Drug-related problems and satisfaction among patients receiving pharmacist-led consultations at the initiation of cardiovascular drugs.

BACKGROUND: Drug-related problems (DRPs) lead to substantial morbidity and mortality and increase healthcare costs. Several interventions have been developed to reduce DRPs and improve the outcome of drug therapy. OBJECTIVE: To investigate DRPs identified through a pharmacist-led intervention and to assess patient satisfaction with the intervention. METHODS: Patients received two pharmacist consultations 1-2 weeks and 3-5 weeks after collecting a new cardiovascular medicine. Information about patient characteristics, beliefs about medicines (BMQ), DRPs, and patient evaluations were collected using questionnaires. RESULTS: Pharmacists identified DRPs among 52.4% and 43.1% of the 633 patients at consultation 1 and 2, respectively. Of the DRPs reported in consultation 1, 43.7% were solved at consultation 2. Among patients with side effects, patients who received advice on managing these in consultation 1 where more likely to have solved problems at consultation 2 (61.2% vs. 42.6%, p = 0.008). Female gender, high BMQ concern and the number of new medicines were associated with DRPs. Patients were highly satisfied with the intervention. Predictors of satisfaction were female gender, older age, higher BMQ necessity, face-to-face consultations, longer duration of consultation 1, and solved problems in consultation 2. CONCLUSIONS: The results indicate that the pharmacist-led follow-up intervention can aid early identification and solving of DRPs in patients prescribed new cardiovascular drugs. Knowledge of factors associated with DRPs and patients' satisfaction may allow further improvement of the intervention.

Pharmacodynamic assessment of mycophenolic acid in resting and activated target cell population during the first year after renal transplantation.

AIMS: To explore the pharmacodynamics of mycophenolic acid (MPA) through inosine monophosphate dehydrogenase (IMPDH) capacity measurement and purine levels in peripheral blood mononuclear cells (PBMC) longitudinally during the first year after renal transplantation (TX). METHODS: PBMC were isolated from renal recipients 0-4 days prior to and 6-9 days, 5-7 weeks and 1 year after TX (before and 1.5 hours after dose). IMPDH capacity and purine (guanine and adenine) levels were measured in stimulated and nonstimulated PBMC. RESULTS: Twenty-nine patients completed the follow-up period, of whom 24 received MPA. In stimulated PBMC, the IMPDH capacity (pmol 10-6 cells min-1 ) was median (interquartile range) 127 (95.8-147) before TX and thereafter 44.9 (19.2-93.2) predose and 12.1 (4.64-23.6) 1.5 hours postdose across study days after TX. The corresponding IMPDH capacity in nonstimulated PBMC was 5.71 (3.79-6.93), 3.35 (2.31-5.62) and 2.71 (1.38-4.08), respectively. Predose IMPDH capacity in nonstimulated PBMC increased with time, reaching pre-TX values at 1 year. In stimulated PBMC, both purines were reduced before (median 39% reduction across days after TX) and after (69% reduction) dose compared to before TX. No alteration in the purine levels was observed in nonstimulated PBMC. Patients needing dose reductions during the first year had lower pre-dose IMPDH capacity in nonstimulated PBMC (1.87 vs 3.00 pmol 10-6 cells min-1 , P = .049) at 6-9 days. CONCLUSION: The inhibitory effect of MPA was stronger in stimulated PBMC. Nonstimulated PBMC became less sensitive to MPA during the first year after TX. Early IMPDH capacity appeared to be predictive of dose reductions.

Effect of a pharmacist-led intervention on adherence among patients with a first-time prescription for a cardiovascular medicine: a randomized controlled trial in Norwegian pharmacies.

