Correlation between Area Under the Concentration-Time Curve of Mycophenolate Mofetil and its Gastrointestinal Tolerability in Renal Transplant Recipients

Introduction

Mycophenolate mofetil (MMF), a selective, non-competitive, reversible inosine monophosphate dehydrogenase (IMPDH) inhibitor, involved in the de-novo pathway of purine biosynthesis, has been approved by the US Food and Drug Administration (US-FDA) for the prevention of renal allograft rejection. It has been integral to the triple drug immunosuppressant regimen for the past 2 to 3 decades.^1,2 It is widely used off-label for various autoimmune diseases, including thrombotic thrombocytopenic purpura, refractory bullous pemphigoid, refractory dermatomyositis, and undifferentiated connective tissue disease (UCTD).³ The drug is available in two formulations, MMF and enteric-coated mycophenolate sodium (EC-MPS). MMF is a prodrug; the mofetil component dissociates to release the active drug, mycophenolic acid (MPA), in the stomach. The EC-MPS has a modified release pattern, and MPA is released in the intestine; hence, it is claimed to have delayed gastrointestinal (GI) absorption and better GI tolerability.

Several studies support this approach. Chan et al. demonstrated that swapping MMF with EC-MPS in renal transplant patients reduced GI symptom burden and improved health-related quality of life (HRQoL).⁴ In contrast Salvadori et al., reported that the safety profile in terms of GI adverse events was equivalent for EC-MPS and MMF.⁵ Similarly, Budde et al. noted that the EC-MPS group had no statistically significantly difference in GI adverse effects compared to MMF patients.⁶

In clinical practice, MMF is typically prescribed at 1000 mg twice daily, with adjustments based on tolerability involving multiple organs such as the gastrointestinal tract and the hematological system.⁷ Common gastrointestinal adverse effects include gastrointestinal discomfort (14-63%), nausea (27-56%), vomiting (29-39%) and diarrhoea (24-53%). Rare adverse events include pulmonary fibrosis, thrombosis, and teratogenic risks. Therapeutic drug monitoring (TDM) is uncommon in India due to limited availability.

While MMF efficacy correlates with drug exposure, GI adverse events may not, though some studies link leukopenia to drug exposure measurable via AUC values.^8,9

The irritant nature of MMF’s metabolites is the probable cause of adverse GI effects. MPA glucuronide MPAG (MPAG, the major active metabolite) and the acyl-glucuronide metabolite of MPA (ac-MPAG) show extensive accumulation in the lumen,¹⁰ decreased GI mucosal turnover, and MMF-induced colitis due to its antimetabolite effects on the enterocytes, which develops months to years after MMF exposure.¹¹

Given the differences in body weight, enterohepatic circulation, and genetic polymorphisms in metabolizing enzymes and transporters (e.g., UGT1A9 and MRP2), it is possible that the pharmacokinetic behavior in Indian subjects is different from that described in Western countries.¹²

The purpose of this study was to ascertain whether GI adverse effects caused by MMF are associated with exposure to MPA or MPAG.

Materials and Methods

This was a prospective study, conducted after obtaining approval from the Institute’s Ethics Committee (IEC), PGIMER, Chandigarh. This study was registered on the Clinical Trials Registry of India (CTRI/2018/09/015853). The ethical norms of ICMR guidelines for Biomedical Research (2017) and Declaration of Helsinki (2024) were strictly adhered to.

The primary objective was to determine whether there is any correlation between the exposure to MPA or MPAG over one dosing interval (AUC_0-12h) and the incidence of drug-related GI adverse events in the renal allograft recipients. The secondary objective was to determine whether there is any correlation between AUC_0-12h of MPA or MPAG and the severity of GI adverse events on the GI symptom rating scale (GSRS) in the above population.

Patients ≥18 years who underwent renal transplantation >4 weeks ago, on triple immunosuppression with therapeutic tacrolimus C₀ levels and good compliance with MMF, were screened at PGIMER, Chandigarh. Those with chronic diarrhea (≥3 episodes for ≥4 weeks), abdominal discomfort, or pain were evaluated, and stool infections were ruled out. Patients with negative stool workup, persistent diarrhea despite MMF dose reduction, and therapeutic tacrolimus levels were invited to participate. After providing written informed consent and agreeing to blood sample collection, eligible patients were enrolled in the study. Patients on EC-MPS, drugs affecting mycophenolate disposition, or with HIV, Hepatitis B or C, malignancies requiring recent treatment, acute systemic infections within 30 days, recent investigational drug use, pregnancy, nursing, or inadequate contraception were excluded.

The control group consisted of participants without GI symptoms. Demographic, clinical details, and recent (≤2 weeks) lab investigations were recorded to exclude patients with abnormal parameters that might affect result interpretation.

