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Ribonucleotide Pro-Moieties for Time-Dependent Drug Release

Ribonucleotide Pro-Moieties for Time-Dependent Drug Release. Amy Davis Raines Laboratory Department of Biochemistry. Metronidazole. Selective uptake by anaerobic bacteria After ingestion, reduction of nitro group to nitroso group by cellular ferredoxin

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Ribonucleotide Pro-Moieties for Time-Dependent Drug Release

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  1. Ribonucleotide Pro-Moieties for Time-Dependent Drug Release Amy Davis Raines Laboratory Department of Biochemistry

  2. Metronidazole • Selective uptake by anaerobic bacteria • After ingestion, reduction of nitro group to nitroso group by cellular ferredoxin • Resulting interaction with bacterial DNA yields chromosomal breakage, increased denaturation • Common side effects include: nausea, diarrhea, and in some cases pancreatitis and CNS damage Eisenstein, Barry I.; Schaechter, Moselio (2007). "DNA and Chromosome Mechanics". In Schaechter, Moselio; Engleberg, N. Cary; DiRita, Victor J. et al.Schaechter's mechanisms of microbial disease. Hagerstown, MD: Lippincott Williams & Wilkins. p. 28.

  3. Typical Absorption of a Drug Pill Dissolves Deaggregation Absorption

  4. Aiding Absorption • Phosphate esters improve the water solubility of therapeutics • Endogenous alkaline phosphatase cleaves the phosphate from the drug http://en.wikipedia.org/wiki/Alkaline_phosphatase Jana,S.; Mandlekar, S.; Marathe, P.; Curr. Med Chem. 2010, 17, 3874.

  5. Ribonuclease 1 • Endogenous enzyme • Found in concentrations of approximately ~0.9 mg/L • Cleaves preferentially at the 3’-OH of pyrimidine bases (thymine, uracil, and cytosine) Landre, J.B.P. Hewett,P.W.; Olivot,J.; Friendi,P.; Ko,Y.; Sachinidis,A.; Moenner,M. J. Cell. Biochem. 2002, 86, 540. http://www.pdb.org/pdb/explore/explore.do?structureId=1dza

  6. Ribonuclease Action

  7. Mechanism of Ribonuclease Roberts, G.C.K.; Dennis, E.A.; Meadows, D.H.; Cohen, J.S.; Jardetzky, O.; Proc. Natl. Acad. Sci.1969, 62, 1151.

  8. Pro-Drug Synthesis

  9. Pro-Drug Synthesis (cont)

  10. NMR Kinetic Assays • Ribonuclease 1 added at biological pH in aqueous media to NMR tube, along with ribonucleotide pro-drug • Tested in the presence of different RNAse 1 concentrations: 0.01mg/mL and 0.1mg/mL • RNAse 1 prefers cytosine over uridine, and does not cleave after purines • Expected Results: RNAse 1 cleaves cytosine prodrug more quickly than uridine and adenosine, respectively

  11. NMR Assay Data

  12. Results • Rapid cleavage occurred for metronidazole attached to cytosine and thymine nucleosides • Adenine pro-drug resulted in drastically reduced rate of cleavage

  13. Anaerobic Bacterial Assays

  14. Results • MIC concentration for pure metronidazole is 0.5-1μg/mL • The MIC for the uridine prodrug is ~40 times that concentration • When incubated with RNAse, the uridine pro-drug has the same MIC as pure metronidazole

  15. Conclusions Cytosine and uridine prodrugs may be a useful strategy for delivering metronidazole Testing in human systems will be necessary to fully determine the efficacy of this prodrug strategy Future directions involve coupling other drugs to ribonucleotide pro-moieties

  16. With the Help of: Professor Ronald Raines and the entire Raines Lab! Mentors: Michael Palte Nicholas McGrath A great big thanks to everyone for their help and support. Thank you to CALS for supporting this symposium.

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