OBJECTIVE: To examine whether a pharmacist-led intervention improves medication adherence among patients who have filled a first-time prescription for a cardiovascular medicine. METHODS: Design: Unblinded randomized controlled trial. SETTING: 67 Norwegian pharmacies, October 2014-June 2015. PARTICIPANTS: 1480 adults with a first-time prescription for a cardiovascular medicine. INTERVENTION: Participants in the intervention group received two consultations with a pharmacist 1-2 and 3-5 weeks after filling the prescription. Participants in the control group received care according to usual practice. MAIN OUTCOME MEASURE: The primary outcome was self-reported adherence as measured by the 8-item Morisky Medication Adherence Scale (MMAS-8), at 7 and 18 weeks after filling the prescription. Adherence from baseline to week 52 was estimated using data from the Norwegian Prescription Database (NPD). KEY FINDINGS: Data from MMAS-8 showed that 91.3% of the patients in the intervention group were adherent after 7 weeks versus 86.8% in the control group (4.5% difference, 95% CI 0.8-8.2, P = 0.017). The corresponding proportions were 88.7% versus 83.7% after 18 weeks (5.0% difference, 95% CI 0.8-9.2, P = 0.021). NPD data (n = 1294) showed no significant difference in adherence after 52 weeks (95% CI -2.0 to 7.8, P = 0.24). However, adherence among statin users (n = 182) was 66.5% in the intervention group versus 57.4% among new statin users in the general population (n = 1500) (difference 9.1%, 95% CI 1.5-16.0, P = 0.019). CONCLUSION: The main outcome measure indicates that a short, structured pharmacist-led intervention may increase medication adherence for patients starting on chronic cardiovascular medication. However, these findings could not be confirmed by the NPD data analysis.

Longitudinal Study of Tacrolimus in Lymphocytes During the First Year After Kidney Transplantation.

INTRODUCTION: Tacrolimus (TAC) is an immunosuppressive drug used after organ transplantation. Dosing is adjusted using whole blood (WB-TAC) measurements. Patients within the therapeutic WB-TAC window still experience rejections and adverse effects. Alternative monitoring methods are therefore warranted. The authors developed a method for measuring TAC in peripheral blood mononuclear cell (PBMC) isolates (PBMC-TAC) and performed a pharmacokinetic study in a cohort of kidney transplant patients during the first year after transplantation. METHODS: PBMCs were isolated from whole blood by gradient centrifugation. After methanol-based extraction, liquid chromatography with tandem mass spectrometry was used to determine TAC in the extract. PBMC-TAC was normalized to the number of cells and alternatively to the protein amount in cells. Predose and postdose (1.5 hours) samples from kidney transplant patients were collected at 1 week, 6 weeks, and 1 year after transplantation. WB-TAC was measured using immunoassay. RESULTS: The PBMC-TAC assay fulfilled the validation criteria of the European Medicines Agency guidelines. Twenty-nine patients completed the study. Predose PBMC-TAC was (median) 23 (1 week), 33 (6 weeks), and 27 pg/10 cells (1 year). Postdose PBMC-TAC was 44, 30, and 27 pg/10 cells at 1 week, 6 weeks, and 1 year after transplantation, respectively. Predose WB-TAC (median) was 5.0, 6.0, and 5.4 mcg/L, and postdose WB-TAC was 10.5, 8.3, and 9.1 mcg/L, respectively, at 1 week, 6 weeks, and 1 year after transplantation. Whole blood and PBMC-TAC correlated at all timepoints (rho 0.40-0.82, P < 0.05) except before dosage at 6 weeks. PBMC-TAC normalized to the number of cells, and the amount of protein was modestly correlated (rho 0.36-0.81, P < 0.056). CONCLUSIONS: The correlation between WB-TAC and PBMC-TAC is modest during the first-year posttransplantation. Normalization of PBMC-TAC to cells or protein may yield different results. PBMC-TAC is increased 1.5 hours after dose at 1 week after transplantation, but not after 6 weeks or 1 year, indicating altered distribution kinetics.

Prednisolone and Prednisone Pharmacokinetics in Pediatric Renal Transplant Recipients-A Prospective Study.

