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The measurement of blood concentration of immunosuppressive drugs is strongly recommended because of the narrow therapeutic range. An important aspect in the therapeutic monitoring of a drug is its possible degradation. This paper is aimed at investigating the stability of two widely-used immunosuppressants, sirolimus and everolimus. Short (storage at 30 degrees C for 3 or 7 days) and long term (storage at -20 degrees C for 0-90 days with a single freeze-thaw cycle) stability of sirolimus and everolimus in whole blood samples from kidney transplant patients were examined by using MEIA and FPIA. Sirolimus and everolimus samples stored at 30 degrees C in light for up to a week showed a decrement in concentration of 5.2 percent and 6.1 percent, respectively. Our findings on long term stability for both sirolimus and everolimus highlight the possibility of storing samples at -20 degrees C for up to 90 days, without the need to use lower storage temperatures. The results have important implications for patients living far from laboratories where drug concentration is measured or when the storing of blood samples is needed for pharmacokinetic studies.

The acronym mTOR defines a family of serine-threonine protein kinase called mammalian target of rapamycin. The major role of these kinases in the cell is to merge extracellular instructions with information about cellular metabolic resources and to control the rate of anabolic and catabolic processes accordingly. In mammalian cells mTOR is present in two distinct heteromeric protein complexes commonly referred to as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), involved in the control of a wide variety of cellular processes. It has been recently reported that compounds acting modulating mTOR activity, beside mediating the well recognized processes exploited in the anticancer and immunosuppressant effects, are provided with neuroprotective properties. In fact, mTOR is involved in the mechanism of PI3K/Akt-induced upregulation of glutamate transporter 1, GLT1, that is linked to several neuronal disorders such as stroke, Alzheimer's disease, and amyotrophic lateral sclerosis. Furthermore, in adult brain mTOR is crucial for numerous physiological processes such as synaptic plasticity, learning, memory, and brain control of food uptake. Moreover, the activation of mTOR pathway is involved in neuronal development, dendrite development and spine morphogenesis.

Immunosuppression has improved graft and recipient survival in transplantation but is associated with possible adverse effects including cardiovascular diseases. The impact of tacrolimus on the lipidic profile has been debated for several years. Twenty-nine kidney transplant recipients on tacrolimus treatment were monitored for six years, and multiple laboratory parameters investigating the lipid asset, as well as glucose profile, were carried out. Tacrolimus has been responsible for significant changes in plasma lipid concentrations only for the first six months, but not for the remaining time of observation. Similarly, in the same periods, glycemic imbalance was highlighted. The liver enzyme activity showed a modest derangement during the tacrolimus treatment, suggesting the presence of lipid accumulation in the liver. Fatty liver reversed in the long term follow-up. Tacrolimus, although it is not a completely safe option in the first months of the immunosuppressive protocols in organ transplanted recipients, still retains a certain role in the long-term post-transplantation immunosuppressive approach with high cardiovascular risks

Gastrointestinal (GI) adverse events in renal transplant patients are a common cause of mycophenolate mofetil (MMF) dose reductions, which result in an increased risk of graft rejection because of a low immunosuppression. This study investigated whether conversion from MMF to enteric-coated mycophenolate sodium (EC-MPS) in renal transplant patients with serious GI side-effects, alleviated these symptoms and allowed administration of higher doses of EC-MPS. Nineteen renal transplant patients with severe MMF-related GI side-effects underwent a progressive reduction in MMF dose until symptoms disappeared. At this point, 12-h AUC(MMF) was evaluated and patients were shifted to an equimolar dose of EC-MPS. The EC-MPS dose was then progressively increased until the highest recommended dose was reached or GI symptoms re-appeared. Four weeks post-conversion, AUC(EC-MPS) was determined. Conversion led to a mean increase in EC-MPS dose of 68% (P < 0.0001), with a corresponding rise in AUC(0-12) (60.5%, P < 0.0006) associated with significant benefits in terms of both quality of life (Kidney Transplant Questionnaire, P < 0.01) and GI symptoms (Gastrointestinal Symptom Rating Scale, P < 0.0001), using validated questionnaires. In five of 19 patients, the EC-MPS dose could not be increased because of the prompt insurgence of GI symptoms. Renal function and biochemical parameters remained stable post-conversion and no rejection episodes occurred. These findings suggest that, in selected patients, EC-MPS may be better tolerated than MMF when GI symptoms are particularly important and permits higher mycophenolic acid exposure, when required.

