Validation of a novel direct method to determine reduced adherence to atorvastatin therapy.

AIMS: Objective methods to determine statin adherence are requested to improve lipid management. We have recently established a method to detect reduced adherence to atorvastatin therapy with cut-off values based on the sum of atorvastatin and its major metabolites in blood. We aimed to validate this method in patients with and without cardiovascular disease, and optimize previous cut-off values. METHODS AND RESULTS: The pharmacokinetic study included 60 participants treated with atorvastatin 20 mg (N=20), 40 mg (N=20), and 80 mg (N=20). Atorvastatin was then stopped and blood samples collected from day zero to day four. Quantification of the parent drug and its metabolites in blood plasma was performed with a liquid chromatography-tandem mass spectrometry assay. The cut-off values for reduced adherence were validated and optimized by calculating diagnostic sensitivity and specificity. Our candidate cut-off value of dose-normalized six-component sum of atorvastatin plus metabolites <0.10 nM/mg provided a sensitivity of 97% and a specificity of 93% for detecting ≥2 omitted doses. An optimized cut-off <0.062 nM/mg provided a sensitivity of 90% and a specificity of 100%. An alternative simplified two-component metabolite sum with cut-off value <0.05 nM/mg provided a sensitivity of 98% and a specificity of 76%. An optimized cut-off <0.02 nM/mg provided a sensitivity of 97% and a specificity of 98%. CONCLUSION: This validation study confirms that our direct method discriminates reduced adherence from adherence to atorvastatin therapy with high diagnostic accuracy. The method may improve lipid management in clinical practice and serve as a useful tool in future studies.

The atorvastatin metabolite pattern in muscle tissue and blood plasma is associated with statin muscle side effects in patients with coronary heart disease; An exploratory case-control study.

Background and aims: Statin-associated muscle symptoms (SAMS) is a prevalent cause of statin discontinuation. It is challenging and time-consuming for clinicians to assess whether symptoms are caused by the statin or not, and diagnostic biomarkers are requested. Atorvastatin metabolites have been associated with SAMS. We aimed to compare atorvastatin pharmacokinetics between coronary heart disease (CHD) patients with and without clinically statin intolerance and statin-dependent histopathological alterations in muscle tissue. Secondarily we aimed to assess genetic variants relevant for the observed pharmacokinetic variables. Methods: Twenty-eight patients with CHD and subjective SAMS were included in the exploratory MUSE biomarker study in 2020. Participants received atorvastatin 40 mg/day for seven weeks followed by no statins for eight weeks. Muscle biopsies and blood were collected at the end of each period. Four patients were categorized as clinically intolerant to ≥3 statins prior to study start whereas four patients had signs of muscle cell damage during treatment. Results: We found significantly lower levels of atorvastatin acids, and higher lactone/acid ratios in the statin intolerant, both in muscle and plasma. With optimal cut-off, the combination of 2-OH-atorvastatin acid and the 2-OH-atorvastatin lactone/acid ratio provided sensitivity, specificity, and predictive values of 100 %. Patients with variants in UGT1A1 and UGT1A3 had higher lactone metabolite levels than those with wild type, both in muscle and plasma. Conclusion: Atorvastatin metabolites appear promising as biomarkers for the identification of clinical statin intolerance in patients with self-perceived SAMS, but the findings have to be confirmed in larger studies.

Clinical performance of volumetric finger-prick sampling for the monitoring of tacrolimus, creatinine and haemoglobin in kidney transplant recipients.

