Document Type

Thesis

Date of Award

5-2024

School/College

College of Pharmacy and Health Sciences (COPHS)

Degree Name

MS Pharmaceutical Science

Committee Chairperson

Song Gao

Committee Member 1

Huan Xie

Committee Member 2

Yun Zhang

Committee Member 3

Mario Hollomon

Keywords

Chrysin, DMPK, metabolism, Mycophenolate mofetil, PKPD, Wogonin

Abstract

Medication-induced diarrhea is a common adverse effect experienced by patients taking immunosuppressant medications used in Organ transplants. However, Mycophenolate mofetil (MMF) is the first-line drug to prevent organ transplant rejection. (Van Gelder & Hesselink, 2015) . MMF’s mechanism of action involves inhibition of inosine-5'-monophosphate dehydrogenase. It is used for the treatment and management of a variety of autoimmune diseases as well as to prevent organ rejection in patients with bone marrow and solid organ transplants; however, over 50% of its adverse effects have been reported to be diarrhea. (Arslan et al., 2007; Farooqi et al., 2020). However, few options exist for managing the unwanted effects of MMF-induced diarrhea, which often leads to a subtherapeutic dose, as seen in alternatives such as Azathioprine (Sekmek et al., 2021) or using a low dose of MMF or withdrawal of an MMF which on the long-term may cause a high incidence of organ rejection, graft failure, and eventual patient mortality (Park et al., 2019). MMF can affect the upper and lower GI tract, which often increases diarrhea intensity if left untreated (Parfitt et al., 2008a). Nevertheless, the area of alleviating MMF-induced diarrhea is grossly understudied; hence, one does not fully understand the precise mechanism underlying GI toxicity. However, enterohepatic recycling after glucuronidation has been implicated. The labile acyl glucuronide (acylMPAG) and the inactive phenolic glucuronide (MPAG) are produced by glucuronidation. Although the main process of MPA detoxification in vivo is the creation of MPAG, research has attempted to connect acyl glucuronide synthesis with adverse drug reactions (ADRs) like MMF-induced diarrhea(Fukushima et al., 2021). For drugs like MMF, it is postulated that glucuronidation in the GI tract is insufficient, and this might result in excessive MPA and acyl MPAG accumulation in the lower GI segments to cause local toxicity, which manifests as diarrhea. In this study, we hypothesized that when we hydrolyze the active moiety of MMF causing MMF-induced diarrhea, that is, prevent MPAG from undergoing enterohepatic recycling to MPA or eventually AcMPAG, using the flavonoids Wogonin and Chrysin. We can successfully alleviate MMF-induced diarrhea and would also evaluate the dose-effect and eventual survival rate. Following an already established diarrhea model, the female Wister Han used were divided into five groups; the first group was administered 70mg/kg/day MMF-only (Control), the other was given 50mg/kg/day of Wogonin (Treatment) and Chrysin (Treatment) separately. The next batch was also dosed with 100mg/kg/day of Wogonin and Chrysin separately. The non-control group (treatment group) received oral gavage of wogonin and chrysin separately at 50 mg/kg per day for two days before co-administering with MMF as pretreatment. From day 3, MMF was administered orally to female Wistar Han rats at 70 mg/kg daily for seven consecutive days as an oral gavage. Subsequently, the indicators of illness activity (body weight, diarrhea score, and survival analysis) were observed. Plasma blood samples were taken on day 9, the seventh day of dosing with MMF, to monitor the PK profile of the various samples. Also, on day 10, GI tissues were collected before diarrhea to quantify tissue drug concentrations of MMF and MPA, MPAG, and AcylMPAG using LC-MS/MS. Wogonin in both groups, orally administered with 100mg/kg/day and 50mg/kg/day of wogonin separately alongside MMF 70mg/kg/day in each batch, alleviated Mycophenolate Mofetil-induced diarrhea damage, which was indicated by reduced weight loss compared to control and diarrhea score, thereby preventing possible mucositis in the small intestine as well as the colon. However, the chrysin batch was dosed orally with 100mg/kg/day and 50mg/kg group differently along with MMF 70mg/kg/day grade 3 and 4 diarrhea. The control group, which was administered only with MMF at 70mg/kg/day, had severe diarrhea and resulted in a nosedived survival rate following Survival analysis. Nevertheless, after determining that wogonin and chrysin majorly influenced the disease activity index and survival index in all the MMF-diarrhea rats, we went further to check the Pharmacokinetic Profile of the five batches of the rat’s plasma and tissues. Blood samples were collected at 0.25-hour, 0.50-hour, 1.00-hour, 2.00-hours, 4.00-hours, 6.00-hours, 8.00-hours, and 24.00 hours and used in the plasma preparation for quantitation. Subsequently, tissues were collected from the liver, colon, and ileum and processed and quantified using LC-MS. In conclusion, a wogonin batch of animals survived throughout the experiment without any significant deaths, which showed that regardless of dose, Wogonin alleviated MMF-induced diarrhea and enhanced the rats' survival. These conclusions confirm our earlier postulation that flavonoids Like Wogonin and Chrysin can alleviate diarrhea in rats that have MMF-induced diarrhea, possibly by inhibiting the formation of toxic metabolites of MMF like MPA and AcylMPAG, thereby enhancing intestinal glucuronidation without significantly affecting the active metabolites MPA, which is in tandem with other reported in-vitro studies. Also, Wogonin has an enhanced local bioavailability in the GI Tract. This insight could be useful in the management of Mycophenolate mofetil-induced diarrhea by mitigating the need to carry out further study on wogonin as an alternative to toxic chemicals in the management of organ MMF-induced diarrhea since it belongs to the naturally occurring flavonoids family, which are considered safe.

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