Impact of imipenem concentration in lung perfusate and tissue biopsy during clinical ex-vivo lung perfusion of high-risk lung donors.

BACKGROUND: Normothermic ex-vivo lung perfusion (EVLP) limits organ donor shortage by potentially using high-risk donor lungs. Microbial burden reduction has been demonstrated after EVLP using antibiotic prophylaxis with imipenem. However, no data have been published on the clinical consequences of the potential residual bacterial burden. METHODS: Imipenem concentration was measured every hour (T0 to T6) in the lung perfusate and at the end of EVLP (Tf) in biopsies. The antimicrobial activity of perfusate at T1 and Tf against E. coli and K. pneumoniae was evaluated. Lungs were distinguished: no bacterial species in recipients and donors (donor-/recipient-); bacterial species isolated from donors and not from recipients (donor+/recipient-); same bacterial species in both recipients and donors (donor+/recipient+). Interleukin 6 (IL-6) and IL-8 concentrations in lung perfusate, clinical pulmonary infection score (CPIS) and primary graft dysfunction (PGD) were evaluated. RESULTS: Imipenem concentration in perfusate decreased over time. T1 and Tf perfusates exhibited bactericidal activity against E. coli and K. pneumoniae. Overall, T1 perfusates yielded higher bactericidal titers (BTs) than Tf. The donor+/recipient+ group (26% of cases) had higher IL-6 and IL-8 in perfusate and higher CPIS. CONCLUSIONS: Recipients with the same bacterial species isolated in their donors had higher risk of pulmonary inflammation and early post-transplant pneumonia. Improvements in antimicrobial strategies during EVLP are warranted to minimize the consequences of donor associated respiratory infection.

Role of vitamin D pathway gene polymorphisms on rifampicin plasma and intracellular pharmacokinetics.

AIM: We retrospectively evaluate the pharmacogenetic role of single nucleotide polymorphisms involved in rifampicin transport (SLCO1B1, MDR1 and PXR genes) and vitamin D (VDR, CYP24A1 and CYP27B1 genes) metabolism and activity on drug plasma and intracellular concentrations. PATIENTS & METHODS: Rifampicin Cmax and Ctrough were measured at weeks 2 and 4 using Ultra-Performance Liquid Chromatography-tandem mass spectroscopy methods. Allelic discrimination was performed by real-time polymerase chain reaction. RESULTS: Twenty-four patients were enrolled. At week 2, OATP1B1 521TT and CYP27B1 +2838CC/CT considering plasma and BsmIAA for intraperipheral blood mononuclear cells Cmax, remained in regression analysis. Concerning week 4, TaqITC/CC and CYP24A1 22776CT/TT were retained in plasma Cmax regression model. CONCLUSION: This study confirms the role of SLCO1B1 and it suggests the involvement of vitamin D pathway gene polymorphisms in rifampicin pharmacokinetics.

Pharmacogenomic influence on sepsis outcome in critically ill patients.

In infectious and inflammatory diseases, pharmacogenetics affects treatment efficacy and toxicity. Moreover, recent studies suggest its important role in predicting the clinical outcome of sepsis. Our aim was to investigate the influence of single nucleotide polymorphisms (SNPs) in genes which we supposed to be involved in linezolid elimination upon sepsis outcome. Fourteen ICU-admitted patients in therapy with intravenous linezolid (600mg q12h) were enrolled and classified into three groups: group 0 for sepsis, 1 for severe sepsis and 2 for septic shock. Genotyping for SNPs in MDR1 3435 rs1045642 C>T, 2677 rs2032582 G>T and 1236 rs1128503 C>T, MRP2 -24 rs717620 G>A and 1249 rs2273697 G>A, MRP4 *879 rs1059751 T>C and 3348 rs1751034 T>C, BCRP1 421 rs2231142 C>A and 1194+928 rs13120400 T>C, -127 rs4149170 G>A and OCT1 480 rs683369 C>G genes was done using real-time PCR allelic discrimination assay. The Mann-Whitney statistical test was used to analyse variables. MDR1 2677 (p= 0.012), MRP2 1249 (p= 0.038), MRP4 *879 (p= 0.032) and 3348 SNPs significantly influenced the sepsis score. Our study, despite its limited sample size, could be decisive for early sepsis prediction and may improve the management of critically ill patients.

Daptomycin Pharmacokinetics and Pharmacodynamics in Septic and Critically Ill Patients.