AIMS: Finger-prick sampling has emerged as an attractive tool for therapeutic drug monitoring and associated diagnostics. We aimed to validate the clinical performance of using two volumetric devices (Capitainer® qDBS and Mitra®) for monitoring tacrolimus, creatinine and haemoglobin in kidney transplant (KTx) recipients. Secondarily, we evaluated potential differences between finger-prick sampling performed by healthcare professionals vs. self-sampling, and differences between the two devices. METHODS: We compared finger-prick and venous sampling in three settings: microsampling performed by healthcare personnel, self-sampling under supervision, unsupervised self-sampling. The finger-prick samples were analysed with adapted methods and results compared to routine method analysis of the venous blood samples. RESULTS: Twenty-five KTx recipients completed the main study and 12 KTx recipients completed a post hoc validation study. For tacrolimus measurements and predicted area under the curve, the proportions within ±20% difference were 79%-96% for Capitainer and 77%-95% for Mitra. For creatinine and haemoglobin, the proportions within ±15% were 92%-100% and 93%-100% for Capitainer and 79%-96% and 67%-92% for Mitra, respectively. Comparing sampling situations, the success rate was consistent for Capitainer (92%-96%), whereas Mitra showed 72%-88% and 52%-72% success rates with samples collected by healthcare personnel and the patients themselves. CONCLUSIONS: Capitainer and Mitra are technically feasible for measuring tacrolimus, creatinine and haemoglobin. In the context of self-sampling, Capitainer maintained consistent sampling success and analytical quality. Implementing volumetric finger-prick self-sampling for the monitoring of tacrolimus, creatinine and haemoglobin may simplify and improve the follow-up of KTx recipients.

Atorvastatin Metabolite Pattern in Skeletal Muscle and Blood from Patients with Coronary Heart Disease and Statin-Associated Muscle Symptoms.

Self-perceived statin-associated muscle symptoms (SAMS) are prevalent, but only a minority is drug-dependent. Diagnostic biomarkers are not yet identified. The local statin exposure in skeletal muscle tissue may correlate to the adverse effects. We aimed to determine whether atorvastatin metabolites in blood reflect the corresponding metabolite levels in skeletal muscle, and whether genetic variants of statin transporters modulate this relationship. We also addressed atorvastatin metabolites as potential objective biomarkers of SAMS. Muscle symptoms were examined in patients with coronary disease and self-perceived SAMS during 7 weeks of double-blinded treatment with atorvastatin 40 mg/day and placebo in randomized order. A subset of 12 patients individually identified with more muscle symptoms on atorvastatin than placebo (confirmed SAMS) and 15 patients with no difference in muscle symptom intensity (non-SAMS) attended the present follow-up study. All received 7 weeks of treatment with atorvastatin 40 mg/day followed by 8 weeks without statins. Biopsies from the quadriceps muscle and blood plasma were collected after each treatment period. Strong correlations (rho > 0.7) between muscle and blood plasma concentrations were found for most atorvastatin metabolites. The impact of the SLCO1B1 c.521T>C (rs4149056) gene variant on atorvastatin's systemic pharmacokinetics was translated into muscle tissue. The SLCO2B1 c.395G>A (rs12422149) variant did not modulate the accumulation of atorvastatin metabolites in muscle tissue. Atorvastatin pharmacokinetics in patients with confirmed SAMS were not different from patients with non-SAMS. In conclusion, atorvastatin metabolite levels in skeletal muscle and plasma are strongly correlated, implying that plasma measurements are suitable proxies of atorvastatin exposure in muscle tissue. The relationship between atorvastatin metabolites in plasma and SAMS deserves further investigation.

Monitoring Simvastatin Adherence in Patients With Coronary Heart Disease: A Proof-of-Concept Study Based on Pharmacokinetic Measurements in Blood Plasma.

BACKGROUND: Poor statin adherence remains a public health concern associated with adverse outcomes. We evaluated the use of pharmacokinetic measurements to monitor adherence to simvastatin in patients with coronary heart disease (CHD). METHODS: Eighteen patients with CHD taking an evening dose of simvastatin 20 mg (n = 7), 40 mg (n = 5), or 80 mg (n = 6) were examined at steady-state pharmacokinetics. Ten patients were instructed to interrupt simvastatin dosing and return for blood sampling for the subsequent 3 days. Dose-normalized plasma concentrations of simvastatin lactone and simvastatin acid and the sum of the 2 were evaluated to discriminate between adherent dosing and dose omission. Bioanalytical quantification was performed using liquid chromatography-tandem mass spectrometry. RESULTS: A simvastatin acid cutoff of 1.0 × 10 -2 nmol -1 ·L -1 ·mg -1 identified 100% of those omitting 2 doses and 60% of those omitting a single dose. Simvastatin acid showed superior ability to discriminate dose omission, as well as the best agreement between samples handled at ambient and cool temperatures (median deviation 3.5%; interquartile range -2.5% to 13%). The cutoff for a morning dose schedule, with a similar ability to discriminate, was estimated at 2.0 × 10 -3 nmol -1 ·L -1 ·mg -1 . CONCLUSIONS: The present method discriminated between adherence and reduced adherence to simvastatin therapy in patients with CHD. Sample handling is feasible for routine practice, and the assessment of adherence can be performed by direct measurement of simvastatin acid in a blood sample, according to defined cutoff values. Further studies validating the cutoff value and utility for clinical application are encouraged.