BACKGROUND: Prednisolone is a standard component of immunosuppressive protocols in renal transplantation (Tx) and despite standardized treatment regimens, adverse side effects are still frequent. The aim of this study was to characterize the pharmacokinetics of prednisolone and prednisone in pediatric renal transplant recipients in the first 52 weeks post Tx, to describe the relationship between prednisolone and prednisone, and to investigate a possible relationship between the development of new-onset diabetes after Tx (NODAT) and glucocorticoid exposure. METHODS: Renal transplant recipients receiving prednisolone (n = 11, age 1-15 years) were included in this prospective open-label, descriptive, nonrandomized, and noninterventional study. Blood samples were drawn pre-Tx and during selected dose intervals (0, 1, 2, 4, 6, and 12 hours postdose; less frequent in children <10 kg) at 1, 2, 3, 4, 12, and 52 weeks post-Tx. Concentrations of prednisolone and cortisol, their inactive keto forms, plus methylprednisolone, were measured using a validated LC-MS/MS method. Genetic variants in the CYP3A4, CYP3A5, ABCB1, and HSD11B2 genes were analyzed using real-time polymerase chain reaction and Sanger sequencing. Correlation with NODAT was investigated. RESULTS: The patients displayed considerable intra- and inter-individual variability in prednisolone exposure, with up to 5-fold differences in the area under the concentration-time curve (AUC). There were up to 7-fold differences in prednisolone/prednisone AUC ratio between patients, and patients experiencing NODAT tended to have a higher ratio (>12) compared with patients without NODAT (<12). Genetic variants in CYP3A5 and ABCB1 were found, but due to the limited study population causality cannot be definitive. CONCLUSIONS: The study suggests that a high prednisolone/prednisone AUC ratio may be a possible risk factor for NODAT. Further studies of individualization of glucocorticoid treatment in pediatric organ Tx are warranted.

NFAT-regulated cytokine gene expression during tacrolimus therapy early after renal transplantation.

AIMS: Despite pharmacokinetic monitoring of calcineurin inhibitors, the long-term outcome after transplantation (Tx) is still hampered by the side effects of these drugs. The aim of the present study was to characterize nuclear factor of activated T cells (NFAT)-regulated gene expression as a potential pharmacodynamic biomarker for further individualization of tacrolimus (Tac) therapy. METHODS: In 29 renal allograft recipients, samples were drawn once pre-Tx, and before and 1.5 h after Tac dosing at approximately 1 week, 6 weeks and 1 year post-Tx. Tac concentrations were measured by immunoassay, while the expression of genes encoding NFAT-regulated cytokines [interleukin 2 (IL2), interferon gamma (IFNG), colony stimulating factor 2 (CSF2)] and cytochrome P450 3A5 (CYP3A5) genotyping were determined by real-time polymerase chain reaction. RESULTS: The cytokine response after Tac dosing varied up to 46-fold between patients and changed significantly with time post-engraftment. Tac concentrations 1.5 h postdose (C1.5 ) >15 µg l-1 were associated with strong cytokine inhibition and residual gene expression (RGE) ≤10%, while lower Tac C1.5 resulted in more variable responses (RGE 2.5-68.7%). Patients with ongoing subclinical acute rejection (n = 5) demonstrated limited cytokine inhibition (RGE 39.7-72.6%), while patients with polyoma virus viraemia (n = 3) had relatively strong inhibition of cytokines (RGE 2.5-32.5%). By contrast, there was no association between Tac exposure and rejection or viraemia. CONCLUSIONS: The findings of our study support the potential of NFAT-regulated gene expression measurements as a pharmacodynamic tool for additional monitoring of Tac therapy, especially in the context of overimmunosuppression and viraemia.

Is toxicity of PMMA (paramethoxymethamphetamine) associated with cytochrome P450 pharmacogenetics?

In 2010-2013, 29 fatal intoxications related to the designer drug paramethoxymethamphetamine (PMMA, 4-methoxymethamphetamine) occurred in Norway. The current knowledge about metabolism and toxicity of PMMA in humans is limited. Metabolism by the polymorphic cytochrome P450 (CYP) 2D6 enzyme to the psychoactive metabolite 4-hydroxymethamphetamine (OH-MA), and possibly by additional enzymes, is suggested to be involved in its toxicity. The aim of this work was to study the association between CYP genetics, PMMA metabolism and risk of fatal PMMA toxicity in humans. The frequency distribution of clinically relevant gene variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A5, and the phenotypic blood CYP2D6 metabolic ratio (OH-MA/PMMA) in particular, were compared in fatal PMMA intoxications (n=17) and nonfatal PMMA abuse controls (n=30), using non-abusers (n=305) as references for the expected genotype frequencies in the Norwegian population. Our study demonstrated that the CYP2D6 enzyme and genotype are important in the metabolism of PMMA to OH-MA in humans, but that other enzymes are also involved in this biotransformation. In the fatal PMMA intoxications, the blood concentrations of PMMA were higher and the CYP2D6 metabolic ratios were lower, than in the nonfatal PMMA abuse controls (median (range) 2.1 (0.03-5.0) vs 0.3 (0.1-0.9) mg/L, and ratio 0.6 (0.0-4.6) vs 2.1 (0.2-7.4) p=0.021, respectively). Overall, our findings indicated that, in most cases, PMMA death occurred rapidly and at an early stage of PMMA metabolism, following the ingestion of large and toxic PMMA doses. We could not identify any genetic CYP2D6, CYP2C9, CYP2C19 or CYP3A5 predictive marker on fatal toxicity of PMMA in humans. The overrepresentation of the CYP2D6 poor metabolizer (PM) genotype found in the nonfatal PMMA abuse controls warrants further investigations.

