Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application.

The World Anti-Doping Agency (WADA) has recently implemented dried blood spots (DBSs) as a matrix for doping control. However, specifications regarding the analysis of the class of prohibited substances called erythropoietin (EPO) receptor agonists (ERAs) from DBSs are not yet described. The aim of this study was to find optimal conditions (sample volume and storage) to sensitively detect endogenous erythropoietin (hEPO) and prohibited ERAs from DBSs and compare detection limits to WADA-stipulated minimum required performance levels (MRPLs) for ERAs in serum/plasma samples. Venous whole blood was spotted onto Whatman 903 DBS cards with primarily 60 µl of blood, but various volumes from 20 to75 µl were tested. All samples were immunopurified with MAIIA EPO Purification Gel kit (EPGK) and analysed with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western blot. Sixty-microliter DBSs allowed the detection of the four main ERAs (BRP, NESP, CERA and EPO-Fc) at concentrations close to WADA's MRPLs described for 500 µl of serum/plasma. Different storage temperatures, from -20°C to 37°C, were evaluated and did not affect ERA detection. A comparison of the detection of endogenous EPO from the different anti-doping matrices (urine, serum and DBSs produced from upper arm capillary blood) from five participants for 6 weeks was performed. Endogenous EPO extracted from DBSs showed intra-individual variations in male and female subjects, but less than in urine. Doping controls would benefit from the stability of ERAs on DBSs: It can be a complementary matrix for ERA analysis, particularly in the absence of EPO signals in urine.

Role of pharmacogenetics in rifampicin pharmacokinetics and the potential effect on TB-rifampicin sensitivity among Ugandan patients.

BACKGROUND: Suboptimal anti-TB drugs exposure may cause multidrug-resistant TB. The role of African predominant SLCO1B1 variant alleles on rifampicin pharmacokinetics and the subsequent effect on the occurrence of Mycobacterium tuberculosis-rifampicin sensitivity needs to be defined. We describe the rifampicin population pharmacokinetics profile and investigate the relevance of SLCO1B1 genotypes to rifampicin pharmacokinetics and rifampicin-TB sensitivity status. METHODS: Fifty patients with TB (n=25 with rifampicin-resistant TB and n=25 with rifampicin-susceptible TB) were genotyped for SLOC1B1 rs4149032 (g.38664C>T), SLOC1B1*1B (c.388A>G) and SLOC1B1*5 (c.521 T>C). Steady state plasma rifampicin levels were determined among patients infected with rifampicin-sensitive TB. Data were analysed using NONMEM to estimate population rifampicin pharmacokinetics as well as the effect of SLOC1B1 genotypes on rifampicin pharmacokinetics and on rifampicin-TB sensitivity status. RESULTS: Overall allele frequencies of SLOC1B1 rs4149032, *1B and *5 were 0.66, 0.90 and 0.01, respectively. Median (IQR) Cmax and Tmax were 10.2 (8.1-12.5) mg/L and 1.7 (1.125-2.218) h, respectively. Twenty-four percent of patients exhibited Cmax below the recommended 8-24 mg/L range. SLOC1B1 genotypes, gender and age did not influence rifampicin pharmacokinetics or TB-rifampicin sensitivity. CONCLUSIONS: Although SLOC1B1 genotype, age and gender do not influence either rifampicin pharmacokinetics or rifampicin-TB sensitivity status, one in every four Ugandan TB patients achieve subtherapeutic plasma rifampicin concentrations.

Intra- and inter- individual rivaroxaban concentrations and potential bleeding risk in patients with atrial fibrillation.

BACKGROUND: Routine laboratory monitoring of rivaroxaban and dose adjustment relating to exposure is currently not recommended. However, in certain clinical situations, assessment of rivaroxaban levels is desirable. OBJECTIVES: To examine inter- and intra-subject plasma rivaroxaban variability in patients with atrial fibrillation (AF) and to correlate these results to clinical outcomes. PATIENTS/METHODS: We included 60 patients with AF treated with rivaroxaban: half on 20 mg daily (R20) and half on 15 mg daily (R15). Three trough and peak blood samples were collected with an interval of 6-8 weeks apart. Plasma rivaroxaban concentration was measured directly by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) and indirectly by anti-Xa for rivaroxaban, prothrombin time (PT), and activated partial thromboplastin time (APTT). RESULTS: Patients on R15 were older (76 ± 6 vs 71 ± 6 years), had lower creatinine clearance (60 ± 26 vs 99 ± 32 mL/min), higher CHADS2 (2.5 ± 1.2 vs 1.8 ± 1.3), all p < 0.01, but had similar rivaroxaban concentrations in trough samples to patients on R20. There was no significant intra-individual variability for trough or peak rivaroxaban concentration assessed by LC-MS/MS, anti-Xa, or PT. Trough rivaroxaban levels determined by LC-MS/MS (48 ± 30 vs 34 ± 26, p = 0.02) and anti-Xa, but not with PT and APTT, were higher in patients with bleeding than in patients without it. CONCLUSIONS: There is a pronounced inter-, but not intra-individual variability in the rivaroxaban trough levels in patients with AF. Assessment of trough rivaroxaban concentration with LC-MS/MS or anti-Xa, but not with APTT or PT, may help to identify patients at increased risk of bleeding.