BACKGROUND AND AIMS: Systemic caffeine clearance is considered the gold standard for phenotyping cytochrome P450 1A2 in epidemiological studies, and has been recommended for the non-invasive assessment of liver function in chronic liver disease. Our aim was to find a valid, simple and reliable alternative to this method, and therefore focused our attention on the measurement of an unique salivary caffeine concentration, without drug exposure. METHODS: Our evaluation included 36 healthy controls, 47 patients with compensated liver cirrhosis of viral origin, and 48 obese and diabetic patients with cryptogenetic (likely metabolic) cirrhosis. All shared the same caffeine consumption habits (regular daily use of caffeinated beverages, mainly coffee). The total overnight salivary caffeine assessment (TOSCA) was determined by using a single-point concentration of salivary caffeine, after an overnight period of abstinence. RESULTS: Daily routine caffeine intake of our population was adequate for studying the TOSCA. This single-point concentration correlated well with caffeine clearance, measured by salivary concentrations of caffeine. Mean TOSCA in cirrhotic patients was significantly higher than in controls (p

Background. Enteric-coated mycophenolate sodium (ECMPS) and mycophenolate mofetil (MMF) are prodrugs of mycophenolic acid (MPA). Although many patients still receive MMF as an inosine monophosphate dehydrogenase inhibitor, EC-MPS could be considered a reliable alternative to MMF in the immunosuppressive protocols of kidney transplant recipients. MPA shows high pharmacokinetic variability and consequently a 12-h area under the curve (AUC0–12) should be used to guide the therapeutic dosage. However, patient compliance and economic costs make MPA AUC0–12 an unpractical approach. Limited sampling strategies or predictive systemic drug exposure equation models based on limited sampling times are available only for MMF but lack for EC-MPS. Methods. The present study enrolled 26 kidney transplant recipients receiving EC-MPS as part of their immunosuppressive therapy. Twenty-six full MPA AUC0–12 were performed. By using multiple stepwise regression analysis, we obtained several predictive equations of MPA systemic exposure in this group of patients. The value of the selected equations was tested in a subsequently enrolled group of 26 kidney transplant recipients. Results. The best equations obtained in the first group of patients were the following: 22.906 1 3.880� C0 1 1.117� C1 1 7.527� C8 (r ¼ 0.901) and 35.064 13.784� C0 1 1.002� C1 1 1.192� C2 (r ¼ 0.846). These equation models showed an optimal agreement between the full AUCs and estimated AUCs by using the validation group of patients. Conclusions. Limited sampling strategies are useful for MPA AUC0–12 estimation in patients receiving EC-MPS and cyclosporine. The choice of one or the other equation model depends on the pharmacokinetic characteristics of the patients, in particular the potential presence of enterohepatic recirculation.

Cigarette smoking condensate (CSC) contains oxidant compounds able to generate superoxide. The aim of the present study was to investigate the effect of the exposure to CSC on: (1) free radical production, (2) the gene expression of the antioxidant enzymes Cu-Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2), Glutathione Peroxidase (GPx), and catalase (CAT), and (3) cell survival in human neuroblastoma SH-SY5Y cells. The results showed that exposure (24 h) to different concentrations (10-150 mu g/ml) of CSC caused a dose dependent cell injury that was coupled to the maximal increase of free radical production. These events were prevented by the addition to the incubation medium of the scavenger Vitamin E (50 mu M). Furthermore, CSC exposure caused a reduction of the gene expression of the antioxidant enzymes SOD1, SOD2, GPx, and CAT that was counteracted by Vitamin E (50 mu M). These results suggest that CSC exposure can induce a free radical overcharge that may be responsible for the inhibition of antioxidant enzymes expression and cell injury in SH-SY5Y human neuroblastoma cells. In fact the scavenger vitamin E can block both cell injury and inhibition of SOD1, SOD2, GPx, and CAT induced by CSC exposure.

Mammalian Target Of Rapamycin (mTOR) inhibitors represent a new class of immunosuppressant drugs extensively used for the prevention and the treatment of graft rejection in organ transplant recipients. Their current use is due to referred low nephrotoxic effects, particularly important in kidney transplanted and/or patients with renal failure. The most representative drugs of such class are Sirolimus (Siro) and Everolimus (Rad). Both drugs show a narrow therapeutic window, therefore, monitoring of whole-blood drug levels is recommended in order to optimize the therapy. Among the available assays, Liquid Chromatography coupled with UltraViolet or Electrospray Tandem Mass Spectrometry methods (LC/UV or LC/ESI-MSMS) are the most accurate and specific ones. A reliable alternative is represented by immunoassays, which offer the opportunity to minimize sample pre-treatment, thus reducing the time between drawing blood sample and measuring the drug concentration, an important aspect in high-throughput analyses. Despite this, a limitation in the use of immunoassays for therapeutic drug monitoring is the lower specifity compared with the chromatographic methods when analysing structurally-related drugs. New insights to optimize mTOR inhibitors regimens seem to be offered by the evaluation of CYP450 3A activity by using the probe drug approach. To such purpose, there are a number of major probe drugs used for in vivo studies including: midazolam, cortisol, lidocaine, nifedipine, dextromethorphan, erythromycin, dapsone and alfentanil. The aim of the present paper is to report the most recent knowledge concerning this issue, supplying a critical and comprehensive review for whom are involved both in the clinical and analytical areas.

