DECREASED PROTEIN BINDING OF MYCOPHENOLIC ACID ASSOCIATED WITH LEUKOPENIA IN A PANCREAS TRANSPLANT RECIPIENT WITH RENAL FAILURE

Abstract

Background. 

Mycophenolate mofetil (MMF) is rapidly hydrolyzed to its active metabolite mycophenolic acid (MPA), which is excreted by the kidney after undergoing glucuronidation to MPAG. MPAG has been shown to accumulate in patients with renal failure. MPA is extensively and avidly bound to human serum albumin. In vitro inhibition of the pharmacologic target, inosine monophosphate dehydrogenase, is dependent on free MPA. It has been demonstrated that high MPAG concentrations decrease MPA protein binding in vitro. In addition, the uremic state is associated with altered protein binding of many drugs.

Methods. 

We assessed free MPA, total MPA, and MPAG kinetics in a patient with renal failure receiving MMF for a pancreas transplant, who presented with signs of MMF toxicity. MPA, MPAG, and free MPA were measured by high performance liquid chromatography and a validated¹⁴C-MPA ultrafiltration methodology.

Results. 

The MPAG area under the concentration curve (AUC) in this patient was extremely high (5899 μg × hr/ml). The total MPA AUC of 36.8 μg× hr/ml was within the range usually obtained in stable renal patients. The free fraction of MPA and the free MPA AUC were markedly elevated (13.8% and 5.07 μg × hr/ml, respectively).

Conclusions. 

Patients with severe renal insufficiency may have markedly increased free MPA levels that may not be reflected in total MPA concentrations. These patients may be at increased risk for MMF-related toxicity.

Mycophenolate mofetil (MMF*) has been demonstrated to decrease the incidence and severity of acute rejection episodes in renal allograft recipients ^{(1, 2)}. MMF is rapidly converted to mycophenolic acid (MPA), a reversible noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) ⁽³⁾. MPA undergoes glucuronidation in the liver to the primary inactive metabolite MPAG, which is excreted by the kidney ⁽⁴⁾. Patients with compromised renal function have elevated MPAG levels as well as highly variable total MPA levels ⁽⁵⁾. MPA is extensively protein bound (≈97%), and it seems that only the free fraction of the drug is available to inhibit IMPDH ⁽⁶⁾. High MPAG levels decrease MPA-albumin binding in vitro and thus increase the free fraction of MPA ⁽⁶⁾. In addition, the uremic state is known to alter the protein binding of many drugs to human serum albumin ⁽⁷⁾. The effect of chronic renal failure on total MPA and free MPA concentrations has not been explored. We present the case of a pancreas-kidney transplant recipient with chronic renal failure secondary to isolated renal allograft rejection who presented with leukopenia, Gram-negative sepsis, and markedly elevated free MPA levels due to a significant decrease in MPA protein binding.

In July 1994, a 34-year-old Caucasian female presented to the transplant clinic at The University of Texas at Houston Health Science Center with a 1-day history of fever and myalgia. The patient had developed end-stage renal disease in 1989 secondary to type I diabetes mellitus. The patient initially was maintained on hemodialysis until she underwent a simultaneous pancreas-kidney transplant in 1992. The initial function of both grafts was excellent and the patient was maintained on cyclosporine (Sandimmune) and prednisone. Three months before this admission, the patient experienced a rise in serum creatinine to 5 mg/dl (calculated creatinine clearance 9 ml/min). A renal transplant biopsy at that time revealed grade III chronic rejection along with grade II acute rejection; a biopsy of the pancreas allograft revealed no acute rejection. A decision was made to not treat the renal allograft rejection with high-dose steroids or antilymphocyte antibody therapy, but rather to initiate MMF at 750 mg b.i.d. for rejection prophylaxis of the pancreas.

The patient was initiated on hemodialysis via a subclavian catheter at this time and did well until this visit. The patient presented with a complaint of fever and myalgia for 1 day. She described no gastrointestinal symptoms and otherwise had no complaints. Her medications at this time included cyclosporine at 225 mg b.i.d., prednisone at 10 mg q.d., MMF at 750 mg b.i.d., calcitrol, and erythromycin. The patient was on no medications known to be associated with leukopenia or myelosuppression. On physical examination, she was noted to have a temperature of 102 ° F, without allograft tenderness or evidence of catheter exit site infection. Laboratory data was notable for a white blood count of 2100/mm³ with 59 polys, 1 monocyte, and 39 lymphocytes. Hemoglobin was 5.4 g/dl and the platelet count was 158,000 mm³. Serum creatinine was 6.7 mg/dl and the serum albumin was 2.7 gm/dl. Blood and urine were sent for bacterial and fungal cultures. Cytomegalovirus early antigen and culture were also sent. The patient was guaiac negative and had no evidence of gastrointestinal bleeding or hemolysis. Initial therapy consisted of broad spectrum antibacterials, ganciclovir, and a transfusion of 3 U of packed red blood cells.