Clinical and psychological factors in coronary heart disease patients with statin associated muscle side-effects.

BACKGROUND: To compare clinical and psychological factors among patients with self-perceived statin-associated muscle symptoms (SAMS), confirmed SAMS, and refuted SAMS in coronary heart disease patients (CHD). METHODS: Data were obtained from a cross-sectional study of 1100 CHD outpatients and a study of 71 CHD outpatients attending a randomized, double-blinded, placebo-controlled, crossover study to test effects of atorvastatin 40 mg/day on muscle symptom intensity. Clinical and psychosocial factors were compared between patients with and without SAMS in the cross-sectional study, and between patients with confirmed SAMS and refuted SAMS in the randomized study. RESULTS: Bilateral, symmetric muscle symptoms in the lower extremities during statin treatment were more prevalent in patients with confirmed SAMS compared to patients with refuted SAMS (75% vs. 41%, p = 0.01) in the randomized study. No significant differences in psychological factors (anxiety, depression, worry, insomnia, type D personality characteristics) were detected between patients with and without self-perceived SAMS in the cross-sectional study, or between patients with confirmed SAMS and refuted SAMS, in the randomized study. CONCLUSIONS: Patients with confirmed SAMS more often present with bilateral lower muscle symptoms compared to those with refuted SAMS. Psychological factors were not associated with self-perceived SAMS or confirmed SAMS. A careful pain history and a search for alternative causes of muscle symptoms are likely to promote communication in patients with SAMS, and may reduce the risk for statin discontinuation.

The relationship between directly measured statin adherence, self-reported adherence measures and cholesterol levels in patients with coronary heart disease.

BACKGROUND AND AIMS: We aimed to determine the relationship between statin adherence measured directly, and by self-report measures and serum cholesterol levels. METHODS: Patients prescribed atorvastatin (N = 373) participated in a cross-sectional study 2-36 months after a coronary event. Self-reported adherence included statin adherence the past week, the 8-item Morisky medication adherence scale (MMAS-8), and the Gehi et al. adherence question. Atorvastatin was measured directly in spot blood plasma by a novel liquid chromatography tandem mass-spectrometry method discriminating adherence (0-1 doses omitted) and reduced adherence (≥2 doses omitted). Participants were unaware of the atorvastatin analyses at study participation. RESULTS: Mean age was 63 (SD 9) years and 8% had reduced atorvastatin adherence according to the direct method. In patients classified with reduced adherence by the direct method, 40% reported reduced statin adherence, 32% reported reduced adherence with the MMAS-8 and 22% with the Gehi question. In those adherent by the direct method, 96% also reported high statin adherence, 95% reported high adherence on the MMAS-8 whereas 94% reported high adherence on the Gehi question. Cohen's kappa agreement score with the direct method was 0.4 for self-reported statin adherence, 0.3 for the Gehi question and 0.2 for the MMAS-8. Adherence determined by the direct method, self-reported statin adherence last week, and the Gehi question was inversely related to LDL-cholesterol levels with a p-value of <0.001, 0.001 and 0.004, respectively. CONCLUSIONS: Plasma-statin measurements reveal reduced adherence with higher sensitivity than self-report measures, relate to cholesterol levels, and may prove to be a useful tool to improve lipid management.

In vitro assessments predict that CYP3A4 contributes to a greater extent than CYP3A5 to prednisolone clearance.

