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CCKK Drug Delivery Technologies
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CCKK Drug Delivery Technologies:
Monoclonal Antibodies for Delivery of Chemotherapy Drugs for Cancer Therapy
Sarah ClawsonPaul Cole
Sarah KatenNell Keith
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Introduction
Project Objective: Improve chemotherapy drug delivery for maximum efficacy with minimal side effects
Disease Considered: Gliomas – common brain tumor
Method of Delivery: Micelle and monoclonal antibody complex
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What is a Monoclonal Antibody?
MAb - an antibody that binds to a single epitope
Stevens, Glen. Brain Tumors: Meningiomas and Gliomas. April 22, 2003. http://www.clevelandclinicmeded.com/diseasemanagement/neurology/braintumor/braintumors.htm
Image Courtesy of: http://www.strayvr.com/FullyHuman.jpg
In tumor cells the epitope is often a protein on the cell surface
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Treatment Options Using MAbs
Janeway, Charles A., et al. Immunobiology. 5th ed. New York: Garland Publishing, 2001.
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Production of Monoclonal Antibodies
Janeway, Charles A., et al. Immunobiology. 5th ed. New York:
Garland Publishing, 2001.
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Immunogenicity in Humans
Most MAbs are produced using murine(mouse) cells
The human body may recognize the MAbs as being foreign. This can result in:
Allergic reactionFailure of treatment
Janeway, Charles A., et al. Immunobiology. 5th ed. New York: Garland Publishing, 2001.
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Engineering MAbs to Reduce Immunogenicity
Graft the antigen binding loop of the mouse antibody to the framework of a human antibody
MAb still has the same antigen/antibody binding specificity
Janeway, Charles A., et al. Immunobiology. 5th ed. New York: Garland Publishing, 2001.
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Malignant Gliomas Tumors
Gliomas are the most common type of primary brain tumors in adults
Anaplastic Astrocytoma (III) Glioblastoma Multiforme (IV)
Conventional therapy:SurgeryExternal-beam radiationChemotherapy
Median survival: 40-60 weeksA Primer of Brain Tumors. American Brain Tumor Association. 1991.
<http://neurosurgery.mgh.harvard.edu/abta/primer.htm#Section6>
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81C6 MAb
81C6 is a monoclonal antibody that binds to tenascin, a tumor-associated extracellular matrix proteinTenascin is found in:
GliomasConnective tissue (trace amounts)Developing organs (trace amounts)
Cokgor, Ilkcan, et al. “Phase I Trial Results of Iodine-131-Labeled Antitenascin Monoclonal Antibody 81C6 Treatment of Patients With Newly Diagnosed
Chiquet-Ehrismann, Ruth. What Distinguishes Tenascin From Fibronectin? The FASEB Journal. Vol. 4. June 1990.
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Blood Brain Barrier
Tight junctions in capillary endothelial cells that prevent molecules from entering into glial cells
Entry is achieved by a molecule’s solubility in lipids or by transporters
Purves, Dale, et al. Neuroscience. 2nd ed. Sinauer Associate, Inc. 2001.
Image courtesy of http://homepage.psy.utexas.edu/homepage/class/Ps
y332/Salinas/Cells/BBB.gif
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How can the Blood Brain Barrier be overcome?
Getting molecules past the blood brain barrier (BBB) can be achieved by attaching the molecule to a vector
The vector can be a modified protein or monoclonal antibody that is normally transported through the blood brain barrier
Pardridge, W.M. “Vector-mediated drug delivery to the brain.” Advanced Drug Delivery Revues. Vol. 36. April 1999.
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Vector Mechanism
Vector aids in transport across the BBB by transcytosis
Transcytosis –transport of substance across epithelium by uptake into and release from coated vesicles
Bickel, Ulrich, et al. Pharmacologic effects in vivo in brain by vector-mediated peptide drug delivery.Proceddings of the National Academy of Science. 1992.
http://images.google.com/imgres?imgurl=http://www.skcc.org/n_images/transcytosis.jpg&imgrefurl=http://www.skcc.org/schnitzer.html&h=253&w=500&sz=118&tbnid=7FtvVYAfLg0J:&tbnh=64&tbnw=126&start=1&prev=/images%3Fq%3Dtranscytosis%26hl%3Den%26lr%3D%26sa%3DG
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Suggested Vector: OX26
OX26 is a MAb
OX26 undergoes receptor-mediated transcytosis
Targets transferrinTransferrin receptor is highly expressed on brain capillary endolthelial cells
Bickel, Ulrich, et al. Pharmacologic effects in vivo in brain by vector-mediated peptide drug delivery.Proceddings of the National Academy of Science. 1992.
