Therapeutic Drug Monitoring and Dosage Adjustments of Immunosuppressive Drugs When Combined With Nirmatrelvir/Ritonavir in Patients With COVID-19.

ABSTRACT: Nirmatrelvir/ritonavir (Paxlovid) consists of a peptidomimetic inhibitor (nirmatrelvir) of the SARS-CoV-2 main protease and a pharmacokinetic enhancer (ritonavir). It is approved for the treatment of mild-to-moderate COVID-19. This combination of nirmatrelvir and ritonavir can mediate significant and complex drug-drug interactions (DDIs), primarily due to the ritonavir component. Indeed, ritonavir inhibits the metabolism of nirmatrelvir through cytochrome P450 3A (CYP3A) leading to higher plasma concentrations and a longer half-life of nirmatrelvir. Coadministration of nirmatrelvir/ritonavir with immunosuppressive drugs (ISDs) is particularly challenging given the major involvement of CYP3A in the metabolism of most of these drugs and their narrow therapeutic ranges. Exposure of ISDs will be drastically increased through the potent ritonavir-mediated inhibition of CYP3A, resulting in an increased risk of adverse drug reactions. Although a decrease in the dosage of ISDs can prevent toxicity, an inappropriate dosage regimen may also result in insufficient exposure and a risk of rejection. Here, we provide some general recommendations for therapeutic drug monitoring of ISDs and dosing recommendations when coadministered with nirmatrelvir/ritonavir. Particularly, tacrolimus should be discontinued, or patients should be given a microdose on day 1, whereas cyclosporine dosage should be reduced to 20% of the initial dosage during the antiviral treatment. Dosages of mammalian target of rapamycin inhibitors (m-TORis) should also be adjusted while dosages of mycophenolic acid and corticosteroids are expected to be less impacted.

Structural aspects of hepatitis E virus.

Hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. Hepatitis E is an enterically transmitted zoonotic disease that causes large waterborne epidemic outbreaks in developing countries and has become an increasing public-health concern in industrialized countries. In this setting, the infection is usually acute and self-limiting in immunocompetent individuals, although chronic cases in immunocompromised patients have been reported, frequently associated with several extrahepatic manifestations. Moreover, extrahepatic manifestations have also been reported in immunocompetent individuals with acute HEV infection. HEV belongs to the alphavirus-like supergroup III of single-stranded positive-sense RNA viruses, and its genome contains three partially overlapping open reading frames (ORFs). ORF1 encodes a nonstructural protein with eight domains, most of which have not been extensively characterized: methyltransferase, Y domain, papain-like cysteine protease, hypervariable region, proline-rich region, X domain, Hel domain, and RNA-dependent RNA polymerase. ORF2 and ORF3 encode the capsid protein and a multifunctional protein believed to be involved in virion release, respectively. The novel ORF4 is only expressed in HEV genotype 1 under endoplasmic reticulum stress conditions, and its exact function has not yet been elucidated. Despite important advances in recent years, the biological and molecular processes underlying HEV replication remain poorly understood, primarily due to a lack of detailed information about the functions of the viral proteins and the mechanisms involved in host-pathogen interactions. This review summarizes the current knowledge concerning HEV proteins and their biological properties, providing updated detailed data describing their function and focusing in detail on their structural characteristics. Furthermore, we review some unclear aspects of the four proteins encoded by the ORFs, highlighting the current key information gaps and discussing potential novel experimental strategies for shedding light on those issues.

Predictive Cardiometabolic Risk Profiling of Patients Using Vascular Age in Liver Transplantation.

Liver transplantation is the last therapeutic option in patients with end-stage liver diseases. The adequate clinical management of transplant-patients impacts their vital prognosis and decisions on many occasions are made from the interaction of multiple variables involved in the process. This work is based on the National Liver Transplantation Program in Uruguay. We performed predictive analysis of cardiometabolic diseases on the transplanted cohort between 2014 and 2019, considering vascular age as a key factor. This aims at classification of the cohort based on the vascular age of the evaluated patients before transplantation for risk-profiling. Predicted high-risk group of the patients showed substantial deterioration of post-transplant health-conditions, including higher mortality rate. In our knowledge, this is the first study in Latin America incorporating vascular age toward predictive analysis of cardiometabolic risk factors in liver transplantations. Predictive risk-modeling using vascular age in a pre-transplantation scenario provides significant opportunity for early prediction of post-transplant risk factors, leading to efficient treatment with anticipation.

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.

