Prospective investigations of concentration–clinical response for immunosuppressive drugs provide the scientific basis for therapeutic drug monitoring

Departments of
¹ Pathology & Laboratory Medicine and
² Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104.
³ St. Barnabas Medical Center, Livingston, NJ.
^a Author for correspondence. Fax 215-662-7529; e-mail shawlmj{at}mail.med.upenn.edu.

Abstract

The performance of prospective concentration–clinical response investigations during the early stages of the development of new therapeutic agents can provide a more rigorous basis for therapeutic drug monitoring than the traditional retrospective review of drug concentrations vs clinical outcome. Here we discuss the application of the multicenter randomized concentration-controlled clinical trial study design, and related study designs, as applied to older commonly used and monitored drugs and to two new immunosuppressant drugs, mycophenolate mofetil and tacrolimus. Such studies can provide a more rigorous basis for assessing the risk/benefit associated with a target drug concentration in the individual patient and for designing future prospective pharmacokinetic and therapeutic drug monitoring investigations.

Traditionally, the clinical response of patients to most new drugs has been evaluated by comparison of a group receiving a single dosage in parallel with either a placebo group or one or more additional dosage groups. Patients are randomized to one of the dosage groups for the duration of the study. Such studies, usually referred to as randomized dose-controlled clinical trials, often lead to large variability in response within comparison groups, because of extensive interpatient variability of both pharmacokinetics (PK)¹ and pharmacodynamics (PD), i.e., clinical response, effectiveness, and undesirable effects. Thus, understanding the factors that account for patients' clinical response variability is hampered by the lack of control of drug concentration when both PK and PD variabilities are considerable (1)(2)(3).

A recommended approach to controlling PK variability among patients during clinical drug trials is performance of prospective concentration-controlled studies. Such investigations can provide the basis for implementation of effective therapeutic drug monitoring (TDM) early after introduction of new medications into clinical practice. This will lead to early utilization of the new drug in the safest and most efficacious manner

Studies with Already Approved Drugs

Before considering the application of this approach to the study of new immunosuppressive drugs, it will be helpful to first review studies performed in already approved medications. An appreciation of the potential value of prospective concentration-controlled clinical trials is apparent on reviewing the recent application of this approach to some of the commonly monitored drugs (Table 1 ). In each instance concentration-controlled studies were performed to address specific questions that arose after years of usage. In most of these studies the results provided practitioners with an improved ability to provide the optimal dosage of the medication in individual patients. Below, we will consider briefly a concentration-controlled study involving lithium to illustrate how this approach works and its potential significance for the practice of TDM.

concentration-controlled study for lithium
A detailed, long-term study of the serum concentration-clinical response relationship of lithium, a drug with a narrow therapeutic index, was recently reported (6). Lithium salts are prescribed for the acute treatment of manic-depression and most commonly for long-term prevention of manic and depressive episodes in patients with bipolar disease (10). For these purposes lithium has proven to be a very effective medication. Throughout its therapeutic history, however, lithium has been dogged by the reputation of being toxic. A major reason for the routine monitoring of the morning 12-h postdose serum concentration has been to reduce the risk for toxic effects by controlling against doses that are too high in the individual patient (11). In 1975, a task force of the American Psychiatric Association suggested a serum lithium target concentration range of 0.7–1.2 mmol/L for maintenance therapy (12). However, 10 years later, an NIH consensus panel recommended a lower maintenance range, 0.6–0.8 mmol/L. An important factor that led to the recommended lower range was concern about the potential for structural and functional impairment of the kidney from chronic exposure to the higher concentrations of lithium (13). Thus, although lithium had been used in clinical practice for many years, along with TDM to guide dosage adjustment, there was a lack of sufficient concentration–clinical response data to permit selection of the optimal target range with confidence.

