Dr. Charles B. Grissom Professor of Chemistry Adjunct Professor of Biochemistry
	Email: grissomc@chem.utah.edu	Phone: (801)-581-4153
	Address: Department of Chemistry, University of Utah	Fax: (801)-585-9134
	315 S. 1400 E., Salt Lake City, UT 84112-0850	Group Phone: (801)581-4175

Targeted Delivery of Cancer Drugs and Diagnostic Agents

Our laboratory has developed a new method to target the delivery of cytotoxic anticancer drugs to tumor cells by using vitamin B-12 as a "Trojan Horse" delivery vehicle. Drug-B12 bioconjugates are synthesized by attaching various cytotoxic warheads to the cobalt atom of cobalamin or a structurally-related corrinoid. The active cytotoxic drug is released spontaneously inside of immature leukemia cells, but no toxicity is observed when receptor-mediated endocytosis is blocked by presaturation of the receptors with vitamin B-12. In solid tumors and all other tissues, the pro-drug is nontoxic until it is activated by photolysis with tissue-penetrating red light, or with sonolysis by focused ultrasound. Our results in vitro and in vivo show this "Trojan Horse" strategy for targeted drug delivery to be a very effective method to increase the therapeutic index of existing anticancer drugs.

Significance. Most of the front-line chemotherapeutic drugs such as doxorubicin, taxol, etoposide, and the alkylating agents have a dose-limiting toxicity that arises from interaction of the drug with an organ system that is remote from the site of cancer. If these cytotoxic drugs could be delivered selectively to cancer cells, their therapeutic index would be greatly increased and the serious side effects of bone marrow supression, cardiotoxicity, nephrotoxicity, hepatotoxicity, neurotoxicity, hair loss, and nausea, might be eliminated. We are attempting to address the problem of non-targeted cytotoxicity by creating an inert prodrug bioconjugate comprised of a cytotoxic drug ‘warhead’ and cobalamin (vitamin B-12) as the carrier.

Cobalamin is an essential micronutrient that is required for human health and, more importantly, is required in large quantities by cells that are replicating DNA prior to cell division. Because cobalamin is only present in small quantities in meat and dairy products, humans have evolved an elaborate mechanism to absorb small quantities from their diet and transport it to rapidly-growing and dividing cells. Cobalamin in the blood is bound by the protein transcobalamin prior to receptor-mediated endocytosis via the megalin receptor.

Uptake of B-12 by Cancer Cells. Rapidly dividing cells require coenzyme B-12 for thymidine synthesis for DNA replication. In chronic myeloid leukemia (CML) a 6-20 fold increase in transcobalamin is sometimes observed with an overall increase in unsaturated B-12 binding capacity of 3-10 fold. In acute promyelocytic leukemia (APL), an even greater increase in transcobalamin levels and unsaturated B-12 binding capacity is often observed. This suggests leukemia cells may accumulate a drug-cobalamin conjugate to the extent necessary for cobalamin-based drug delivery to be effective. Solid tumors also increase their uptake of cobalamin. In some patients with tumors, up to a 50-fold increase in the major cobalamin transport proteins has been observed.

Light-Triggered Drug Release. The inert cobalamin-drug bioconjugate can be activated by visible light by cleaving the Co-C bond, thereby releasing the active drug in the irradiated tissue. Human tissue is partially translucent to light in the range of 610-750 nm. Very little of this light is absorbed, so that no heat is felt if only red light is used to illuminate the skin, and the intensity is diminished only by scattering.

Ultrasound-Triggered Drug Release. Cobalamin bioconjugates are unique among prodrugs, since they can also be activated by ultrasound to cleave the C-Co bond, thereby allowing release of the drug deep within tissue that is not as easily accessible with red light. Sonolysis of aqueous solutions produces a high concentration of hydroxyl radicals and hydrogen atoms. These reactive oxidizing and reducing species are responsible for initiating most reactions in aqueous solvents, including cleavage of the Co-C bond and activation of the prodrug to release the active chemotherapeutic agent.

Projects for Graduate Students Graduate students have the ability to define their own project within the spectrum of cobalamin-targeted drug delivery. Students can design, synthesize, and test new bioconjugates of cancer drugs. Since this project is a collaboration with research groups in the Pharmacology and Pathology Departments of the University of Utah Medical School, students can participate in every aspect of therapeutic drug development and biological evaluation. This unusual opportunity in an academic setting provides a broad graduate education, with solid training in biological chemistry.

Enzyme Mechanisms, Vitamin B-12, and Magnetic Field Effects

Our laboratory is also studying the mechanism of enzymes with radical pair intermediates. Towards this end, we have developed new methods for probing the mechanism of biological and chemical reactions with DC magnetic fields. Only reactions that produce a radical pair intermediate are susceptible to the influence of an external magnetic field on the reaction rate.

Vitamin B-12 Dependent Enzymes

In 1994, we reported the first magnetic field effect on an enzymatic reaction (B-12 dependent ethanolamine ammonia lyase). This finding is significant because it confirms the existence of a radical pair intermediate and it provides a mechanism by which magnetic fields can interact with biological systems. We have extended our studies to horseradish peroxidase, a heme-containing enzyme that is also magnetic field dependent.

Photochemistry of Vitamin B-12

We are also interested in the properties of coenzyme B-12 that make it a good initiator of radical reactions. Towards this end, we have carried out picosecond and nanosecond laser flash photolysis studies of the B-12 cofactor.

Our laboratory uses standard biochemical techniques for the characterization of enzymatic reactions including enzyme kinetics, isotope effects, stopped-flow kinetics, and spectroscopic methods. We occasionally employ picosecond and nanosecond laser flash photolysis and other rapid reaction (and detection) methods to monitor transient intermediates. Within our lab, we routinely synthesize small organic molecules that are analogues of substrates, isotopically-labeled substrates, or unusual alkyl derivatives of B-12. Scientists from this lab will be well prepared for careers in either industry or academia.