Prodrug strategy , drug development

1. 1
Sharif University of Technology
Department of Chemistry
Ph.D. Seminar I
By: Mehdi Akhlaghi
Supervisor: Prof. Ali Pourjavadi
Kimia hall
Tuesday, June 21, 2011 (3:00 p.m.)
31 khordad 1390
Drug Discovery and Development:
Prodrug Strategy to Increase
Success

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How are drugs discovered and developed?How are drugs discovered and developed?
Prodrug strategy will increase success of process ?Prodrug strategy will increase success of process ?
Drug Discovery and
Development
By: Mehdi Akhlaghi Supervisor: Dr. Pourjavadi

3. 3
FDA Definition of a Drug
Any chemical agent which effects any biological processAny chemical agent which effects any biological process
““ An active ingredient that is intended to furnish pharmacological activity orAn active ingredient that is intended to furnish pharmacological activity or
other direct effect in the diagnosis, cure, mitigation, treatment, orother direct effect in the diagnosis, cure, mitigation, treatment, or
prevention of a disease, or to affect the structure of any function of theprevention of a disease, or to affect the structure of any function of the
human body, but does not include intermediates used in the synthesis ofhuman body, but does not include intermediates used in the synthesis of
such ingredient.”such ingredient.”

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5,000–10,000
Screened
250
Enter Preclinical
Testing
5
Enter
Clinical
Testing
1
Approved by
the FDA
16
14
12
10
8
6
4
2
0
Net Cost: $802 million
invested over 15 yearsSource: DiMasi et al. 2003, Tufts
Years
Discovery:
(2-10 years)
Phase I: 20-80 healthy
volunteers to determine
safety & dosage
Phase III: 1000-5000
volunteers to monitor
adverse reactions to
long-term use
Phase II: 100-
300 volunteers
to look for
efficacy & side
effects
FDA Review
ApprovalAdditional
post-market
testing
Preclinical:
laboratory &
animal tests
Compound Success
Rates by Stage
R&D Is Risky & Costly

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Choosing a Disease
• Most research is carriedMost research is carried
out on diseases whichout on diseases which
afflict “first world”afflict “first world”
countries: (e.g. cancer,countries: (e.g. cancer,
cardiovascular diseases,cardiovascular diseases,
depression, diabetes, flu,depression, diabetes, flu,
migraine, obesity).migraine, obesity).

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Identifying a Drug Target
• Drug Target = specific macromolecule, orDrug Target = specific macromolecule, or
biological system, which the drug willbiological system, which the drug will
interact with.interact with.

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Proposing new drug (Drug Discovery)
Drug source and selection approachesDrug source and selection approaches
• Irrational approachIrrational approach
• Rational approachRational approach
• Antisense ApproachAntisense Approach
• RNAi ApproachRNAi Approach
• BiologicsBiologics
• Gene TherapyGene Therapy
• ..
• ..
• .. O
HO
H
N
HO
CH3
O
O
H
N
O
CH3
O CH3
O
CH3
O
CH3O
H
N
HO
CH3
codeine
heroin
morphine

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In silico methods
http://www.click2drug.org/

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Choosing the Bioassay
• Definitions:Definitions:
• In vitroIn vitro:: In an artificial environment, as in aIn an artificial environment, as in a
test tube or culture mediatest tube or culture media
• In vivoIn vivo:: In the living body, referring to testsIn the living body, referring to tests
conducted in living animalsconducted in living animals
• Ex vivoEx vivo:: Usually refers to doing the test on aUsually refers to doing the test on a
tissue taken from a living organism.tissue taken from a living organism.

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What is Pharmacology ?
PharmacologyPharmacology
Pharmacokinetics Pharmacodynamics
What the body does to drug What the drug does to body
ToxicologyToxicology,, carcinogenicity, mutagenicitycarcinogenicity, mutagenicity
Animal Tests, In Vitro Assays, and In Silico MethodsAnimal Tests, In Vitro Assays, and In Silico Methods
Formulations and Delivery SystemsFormulations and Delivery Systems

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Pharmacokinetics and Pharmacodynamics

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What the body does to the drug
- Absorption
- Distribution
-Metabolism
- Excretion
Pharmacokinetics
Bioavailablity <1Bioavailablity =1

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Distribution
•Passive diffusion
•Facilitated diffusion
•Active transport