To scientifically investigate this problem and to provide a practical guideline for "best practice" lithium TDM, a randomized double-blind trial was conducted, comparing two different lithium concentration ranges for maintenance treatment of bipolar disease (6). Patients with manic-depression were randomized to either a "standard dose" therapeutic range of 0.8–1.0 mmol/L or a "low dose" range of 0.4–0.6 mmol/L and treated for a total of 150 weeks. The clinical responses measured were the relapse rate for manic-depression and the incidence rate of side effects. Serum lithium was measured in morning 12-h postdose samples every 2 months. For continual monitoring of the analytical performance of the lithium measurements, every 10th sample was sent to a referee laboratory. The concentration ranges achieved (mean ± SD) were 0.83 ± 0.11 mmol/L for the standard dose range and 0.54 ± 0.12 mmol/L for the low dose range.

At the conclusion of the study the rate of relapse was 6 of 47 (13%) in the standard dose range patients and 18 of 47 (38%) in the patients assigned to the low dose range. That is, the risk for relapse in the low range patients was 2.6 times higher than that in the standard range group. Side effects, however, were more frequent in the high range group. The conclusions drawn from this study were that doses resulting in serum lithium concentrations of 0.8–1.0 mmol/L are more effective in treating bipolar disorder than those that result in lower concentrations, although the higher concentrations are indeed associated with a higher incidence of side effects. These results, together with evidence that the concern about lithium nephrotoxicity may have been overemphasized, provided support for physicians' use of serum lithium concentrations of 0.8–1.0 mmol/L in patients with bipolar disease who do not respond well to doses that produce low range concentrations. Thus, judicious use of lithium TDM provided assessment of the risks for relapse as well as for toxic side effects. As with all medications, attaining the best outcome requires educating the patient as to the rationale for the selected regimen and the importance of compliance.

concentration-controlled clinical trials of cyclosporine
During the past 16 years, the pharmacological manipulation of the immune system in the field of solid-organ transplantation has undergone remarkable improvements. For example, 1-year kidney allograft survival now approaches 95% for transplanted living related kidneys and 85–90% for cadaveric kidneys (14), whereas before the introduction of cyclosporine (CsA), 1-year graft survival was ~60%. This success has been achieved without increasing the infectious risks for immunosuppressed patients.

The formal investigation of the PK and TDM of immunosuppressive agents is a topic of considerable interest and importance in the field of transplant medicine. This interest began early in the "CsA era" of immunosuppressive therapy in 1983. The following are major developments in the practice of CsA TDM:

1) CsA is the first immunosuppressive agent to receive serious attention regarding PK, PD, and TDM. At least 6 task forces or consensus conferences have considered the issues involved in CsA TDM (15)(16)(17)(18)(19)(20).

2) Most centers use two target ranges, one for initial therapy (usually up to 6 months posttransplant) and the second, lower, target range for maintenance therapy thereafter.

3) The target ranges vary with the analysis method, transplant type, and transplant center philosophy on desired intensity of immunosuppression.

4) The ranges were developed by retrospective review of CsA concentration data and correlation with clinical events from single-center studies.

5) The vast majority of centers use a predose trough blood sample for CsA analysis.

6) Some centers use CsA area under the curve (AUC) for concentration vs time, as derived from selected timed samples collected within the usual 12-h dose interval. This practice is based on their conclusion that trough concentrations correlate poorly with the AUC, and thereby do not adequately reflect CsA exposure, whereas improved correlation with clinical effects of the drug are achieved by evaluating total exposure (i.e., AUC) (21). A practical limitation of this approach has been the necessity to collect several timed samples up to and including a 9–10-h sample (17)(20).

Measurement of whole-blood concentrations of CsA is a standard of practice in virtually all transplant centers. Because of the considerable variability in the bioavailability, metabolism, and excretion of CsA in transplant patients and because of the drug's narrow therapeutic index, dosage individualization based on blood CsA concentration as a guide is required to reduce the risk for either underdosage or toxicity (20). It is generally recognized, however, that without the benefit of prospective concentration-controlled studies performed with validated analytical methodology for CsA in multiple centers, the risk/benefit ratio for specific concentrations of the drug in specific patient groups is lacking. With the benefit of the many years of accumulated experience from TDM of CsA, however, has come a much better appreciation of the types of studies needed to provide more effective TDM of new immunosuppressive agents.

