Pierre Wallemacq

Pharmacokinetics of tacrolimus (FK506) in paediatric liver transplant recipients

SummaryThe pharmacokinetics of intravenous and oral tacrolimus was assessed in paediatric liver transplant patients at two centers in Europe. Sixteen patients, age 0.7 to 13 years, participated in the study; 12 patients were evaluable for intravenous pharmacokinetics, and 16 for oral. Intravenous tacrolimus was given as a continuous 24 h infusion (mean 0.037±0.013 mg/kg/day), and oral tacrolimus was given in 2 doses per day (mean 0.152±0.015 mg/kg). Whole blood samples for the intravenous pharmacokinetic profile were taken before initiation of the first infusion, 4, 8, 12 and 24 h post-infusion, and every 24 h thereafter until intravenous administration was discontinued. During the 12 h wash-out period between intravenous and oral administration, samples were taken every 3 h. Samples for the oral pharmacokinetic profile were taken immediately before the first oral dose and 0.5, 0.75, 1, 2, 2.5, 3, 4, 6, 8, 10 and 12 h post-administration. Non-compartmental procedures were used to characterise the pharmacokinetic parameters. Mean estimates for clearance and terminal half-life were 2.3±1.2 ml/min/kg and 11.5±3.8 h, respectively, following intravenous tacrolimus. The mean bioavailability of oral tacrolimus was 25±20%. A strong correlation was observed between AUC and trough whole blood levels of tacrolimus (r=0.90). The clearance was approximately 2-fold higher than that previously observed in adults; this could explain the higher dosage requirements in children.

Hyperhomocysteinemia and venous thromboembolism: a risk factor more prevalent in the elderly and in idiopathic cases.

Fasting plasma homocysteine level and the related clinical findings were analysed in 240 consecutive patients with venous thromboembolism. Hyperhomocysteinemia, defined as a plasma level above 20 micromol/l (corresponding to the percentile 95th in the controls), was present in 11.2% of the patients. Plasma homocysteine level was similar in patients presenting with either deep venous thrombosis, pulmonary embolism or both conditions. It was significantly higher in patients with primary (unprovoked) VTE than in patients with secondary disease (associated with at least one risk factor): 12.3 vs. 9.55 micromol/l (p < 0.005). Mean homocysteine was higher in male than in female patients (14.51 vs. 12.9 micromol/l, p < 0.05) and increased significantly with age. Hyperhomocysteinemia was more frequent in patients with relapsing disease (14 of 76, 18.4%) than in those presenting with a single episode (13 of 164, 7.9%) (p = 0.034). Furthermore, hyperhomocysteinemia was correlated with reduced protein C level (p = 0.013). In a multivariate analysis, two factors were significantly associated with hyperhomocysteinemia: older age (p < 0.0001) and idiopathic occurrence (p < 0.02). Since the frequency of homozygous MTHFR thermolabile variant was rather similar in patients and controls, testing for C677T mutation was not helpful in screening VTE patients. However, the homozygous mutation was significantly more prevalent among hyperhomocysteinemia patients, confirming its role in the genesis of hyperhomocysteinemia. According to its prevalence, to the putative role in venous and arterial disease and the availability of an effective and low-cost corrective therapy, hyperhomocysteinemia deserves interest, especially in the elderly and in the patients with idiopathic VTE disease.

Glycosomal ABC transporters of Trypanosoma brucei : characterisation of their expression, topology and substrate specificity.

Metabolism in trypanosomatids is compartmentalised with major pathways, notably glycolysis, present in peroxisome-like organelles called glycosomes. To date, little information is available about the transport of metabolites through the glycosomal membrane. Previously, three ATP-binding cassette (ABC) transporters, called GAT1-3 for Glycosomal ABC Transporters 1 to 3, have been identified in the glycosomal membrane of Trypanosoma brucei. Here we report that GAT1 and GAT3 are expressed both in bloodstream and procyclic form trypanosomes, whereas GAT2 is mainly or exclusively expressed in bloodstream-form cells. Protease protection experiments showed that the nucleotide-binding domain of GAT1 and GAT3 is exposed to the cytosol, indicating that these transporters mediate the ATP-dependent uptake of solutes from the cytosol into the glycosomal lumen. Depletion of GAT1 and GAT3 by RNA interference in procyclic cells grown in glucose-containing medium did not affect growth. Surprisingly, GAT1 depletion enhanced the expression of the very different GAT3 protein. Expression knockdown of GAT1, but not GAT3, in procyclic cells cultured in glucose-free medium was lethal. Depletion of GAT1 in glucose-grown procyclic cells caused a modification of the total cellular fatty-acid composition. No or only minor changes were observed in the levels of most fatty acids, including oleate (C18:1), nevertheless the linoleate (C18:2) abundance was significantly increased upon GAT1 silencing. Furthermore, glycosomes purified from procyclic wild-type cells incorporate oleoyl-CoA in a concentration- and ATP-dependent manner, whilst this incorporation was severely reduced in glycosomes from cells in which GAT1 levels had been decreased. Together, these results strongly suggest that GAT1 serves to transport primarily oleoyl-CoA, but possibly also other fatty acids, from the cytosol into the glycosomal lumen and that its depletion results in a cellular linoleate accumulation, probably due to the presence of an active oleate desaturase. The role of intraglycosomal oleoyl-CoA and its essentiality when the trypanosomes are grown in the absence of glucose, are discussed.