Drug target molecules to guide immunosuppression.

The individual and interindividual variability of response to immunosuppressants combined with the prevailing concept of lifelong immunosuppression following any organ transplantation motivates the search for methods to further individualize such therapy. Traditional therapeutic drug monitoring, adapting dose according to concentrations in blood, targets the pharmacokinetic variability. It has been increasingly recognized, however, that there is also a considerable variability in the response to a given concentration. Attempts to overcome this variability in response include the efforts to identify relevant targets and methods for pharmacodynamic monitoring. For several of the currently used immunosuppressants there is experimental data suggesting markers that are relevant as indicators for individual monitoring of the effects of these drugs. There are also some clinical data to support these approaches; however what is generally missing, are studies that in a prospective manner demonstrates the benefits and effects on outcome. The monitoring of antithymocyte globulin by lymphocyte subset counts is actually the only well established example of pharmacodynamic monitoring. For drugs such as MPA and mTOR inhibitors, there are candidates such as IMPDH activity expression and p70SK6 phosphorylation status, respectively. The monitoring of CNIs using assays for NFAT RGE, either alone or combined with concentration measurements, is already well documented. Even here, some further investigations relating to the categories of organ transplant, combination of immunosuppressants etc. will be requested. Although some further standardization of the assay is warranted and there is a need for specific recommendations of target levels and how to adjust dose, the NFAT RGE approach to pharmacodynamic monitoring of CNIs may be close to implementation in clinical routine.

Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

MAIN PROBLEM: Islet transplantation has become a promising treatment for type 1 diabetes. However, immunosuppressive drugs used today cause islet deterioration and modification strategies are necessary. But little is known about pharmacokinetics interactions and intracellular concentrations of immunosuppressive drugs in human islets. METHODS: We determined the pharmacokinetics of tacrolimus and sirolimus in islets by measuring intracellular concentration after exposure alone or in combination at two different doses up to 48 h. A quantification technique established in our laboratory using a Micromass Quattro micro API MS/MS-instrument with electrospray ionization was used. Islets function was measured by oxygen consumption rates. Presence of drug transporters OATP1B1 and ABCB1 and metabolizing enzyme CYP3A4 in islets were quantified using real-time quantitative PCR. RESULTS: Islets incubated with tacrolimus and sirolimus had a significant decrease in intracellular concentration of sirolimus compared to sirolimus alone. Reduced intracellular sirolimus concentration was followed by increased p70S6k phosphorylation suggesting preservation of the mTOR-signaling pathway. Drug transporters OATP1B1 and ABCB1 and enzyme CYP3A4 were expressed in human islets, but were not involved in the reduced sirolimus concentration by tacrolimus. CONCLUSION: These findings provide new knowledge of the drug interaction between tacrolimus and sirolimus, suggesting that tacrolimus has an inhibitory effect on the intracellular concentration of sirolimus in human islets.

Improved Tacrolimus Target Concentration Achievement Using Computerized Dosing in Renal Transplant Recipients--A Prospective, Randomized Study.

