Michael Oellerich

Opportunities to optimize tacrolimus therapy in solid organ transplantation: report of the European consensus conference.

In 2007, a consortium of European experts on tacrolimus (TAC) met to discuss the most recent advances in the drug/dose optimization of TAC taking into account specific clinical situations and the analytical methods currently available and drew some recommendations and guidelines to help clinicians with the practical use of the drug. Pharmacokinetic, pharmacodynamic, and more recently pharmacogenetic approaches aid physicians to individualize long-term therapies as TAC demonstrates a high degree of both between- and within-individual variability, which may result in an increased risk of therapeutic failure if all patients are administered a uniform dose. TAC has undoubtedly benefited from therapeutic drug monitoring, but interpretation of the blood concentration is confounded by the relative differences between the assays. Single time points, limited sampling strategies, and area under concentration-time curve have all been considered to determine the most appropriate sampling procedure that correlates with efficacy. Therapeutic trough TAC concentration ranges have changed since the initial introduction of the drug, while still maintaining adequate immunosuppression and avoiding drug-related adverse effects. Pharmacodynamic markers have also been considered advantageous to the clinician, which may better reflect efficacy and safety, taking into account the between-individual variability rather than whole blood concentrations. The choice of method, differences between methods, and potential pitfalls of the method should all be considered when determining TAC concentrations. The recommendations of this consensus meeting regarding the analytical methods include the following: encourage the development and promote the use of analytical methods displaying a lower limit of quantification (1 ng/mL), perform careful validation when implementing a new analytical assay, participate in external proficiency testing programs, promote the use of certified material as calibrators in high-performance liquid chromatography with mass spectrometric detection methods, and take account of the assay and intermethod bias when comparing clinical trial outcomes. It is also important to consider that TAC concentrations may also be influenced by other factors such as specific pharmacokinetic characteristics associated with the population, drug interactions, pharmacogenetics, adverse events that may alter TAC concentrations, and any change in the oral formulation that may result in pharmacokinetic changes. This meeting emphasized the importance of obtaining multicenter prospective trials to assess the efficacy of alternative strategies to TAC trough concentrations whether it is other single time points or area under the concentration-time curve Bayesian estimation using limited sampling strategies and to select, standardize, and validate routine biomarkers of TAC pharmacodynamics.

Current issues in therapeutic drug monitoring of mycophenolic acid : Report of a roundtable discussion

This review is based on a roundtable discussion held under the auspices of F. Hoffman-LaRoche in Nice, France, in December 1999. The meeting drew together laboratory scientists and clinicians with expertise in the measurement of mycophenolic acid (MPA) and the application of these measurements as a guide to therapy with mycophenolate mofetil (MMF). Three of those presenting data at the meeting had been members of a recent consensus panel on therapeutic drug monitoring (TDM) of MPA. The purpose of the meeting was to update members of the Roche Development Team on the latest critical thinking regarding the advantages and limitations of applying TDM to transplant patients receiving mycophenolate mofetil, and to provide a basis for a consensus on MPA therapeutic drug monitoring. Short presentations were made by each participant, followed by open discussions. Representatives of Roche who attended the meeting included Etienne Dumont, MD, Samson Fung, MD, Hari Kumar, PhD, Richard D. Mamelok, MD, Eleanor L. Ramos, MD, and Mandy Rees, BS. The proceedings were recorded and transcribed by Phase Five Communications. Subsequently, the transcript was made into the following review article, and the latest findings from recent publications were incorporated into the text. The final document is intended as an introduction to the topic of TDM of MPA and provides a summary of what was discussed at the Nice meeting. It also includes recommendations for TDM strategies based on the current pharmacokinetic and pharmacodynamic data for MPA.

Acyl glucuronide drug metabolites: toxicological and analytical implications.