Resultados del Programa Nacional de Trasplante Hepático del Uruguay a 10 años de su inicio / Results of the National Program of Liver Transplantation in Uruguay 10 years after it was initiated / Resultados do Programa Nacional de Transplante de Fígado do Uruguai 10 anos após seu início

Resumen: Introducción: el trasplante hepático (TH) constituye el tratamiento de elección en pacientes con enfermedades hepáticas severas e irreversibles, sin opción de tratamientos alternativos eficaces. La medición de indicadores de calidad permite detectar problemáticas susceptibles de ser mejoradas a fin de optimizar los resultados. Objetivo: presentar los resultados del Programa Nacional de Trasplante Hepático (PNTH) del Uruguay a 10 años de su implementación y compararlos con los estándares de calidad internacionales. Material y método: estudio retrospectivo de los TH realizados del 14/7/2009 al 14/7/2019. Resultados: N: 190 TH. Edad promedio: 45 años. Sexo: 60% hombres. MELD promedio al TH: 21. Principales indicaciones: cirrosis (59%) y hepatocarcinoma (21%). Mortalidad posoperatoria: 7,4% y perioperatoria: 2,1% (estándares <10% y 1%). Tasa de retrasplante: precoz 3,7% y tardío 4,2% (estándares <5% y 8%). Tasa de reintervención: 13,1% (estándar <10%) y de no función primaria: 2,6% (estándar <2%). Sobrevida: 86,6% al año, 81,8% a 3, 77,4% a 5 y 63,2% a 10 años (estándares >80, 75, 70 y 60%). Pacientes evaluados en menos de 30 días: 47% (estándar >75%). Tasa de hígados no implantados sin causa objetiva: 0,5% (estándar <1%). El 86% de los usuarios expresaron satisfacción (estándar >80%). Mortalidad en lista: 19% (estándar <15%). Mortalidad precoz con hígado funcionante: 1% (estándar <1%). Conclusiones: el PNTH del Uruguay cumple con la mayoría de los indicadores de calidad, presentando resultados en sobrevida por encima de los estándares internacionales.

Summary: Introduction: liver transplantation constitutes the first therapy chosen by patients with severe and irreversible liver conditions, when no effective alternative options are available. Measurement of quality indicators allow for the detection of problems that may be solved in order to optimize results. Objective: to present the results obtained in the National Program of Liver Transplantation in Uruguay, 10 years after its implementation and to compare them to international quality standards. Method: retrospective study of liver transplantations performed from July 14, 2009 through July 14, 2019. Results: N: 190 Liver transplantations (LT). Average age: 45 years old. Gender: 60% male. MELD average MELD (Model for End-stage Liver Disease) upon LT: 21. Main indications: cirrhosis he(59%) y hepatocarcinoma (21%). Post-surgery mortality: 7.4% and peri-operative mortality 2.1% (standards <10 and 1%). Re-transplantation rate: early 3.7% and late 4.2% (standards <5% and 8%). Reoperation rate: 13.1% (standard <10%) and of non-primary function: 2.6% (standard <2%). Survival: 86.6% per year, 81.8% after 3 years, 77.4% after 5 and 63.2% after 10 years (standards >80, 75, 70 and 60%). Patients assessed in less than 30 days: 47% (standard >75%). Non-implanted livers with no objective cause rate: 0.5% (standard <1%). 86% of users stated they were satisfied (standard >80%). Mortality in the waiting list: 19% (standard <15%). Early mortality with functioning liver: 1% (standard <1%). Conclusions: national Program of Liver Transplantation in Uruguay meets most quality indicators standards, evidencing survival results that are above international standards.

Resumo: Introdução: o transplante de fígado (TH) é o tratamento de escolha em pacientes com doenças hepáticas graves e irreversíveis, sem a opção de tratamentos alternativos eficazes. A medição de indicadores de qualidade permite detectar problemas que podem ser melhorados para otimizar os resultados. Objetivo: apresentar os resultados do Programa Nacional de Transplante de Fígado (PNTH) do Uruguai 10 anos após sua implantação e compará-los com os padrões internacionais de qualidade. Materiais e métodos: estudo retrospectivo do HT realizado de 14/07/2009 a 14/07/2019. Resultados: N: 190 TH. Idade média: 45 anos. Sexo: 60% homens. Escala MELD média no TH: 21. Principais indicações: cirrose (59%) e hepatocarcinoma (21%). Mortalidade pós-operatória: 7,4% e peri-operatória 2,1% (padrões <10 e 1%). Taxa de retransplante: 3,7% inicial e 4,2% tardio (padrão <5% e 8%). Taxa de reintervenção: 13,1% (padrão <10%) e não função primária: 2,6% (padrão <2%). Sobrevivência: 86,6% em 1 ano, 81,8% em 3, 77,4% em 5 e 63,2% em 10 anos (padrões> 80, 75, 70 e 60%). Pacientes avaliados em menos de 30 dias: 47% (padrão> 75%). Taxa de fígados não implantados sem causa objetiva: 0,5% (padrão <1%). 86% dos usuários expressaram satisfação (padrão> 80%). Mortalidade em lista de espera: 19% (padrão <15%). Mortalidade precoce com fígado funcionante: 1% (padrão <1%). Conclusões: o PNTH do Uruguai cumpre a maioria dos indicadores de qualidade, apresentando resultados de sobrevivência acima dos padrões internacionais.