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What is a Micelle?
Globular structure made of a charged head group with a lipid tail
Hydrocarbon tails located on the inside of the micelle to reduce interactions with water
Berg, Jeremy, et al. Biochemistry. W.H. Freeman and Co. 2002.
Cross-sectional View of a Micelle
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Micelle and MAbs For Drug Delivery: Immunomicelle
StructureMAb attached to the head group of micelle Toxin will be stored inside the micelle
Toxin DeliveryImmunomicelle will be transported across the BBB by vector mechanismToxin delivered to the tumor when immunomicelle is engulfed
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Previous Studies: Immunoliposomes
Huwyler et al. utilized immunoliposomesDeliver the daunomycin to the rat brainLiposome did not cross the BBB without a vectorDrug delivery successful when the vector OX26 was usedHigher density of vector more immunoliposomes cross BBB
Huwyler, Jorg, et al. Brain Drug Delivery of Small Molecules Using Immunoliposomes. Proceedings of the National Academy of Sciences. Neurobiology. 1996.
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How does a Micelle Compare to a Liposome?
Micelles and Liposomes can be made of the same material
Hydrophilic environment inside the liposome and hydrophobic environment inside the micelle
Superficially micelles and liposomes are indistinguishable
More toxin can be inserted inside a micelle than a liposome of the same diameter
Pictures Courtesy of: Berg, Jeremy, et al. Biochemistry. New York. W.H. Freeman and Co.: 2002
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Investigation of Immunomicelles
Torchilin et al. used immunomicelles loaded with Taxol® to treat Lewis lung caracinoma
Drug delivery by cell engulfing the micelle
Micelle made of polyethylene glycol–phosphatidylethanolamine conjugates
Torchilin, Vladimir P., et al. “Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs.” Proceeding of the National Academy of Sciences. May 13, 2003.
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Investigation of Immunomicelles
Use amphiphilic derivative of PEG: pNP-PEG-DOPE pNP-PEG-DOPE readily incorporates into the micelle Binds primary amino group ligands via water-exposed pNP groups
Forms stable, nontoxic urethane bondsSeveral dozen MAbs can be attached to one micelle
Torchilin, Vladimir P., et al. “Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs.” Proceeding of the National Academy of Sciences. May 13, 2003.
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Micelle-Antibody Attachment
MicelleMicelle-Antibody
attachment reaction
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Size Considerations with Drug Delivery to Tumors
Diffusion and accumulation inside the tumor depend on the cutoff size of the tumor blood vessel arrangement
This cutoff size varies for different tumors
MAbs attached to micelles does not significantly affect the size of the micelle
Torchilin, Vladimir P., et al. “Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs.” Proceeding of the National Academy of Sciences. May 13, 2003.
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Selected Drug for Encapsulation
Drug of choice for micelle delivery is Temodar®
Similar side effects as other chemotherapeutic agentsSmaller molecular diameterActs through alkylation of DNA of replicating cellsStandard dosages of 100 and 500 mg daily
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Blood Concentration Model
Generally, the concentration of a drug in the body is modeled by a half life function with the equation
Separating variables and integrating yields
bloodblood kCdt
dC−=
ktC
Cblood −=)ln(0
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Temodar® Blood Concentration Model
The half life of Temodar® is 1.8 hours
Half life can be used to find a k value of 0.38hr-1
The final model is described by the equation
Temodar Product Information, Schering Corporation, 2003
tblood eC
C 38.0
0
−=
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Micelle Blood Concentration Model
Micelle Half Life Model Assumptions:20-25% of the blood in body is received by kidneys10-15% of blood received is cleaned50% micelles removed from the blood in each circulationBlood is recirculated through the body every minute
Micelle half life is 50 min.