Studies with New Drugs

Two concentration-controlled clinical studies, one involving mycophenolate mofetil (MMF), the other tacrolimus, were recently completed. These studies will, it is hoped, provide a more rigorous and scientifically sound basis for developing the clinical use and TDM of these drugs.

mycophenolate mofetil
MMF, the morpholinoethyl ester prodrug form of mycophenolic acid (MPA), was approved in 1995 for use, in combination with CsA and prednisone, in preventing rejection in renal transplant patients. Clinical trials are underway in heart (MMF CsA steroid) and liver (MMF tacrolimus steroid) transplant patients. The triple drug combination of MMF, tacrolimus, and prednisone is under evaluation in renal and liver transplant patients as well. MPA selectively and reversibly inhibits inosine monophosphate dehydrogenase (IMPDH), an enzyme that plays a pivotal role in synthesis of new DNA. IMPDH is the first of two enzymes responsible for the conversion of inosine monophosphate to guanosine monophosphate. Activated T cells are exquisitely dependent on this pathway for synthesis of new DNA (22)(23). The arrest of the proliferative response at the G₁/S interface of the cell cycle occurs when guanine nucleotide pools are diminished (see Fig. 1) by the inhibitory action of MPA on IMPDH in proliferating T cells in vitro. MPA does not inhibit the early signal transduction effects inhibited by CsA. The inhibitory effect of MPA is therefore thought to be additive to that of CsA.

The following characteristics of the metabolism and pharmacokinetics of MPA are important to be aware of in considering the design and results of concentration-controlled studies of MMF (24)(25)(26):

1) MMF is not measurable in plasma at any time after oral administration, owing to rapid conversion to MPA by widely distributed esterases; MMF, therefore, is not measured in concentration-controlled studies.

2) The primary metabolite of MPA is the pharmacologically inactive mycophenolic acid glucuronide (MPAG), which is excreted renally (27)(28)(29). Although MPAG is commonly measured in PK studies, no studies have shown that its concentration relates to either toxicity or acute rejection.

3) In vitro studies have shown that MPA is extensively and tightly bound to human serum albumin, the average bound fraction being 97.5% in individuals with normal renal function. The pharmacologically active fraction, however, is free MPA, according to in vitro studies, which have shown the dependence of (a) suppression of T cell proliferation and (b) inhibition of the pharmacological target, human isoform II of IMPDH, on free MPA concentration (34).

4) In healthy subjects and stable renal transplant patients, the plasma MPA concentration–time profile for a single dose of oral MMF, after an overnight fast, shows a rapid increase and a sharp peak after ~1 h, followed by an initial rapid decrease, then a secondary peak at 6–12 h. This pattern is probably attributable to an enterohepatic pathway involving MPAG passage into the gastrointestinal tract via biliary excretion, conversion to MPA via glucuronidase action in gut flora, and reabsorption of the latter into the general circulation.

As is the case for CsA and tacrolimus, there is substantial interpatient PK variability for MPA. Although the bioavailability of MPA is reportedly high—94%—in healthy subjects and renal transplant patients (24), the 12-h dose interval MPA AUC shows a >10-fold range for renal transplant patients on a fixed MMF dose of 2 g/day (24). Correction of the AUC values for patient weight did not lessen the magnitude of the AUC range, which suggests that a possible reason for the latter observation could be a lack of correlation of the enterohepatic circulation pathway with subject weight (24). One approach to decreasing the interpatient MPA variance and risk for acute rejection is dosage adjustment to achieve an appropriate MPA AUC target concentration. Descriptions of studies designed to accomplish this goal follow.

