbers werelimited, TAOC was positively associated with renal function in patients treated with tacrolimus.

Industrial wastewater as an external carbon source for optimization of nitrogen removal at the Wschod WWTP in Gdansk (Poland).

Carbon source alternatives for denitrification belong to the highest research area priorities as they allow to optimize N removal within the existing capacities. In particular, some food industry effluents appear to be good candidates for such alternatives due to their high C/N ratios and high content of readily biodegradable organic fraction. The aim of this study was to determine the immediate effects of dosing different types of industrial wastewater on the denitrification capability of process biomass originating from the "Wschod" WWTP in Gdansk (northern Poland). Three types of industrial wastewater (effluents from a distillery, brewery and fish-pickling factory) were tested in two kinds of batch experiments. The results of this study revealed that the investigated industrial wastewater can be a potential external carbon source to improve denitrification efficiency. The observed single nitrate utilization rates (NURs) were ranging from 2.4 to 6.0 g N/(kg VSS.h) and were comparable to the rates associated with the utilization of readily biodegradable COD in the settled wastewater. When the NURs were measured during anoxic P uptake, the P uptake rates did not appear to be adversely affected by the addition of any carbon source.

Partition coefficients of some purine derivatives and its application to pharmacokinetics.

Metazathioprine (MAZA), a methylated derivative of azathioprine (AZA), demonstrated the greatest values of apparent and specific partition coefficients in n-octanol/phosphate buffer at pH 5.7 and pH 7.4 among purine derivatives such as 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and AZA. Introduction of a methyl group into the imidazole ring of AZA increases lipophilic properties of MAZA compared to AZA. Mass balance of purine derivatives in n-octanol and in phosphate buffer indicated their chemical stability in those media.

The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance.

Hypersensitive resistance (HR) is an efficient defense strategy in plants that restricts pathogen growth and can be activated during host as well as non-host interactions. HR involves programmed cell death and manifests itself in tissue collapse at the site of pathogen attack. A novel hypersensitivity gene, Ny-1, for resistance to Potato virus Y (PVY) was revealed in potato cultivar Rywal. This is the first gene that confers HR in potato plants both to common and necrotic strains of PVY. The locus Ny-1 mapped on the short arm of potato chromosome IX, where various resistance genes are clustered in Solanaceous genomes. Expression of HR was temperature-dependent in cv. Rywal. Strains PVYO and PVYN, including subgroups PVYNW and PVYNTN, were effectively localized when plants were grown at 20 degrees C. At 28 degrees C, plants were systemically infected but no symptoms were observed. In field trials, PVY was restricted to the inoculated leaves and PVY-free tubers were produced. Therefore, the gene Ny-1 can be useful for potato breeding as an alternative donor of PVY resistance, because it is efficacious in practice-like resistance conferred by Ry genes.

Attempts on freezing the Greylag (Anser anser L.) gander semen.

Semen of Greylag (Anser anser L.) ganders was frozen according to a method previously elaborated by the authors for freezing the White Koluda gander semen. Semen was collected from five to eight Greylag ganders, twice a week during three succeeding reproductive cycles, by dorso-abdominal massage. Semen samples were diluted in the ratio of 1:1 or 2:1 (two parts semen: one part diluent) with EK diluent, supplemented by 6% DMF, equilibrated and pre-frozen to -140 degrees C at a rate 60 degrees C/min, before being transferred into liquid nitrogen container. Semen samples thawed in a water bath of 60 degrees C were used for twice a week insemination in a volume of 200 microl. Three Greylag and three White Koluda geese were involved in frozen-thawed semen fertilizing ability test. The reproductive cycle of wild geese lasts usually about 6-7 weeks. The ejaculate volume (30-140 microl) and sperm concentration (10x10(6) to 150x10(6) ml(-1)) are much lower than these of domestic ganders, but spermatozoa morphology is similar, particularly while compared to 1-year-old White Koluda ganders semen. There are about 90% of live spermatozoa and about 30% of live morphologically normal cells in Greylag gander fresh semen. The Greylag gander spermatozoa susceptibility to cryopreservation procedure is as high as in domestic ganders. Dilution ratio 2:1 resulted in higher number of live spermatozoa, which withstood cryoinjury stress. In relation to fresh semen about 60% of spermatozoa remained intact (on the basis of light microscope examination) in the frozen-thawed semen. Insemination of frozen-thawed semen resulted in 37.5% of fertile eggs in Greylag and 25.0% in White Koluda geese. Low fertility rate was caused by an insufficient number of live normal spermatozoa used for insemination (about three million in every dose).