BACKGROUND: Early after renal transplantation, it is often challenging to achieve and maintain tacrolimus concentrations within the target range. Computerized dose individualization using population pharmacokinetic models may be helpful. The objective of this study was to prospectively evaluate the target concentration achievement of tacrolimus using computerized dosing compared with conventional dosing performed by experienced transplant physicians. METHODS: A single-center, prospective study was conducted. Renal transplant recipients were randomized to receive either computerized or conventional tacrolimus dosing during the first 8 weeks after transplantation. The median proportion of tacrolimus trough concentrations within the target range was compared between the groups. Standard risk (target, 3-7 µg/L) and high-risk (8-12 µg/L) recipients were analyzed separately. RESULTS: Eighty renal transplant recipients were randomized, and 78 were included in the analysis (computerized dosing (n = 39): 32 standard risk/7 high-risk, conventional dosing (n = 39): 35 standard risk/4 high-risk). A total of 1711 tacrolimus whole blood concentrations were evaluated. The proportion of concentrations per patient within the target range was significantly higher with computerized dosing than with conventional dosing, both in standard risk patients (medians, 90% [95% confidence interval {95% CI}, 84-95%] vs 78% [95% CI, 76-82%], respectively, P < 0.001) and in high-risk patients (medians, 77% [95% CI, 71-80%] vs 59% [95% CI, 40-74%], respectively, P = 0.04). CONCLUSIONS: Computerized dose individualization improves target concentration achievement of tacrolimus after renal transplantation. The computer software is applicable as a clinical dosing tool to optimize tacrolimus exposure and may potentially improve long-term outcome.

Glutathione Transferase Gene Variants Influence Busulfan Pharmacokinetics and Outcome After Myeloablative Conditioning.

BACKGROUND: Busulfan (Bu) and cyclophosphamide (Cy) are frequently included in conditioning regimens before hematopoietic stem cell transplantation (HSCT). Both drugs are detoxified by glutathione transferases (GST), and GST gene variants may explain some of the interindividual variability in pharmacokinetics and drug toxicity. METHODS: The study investigated adult patients (n = 114) receiving oral Bu pre-HSCT. Bu doses were adjusted to obtain an average steady-state concentration (Css) of 900 mcg/L. RESULTS: Median first dose Bu Css was 1000 mcg/L (600-1780 mcg/L). Patients carrying 1 and 2 GSTA1*B (rs3957357) alleles demonstrated median 12% and 16% higher Bu Css (P ≤ 0.05). Bu exposure (average Css; odds ratio = 1.009, 95% confidence interval = 1.002-1.017, P = 0.013) and GSTM1 gene copy number (odds ratio = 17.1, 95% confidence interval = 1.46-201, P = 0.024) were significant predictors of mortality ≤30 days. The mortality was 25% versus 2% among carriers of 2 versus no GSTM1 copies (P = 0.021). Mortality ≤3 months was associated with higher first dose Bu exposure (1090 versus 980 mcg/L, P = 0.021). GSTM1 expression and high Bu exposure may increase Cy toxicity by reducing intracellular glutathione. CONCLUSIONS: GST genotyping before HSCT may allow better prediction of Bu pharmacokinetics and drug toxicity, and thereby improve outcome after BuCy conditioning.

Use of generic tacrolimus in elderly renal transplant recipients: precaution is needed.

BACKGROUND: Proper bioequivalence studies comparing original with generic immunosuppressive drugs in patients are limited, especially in the increasing population of elderly renal transplant recipients. We performed an open-label, single-center, prospective, randomized, cross-over study and compared steady-state pharmacokinetics (PK) of a generic tacrolimus (Tacni) formulation with the original (Prograf) in renal transplant recipients older than 60 years. METHODS: Twenty-eight patients, with a median age of 69 years (range, 60 to 78), were randomized at time of transplantation to receive original or generic tacrolimus, and 25 (21 men, 4 women) provided two evaluable 12-hr PK profiles. The PK investigations were performed in a stable phase approximately 6 and 8 weeks postengraftment. After the first PK investigation, tacrolimus formulations were switched (1:1 dose ratio). RESULTS: Generic tacrolimus did not meet the bioequivalence criteria; the area under the curve(0-12) ratio of generic-original tacrolimus formulation was 1.17 (90% confidence interval, 1.10-1.23) and the Cmax ratio was 1.49 (90% confidence interval, 1.35-1.65). The generic formulation also showed a shorter time to C(max) (T(max)) (P=0.04). Importantly, the lack of bioequivalence was not reflected in the standard monitoring parameter, trough concentrations (P=0.80). CONCLUSION: Generic tacrolimus (Tacni) was not found to be bioequivalent to the original formulation in elderly renal transplant recipients. The significantly higher systemic exposure of tacrolimus, despite similar trough concentrations, may in the long run increase the risk of adverse effects.

