SUPPLEMENT

Next Level of Immunosuppression:Drug/Immune MonitoringJosh LevitskyDivision of Hepatology and Comprehensive Transplant Center, Northwestern UniversityFeinberg School of Medicine, Chicago, IL

Key Points1. Current immunological monitoring relies heavilyon clinical judgment and therapeutic drug levels anddoes not adequately assess the functional or donor-specific immunosuppression (IS) status of recipientsof liver transplantation (LT).2. Trough levels of drugs are arbitrary and are moreclinically relevant for preventing supratherapeutic orsubtherapeutic dosing and blood concentrations andfor more closely monitoring at-risk populations(children, the elderly, and patients with organ dys-function). The AUC or the post-dose levels may bemore precise, but they have not been used exten-sively by transplant centers.3. Data on drug/immune monitoring specific to LTare fairly limited; therefore, clinical practice is oftenborrowed from experiences with nonhepatic trans-plantation (mainly renal transplantation).4. The monitoring of drug levels in patients takinggeneric immunosuppressants is challenging becausethe formulations may change with each prescription.The monitoring of drug or antibody levels is not yetclinically available for biological therapies (induction,lymphocyte-depleting, and maintenance agents).5. Polymorphisms in drug metabolism (cytochromeP450 and P-glycoprotein) may be useful in selectingthe initial and maintenance dosages of immunosup-pressants and in preventing complications from over-or underimmunosuppression.6. Future immune monitoring assays should befocused on genomic or immunological predisposi-

tions and on specific reactivities to donor antigensto guide the appropriate dosing and minimization ofIS after LT. Liver Transpl 17:S60-S65, 2011. VC 2011AASLD.

Received June 1, 2011; accepted July 11, 2011.

Initial and maintenance immunosuppression (IS)therapies have directly led to improved long-termpatient and graft survival after liver transplantation(LT). However, with patients living longer after LT,maintenance IS is contributing significantly to healthcare costs and late morbidity. The adverse effects ofIS therapy [particularly the calcineurin inhibitors(CNIs) tacrolimus and cyclosporine] include increasedrisks of cardiovascular disease, metabolic syndrome,bone loss, opportunistic and community-acquiredinfections, malignancies, and chronic kidney disease.1

To lessen these effects, clinicians often empiricallyattempt to minimize CNI dosages by trial and error, orthey use alternative IS agents [antimetabolites suchas azathioprine and mycophenolic acid (MPA) andmammalian target of rapamycin (mTOR) inhibitorssuch as sirolimus and everolimus] because of the lowsuccess rate of complete IS withdrawal.2 Currently,the IS status of individual patients depends heavilyon subjective clinical characteristics (history of infec-tions and malignancies versus rejection) and on arbi-trary ranges for IS trough concentrations. Althoughnothing can replace the clinical judgment of an experttransplant clinician for pooling together data to

Abbreviations: ABC, adenosine triphosphate–binding cassette; ATP, adenosine triphosphate; AUC, area under the curve; CNI,calcineurin inhibitor; CYP3A, cytochrome P450 3A; FOXP3, forkhead box P3; IS, immunosuppression; LT, liver transplantation;MPA, mycophenolic acid; MPAG, mycophenolic acid glucuronide; mTOR, mammalian target of rapamycin.

Potential conflict of interest: Nothing to report.

Address reprint requests to Josh Levitsky, M.D., M.S., Division of Hepatology and Comprehensive Transplant Center, Northwestern UniversityFeinberg School of Medicine, 676 North St. Clair Street, Suite 1900, Chicago, IL 60611. Telephone: 312-695-9286; FAX: 312-695-0036; E-mail:j-levitsky@northwestern.edu

DOI 10.1002/lt.22385View this article online at wileyonlinelibrary.com.LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

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individualize IS therapy, more objective assessmentsof the net IS state that can be used in clinical trans-plantation are greatly needed. In addition, becausedata on IS drug therapy and monitoring have beenborrowed from experiences with renal transplantation(for which the patient populations, pharmacokinetics,and disease states are different), more data specific tothe LT population are needed. This review is focusedon the current state of drug/immune monitoring,individualized therapy, and the potential applicationsof currently available immune assays and those indevelopment.