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Drug Metabolism
• Most metabolic products areMost metabolic products are less pharmacologically activeless pharmacologically active
• Close relationshipClose relationship between thebetween the biotransformation of drugsbiotransformation of drugs
andand normal biochemical processesnormal biochemical processes occurring in the body:occurring in the body:
• Metabolism of drugs involves many pathways associated with theMetabolism of drugs involves many pathways associated with the
synthesis of endogenous substrates such as steroid hormones, cholesterolsynthesis of endogenous substrates such as steroid hormones, cholesterol
and bile acidsand bile acids
• Many of the enzymes involved in drug metabolism are principallyMany of the enzymes involved in drug metabolism are principally
designed for the metabolism of endogenous compoundsdesigned for the metabolism of endogenous compounds
• These enzymes metabolize drugs only because theThese enzymes metabolize drugs only because the drugs resemble thedrugs resemble the
natural compoundnatural compound

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Phases of Drug Metabolism• Phase I ReactionsPhase I Reactions
• Convert parent compound into a more polar (=hydrophilic) metabolite byConvert parent compound into a more polar (=hydrophilic) metabolite by
adding or unmasking functional groupsadding or unmasking functional groups (-OH, -SH, -NH(-OH, -SH, -NH22, -COOH, etc.), -COOH, etc.)
• Often these metabolites are inactiveOften these metabolites are inactive
• May be sufficiently polar to be excreted readilyMay be sufficiently polar to be excreted readily
• Phase II ReactionsPhase II Reactions
• Conjugation with endogenous substrateConjugation with endogenous substrate to further increase aqueousto further increase aqueous
solubilitysolubility
• Conjugation with glucuronideConjugation with glucuronide, sulfate, acetate, amino acid, sulfate, acetate, amino acid
Liver is principal site of drug metabolism:Liver is principal site of drug metabolism:
• Other sites include the gut, lungs, skin and kidneysOther sites include the gut, lungs, skin and kidneys
• For orally administered compounds, there is theFor orally administered compounds, there is the
““First Pass Effect”First Pass Effect”
• Intestinal metabolismIntestinal metabolism
• Liver metabolismLiver metabolism
• Enterohepatic recyclingEnterohepatic recycling
• Gut microorganisms - glucuronidasesGut microorganisms - glucuronidases

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Drug Metabolism - Phase I
• Phase I ReactionsPhase I Reactions
• OxidationOxidation
• ReductionReduction
• Hydrolytic cleavageHydrolytic cleavage
• Alkylation (Methylation)Alkylation (Methylation)
• DealkylationDealkylation
• Ring cyclizationRing cyclization
• N-carboxylationN-carboxylation
• DimerizationDimerization
• TransamidationTransamidation
• IsomerizationIsomerization
• DecarboxylationDecarboxylation

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Drug Metabolism - Phase II
• Conjugation reactionsConjugation reactions
• GlucuronidationGlucuronidation by UDP-Glucuronosyltransferase:by UDP-Glucuronosyltransferase:
(on -OH, -COOH, -NH(on -OH, -COOH, -NH22, -SH groups), -SH groups)
• SulfationSulfation by Sulfotransferase:by Sulfotransferase:
(on -NH2, -SO(on -NH2, -SO22NHNH22, -OH groups), -OH groups)
• AcetylationAcetylation by acetyltransferase:by acetyltransferase:
(on -NH(on -NH22, -SO, -SO22NHNH22, -OH groups), -OH groups)
• Amino acid conjugationAmino acid conjugation
(on -COOH groups)(on -COOH groups)
• Glutathione conjugationGlutathione conjugation by Glutathione-S-transferase:by Glutathione-S-transferase:
(to epoxides or organic halides)(to epoxides or organic halides)
• Fatty acid conjugationFatty acid conjugation
(on -OH groups)(on -OH groups)
• Condensation reactionsCondensation reactions

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Excretion

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What the drug does to the body
- Drug receptors
- Effects of drug
- Responses to drugs
- Toxicity and adverse effects of drugs
Pharmacodynamics
Target validation
• Microarray for disease target identifi cation
• Radioligands
• High throughput screening
• Combinatorial chemistry
• Structure – activity relationships: X - ray crystallography, nuclear magnetic resonance,
computational chemistry
• Genomics and proteomics
• Metabolomics
• Systems biology
• Nanotechnology
• Bioinformatics: data mining
• Recombinant DNA technologies

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IND Review Process (when Preclinical research finished)

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NDA Review Process

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• To increase successTo increase success
• To increase bioavailabilityTo increase bioavailability
• To pass liver metabolism “First passTo pass liver metabolism “First pass
effect”effect”
• To increase Half LifeTo increase Half Life
• To increase the target to non-target uptakeTo increase the target to non-target uptake
Prodrug can be a
choice

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What is the Prodrug?
• Prodrug - a pharmacologically inactive compound that isProdrug - a pharmacologically inactive compound that is
converted to an active drug by a metabolic biotransformationconverted to an active drug by a metabolic biotransformation
(enzymatic or chemical hydrolysis)(enzymatic or chemical hydrolysis)
• Ideally,Ideally, conversion occurs as soon as the desired goal forconversion occurs as soon as the desired goal for
designing the prodrug is achieved.designing the prodrug is achieved.
• Prodrugs currently constituteProdrugs currently constitute 5%5% of known drugs and a larger percentage ofof known drugs and a larger percentage of
new drugsnew drugs

