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.

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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.

1 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 UDPglucuronosyltransferases. 2 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. 3 All microsomes investigated formed both MPAG and AcMPAG. Whereas the e ciency of MPAG formation was greater with HKM compared to HLM, AcMPAG formation was greater with HLM than HKM. HIM showed the lowest glucuronidation e ciency and the greatest interindividual variation. The capacity for MPAGls formation was highest in HKM, while no glucoside was detected with HIM. 4 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. 5 All recombinant UGTs investigated were able to glucuronidate MPA with K M values ranging from 115.3 to 275.7 mM l 71 and V max values between 29 and 106 pM min 71 mg protein 71 . 6 Even though the liver is the most important site of MPA glucuronidation, extrahepatic tissues particularly the kidney may play a signi®cant role in the overall biotransformation of MPA in man. Only kidney microsomes formed a putative acyl glucoside of MPA.

Background: Tacrolimus is metabolized predominantly to 13-O-demethyltacrolimus in the liver and intestine by cytochrome P450 3A (CYP3A). Patients with high concentrations of CYP3A5, a CYP3A isoenzyme polymorphically produced in these organs, require higher doses of tacrolimus, but the exact mechanism of this association is unknown. Methods: cDNA-expressed CYP3A enzymes and a bank of human liver microsomes with known CYP3A4 and CYP3A5 content were used to investigate the contribution of CYP3A5 to the metabolism of tacrolimus to 13-O-demethyltacrolimus as quantified by liquid chromatography-tandem mass spectrometry. Results: Demethylation of tacrolimus to 13-O-demethyltacrolimus was the predominant clearance reaction. Calculated K m and V max values for CYP3A4, CYP3A5, and CYP3A7 cDNA-expressed microsomes were 1.5 mol/L and 0.72 pmol ⅐ (pmol P450) ؊1 ⅐ min ؊1 , 1.4 mol/L and 1.1 pmol ⅐ (pmol P450) ؊1 ⅐ min ؊1 , and 6 mol/L and 0.084 pmol ⅐ (pmol P450) ؊1 ⅐ min ؊1 , respectively. Recombinant CYP3A5 metabolized tacrolimus with a catalytic efficiency (V max /K m ) that was 64% higher than that of CYP3A4. The contribution of CYP3A5 to 13-O-demethylation of tacrolimus in human liver microsomes varied from 1.5% to 40% (median, 18.8%). There was an inverse association between the contribution of CYP3A5 to 13-O-demethylation and the amount of 3A4 protein (r ‫؍‬ 0.90; P <0.0001). Mean 13-O-demethylation clearances in CYP3A5 high and low expressers, estimated by the parallel-tube liver model,