erika snow mentoring professor - dr. john mata. caused by an airborne bacterium tuberculosis ...

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