Clinical investigations of the radiation protector S 2 (3 aminopropylamino)ethyl phosphorothioic acid (amifostine, WR 2721, ethyol) with radiotherapy are based on the rationale that amifostine can protect most normal tissues, except the brain and the spinal cord, against the harmful effects of ionizing radiation and chemotherapeutic agents without appreciable protection of certain tumors. The active species involved in the protective effects of amifostine is its enzymatic dephosphorylation product, 2 (3 aminopropylamino) ethanethiol (WR 1065). Preferential protection of the host tissues is not always the case. There are examples where the drug offers equal or greater protection to the tumor compared to the host.
Pharmacokinetics of these compounds would be useful in devising treatment strategies. Preliminary results suggest that both in vivo and in vitro 31Phosphorus nuclear magnetic resonance spectroscopy (31P-NMR) can be used for detecting the presence of amifostine and for following its dephosphorylation. Drug screening studies have identified an analog of amifostine, sodium hydrogen S (3 amino 2 hydroxypropyl) phosphorothioate (WR 77913) as a promising radiation protector. 31P NMR would complement the other existing methods for studying the pharmacokinetics of phosphorothioates. The potential of in vivo 31P NMR for monitoring of phosphorothioates in tumor and normal tissues of live mice are evaluated along with any potential influence of amifostine on the antitumor efficacy of cisplatin and paclitaxel.
Aim 1: In vivo Studies — Surface coils are used to obtain 31P-NMR spectra of muscle and solid tumors growing in live mice. Spectra are recorded before and after administration of graded doses (up to half the maximum tolerated dose (MTD)) of WR 2721 and WR 77913 to tumor bearing mice. A series of spectra are recorded at different times after administration of the drug, in order to monitor its appearance and disappearance from the muscle and tumor in the intact animal. Human lung cancer cell lines such as H125, H661, A549, and A549-DDP, known to differ in their drug susceptibility, is grown as solid tumors in nude mice.
Aim 2: In vitro Studies — In vitro 31P-NMR to estimate the dephosphorylation of WR-2721 by normal tissue and tumors. The dephosphorylation activity will be determined in homogenates of lung, muscle, kidney, liver, and spleen as well as a variety of tumor cells differing in their phosphatase activity and drug sensitivity. Clonogenicity and apoptosis assays are utilized to study the influence of amifostine and WR-1065 on the cell death caused by paclitaxel and cisplatin in lung cancer cell lines.
Aim 3 — Study the differential display of the proteins expressed by lung cancer cells that differ in their drug sensitivities, treated in vitro with and without WR-1065 (which is the active metabolite of amifostine). Two dimensional gel electrophoresis and MALDI-TOF is used to identify the differentially expressed proteins. This project utilizes resources of the BNMR Laboratory.