Infections, including sepsis, are associated with high mortality rates in critically ill patients in the intensive care unit (ICU). Appropriate antibiotic selection and adequate dosing are important for improving patient outcomes. Daptomycin is bactericidal in bloodstream infections caused by Staphylococcus aureus and other Gram-positive pathogens cultured in ICU patients. The drug has concentration-dependent activity, and the area under the curve/minimum inhibitory concentration ratio is the pharmacokinetic/pharmacodynamic (PK/PD) index that best correlates with daptomycin activity, whereas toxicity correlates well with daptomycin plasma trough concentrations (or minimum concentration [C min]). Adequate daptomycin exposure can be difficult to achieve in ICU patients; multiple PK alterations can result in highly variable plasma concentrations, which are difficult to predict. For this reason, therapeutic drug monitoring could help clinicians optimize daptomycin dosing, thus improving efficacy while decreasing the likelihood of serious adverse events. This paper reviews the literature on daptomycin in ICU patients with sepsis, focusing on dosing and PK and PD parameters.

Ethambutol plasma and intracellular pharmacokinetics: A pharmacogenetic study.

We evaluated ethambutol plasma and intracellular pharmacokinetic according to single nucleotide polymorphisms in ABCB1, OATP1B1, PXR, VDR, CYP24A1 and CYP27B1 genes. Mycobacterium tubercolosis infected patients were enrolled. Standard weight-adjusted antitubercular treatment was administered intravenously for 2 weeks and then orally. Allelic discrimination was performed by real-time PCR. Ethambutol plasma and intracellular concentrations were measured by UPLC-MS/MS methods. Twenty-four patients were included. Considering weeks 2 and 4, median plasma Ctrough were 73 ng/mL and 247 ng/mL, intracellular Ctrough were 16,863 ng/mL and 13,535 ng/mL, plasma Cmax were 5627 ng/mL and 2229 ng/mL, intracellular Cmax were 133,830 ng/mL and 78,544 ng/mL. At week 2, ABCB1 3435 CT/TT (p=0.023) and CYP24A1 8620 AG/GG (p=0.030) genotypes for plasma Ctrough, BsmI AA (p=0.036) for intracellular Ctrough and BsmI AA (p<0.001) and ApaI AA (p=0.048) for intracellular Cmax, remained in linear regression analysis as predictive factors. Concerning week 4 only ABCB1 3435 CT/TT (p=0.035) and Cdx2 AG/GG (p=0.004) genotypes for plasma Ctrough and BsmI AA (p=0.028) for plasma Cmax were retained in final regression model. We reveal, for the first time, the possible role of single nucleotide polymorphisms on ethambutol plasma and intracellular concentrations; this may further the potential use of pharmacogenetic for tailoring antitubercular treatment.

Physical and Chemical Stability of Urapidil in 0.9% Sodium Chloride in Elastomeric Infusion Pump.

Urapidil is an antihypertensive agent, usually administered through intravenous bolus injection, slow-intravenous infusion, or continuous-drug infusion by perfusor. Since to date no evidences are available on drug stability in elastomeric pumps, patients have to be hospitalized. The purpose of this study was to validate an ultra-performance liquid chromatographic method to evaluate urapidil stability in an elastomeric infusion pump, in order to allow continuous infusion as home-care treatment. Analyses were conducted by diluting urapidil in an elastomeric pump. Two concentrations were evaluated: 1.6 mg/mL and 3.3 mg/mL. For the analyses, a reverse-phase ultra-performance liquid chromatographic- photodiode array detection instrument was used. Stressed degradation, pH changes, and visual clarity were used as stability indicators up to 10 days after urapidil solution preparation. The drug showed no more than 5% degradation during the test period at room temperature. No pH changes and no evidences of incompatibility were observed. Stress tests resulted in appreciable observation of degradation products. Considering the observed mean values, urapidil hydrochloride in sodium chloride 0.9% in elastomeric infusion pumps is stable for at least 10 days. These results indicate that this treatment could be administered at home for a prolonged duration (at least 7 days) with a satisfactory response.

A probable drug-to-drug interaction between voriconazole and haloperidol in a CYP2C19 poor metabolizing patient. [corrected].

We present a case of Aspergillus fumigatus renal abscess treated with voriconazole. Following haloperidol treatment we observed an unexpected increase in voriconazole--trough concentrations and liver function tests. CYP2C19*2 loss of function allele was stated and the introduction of haloperidol, a weak CYP3A4 inhibitor, probably explains this interaction. [corrected]. Therapeutic drug monitoring and CYP2C19 genotyping may be suggested when administering voriconazole to complex patients.

A UPLC-MS-MS method for the simultaneous quantification of first-line antituberculars in plasma and in PBMCs.