Is plasma and urine neutrophil gelatinase-associated lipocalin (NGAL) determination in donors and recipients predictive of renal function after kidney transplantation?

OBJECTIVESnDelayed graft function (DGF) is still a major issue in kidney transplantation. Plasma and urine neutrophil gelatinase-associated lipocalin (NGAL) were evaluated in a population of kidney donors and recipients to investigate their performance to predict early renal function.nnnDESIGN AND METHODSnPlasma (pNGAL) and urine (uNGAL) samples were obtained from donors before organ procurement, and from recipients before transplantation, and then 6, 24 and 48h after the procedure. Kidney transplantations were performed from both living donors (LDs, n=17) and deceased donors (DDs, n=80). Recovery of renal function was evaluated as the time to reach serum creatinine <2mg/l or glomerular filtration rate (GFR)>40mL/min. Logistic regression was used to assess the ability of different variables to predict the occurrence of DGF.nnnRESULTSnPlasma NGAL levels were significantly lower in LDs than in DDs. No episodes of DGF were recorded among LD kidney recipients, but DGF was observed in 25% of patients in the DD group. There was no correlation between donor pNGAL and uNGAL values and the occurrence of post-transplant DGF. Recipient pNGAL performed better than uNGAL in terms of predicting DGF occurrence. Donor pNGAL and uNGAL values did not influence the time needed to reach serum creatinine levels of <2mg/dl after transplantation. When time to reach eGFR of >40mL/min is considered, only donor uNGAL seems to be a predictor of graft function recovery. However, recipient pNGAL values obtained 24 and 48h after transplantation, but not uNGAL values, were found to be a significant predictor of graft function recovery.nnnCONCLUSIONSnPlasma NGAL level determination in recipients, but not in donors, proved to be a reliable predictor of DGF occurrence and renal function restoration, but too long for an interval to be able to compete with biomarkers currently used in clinical practice.

Genetic and chemical evaluation of Trypanosoma brucei oleate desaturase as a candidate drug target.

Background Trypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites. Methodology Cultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied. Principal Findings Growth of PCF T. brucei was totally inhibited by 100 µM of 12-TS and 13-TS, with EC50 values of 40±2 and 30±2 µM, respectively. The BSF was more sensitive, with EC50 values of 7±3 and 2±1 µM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itself and not only fluidity could be essential for normal membrane functionality. A similar deleterious effect was found after RNAi in BSF, even with a mere 8% reduction of OD activity, indicating that its full activity is essential. Conclusions/Significance As OD is essential for trypanosomes and is not present in mammalian cells, it is a promising target for chemotherapy of African trypanosomiasis.

Life-Threatening Dextromethorphan Intoxication Associated with Interaction with Amitriptyline in a Poor CYP2D6 Metabolizer: A Single Case Re-exposure Study.

We report a case of life-threatening intoxication and a controlled re-exposure study to dextromethorphan. A 60-year-old man developed postsurgical neuropathic cervical pain treated by hydromorphone, gabapentin, clonazepam, and amitriptyline. He received a dextromethorphan preparation for a catarrhal syndrome. Two days later, he was admitted into an emergency department in a profound coma. Thirty-six hours later, after withdrawal of all drugs, the situation normalized. A genotyping for UDP-glucuronyltransferase 1A1 and CYP2D6 was followed by a re-exposure study. During the three days, vital parameters and side effects of drugs were prospectively recorded. The second day, dextromethorphan was introduced. No significant impairment in parameters nor influence on analgesic efficacy were noted. Dextromethorphan concentrations suggested an accumulation without reaching any steady state. Somnolence was noted for plasma concentrations around 100ng/mL. The CYP2D6*4 variant leading to a poor metabolizer phenotype was found. Moreover, this phenotype was potentially aggravated by amitriptyline intake. This study allowed the identification and the confirmation of the cause of the coma. In conclusion, it is probably wise to recommend avoiding dextromethorphan in patients taking tricyclic antidepressants or another inhibitor of CYP2D6. Drug-drug interactions are probably underdiagnosed and underreported, and drugs considered as safe may induce serious complications.

Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. bruceis survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC(50)) of PCF was 1.0 ± 0.2 μM for Isoxyl and 5 ± 2 μM for 10-TS, whereas BSF appeared more susceptible with EC(50) values 0.10 ± 0.03 μM (Isoxyl) and 1.0 ± 0.6 μM (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents survival.

Clinically unexpected cyclosporine levels using the ACMIA method on the RXL dimension analyser

Safe use of cyclosporine (CsA) in solid organ transplantation relies on regular whole-blood drug monitoring. Several promising immmunoassays, e.g. the antibody-conjugated magnetic immunoassay (ACMIA) method, were developed and commercialized during recent years to compete with liquid chromatography coupled to tandem mass spectrometry, which remains the reference method but is labor-intensive. We describe the occurrence of interference in the monitoring of whole-blood CsA after transplantation when using the ACMIA method and discuss the potential mechanisms involved in such interference. Clinically unexpected results of whole-blood CsA require immediate reassessment by another technique to prevent the risk of CsA underdosage and graft rejection.

Extended release tacrolimus and antiretroviral therapy in a renal transplant recipient: so extended!

HIV-infected patients are at risk of developing end-stage renal disease. In selected patients with controlled disease, kidney transplantation (KT) has been recognized during last years as a safe and effective treatment [1]. Highly active antiretroviral therapy (HAART) drugs can interfere with immunosuppressive agents, and this has to be taken into account when adjusting immunosuppression regimen. We report the case of an HIV1-infected patient who developed a severe interaction between extended-release (ER) tacrolimus and lopinavir boosted by ritonavir (lopinavir/r) shortly after KT. This resulted in sustained tacrolimus overdosage, surprisingly well tolerated by the renal graft. Careful therapeutic drug monitoring and pharmacokinetics follow-up, led to a drastic reduction of ER tacrolimus dosage administered on a once weekly basis. A 45-year-old man with end-stage renal disease secondary to cidofovir toxicity, given for CMV retinitis, underwent KT from a heart-beating deceased-donor after 5 years on haemodialysis. His past medical history included HIV-1 infection diagnosed 22 years earlier, without hepatitis B or C virus coinfection. CD4 cell count and viral load were 516/ll and below 50 copies/ll, respectively, 3 months before KT. Recovery of diuresis was immediate following KT and serum creatinine rapidly decreased from 9.97 mg/dl at admission to 1.06 mg/dl at day 7. Immunosuppressive regimen included ER tacrolimus 0.2 mg/kg o.d (Advagraf , Astellas Pharma Europe, Staines, UK), mycophenolate mofetil 500 mg b.i.d. (Cellcept , Roche, Basel, Switzerland) and prednisolone. At the time of KT, HAART included lopinavir/r 600/150 mg b.i.d., lamivudine 300 mg o.d., raltegravir 400 mg b.i.d. and nevirapine 200 mg b.i.d. Acetylsalicylic acid and prophylactic trimethoprime–sulfamethoxazole 800–160 mg three times a week were added in the early post-operative period. At day 3 post-KT, tacrolimus dosage, performed with Chemiluminescent Microparticle Immuno-Assay on the Architect analyser (Abbott Diagn., Chicago, IL, USA), had to be reduced because of trough levels >30 ng/ ml and completely withdrawn at day 7 (Fig. 1). On day 15, the patient developed fungal and herpetic oesophagitis treated with oral fluconazole 50 mg o.d. (for 10 days), aciclovir 800 mg b.i.d. and pantoprazole 40 mg o.d. Despite tacrolimus discontinuation, whole blood levels remained >30 ng/ml until day 30, with a value of 47.4 ng/ml obtained by dilution on day 24. On day 47, 40 days after drug discontinuation, trough level was

Quantification of serum free light chain kappa and lambda by the SPAPLUS analyser.

OBJECTIVEnClinical assessment of the SPAPLUS® system for the determination of the serum free light chains kappa (κ FLC) and lambda (λ FLC) compared to the BNII®.nnnDESIGN AND METHODSn126 serum specimens from our routine activity were analysed on two different analysers: the BNII® (immunonephelometry, Siemens) and the SPAPLUS® (turbidimetry, Binding Site). We compared the absolute values of the serum κ FLC and λ FLC, as well as the FLC κ/λ ratio on both analysers. These results were further evaluated together with the clinical history of the patients.nnnRESULTSnRegression analysis between the BNII® and the SPAPLUS® for κ FLC and λ FLC did not display any significant differences between both methods in the normal and pathological ranges. Nevertheless, some differences have been observed for some patients in the absolute value of the involved light chain, with potential clinical implications.nnnCONCLUSIONnThe results show overall good concordance between both methods. However, it is recommended that the monitoring of patients affected by monoclonal gammapathies by measuring FLC, be performed in the same laboratory and by the same method. Moreover, the FLC results should always be interpreted together with other laboratory tests taking into account the patients diagnosis.