tetrahydrouridine and Carcinoma--Squamous-Cell

The study's goals were as follows: (1) to extend our past findings with rodent tumors to human tumors in nude mice, (2) to determine if the drug protocol could be simplified so that only CldC and one modulator, tetrahydrouridine (H4U), would be sufficient to obtain efficacy, (3) to determine the levels of deoxycytidine kinase and dCMP deaminase in human tumors, compared to adjacent normal tissue, and (4) to determine the effect of CldC on normal tissue radiation damage to the cervical spinal cord of nude mice.. The five human tumors used were as follows: prostate tumors, PC-3 and H-1579; glioblastoma, SF-295; breast tumor, GI-101; and lung tumor, H-165. The duration of treatment was 3-5 weeks, with drugs administered on Days 1-4 and radiation on Days 3-5 of each week. The biomodulators of CldC were N-(Phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartyl transcarbamoylase, 5-fluorodeoxycytidine (FdC), resulting in tumor-directed inhibition of thymidylate synthetase, and H4U, an inhibitor of cytidine deaminase. The total dose of focused irradiation of the tumors was usually 45 Gy in 12 fractions.. Marked radiosensitization was obtained with CldC and the three modulators. The average days in tumor regrowth delay for X-ray compared to drugs plus X-ray, respectively, were: PC-3 prostate, 42-97; H-1579 prostate, 29-115; glioblastoma, 5-51; breast, 50-80; lung, 32-123. Comparative studies with PC-3 and H-1579 using CldC coadministered with H4U, showed that both PALA and FdC are dispensable, and the protocol can be simplified with equal and possibly heightened efficacy. For example, PC-3 with X-ray and (1) no drugs, (2) CldC plus the three modulators, (3) a high dose of CldC, and (4) escalating doses of CldC resulted in 0/10, 3/9, 5/10, and 6/9 cures, respectively. The tumor regrowth delay data followed a similar pattern. After treating mice only 11/2 weeks with CldC + H4U, 92% of the PC-3 tumor cells were found to possess CldU in their DNA. The great majority of head-and-neck tumors from patient material had markedly higher levels of dC kinase and dCMP deaminase than found in adjacent normal tissue. Physiologic and histologic studies showed that CldC + H4U combined with X-ray, focused on the cervical spinal cord, did not result in damage to that tissue.. 5-CldC coadministered with only H4U is an effective radiosensitizer of human tumors. Ninety-two percent of PC-3 tumor cells have been shown to take up ClUra derived from CldC in their DNA after only 11/2 weeks and 2 weeks of bolus i.p. injections. Enzymatic alterations that make tumors successful have been exploited for a therapeutic advantage. The great electronegativity, coupled with the relatively small Van der Waal radius of the Cl atom, may result in CldC's possessing the dual advantageous properties of FdC on one hand and BrdU and IdU on the other hand. These advantages include autoenhancing the incorporation of CldUTP into DNA by not only overrunning but also inhibiting the formation of competing TTP pools in tumors. A clinical trial is about to begin, with head-and-neck tumors as a first target of CldC radiosensitization.

Topics: Animals; Breast Neoplasms; Carcinoma, Squamous Cell; Combined Modality Therapy; DCMP Deaminase; Deoxycytidine; Deoxycytidine Kinase; Female; Glioblastoma; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms; Prostatic Neoplasms; Radiation-Sensitizing Agents; Radiotherapy Dosage; Spinal Cord; Tetrahydrouridine