Although glucuronidation is generally considered a detoxification route of drug metabolism, the chemical reactivity of acyl glucuronides has been linked with the toxic properties of drugs that contain carboxylic acid moieties. It is now well documented that such metabolites can reach appreciable concentrations in blood. Furthermore, they are labile, undergo hydrolysis and pH-dependent intramolecular acyl migration to isomeric conjugates of glucuronic acid, and may react irreversibly with plasma proteins, tissue proteins, and with nucleic acids. This stable binding causes chemical alterations that are thought to contribute to drug toxicity either through changes in the functional properties of the modified molecules or through antigen formation with subsequent hypersensitivity and other immune reactions. Whereas in vitro data on the toxicity of acyl glucuronides have steadily accumulated, direct evidence for their toxicity in vivo is scarce. Acyl glucuronides display limited stability, which is dependent on pH, temperature, nature of the aglycon, and so on. Therefore, careful sample collection, handling, and storage procedures are critical to ensure generation of reliable pharmacologic and toxicologic data during clinical studies. Acyl glucuronides can be directly quantified in biologic specimens using chromatographic procedures. Their adducts with plasma or cell proteins can be determined after electrophoretic separation, followed by blotting. ELISA techniques have been used to assess the presence of antibodies against acyl glucuronide–protein adducts. This review summarizes the most recent evidence concerning biologic and toxicologic effects of acyl glucuronide metabolites of various drugs and discusses their relevance for drug monitoring. A critical evaluation of the available methodology is included.

Comparing Mycophenolate Mofetil Regimens for de Novo Renal Transplant Recipients: The Fixed-Dose Concentration-Controlled Trial

Background. Fixed-dose mycophenolate mofetil (MMF) reduces the incidence of acute rejection after solid organ transplantation. The Fixed-Dose Concentration Controlled trial assessed the feasibility and potential benefit of therapeutic drug monitoring in patients receiving MMF after de novo renal transplant. Methods. Patients were randomized to a concentration-controlled (n=452; target exposure 45 mg hr/L) or a fixed-dose (n=449) MMF-containing regimen. The primary endpoint was treatment failure (a composite of biopsy-proven acute rejection [BPAR], graft loss, death, or MMF discontinuation) by 12 months posttransplantation. Results. Mycophenolic acid (MPA) exposures for both groups were similar at most time points and were below 30 mg hr/L in 37.3% of patients at day 3. There was no difference in the incidence of treatment failure (25.6% vs. 25.7%, P=0.81) or BPAR (14.9% vs. 15.5%, P>0.05) between the concentration-controlled and the fixed-dose groups, respectively. We did find a significant relationship between MPA-area under the concentration–time curve on day 3 and the incidence of BPAR in the first month (P=0.009) or in the first year posttransplantation (P=0.006). For later time points (day 10, month 1) there was no significant relationship between area under the concentration–time curve and BPAR (0.2572 and 0.5588, respectively). Conclusions. There was no difference in the incidence of treatment failure between the concentration-controlled and the fixed-dose groups. The applied protocol of MMF dose adjustments based on target MPA exposure was not successful, partly because physicians seemed reluctant to implement substantial dose changes. Current initial MMF doses underexpose more than 35% of patients early after transplantation, increasing the risk for BPAR.

Identification of glucoside and carboxyl‐linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil

Mycophenolic acid (MPA), is primarily metabolized in the liver to 7‐O‐MPA‐β‐glucuronide (MPAG). Using RP‐h.p.l.c. we observed three further MPA metabolites, M‐1, M‐2, M‐3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP‐glucuronic acid, UDP‐glucose or NADPH; (C) hydrolysis of metabolites was investigated using β‐glucosidase, β‐glucuronidase or NaOH; (D) cross‐reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). Mass spectrometry of M‐1, M‐2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. Incubation of microsomes with MPA and UDP‐glucose produced M‐1, with MPA and UDP‐glucuronic acid MPAG and M‐2 were formed, while with MPA and NADPH, M‐3 was observed. β‐Glucosidase hydrolysed M‐1 completely. β‐Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M‐2 was reduced by approximately 30%. Only M‐2 was labile to alkaline treatment. M‐2 and MPA but not M‐1 and MPAG cross‐reacted in the EMIT assay. These results suggest that: (i) M‐1 is the 7‐OH glucose conjugate of MPA; (ii) M‐2 is the acyl glucuronide conjugate of MPA; (iii) M‐3 is derived from the hepatic CYP450 system.