Biopharmaceutical characterization of some synthetic purine drugs.

Hydrophilic-lipophilic properties (water solubility, n-octanol/water partition coefficient, transport across membranes) of some mercaptopurines (6-MP, 6-TG, AZA and a new AZA derivative--metazathioprine (MAZA) were determined. MAZA is the most lipophilic compound due to low aqueous solubility and high n-octanol/water partition coefficient. The fluxes from the donor medium into the membrane and from the membrane into the acceptor medium are highest for MAZA as well. The partition coefficients of the other purines decrease in the order: AZA > 6-TG > 6-MP.

Mechanism of activation of an immunosuppressive drug: azathioprine. Quantum chemical study on the reaction of azathioprine with cysteine.

Azathioprine is an important drug used in the therapy of autoimmune disorders and in preventing graft rejection. Its molecule is composed of two main moieties: mercaptopurine and imidazole derivative. It is an immunosuppressive agent whose biological activity results from its in vivo mercaptolysis mediated by a nucleophilic attack on the C(5i) atom of imidazole ring of the azathioprine molecule. Solvation model SM5.4 with the PM3 Hamiltonian have been applied to model the reaction of azathioprine with cysteine. The employed quantum mechanical method shed new light on the mechanism of the reaction of azathioprine with cysteine in aqueous solution. The obtained results indicated that the first step in the reaction most likely involves the nucleophilic attack of the COO(-) of cysteine on the C(5i) atom of the imidazole ring of azathioprine, followed by a subsequent intramolecular attack of the SH group of the cysteine residue. It was shown that biogenic thiols such as glutathione or cysteine facilitate the first and crucial step of azathioprine metabolism, due to the presence of COO(-), SH, and NH(3)(+) groups in their molecules.

Metabolites of mercaptopurine in red blood cells: a relationship between 6-thioguanine nucleotides and 6-methylmercaptopurine metabolite concentrations in children with lymphoblastic leukemia.

Intracellular concentrations of 6-mercaptopurine metabolites, i.e. of 6-thioguanine nucleotides (6-TGN) and of 6-methylmercaptopurine metabolites (6-mMP) were analysed in red blood cells (RBC) of 19 children with acute lymphoblastic leukemia (ALL), the subjects of a maintenance chemotherapy of their first remission. Interpatient variations in concentrations of both metabolites were high; concentrations of 6-TGN varied from <60 to 833 pmol/8x10(8) RBC (median value, 144) and those of 6-mMP metabolites from <150 to 19000 pmol/8x10(8) RBC (median value, 3250). In two patients, 6-TGN appeared at concentrations below the limits of assay sensitivity, and 6-mMP metabolites were not detected. In another child the concentrations of both metabolites were at the limit of the assay sensitivity. In three other children the concentrations of both metabolites were below the median value of the group. In the analysed group of children, significant correlations were found between the white cell count (WBC) and RBC 6-TGN (r(s)=-0.72, P<0. 005) as well as between the neutrophil count and RBC 6-TGN (r(s)=-0.60, P<0.01). No significant correlation was found between the concentrations of 6-TGN and 6-mMP metabolites. The monitoring of concentrations of 6-TGN as well as of 6-mMP metabolites allows an early identification of patients who are at an increased risk of the disease relapse as indicated by the low levels of either 6-TGN itself or of its two metabolites.

Determination of 6-thioguanine and 6-methylmercaptopurine metabolites in renal transplantation recipients and patients with glomerulonephritis treated with azathioprine.

