*3.3.1. Why TDM for immunosuppressants?*

Anyone involved in the utilization of information derived from TDM must always bear in mind that the interpretation of plasma drug concentration must always be carried out in conjunction with an assessment of the clinical status of the patient. Therapeutic ranges should more correctly be described as optimal concentrations. According to the definition mentioned above, the therapeutic range (optimal concentration) of a drug is that concentration of drug present in plasma or some other biologic fluid or tissue that provides the desired therapeutic response in most patients. The severity of the disease process determines the amount of drug necessary to achieve a given therapeutic effect. Thus, it is quite possible that a patient may achieve the desired therapeutic effect at a plasma concentration well below the optimal range. Conversely, some patients will not achieve the desired therapeutic effect even when plasma concentrations are elevated into the toxic range. If the desired therapeutic effect is achieved at suboptimal plasma concentrations, every attempt should be made to avoid the prescription of additional drugs simply to increase the plasma concentration into what is commonly referred to as the therapeutic range. Obviously, the interpretation of plasma drug concentration must take into account the various factors that can alter the steady state plasma concentrations

Individualizing a patient's drug therapy to obtain the optimum balance between therapeutic efficacy and the occurrence of adverse events is the physician's goal. However, achieving this goal is not always straight forward, being complicated by within and between patient variability in both pharmacokinetics and pharmacodynamics. In the early 1960's new analyt‐ ical techniques became available allowing the measurement of the low drug concentrations seen in biological fluids during drug treatment. This offered the opportunity to reduce the pharmacokinetic component of variability by controlling drug therapy using concentrations in the body rather than by dose alone. This process became known as therapeutic drug

The aim of TDM is to optimize pharmacotherapy by maximizing therapeutic efficacy, while minimizing adverse events, in those instances where the blood concentration of the drug is a better predictor of the desired effect(s) than the dose. The reasons why these principles have gained wide acceptance include the following: (1) although imperfect, a better relationship often exists between the effect of a given drug and its concentration in the blood than between the dose of the drug and the effect; (2) a thorough understanding of pharmacokinetics, i.e., the processes of drug absorption, distribution, metabolism, and drug excretion in individual patients and in patient populations is available; and (3) the development of reliable and relatively easy to use drug-monitoring assays. In addition, TDM can also be useful in cases in which compliance is in question, where it is not clear if the right drug is being taken, where dosage adjustment is required as a result of drug–drug or drug–food interactions, and where

TDM is more than simply the analysis of a single drug concentration in the blood of a patient and a report of this number. It also comprises interpretation of the value measured using the mathematical (pharmacokinetic) principles mentioned above, drawing the appropriate

achieved on a given dosage form [54].

monitoring [55].

intoxication is suspected.

**3.3. Therapeutic Drug Monitoring (TDM)**

320 Current Issues and Future Direction in Kidney Transplantation

For a drug to be a suitable candidate for therapeutic drug monitoring it must satisfy the following criteria [55]:


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 supratherapeutics and subtherapeutics drug concentrations can have devastating results. At subtherapeutics drug concentrations, the transplant recipient is at risk for allograft rejection. At supratherapeutics drug concentrations, the patient is at risk for over-immunosuppression which can potentially lead to infection or drug specific side effects. It is it known that neurological and gastrointestinal side effects occur more frequently at higher concentrations of TRL [53]. Immunosuppressants display significant interindividual variability in plasma drug concentrations, which creates the demand for TDM when such drugs are used.

#### *3.3.2. Factors contributing to the variability*

Immunosuppressants display significant interindividual variability in plasma drug concen‐ trations, 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 include drug-nutrients 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. Renal transplant patients may have reduced oral bioavailability for TRL. When given with meals, especially with high fat content food, oral bioavailability of TRL decreases [57].

ic differences. Bioavailability after oral drug dosing, for CYP3A substrates in particular, may be somewhat higher in women compared to men [63]. It is known that MPA is pri‐ marily metabolized in the liver to its MPAG derivative. Morissette et al., found that men treated with MMF and TRL showed a lower ratio than patients treated with this couple of drugs, confirming that TRL inhibits glucuronidation of MPA. Because MPAG can fa‐ vor the elimination of MPA, they concluded that gender differences and cotreatment with TRL must be taken into consideration when MMF is being administered [64]. Velickovic et al., investigated the gender differences in pharmacokinetics of TRL, their result show remarkable gender-related differences between women and men after the first oral dose among kidney transplant recipients on quaternary immunosuppressive therapy, including

