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Erika Snow
Mentoring Professor - Dr. John Mata
Tuberculosis
http://www.healthjockey.com/2009/03/
20/ultraviolet-light-may-curbtuberculosis-transmission/
•Caused by an airborne bacterium
Multi-drug resistant TB
•1/3 ofNeed/urgency
the world’s population
to developinfected
alternative drug
regimens for treatment
•Annual rate 9 million people per year
•1.7 million people die each year
• Treatment
Biodegradable
and prevention
microspheres
• RSV
in infants
– small dose
Chemical
structure
over
long period time
modifications
• RSV
Drug
in complexes
adults - ineffective
http://www.wellnessctr.org/body.cfm?i
d=99&chunkiid=120795
Solution: increase and retain local concentration
•Low systemic bioavailability
•Low toxicity
•High efficacy
•Long residency time
•Easily delivered to the
affected areas
http://www.homelanddefense4u.com/images/Human%20
Lungs%20%2057577495.gif
•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
•Cancerform
therapies,
HIV antiretroviral
therapies,
viral
•Active
can interfere
with a pathogen’s
life cycle
lung infections
•Inhibit RNA/DNA polymerase
•Mimic natural DNA and RNA precursors of specific
•DNA chain termination
pathogens
•Induce damage through replacement of
natural nucleic genomic sequences
•Investigate efficacy of new inhalation therapy treatment
•Demonstrate efficacy of polymer pr0-drug
•To determine dosages, toxicity, and absorption patterns to
be used in further testing models
•Construct and validate inhalation apparatus
•Develop new treatment platform
•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-fluoroadenosine
2-methyladenosine
Properties
•Have specific affinity to one or more enzymes present in
M. tuberculosis
NH2
OO
NH2
O
N
N
O-
P
O
N
O
N
O
H
H
O
OH
H
NH2
H H
O
N
N
O
N
P
N
O
H
O
OH
H
P
N
H
H
O
H
H
8
NH2
H
8
N
O
N
P
OH
2-fluoroadenosine
O
O
N
O
2-methyladenosine
H
H
OH
OH
H
N
F
O
N
O-
N
CH3
NH2
H
N
OH
O
N
H
F
H
H
O
O
N
O
NH2
O-
P
N
CH3
H
H
N
O
O
N
O
P
N
CH3
2-methyladenosine polymer
F
O
H
H
OH
H
H
H
N
N
H
2-fluoroadenosine polymer
Properties
SeveralPharmaceutically
active drug
units
incorporated
intoanalogs
single synthetic
Susceptible
active
to
nucleoside
hydrolysis
Longer
No
carrier
residency
molecule
time
polymer molecule
5-fluorouridine monomer and polymer (10 subunits)
http://www.bio-world.com/productinfo/4_847_50_352/125261/Fluorouridine.html
Characterized
through chromatography
Similar pharmacokinetic
properties
and NMR spectroscopy
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:
• In progress
Brdu Assay:
• No results
•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:
•10 mL water in pump
•Nine minutes
•Vary number of open cones
Water vapor trial 2:
•Four minutes
•Cotton ball in each cone
Water vapor trial 1:
•Calculate
total water lost
0.4
0.994
•Estimate the flow per coneR =per
minute
Flow rate in (ml/min)
2
0.3
0.2
0.1
0.0
0
1
2
3
4
5
6
cones available
7
8
9
Water vapor trial 2:
•Calculate water collected
Average = 0.04322 g/4 min
St. Dev. = 0.00607 g/4 min
•Estimate the mean
vapor mass
Cone Number
High speed photomicroscopic analysis
Synthesis of 2-fluoroadenosine polymer
H2N
(Bz)2N
N(Bz)2
NHBz
NHBz
N
N(Bz)
N 2N(Bz)
N
2
N
N
N
OBz
N
N
F
N
-
O
OBzOH N
H
(1) TBAF, THF, DMF
NN
O
HO
H
N
N
(Pr)2N
N
PCNET-O
O H
O
HO
O
H
N
N
H
H
H
O
N
N
H
DMTO
H
N
N
H2N
N
H
N
N
H
N
O
H
N
N
O
H H
N
N
OH
O
H
F
N
P (2) TBAN,
O
N CH2CL2 F NO2
TFFA,
O -O P OBzO OBz
F
CNET-O
H
H
H
H
H
H
H
H
H
BzO DMTOBzO H
BzO
BzO
N
O
(1)(1)
NH3,
MeOHpyridine
BzCl,
H
NO2
(Pr)2N H OP
OBz
N
(Bz)2N
O
H
N
N
O
H
F
N
H
H
H
HOH
H
HOH
H
BzO BzO BzO BzO
Create
protected,
nitrated
intermediate
Phosphoramidite
building
block
Fluorinate
monomer
compound
Deprotection
reaction
which
yields final
product:
Standard protocol
(Glen
Research)
method
for
2-fluoroadenosine
polymer
oligonucleotide
synthesis to create
10-subunit 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
•Approximately equal distribution of vaporized particles
•Calculate expected dosages
Particle Size
•5 micron average particle size
•Increased efficacy
•Decreased cost of treatment
•Decreased toxicity
Theoretical Scheme
Solutions
•Protect active monomer = building block
•Standard oligonucleotide synthesis protocol
Oligonucleotide
synthesis 1st step
•Deprotect
polymer
Building blocks not fluorinated
Possible Problems
•Fluorinated monomer compounds unstable
•Harsh conditions and chemicals
• 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