Research Project
6887063
DESCRIPTION (provided by applicant): Numerous studies have demonstrated that homocysteine (HCY) is a major risk factor in heart disease and stroke. Recent studies have shown that lowering HCY levels with large doses of folate have reduced the incidence of cardiovascular disease. However, in end-stage renal disease and other diseases with very high levels of HCY, vitamin therapy has not shown to be adequate to reduce HCY levels or reduce the elevated risk of cardiovascular disease. A mouse model of homocysteinemia has been previously developed by Dr. Maeda's laboratory by introducing a mutation in the gene for cystathionine-beta-synthase (CBS) which is the rate limiting enzyme in homocysteine transulfuration. Diets elevated in methionine and deficient in folate have induced atherosclerotic disease in CBS heterozygous (+/-) mice by elevating HCY as demonstrated in the laboratory of Dr. Lentz. These animal models can directly demonstrate the pathogenic effects of HCY. This application will utilize the CBS-deficient mice to determine the efficacy of the enzyme recombinant methionine (homocysteine)-alpha, gamma-lyase (rMETase) to decrease the elevated levels of HCY in these models. rMETase has been cloned from Pseudomonas putida in Escherichia coli. The enzyme is a homotetrameric pyridoxal 5'phosphate enzyme of 172 kDa molecular mass. In order to prolong the half-life of the enzyme that would extend the in vivo period of depletion of plasma homocysteine and attenuate antigenicity, METase-HCYase was coupled with methoxypolyethylene glycol succinimidyl glutarate-5000 (MEGC-PEG-5000). PEG/rMETase which had a half-life increase of up to 20-fold and methionine depletion time increase up to 10-fold compared to unmodified rMETase. MEGC-PEG-rMETase had reduced antigenicity compared to rMETase in mice and monkeys indicating the possibility of chronic use of the enzyme to decrease elevated HCY. CBS+/- mice were fed a high-HCY diet containing low levels of folate for 3 months resulting in HCY levels reaching approximately 200 mu M. After treatment with MEGC-PEG-rMETase for one hour, the tHCY levels were depleted to approx 10 mu M, which is in the normal range, thereby demonstrating the proof-of-principle of rMETase as a homocysteine-lowering enzyme. The overall aim of this application is: (1) Determine efficacy of MEGC-PEG-rMETase to chronically lower HCY in the hyperhomocysteinemic heterozygous cystathionine beta-synthase (CBS+/-) mouse model on a high-methionine, low-folate diet; (2) Determine toxicity of MEGC-PEG-rMETase during chronic HCY depletion in the hyperhomocysteinemic CBS+/- mouse model on the high-methionine, low-folate diet. The commercial aim of this Phase I application and subsequent Phase II application is to develop MEGC-PEG-rMETase as a therapeutic to target end-stage renal disease patients and other patients with hyperhomocysteinemia that do not sufficiently responded to vitamin therapy. The Phase II application will determine if MEGC-PEG-rMETase treatment can increase the survival of homozygous CBS -/- mice and other mouse models of hyperhomocysteinemia with shortened life-span. Phase II results will lead to an IND for MEGC-PEG-rMETase for end stage renal disease patients and other patients with intractable hyperhomocysteinemia and high cardiovascular disease mortality.
