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1 Educational Forum Therapeutic drug monitoring of immunosuppressants: An overview Sukhpreet, P. Tiwari Department of Pharmacy Practice, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, India Received: Revised: Accepted: Correspondence to: P. Tiwari ABSTRACT Individualizing drug therapy to obtain an optimum balance between therapeutic efficacy and occurrence of adverse events is one of the main goals for the physician. Intra- and interpatient variability makes this difficult to achieve. This variability leads to variation in drug concentration at the site of action, creating the need for individualization of the dose for each patient. [1] In case of organ transplantation, cyclosporine became the first critical dose immunosuppressant drug that required a routine measurement. Since that time, there has been an expanding range of new immunosuppressant drugs for the prophylaxis of rejection in solid organ transplant recipients. Tacrolimus, sirolimus, everolimus and mycophenolic acid are also registered immunosuppressants. Many of these drugs require measurement of concentration with subsequent dosage adjustment. With the introduction of each of these drugs, new challenges have arisen for the laboratory scientist performing therapeutic drug monitoring (TDM). The measurement of low drug concentrations seen in biological fluids during drug treatment became possible by adopting new analytical technology in the early 1960s. [2] The first half of this article is devoted to the need for TDM of immunosuppressants while the latter half revolves around the analytical and clinical aspects of four most commonly used immunosuppressant drugs. Why TDM for Immunosuppressants? Immunosuppressant drugs have contributed significantly to the success of organ transplantation. Therapeutic drug monitoring is an integral part of transplant protocols. Immunosuppressants require therapeutic drug monitoring because of their narrow therapeutic index and significant variability in blood concentrations between individuals. The variability in blood concentrations can be because of factors like drug-nutrient interactions, gender influence and polymorphism. Drug monitoring is widely practised especially for cyclosporine, tacrolimus, sirolimus and mycophenolic acid. The accuracy and specificity of the drug measurements are fundamental to the clinical interpretation of the concentration data. The issues surrounding the measurement and interpretation of immunosuppressant drug levels such as body fluids used for drug estimation, protein binding, sampling time and method for assay of the drug are discussed in detail in the article. KEY WORDS: Cyclosporine, mycophenolic acid, sirolimus, tacrolimus, therapeutic drug monitoring The most commonly used immunosuppressants require TDM because of their narrow therapeutic index and significant variability in blood concentrations between individuals. In transplant recipients, both supratherapeutic and subtherapeutic drug concentrations can have devastating results. For example, the therapeutic index of tacrolimus is narrow with a targeted therapeutic range of 5-20 ng/ml. At subtherapeutic drug concentrations, the transplant recipient is at risk for allograft rejection. At supratherapeutic drug concentrations, the patient is at risk for over-immunosuppresion which can potentially lead to infection or drug-specific side effects. It is known that neurological and gastrointestinal side effects occur more frequently at higher concentrations of tacrolimus. [3] Immunosuppressants display significant interindividual variability in plasma drug concentrations, which creates the demand for TDM when such drugs are used. It is appropriate to look into the multitude of factors that contribute to the interindividual variability. Some of the factors discussed in this article are drug-nutrient interactions, drug-disease interactions, renal insufficiency, inflammation and infection, gender, age, polymorphism and liver mass. Drug-nutrient interactions are becoming very widely appreciated. The metabolism of drugs sometimes also depends on the type of diet taken by the patients. For example, widely consumed grape fruit juice affects the metabolism of many drugs. Several studies have demonstrated that grapefruit juice can increase plasma concentration of cyclosporine (CsA) by 66