Improved prediction of tacrolimus concentrations early after kidney transplantation using theory-based pharmacokinetic modelling.

AIMS: The aim was to develop a theory-based population pharmacokinetic model of tacrolimus in adult kidney transplant recipients and to externally evaluate this model and two previous empirical models. METHODS: Data were obtained from 242 patients with 3100 tacrolimus whole blood concentrations. External evaluation was performed by examining model predictive performance using Bayesian forecasting. RESULTS: Pharmacokinetic disposition parameters were estimated based on tacrolimus plasma concentrations, predicted from whole blood concentrations, haematocrit and literature values for tacrolimus binding to red blood cells. Disposition parameters were allometrically scaled to fat free mass. Tacrolimus whole blood clearance/bioavailability standardized to haematocrit of 45% and fat free mass of 60 kg was estimated to be 16.1 l h−1 [95% CI 12.6, 18.0 l h−1]. Tacrolimus clearance was 30% higher (95% CI 13, 46%) and bioavailability 18% lower (95% CI 2, 29%) in CYP3A5 expressers compared with non-expressers. An Emax model described decreasing tacrolimus bioavailability with increasing prednisolone dose. The theory-based model was superior to the empirical models during external evaluation displaying a median prediction error of −1.2% (95% CI −3.0, 0.1%). Based on simulation, Bayesian forecasting led to 65% (95% CI 62, 68%) of patients achieving a tacrolimus average steady-state concentration within a suggested acceptable range. CONCLUSION: A theory-based population pharmacokinetic model was superior to two empirical models for prediction of tacrolimus concentrations and seemed suitable for Bayesian prediction of tacrolimus doses early after kidney transplantation.

The influence of CYP3A, PPARA, and POR genetic variants on the pharmacokinetics of tacrolimus and cyclosporine in renal transplant recipients.

PURPOSE: Tacrolimus (Tac) and cyclosporine (CsA) are mainly metabolized by CYP3A4 and CYP3A5. Several studies have demonstrated an association between the CYP3A5 genotype and Tac dose requirements. Recently, CYP3A4, PPARA, and POR gene variants have been shown to influence CYP3A metabolism. The present study investigated potential associations between CYP3A5*3, CYP3A4*22, PPARA c.209- 1003G>A and c.208+3819A>G, and POR*28 alleles and dose-adjusted concentrations (C/D) of Tac and CsA in 177 renal transplant patients early post-transplant. METHODS: All patients (n=177) were genotyped for CYP3A4*22, CYP3A5*3, POR*28, PPARA c.209-1003G>A, and PPARA c.208+3819A>G using real-time polymerase chain reaction (PCR) and melting curve analysis with allelespecific hybridization probes or PCR restriction fragment length polymorphisms (RFLP) methods. Drug concentrations and administered doses were retrospectively collected from patient charts at Oslo University Hospital, Rikshospitalet, Norway. One steady-state concentration was collected for each patient. RESULTS: We confirmed a significant impact of the CYP3A5*3 allele on Tac exposure. Patients with POR*28 and PPARA variant alleles demonstrated 15 % lower (P=0.04) and 19 % higher (P=0.01) Tac C0/D respectively. CsA C2/D was 53 % higher among CYP3A4*22 carriers (P=0.03). CONCLUSION: The results support the use of pre-transplant CYP3A5 genotyping to improve initial dosing of Tac, and suggest that Tac dosing may be further individualized by additional POR and PPARA genotyping. Furthermore, initial CsA dosing may be improved by pre-transplant CYP3A4*22 determination.

A taste of individualized medicine: physicians' reactions to automated genetic interpretations.

The potential of pharmacogenomics is well documented, and functionality exploiting this knowledge is about to be introduced into electronic medical records. To explore physicians' reactions to automatic interpretations of genetic tests, we built a prototype with a simple interpretive algorithm. The algorithm was adapted to the needs of physicians handling immunosuppressive treatment during organ transplantation. Nine physicians were observed expressing their thoughts while using the prototype for two patient scenarios. The computer screen and audio were recorded, and the qualitative results triangulated with responses to a survey instrument. The physicians' reactions to the prototype were very positive; they clearly trusted the results and the theory behind them. The explanation of the algorithm was prominently placed in the user interface for transparency, although this design led to considerable confusion. Background information and references should be available, but considerably less prominent than the result and recommendation.