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323

Likewise, age can also contribute to interindividual difference in immunosuppressant plasma concentration. Pharmacokinetic parameters observed in adults may not be applicable to children, especially to the younger age groups. In general, patients younger than 5 years of age show higher clearance rates regardless of the organ transplanted or the immunosuppres‐ sive drug used [66]. Young children (1–6 years of age) appear to need higher doses per kilogram body weight of TRL than older children and adults to maintain similar trough concentrations. The reason for this age-related faster clearance rate is unknown [67]. Pediatric transplant recipients require higher doses of CsA to maintain blood concentrations equal to those found in adults [68]. Studies using intravenous CsA demonstrate that this is not because of any

Polymorphism has demonstrated functional consequences of many drug metabolizing enzymes. For example, CsA is known substrate for CYP3A4/5 and P-gp. CYP3A5 is one of the main CYP3A enzymes and its expression is clearly polymorphic and shows ethnic dependence. TRL is primarily metabolized by cytochrome P450(CYP)3A enzymes in the gut wall and liver. It is also a substrate for P-gp, which counter-transports diffused TRL out of intestinal cells and back into the gut lumen. Age-associated alterations in CYP3A and P-gp expression and/or activity, along with liver mass and body composition changes would be expected to affect the pharmacokinetics of TRL in the elderly [70]. The importance of interethnic differences in the pharmacokinetics of immunosuppressants has been recognized as having a significant impact on the outcome of transplantation. In a retrospective analysis Fitzimmons et al., found that the oral bioavailability of TRL in African American healthy volunteers and kidney transplant patients was significantly lower than in non-African Americans, but there was no statistically significant difference in clearance [71]. These results were confirmed in a healthy volunteer study. The absolute oral bioavailability of TRL in African American and Latin American subjects was significantly lower than in Caucasians. The results suggested that the observed ethnic differences in TRL pharmacokinetics were, instead, related to differences in intestinal P-glycoprotein-mediated efflux and CYP3A-mediated metabolism rather than differences in

Other ethnic groups such as the Japanese populations are not different from the Caucasian population because their transplant outcomes were comparable under usual TRL dosages [73]. All this factors contribute to the variability of immunosuppressant concentrations which has

TRL, MMF, methylprednisolone and basiliximab [65].

metabolic differences, as CsA clearance is not related to age [69].

hepatic elimination [72].

To avoid the possible effect of food on TRL bioavailability, the drug should be given at a constant time in relation to meals. Several studies have demonstrated that grapefruit juice can increase plasma concentrations of CsA by inhibiting CYP3A-mediated metabolism and by increasing drug absorption via inhibition of P-glycoprotein (P-gp) efflux transporters. Also, oral TRL should not be taken with grapefruit juice since this vehicle inhibits CYP3A4 and/or P-gp contained in the gastrointestinal tract and markedly increases bioavailability. Similarly, drug disease interactions can also contribute to interindividual variability in plasma concen‐ tration of immunosuppressants. Renal insufficiency can result in an altered free fraction of MPA due to the reduction in protein binding. MMF is rapidly converted to its active form, MPA, upon reaching the systemic circulation. MPA is metabolized to its glucuronide metab‐ olite, MPA glucuronide (MPAG), by glucoronyl transferases in the liver and possibly else‐ where. MPAG is then excreted by the kidney. MPA is extensively and avidly bound to serum albumin. Previous studies have demonstrated that it is only the free (non-protein-bound) fraction of MPA that is available to exert its action. *In vivo* and *in vitro* studies demonstrate that renal insufficiency decreases the protein binding of MPA and increases free drug concentra‐ tions. This decrease in protein binding seems to be caused both by the uremic state itself and by competition with the retained metabolite MPAG. The disposition of MPA in patients with severe renal impairment may be significantly affected by this change in protein binding [58]. The concomitant administration of TRL and nonsteroidal anti-inflammatory drugs has been described as a possible cause of increased TRL nephrotoxicity because of the reduction of vasodilator prostaglandin synthesis through a blockade of the enzyme cyclo-oxygenase. Coadministration of ibuprofen and TRL has resulted in acute renal failure. Drugs such as aminoglycosides, cotrimoxazole (trimethoprim/sulfamethoxazole), amphotericin B and aciclovir, which cause significant renal dysfunction on their own, may also enhance TRL nephrotoxicity in the absence of careful monitoring of both renal function and drug concen‐ trations [59].

It has been demonstrated that the *in vitro* metabolism of CsA in human liver microsomes was significantly reduced by TRL [60]. Interaction between MMF and TRL or CsA is probably related to a possible inhibitory effect of TRL on MPA metabolism and an inhibition of the enterohepatic recirculation of MPA by CsA, resulting in a substantial reduction in the MMF dosage when associated with TRL as compared with CsA. This has been reported in pediatric renal allograft patients and animal models [61,62].

