erika snow mentoring professor - dr. john mata. caused by an airborne bacterium tuberculosis ...
TRANSCRIPT
Erika SnowMentoring Professor - Dr. John
Mata
•Caused by an airborne bacterium
Tuberculosis
http://www.healthjockey.com/2009/03/20/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
http://www.wellnessctr.org/body.cfm?id=99&chunkiid=120795
• 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
http://www.homeland-defense4u.com/images/Human%20Lungs%20%2057577495.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
PropertiesSeveral active drug units incorporated into single
synthetic polymer molecule
N
NN
N
NH2
O
OHO
HH
HH
O
PO
O-
O-
CH3
N
NN
N
NH2
O
OHO
HH
HH
O
PO O-
CH3
N
NN
N
NH2
O
OHOH
HH
HH
O
PO O-
CH3
8
N
NN
N
NH2
O
HOH
HH
HH
OF
P OHO
O
N
NN
N
NH2
O
H
HH
HH
OF
P OHO
O
N
NN
N
NH2
O
H
HH
HH
O
F
P OO
O
8
Longer residency time Pharmaceutically 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
http://www.bio-world.com/productinfo/4_847_50_352/125261/Fluorouridine.html
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
0 1 2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
cones available
Flo
w r
ate
in (
ml/
min
)
•Calculate total water lost •Estimate the flow per cone per minute
Water vapor trial 1:
R2= 0.994
Water vapor trial 2:•Calculate water collected •Estimate the mean vapor mass
Average = 0.04322 g/4 min
St. Dev. = 0.00607 g/4 min
Cone Number
High speed photomicroscopic analysis
Synthesis of 2-fluoroadenosine polymer
N
NN
N
NHBz
O
HDMTO
HH
HH
OP(Pr)2N
CNET-O
Phosphoramidite building block
N
NN
N
NHBz
O
HDMTO
HH
HH
OP(Pr)2N
CNET-O
(1) BzCl, pyridine
(2) TBAN, TFFA, CH2CL2
N
NN
N
N(Bz)2
O
BzOBzO
HH
HH
OBz
NO2
Create protected, nitrated intermediate
N
NN
N
N(Bz)2
O
BzOBzO
HH
HH
OBz
NO2
(1) TBAF, THF, DMF
N
NN
N
N(Bz)2
O
BzOBzO
HH
HH
OBz
F
Fluorinate monomer compound Standard protocol (Glen Research) method for
oligonucleotide synthesis to create 10-subunit polymer
N
NN
N
H2N
O
OHO
HH
HH
HO
F
N
NN
N
H2N
O
OHOH
HH
HH
O
F
P
O
-O
N
NN
N
(Bz)2N
O
BzOO
HH
HH
OBz
F
N
NN
N
(Bz)2N
O
BzOBzO
HH
HH
O
F
P
O
-O
(1) NH3, MeOH
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 1st 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