OBJECTIVES: TB is currently the second cause of death among patients affected with infectious diseases. Quantification of drug levels in plasma and in cells where Mycobacterium tuberculosis persists and grows may be useful in understanding the appropriateness of dosage regimens. We report a new and fully validated chromatographic method to quantify first-line antituberculars in plasma and PBMCs. The method was used for plasma and cell quantification of antituberculars in patients undergoing treatment with standard oral therapy. METHODS: Ethambutol, isoniazid, pyrazinamide and rifampicin were extracted from plasma and PBMCs using two separate and optimized procedures; analysis was performed using UPLC coupled with a mass-mass detector system (UPLC-MS-MS). Antitubercular levels in patients were assayed at the end of the dosing interval (C trough) and 2 h post-dose (C max). RESULTS: The method was accurate and precise. Recovery and the matrix effect were reproducible. While rifampicin intracellular concentrations were similar to plasma values (median intra-PBMC C max = 7503 ng/mL versus median plasma C max = 6505 ng/mL), isoniazid and pyrazinamide intracellular concentrations were lower than plasma values (median intra-PBMC C max = 12 ng/mL versus median plasma C max = 3258 ng/mL for isoniazid and median intra-PBMC C max = 2364 ng/mL versus median plasma C max = 26 988 ng/mL for pyrazinamide) and ethambutol intracellular concentrations were significantly higher than plasma values (median intra-PBMC C max = 73 334 ng/mL versus median plasma C max = 2244 ng/mL). CONCLUSIONS: The method was suitable for both therapeutic drug monitoring and for pharmacokinetic analysis. Should the clinical usefulness of measuring antitubercular drug intracellular concentrations be confirmed, this method could be useful to enhance the clinical application of intra-PBMC evaluation.

Development and validation of a HPLC-UV method for the quantification of antiepileptic drugs in dried plasma spots.

BACKGROUND: Therapeutic drug monitoring (TDM) of antiepileptic drugs is widely used in clinical practice to optimise therapy, but it is limited by technical problems and cost considerations. The aim of the present study was: 1) to validate a chromatographic method for the concomitant determination of levetiracetam, lamotrigine, ethosuximide, felbamate, rufinamide, zonisamide and monohydroxycarbamazepine; 2) to develop it for dried plasma spot (DPS) assessing its reliability against the classical determination from plasma; and 3) test its clinical application. METHODS: Extraction of plasma samples and DPS was done by simple precipitation. Chromatographic analysis was performed using high performance liquid chromatography with ultraviolet detection. After validation, both methods were applied for the quantification of plasma samples from patients on antiepileptic therapy. RESULTS: Mean inter- and intra-day accuracy and precision were <15% for all compounds both in plasma and in DPS samples. DPS samples were considered stable under tested conditions. Measurements between plasma and DPS samples appeared related (p<0.0001). Bland-Altman analysis revealed accordance in lamotrigine values with mean overestimation of concentration for DPS sample of 2.8%. Also for monohydroxycarbamazepine data the agreement was acceptable (mean overestimation of 9.2%). For levetiracetam mean difference was 7.6%, while for ethosuximide mean percentage difference was 20.6%. CONCLUSIONS: The developed methods simplify TDM of antiepileptic drugs. This is particularly relevant for the method on dried spot sample devices because it facilitates further sample handling, stability and shipments making the management of therapies in epileptic patients easier also in hospitals devoid of a dedicated laboratory.

Vitamin D pathway gene variants and HCV-2/3 therapy outcomes.

BACKGROUND: The combination of ribavirin and pegylated interferon-α is considered the standard of care for HCV-2/3 genotypes. The immune system plays a key role in the achievement of a sustained virological response (SVR). Vitamin D seems to influence antiviral response in chronic hepatitis C and its pathway is controlled by polymorphic genes such as CYP27B1, CYP24A1 and VDR. In this study, we have investigated the correlation among the treatment outcomes and single nucleotide polymorphisms (SNPs) in the above-mentioned genes and IL28B genes. METHODS: A total of 112 HCV-2/3 patients treated with interferon plus ribavirin were retrospectively studied; allelic discrimination was performed by real-time PCR. RESULTS: CYP24A1rs2585428, IL28Brs12979860 and rs8099917 SNPs affected treatment failure and body mass index (BMI), Metavir score, IL28Brs8099917TT and CYP24A1rs2585428GG were the only factors able to predict it. SVR was predicted by Metavir score, HCV RNA at baseline and early virological response (EVR). IL28Brs12979860 SNP and HCV RNA were also related to rapid virological response. EVR was predicted by BMI, Metavir score and CYP24A1rs2585428 SNP. IL28Brs8099917TT and FokITT were relapse prediction factors. CONCLUSIONS: In addition to non-genetic factors, SNPs in the vitamin D pathway seem to have a role in HCV-2/3 therapy outcomes. This study reveals the likely usefulness of pharmacogenetic-based ribavirin and interferon therapy to help identify patients for whom therapy could be successful or not, also considering new future expensive therapy options. To date, no similar data were published on these viral genotypes, but further studies in different and bigger cohorts are needed.