Gender also influences drug concentration. Biologic differences exist between men and women that can result in differences in responses to drugs. Both pharmacokinetic and pharmacodynamic differences between the sexes exist, with more data on pharmacokinet‐ ic differences. Bioavailability after oral drug dosing, for CYP3A substrates in particular, may be somewhat higher in women compared to men [63]. It is known that MPA is pri‐ marily metabolized in the liver to its MPAG derivative. Morissette et al., found that men treated with MMF and TRL showed a lower ratio than patients treated with this couple of drugs, confirming that TRL inhibits glucuronidation of MPA. Because MPAG can fa‐ vor the elimination of MPA, they concluded that gender differences and cotreatment with TRL must be taken into consideration when MMF is being administered [64]. Velickovic et al., investigated the gender differences in pharmacokinetics of TRL, their result show remarkable gender-related differences between women and men after the first oral dose among kidney transplant recipients on quaternary immunosuppressive therapy, including TRL, MMF, methylprednisolone and basiliximab [65].

look into the multitude of factors that contribute to the interindividual variability. Some of the factors include drug-nutrients 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. Renal transplant patients may have reduced oral bioavailability for TRL. When given with meals, especially with high fat content food, oral

To avoid the possible effect of food on TRL bioavailability, the drug should be given at a constant time in relation to meals. Several studies have demonstrated that grapefruit juice can increase plasma concentrations of CsA by inhibiting CYP3A-mediated metabolism and by increasing drug absorption via inhibition of P-glycoprotein (P-gp) efflux transporters. Also, oral TRL should not be taken with grapefruit juice since this vehicle inhibits CYP3A4 and/or P-gp contained in the gastrointestinal tract and markedly increases bioavailability. Similarly, drug disease interactions can also contribute to interindividual variability in plasma concen‐ tration of immunosuppressants. Renal insufficiency can result in an altered free fraction of MPA due to the reduction in protein binding. MMF is rapidly converted to its active form, MPA, upon reaching the systemic circulation. MPA is metabolized to its glucuronide metab‐ olite, MPA glucuronide (MPAG), by glucoronyl transferases in the liver and possibly else‐ where. MPAG is then excreted by the kidney. MPA is extensively and avidly bound to serum albumin. Previous studies have demonstrated that it is only the free (non-protein-bound) fraction of MPA that is available to exert its action. *In vivo* and *in vitro* studies demonstrate that renal insufficiency decreases the protein binding of MPA and increases free drug concentra‐ tions. This decrease in protein binding seems to be caused both by the uremic state itself and by competition with the retained metabolite MPAG. The disposition of MPA in patients with severe renal impairment may be significantly affected by this change in protein binding [58]. The concomitant administration of TRL and nonsteroidal anti-inflammatory drugs has been described as a possible cause of increased TRL nephrotoxicity because of the reduction of vasodilator prostaglandin synthesis through a blockade of the enzyme cyclo-oxygenase. Coadministration of ibuprofen and TRL has resulted in acute renal failure. Drugs such as aminoglycosides, cotrimoxazole (trimethoprim/sulfamethoxazole), amphotericin B and aciclovir, which cause significant renal dysfunction on their own, may also enhance TRL nephrotoxicity in the absence of careful monitoring of both renal function and drug concen‐

It has been demonstrated that the *in vitro* metabolism of CsA in human liver microsomes was significantly reduced by TRL [60]. Interaction between MMF and TRL or CsA is probably related to a possible inhibitory effect of TRL on MPA metabolism and an inhibition of the enterohepatic recirculation of MPA by CsA, resulting in a substantial reduction in the MMF dosage when associated with TRL as compared with CsA. This has been reported in pediatric

Gender also influences drug concentration. Biologic differences exist between men and women that can result in differences in responses to drugs. Both pharmacokinetic and pharmacodynamic differences between the sexes exist, with more data on pharmacokinet‐

bioavailability of TRL decreases [57].

322 Current Issues and Future Direction in Kidney Transplantation

trations [59].

renal allograft patients and animal models [61,62].

Likewise, age can also contribute to interindividual difference in immunosuppressant plasma concentration. Pharmacokinetic parameters observed in adults may not be applicable to children, especially to the younger age groups. In general, patients younger than 5 years of age show higher clearance rates regardless of the organ transplanted or the immunosuppres‐ sive drug used [66]. Young children (1–6 years of age) appear to need higher doses per kilogram body weight of TRL than older children and adults to maintain similar trough concentrations. The reason for this age-related faster clearance rate is unknown [67]. Pediatric transplant recipients require higher doses of CsA to maintain blood concentrations equal to those found in adults [68]. Studies using intravenous CsA demonstrate that this is not because of any metabolic differences, as CsA clearance is not related to age [69].