This is used to find a k of 0.83hr-1
Koeppen, Bruce M. and Bruce A. Stanton, Renal Physiology, ed. 3, St. Louis, MO, 2001
processedreceivedcleaned %%% ×=
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Why is Micelle better than Temodar® alone?
00.10.20.30.40.50.60.70.80.9
1
0 2 4 6 8 10
time (hr)
C/C
o OralDosageHalf LifeModelMicelle HalfLife Model
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Micelle Concentration Model Considerations
Model is accurate for determining the blood concentration of an oral dosage
Micelles will be injected to deliver the micelles directly to the tumor in the brain
Additional model needed to determine how the concentration of micelles in brain changes with time
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Injection Delivery and Dosage Model
Drug is administered by injection into the internal carotid artery (ICA)Average flow rate within carotid artery is 370 mL/minAssume all of drug enters artery in 5 seconds
Drug concentration = dosage/(flowrate*time)
Image courtesy of www.pennhealth.com/
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Injection Delivery and Dosage Model
Estimated number of capillaries in the portion of interest in the brain can be used to determine the amount of blood contained in each mircovesselAmount of blood in capillaries can be used to determine amount of drug per capillary
capillarydelivereddrugionconcentratplug
scapillarieofnumberICAinvolumeplug
=×
Blinkov, Samuil M., and Il-ya I. Glezer, eds. The Human Brain in Figures and Tables: A Quantitative Handbook. Moscow: Basic Books, Inc, 1968
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Injection Delivery and Dosage Model
Assumptions50% of initially injected drug will penetrate BBB50% of drug that crosses will bind to tumor cells 3000 capillaries/mm3 brain/tumor tissue
Blinkov, Samuil M., and Il-ya I. Glezer, eds. The Human Brain in Figures and Tables: A Quantitative Handbook. Moscow: Basic Books, Inc, 1968
tissuetumorvolumedelivereddrug
capillarydelivereddrug
tissuetumorvolumescapillarie
=×
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Injection Delivery Standard Curve
Drug Delivered upon Initial Injection (350 mg dosage)
0
0.5
1
1.5
2
0 20 40 60 80
Tumor Volume (cc)
Drug
Del
iver
ed (m
g)
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Why is Injection Superior to Oral Dosage?
Delivery model shows a 250 fold efficacy improvement from injection over oral delivery
Dosage can be kept to a minimum with equal tumor reduction as current treatments, which will subsequently lower side effects
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What are the Applications of the Dosage Model?
Dosages can be determined for individual patients based on tumor size and location
Necessary dosage for effective treatment can then be incorporated into the blood concentration model in order to determine dosage regimen
Initial and final brain concentrations can be used to estimate parameters for brain elimination models
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Brain Concentration Model
Analyzed as a two compartment membrane model
k values are rate constants, X values are concentrations
Compartment 1: Brain
Compartment 2:Rest of Body
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Brain Concentration Model
www.4um.com/tutorial/science/pharmak.html
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Brain Concentration Model
Plasma Concentration in brain decays bi-exponentially with time and will fit the following equation:
T is timeA1 and B1 are the intercept constantsα and β are the hybrid rate constants (units T-1)
Welling, Peter G., Pharmacokinetics: Processes and Mathematics, American Chemical Society, Washington DC, 1986
TTbrain eBeAC βα −− += 11
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Brain Concentration Model Derivation
α and β are functions of phase half life
The half life for the elimination phase was assumed to be the half life of the micelle in the body in the oral dosage model
β
β)()2ln(
2/1t=
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Brain Concentration Model Derivation
Half life for redistribution phase is determined from the injection dosage model
Mass balances on brain/body system were used to determine how the concentrations in the body and brain change during this phase to determine half life
α
α)()2ln(
2/1t=
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Brain Concentration Model Derivation
Alpha Equilibration Model
0
0.2
0.4
0.6
0.8
1
0 2 4 6
time (min)
Conc
entra
tion
(mg/
mL
Alpha Phase (brain)Alpha Phase (body)
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A1 was determined from concentration of drug leaving brain after tumor delivery
B1 was determined from estimate of theoretical body concentration as a function of mass of drug exiting the brain after initial injection
Brain Concentration Model Derivation
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Elimination Model Rate Constants
From model parameters, k-values can be determined
Compartment 1: Brain
Compartment 2:Rest of Body
5.36 hr-1
61.1 hr-1
0.87 hr-1
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Brain Concentration Model
0.01
0.1
1
10
0 0.5 1 1.5
Time (hr)
Bra
in C
once
ntra
tion
(mg/
mL
Redistribution Phase
Elimination Phase
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Pre-FDA Model Verification
Before FDA testing begins, this model will be verified by animal testing
Determine % micelles eliminated from the blood by kidneysDetermine percent of micelle to cross BBBDetermine percent of micelle to bind to cancer cellsDetermine rate constants for the redistribution phase and elimination phase from experimental half life results
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FDA Approval
There are 3 major phases of FDA approval for a new drug or therapeutic:
Pre Clinical Trials (~13 years)Clinical Trials (~8.5 years)Validation (~2 years)
The FDA approval process will take around 23 years
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What occurs during Pre-FDA Testing?