The MMF randomized concentration-controlled clinical trial.
A retrospective statistical evaluation of MPA dose-interval AUC data in relation to the incidence of acute rejection was performed in patients enrolled in an MMF Japanese renal transplant clinical trial (24)(31). The study patients were randomized to one of several doses of MMF, in addition to receiving CsA doses guided by blood concentration monitoring and empiric doses of prednisone. A significant correlation (P <0.001) was observed between risk for rejection (relative to the risk with no MMF) and the natural log of the dose-interval MPA AUC, but not to MMF dose (24). This initial retrospective finding was confirmed after review of other clinical trial PK–PD study data (24). The observation of a statistically strong correlation between relative risk for acute rejection and the natural log of dose-interval MPA AUC led to a prospective multicenter randomized concentration-controlled clinical trial in renal transplant patients, sponsored by Roche global development. The patients (n = 150) from a total of seven centers in Belgium and the Netherlands were randomized to low, intermediate, and high target MPA AUC values. A strategy was developed and implemented to permit continual adjustment of dosing to maintain the target AUC values throughout the 6-month study period; acute rejection incidence and other outcomes were determined. A validated HPLC method in a central laboratory was used to measure MPA plasma concentrations throughout this study. This method had been in use in at least five contract laboratories and two North American research laboratories over a 4-year period. The interassay reproducibility (CV) ranged from <5% to 11% for assaying plasma pools with added MPA concentrations ranging from 0.2 to 36 mg/L. Comparison of the HPLC method with an HPLC-MS procedure provided excellent agreement between the two methods (M. Korecka, personal communication).

This prospective concentration–clinical response study confirmed the hypothesis of a strong statistically significant relationship (P <0.001) between rejection risk and MPA AUC but not MMF dose (32). The incidence of acute rejection for the three target MPA AUC values is summarized in Table 2 . The results of this study will be reported more fully in the literature and at scientific meetings. In the field of transplant medicine this is a landmark study for providing the scientific basis for relating the PK of an immunosuppressive drug (MPA AUC) to the patient's risk for rejection. We believe this will serve as the basis for developing prospective investigations of adjusting MMF dosage to achieve a target MPA AUC or abbreviated AUC to assure that optimal immunosuppression is attained from this drug in the individual patient while, one hopes, minimizing the risk of toxicity. One such study, described next, is being conducted at the University of Pennsylvania Medical Center.

Prospective PK and TDM study of MPA in renal transplant patients.
Because control of the natural PK variability of MPA can be achieved by means of dosage adjustment to achieve a target MPA AUC, we decided to investigate, prospectively, the use of a 2-h abbreviated MPA AUC vs predose MPA plasma concentrations to control the intrapatient variance of MPA AUC. The major features of this prospective PK and TDM study are summarized in Table 3 . For each study patient, abbreviated 2-h MPA AUC values are determined at days 4, 7, 11–14, 28, and 90. The MMF dosage is adjusted to achieve target MPA AUC or predose concentration values. The abbreviated MPA AUC correlates very well with the full 12-h AUC but is much more practical to perform in the clinical setting (Table 3 ). The incidence of such clinical outcomes as acute rejection and side effects will be assessed through 3 months after transplant surgery in each study patient.

The transplant community is interested in developing TDM of MPA for several reasons. Given the >10-fold variation in MPA AUC in the early posttransplant period when the risk for acute rejection is greatest, and the well-documented evidence for a relationship between MPA AUC and risk for acute rejection, measurement of this PK parameter can be valuable for the following reasons:

(a) to establish the adequacy of MPA concentrations early after transplant surgery;

(b) to provide a basis for how flexible the practitioner can be in MMF dose reduction to avoid side effects; and

(c) to establish a baseline for determination of dose reduction of concomitant immunosuppressive drugs for maintenance immunosuppression.

