Erika Snow Mentoring Professor - Dr. John Mata

Caused by an airborne bacterium Tuberculosis 20/ultraviolet-light-may-curb- tuberculosis-transmission/ 1/3 of the world’s population infected Annual rate 9 million people per year 1.7 million people die each year Multi-drug resistant TB Need/urgency to develop alternative drug regimens for treatment

d=99&chunkiid= Treatment and prevention RSV in infants – small dose over long period time RSV in adults - ineffective Solution: increase and retain local concentration Biodegradable microspheres Chemical structure modifications Drug complexes

Low systemic bioavailability defense4u.com/images/Human%20 Lungs%20% gif Low toxicity High efficacy Long residency time Easily delivered to the affected areas

Bioactive agent delivered directly to the affected area Drug susceptible to hydrolysis Agent released in a sustained fashion into local circulation Avoid toxic concentrations within systemic circulation

DNA chain termination Cancer therapies, HIV antiretroviral therapies, viral lung infections Active form can interfere with a pathogen’s life cycle Mimic natural DNA and RNA precursors of specific pathogens Inhibit RNA/DNA polymerase Induce damage through replacement of natural nucleic genomic sequences

Investigate efficacy of new inhalation therapy treatment Demonstrate efficacy of polymer pr0-drug Construct and validate inhalation apparatus Develop new treatment platform To determine dosages, toxicity, and absorption patterns to be used in further testing models

Polymer pro-drug will have slow, sustained release from apical side Remain in lung cells for longer period of time Inhalation apparatus will produce particles of appropriate size at a theoretically efficient flow rate Synthesis scheme for polymer of 2-methyladenosine pro-drug

2-methyladenosine 2-fluoroadenosine Properties Have specific affinity to one or more enzymes present in M. tuberculosis

2-methyladenosine 2-fluoroadenosine Properties Several active drug units incorporated into single synthetic polymer molecule Longer residency timePharmaceutically active nucleoside analogsSusceptible to hydrolysisNo carrier molecule 2-methyladenosine polymer 2-fluoroadenosine polymer

5-fluorouridine monomer and polymer (10 subunits) Characterized through chromatography and NMR spectroscopy Similar pharmacokinetic properties

Experimental Procedure: Calu-3 lung carcinoma cells were grown in Transwell ™ plates Testing solutions of monomer and polymer were prepared and allowed to efflux through the cell layer Samples collected at thirty minute intervals and stored for HPLC analysis

Measure the affect of varying concentrations Experimental Procedure: Calu-3 cells grown in 96 Transwell ™ plates Treated with monomer or polymer solution with serial dilution Brdu assay with absorption analysis

HPLC Analysis: Brdu Assay: No results In progress

Apparatus design Flow rate determination Particle size analysis

Directed-flow, nose only chambers Deliver efficient concentrations inhaled prodrug to the lungs

Water vapor trial 1: Nine minutes Vary number of open cones 10 mL water in pump Water vapor trial 2: Four minutes Cotton ball in each cone

Calculate total water lost Estimate the flow per cone per minute Water vapor trial 1: R 2 = 0.994

Water vapor trial 2: Calculate water collected Estimate the mean vapor mass Average = g/4 min St. Dev. = g/4 min Cone Number

High speed photomicroscopic analysis

Synthesis of 2-fluoroadenosine polymer Phosphoramidite building block Create protected, nitrated intermediate Fluorinate monomer compound Standard protocol (Glen Research) method for oligonucleotide synthesis to create 10-subunit polymer Deprotection reaction which yields final product: 2-fluoroadenosine polymer

Expected Results 5-fluorouridine pro-drug slowly efflux across epithelial cell monolayer Effective permeability (Pe) Polymer lower Pe Increased active drug concentrations Smaller, less frequent doses

Possible Explanations Antibodies did not bind Concentrations Solutions Further dilution Alternative assays

Flow Rate Inverse relationship Non-significant variability Particle Size 5 micron average particle size Increased efficacy Approximately equal distribution of vaporized particles Calculate expected dosages Decreased cost of treatment Decreased toxicity

Theoretical Scheme Protect active monomer = building block Standard oligonucleotide synthesis protocol Fluorinated monomer compounds unstable Harsh conditions and chemicals Deprotect polymer Possible Problems Solutions Oligonucleotide synthesis 1 st step Building blocks not fluorinated

Complete in vitro experiments and analysis Toxicity, dosages, absorption patterns Synthesize pro-drugs Evaluate efficacy/activity of pro-drugs Multiple strains Macrophage test system In vitro efficacy testing

Howard Hughes Medical Institute URISC Dr. John Mata Wanda Crannell Dr. Kevin Ahern Dr. Luiz Bermudez Dr. Katharine Field