Because several steroid hormones are metabolized to their respective 6ß-hydroxy forms by CYP3A4 and CYP3A5, these isoenzymes have been assumed to metabolize the immunosuppressive drug prednisolone, with conflicting results in the literature with respect to their relative importance. A direct study of the metabolism of prednisolone by microsomal CYP3A4 and CYP3A5 is missing. The aim of this in vitro study was to investigate the relative importance of recombinant CYP3A4 and recombinant CYP3A5 in the metabolism of prednisolone and to compare the extent of formation of 6ß-OH-prednisolone by the two enzymes. Through in vitro incubations using rCYP3A4 and rCYP3A5 enzymes, intrinsic clearance (CLint ) of prednisolone was determined by the substrate depletion approach. Formation of the metabolite 6ß-OH-prednisolone by rCYP3A4 and rCYP3A5, respectively, was compared. Prednisolone concentrations were measured, and its metabolite 6ß-OH-prednisolone was identified using a HPLC-MS/MS in-house method. CLint for prednisolone by rCYP3A5 was less than 26% relative to rCYP3A4. Formation of 6ß-OH-prednisolone by rCYP3A5 was less than 11% relative to rCYP3A4. The study indicates that 6ß-hydroxylation of prednisolone assessed in vitro in recombinant CYP enzymes depends on rCYP3A4 rather than rCYP3A5 and that CYP3A5 may be responsible for the formation of other prednisolone metabolite(s) in addition to 6ß-OH-prednisolone.

Fast and reliable quantification of busulfan in blood plasma using two-channel liquid chromatography tandem mass spectrometry: Validation of assay performance in the presence of drug formulation excipients.

A fast and reliable method based on two-channel liquid chromatography coupled to tandem mass spectrometry was developed and successfully validated for quantification of busulfan. The drug vehicle polyethylene glycol 400 was quantified simultaneously in patient samples. The sample preparation consisted of simple protein precipitation using a mixture of methanol and zinc sulphate containing busulfan-d8 as internal standard. Chromatographic separation was performed on a short biphenyl column (30 mm × 3.0 mm, 5 µm particles) using a step gradient from 30 % to 85 % methanol, ensuring co-elution of the analyte and internal standard. Quantification was performed using the mass transition of 264.1 > 151.1 for busulfan and 272.1 > 159.1 for the internal standard. Using only 20 µL of plasma sample, the lower limit of quantification was 25 ng/mL. Signal to noise ratio at the lower limit of quantification exceeded 300. The assay performance was not adversely affected by matrix effects originating from drug formulation excipients or other sample components. The coefficient of variation was ≤4 % and the mean accuracy 101-108 % across the calibration range 25-5 000 ng/mL. Chromatographic run time was 2 min and 8 s, allowing an effective run-time of 1 min and 10 s when using two alternating LC-channels. The assay has been implemented in routine practice with accreditation according to the ISO 15189 standard, and performs well in external quality control assessments. We present for the first time that shortly after an IV infusion of busulfan, the plasma levels of polyethylene glycol 400 may be in the range of 400-800 mg/L. The presence of these levels of detergent in patient samples may have detrimental effects on assay performance in LC-MS/MS, not limited to busulfan assays. This may be a concern for any LC-MS/MS analysis performed on samples collected within the first 24 h after an IV infusion of busulfan.

Personalized Therapy for Mycophenolate: Consensus Report by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.

ABSTRACT: When mycophenolic acid (MPA) was originally marketed for immunosuppressive therapy, fixed doses were recommended by the manufacturer. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA area under the curve based on the measurement of drug concentrations in only a few samples. This approach is feasible in the clinical routine and has proven successful in terms of correlation with outcome. However, the search for superior correlates has continued, and numerous studies in search of biomarkers that could better predict the perfect dosage for the individual patient have been published. As it was considered timely for an updated and comprehensive presentation of consensus on the status for personalized treatment with MPA, this report was prepared following an initiative from members of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Topics included are the criteria for analytics, methods to estimate exposure including pharmacometrics, the potential influence of pharmacogenetics, development of biomarkers, and the practical aspects of implementation of target concentration intervention. For selected topics with sufficient evidence, such as the application of limited sampling strategies for MPA area under the curve, graded recommendations on target ranges are presented. To provide a comprehensive review, this report also includes updates on the status of potential biomarkers including those which may be promising but with a low level of evidence. In view of the fact that there are very few new immunosuppressive drugs under development for the transplant field, it is likely that MPA will continue to be prescribed on a large scale in the upcoming years. Discontinuation of therapy due to adverse effects is relatively common, increasing the risk for late rejections, which may contribute to graft loss. Therefore, the continued search for innovative methods to better personalize MPA dosage is warranted.