In our experience thus far with the 30 patients enrolled (as of 9/15/97), we believe the approach to dose individualization we are evaluating is practical and provides the ability to effectively individualize MMF dosage in the early posttransplant period, the period in which the risk for acute rejection is greatest. Furthermore, we hope to gain greater insight into the potential clinical significance of free MPA concentrations in patient populations in which an increased free drug concentration may be prolonged, such as renal failure patients.

tacrolimus
Tacrolimus (FK-506; Prograf) was approved in 1994 by the US Food and Drug Administration for prophylaxis of organ rejection in patients receiving allogeneic liver transplants; it is usually used in combination with steroids. Tacrolimus is being evaluated in patients who receive other solid-organ transplants and in combination with other immunosuppressive agents, particularly MMF. Like CsA, tacrolimus is thought to exert its pharmacologic effects by inhibiting the phosphatase calcineurin. Both CsA and tacrolimus bind to a family of proteins known as immunophilins, termed cyclophilin and FK-binding protein, respectively. The complex of tacrolimus–FK-binding protein is postulated to inhibit calcineurin, thereby inhibiting activation of a transcription factor, NF-AT, which is an essential factor for early T-cell activation, including interleukin-2 gene synthesis.

Tacrolimus is metabolized by the same cytochrome p450 3A enzyme family responsible for the biotransformation of CsA, sirolimus, and prednisone in enterocytes and liver (33) to at least nine O-demethylated or hydroxylated metabolites. Nevertheless, these metabolites do not accumulate in blood in most transplant patients to the extent observed for CsA, and the metabolite bias observed with the immunoassays widely used for tacrolimus measurement in whole blood does not appear to be as problematic for tacrolimus as for CsA. However, more study data in different patient populations will be needed to rigorously determine metabolite bias and risk for inappropriate dosage adjustment in patients with high concentrations of pharmacologically inactive (but immunologically cross-reactive) metabolite in the presence of therapeutic concentrations of the active parent drug. Good correlation between the morning predose concentration and the morning dose–interval tacrolimus AUC has been reported (33) in liver transplant patients. This is the basis for the current use of the morning predose trough concentration as an index of tacrolimus exposure. Again, additional studies in liver transplant patients and other transplant patient populations should be performed to confirm this finding.

Prospective PK and TDM study for tacrolimus.
One hundred twenty renal transplant patients were enrolled in an open label clinical trial that included five transplant centers (34)(35). The patients were randomized to one of three target predose tacrolimus blood concentration ranges: low, middle, or high. Each participating center used quadruple drug therapy, i.e., induction with antilymphocyte globulin and maintenance immunosuppression with tacrolimus, azathioprine, and prednisone (34)(35). At the conclusion of the 42-day postsurgery study period, the incidences of acute rejection and of toxicity requiring tacrolimus dosage adjustment were the outcomes measured.

Each participating center used an ELISA or in some cases the original Abbott IMx microparticle-enhanced immunoassay method for tacrolimus measurement. Dosage adjustments were made by each participating transplant team according to their prior experience. In this trial the interassay CVs determined in tacrolimus-supplemented pools of human blood were: 6.3–23.1% at 4 µg/L; 7.1–19% at 15 µg/L; and 4.5–14.2% at 60 µg/L. Besides appreciable variance in measured tacrolimus concentrations, presumably because of the use of two methods for analysis in multiple laboratories, another limitation of this study was the use of the lowest tacrolimus concentration during the 7 days before rejection and the highest concentration during the 7 days prior to toxicity for determining the statistical correlation between the concentration and risk for rejection. Nevertheless, posthoc analysis of the data, summarized in Table 4 , showed that in renal transplant patients, the relationship between increasing blood concentration of tacrolimus and (a) the decreasing rate of rejection and (b) the increasing rate of toxicity were both statistically significant (34)(35).