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.

Activity of the Calcineurin Pathway in Patients on the Liver Transplantation Waiting List: Factors of Variability and Response to Tacrolimus Inhibition.

BACKGROUND: We sought to evaluate, in patients on a liver transplantation waiting list, potential biomarkers of the base calcineurin pathway activity with use of a new model of nonstimulated peripheral blood mononuclear cells (PBMC) and ex vivo response to tacrolimus (TAC). METHODS: The calcineurin pathway activity was explored ex vivo in stimulated and nonstimulated PBMC from 19 patients. The inhibition of NFAT1 translocation to PBMC nuclei, expression of intracellular IL-2, and membrane CD25 in different T-cell subsets were measured by multiparametric flow cytometry before and after exposure to TAC. We also studied the influence on the individual response of polymorphisms in 3 key genes of the calcineurin pathway: PPIA, PPP3CA, and IL2RA. RESULTS: All pharmacodynamics profiles closely fitted an I/Imax sigmoid model. Interindividual variability was higher in nonstimulated than in stimulated conditions, as well as in the presence of TAC. IL-2+CD8+ cells at TAC Imax showed the highest interindividual variability, suggesting its usefulness as a biomarker of individual TAC effects integrating many different sources of regulation and variability. Moreover, in the absence of TAC, patients with end-stage liver disease exhibited lower NFAT1 translocation and T-cell activation than healthy volunteers from a previous study under similar conditions. Multivariate statistical analysis showed strong and significant associations between TAC pharmacodynamic parameters and 2 polymorphisms in the gene-coding cyclophilin A (rs8177826 and rs6850). CONCLUSIONS: We show the feasibility of using nonstimulated PBMCs to explore the calcineurin pathway under more physiologic conditions and point toward potential biomarkers for TAC pharmacodynamic monitoring. ClinicalTrials.gov Identifier: NCT01760356.

Actualización acerca de colistina (polimixina E): aspectos clínicos, PK/PD y equivalencias / Colistin: an update on the antibiotic: clinical aspects PK/PD indices and equivalences

En los últimos diez años, en esta era posantibiótica, la colistina ha resurgido como un último recurso frente a infecciones por patógenos como Pseudomonas aeruginosa, Acinetobacter baumannii complex y enterobacterias productoras de carbapenemasas. Cayó en desuso previamente dados sus efectos adversos potencialmente graves, como la nefro y neurotoxicidad, pero hoy revive como parte fundamental de los planes antibióticos frente a patógenos extremadamente resistentes. El aumento de su uso conlleva a la emergencia de resistencia, encontrándonos frente a una situación potencialmente catastrófica, en particular dada la recientemente descrita presencia de plásmidos transferibles entre especies conteniendo genes que confieren resistencia a colistina (gen mcr-1), mecanismo detectado también en nuestro país. En la presente revisión, basados en los conocimientos actuales sobre la farmacocinética y la farmacodinamia, se describe en detalle la dosificación apropiada con necesidad de realizar dosis carga para alcanzar los niveles terapéuticos adecuados, así como aspectos prácticos de la administración en casos de meningitis/ventriculitis posquirúrgica y del empleo por vía nebulizada para el tratamiento de la neumonía asociada a la ventilación. Se destaca la necesidad de su uso combinado con otro antibiótico activo in vitro, en particular en pacientes críticos y en aquellos con un clearance de creatininemia mayor a 80 ml/min. La biterapia es necesaria en particular si la concentración inhibitoria mínima (CIM) del patógeno es mayor a 1 mg/l, debido al riesgo de subdosificación y emergencia de resistencia intratratamiento. En una segunda sección se aborda la complejidad de la dosificación en función de las distintas presentaciones comercializadas a nivel nacional que han conducido a errores en la posología, con un riesgo mayor de eventual toxicidad. Con el objetivo de mejorar la comprensión referente a la rotulación de la dosis a administrar de colistina se revisaron los insertos y envases primarios de las presentaciones de colistina comercialmente disponibles en Uruguay, a efectos de solicitar formal y documentalmente a las empresas farmacéuticas representantes, se expidan en cuanto a los contenidos de colistina (droga activa) y de su profármaco, el colistimetato sódico (CMS). Finalmente se elaboraron una serie de recomendaciones en cuanto a la información que a entender de los autores debieran exhibir las distintas presentaciones de colistina comercialmente disponibles para no inducir a errores en la prescripción.