2 inhibiting CYP3A-mediated metabolism and by increasing drug absorption via inhibition of P-glycoprotein efflux transporters. [4,5] Hence, drug concentration needs to be monitored if the patient is consuming grape fruit juice and CsA concomitantly. Similarly, drug-disease interactions can also contribute to interindividual variability in plasma concentration of immunosuppressants. For example, renal insufficiency can result in an altered free fraction of mycophenolic acid (MPA) due to the reduction in protein binding. [6] Moreover, inflammation and infection are generally associated with a decline in cytochrome P450 enzyme activity. Hence, the quantum of drugs metabolized by this enzyme will increase CsA concentrations. [7] Gender also influences drug concentration. For example, the rate of MPA metabolism is higher in male kidney transplant recipients. This is secondary to a difference in glucuronidation between the two sexes. [8] Likewise, age can also contribute to interindividual difference in immunosuppressant plasma concentration. For instance, pediatric transplant recipients require higher doses of CsA to maintain blood concentrations equal to those found in adults. [9] Early studies on the effect of age with cyclosporine demonstrated that there was no difference in the pharmacokinetics of oral CsA with age. [9] However, it is possible that this apparent lack of difference with CsA may variability of immunosuppressant concentrations which has to be maintained within therapeutic range in order to achieve the optimal benefit of drug therapy, rendering TDM necessary for these drugs. Drug-Specific Scenarios In this section, issues like body fluid used for drug estimation, protein binding, sampling time and method for assay of the drug (and its metabolites) are summarized [Table 1] for cyclosporine, mycophenolic acid, tacrolimus and sirolimus. Cyclosporine Cyclosporine is a cyclic peptide immunosuppressant used to prevent graft rejection in solid organ transplant patients. CsA has been used in patients since 1978 and has proven to be effective in improving the long-term survival of patients with renal and other solid organ transplants. [12] Earlier, both blood and plasma were used to measure CsA concentration. Currently, the sample matrix issue has been resolved by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDM-CT) by considering whole blood as the matrix for estimation of CsA. [13] An alternative method using saliva as the matrix for therapeutic monitoring of CsA in patients [14] has been reported by Mendonza et al. This can be used in mask a complex interplay of age-related changes. patients with difficult venous access and in pediatrics and adult Polymorphism has demonstrated functional consequences patients suffering from gingival hyperplasia. The relevance of of many drug metabolizing enzymes. For example, CsA is a saliva concentration monitoring in CsA TDM requires further known substrate for CYP3A4/5 and P-glycoprotein. CYP3A5 investigation. Finally, the correlation between saliva and total is one of the main CYP3A enzymes and its expression is as well as unbound concentrations of CsA in plasma needs to clearly polymorphic and shows ethnic dependence. High levels be established. of metabolically active CYP3A5 are expressed by 45-73% CsA is more than 99% bound to blood cells and plasma of African-American but by only 5-15% of Caucasians and proteins, leaving a very small fraction of unbound (free) drug. Japanese. The pharmacological action of a highly protein-bound drug CYP3A5*3C polymorphism significantly affects the area is dependent on the drug concentration at the receptor site, under the curve (AUC) of oral CsA in healthy subjects as which is directly related to the concentration of unbound drug high CYP3A5 activity may increase first-pass metabolism in in plasma. [14] There are now at least seven different assay African Americans as compared to Caucasians. [10] Several systems used for the TDM of total CsA, viz., HPLC-UV, HPLCstudies have demonstrated that liver mass correlates with MS, immunoassays (enzyme, enzyme donor, radiolabeled and body mass. Liver mass itself is a primary determinant in the fluorescence). [15] Although immunoassays are predominantly metabolism of drugs with low extraction (or high bioavailability). used throughout the world, HPLC-UV is considered as the gold While many immunosuppressants display low-to-moderate standard for CsA measurement. [13] Although the trough CsA bioavailability, there is a great interindividual variability in concentration is well within the analytical range of HPLC-MS, their bioavailability, suggesting that body weight and / or liver there are few reported HPLC-MS methods. HPLC-MS has been mass may be an important source of interindividual variability used where sample requirements are limited, such as biopsy in drug concentration. [11] All these factors contribute to the or blood samples from pediatric patients. [2] Table 1 Principle features of therapeutic drug monitoring of immunosuppressant agents Feature Tacrolimus Sirolimus Mycophenolate mofetil Cyclosporine Oral bioavailability 4-93% ~15% 94% 10-46% Time to peak concentration (hours) Plasma protein binding 88% 92% 97% 91-95% Metabolism Intestinal and Intestinal and Hepatic Hepatic hepatic CYP3A4 hepatic CYP3A4 glucuronidation CYP3A4/5 Preferred analytical method MEIA HPLC-MS and HPLC HPLC HPLC-UV Matrix for concentration measurement Whole blood Whole blood Plasma Plasma Therapeutic trough concentration range 5-20 g/l 5-15 g/l mg/l mcg/l 67