Testing in mice to determine treatment safety and efficacy
Testing to confirm drug delivery mechanism parameters
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Pre-FDA Testing Flow
Chart
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Pre-FDA Funding
Major source of funding for Pre-FDA testing is the National Institute of Health (NIH)These grants are renewable annuallyPre-FDA testing will be performed in conjunction with universities
Grant Average Amount per fiscal year
Small Business Technology Transfer (Phase I)
$140,700
Small Business Technology Transfer (Phase II)
$318,492
Small Business Innovation Research (Phase I)
$149,261
Small Business Innovation Research (Phase II)
$425,517
Animal (Mammalian and Non-mammalian) Model, and Animal and Biological Material Resource
$716,044
Biotechnology Resource Grant Program
$1,628,377
Exploratory Grants $1,134,298
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What occurs during FDA Clinical Trials?
Phase I TrialsDetermine immediate treatment safety and dosage1½ Years
Phase II TrialsDetermine potential short-term side effects 3 Years
Phase III TrialsDetermine potential long-term side effects4 Years
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Micelle Begins Approval Process
Phase I Trials
(1½ Years)
Safe Unsafe
Project FailsPhase II
Trials(3 Years)
More effective with lower side effects
than current treatments.
Same effectiveness with lower side
effects than current treamtents.
More effective with more side effects
than current treatments.
Less effective with more side effects
than current treatments.
Project FailsSide Effects
UnacceptableSide Effects Acceptable
Project FailsPhase III
Trials (4 Years)
Long term severe side effects, with
more effectiveness
Long term mild side effects, with
more effectiveness
Long term mild side effects, with
same effectiveness.
Long term severe side effects, with
less effectiveness.
Project Fails Project Fails
Approved
FDA Clinical Trials Flow
Chart
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FDA Approval
At the completion of clinical trials a New Drug Application (NDA) is filed with the FDA
The final stage of FDA approval is the review and post-marketing analysis by the FDA
At the end of this period, one drug is approved out of many that enter the FDA approval process
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MAb Market in Cancer Therapy
The market for cancer treatments is growing at a tremendous rate
In 1997 the first MAb for cancer was approved by the FDA (Rituxan)
Annual average growth over 2003 and 2004 for the MAb market in cancer therapy is 60%
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MAb Market in Cancer Therapy
Revenues for MAb products increased by $2 billion since 2001
$3 billion in revenue in 2003 for MAb’s for Cancer Therapy
By 2008 Projected Sales are more than $12 billion.
Image courtesy of: Elder, Melissa. Monoclonal Antibodies for Cancer. Biopharm International. Volume 17, Number 11. pp 66. Advanstar Communications Publication. November 2004.