Simultaneous quantification of IMPDH activity and purine bases in lymphocytes using LC-MS/MS: assessment of biomarker responses to mycophenolic acid.

BACKGROUND: The development of biomarkers describing the individual responses to the immunosuppressant mycophenolic acid (MPA) has focused on the target enzyme activity [inosine 5'-monophosphate dehydrogenase (IMPDH)]. An extended strategy is to quantify the metabolic consequences of IMPDH inhibition. The aim of this study was to develop an assay for quantification of IMPDH activity and related purine bases and to provide preliminary data on the behavior of these biomarkers during clinical exposure to MPA. METHODS: Liquid chromatography-mass spectrometry was used to determine xanthine (IMPDH activity in incubated cell lysate), hypoxanthine, guanine, and adenine derived from free nucleotides in lymphocytes. Analytical performance was assessed, and the biomarkers were examined in CD4⁺ cells from 2 groups: Healthy individuals in a single-dose MPA study (n = 5) and liver transplant recipients on MPA therapy (n = 15). RESULTS: Coefficients of variation between series were below 10% and 15% for measurement of the purines and IMPDH activity, respectively. Although IMPDH was inhibited, the purine levels increased in response to MPA in 3 of the 5 healthy individuals, and this positive response seemed to be associated with IMPDH1 c.579 + 119 G/G and c.580 - 106 G/G. In the liver transplant study, guanine was not reduced in response to the transient drop in IMPDH activity after MPA dosing. However, there were trends toward decrease in guanine and elevation of hypoxanthine during prolonged MPA therapy. The guanine/hypoxanthine ratio (median) was 37% lower and the adenine level was 21% lower at day 17 compared with day 4 after transplantation. CONCLUSIONS: The assay allows precise quantification of IMPDH activity, hypoxanthine, guanine, and adenine in lymphocytes. Some individuals may possess a counteracting purine response to the MPA-mediated inhibition of IMPDH. Reduction of the guanine/hypoxanthine ratio may be related to prolonged inhibition of IMPDH and seems as an intriguing pharmacodynamic biomarker for MPA.

Importance of hematocrit for a tacrolimus target concentration strategy.

PURPOSE: To identify patient characteristics that influence tacrolimus individual dose requirement in kidney transplant recipients. METHODS: Data on forty-four 12-h pharmacokinetic profiles from 29 patients and trough concentrations in 44 patients measured during the first 70 days after transplantation (1,546 tacrolimus whole blood concentrations) were analyzed. Population pharmacokinetic modeling was performed using NONMEM 7.2®. RESULTS: Standardization of tacrolimus whole blood concentrations to a hematocrit value of 45 % improved the model fit significantly (p<0.001). Fat-free mass was the best body size metric to predict tacrolimus clearance and volume of distribution. Bioavailability was 49 % lower in expressers of cytochrome P450 3A5 (CYP3A5) than in CYP3A5 nonexpressers. Younger females (<40 years) showed a 35 % lower bioavailability than younger males. Bioavailability increased with age for both males and females towards a common value at age >55 years that was 47 % higher than the male value at age <40 years. Bioavailability was highest immediately after transplantation, decreasing steeply thereafter to reach its nadir at day 5, following which it increased during the next 55 days towards an asymptotic value that was 28 % higher than that on day 5. CONCLUSIONS: Hematocrit predicts variability in tacrolimus whole blood concentrations but is not expected to influence unbound (therapeutically active) concentrations. Fat-free mass, CYP3A5 genotype, sex, age and time after transplant influence the tacrolimus individual dose requirement. Because hematocrit is highly variable in kidney transplant patients and increases substantially after kidney transplantation, hematocrit is a key factor in the interpretation of tacrolimus whole blood concentrations.

Tacrolimus exposure and mycophenolate pharmacokinetics and pharmacodynamics early after liver transplantation.