3 A key development that has implications for both analytical a higher bias has been noted in samples collected to 2-4 hours methodology and approaches to monitoring CsA has been the oral dosing. The principal reason for this bias appears to be introduction of the microemulsion formulation of the drug. [16] cross-reactivity with a recently identified metabolite of MPA, The microemulsion produces a more consistent concentration the acyl glucuronide, usually known as MPAG. This metabolite profile during the absorption phase with most of the intrasubject should not be confused with the phenolic glucuronide. MPAG and intersubject variability occurring during the first four hours does not cross react with the antibody used in the EMIT assay. after dosing. [17] The currently available evidence suggests the Therefore, EMIT is the preferred method for estimation. Plasma use of measurement of CsA at two hours after dosing (C2) samples should be stored carefully as MPAG may be unstable as a remedy for the variability of exposure during the first in plasma, breaking down to MPA. four hours after dosing. [18,19] The measurement of CsA in C2 The time commitment for laboratory staff would be samples does present some methodological problems to many considerably reduced in the case of the EMIT-MPA assay laboratories using immunoassay techniques and possibly those compared with the HPLC assay. [23,24] In case of altered binding using HPLC. A survey conducted by IFCC / IATDM-CT had shown of MPA, the unbound concentration needs to be monitored. that respondents report target concentrations of C2 as high Sometimes, the unbound MPA concentration is below the as 2100 mcg/l, a concentration higher than the top calibrator sensitivity of the EMIT assay and difficult to measure by of the immunoassay. [13] As a result, sample dilution becomes conventional HPLC techniques. This problem was overcome by necessary. New control procedures are required for laboratories one center by developing an HPLC assay with MS detection for adopting this monitoring strategy. In light of this problem, Keevil the measurement of unbound MPA [25] while another group opted [20] et al. reported an assay using HPLC-MS / MS that has a wide to modify the EMIT assay to allow the unbound concentration analytical range ( µg/l). The method required simple to be measured. [26] Clinical interpretation for effective TDM is protein precipitation and had an injection-to-injection cycle based on the monitoring of free MPA concentrations but as time of 1.5 minutes, which is suitable for TDM. mentioned above, measurement of this parameter may not be Mycophenolic acid possible for all laboratories because of constraints in assay Mycophenolic acid is the active immunosuppressant sensitivity. metabolite of the prodrug mycophenolate mofetil (MMF). MMF The trough plasma concentration monitoring of MPA and its has been shown to be effective in reducing the rate of acute metabolites may not provide a useful clinical tool for guiding rejection. It is widely used in kidney transplantation and is MMF dose adjustments to avoid drug-related toxicity. [27] The indicated for heart transplantation. [21] MMF was approved for AUC for MPA is the most reliable predictor of risk for acute use in renal transplant patients in 1995, heart transplantation rejection. [28] In general, AUC data have been associated more in 1998 and liver transplantation in 2000 as an alternative significantly with clinical efficacy than trough concentration to azathioprine. MMF also has been used in association with data. [13] A full MPA AUC typically requires at least eight blood tacrolimus and sirolimus and is gaining importance as a primary samples during a 12 hour dose interval. In clinical practice, component in long-term immunosuppressant regimens. [3] The this is impractical; therefore, abbreviated sampling schemes pharmacokinetics of MPA shows large intraindividual and involving the collection of three to five plasma samples are interindividual variability. under investigation. In clinical studies, good correlations have Plasma is the preferred matrix for MPA measurement. In been noted between estimates of 12 hour