THERAPEUTIC DRUG MONITORING

Assay Methods

Because tacrolimus (>90%) and cyclosporine (>50%)are concentrated in erythrocytes, whole blood is usedto measure the trough levels of both CNI agents.3 Mostcenters use an enzyme-linked immunosorbent assay tomeasure tacrolimus and cyclosporine levels, althoughother methods such as microparticle enzyme-linkedimmunoassays and the enzyme-multiplied immunoas-say technique can be used; however, they may overes-timate concentrations in anemic patients. Anothermethod, high-performance liquid chromatography, wasdeveloped for evaluating 4 cyclosporine A degradationproducts (ID-005-95, cyclosporine H, isocyclosporineA, and isocyclosporine H) and 2 compounds (cyclospo-rine B and cyclosporine G); equivalent results werefound for Neoral and its generic formulations.4 Thistype of assay might provide more precision and accu-racy through the analysis of breakdown productsrather than drug levels. MPA and its inactive glucuro-nide [mycophenolic acid glucuronide (MPAG)] are usu-ally assessed in plasma with high-performance liquidchromatography, but other methods such as theenzyme-multiplied immunoassay technique and morespecific automated methods have been used for freeconcentration determinations.5,6 Like CNIs, the mTORinhibitors sirolimus and everolimus are distributedmainly in red blood cells (95%); approximately 40% isbound to lipoproteins, 60% remains unbound, and<5% is bound to plasma proteins.7 Thus, whole bloodis considered the best matrix for monitoring. Liquidchromatography with either tandem mass spectrome-try or ultraviolet detection, enzyme-linked immunosor-bent assays, microparticle enzyme-linked immunoas-says, and fluorescence polarization immunoassays areamong the methods used for measuring mTOR inhibi-tor levels, although liquid chromatography with massspectrometry or ultraviolet detection yields resultsapproximately 20% less accurate than those of micro-particle enzyme-linked immunoassays.8

Maintenance Agents

For nearly all standard maintenance agents used inLT patients, there are assays available for therapeuticdrug monitoring, which is mainly based on the pre-

dose trough levels. Trough levels alone have littlevalue for assessing the overall IS status, and theyneed to be placed in the context of clinical scenarios.Clinical symptoms that correlate with drug levels (ie,headaches, tremors, and gastrointestinal symptomsrelated to high CNI levels) are mainly related to supra-therapeutic levels and will often improve with dosagereductions or temporary discontinuation. The appro-priate therapeutic target levels of drugs at varioustime periods after LT are controversial because stand-ard protocols that are helpful in managing large num-bers of recipients are not targeted at specific clinicalscenarios and are not personalized unless the clini-cian does so. Because agents such as sirolimus haveprolonged half-lives and large interpatient and intra-patient variability, therapeutic drug monitoring isusually performed, but as with CNIs, it does not pro-vide well-defined therapeutic ranges and mainly corre-lates high and low levels with clinical toxicity (ie, hy-perlipidemia, leukopenia, thrombocytopenia, anemia,and oral ulcers) and rejection, respectively.7 Adverselaboratory and clinical symptoms can be easily moni-tored by laboratory tests and clinical assessmentsand then later correlated with drug levels for patientmanagement (ie, rather than the reverse). Therefore,drug trough levels tend to be used as a supportiveguide rather than the focal point for the clinician whois managing IS in transplant patients.