In the recent 10 years, in this post-antibiotic era, colisitin has reappeared as the last resource to face infections caused by Pseudomonas aeruginosa, Acinetobacter baumannii complex and carbapenemase-producing enterobacteriaceae. At some point the use of colistin was discontinued given its potentially severe side effects, such as nephro and neurotoxicity , although it reemerges today as an essential part of antibiotic plans when facing extremely resistant pathogens. This increase in the use of colistin has led to an antibiotic resistance emergency and thus today we face a potentially catastrophic situation. This happens in particular in connection with the recently described presence of transferable plasmids among species containing genes that confer resistance to colistin (gen mcr-1), a mechanism also identified in our country. This review describes in detail the right dosing with the need to make load doses to achieve the appropriate therapeutic levels, based on current knowledge on pharmacokinetics and pharmacodynamics, as well as practical aspects in the administration of the drug in cases of postsurgical meningitis/ventriculitis and the use by nebulization for the treatment of ventilation associated pneumonia. The study points out the need to use colistin along with another in-vitro active antibiotic, especially in critical patients and those with a creatinine clearance over 80 ml/min. Dual pharmacotherapy is necessary in particular if the pathogen´s minimuminhibitory concentration (MIC) is higher than 1 mg/min, due to intra-treatment risk of sub-dosing and resistance emergency. Likewise, in second section, the study addresses the complex nature of dosing given by the different presentations available in the market at the national level, what has resulted in dosage errors, and thus a higher risk of toxicity. The insets and primary packaging of colistin presentation available in the Uruguayan marketing were reviewed with the purpose of improving understanding in connection with the labeling of the doses to be administered. With that information, the pharmaceutical industries agents will be formally asked in writing to inform the colistin content (active drug) and its prodrug colistimethate sodium. Last, a number of recommendations were prepared as to the Information the authors understand should appear in the different presentations of colistin that is available in the market, to avoid prescription errors.

Nos últimos 10 anos, nesta era pós-antibiótica, a colistina reapareceu como um último recurso para enfrentar infecções por patógenos como Pseudomonas aeruginosa, Acinetobacter baumannii complexo e enterobactérias produtoras de carbapenemases. Durante um período não foi utilizada devido a seus efeitos adversos potencialmente graves como a nefro e a neurotoxicidade, porém atualmente é parte fundamental da antibioticoterapia nos casos de patógenos extremadamente resistentes. O aumento de seu uso levou ao aparecimento de resistência e como consequência, de uma situação potencialmente catastrófica, especialmente a recentemente descrita presença de plasmídeos transferíveis entre espécies que contém genes que conferem resistência a colistina (gen mcr-1), mecanismo detectado também no Uruguai. Nesta revisão, baseados nos conhecimentos atuais sobre a farmacocinética e farmacodinâmica, descreve-se detalhadamente a dosagem apropriada com necessidade de realizar doses carga para alcançar os niveles terapêuticos adequados, e os aspectos práticos da administração nos casos de meningite/ventriculite pós-operatória e de seu emprego na nebulização para o tratamento da pneumonia associada à ventilação. Destaca-se a necessidade de seu uso combinado com outro antibiótico ativo in vitro, especialmente em pacientes críticos e naqueles com clearance de creatininemia maior a 80 ml/min. A bi terapia é necessária principalmente se a concentração inibitória mínima do patógeno é maior a 1 mg/l, devido ao risco de subnotificação e o aparecimento de resistência intratratamiento. Na segunda parte, discute-se a complexidade da dosagem em função das diferentes apresentações comercializadas no país que levaram a erros na posologia, com um maior risco de toxicidade. Buscando melhorar a compreensão dos aspectos relacionados a rotulação da dose de colistina revisaram-se as bulas e as embalagens primárias das apresentações de colistina comercialmente disponíveis no Uruguai, para solicitar formal e documentalmente às empresas farmacêuticas representantes, informação sobre a dosagem de colistina (droga ativa) e de seu precursor o colistimetato de sódio (CMS). Finalmente elabora-se uma série de recomendações com relação à informação que os autores consideram que deveria ser oferecida nas diferentes apresentações de colistina comercialmente disponíveis para não induzir a erros na prescrição.