AUCs using samples plasma samples, MPA concentration remains constant for as collected at three, four or five time points and full 12-hour AUC long as eight hours at room temperature, for at least 96 hours determinations. The measurement of AUC does incur some at 4ºC and for at least 11 months at -20ºC. The recommended logistic and cost problems. [21] anticoagulant for the plasma samples is ethylene diamine Tacrolimus tetraacetic acid (EDTA). [21] MPA is extensively bound to albumin Tacrolimus is an immunosuppressant agent that has with a range of protein binding of 97-99% in patients with emerged as a valuable therapeutic alternative to cyclosporine normal renal and liver function. In stable transplant patients, following solid organ transplantation. Tacrolimus has a the MPA free fraction ranges from 1-3%. [22] Factors that alter narrow therapeutic window, displays large interindividual and protein binding can affect the relationship between total and intraindividual pharmacokinetics variability and is susceptible free concentrations. As significant changes in the free fraction to cytochrome P450-mediated drug interactions. Thus, it of MPA occur as a result of factors such as changes in albumin is recommended that dosing of this agent be guided by concentration, displacement of bound MPA from albumin, renal routine TDM of whole blood trough concentrations. [29] Several failure or hyperbilirubinemia, there has been interest in the research groups support monitoring tacrolimus concentration measurement of the unbound concentration of MPA in plasma. in plasma [30-32] but other results support the use of whole Under circumstances of altered binding, the interpretation of blood. [33,34] However, only a few studies have reported a total MPA plasma concentrations must take into account the significant correlation between tacrolimus blood concentration unbound plasma concentration of MPA. [21] and the incidence of graft rejection. [34,35] Although a relatively simple HPLC method is available Optimizing tacrolimus TDM requires a detailed understanding to measure drug concentrations, many centers opt to use of the drug distribution in recipients to identify a potentially an immunoassay. The enzyme-multiplied immunoassays better predictor of clinical outcome. [36] Warty et al. [37] studied technique (EMIT) is available for commercial assays. There the distribution of tacrolimus in 13 transplant recipients and is a recurring assay bias against chromatographic techniques reported that in plasma, 64 ± 8.0% of tacrolimus binds to with an estimated positive bias averaging about 20% although lipoprotein-deficient plasma (LPDP), 21 ± 8% associates with 68

4 high density lipoprotein (HDL), 3 ± 3% associates with low density lipoprotein (LDL) and 11 ± 3% associates with very low density lipoprotein (VLDL). Piekoszewski et al. [38] have reported plasma protein binding of tacrolimus to be 72 ± 3.6% in a group of eight liver transplant recipients. The above two studies show that distribution and protein binding of tacrolimus vary significantly leading to changes in its unbound concentrations. The conflicting evidence of a relationship between trough blood tacrolimus concentration and clinical outcome complicates TDM of tacrolimus. The trough concentration stable in blood samples at temperatures up to 30ºC, instability of the drug in blood samples kept at temperatures above 35ºC has been noted. Thus, when samples are transported for long distances for analysis in central laboratories, refrigerated containers should be used. [13] The peak concentrations are reached in approximately 1.4 ± 1.2 hours. Studies have shown a correlation between the trough concentration and AUC. Thus, sirolimus trough concentrations may be useful for guiding therapy. The frequency of monitoring is however less often that the other drugs considered in this review because it has a relatively long half life of about 60 hours. [13] (achieved 12 hours after dose administration) monitoring is still Currently, there is no commercial immunoassay method for the mainstay of tacrolimus measurement, based on the belief sirolimus. The two major techniques for the quantification that this measurement is a good reflection of the total drug of circulating sirolimus levels are HPLC-UV and HPLC-MS. exposure. [13] Recent data question the predictive value of a single The current HPLC-UV method requires large sample volumes trough concentration and suggest that a measurement made which are unfavorable for pediatric patients and tend to involve at an additional time point during the absorption phase would elaborate and labor-intensive extractions resulting in lengthy be useful. [13] If the samples