To further define targeted dosing and individualizedtherapy, there have been efforts to perform AUCassessments as better measures of drug exposure thanpredose trough concentrations. Large clinical trials ofLT patients treated with cyclosporine have demon-strated low rates of underimmunosuppression (rejec-tion) and overimmunosuppression (nephrotoxicity)complications with monitoring based on either AUC0-4

or the concentration 2 hours after administration.9-11

However, the monitoring of the 2-hour concentrationwith cyclosporine has been implemented only at asmall number of centers, likely because insufficientcomparative data support the monitoring of the 2-hourconcentration versus the predose trough concentra-tion. Therapeutic drug monitoring for MPA is also con-troversial. AUC-measured plasma concentrations ofMPA may be useful for predicting rejection, but wideAUC variations occur between different individuals,types of recipients, concurrent IS therapies, and clini-cal conditions (liver and kidney dysfunction).12 In addi-tion, the correlation between MPA exposure (by theAUC or MPAG concentration) and toxicity (gastrointes-tinal toxicity, hematological toxicity, infections, andmalignancies) is poor, and this would be betterassessed by free MPA levels, although they are moredifficult to measure than protein-bound plasma con-centrations.13 One study correlated predose MPA con-centrations with adverse events and rejection in LTrecipients, and it was concluded that the MPA thera-peutic range should be 1 to 3.5 mg/L.14 However, themonitoring of MPA levels has not gained wide accep-tance because of the cost and the lack of convincingclinical utility.

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Patients at the extremes of age are at greaterrisk for IS drug toxicity and rejection. The pharmaco-kinetic parameters of adults may not be applicable tochildren and especially to those less than 5 years oldwho demonstrate greater clearance rates and the needfor higher IS dosages.15 Children are also more proneto gastroenteritis, which can result in malabsorptionand rejection, and they may require dose adjustmentswith growth and time after transplantation. At theother extreme, the elderly have decreased immunores-ponsiveness, have a higher risk of drug interactionsbecause of polypharmacy and comorbidities, andhave greater interindividual and intra-individualpharmacokinetic variability.16 The absorption, distri-bution volume (changes in body fat and plasma pro-teins), metabolism [cytochrome P450 3A (CYP3A) ac-tivity and P-glycoprotein levels], and elimination ofdrugs may all be affected by advancing age, althoughthere is a paucity of pharmacokinetic data for elderlyrecipients. Although this has not been proven, less in-tensive IS therapy (ie, lower dosages and less use ofcombined drug therapies) may be preferable in the el-derly. At both age extremes, therapeutic drug moni-toring appear to be more important because of thephysiological changes related to growth and aging,although these populations and particularly LT recipi-ents are the least well studied.

Induction/Biological Agents

With the growing interest in the use of induction andother antibody therapies in LT recipients to reducethe toxicity of maintenance agents, drug monitoringhas become even more challenging. Other than totallymphocyte and neutrophil counts/percentages andassays for monitoring infections (cytomegalovirus andEpstein-Barr virus), there are no commercially devel-oped assays for measuring the levels of or exposure tolymphocyte-depleting antibodies (antithymocyte glob-ulin, OKT3, and alemtuzumab) and non–lymphocyte-depleting antibodies (interleukin-2 receptor inhibitorsand cytotoxic T lymphocyte antigen 4 immunoglobu-lin). This is important because many of these agentshave been associated with an increased risk of infec-tious and neoplastic complications. It may be possibleto measure serum or plasma free antibody levels andcomplexes (eg, alemtuzumab and other humanizedantibodies), although these assays are not yet avail-able for clinical use.17 Even though these agents arepromising, there are many unresolved issues, includ-ing their long half-lives, their long-term safety and ef-ficacy, and the lack of monitoring tools for modulatingtheir use over time.