Polymorphism has demonstrated functional consequences of many drug metabolizing enzymes. For example, CsA is known substrate for CYP3A4/5 and P-gp. CYP3A5 is one of the main CYP3A enzymes and its expression is clearly polymorphic and shows ethnic dependence. TRL is primarily metabolized by cytochrome P450(CYP)3A enzymes in the gut wall and liver. It is also a substrate for P-gp, which counter-transports diffused TRL out of intestinal cells and back into the gut lumen. Age-associated alterations in CYP3A and P-gp expression and/or activity, along with liver mass and body composition changes would be expected to affect the pharmacokinetics of TRL in the elderly [70]. The importance of interethnic differences in the pharmacokinetics of immunosuppressants has been recognized as having a significant impact on the outcome of transplantation. In a retrospective analysis Fitzimmons et al., found that the oral bioavailability of TRL in African American healthy volunteers and kidney transplant patients was significantly lower than in non-African Americans, but there was no statistically significant difference in clearance [71]. These results were confirmed in a healthy volunteer study. The absolute oral bioavailability of TRL in African American and Latin American subjects was significantly lower than in Caucasians. The results suggested that the observed ethnic differences in TRL pharmacokinetics were, instead, related to differences in intestinal P-glycoprotein-mediated efflux and CYP3A-mediated metabolism rather than differences in hepatic elimination [72].

Other ethnic groups such as the Japanese populations are not different from the Caucasian population because their transplant outcomes were comparable under usual TRL dosages [73]. All this factors contribute to the 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.

therapeutic concentrations in blood are 200-800 ng/mL, and by polyclonal fluorescence polarization immunoassay (monoclonal TDx assay, Abbott Diagnostics®), or polyclonal radioimmunoassay (various manufacturers), the level of therapeutic concentrations in plasma

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The first steps towards the development of a more precise monitoring strategy for CsA resulted from the landmark studies by Lindholm and Kahan and Kahan et al., which identified a link between the pharmacokinetics of CsA and clinical outcomes in the individual transplant recipient [80,81]. The area under the concentration-time curve for CsA over a 12-hour drug administration interval (AUC0-12h) was a more precise predictor of graft loss and incidence of acute rejection than other parameters, including the C0. Since then, subsequent studies on the pharmacokinetics of CsA in renal transplant patients have identified that intrapatient varia‐ bility in AUC values over time was directly correlated with the risk of chronic rejection [77,82].

Proper calculation of AUC requires administration of a dose, followed by blood collection according to an intensive sampling strategy. Concentration values obtained are used to calculate AUC, usually by the trapezoidal method [78]. Some advantages of AUC monitoring are that it is the most precise indicator of drug exposure, can characterize abnormal absorption patterns, appears to be a predictor of clinical outcomes, generates a concentration-time profile, allows calculation of oral pharmacokinetic parameters, and reduces the problems associated

Despite its appealing potential advantages, the major disadvantage of AUC monitoring is its inherent need for multiple blood samples. The increased number of samples required, makes AUC monitoring impractical for routine clinical use, more expensive in the short term because of increased sample collection, analysis and interpretation of results, and inconvenient for patients, especially those in an outpatient setting [77,85]. AUC has been advocated as a better parameter to monitor than trough concentrations, because trough concentrations give no indication of exposure to CsA. For example, 2 patients could have the same trough concen‐ tration, but one could have a much lower AUC and, therefore, exposure to CsA. Unfortunately, AUC monitoring is not clinically feasible because of the added time, expense and inconven‐ ience required to collect a sufficient number of samples to properly calculate AUC. Although the full AUC for CsA has been demonstrated as being a sensitive monitoring tool, there may be an alternative approach to the determination of the degree and variability of CsA exposure

This approach, which is termed 'absorption profiling', has the underlying rationale that the 4 hour absorption phase following administration provides measurements that are more informative than C0 monitoring in the assessment of likely CsA exposure and subsequent clinical response [86,87]. AUC0-4h monitoring is a sensitive tool used to optimize CsA immu‐ nosuppression in renal transplant recipients. However, the tool is not practical in the clinical

are 100-400 ng/mL [79].

*4.1.1.2. Area under the blood concentration-time curve (AUC)*

with laboratory errors and single concentrations [74,83,84].

*4.1.1.3. Two hours post dose concentration monitoring (C2)*

in the individual patient [77,83].