Multicenter prospective analytical and clinical outcome study of a diagnostic immunoassay for measuring tacrolimus.
Another type of multicenter prospective concentration–clinical response study, a modified version of the concentration-controlled study design, is the collection of drug concentration and clinical response data according to a formal research protocol based on clinical criteria and a broadly defined target range for making dosage adjustments. In an open label multicenter prospective study evaluating the PRO-Trak II ELISA method (Incstar Corp.) for tacrolimus measurement in liver transplant patients, 111 adult liver transplant patients were enrolled at six US centers. Each patient was studied prospectively for 12 weeks according to a protocol that included regular performance of and charting of clinical and laboratory tests and evaluations and a strict quality-control program for continually documenting analytical performance throughout the study. A subset of 150 blood samples from 50 study patients were analyzed by a reference HPLC/MS/MS method. To our knowledge, this is the first study of this kind of prospective evaluation of a TDM test commercial kit method. The full details of this study will be described and discussed at future scientific meetings and in the peer-reviewed literature.

One of the important outcomes of this study was the finding of statistically significant relationships between increasing trough concentrations of tacrolimus and (a) decreasing risk for rejection, based on the lowest blood concentration during the preceding 0 to 7 days, and (b) increasing risk for nephrotoxicity, based on the highest blood concentration during the preceding 7 days. Further analyses of these data, when fully reported, will provide the transplant team with more-precise risk/benefit information for target tacrolimus concentrations than currently available. Future studies will be required to compare the relative merits and deficiencies of each type of concentration–clinical response study design, including cost–benefit.

It is hoped that these concentration–clinical response studies will contribute to the development of a higher standard for evaluation of TDM test methods. Systematic, protocol-driven multicenter prospective investigations show promise for providing clearer and more rigorous definitions of risk/benefit for target concentrations of immunosuppressive drugs, and systematically define the performance characteristics of the analytical methods used for measuring immunosuppressive drugs.

Future Directions

The performance of prospective studies of the relationship between drug concentration and clinical response for some of the older common medicines and, more recently, for the immunosuppressive drugs MMF and tacrolimus, has provided a more rigorous scientific basis for the application of effective TDM programs for these agents. Perhaps the new microemulsion formulation of CsA, with a PK profile more predictable than that of the older corn oil formulation, will lend itself more readily to the more rigorous concentration–control design successfully used for MMF and tacrolimus (36).

This approach can be of considerable benefit not only for other new drugs but also for other older commonly used medications. A good example is digoxin, which is widely used in the treatment of congestive heart failure, ranks as the fifth most often prescribed drug in the US, and is still the number one drug for which serum concentrations are monitored (37). As highlighted by recent studies (38)(39), steady-state digoxin trough concentrations within and below the lower half of the 0.9–2 ng/L therapeutic range, as are commonly achieved in outpatients, provide hemodynamic and autonomic benefit in congestive heart failure patients as effective as the concentrations in the upper half of the range, while decreasing the risk for toxicity. The digoxin therapeutic range in current use was derived empirically with the use of older and more-interference-prone RIAs (40). This, together with the fact that drug regimens used today for treatment of congestive heart failure frequently also include an angiotensin-converting enzyme inhibitor and a diuretic, and thereby provide a different pharmacological milieu from that in earlier studies, are additional important reasons why such a study could provide a more scientific foundation on which to base digoxin TDM in contemporary clinical practice.

A final recommendation for future consideration pertains especially to the immunosuppressive drugs because they are generally used in combination. Ideally, for situations in which combination therapy provides the best clinical response, concentration-controlled studies would assess several concentration ranges of each agent in the presence of several target ranges of the other agent to determine the best set of concentrations of the combination for specific transplant patient populations.

Footnotes

¹ Nonstandard abbreviations: PK, pharmacokinetics; PD, pharmacodynamics; CsA, cyclosporine; AUC, area under the (drug concentration–time) curve; MMF, mycophenolate mofetil; MPA, mycophenolic acid; IMPDH, inosine monophosphate dehydrogenase; MPAG, mycophenolic acid glucuronide.

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