Pharmacokinetic and metabolic investigations of mycophenolic acid in pediatric patients after renal transplantation : Implications for therapeutic drug monitoring

The need for mycophenolic acid (MPA) monitoring is still under discussion. Key issues for the PK/PD relationships of this drug are: the role of metabolites, the usefulness of AUC versus predose levels, and the need to monitor the free concentration of MPA (f-MPA). Recent advances have revealed that, in addition to 7-O-MPAG, three additional MPA metabolites are present in the plasma of transplant recipients. One of these metabolites (M-2), identified as an acyl glucuronide of MPA, was found to inhibit IMPDH-II in vitro. This active metabolite was also found to cross-react in the Emit assay for MPA. In an ongoing multicenter study, the authors are evaluating the relevance of monitoring total (t-MPA) and free mycophenolic acid (f-MPA) in pediatric renal transplant recipients. As in adults, a time-dependent increase of t-MPA-AUC(0-12h) within the first 3 months posttransplant (35 versus 64 mg x h/L, [corrected] 3 weeks versus 3 months respectively; daily dosage: 0.6 g/m2 bid) was seen. Receiver operating characteristics curve analyses were used to test the ability of predose levels or AUC(0-12h) to discriminate between cases with no complications and those with acute rejection, adverse events (severe infections, leukopenia), or gastrointestinal disorders observed during the early posttransplant course. In agreement with observations in adults, a significant (p = 0.001) association was observed between AUC(0-12h) and acute rejection. A t-MPA-AUC(0-12h) of approximately 30-60 mg x h/L [corrected], as determined by HPLC, seems to be a reasonable target for the early posttransplant period. It remains to be elucidated whether regular predose level monitoring may be of more practical value. A higher incidence of rejection was observed at predose MPA concentrations < or = 1 mg/L, as measured by HPLC. In contrast to t-MPA, f-MPA-AUC(0-12h) was significantly related to severe infections and leukopenia. The risk for severe adverse events was increased at f-MPA- AUC(0-12h) values > or =600 microg x h/L [corrected]. On the basis of these data and the observed variability in the pharmacokinetics of MPA, the development of monitoring strategies for this drug appears to be promising.

New developments in the immunosuppressive drug monitoring of cyclosporine, tacrolimus, and azathioprine

The calcineurin inhibitors cyclosporine and tacrolimus form the cornerstones of most immunosuppression protocols. Because of their variable pharmacokinetics, and their narrow therapeutic indices, post-transplant immunosuppressive drug monitoring is an essential part of patient care to minimize the risks of toxicity or acute rejection. Furthermore, a reduction in the rate of acute rejection has been shown to result in a lower rate of graft loss due to chronic rejection. The introduction of the microemulsion formulation of cyclosporine with its more consistent bioavailability has renewed interest in the use of alternative sampling strategies to the trough cyclosporine concentration. Both pharmacokinetic and pharmacodynamic considerations support the concept that determination of cyclosporine during the absorption phase (0-4 h) might offer a better prediction of cyclosporine immunosuppressive efficacy. Initial investigations suggest that monitoring a 2-h postdose concentration C(2) may provide a more efficacious alternative to trough monitoring for optimizing therapy with Neoral. Tacrolimus has a 10- to 100-fold greater in vitro immunosuppressive activity compared with cyclosporine. Consistent with its greater potency, therapeutic whole blood trough concentrations for tacrolimus are around 20-fold lower than the corresponding cyclosporine concentrations. The correlation between toxicity and tacrolimus trough concentrations appears to be stronger than that for acute rejection. The results from a concentration-ranging trial in primary kidney-transplantation and liver-transplantation trials all found a significant relationship between toxicity and tacrolimus trough levels. Azathioprine is converted in vivo to 6-mercaptopurine, which is subsequently metabolized to the pharmacologically active 6-thioguanine nucleotides. The latter are also responsible for the cytotoxic side effects. Reliance on blood counts to monitor azathioprine therapy can be misleading, and they do not provide information on immunosuppresive efficacy. More pertinent information can be obtained through the measurement of thiopurine S-methyltransferase activity and the quantification of intracellular 6-thioguanine nucleotides concentrations in red blood cells. Prospective studies have demonstrated the clinical utility of determining 6-thioguanine nucleotides to individualise immunosuppressive therapy with azathioprine not only in the field of transplantation, but also in inflammatory bowel disease.