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The barriers!!!
•Low oral absorption properties
• Lack of site specificity
• Chemical instability
• Toxicity
• Bad taste
• Bad odour
• Pain at application site

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Why to use Prodrugs?
•Increased Solubility
Improve patient acceptability (decrease pain on injection)
•improve chemical stability
•minimize toxicity and side effects
•Improved Permeability and Bioavailability
Alter or improve absorption.
•Prolonged Half-Life
Alter biodistribution
Alter metabolism
elimination
Tissue-Targeted Delivery

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characteristics of prodrugs
• It should not have intrinsic pharmacologicalIt should not have intrinsic pharmacological
activity.activity.
• The prodrug must be readily transported toThe prodrug must be readily transported to
site of action.site of action.
• The prodrug must be selectively cleaved toThe prodrug must be selectively cleaved to
active drug utilizing specific enzymes.active drug utilizing specific enzymes.

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Classification of Prodrugs
1- Carrier linked prodrug
2- Bioprecursor

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2-Bioprecursor prodrugs
 It does not contain a temporary linkage between the activeIt does not contain a temporary linkage between the active
drug & a carrier moiety but designed from a moleculardrug & a carrier moiety but designed from a molecular
modification of the active principle itself.modification of the active principle itself.
 It is a compound that is converted to active drug by aIt is a compound that is converted to active drug by a
Metabolic biotransformation.Metabolic biotransformation.
 Types of activation-Types of activation-
 OxidaOxidation (most common method)tion (most common method)
 ReductionReduction
 Phosphorylation (For antiviral agents)Phosphorylation (For antiviral agents)

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Oxidation Example –
Nabumetone, which is a Non-steroidal anti-inflammatory
prodrug used in arthritis
CH3
O
CH3
O
OH
CH3
O
O
series of oxidative
decaboxylation
Active form of the drug
that inhibits Prostaglandin
biosynthesis by cyclo-oxygenase
Nabumetone

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1- Carrier linked prodrug
Contain a group that can be easily removed enzymatically toContain a group that can be easily removed enzymatically to
reveal the true drugsreveal the true drugs
Ideally the group removed is pharmacologicallyIdeally the group removed is pharmacologically
inactive and nontoxic while the connecting bond must be labileinactive and nontoxic while the connecting bond must be labile
for efficient activation in vivofor efficient activation in vivo
Bipartite-Bipartite- Composed of one carrierComposed of one carrier
groupgroup attached to the drugsattached to the drugs
Tripartite-Tripartite- Carrier group is attached viaCarrier group is attached via
linker to druglinker to drug
Mutual ProdrugsMutual Prodrugs-- Two drugs linkedTwo drugs linked
togethertogether

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• Mutual prodrugMutual prodrug
• In such type of prodrug two pharmacologicallyIn such type of prodrug two pharmacologically
active agents are coupled to form a singleactive agents are coupled to form a single
molecule which acts as carrier for others.molecule which acts as carrier for others.
• Eg.benorylate is a mutual prodrug of AspirinEg.benorylate is a mutual prodrug of Aspirin
& Paracetamol.& Paracetamol.
C
O
O
NH C
O
CH3OCOCH3
COOH
OCOCH3
OH
NHCOCH3
+
prodrug
ParacetamolAsprin
Benorylate

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Carrier linked prodrug(bi & tri partite)
• Targeting-ligand conjugated prodrug
Antibody-drug conjugate
Peptide–drug conjugate
...
• Membrane transporter-associated prodrug
• Polymeric prodrug
PEG–drug conjugate
HPMA–drug conjugate
PLGA- –drug conjugate
• Enzyme cleavable prodrug
Antibody-directed enzyme prodrug therapy (ADEPT)
Gene-directed enzyme prodrug therapy (GDEPT)
Directly or
by spacer conjugated
×