Tacrolimus Measured in Capillary Volumetric Microsamples in Pediatric Patients-A Cross-Validation Study.

BACKGROUND: Therapeutic drug monitoring of tacrolimus (Tac) is mandatory in solid organ transplant (SOT) recipients. Finger-prick microsampling is more flexible and tolerable during the therapeutic drug monitoring of tacrolimus and has been shown to be applicable in adult SOT recipients. In this study, a previously validated method applying volumetric absorptive microsampling (VAMS) to measure Tac in adults was cross-validated in a pediatric population. METHODS: Patients with SOT scheduled for standard posttransplant follow-up visits were recruited. Blood samples were obtained by trained phlebotomists using standard venipuncture and capillary microsampling, before the morning dose of Tac as well as 2 and 5 hours after dosing. Tac concentrations were quantified using liquid chromatography-tandem mass spectrometry. Concordance between Tac concentrations obtained with venipuncture and VAMS was evaluated using Passing-Bablok regression, calculation of absolute and relative differences, and percentage of samples within ±20% and ±30% difference. RESULTS: A total of 39 SOT patients aged 4-18 years (22 male) were included. The median (range) predose venous blood concentration was 4.8 (2.6-13.6) mcg/L, with a difference between VAMS and venous blood samples of -0.2 ± 0.7 mcg/L. The relative mean difference was -1.3% [95% confidence interval (CI), -5.9% to 3.4%]. Ninety-two percent and 97% of the sample pairs demonstrated differences within ±20% and ±30%, respectively. Postdose (2 hours and/or 5 hours, n = 17) median concentration in venous blood was 7.9 (4.8-19.2) mcg/L. The difference between VAMS and venous blood samples was 0.1 ± 1.0 mcg/L, with a relative mean difference of -2.5% (95% confidence interval, -8.8% to 3.8%). Eighty-eight percent of the postdose sample pairs were within ±20% difference, and all were within ±30% difference. CONCLUSIONS: Tac concentrations can be accurately measured using VAMS technology in pediatric SOT recipients. This makes home-based Tac monitoring feasible in the pediatric population.

Effect of atorvastatin on muscle symptoms in coronary heart disease patients with self-perceived statin muscle side effects: a randomized, double-blinded crossover trial.

AIMS: To estimate the effect of atorvastatin on muscle symptom intensity in coronary heart disease (CHD) patients with self-perceived statin-associated muscle symptoms (SAMS) and to determine the relationship to blood levels of atorvastatin and/or metabolites. METHODS AND RESULTS: A randomized multi-centre trial consecutively identified 982 patients with previous or ongoing atorvastatin treatment after a CHD event. Of these, 97 (9.9%) reported SAMS and 77 were randomized to 7-week double-blinded treatment with atorvastatin 40 mg/day and placebo in a crossover design. The primary outcome was the individual mean difference in muscle symptom intensity between the treatment periods, measured by visual-analogue scale (VAS) scores. Atorvastatin did not affect the intensity of muscle symptoms among 71 patients who completed the trial. Mean VAS difference (statin-placebo) was 0.31 (95% CI: -0.24 to 0.86). The proportion with more muscle symptoms during placebo than atorvastatin was 17% (n = 12), 55% (n = 39) had the same muscle symptom intensity during both treatment periods whereas 28% (n = 20) had more symptoms during atorvastatin than placebo (confirmed SAMS). There were no differences in clinical or pharmacogenetic characteristics between these groups. The levels of atorvastatin and/or metabolites did not correlate to muscle symptom intensity among patients with confirmed SAMS (Spearman's rho ≤0.40, for all variables). CONCLUSION: Re-challenge with high-intensity atorvastatin did not affect the intensity of muscle symptoms in CHD patients with self-perceived SAMS during previous atorvastatin therapy. There was no relationship between muscle symptoms and the systemic exposure to atorvastatin and/or its metabolites. The findings encourage an informed discussion to elucidate other causes of muscle complaints and continued statin use.

Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing.

Gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC) is attributed to cancer cell-intrinsic drug processing and the impact of the tumor microenvironment, especially pancreatic stellate cells (PSCs). This study uses human PDAC-derived paired primary cancer cells (PCCs) and PSCs from four different tumors, and the PDAC cell lines BxPC-3, Mia PaCa-2, and Panc-1, to assess the fate of gemcitabine by measuring its cellular uptake, cytotoxicity, and LC-MS/MS-based metabolite analysis. Expression analysis and siRNA-mediated knockdown of key regulators of gemcitabine (hENT1, CDA, DCK, NT5C1A) was performed. Compared to PSCs, both the paired primary PCCs and cancer cell lines showed gemcitabine-induced dose-dependent cytotoxicity, high uptake, as well as high and variable intracellular levels of gemcitabine metabolites. PSCs were gemcitabine-resistant and demonstrated significantly lower drug uptake, which was not influenced by co-culturing with their paired PCCs. Expression of key gemcitabine regulators was variable, but overall strong in the cancer cells and significantly lower or undetectable in PSCs. In cancer cells, hENT1 inhibition significantly downregulated gemcitabine uptake and cytotoxicity, whereas DCK knockdown reduced cytotoxicity. In conclusion, heterogeneity in gemcitabine processing among different pancreatic cancer cells and stellate cells results from the differential expression of molecular regulators which determines the effect of gemcitabine.

Fasting Status and Circadian Variation Must be Considered When Performing AUC-based Therapeutic Drug Monitoring of Tacrolimus in Renal Transplant Recipients.

Therapeutic drug monitoring (TDM) is mandatory for the immunosuppressive drug tacrolimus (Tac). For clinical applicability, TDM is performed using morning trough concentrations. With recent developments making tacrolimus concentration determination possible in capillary microsamples and Bayesian estimator predicted area under the concentration curve (AUC), AUC-guided TDM may now be clinically applicable. Tac circadian variation has, however, been reported, with lower systemic exposure following the evening dose. The aim of the present study was to investigate tacrolimus pharmacokinetic (PK) after morning and evening administrations of twice-daily tacrolimus in a real-life setting without restrictions regarding food and concomitant drug timing. Two 12 hour tacrolimus investigations were performed; after the morning dose and the following evening dose, respectively, in 31 renal transplant recipients early after transplantation both in a fasting-state and under real-life nonfasting conditions (14 patients repeated the investigation). We observed circadian variation under fasting-conditions: 45% higher peak-concentration and 20% higher AUC following the morning dose. In the real-life nonfasting setting, the PK-profiles were flat but comparable after the morning and evening doses, showing slower absorption rate and lower AUC compared with the fasting-state. Limited sampling strategies using concentrations at 0, 1, and 3 hours predicted AUC after fasting morning administration, and samples obtained at 1, 3, and 6 hours predicted AUC for the other conditions (evening and real-life nonfasting). In conclusion, circadian variation of tacrolimus is present when performed in patients who are in the fasting-state, whereas flatter PK-profiles and no circadian variation was present in a real-life, nonfasting setting.

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.

Comparing the Effects of the mTOR Inhibitors Azithromycin and Rapamycin on In Vitro Expanded Regulatory T Cells.