Infection occurs frequently in the organ transplant recipients during the post-transplant period because of immunosuppression. Therefore, prophylactic antimicrobial agents are often used. The azole antifungals, widely prescribed prophylactically, are known to have many drug-drug interactions. This report presents a case of drug-drug interaction between voriconazole and tacrolimus in a kidney transplant recipient. Voriconazole treatment led to a dramatic increase in tacrolimus concentration that required its discontinuation in spite of the manufacturer's guidelines that recommend a reduction of tacrolimus dosage by one-third. The present drug-drug interaction can be attributed to a strong inhibitory effect on cytochrome P450-3A4 activity by voriconazole. When voriconazole and tacrolimus are coadministered, close monitoring of tacrolimus blood levels is recommended as the rule-of-thumb reduction of tacrolimus dose by one-third may not be satisfactory.

This study investigates the potential pharmacokinetic interactions between an antimicrobial agent, moxifloxacin, and 2 immunosuppressant drugs, cyclosporine and tacrolimus, in kidney transplant recipients. Twenty-two kidney transplant patients needing antibiotic therapy for urinary tract infections are enrolled. Eleven patients are under cyclosporine treatment and the other 11 patients are under tacrolimus treatment. Because the urinary tract infections are caused by gram-negative aerobes sensitive to moxifloxacin, this antibiotic is administered by oral route at a dose of 400 mg/d for 1 week; in each patient pharmacokinetic studies are carried out before and at the seventh day of therapy. For both immunosuppressors, none of the pharmacokinetic parameters investigated show statistically significant differences between values obtained before and during treatment with moxifloxacin. In fact, the concentration-time profiles of monoclonal cyclosporine, polyclonal cyclosporine, and tacrolimus are not significantly different before and during the antimicrobial therapy. The results of the present study rule out interference of moxifloxacin with both cyclosporine and tacrolimus kinetics and indicate that the concomitant administration of the fluoroquinolone and cyclosporine or tacrolimus does not require modifications of the dosages of 2 immunosuppressant drugs.

Aim: Liver damage due to facultative hepatotoxins is scarcely foreseeable. We evaluated the prevalence of acute drug-induced liver injury (DILI) in a specific setting, assessing eventual interactions with pre-existing hepatic illnesses. Methods: The research was carried out in an Italian tertiary care hospital, by analyzing 248 patients with non-advanced liver disease, divided into two well-matched groups: 174 patients (median age 53, 94 females) with hepatitis C virus-related chronic hepatitis; and 74 (median age 55, 39 females) with non-alcoholic fatty liver disease (NAFLD). Results: Six patients (2.4% of the whole population) belonging to the NAFLD group (χ2-test, P = 0.004) suffered from acute hepatoxicity related to the following drugs, that is antihypertensive, acting on platelet aggregation, antimicrobial, non-steroidal anti-inflammatory and proton pump inhibitor. The NAFLD presence was an independent risk factor in determining drug-related acute hepatitis, with an odds ratio of 3.95 (95% confidence intervals: 11.48–1.35). Central obesity was relevant in every patient with acute toxicity. Alcohol consumption and drug association did not influence the acute drug-induced liver damage. Conclusion: NAFLD conveys a nearly fourfold increase of DILI risk in obese middle-aged patients. NAFLD, characterized by mitochondrial dysfunction, could predispose to drug-induced hepatotoxicity that probably shares the same pathophysiological mechanism.

The possible cross-reactivity of immunoassays with structurally-related drugs was investigated. Innofluor® Certican® (FPIA) calibrators were measured by using IMx® Sirolimus assay (MEIA) and MEIA Sirolimus calibrators were analysed by using FPIA Certican® assay. Drug concentrations were measured in 95 and 100 samples from renal transplanted patients (RTP) on sirolimus or everolimus treatment by using immunoassays and LC/ESI-MSMS. A high cross-reactivity was found both for MEIA and FPIA. High correlation degrees, confirmed by the Bland-Altman and the Eksborg tests, were found between drug concentrations measured in real samples by both immunoassays (r=0.909 and r=0.970, respectively). LC/ESI-MSMS analysis of samples containing sirolimus showed no positivity for everolimus. Similarly, samples from patients on treatment with everolimus resulted negative as far as regards sirolimus. MEIA and FPIA could be considered mutually reliable and accurate alternatives for the specific-drug immunoassay. It should be noticed that in patients switching from one drug to the other unreal overestimation of the blood levels of the current administered immunosuppressant can occur.