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Past Research

Cytochlor
A promising new treatment in the battle against cancer.

RGI, in conjunction with collaborators at the University of Miami, developed a new cancer drug, Cytochlor. The National Cancer Institute at the National Institutes of health recently approved it for use in human patients in Phase I Clinical trials. Cytochlor is unique in that it was killing only cancer cells by exploiting an enzyme found in higher levels within tumor cells. Cytochlor acts like a smart missile; the drug will seek out nests of cancer cells. Once Cytochlor enters the cancer cell it breaks down the DNA within the cell.

Tumor cells are also treated with low levels of radiation causing further break down of the DNA. This one-two-punch damages the cells DNA so much that the cell commits suicide. Non-cancer cells are not damaged and are left in a normal state of health. In pre-clinical trials, Cytochlor had an 80% cure rate and a 0% mortality rate. This is an amazing statistic that has instilled hope in the scientific community. Another promising aspect of Cytochlor is that the radiation required to be effective is 3 to 4 times less than that of radiation therapy alone. This allows the subject to avoid the major negative side effects associated with radiation therapy.

Technical discussion

Advantages of CldC + Tetrahydrouridine (H4U) Radiosensitization over IdU or BrdU conversion to a radiosensitizer in human tumors due to elevation of one of 3 key enzymes: deoxycytidine kinase, cytidine- and dCMP-deaminase. In contrast, IdU is incorporated into any tissue with rapid cell kinetics, including bone marrow and intestine. CldC is not toxic at radiosensitizing doses. The NCI has confirmed this in mice, dogs and monkeys. Radiation of tumors does not result in significant weight loss. In comparative studies, IdU at its maximum tolerated dose was far less effective than CldC versus mouse and human tumors.

CldC in not catabolized; it acts as a prodrug for the ultimate radiosensitizer, CldUTP. Recent studies show that the catabolic enzyme thymidine phosphorylase is elevated 15-fold in invasive human tumors. CldC circumvents this catabolic enzyme. H4U increases the half-life of CldC as shown in mice and monkeys. CldC is metabolized towards the ultimate radiosensitizer by two distinct pathways: therefore, mutation to resistance is remote it would require two independent mutations in the same cell.

An anabolite of IdU or BrdU: IdUMP or BrdUMP, respectively, is dehalogenated by thymidylate synthetase. This degrades the radiosensitizer and renders it ineffective. Indeed, the dehalogenated product (dUMP) competes with IdU or BrdU for incorporation into DNA. In contrast, this enzyme does not degrade CldUMP formed from CldC. I and Br are toxic to the patient affecting the thyroid and brain, respectively.

The formation of the ultimate radiosensitizer, CldUTP, is assured not only because the levels of deoxycytidine kinase and dCMP deaminase are elevated in tumor, but also because the affinity of CldCMP for dCMP kinase is low. This means that CldCMP is preferentially deaminated to CldUMP rather than phosphorylated to CldCDP. Thus, CldCMP is diverted to the radiosensitization pathway in tumors.

Although CldUMP is not as effective as FdUMP as an inhibitor of thymidylate synthetase (TS), it is a reasonably good inhibitor, especially when compared to IdUMP and BrdUMP. Therefore, the well-tolerated high doses of CldC not only will overrun the competition of TTP but also will inhibit the formation of TTP.  By lowering competing TTP pools, CldUMP derived from CldC can cause nucleoside pool imbalances that result in DNA strand breaks, which enhances tumor-directed radiosensitization. Lowering TTP pools result in the activation of dCMP deaminase.

CldC is converted to CldUTP, which up-regulates the enzyme that activates CldC. In contrast, IdUTP and BrdUTP inhibit thymidine kinase, the enzyme that activates IdU and BrdU. Therefore, pharmacologically effective doses more readily occur with CldC than with IdU or BrdU. CldC enhances its own incorporation into DNA.

CldC, when anabolized to CldUTP, inhibits nucleoside diphosphate reductase. This lowers competing metabolites that interfere with the incorporation of CldUTP into DNA. The down regulation of the reductase results in decreasing dCTP levels, which, in turn, increases the activity of deoxycytidine kinase, the enzyme which initially activates CldC. Thus, CldC enhances its own incorporation into DNA by different mechanisms.

Radiation has been shown to increase the levels of a) deoxycytidine kinase, the first enzyme that activates CldC and b) increase the levels of nucleoside diphosphate reductase perhaps for purposes of repair. This, inhibition of the reductase by CldUTP may interfere with this protective response by the target cells. Inhibition of the reductase results in metabolite pool imbalances that could lead to a substantial number of DNA single strand breaks and apoptosis.

CldU derived from CldC and incorporated into DNA may be removed by a repair mechanism involving uracil-N-glycosidase and an apyrimidinic endonuclease, as is the case with FdU. This also leads to tumor-directed single strand breaks.

The inhibition of thymidylate synthetase in tumors leads to the incorporation of uracil into tumor DNA. The incorporation of uracil, (and CldU) into DNA results in additional DNA-strand breaks as a consequence of DNA repair. Thus, in addition to strand breaks that occur as a consequence of X-irradiation, three different biochemical mechanisms result in tumor-directed DNA strand breaks. These breaks persist because the repair capacity of the tumor cell is over-run.

When CldC is selectively incorporated into DNA, the DNA becomes hypomethylated (FdC in DNA displays this property). The expression of tumor surface antigens, to which the host mounts an immune response, has been demonstrated with 5-azacytidine

Hypomethylation may a) turn on silenced tumor suppressor genes or invasion suppressor genes (like E-cadherin) b) prevent tumor genetic instability by removing hot spots of mutation and c) restore activity of silenced repair enzymes such as methylguanine DNA transferase so that aggressive subclones of the tumor do not develop.

This strategy may allow the radiation therapist to achieve a tumor kill that would be obtained with > 60 Gy with only 20 Gy without damage to normal tissue or alternatively, obtain a tumor kill equivalent to > 180 Gy with only 60 Gy.

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