Adoptive transfer of autologous polyclonal regulatory T cells (Tregs) is a promising option for reducing graft rejection in allogeneic transplantation. To gain therapeutic levels of Tregs there is a need to expand obtained cells ex vivo, usually in the presence of the mTOR inhibitor Rapamycin due to its ability to suppress proliferation of non-Treg T cells, thus promoting a purer Treg yield. Azithromycin is a bacteriostatic macrolide with mTOR inhibitory activity that has been shown to exert immunomodulatory effects on several types of immune cells. In this study we investigated the effects of Azithromycin, compared with Rapamycin, on Treg phenotype, growth, and function when expanding bulk, naïve, and memory Tregs. Furthermore, the intracellular concentration of Rapamycin in CD4+ T cells as well as in the culture medium was measured for up to 48 h after supplemented. Treg phenotype was assessed by flow cytometry and Treg function was measured as inhibition of responder T-cell expansion in a suppression assay. The concentration of Rapamycin was quantified with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Azithromycin and Rapamycin both promoted a FoxP3-positive Treg phenotype in bulk Tregs, while Rapamycin also increased FoxP3 and FoxP3+Helios positivity in naïve and memory Tregs. Furthermore, Rapamycin inhibited the expansion of naïve Tregs, but also increased their suppressive effect. Rapamycin was quickly degraded in 37°C medium, yet was retained intracellularly. While both compounds may benefit expansion of FoxP3+ Tregs in vitro, further studies elucidating the effects of Azithromycin treatment on Tregs are needed to determine its potential use.

Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report.

Ten years ago, a consensus report on the optimization of tacrolimus was published in this journal. In 2017, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicity (IATDMCT) decided to issue an updated consensus report considering the most relevant advances in tacrolimus pharmacokinetics (PK), pharmacogenetics (PG), pharmacodynamics, and immunologic biomarkers, with the aim to provide analytical and drug-exposure recommendations to assist TDM professionals and clinicians to individualize tacrolimus TDM and treatment. The consensus is based on in-depth literature searches regarding each topic that is addressed in this document. Thirty-seven international experts in the field of TDM of tacrolimus as well as its PG and biomarkers contributed to the drafting of sections most relevant for their expertise. Whenever applicable, the quality of evidence and the strength of recommendations were graded according to a published grading guide. After iterated editing, the final version of the complete document was approved by all authors. For each category of solid organ and stem cell transplantation, the current state of PK monitoring is discussed and the specific targets of tacrolimus trough concentrations (predose sample C0) are presented for subgroups of patients along with the grading of these recommendations. In addition, tacrolimus area under the concentration-time curve determination is proposed as the best TDM option early after transplantation, at the time of immunosuppression minimization, for special populations, and specific clinical situations. For indications other than transplantation, the potentially effective tacrolimus concentrations in systemic treatment are discussed without formal grading. The importance of consistency, calibration, proficiency testing, and the requirement for standardization and need for traceability and reference materials is highlighted. The status for alternative approaches for tacrolimus TDM is presented including dried blood spots, volumetric absorptive microsampling, and the development of intracellular measurements of tacrolimus. The association between CYP3A5 genotype and tacrolimus dose requirement is consistent (Grading A I). So far, pharmacodynamic and immunologic biomarkers have not entered routine monitoring, but determination of residual nuclear factor of activated T cells-regulated gene expression supports the identification of renal transplant recipients at risk of rejection, infections, and malignancy (B II). In addition, monitoring intracellular T-cell IFN-g production can help to identify kidney and liver transplant recipients at high risk of acute rejection (B II) and select good candidates for immunosuppression minimization (B II). Although cell-free DNA seems a promising biomarker of acute donor injury and to assess the minimally effective C0 of tacrolimus, multicenter prospective interventional studies are required to better evaluate its clinical utility in solid organ transplantation. Population PK models including CYP3A5 and CYP3A4 genotypes will be considered to guide initial tacrolimus dosing. Future studies should investigate the clinical benefit of time-to-event models to better evaluate biomarkers as predictive of personal response, the risk of rejection, and graft outcome. The Expert Committee concludes that considerable advances in the different fields of tacrolimus monitoring have been achieved during this last decade. Continued efforts should focus on the opportunities to implement in clinical routine the combination of new standardized PK approaches with PG, and valid biomarkers to further personalize tacrolimus therapy and to improve long-term outcomes for treated patients.