Generics

Within the last 5 years, 2 of the most common IStherapies in solid organ transplantation, tacrolimusand mycophenolate mofetil, have lost their patent pro-tection along with cyclosporine. Therefore, severalgeneric companies are now manufacturing these

agents. Because of the significantly lower costs, insur-ance companies provide financial incentives forgeneric drug use via higher copayments for brandname agents. The US Food and Drug Administrationrequires only that generic formulations fall within the80% to 125% range for bioequivalence, and it has notgenerally taken into account the narrow therapeuticwindow of IS therapy in its recommendations. Trans-plant clinicians are now faced with the dilemma ofpatients who want to switch to generic drugs for logi-cal financial reasons but risk the possibility of drugtoxicity or rejection due to drug formulation variabilityand narrow therapeutic windows. The question iswhether these concerns are fact or fiction. Cangeneric drugs be used safely in LT recipients just asthey are in patients with other common conditions(eg, hypertension and diabetes)? At this juncture,there are 2 major issues with generic IS: (1) the lackof data for all transplant recipients and for LT recipi-ents in particular and (2) the potential for the variabil-ity in every new prescription to lead to swings in ISlevels. The data on the equivalency of generic andbranded formulations are limited and show mixedresults. Most studies have been performed in healthyvolunteers or renal transplant recipients. Moreover,bioequivalent studies are performed only in a limitednumber of healthy volunteers whose pharmacoki-netics may be markedly different from those expectedin LT recipients. For CNI therapy, some studies haveshown equivalence or a lack of inferiority,18,19

whereas others have reported more significant con-cerns and the need for dose adjustments and closemonitoring.20,21 Some studies comparing generic andbrand name formulations of mycophenolate mofetilhave shown reasonable bioequivalence.18,22 The factthat one prescription’s formulation can be different interms of drug bioavailability is less of a concernbecause a steady state can be addressed and moni-tored; the real problem is that every new prescription(typically with 30- or 90-day refills) can lead to multi-ple formulation changes. The need for closer bloodmonitoring or higher doses due to this variabilitymay paradoxically increase the cost and inconven-ience.23 Although no firm recommendations can bemade at this point, patients should be warned of thepotential for complications (toxicity and rejection)and the need for closer blood level monitoring withevery new generic prescription. At Northwestern Uni-versity, we empirically recommend laboratory valuesand trough levels every 1 to 2 weeks until a steadystate has been achieved whenever a new generic pre-scription is filled. These monitoring approaches,which we feel are necessary because of the risks,clearly burden patients and may paradoxicallyincrease health care costs.

Pharmacogenetics/Dynamics

There has been a growing interest in the effects ofgenetic variations of drug-metabolizing enzymes andspecifically the CYP3A enzymes (CYP3A4 and

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CYP3A5) on drug exposure in LT recipients. Theseenzymes are responsible for the metabolism of CNIand mTOR therapies as well as multiple other phar-macological agents (Table 1). Variations in the DNAsequence at a single nucleotide (ie, single-nucleotidepolymorphisms) have been reported to significantly al-ter drug metabolism and may provide the ability topersonalize drug dosing. Two single-nucleotide poly-morphisms (CYP3A4*1B and CYP3A5*1) are morecommon in African Americans than Caucasians orAsians,24 although only the CYP3A5 polymorphisms(CYP3A5*1) are associated with higher tacrolimusclearance, lower trough levels, and more than 2-foldgreater dose requirements.25 These polymorphismshave been tested mainly in renal transplant recipientsand require confirmation in LT patients.26

Recipients also receive numerous medications thatare substrates for P-glycoprotein, which is a cellularcarrier and membrane transporter involved in thepharmacokinetics/dynamics of many drugs, includingCNIs, mTORs, and MPA. The gene encoding P-glyco-protein is adenosine triphosphate–binding cassette B1(ABCB1), which has 3 single-nucleotide polymor-phisms associated with altered activity. These varia-tions may occur at the level of drug absorption, distri-bution, metabolism (P-glycoprotein and CYP3Aisoform associations), and/or elimination (smallbowel, bile canaliculus, and renal tubule P-glycopro-tein). A recent study has demonstrated a significantassociation between genetic polymorphisms of ABCB1and tacrolimus-associated nephrotoxicity in pediatricLT recipients.27 Another gene, ABCC2, encodes multi-drug resistance–associated protein 2 transporters,which are important for small bowel absorption ofMPA and the movement of MPAG into bile. Polymor-phisms of ABCC2 (C-24T) may be associated with gas-trointestinal intolerance related to MPA.