The metabolism of azathioprine (AZA) was studied by monitoring the concentrations of red blood cell (RBC) 6-thioguanine nucleotides (6-TGN) and of 6-methylmercaptopurine metabolites (6-mMP) in 27 renal transplantation recipients and in 10 patient subjects with glomerulonephritis (GN). Concentrations of 6-TGNs and 6-mMP metabolites were measured using high-performance liquid chromatography (HPLC). Six patients from the group of renal transplantation recipients were also administered allopurinol. Median values of RBC 6-TGN and of 6-mMP metabolites concentrations in 21 renal transplantation recipients (without allopurinol) were 122 pmol/8x10(8) RBCs (range, <60-298) and 280 pmol/8x10(8) RBC (range, <150-1330), respectively; there was no correlation between concentrations of 6-TGN and of 6-mMP metabolites. The group of 21 renal transplantation recipients received different AZA doses (100 or 50 mg/d) related to clinical symptoms of AZA intolerance. The median values of 6-TGN concentrations in these subgroups were 131 and 122 pmol/8x10(8) RBCs and were not significantly different. Median values of 6-TGN concentrations in patients given allopurinol were significantly higher, despite AZA dose reduction, compared with the group without allopurinol and were equal to 363 and 122 pmol/8x10(8) RBC, p < 0.004, respectively. No significant differences were found between the concentrations of 6-mMP metabolites in either group. In the group of renal transplantation recipients, a significant correlation between white blood cell (WBC) count and 6-TGN concentration was established (r(s) = -0.59, p < 0.005). In the group of GN patients, the median values of 6-TGN and of 6-mMP metabolites concentrations were 108 pmol/8x10(8) RBCs (range, 0-297) and 420 pmol/8x10(8) RBC (range, 0-1440), respectively. There were no significant correlations between either the WBC count and 6-TGN concentrations or between 6-TGN concentrations and 6-mMP metabolites. We expect the results of our study to provide indications for better individualization of AZA therapy.

Tobacco veinal necrosis determinants are unlikely to be located within the 5' and 3' terminal sequences of the potato virus Y genome.

Three potato virus Y isolates, representatives of distinct PVY groups, identified in potato fields in northern Poland were submitted to biological and molecular analysis. Phenotypically, two isolates, PVYN-Ny and PVYN-Wi, belong to the necrotic strain and the third one (PVYO-LW) to the common strain. PVYN-Wi, however, did not react with monoclonal antibodies directed against the necrotic strain isolates which recognise PVYN-Ny. To characterise the isolates, coat protein genes were sequenced and compared with sequences from databases. The necrotic PVYN-Wi isolate showed 99% amino acid homology with the common one-PVYO-LW and significantly differed from the second necrotic isolate (PVYN-Ny). Sequence based homology matrix and phylogenetic analysis lead to classification of PVYN-Ny into group I, encompassing solely necrotic strain isolates, whereas PVYN-Wi falls into a phenotypically heterogeneous group II. The sequence analysis allowed for identification of putative group I-specific epitopes. 3'NTR (non-translated region) sequences were identical for PVYN-Wi and PVYO-LW. The 5'NTR, P1 gene, coat protein gene and 3'NTR sequences of the common (PVYO-LW) and the necrotic (PVYN-Wi) isolates are 99-100% homologous. This suggests that tobacco veinal necrosis determinants are located outside the 3' and 5' terminal sequences of the PVY genome.

The importance of the phenyl-tropolone 'aS' configuration in colchicine's binding to tubulin.

Measuring ellipticities of (+/-)-colchicine and (+/-)-deacetamidocolchicine in the presence of tubulin afforded net positive CD bands with maxima at 340 nm resulting from reduction of the negative ellipticities upon binding of (-) enantiomers to the protein. Results of optical studies together with earlier NMR conformational analysis of these molecules substantiate the hypothesis that colchicinoids bind to tubulin with the phenyl-tropolone moiety in the 'aS' configuration. Natural colchicine which binds to tubulin, therefore, should be referred to as (-)-(aS,7S)-colchicine.

Kinetics of azathioprine metabolism in fresh human blood.

Azathioprine (AZA) is transformed in the whole fresh human blood in vitro to 6-mercaptopurine (6-MP). The rate of the above reaction was followed as a function of time at 25, 30 and 37 degrees C. Pseudo-first-order rate constants and thermodynamic parameters were calculated. The statistical evaluation of the parameters calculated was provided. Half-life time of 6-MP formation in blood from AZA at e.g. 37 degrees C was equal to 28.9 +/- 2.8 min.

Pharmacokinetics of elimination of 6-mercaptopurine in the urine of children with acute lymphoblastic leukemia.

6-Mercaptopurine (6-MP)-induced sodium azide--iodine reaction was adapted for determination of 6-MP in urine of four children treated with single oral doses of the drug in tablets. Open one-compartment body model was assumed, and first-order elimination rate constants (K) and biological half-life times (t0,5) were calculated, and their precision was determined by statistical treatment.