The next morning, following an overnight fast, the patient underwent a full pharmacokinetic profile of MPA, MPAG, free MPA, and cyclosporine. At 9:00 a.m., plasma was prepared from blood samples (EDTA, Vacutainer tubes) drawn at 0, 20, 40, 75, 120, 240, 360, 480, and 720 min after the patient had taken 750 mg of MMF and 225 mg of Sandimune. Total MPA, MPAG, and free MPA were measured by validated high performance liquid chromatograph methods and a validated¹⁴ C-MPA ultrafiltration method ⁽⁶⁾. Cyclosporine whole blood concentrations were determined by a monoclonal fluorescence polarization immunoassay. Results of the MPA, MPAG, and free MPA along with data from stable renal transplant patients with varying degrees of renal function are presented in Table 1.Figure 1 depicts the pharmacokinetic curve for MPA, MPAG, and free MPA in this patient. The cyclosporine 12-hr area under the concentration curve was 4900 ng × hr/ml and the trough level was 240 ng/ml (both within therapeutic range for our institution). MMF was discontinued after this dose. Subsequently the patient's blood cultures grewKlebsiella pneumoniae, and antibiotics were continued for 14 days. The white blood count slowly increased off MMF, and at 1 week was 5000/mm³ with a stable hemoglobin of 10 gm/dl. The patient's cytomegalovirus early antigen and culture were negative.

Table 1: MMF pharmacokinetic data for patient and comparison group of stable renal transplant recipients

Figure 1: The pharmacokinetic curve for MPA (□), MPAG (○), and free MPA (⋄).

It has been reported previously that MPAG may accumulate in patients with renal failure ^{(8, 9)}. To our knowledge, the disposition of free MPA in patients with severe chronic renal insufficiency has not been documented in the literature. Preliminary data indicate that the free fraction of MPA may be elevated in patients experiencing delayed graft function.⁴ It has been shown that, in vitro, the inhibition of pure human IMPDH and of activated lymphocytes is dependent on free MPA ⁽⁶⁾. It is interesting to note that although our patient's free MPA levels were markedly elevated, the total MPA (free plus protein bound) was not beyond the range previously reported in stable renal transplant patients with and without renal insufficiency⁽⁸⁾. As expected, MPAG levels were markedly elevated.

Although MMF is normally administered as a fixed dose and levels of MPA are not routinely performed, it is likely that MPA levels or free MPA levels correlate to clinical effect. In fact, a randomized concentration control trial has been completed, and as of this date results are being actively analyzed.

The reason for this patient's markedly elevated free MPA may be multifactorial-MPAG levels greater than 100 μg/ml have been associated with decreased MPA-albumin binding ⁽⁶⁾. Second, patients with chronic renal failure may have altered drug-protein binding characteristics. Finally, our patient's hypoalbuminemia may also have contributed to the increased free fraction of drug. A second interesting finding in our patient was the relatively flat pharmacokinetic curve found in this patient in marked contrast to the rapid and rather steep profile seen in most patients. The reason for our patient's flat MPA pharmacokinetic curve is not clear. Whether this is also a feature of patients with chronic renal failure will need to be explored.

Although we believe that it is likely, we cannot conclusively state that this patient's leukopenia was causally related to her elevated free MPA concentrations nor that her Gram-negative sepsis was related to her leukopenia. This should not detract from the finding of markedly decreased MPA protein binding with concomitantly increased free MPA levels seen in this patient. Given the fact that both MPAG and hypoalbuminemia decrease MPA binding to albumin, our observation is not a totally unexpected finding in a patient (with renal failure) maintained on MMF for any extended period of time. This patient did not develop any gastrointestinal disturbance, and one can speculate that local MPAG/MPA levels may play a more important role in this toxicity than systemic levels.

The relationship of free MPA concentrations and clinical response requires further study. If a relationship is indeed present, special consideration may be needed in patients with chronic renal failure and severe hypoalbuminemia. This might include not only measurements of total MPA, but also of free MPA concentrations in this special patient group.