Voriconazole and atazanavir: a CYP2C19-dependent manageable drug-drug interaction.

AIM: To investigate the pharmacokinetics of voriconazole when administered to HIV-positive patients receiving treatment with atazanavir-containing therapies according to CYP2C19 genotype. MATERIALS & METHODS: We describe four HIV-positive patients with pulmonary aspergillosis treated with voriconazole and atazanavir-based regimens (with or without ritonavir). They were managed by assessing their CYP2C19 genotype (CYP2C19*2, rs4244285, G>A, real-time PCR) and therapeutic drug monitoring (HPLC-based validation methods). RESULTS & CONCLUSION: Voriconazole exposure was variable but Ctrough levels were above 1000 ng/ml in all patients; one CYP2C19 intermediate metabolizer required lower doses of voriconazole (50 mg twice daily) to obtain satisfactory drug concentrations. Atazanavir and ritonavir plasma levels were moderately reduced (area under the curve: -23 and -26%, respectively); raltegravir exposure seemed increased by voriconazole administration (area under the curve: 2.5-fold higher) in a single subject. Coadministration of atazanavir and voriconazole may be feasible in selected HIV-positive patients; therapeutic drug monitoring and CYP2C19 genotyping may optimize exposure of both drugs.

A 30-years review on pharmacokinetics of antibiotics: is the right time for pharmacogenetics?

Drug bioavailability may vary greatly amongst individuals, affecting both efficacy and toxicity: in humans, genetic variations account for a relevant proportion of such variability. In the last decade the use of pharmacogenetics in clinical practice, as a tool to individualize treatment, has shown a different degree of diffusion in various clinical fields. In the field of infectious diseases, several studies identified a great number of associations between host genetic polymorphisms and responses to antiretroviral therapy. For example, in patients treated with abacavir the screening for HLA-B*5701 before starting treatment is routine clinical practice and standard of care for all patients; efavirenz plasma levels are influenced by single nucleotide polymorphism (SNP) CYP2B6-516G>T (rs3745274). Regarding antibiotics, many studies investigated drug transporters involved in antibiotic bioavailability, especially for fluoroquinolones, cephalosporins, and antituberculars. To date, few data are available about pharmacogenetics of recently developed antibiotics such as tigecycline, daptomycin or linezolid. Considering the effect of SNPs in gene coding for proteins involved in antibiotics bioavailability, few data have been published. Increasing knowledge in the field of antibiotic pharmacogenetics could be useful to explain the high drug inter-patients variability and to individualize therapy. In this paper we reported an overview of pharmacokinetics, pharmacodynamics, and pharmacogenetics of antibiotics to underline the importance of an integrated approach in choosing the right dosage in clinical practice.

Therapeutic drug monitoring of intracellular anti-infective agents.

Many microorganisms, including viruses, some bacteria and fungi, replicate within the cells. Therefore, the efficacy of therapy and the selection of resistances could be related to intracellular concentration of the drugs and to their ability to cross biological membranes and penetrate into various tissue compartments. The efficacy of treatment may be limited by pharmacological factors. Dose-response relationship exists for many agents, and failure to maintain adequate concentrations may allow the development of viral or bacterial resistance, thereby decreasing the probability of response of current and subsequent therapies. The major target of antivirals and many other anti-infective agents is within infected cells. Therefore, clinical outcome ultimately should be related to intracellular drug concentrations. Intracellular pharmacokinetics provides information regarding drug disposition in a compartment where microorganism replication occurs and combined with plasma data may be useful in understanding therapeutic failure in relation to cellular resistance. With a focus on possible methodological biases, this review reports the current state of the art in intracellular, particularly in peripheral blood mononuclear cells, therapeutic drug monitoring of the following anti-infective drugs: antivirals, antifungals and antibiotics. Although measurement of intracellular concentrations needs to be still standardized focusing on each single drug, this review showed some relationships between intracellular concentrations of few anti-infective drugs and their efficacy and/or toxicity. Such relationships should be interpreted with caution, as intracellular concentrations reflect the total amount of drug within the cell and not the effective unbound fraction. The number of clinical studies in that area is, however, rather limited, and not always adequately designed. Then, intracellular drug determination has to be considered a test for research only and not to be carried out as routine.