Our approach to overcome the problem of rapid catabolism and general toxicity encountered with 5-halogenated analogues of deoxyuridine (5-bromo, chloro or iododeoxyuridine), which has limited their use as tumor radiosensitizers, is to utilize 5-chlorodeoxycytidine (CldC) with tetrahydrouridine (H4U). We propose that CldC, coadministered with H4U, is metabolized in the following manner: CldC----CldCMP----CldUMP---- ----CldUTP----DNA. All the enzymes of this pathway are elevated in many human malignant tumors and in HEp-2 cells. In X irradiation studies with HEp-2 cells, limited to 1 or 2 radiation doses, we have obtained 3.0 to 3.8 apparent dose enhancement ratios (these represent upper limits) when cells are preincubated with inhibitors of pyrimidine biosynthesis: N-(Phosphonacetyl)-L-aspartate (PALA) and 5-fluorodeoxyuridine (FdU) or 5-fluorodeoxycytidine (FdC) + H4U. Optimum conditions for radiosensitization are: PALA (0.1 mg/ml) 18-20 hr prior to FdU (0.1 microM) or FdC (0.02 microM) + H4U (0.1 mM) followed 6 hr later by CldC (0.1-0.2 mM) + H4U (0.1 mM) for 56-68 hr. Viabilities of 10 +/- 4% to 15 +/- 1% (+/- S.E.) were obtained for drug-treated unirradiated cells. Enzymatic studies indicate that this toxicity may be tumor selective. CldC + H4U alone (at these concentrations) results in 20% substitution of CldU for thymidine in DNA (determined by HPLC analysis). Preliminary toxicity studies indicate that mice will tolerate treatment protocols involving a single dose of PALA (200 mg/kg) followed by a dose of FdU (50 mg/kg) and 3 cycles of CldC (500 mg/kg) + H4U (100 mg/kg) at 10 hour intervals, with marginal weight loss (4%). In this approach we seek to obtain preferential conversion of CldC to CldUTP at the tumor site by taking advantage of quantitative differences in enzyme levels between tumors and normal tissues.

Topics: Animals; Aspartic Acid; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Deoxycytidine; Dose-Response Relationship, Radiation; Floxuridine; Humans; Laryngeal Neoplasms; Mice; Organophosphorus Compounds; Phosphonoacetic Acid; Radiation-Sensitizing Agents; Tetrahydrouridine; Uridine

In view of the 20- to 80-fold elevation of deoxycytidine-5'-phosphate (dCMP) deaminase in many human malignant tumors, we have utilized 5-fluorodeoxycytidine ( FdCyd ) coadministered with tetrahydrouridine ( H4Urd ) as a combination of antitumor agents against two murine solid tumors which possess high levels of dCMP deaminase. This approach is based on our past studies in which we demonstrated that FdCyd is an excellent substrate for mammalian 2'-deoxycytidine kinase, and that H4Urd increases the toxicity of FdCyd in the mouse. Cell culture studies utilizing 2'- deoxytetrahydrouridine which inhibits cytidine deaminase and as 2'- deoxytetrahydrouridine -5'-monophosphate inhibits dCMP deaminase, provide indirect evidence for the pathway that we had proposed in the past, 2'- Deoxytetrahydrouridine antagonized the toxicity of FdCyd to a greater extent than did H4Urd and showed marked antagonism in cytidine deaminase-deficient cells. Cell lines lacking both cytidine and 2'-deoxycytidine-5'-monophosphate deaminase were markedly resistant to FdCyd . Thymidine and deoxyuridine antagonized toxicity in a manner consistent with the proposed pathway of anabolism of FdCyd and consistent with its resulting in the inhibition of thymidylate synthetase. We have established the efficacy of FdCyd + H4Urd chemotherapy utilizing adenocarcinoma 755 and Lewis lung carcinoma in C57BL X DBA/2 F1 mice. An example of an optimum schedule versus Lewis lung carcinoma is FdCyd , 10 to 12 mg/kg, plus H4Urd , 25 mg/kg, coadministered simultaneously, once per day on Days 1 to 7 after tumor implantation. Tumor inhibitions on Days 12, 14, and 16 were 95, 90, and 80%, respectively, with 8% maximum weight loss. Comparative studies were undertaken only with Lewis lung carcinoma and it was established that FdCyd + H4Urd surpasses the efficacies of 5-fluorouracil and 5-fluorodeoxyuridine as well as FdCyd when administered without H4Urd . We propose that the administration of FdCyd with H4Urd can result in preferential, tumor-directed conversion of a nontoxic nucleoside analogue to a toxic antimetabolite by an enzyme that is markedly elevated in human tumor tissue. The analogues of deoxycytidine are resistant to catabolism and are anabolized by a different subset of enzymes than are 5-fluorouracil or 5-fluorodeoxyuridine; therefore, it is a novel approach. Not only are there intrinsic selectivity, metabolic stability, and the advantages that accrue from prodrug therapy in this strategy, but in a

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Ehrlich Tumor; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Cricetinae; Cricetulus; DCMP Deaminase; Deoxycytidine; DNA Replication; Drug Evaluation, Preclinical; Female; Humans; Leukemia L1210; Leukemia, Experimental; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Nucleotide Deaminases; Ovary; Tetrahydrouridine; Uridine