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Facilities
The facilities needed are:Laboratories (for Quality Control and research)WarehousesManufacturing plants
MAb and Vector ProductionMicelle Production
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Cost Estimation
FCI: $31.3 millionTCI: $45 millionManufacturing are assumed as $1,000/g MAb produced per yearProduct will sell for around $15,000/per treatment
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Plant Location
Top 50 cancer research hospitals in the US plotted on map
33 of the top 50 hospitals are east of the Mississippi River
3 of top 5 hospitals are in New England from Washington D.C to Boston
Best Hospitals 2004: Cancer. USNews.com. http://www.usnews.com/usnews/health/hosptl/rankings/specihqcanc.htm
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Cancer Research Locations
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Plant Location
Best place to build project plant is outside New York City9 top 50 hospitals are within 200 miles of New York CityMarket strategy will be to supply these 9 hospitals with treatment
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Incidence of Gliomas in the US
Population of the US determined by report issued by the Census Bureau
295 million as of January 1, 2005Population growth of 1%/year
2 to 3 cases per 100,000 people per yearCases in US per year: 5900-8850
Mean number of patients per year assumed to be 7350
Stevens, Glen. Brain Tumors: Meningiomas and Gliomas. April 22, 2003. http://www.clevelandclinicmeded.com/diseasemanagement/neurology/braintumor/braintumors.htm
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Inferiority Function
For a given market, two products of equal quality and prominence follow the equation:
p1d1 = p2d2
Two products of equal quality and unequal prominence follow the equation:
p1d1 = αp2d2
Knowledge multiplier α is function of marketing expenditure
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Minimum Proposed Marketing
Marketing strategy is geared toward the 9 cancer research centers located near suggested plant locationDuring Pre-FDA testing and Phase I and II Trials $150,000/year will be spent on marketing to oncologists at these hospitalsMarketing will increase to $400,000/year when Phase III Clinical Trials begin
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Potential Demand Model
An increase in post-production marketing expeditures causes a negligible difference in rate of increase of demandVaried pre-production marketing spending changes the initial percent of potential demand Effects of increased marketing modeled with inferiority function α(t)
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Inferiority Function Plot
0%
20%
40%
60%
80%
100%
120%
1 2 3 4 5 6 7
Time (Years)
Dem
and
Minimal Marketing
$500,000
$1,000,000
$1,825,000
$2,250,000
Maximum Demand
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Superiority Function
For a given market, two products of unequal quality and prominence will follow the equation:
βp1d1 = αp2d2
Treatment demand is a function of treatment effectiveness and side effects
Increased treatment effectiveness decreases βDecreased treatment side effects decreases β
25% demand increase assigned to each facet of product superiority in financial analysis
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Financial Analysis
Treatment cost to recover the investment in 3 years is based on the most likely successful pathway in FDA testing
Treatment cost determined as $9,000 per patient based on assumed sales of 7350
Treatment cost fixed at $15,000 to ensure an acceptable profit margin
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Expected NPV Risk Curves
0.00%
25.00%
50.00%
75.00%
100.00%
-$15,000,000 $15,000,000 $45,000,000 $75,000,000 $105,000,000Expected NPV
Prob
abili
ity
Minimal R&D Investment
Increased Pre-FDASpendingIncreased FDA Spending
Increased Pre-FDA & FDASpending
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Uncertainties in Financial Analysis
Number of Sales per Year
Facility Costs
Pathway Probabilities
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NPV Distributions of Successful Pathways
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Conclusions
Financial Analysis indicates that the profitability for this project is high
Potential losses are minimal when compared to the potential gains
CCKK recommends that this project be commercialized as soon as possible
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Questions?
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Synthesis of pNP-PEG-PE
Add PE to a 10x excess of PEG-(pNP)2 in chloroform in the presence of triethylamineRemove organic solventsSeparation from free PEG and pNP on using a CL-4B columnFreeze dry the pNP-PEG-PEExtract using chloroform (storage should be at -80ºC)
Torchilin, Vladimir, et al. TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. National Academy of Sciences. 2001
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Loading the Micelle with Toxin
Prepare lipid film by putting the mixed solution of PEG-PE/pNP-PEG-PE under vacuumAdd drug dissolved in methanol to chloroform solution of the pNP-PEG-PE/PEG-PE solutionRehydrate at 50ºC in a 5mM sodium citrate buffered saline at pH 5.0 and vortex for 5 minutes to reform micelles
Torchilin, Vladimir P., et al. “Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs.” Proceeding of the National Academy of Sciences. May 13, 2003.
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Attaching MAb to Micelle
Procedure:Add 1mg protein per 10mg of pNP-PEG-PEIncrease pH to 8.5Incubate 2 hours to attach MAb and hydrolyze pNPPurification - gel filtration chromatography
Yield: 60% Torchilin, Vladimir P., et al. “Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs.” Proceeding of the National Academy of Sciences. May 13, 2003.