Participants were instructed to report on a specified day for sample collection and have a standardized breakfast at least 2 hours before drug administration. Blood samples (2 mL) were collected in heparinized tubes as trough samples (C₀), followed by samples at 1 hour (C₁), 2 hours (C₂), 4 hours (C₄), and 6 hours (C₆) post-MMF administration. A five-minute relaxation window ensured timing accuracy.

Plasma MPA concentrations were measured using a validated high-performance liquid chromatography (HPLC)-UV method (LC20AD Shimadzu). Sample preparation involved protein precipitation with acetonitrile (ACN) containing carbamazepine (10 µg/mL) as the internal standard. MPA separation was achieved on a C18 column (150 mm × 4.6 mm, 5 μM) using an isocratic mobile phase of ACN:20 mM phosphate buffer (62:38, pH 2.3–2.4). The HPLC conditions included a 215 nm detection wavelength, 25 µL injection volume, 2 mL/min flow rate, and 55°C column temperature. MPA and CBZ eluted at 7.2 and 3.5 minutes, respectively. The calibration range was 0.39–50 µg/mL (R² > 0.999), with LOD and LLOQ of 0.1 µg/mL and 0.39 µg/mL, respectively. Mean recovery and accuracy were 90.5% and 97.6%, respectively, with precision ≤15% for QC samples.

The AUC_0-12 for MPA was calculated using the limited sampling formula: AUC_0-12 = AUC_0-6h + (3 × C₆) + (3 × C₀). AUC_0-6h was determined using the trapezoidal rule. The therapeutic range for MPA is 30–60 mg·h/L.

Results

A total of 62 patients were screened. Of them, 16 were excluded because of refusal to give consent for multiple sampling (n=10), and six were excluded as they were shifted to EC-MPS formulation (n=6). The remaining 46 patients were grouped according to whether they had or did not have diarrhea. A total of 30 patients were in the chronic diarrhea group and 10 in the never-diarrhea group after excluding those who refused to give consent on the day of sampling (n=4) and those who discontinued MMF (n=2) [Figure 1].

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Figure 1:

Flow of the study. EC-MPS: Enteric coated mycophenolate sodium, MMF: Mycophenolate mofetil, AUC: Area under the concentration-time curve, MPA: Mycophenolic acid, MPAG: Mycophenolic acid glucuronide, HPLC-UV: High-performance liquid chromatography-ultraviolet.

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The comparative demographics of patients is presented in Table 1. There was no significant difference between the groups in terms of age, BMI, and sex distribution. Both groups consisted of relatively young patients (33-40 years old), with a male preponderance. The more frequent primary cause of ESRD in both groups was CKD of unknown origin; 40% and 33% in the never- and chronic diarrhea groups. Additionally, 17% of hypertension was the cause in the latter. All the patients were on a triple immunosuppression maintenance regimen comprising tacrolimus, mycophenolate, and steroids. Tacrolimus trough concentration (C₀) was similar in both groups. The chronic diarrhea group had a lower average MMF dose, which may reflect dose adjustments made due to gastrointestinal tolerance. However, the tacrolimus dose (0.1-0.2 mg/kg/day) was also relatively smaller in this group. There was a notable difference in GSRS scores (P <0.001) (13.4 ± 3.3 in the chronic diarrhea group and 1.6 ± 2.1 in the never-diarrhea group), with a significantly higher symptom burden in the chronic diarrhea group, underscoring a greater impact of GI symptoms in these patients. No subject developed leukopenia denoting the absence of infection in the preceding four weeks. The serum creatinine was in the range of 0.6 to 1.8 mg/dL, and graft rejection did not occur.

Figure 2 shows considerable variation in the AUC of MPA, ranging from 45.1 to 188.0 and 42.41 to 115.43 in the diarrhea and control groups, respectively. Similarly, the AUC of MPAG varied widely, from 190.23 to 613.72 and 130.82 to 482.84 in the diarrhea and control groups, respectively.

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Figure 2:

Comparison of AUC of MPA (blue) and MPAG (orange) in chronic-diarrhea group and never-diarrhea group. AUC: Area under the concentration-time curve, MPA: Mycophenolic acid, MPAG: Mycophenolic acid glucuronide.

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Discussion

Our study findings reveal no significant correlation between MPA or MPAG exposure and chronic diarrhea status in RTRs. This contrasts with studies such as those by van Gelder et al., which report a link between higher MPA AUC within the first six months post-transplantation and an elevated risk of adverse events.¹³ Sarangi and colleagues further supported that maintaining MPA AUC levels between 30-60 mg.h/L was associated with a lower GI adverse event profile, based on scores from the GSRS and the GI Quality of Life Index (GIQLI).¹⁴ We used the GSRS scale, a validated tool for assessing the severity and impact of GI symptoms, which provides insight into the patient’s experience of discomfort, which is critical for understanding treatment tolerability in the transplant population.¹⁵ The significant difference corroborates the sensitivity and specificity of the tool.

