Imoh Etim

Document Type

Dissertation

Date of Award

5-2023

School/College

College of Pharmacy and Health Sciences (COPHS)

Degree Name

Ph.D. in Pharmaceutical Science

Committee Chairperson

Song Gao

Committee Member 1

Dong Liang

Committee Member 2

Huan Xie

Committee Member 3

Yun Zhang

Committee Member 4

Ming Hu

Keywords

• Diarrhea • Inflammatory cytokines • Irinotecan (CPT-11) • SN-38 • SN-38G • Tight junction proteins

Abstract

Chemotherapy-induced diarrhea is a frequent side effect that occurs with several chemotherapeutic agents. However, it is an understudied area in the management of cancer. This concern is significant with irinotecan, a camptothecin derivative. Irinotecan hydrochloride (CPT-11) is a prodrug that is hydrolyzed to SN-38, a potent topoisomerase 1 inhibitor used as the first-line agent for the treatment of metastatic colorectal cancer. However, there is a concern regarding gastrointestinal toxicity, especially diarrhea. Many patients have reported experiencing diarrhea, and severe diarrhea episodes (grades 3 and 4) have been recorded in about 40 % of patients (J. J. Lee & Sun, 2016). Several medications have been used to attenuate the diarrhea episodes, ranging from nonpharmacological such as using probiotics, glutamine, and activated charcoal, to pharmacological approaches, such as loperamide and octreotide, and diphenoxylate-atropine (Koselke, Elizabeth; Kraft, 2012). Yet, the challenge persists as patients are administered this chemotherapeutic agent; irinotecan does not seem to respond to these anti-diarrheal medications. Researchers have well studied the mechanism and disposition of the prodrug irinotecan; after parenteral administration of irinotecan, it is hydrolyzed by carboxylesterase enzyme to the active moiety SN-38 which is further conjugated to the inactive metabolite SN-38G in the presence of UGT. However, this conjugated form of the drug SN-38G can be deconjugated back to the active drug SN-38 in the presence of β-GUS, produced in the intestine. Thus, constant accumulation of the active drug in the intestinal lumen results in intestinal epithelial injury leading to severe diarrhea. Therefore, this study aims to utilize locally bioavailable naturally occurring flavonoids (wogonin and chrysin) to alleviate irinotecan-induced diarrhea. Before starting the experimental plan, we had to establish a diarrhea model. Firstly, we fed the mouse with regular diets for two weeks; after that, we administered CPT-11 at doses of 50 mg/kg, 60 mg/kg, and 75 mg/kg. However, the animals experienced only grade 1 and 2 diarrhea at those doses. We then decided to change their animal feed to a special diet, which has been reported to contain fewer phytoestrogens and fibers. We repeated similar doses, and at 75 mg/kg, the animals had severe diarrhea (grade 4); thus, we established the diarrhea model at 75 mg/kg dose. Prior to the administration of CPT-11, the animals were divided into three groups, naïve (blank), control (irinotecan-only), and treatment group (irinotecan and oral flavonoids). The treated group was pretreated with oral gavage of wogonin/chrysin at 100 mg/kg per day for three days before co-administering with CPT-11. CPT-11 was administered intraperitoneally (I.P) to mice at a dose of 75 mg/kg per day for six consecutive days as a bolus injection, and then the disease activity indexes (i.e., body weight, diarrhea score, and survival analysis) were monitored. GI tissues were also collected on day 4 (before diarrhea) to quantitate tissue drug concentrations of SN-38 and SN-38G using LC-MS/MS, alongside histological evaluation and using enzyme-linked immune sorbent assay (ELISA) to document inflammatory markers. In addition, a xenograft mouse model study was done using immunocompromised nude mice to evaluate the possible drug-drug interaction of the oral flavonoids in the anti-tumor activity of CPT-11. Oral flavonoids (wogonin/chrysin) alleviated irinotecan-induced diarrhea damage by reducing weight loss (steady body weight) and diarrhea score (grade 1) and attenuating mucositis in the small intestine and colon. The chemotherapy-only administered group experienced severe diarrhea (grade 4) and weight loss of about a 20 % decrease. Similarly, with the survival analysis, the oral flavonoids treated group all survived (100 %) both male and female, but in the irinotecan group, only 40 % of female and 60 % of male mice survived. Histological analysis confirmed that the oral flavonoids prevented short, scanty, and denuded villi in the ileum and colon. Moreover, oral flavonoid treatment mitigated irinotecan-induced oxidative stress by downregulating IL-1β, IL-6, IL-18, TNFα, and IFNα. Compared with the control group (irinotecan only), the oral flavonoid groups (irinotecan co-administered) decreased the expression of IL-1β by 2-folds in the small intestine and 1.5 folds in the colon. With IL-6, we also observed a similar trend in both small intestine and colon. Interestingly, the expression of IL-18 was significantly downregulated in the small intestine, with almost a 4-fold decrease and a 2-fold decrease in the colon. The expressions of TNFα and IFNα were downregulated considerably, with about a 2-fold decrease in the oral flavonoid-treated group showing promising potential in reducing the expression of inflammatory cytokines in irinotecan-exposed mice. To evaluate the epithelial tight junction barrier, we use an ELISA kit to detect the expression of tight junction proteins ZO-1 and occludin. We observed that the oral flavonoids prevented the disruption of the tight junction ZO-1 and occludin, especially in the small intestine, in about a 2-folds increase. Having determined that the oral flavonoids significantly impacted the disease activity index, inflammatory cytokines, and tight junction proteins in irinotecan-exposed mice, we decided to see if the coadministration of oral flavonoids and irinotecan could impact the efficacy of the chemotherapy agent. We established a xenograft mouse model using immunocompromised nude mice. We inoculated the nude mice with 2 million cells/mL of HT-29 colon cell subcutaneously into their upper right limb, and when the tumor-bearing mice tumor growth was 150 mm^3, we started the coadministration of the irinotecan and oral flavonoids for seven days. After the study, we excised the liver, small intestine, and colon tissues. The results showed that with the oral flavonoids treatment both at low (50 mg/kg) and high (75 mg/kg) doses of irinotecan, there was no significant impact of wogonin/chrysin on the PK of irinotecan on day eight, as the AUC was within ±1 fold for the control and the treated group. We randomly excised the tumor after 6 hours of dose administration of irinotecan, and we observed no significant difference between the irinotecan-only group and the treated group. The drug concentration of CPT-11 and SN-38 was within ±1 fold for the control and the treated group. Also, the oral flavonoids did not interfere with the anti-tumor activity of CPT-11 as the tumor volumes on the last day of study for irinotecan only, low treated dose, and the high treated dose was 1133 mm^3, 550 mm^3, and 578 mm^3, respectively for the female group, while for the male group was 436 mm^3, 621 mm^3 and 368 mm^3 respectively. This result shows that the female group responded to both irinotecan and oral flavonoids, inferring some gender differences. Interestingly, the oral flavonoids-treated group showed no significant difference to the control group (irinotecan only) in SN38-G tissue drug concentration in the duodenum and jejunum as the oral flavonoid-treated group showed promising potential to alleviate irinotecan-induced diarrhea. In conclusion, we can clearly show that the upregulation of inflammatory cytokines and tight junctions proteins upregulation are significant factors in the development of irinotecan-induced diarrhea, and its manipulation can result in alleviating irinotecan-induced diarrhea by using locally bioavailable naturally occurring flavonoids. This knowledge can be helpful in the management of chemotherapy-induced diarrhea by mitigating this dose-limiting gastrointestinal toxicity.

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