BACKGROUND: Mycophenolic acid (MPA) and tacrolimus play important roles in immunosuppressive therapy after solid organ transplantation (Tx) and show large intra- and interindividual pharmacokinetic (PK) variabilities. The purpose of this study was to describe the intra- and interindividual variabilities of MPA and tacrolimus PKs during the first 3 weeks after adult liver transplantation. Furthermore, inosine monophosphate dehydrogenase activity was investigated. MATERIALS: This study describes PK and pharmacodynamic parameters of MPA and the PKs of tacrolimus in 16 liver transplant recipients, in 4 follow-up periods (I-IV). RESULTS: The area under the concentration-time curve (AUC(0-12 hours)) for tacrolimus was low early after Tx (eg, median 78.6 around day 4) and variable in all 4 periods ranging from 3.8 to 267 µg h/L, whereas the predose concentrations (C0) were 0.0-17.9 µg/L. From periods I to IV, the tacrolimus dose was doubled and the median dose per body weight-adjusted AUC(0-12 hours) increased by 123% (P = 0.017). The AUC(0-12 hours) of MPA was in the range 8.6-57.4 mg h/L, with median values from 21.9 to 27.8 mg h/L, whereas C0 was between 0.0 and 7.3 mg/L in the 4 periods (medians from 1.2 to 1.6 mg/L). The maximum inhibition of inosine monophosphate dehydrogenase within a dose interval ranged from 9.5% to 100%. CONCLUSIONS: This study confirmed the large variability in the PKs of tacrolimus and MPA in liver transplant recipients. In particular, the MPA AUC(0-12 hours) was consistently low in all 4 periods. We also observed a low tacrolimus exposure during the first days after transplant compared with the following weeks.

Inclusion of CYP3A5 genotyping in a nonparametric population model improves dosing of tacrolimus early after transplantation.

Following organ engraftment, initial dosing of tacrolimus is based on recipient weight and adjusted by measured C(0) concentrations. The bioavailability and elimination of tacrolimus are affected by the patients CYP3A5 genotype. Prospective data of the clinical advantage of knowing patient's CYP3A5 genotype prior to transplantation are lacking. A nonparametric population model was developed for tacrolimus in renal transplant recipients. Data from 99 patients were used for model development and validation. A three-compartment model with first-order absorption and lag time from the dosing compartment described the data well. Clearances and volumes of distribution were allometrically scaled to body size. The final model included fat-free mass, body mass index, hematocrit, time after transplantation, and CYP3A5 genotype as covariates. The bias and imprecision were 0.35 and 1.38, respectively, in the external data set. Patients with functional CYP3A5 had 26% higher clearance and 37% lower bioavailability. Knowledge of CYP3A5 genotype provided an initial advantage, but only until 3-4 tacrolimus concentrations were known. After this, a model without CYP3A5 genotype predicted just as well. The present models seem applicable for clinical individual dose predictions but need a prospective evaluation.

A novel mucopolysaccharidosis type I associated splice site mutation and IDUA splice variants.

Mucopolysaccharidosis type I is an autosomal recessive disorder caused by deficiency of α-l-iduronidase, encoded by the IDUA gene. More than 100 disease causing mutations have been reported in the gene, resulting in a wide range of phenotypes. Here we describe a previously unreported IDUA splice site mutation (NG_008103.1:g.21632G>C; NM_000203.3:c.1727+3G>C) causing a Hurler phenotype in a patient heterozygous for the common p.Q70X (NG_008103.1:g.5862C>T) mutation. Sequence analysis of IDUA transcripts demonstrated that the g.21632G>C mutation results in aberrant splicing of intron 12 (NM_000203.3:c.1727_1728insGTCC), introducing a frame shift and premature termination codon (NP_000194.2:p.Cys577SerfsX15). Gene expression studies suggest that the deleterious effect of the mutation is primarily due to a C-terminal truncation of the encoded polypeptide. Furthermore, we observed that both normal and mutant IDUA alleles give rise to alternatively spliced transcripts in leukocytes. Exclusion of exon 4 appeared to be the predominant alternative splicing event, probably resulting in polypeptides lacking iduronidase activity. The Hurler patient demonstrated exon 4 skipping in 5.6% of IDUA transcripts, while exon 4 skipping ranged 25-34% of transcripts among healthy individuals (n=5). Alternative splicing might represent a mechanism for regulation of this enzyme, and the lower level of exon 4 skipping in the patient might be a response to intracellular accumulation of iduronidase substrates. Molecular characterization of IDUA mutations and splicing may assist early prediction of mucopolysaccharidosis type I phenotypes and increase the understanding of disease mechanisms. This is important considering the choice of current treatment options and for the development of future therapies.