The Opticept Trial found that concentration-controlled MMF with reduced-dose CNI (Group A) had a 22.6% treatment failure rate compared to 27.9% in the fixed-dose MMF with standard-dose CNI group (Group C), confirming noninferiority. The lower AUC group also showed a 12.3% increase in renal function (eGFR) vs. 8.2% in the control group, along with fewer adverse event-related MMF withdrawals (7.4% vs. 14%).¹⁶

Our findings add to this varied landscape, suggesting substantial inter-individual variability in MPA and MPAG levels among patients with and without GI intolerance, and that neither MPA nor MPAG exposure levels appear to predict GI-related adverse events. This suggests that factors beyond MPA exposure might contribute to GI side effects in transplant recipients, warranting further investigation.¹⁷ To address GI intolerance, EC-MPS emerged as an alternative to MMF. EC-MPS releases MPA in the small intestine’s alkaline environment, which may reduce GI adverse events. Dissolution studies of EC-MPS demonstrate maximal MPA release at pH 6.0-6.8, offering bioequivalent exposure to MMF 1 g twice daily with a similar safety profile.¹⁸ Studies have shown significant improvement in GI symptoms and quality of life for patients transitioned from MMF to EC-MPS, with sustained benefits over time. For instance, Bolin et al.¹⁹ and Salvadori et al.⁵ reported that patients switching to EC-MPS experienced similar therapeutic efficacy and safety, with a reduced need for dose adjustments due to GI side effects. Importantly, reductions in GSRS scores after the switch to EC-MPS reflect alleviation of GI discomfort, underscoring the benefits of this formulation for patients experiencing intolerance with MMF.^5,19 However, to avoid ambiguity and to keep the sample collection schedule uniform for the patients, we did not include the EC-MPS group, though some patients on EC-MPS did complain of diarrhea.

One potential factor in MPA-related toxicity is its acyl-glucuronide metabolite, Ac-MPAG, formed by UGT2B7, which may promote cytokine release and link to GI toxicity. Both Ac-MPAG and MPAG are substrates for OATPs, accumulating in cells expressing OATP1B3 and OATP1B1. However, a study by Raggi MC et al. found no correlation between Ac-MPAG levels and GI adverse effects. Our study did not observe a significant difference in MPAG levels between groups, though we could not assess Ac-MPAG.²⁰

In addition to the pharmacological factors, emerging evidence suggests that post-transplant diarrhea may be linked to changes in the gut microbiome.²¹ Studies have shown that RTRs experiencing post-transplant diarrhea have lower diversity in commensal bacterial taxa, fewer metabolism-associated bacterial genes, and a distinct microbiome composition characterized by increased Proteobacteria and decreased Actinobacteria.²² This dysbiosis includes a reduction in butyrate-producing bacteria, a disruption that contrasts with the microbiome of healthy controls and may play a role in the development of GI symptoms.^23,24

Zhang P et al., demonstrated that probiotic treatment significantly reduced the severity of MPA-induced colitis in mice. Probiotics improved symptoms by restoring weight, reducing stool scores, and maintaining colon length. They enhanced intestinal barrier integrity by increasing proteins like ZO-1, Occludin, and sIgA and helped correct gut microbiota imbalances, restoring Bacteroidetes and reducing Firmicutes. The Clostridiales bacteria, elevated in the MPA group, emerged as a potential therapeutic target. Future research may focus on targeted probiotics and microbiota transplantation as strategies for treating MPA-induced colitis.²⁵ Nevertheless, the Indian patients differ significantly from the rest of the world due to different dietary patterns and environmental exposure. The MMF-induced diarrhea due to gut dysbiosis needs further exploration in our population.

This study has a few limitations, such as a limited sample size, and the large variation in MPA and MPAG levels. However, the clinical phenotype of our patients with repeated episodes of diarrhea and a negative stool study was robust enough to label them as diarrhea and non-diarrhea, adding to the strength of the assessment.

In summary, our findings support the notion that MPA and MPAG levels alone are not predictive of GI adverse events in renal transplant patients with chronic diarrhea. The substantial variability observed in MPA exposure highlights the complexity of managing GI adverse effects and the need to explore alternative risk factors, including metabolic byproducts and microbiome alterations. Future studies with a larger number of patients could focus on the gut microbiome to better understand and manage GI toxicity in renal transplant recipients.