Glucuronide and glucoside conjugation of mycophenolic acid by human liver, kidney and intestinal microsomes

Mycophenolic acid (MPA) is primarily metabolized to a phenolic glucuronide (MPAG) as well as to two further minor metabolites: an acyl glucuronide (AcMPAG) and a phenolic glucoside (MPAG1s). This study presents investigations of the formation of these metabolites by human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as by recombinant UDP‐glucuronosyltransferases. HLM (n=5), HKM (n=6), HIM (n=5) and recombinant UGTs were incubated in the presence of either UDP‐glucuronic acid or UDP‐glucose and various concentrations of MPA. Metabolite formation was followed by h.p.l.c. All microsomes investigated formed both MPAG and AcMPAG. Whereas the efficiency of MPAG formation was greater with HKM compared to HLM, AcMPAG formation was greater with HLM than HKM. HIM showed the lowest glucuronidation efficiency and the greatest interindividual variation. The capacity for MPAGls formation was highest in HKM, while no glucoside was detected with HIM. HKM produced a second metabolite when incubated with MPA and UDP‐glucose, which was labile to alkaline treatment. Mass spectrometry of this metabolite in the negative ion mode revealed a molecular ion of m/z 481 compatible with an acyl glucoside conjugate of MPA. All recombinant UGTs investigated were able to glucuronidate MPA with KM values ranging from 115.3 to 275.7 μM l−1 and Vmax values between 29 and 106 pM min−1 mg protein−1. Even though the liver is the most important site of MPA glucuronidation, extrahepatic tissues particularly the kidney may play a significant role in the overall biotransformation of MPA in man. Only kidney microsomes formed a putative acyl glucoside of MPA.

Twelve-month evaluation of the clinical pharmacokinetics of total and free mycophenolic acid and its glucuronide metabolites in renal allograft recipients on low dose tacrolimus in combination with mycophenolate mofetil

Background: The establishment of a rationale for therapeutic drug monitoring for mycophenolic acid (MPA) and outlining a therapeutic window remains a challenging task in renal transplantation. Furthermore, the pharmacokinetic characteristics of free and total MPA and its glucuronides depend directly or indirectly on graft function and the type of co‐administered calcineurin‐inhibitor. Methods: The authors conducted a prospective 12‐month multicenter pharmacokinetic study on MPA (MPA, free MPA, free fraction MPA) and its metabolites (MPAG, Acyl‐MPAG). The aim of this study was to examine the long‐term pharmacokinetic characteristics of MMF when combined with tacrolimus in renal allograft recipients and to identify a possible relationship between these pharmacokinetic parameters and clinical outcome parameters. Results: They have demonstrated that in renal transplant recipients MPA, free MPA, Acyl‐MPAG and MPAG have a particular pharmacokinetic profile when combined with tacrolimus which differs from the combination with CsA. They could not establish a relationship between pre‐dose trough concentration of MPA and its metabolites and clinical efficacy endpoints and drug‐related adverse events, except for anemia. Conclusions: These findings suggest that trough plasma concentration monitoring of MPA and its metabolites might not provide a useful clinical tool for guiding MMF dose adjustments to avoid drug‐related toxicity. More extensive pharmacokinetic measurements like area under the concentration curves might be necessary for routine therapeutic drug monitoring of MMF.

Induction of cytokine release by the acyl glucuronide of mycophenolic acid: a link to side effects?

OBJECTIVES We have identified an acyl glucuronide (M-2) of the immunosuppressant mycophenolic acid (MPA). Acyl glucuronides have toxic potential and may contribute to drug toxicity. Whether acyl glucuronides are able to induce release of proinflammatory cytokines is unknown. Gastrointestinal disturbances have been observed during MPA therapy and may involve an inflammatory reaction. This study investigated whether M-2 can induce IL-6 and TNF-alpha release as well as gene expression of these cytokines in leukocytes. DESIGN AND METHODS M-2 was produced by incubation of MPA with human liver microsomes. Human mononuclear leukocytes were incubated in the presence of M-2. Concentrations of IL-6 and TNF-alpha were measured by ELISA. Expression of mRNA was determined by quantitative RT-PCR. RESULTS Incubation of 3 x 10(6) cells with M-2 resulted in a time and dose dependent release of cytokines, whereas MPA or its phenolic glucuronide MPAG were without effect. Cytokine liberation depended on mRNA induction. Response to M-2 showed much inter individual variability (30-fold for IL-6, 3-fold for TNF-alpha). CONCLUSIONS If M-2 promotes release of cytokines in vivo, these may mediate some of the toxic actions of MPA.