Barcelona Consensus on Biomarker-Based Immunosuppressive Drugs Management in Solid Organ Transplantation.

With current treatment regimens, a relatively high proportion of transplant recipients experience underimmunosuppression or overimmunosuppression. Recently, several promising biomarkers have been identified for determining patient alloreactivity, which help in assessing the risk of rejection and personal response to the drug; others correlate with graft dysfunction and clinical outcome, offering a realistic opportunity for personalized immunosuppression. This consensus document aims to help tailor immunosuppression to the needs of the individual patient. It examines current knowledge on biomarkers associated with patient risk stratification and immunosuppression requirements that have been generally accepted as promising. It is based on a comprehensive review of the literature and the expert opinion of the Biomarker Working Group of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology. The quality of evidence was systematically weighted, and the strength of recommendations was rated according to the GRADE system. Three types of biomarkers are discussed: (1) those associated with the risk of rejection (alloreactivity/tolerance), (2) those reflecting individual response to immunosuppressants, and (3) those associated with graft dysfunction. Analytical aspects of biomarker measurement and novel pharmacokinetic-pharmacodynamic models accessible to the transplant community are also addressed. Conventional pharmacokinetic biomarkers may be used in combination with those discussed in this article to achieve better outcomes and improve long-term graft survival. Our group of experts has made recommendations for the most appropriate analysis of a proposed panel of preliminary biomarkers, most of which are currently under clinical evaluation in ongoing multicentre clinical trials. A section of Next Steps was also included, in which the Expert Committee is committed to sharing this knowledge with the Transplant Community in the form of triennial updates.

Analytical Aspects of the Implementation of Biomarkers in Clinical Transplantation.

In response to the urgent need for new reliable biomarkers to complement the guidance of the immunosuppressive therapy, a huge number of biomarker candidates to be implemented in clinical practice have been introduced to the transplant community. This includes a diverse range of molecules with very different molecular weights, chemical and physical properties, ex vivo stabilities, in vivo kinetic behaviors, and levels of similarity to other molecules, etc. In addition, a large body of different analytical techniques and assay protocols can be used to measure biomarkers. Sometimes, a complex software-based data evaluation is a prerequisite for appropriate interpretation of the results and for their reporting. Although some analytical procedures are of great value for research purposes, they may be too complex for implementation in a clinical setting. Whereas the proof of "fitness for purpose" is appropriate for validation of biomarker assays used in exploratory drug development studies, a higher level of analytical validation must be achieved and eventually advanced analytical performance might be necessary before diagnostic application in transplantation medicine. A high level of consistency of results between laboratories and between methods (if applicable) should be obtained and maintained to make biomarkers effective instruments in support of therapeutic decisions. This overview focuses on preanalytical and analytical aspects to be considered for the implementation of new biomarkers for adjusting immunosuppression in a clinical setting and highlights critical points to be addressed on the way to make them suitable as diagnostic tools. These include but are not limited to appropriate method validation, standardization, education, automation, and commercialization.

Tacrolimus pharmacodynamics and pharmacogenetics along the calcineurin pathway in human lymphocytes.

BACKGROUND: Although therapeutic drug monitoring has improved the clinical use of immunosuppressive drugs, there is still interpatient variability in efficacy and toxicity that pharmacodynamic monitoring may help to reduce. To select the best biomarkers of tacrolimus pharmacodynamics, we explored the strength and variability of signal transduction and the influence of polymorphisms along the calcineurin pathway. METHODS: Peripheral blood mononuclear cells from 35 healthy volunteers were incubated with tacrolimus (0.1-50 ng/mL) and stimulated ex vivo. Inhibition of NFAT1 (nuclear factor of activated T cells 1) translocation to the nucleus and intracellular expression of interleukin-2 in CD4(+) and CD8(+) T cells and the surface activation marker CD25 in CD3(+) cells were measured by flow cytometry. We sequenced the promoter regions of immunophilins and calcineurin subunits and characterized selected single nucleotide polymorphisms in the genes of the calcineurin pathway with allelic discrimination assays. RESULTS: All responses closely fitted an I/Imax sigmoid model. Large interindividual variability (n = 30) in I0 and IC50 was found for all biomarkers. Moreover, strong and statistically significant associations were found between tacrolimus pharmacodynamic parameters and polymorphisms in the genes coding cyclophilin A, the calcineurin catalytic subunit α isoenzyme, and CD25. CONCLUSIONS: This study demonstrates the consistency and large interindividual variability of signal transduction along the calcineurin pathway, as well as the strong influence of pharmacogenetic polymorphisms in the calcineurin cascade on both the physiological activity of this route and tacrolimus pharmacodynamics.