Other genetic polymorphisms, such as those usedin commercially available assays for thiopurine

S-methyltransferase deficiency, are associated withpancytopenia due to drugs less commonly used intransplantation: azathioprine and 6-mercaptopurine.There also may be an immunogenetic (human leuko-cyte antigen) predisposition to drug toxicity, althoughthis is not well explored in transplantation. Recently,a unique approach involving pharmacodynamic drugmonitoring was used to correlate drug levels with theactual biological or cellular activity of IS therapy.28 Inthat study, there was a significant negative correlationbetween the peak levels of cyclosporine and tacroli-mus (P < 0.001, r ¼ �0.6982) and the residual geneexpression of all genes regulated by nuclear factor ofactivated T cells.

Overall, none of the pharmacogenetic/dynamicassays have moved into the clinical setting, but theyappear to be promising for the prediction of dosingrequirements and for the monitoring of adverseeffects. In addition, these polymorphisms that affectliver drug metabolism originate from the donor. Theseanalyses are challenging in LT recipients versus othersolid organ transplant recipients because they requirethe availability of donor cells for the analysis ofgenetic variations.

COMMERCIALLY AVAILABLE IMMUNEMONITORING

Assessing the net IS state in an LT recipient is amajor challenge, and subjective clinical parameterscurrently outweigh any single laboratory measure.Therefore, there is a high level of interest in the devel-opment of specific immune monitoring assays thatcan be used to prospectively assess recipients onlong-term maintenance IS. There is one commerciallyavailable test, the Cylex ImmuKnow assay, that mayprovide a global assessment of immune function andallow for more accurate, individualized IS therapyadjustments than clinical assessments and drugtrough levels. This whole blood assay quantifies theamount of adenosine triphosphate (ATP) produced byCD4þ T cells in response to non–donor-specific mito-genic stimulation (phytohemagglutinin-L) in vitro. Ameta-analysis of solid organ transplantation studiesusing this assay revealed high correlations betweenlower and upper ranges of ATP responses and infec-tious complications and acute cellular rejection,respectively, with the target level of immune functionin the middle (ATP level ¼ 280 ng/mL).29 This assayappears to be more helpful in assessing overimmuno-suppressed states (viral hepatitis recurrence andinfections) than underimmunosuppressed states(rejection).30 Much of the recent literature has sup-ported the use of ATP assays in LT for distinguishinghepatitis C virus recurrence from rejection, particu-larly when liver biopsy cannot make the distinction orshows features of both.31 Hepatitis C virus–positiverecipients have low ATP responses at the baseline(right after LT) and when recurrent disease is con-firmed on biopsy.32 In addition, low ATP productionhas been correlated with the more rapid development

TABLE 1. Interactions Between IS Agents and Other

Medications

Drugs Increasing CNI and mTOR Trough

Concentrations

Macrolides: clarithromycin, erythromycin, andazithromycinAntifungals: fluconazole, ketoconazole, itracona-zole, voriconazole, and clotrimazoleCalcium channel blockers: verapamil, diltiazem,and nifedipineOthers: metoclopramide, danazol, human immuno-deficiency virus/hepatitis C virus protease inhibi-tors, and grapefruit juice

Drugs Decreasing CNI and mTOR Trough

Concentrations

Antibiotics: rifampin, rifampicin, and rifabutinAnticonvulsants: phenytoin, phenobarbital, andcarbamazepineOthers: St. John’s wort

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of fibrosis, likely because of overimmunosuppressionand a lack of virological control.33 The degree to whichcenters are using this assay to monitor their patientswith and without hepatitis C virus after LT is notknown, although the assay is likely being increasinglyused as an adjunct in specific clinical scenarios.

