Published online Mar 14, 2018. doi: 10.3748/wjg.v24.i10.1093
Peer-review started: December 12, 2017
First decision: December 27, 2017
Revised: December 31, 2017
Accepted: January 24, 2018
Article in press: January 24, 2018
Published online: March 14, 2018
Defects in the innate immune system are key factors in the pathogenesis of inflammatory bowel disease (IBD). The most commonly used immunomodulators are azathioprine (AZA) or 6-mercaptopurine; however, approximately 10% of patients exhibit intolerance to these drugs, resulting in either withdrawal or prescription of an alternative immunomodulator. Discovered by Gosio in 1893, mycophenolic acid (MPA) was the first antibiotic to be synthesized in a pure and crystalline form. It was used as an immunosuppressant to prevent rejection in organ transplantation, but poor oral bioavailability of MPA was an issue of serious concern. To overcome this setback, mycophenolate mofetil (MMF), the only marketed prodrug of MPA was introduced in 1995 (Trade name: Cellcept) which was used in the treatment of refractory Crohn’s disease. Unfortunately MMF also induced GI side effects such as diarrhoea and local gut toxicity leading to poor patient compliance. The role of MMF as an immunomodulator in managing IBD is yet to be fully defined. It is reported in the literature that it may represent a promising treatment for inducing and maintaining remission in IBD patients intolerant of thiopurines. It may be of more value and relevance in ulcerative colitis, as few alternative proven therapies are available.
Hence, it is necessary to design novel colon-targeted prodrugs of MPA with improved bioavailability, lower gastrointestinal (GI) side effects and enhanced efficacy. Many derivatives of MPA have been patented but none has cleared the clinical trials. Therefore, in the present study, colon-specific mutual prodrugs were synthesized by the formation of a covalent amide linkage between MPA and aminosugars. To confirm attainment of an effective concentration of MPA in the colon, in vivo pharmacokinetic studies were performed. A 2,4,6-trinitrobezenesulfonic acid (TNBS)-induced colitis model in Wistar rats was used for biological screening of prodrugs and to confirm their mitigating effect on colonic inflammation. The present study underlines the potential of colon-targeting co-therapy of IBD using a novel strategy of delivering MPA with aminosugars simultaneously at the site of action without causing the GIT distress associated with MMF therapy.
Immunosuppressants such as AZA are an inherent constituent of therapy for 50% of patients with Crohn’s disease (CD) who develop steroid-dependent or refractory disease. MMF is of proven efficacy and safety in transplantation and in some autoimmune disorders. It has been reported that both AZA and MMF are effective in inducing remission, but AZA seems to be more effective in maintaining remission while onset of the therapeutic effect is delayed less under MMF treatment. Both drugs have steroid-sparing potential, which is delayed under AZA. It seems that AZA is still the immunosuppressant of choice in chronic active CD, but MMF is a reasonable alternative in patients who do not tolerate AZA. One of the major side effects of MMF is gastric distress and diarrhoea that may worsen the symptoms of IBD. The morpholino moiety in MMF is implicated in these side effects. This particular observation inspired us to design codrugs of MPA; replacing the morpholino moiety of MMF with aminosugars which could help to maintain the integrity of the colonic mucosal wall. It was envisaged that these prodrugs might find applicability in those cases of CD which are intolerant to AZA.
The main objectives of this work were to minimize gastric discomfort caused by MMF, and improve the bioavailability and efficacy of MPA in the management of IBD. As gastric side effects are known to be caused by the morpholino moiety of MMF, replacing this moiety with nontoxic aminosugars may have a mitigating effect on inflammation, a stabilizing effect on the colonic mucosal layer and the requisite hydrophilic nature that would impart the desired aqueous solubility to MPA ensuring its efficient delivery to the colon. As anticipate, effective targeting of MPA to the colon using glucosamine and galactosamine as carriers was achieved due to enhanced aqueous solubility of the prodrugs and a resistant amide linkage between MPA and the aminosugars. MMF-related diarrhoea was not evident in the prodrug-treated groups, which was one of the important objectives of the present study. Site-specific delivery of MPA resulted in improved bioavailability of MPA. Future research should be directed at investigating the potential of these prodrugs on refractory CD and for inducing and maintaining remission in IBD patients intolerant of thiopurines.
A clean, single step synthesis of target codrugs was achieved through optimization of the EDCI coupling reaction to avoid complex purification procedures. The synthesized compounds were extensively characterized by spectral analysis. New HPLC methods were developed and validated for simultaneous estimation of MPA and aminosugars in the presence of intact codrugs in order to study the release profiles of codrugs in buffers of various pH, homogenates of gastro-intestinal tract, faecal matter, rat blood, urine and faeces. The TNBS-induced experimental colitis model was optimized to investigate the mitigating effect of codrugs of MPA and aminosugars in comparison to standard drugs (MPA and MMF) and physical mixtures (MPA plus aminosugars) to prove the codrug hypothesis. All data were analysed using relevant statistical tests.
Colon-targeted release of MPA, absence of gastric distress, diarrhoea, maintenance of the integrity of colonic mucosa by released aminosugars and a significant marked amelioration of TNBS-induced colitis in Wistar rats as compared to MPA were the promising outcomes of this study. These codrugs should be explored further as an alternative to MMF for inducing and maintaining remission in IBD patients, those intolerant to thiopurines as well as those with refractory CD.
A comparative analysis of efficacy of these codrugs against the established anticolitics such as aminosalicylates and corticosteroids is required. In addition, screening of combinations of these prodrugs with established therapies would help to prove their potential in the management of IBD. Acute toxicity and estimation of various pro-inflammatory mediators such as interleukins, tumour necrosis factor-α and myeloperoxidase enzyme are several studies which are underway.
In the present work, the morpholino moiety of MMF was replaced by novel aminosugars which were tethered with MPA as a literature review revealed that glucosamine and galactosamine play a vital role in resisting chemical attack and improving the tenacity of colon mucus. The novel strategy of codrug therapy proved to be beneficial in terms of lowering the disease activity, colon to body weight ratio and markedly improving the degenerated colon morphology induced by TNBS. MMF-related gut toxicity and diarrhoea were not evident with these codrugs, which was a very promising outcome. This study underlined the utility of aminosugars in maintaining the integrity of colonic mucosa and supported the significant role of an abnormal immune response in the pathophysiology of IBD. These codrugs have the potential to be screened further to determine their efficacy in refractory CD and in the induction and maintenance of IBD remission in patients who are intolerant to thiopurines or MMF.
A mutual prodrug approach was proven to be superior to physical mixtures of two active drugs i.e., MPA and aminosugars in this study as the efficacy of the codrugs was found to be significantly better than physical mixtures. There is a scope to explore the possibility of conjugation of other nontoxic, biocompatible carrier moieties and established drugs with MPA for a synergistic advantage over administering them in the form of a physical combination.