Oil-Fortified Maize Porridge Increases Absorption of Lumefantrine in Children with Uncomplicated Falciparum Malaria.

Artemether-lumefantrine (AL) is a first-line treatment for uncomplicated malaria. Absorption of lumefantrine (LUM) is fat dependent, and in children, intake is recommended with milk. We investigated whether oil-fortified maize porridge can be an alternative when milk is not available. In an open-label pharmacokinetic study, Ugandan children <5 years with uncomplicated Plasmodium falciparum malaria were randomized to receive standard six-dose AL treatment [one tablet (20 mgA/120 mg LUM) if <15 kg and two tablets if >15 kg] with milk (A) or maize porridge plus oil (B). Parametric two-sample t-test was used to compare relative oral LUM bioavailability. The primary end-point was LUM exposure till 8 hr after the first dose (AUC0-8 hr ). Secondary outcome included day 7 concentrations (d7LUM ), LUM exposure between days 7 and 28 (AUCd7-28 ) and day 28 PCR-adjusted parasitological response. Evaluable children (n = 33) included 16 in arm A and 17 in arm B. The AUC0-8 hr was comparable between A and B [geometric mean (95% CI): 6.01 (3.26-11.1) versus 6.26 (4.5-8.43) hr*µg/mL, p = 0.9]. Less interindividual variability in AUC0-8 hr was observed in B (p = 0.01), but d7LUM and AUCd7-28 were comparable. Children receiving two tablets had significantly higher exposure than those receiving one tablet [median d7LUM (505 versus 289 ng/mL, p = 0.02) and AUCd7-28 (108 versus 41 hr*µg/mL, p = 0.006)]. One parasitological failure (d28 recrudescence) was observed. Our findings suggest that oil-fortified maize porridge can be an alternative to milk in augmenting absorption of LUM. The lower LUM exposure observed in children dosed with one AL tablet needs further attention.

Pronounced Interindividual But Not Intraindividual Variation in Tamoxifen and Metabolite Levels in Plasma During Adjuvant Treatment of Women With Early Breast Cancer.

BACKGROUND: Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and estrogen receptor-positive tumors. More than 20% of patients relapse despite treatment. The drug is usually dosed 20 mg/d irrespective of interindividual variation in drug clearance. To study interindividual and intraindividual variation in plasma levels we measured tamoxifen and metabolite levels in plasma on 2 occasions, with at least 4 weeks in between, of 39 women (19 premenopausal and 20 postmenopausal women) on adjuvant treatment (20 mg/d) of early breast cancer. METHODS: We used an ultra-performance liquid chromatography with a mass spectrometry detection method for identification and quantification of tamoxifen, N-desmethyltamoxifen, 4-OH-tamoxifen, and endoxifen. Follicle-stimulating hormone, luteinizing hormone, and estradiol levels were also measured. RESULTS: The plasma concentrations of tamoxifen and its metabolites showed a pronounced interindividual variation, whereas intraindividual concentrations were rather stable. Despite the same dosage, interindividual tamoxifen concentrations varied from 51 to 307 ng/mL (124 ± 57, mean ± SD) and endoxifen values showed a range from 3.2 to 19 ng/mL (10.4 ± 5.2, mean ± SD), that is, 6-fold variation for both. CONCLUSIONS: Large interindividual variation of tamoxifen and endoxifen with stable intraindividual levels, and too low levels of endoxifen in a considerable proportion of patients strongly support that therapeutic drug monitoring and individualized dosing could lead to optimal exposure and hopefully better outcome. A randomized outcome study between conventional dosing and therapeutic drug monitoring-guided dosing is needed to show whether this approach works.

Ganciclovir concentrations in the cerebral extracellular space after valganciclovir treatment; a case study.

Nearly all glioblastomas (GBMs), brain tumours with very poor prognosis, are infected with human cytomegalovirus (CMV). The anti-CMV drug valganciclovir (VGCV) has shown promise as a treatment option for patients with GBM, but its penetration into the central nervous system (CNS) is unknown. Here we describe a patient with GMB receiving VGCV in whom an intracerebral microdialysis catheter was implanted and ganciclovir (GCV) concentrations in brain extracellular fluid (BECF) and serum were monitored. GCV was rapidly absorbed. Cmax values (at 3 h) in serum and BECF were 19.6 and 10.2 µmol/L, T½ values were 3.2 and 4.5 h, and plasma and BECF AUC0-∞ values were 90.7 and 75.9 µmol h/L, respectively. Thus, VGCV treatment results in significant intracerebral levels of GCV that may be sufficient for therapeutic effects. Further studies of this drug in patients with GBM are warranted.

Posaconazole concentrations in human tissues after allogeneic stem cell transplantation.

Few data have been published regarding posaconazole tissue concentrations in humans. We analyzed tissue concentrations in biopsy specimens taken at autopsy from seven patients who received posaconazole prophylaxis because of graft-versus-host disease. The results were compared to plasma concentrations collected before death. Tissue concentrations suggestive of an accumulation of posaconazole were found in the heart, lung, liver, and kidney but not in the brain.