Document Type

Dissertation

Date of Award

5-2021

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

Ming Hu

Committee Member 4

Mahmoud Saleh

Keywords

Beta-glucuronidase, Chemotherapy-induced Diarrhea, Irinotecan, XCHT

Abstract

Chemotherapy-induced diarrhea is a common side effect but is an understudied area in cancer management. This problem is especially significant with irinotecan hydrochloride (CPT-11), a prodrug of SN-38 (7-ethyl-10-hydroxy camptothecin) used in treating metastatic colon cancer as well as other types of cancers (e.g., lung, pancreatic). It is reported that more than 80% of patients treated with irinotecan experienced diarrhea, with up to 40% experiencing severe (grade 3 and 4) diarrhea. Different anti-diarrhea medications (e.g., loperamide, octreotide, tincture of opium) have been recommended, but diarrhea is still a major concern in many patients treated with irinotecan as they do not respond well to these treatments. The disposition of irinotecan has been well studied. After being administered through intravenous infusion, irinotecan is mainly activated to SN-38 by carboxylesterase (CE) and then detoxified to SN-38 glucuronide (SN-38G) by UDP-glucuronosyltransferase (UGT) in the liver. Irinotecan and its metabolites are secreted into the intestine through biliary excretion, where SN-38G can be hydrolyzed back to SN-38 through the action of β-glucuronidase (β-GUS) produced by the intestinal bacteria. Accumulation of SN-38 in the intestinal tract then causes intestinal mucosal injury, resulting in delayed-onset diarrhea. Therefore, the purpose of this study is to determine the role of intestinal bacterial β-glucuronidase (β-GUS) in irinotecan-induced diarrhea.

Glucuronide hydrolysis by bacterial β-glucuronidase (β-GUS) is a well-known reaction. Typically, substrates will be incubated with fecal enzymes prepared from feces to determine bacterial β-glucuronidase (β-GUS) activity. Different methods have been reported for fecal enzyme preparation and different conditions have been used in incubating substrates with fecal enzymes. However, the method for enzyme preparation and the reaction conditions were not standardized and different conditions may affect the GUS activity. Therefore, we first used a standard GUS substrate pNPG and a natural glucuronide wogonoside as the substrate to determine how enzyme preparation procedure and reaction conditions will affect GUS activity. Mouse, rat, and human feces were tested. Fecal S9 fractions were prepared with sonication and without sonication (suspension). Different reaction conditions including, buffer pH, Mg2+ concentration, and feces collection time were tested. The relative reaction activity of pNPG, reaction rates, and reaction kinetics for wogonoside were calculated. The results showed that sonication increased total protein yield during enzyme preparation. Fresh feces showed the highest hydrolysis activities when compared to feces collected after 24hrs and after 7 days. The pH of the reaction system increased the activity in 0.69-1.32, 2.9-12.9, and 0.28-1.56 folds for mice, rats, and human at three different concentrations of wogonoside, respectively. The Vmax for wogonoside hydrolysis was 2.37±0.06, 4.48±0.11, and 5.17±0.16 μmol/min/mg and Km was 6.51±0.71, 3.04±0.34, and 0.34±0.047 μM for mouse, rat, and human, respectively. The inter-individual difference was significant (4-6 folds) using inbred rats as the model animal. Therefore, for an optimized hydrolysis reaction, sonication should be included in the preparation of the enzyme, fresh feces should be used to avoid activity loss and the buffer pH should be appropriate according to the species of the animal being used.

To determine if SN-38G hydrolysis by bacterial GUS activity can be altered, we prepared fecal enzymes using feces collected from rats at different conditions including F344 rats at different ages (4 and 10 weeks old), different breed of rats (Pirc and F344), rats before and after irinotecan administration. The results showed that GUS activity is increased after the administration of irinotecan. Younger showed higher GUS activity when compared to the older ones and the increased in GUS activity noticed in the young rats increased by two folds after administration of Irinotecan. We, therefore, suspect that age may have a synergistic effect on diarrhea induced by CPT-11. Pirc rats, a type of rat that is known for inflammation of their colon mucosa showed higher GUS activity when compared with the wildtype (F344). This might be the reason why Pirc rat have a high incidence of diarrhea when CPT-11 is administered to them.

Having determined that hydrolysis of SN-38G by bacterial GUS can be altered, we decided to see if this manipulation can result in reduced incidence of irinotecan-induced diarrhea. We established an irinotecan-induced diarrhea model using F344 rats. We used an herbal formula Xiao-Chai-Hu-Tang (XCHT) to treat the rats 3 days prior to CPT-11 injection and their fecal samples were collected for 9 days afterward. The results showed that with XCHT treatment (1.8g/kg p.o.), bacterial GUS against SN-38G hydrolysis was significantly decreased (Vmax 0.4umol/min/mg) when compared to that of the rats without XCHT treatment (Vmax 1.3umol/min/mg). In vitro study also showed that XCHT can also inhibit GUS activity. Toxicity results showed that with XCHT treatment, irinotecan-induced diarrhea was attenuated. Rats given XCHT treatment showed only grade 1 diarrhea for up to 9 days after CPT-11 injection and rats without XCHT treatment showed severe diarrhea (grade 3 and 4) by day 5 after CPT-11 injection. Therefore, it can be said that XCHT alleviates diarrhea by reducing the amount of GI microflora available to deconjugate SN-38G to SN-38.

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