Date of Award

May 2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Shama Mirza

Committee Members

Alexander Arnold, Ava Udvadia, Xiaohua Peng, Nicholas Silvaggi

Keywords

5-Fluorouracil, ARN14988-M, Carmofur, DMPK, Glioblastoma, Mass spectrometry

Abstract

Glioblastoma is the most aggressive solid tumor of the brain. Prior studies from our lab found that carmofur, also known as 1-hexylcarbanoyl 5-fluorouracil (HCFU) is a promising candidate in suppressing and killing tumor cell lines compared to the FDA-approved drug temozolomide. Originally developed as pro-drug of 5-fluorouracil (5-FU), carmofur has been in use in clinic for the treatment of colorectal cancer for over four decades in Japan. Having it used in clinics, it has a high probability of repurposing for diseases with no cure such as glioblastoma. Carmofur has also been found effective against other cancers such as head and neck, breast, cervical, pancreatic, gastrointestinal, ovarian, bladder, gastric, and solid tumors of the brain. Several recent studies also found carmofur efficacious against Krabbe disease, lipopolysaccharide-induced acute lung injury and as a potential lead inhibitor of the main protease (MPRO) of SARS-CoV-2 etc. However, there is no efficient analytical method for the qualitative and quantitative bioanalysis of carmofur to support further pre-clinical and clinical studies.

First, we have described the development of a liquid-liquid extraction technique to extract carmofur from mouse plasma. We have also developed a sensitive and accurate LC-MS/MS method to quantify carmofur in mouse plasma with an application to a pharmacokinetic study. The Cmax, Tmax, and T1/2 was found 10.28±1.53 μg.mL-1, 5 min, and 9.6 min respectively.

Second, we performed a LC-MS/MS based parallel artificial membrane permeability assay (PAMPA) to understand the membrane and blood-brain-barrier (BBB) permeability of two compounds, carmofur and ARN14988-M, a potential metabolite of ARN14988, another promising acid ceramidase inhibitor. Our study indicates that both analytes are likely to cross BBB through passive diffusion which is an important consideration to drugs targeting glioblastoma.

Third, we have developed an analyte extraction technique to simultaneously extract carmofur and 5-FU. We also validated a LC-MS/MS method to quantify carmofur and 5-FU in mouse brain, liver, and kidney tissues after intraperitoneal injection of carmofur solution. Carmofur was found to cross the BBB which confirms our observations from in vitro study described in chapter 3.Carmofur also immediately distributes among the tissues with a Tmax of 5 min. Tmax for 5-FU was found 20 min in mouse plasma, brain, kidney, and 40 min for liver.

Lastly, we used imaging mass spectrometry (MALDI-TOF-MS) to understand the spatial distribution of carmofur/5-FU in mouse brain. We found that carmofur distributes itself throughout the brain tissue which is important for glioblastoma drug development given that the tumor can grow any location of the brain.

Available for download on Friday, May 31, 2024

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