FUTURE OF IMMUNE MONITORING

Lessons From Spontaneously Tolerant

Recipients

Transplant researchers have obtained samples fromspontaneously or research protocol–tolerant patients,and their tolerant phenotype has allowed the testingof bioassays as putative in vivo markers of immunore-gulation or alloreactivity. Although the major focus ofthese studies has been the determination of tolerancesignatures, the assays could alternatively be used toimmunologically monitoring patients for over/under-immunosuppression and for more specifically direct-ing the need for IS increases or weaning. The majorcandidates are blood immunophenotypic assays(CD4þCD25high FOXP3+ cells and Vd1/Vd2 cellratios), cytokine gene profiles (natural killer, cd T cell,and CD8þ receptor genes), and genomic microarrays.Studies have shown that operationally tolerant adultLT recipients have significantly higher numbers of pe-ripheral blood CD4þCD25high T cells and cd T cells(Vd1/Vd2 ratios) than nontolerant recipients orhealthy individuals.34 Blood FOXP3 in conjunctionwith CD4þCD25high T cells were observed at a higherfrequency in LT recipients undergoing successfulwithdrawal versus those developing rejection.35 Morerecently, gene expression profiling of tolerant liverrecipients demonstrated a unique signature involvingcd T cell receptors, natural killer cell receptors, andcell proliferation arrest proteins.34 The same groupfound similar gene signatures (natural killer, cd, andCD8þ cells) and immunophenotypic signatures(CD4þCD25þ cd T cells of the Vd1þ subset) in LTrecipients who were successfully withdrawn from ISversus healthy controls and recipients who rejectedwith weaning.36 Sequential data that directly supportthe ability of these assays to monitor the net IS statehave not yet been published, and the assays are notyet commercially available in clinical settings. How-ever, they appear to be ready for testing as immunemonitoring predictors in prospective studies. As such,patients could be characterized more specifically byfavorable or unfavorable characteristics and bioas-says. Thus, safer IS modifications would be possible,and the risks of rejection with minimization could bereduced.

Donor-Specific Immune Monitoring

Ideally, immune monitoring for transplantationshould be donor-specific: an assay should be able totest donor-specific hyporesponsiveness and the pres-ervation of immunity against nondonor antigens

(infections and tumors). This may now be more feasi-ble with donor-specific immune monitoring assays,which more clearly detect states favoring alloimmunequiescence over reactivity (ie, whether they are due topartial clonal deletion/exhaustion or an active regula-tory process).2 These functional assays (mixed lym-phocyte reactions, enzyme-linked immunosorbentspot assays, trans vivo delayed-type hypersensitivityassays, cell-mediated lympholysis, cytokine kineticsassays, and interferon-c kinetics) might then be usedto evaluate whether changes in IS therapy should bemade. This could allow minimization in those withfavorable donor-specific responses and prevent suchinterventions or even optimize IS therapy in thosewith immunoreactivity. The major limiting factor isthe requirement for available donor cells, which aredifficult to obtain in cadaveric LT unless cells fromdonor lymph nodes or spleen tissue can be harvested,isolated, and cryopreserved for future donor-specificassays. Others have previously reported an assaytesting the activation status of allospecific peripheralblood lymphocytes against allogeneic graft–derivedfibroblasts versus third-party antigens by 3H-thymi-dine incorporation.37 This test was able to detect allo-graft rejection with high specificity and sensitivity.However, this assay has not been tested in clinicaltransplantation and still requires the preoperativestorage of donor cells (graft-derived fibroblasts).

In conclusion, past experiences can indeed providevaluable lessons when we are considering futureapproaches to IS monitoring. In the next era, weshould strive for more personalized IS approaches sothat we can increase our ability to monitor the netIS state on the basis of precisely determined, highlyobjective immunological traits, genetics, andbiomarkers.

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