collected during the absorption turnaround times. While extraction for HPLC-MS methods tend phase are to be analyzed, the suitability of the commonly used to be simpler which also have shorter sample analysis times, methods needs to be considered, both in terms of calibration the equipment necessary for such methods is expensive and range and the effects of tacrolimus metabolites on performance not available to most clinical laboratories. [42] of the assays. A synergistic effect between sirolimus and cyclosporine A recent study by Venketaramanan [39] used the enzyme- is reported. Both sirolimus and CsA are metabolized by linked immunosorbent assay (ELISA) to measure tacrolimus CYP3A4. If these two drugs are taken concomitantly, the trough concentrations in samples. The trough concentration of sirolimus concentration is significantly higher than if sirolimus tacrolimus alone was not able to discriminate between graft is adminsitered four hours after CsA. Finally, the interpretation rejection and other causes of liver dysfunction. However, the of sirolimus concentration data is based almost entirely on risk of acute rejection at a tacrolimus concentration of 5 ng/ml its use with CsA. Also, the current target concentrations for was approximately twice that at tacrolimus concentrations and the use of sirolimus in combination with tacrolimus after was ultimately related to tacrolimus-induced nephrotoxicity. liver transplantation is based on those used for sirolimus in A number of analytical methods including radioreceptor combination with CsA. [13] Additionally, although one of the monitoring, enzyme immunoassays, high performance liquid adverse effects related to sirolimus blood concentrations chromatography tandem mass spectrometry (HPLC/MS) is hyperlipidemia, there are no reports of pharmacokinetic and bioassays, have been developed for the measurement of interaction between sirolimus and statins. [44] tacrolimus concentrations. [29,40] Tacrolimus immunoassays (ELISA and microparticle Conclusions enzyme immunoassay, MEIA) overestimate results in patient The appropriate use of TDM may minimize the risk of rejection samples because the antibody cross-reacts with metabolites after transplantation. The monitoring of immunosuppressant of the parent drug. [41] An alternative to immunoassays for the drugs is still an evolving field. The value of monitoring can be measurement of tacrolimus is liquid chromatography-tandem enhanced by attention to both the choice of monitoring strategy mass spectrometry (LC-MS/MS). LC-MS/MS is specific for the and adherence to accurate sample timing. Improvement measurement of the parent drug and thus avoids the problem in bioanalytics will allow more sensitive and selective of metabolite interference. It is important to note that there is measurements of these drugs. The growth in the use of evidence to suggest that ELISA measurements of tacrolimus HPLC with MS detection holds the possibility of increased were not automatically interchangeable with measurements selectivity for the measurement of these compounds as well obtained by LC-MS/MS [29] The initial cost and thus lack as allowing the simultaneous measurement of several coprescribed immunosuppressant drugs. [45] As a result, it will be of availability of HPLC-mass spectrometers in the many laboratories, limits the widespread use of the technique. possible to investigate the relationship between selective drug Sirolimus It is a new macrolide immunosuppressant derived from Streptomyces hygroscopicus. Sirolimus is poorly absorbed from the gastrointestinal tract and the oral bioavailability measurements and clinical outcome and to more easily assess the impact of genetic factors on drug metabolism. References is approximately 15%. [42] Clinical studies have shown that monitoring blood sirolimus levels is necessary for successful patient management. Whole blood has been established as 1. Karaalp A, Demir D, Goren MZ, Akc A, ISkender E, Yananl HR, et al. Therapeutic drug monitoring of immunosuppressant drugs in Marmara University Hospital. Ther Drug Monit 2004;26: Taylor PJ. Therapeutic drug monitoring of immunosuppressant drugs by high the matrix for routine monitoring. The drug is sequestered performance liquid chromatography-mass spectrometry. Ther Drug Monit into erythrocytes to such an extent (approx. 95%) that 2004;26: measurements in plasma would be virtually impossible by the 3. Johnson HJ, Heim-Duthoy KL. Renal Transplantation. In: Dipiro JT, Talbert RL, Yee published technique. [43] Although the drug has been shown to be GC, Matzke GR, Wells BG, Posey LM, editors. Pharmacotherapy: A Pathophysi- 69

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