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Prodrugs for Site Specificity (targeted therapy)
• Site specific delivery is a ultimate goal in all drug delivery research program,Site specific delivery is a ultimate goal in all drug delivery research program,
where optimal therapeutic benefit of a drug is obtained & unwanted effect arewhere optimal therapeutic benefit of a drug is obtained & unwanted effect are
minimized.minimized.
• It is desirable forIt is desirable for highly toxic compoundhighly toxic compound such as employed in a cancer.such as employed in a cancer.
• The main aim of Prodrug for Site Specificity is to achieve very precise andThe main aim of Prodrug for Site Specificity is to achieve very precise and
direct effect at the target with minimal effect on rest of the body.direct effect at the target with minimal effect on rest of the body.
• One important parameter in prodrugs for site specificity is the DrugOne important parameter in prodrugs for site specificity is the Drug
therapeutic index.therapeutic index.
 A drug after its absorption into systemic circulation gets distributedA drug after its absorption into systemic circulation gets distributed intointo
target as well as non-target site.target as well as non-target site.
 The distribution to non-targeted tissue may leads to undesirable toxic effectThe distribution to non-targeted tissue may leads to undesirable toxic effect
and also insufficient concentration to the target site.and also insufficient concentration to the target site.
 If the target is too long and take more time for distribution the drug may getIf the target is too long and take more time for distribution the drug may get
eliminated without reaching such a site.eliminated without reaching such a site.
 To minimize such a problems in a targeted drug delivery prodrug approachTo minimize such a problems in a targeted drug delivery prodrug approach
has been used.has been used.

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 Novel prodrugs with modified properties has been designed
which preferentially achieve higher concentration of
biotransformed drug at the desired targeting sites such as-
 Brain targeting
 Kidney targeting
 Liver targeting
 Virus targeting
 Tumor targeting
 Lymphatic targeting
 Colon targeting

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Targeting to brain
 In a brain targeting, delivery of drug is limited by Blood Brain BarrierIn a brain targeting, delivery of drug is limited by Blood Brain Barrier
(BBB).(BBB).
 The Blood Brain Barrier can allowsThe Blood Brain Barrier can allows only small and lipid solubleonly small and lipid soluble molecules,molecules,
which can diffuse the BBB from systemic circulation.which can diffuse the BBB from systemic circulation.
 But the larger, more water soluble and ionic moleculesBut the larger, more water soluble and ionic molecules do notdo not readily crossreadily cross
BBB.BBB.
 The BBB allows only lipophilic molecules to enter brain, on this basisThe BBB allows only lipophilic molecules to enter brain, on this basis
Bodor and Co-workers (1981) developedBodor and Co-workers (1981) developed Dihydropyridine-pyridiniumDihydropyridine-pyridinium
type redox system for Brain specific sustain drug delivery.type redox system for Brain specific sustain drug delivery.

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Targeting to Tumor
 Tumor cells contains a higher concentration of enzyme
phosphates, amides than do normal cells.
 Because of higher growth rates associated with tumor cells.
 For tumor drug delivery firstly studied prodrug activating
enzyme (extracellular or intracellular)
 For selective activation of prodrugs in tumor cells
Two steps:
 Selecting a specific linker based on tumor cell specific enzyme.
 Incorporate a prodrug-activating enzyme into a target tumor
cell.

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Criteria for Success With Enzyme-Prodrug Therapies
 The prodrug-activating enzyme is either nonhuman or a
human protein.
 It should be a good substrate for the incorporated enzyme but
not be activated by endogenous enzyme outside tumor cell.
 Prodrug must be able to cross tumor cell membranes.
 Prodrug have low cytotoxicity and drug have high
cytotoxicity.
 The half-life of the active drug is long enough for bystander
killing effect but short enough to avoid leaking out of tumor
cells.

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Antibody targeted drugs as cancer
therapeutics
NATURE REVIEWS | DRUG DISCOVERY VOLUME 5 | FEBRUARY 2006 | 147

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Antibody targeted drugs as cancer
therapeutics-continued

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pH-labile Hydrazone linkers

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PSA labile spacer

50. 50

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Antibody-Directed Enzyme Prodrug
Therapy (ADEPT)
Administration of
Antibody-enzyme conjugate
Administration of
prodrug
Prodrug
Enzyme
Drug
Tumor cell
 An approach for site-specific delivery of cancer drugs

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 An antibody-enzyme conjugate is administered which
binds to the surface of the tumor cells.
 The antibody used has been targeted for the particular
tumor cell.
 After the antibody-enzyme has localized within the tumor
cell and the excess conjugate is cleared from the blood and
normal tissues get enough time to clear.
 After the prodrug is administered, The Ab-E conjugated at
the tumor cell surface catalyzes the conversion of the
prodrug to the drug when it reaches the tumor cell.

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Example:
 Delivery of Nitrogen mustard as a Glutamic acid conjugate, after
administration of humanized monoclonal antibody conjugated to
the bacterial enzyme carboxypeptidase G2.
 Prodrug-activating enzyme in ADEPT is a bacterial enzyme.
I
N
I
O N
H
O
CO2
H
CO2
H
I
N
OH
I
Carboxypeptidase G2
Nitrogen mustard as a Glutamic acid conjugate Activated Nitrogen mustard
L-Glu + CO2+

54. 54
Gene-Directed Enzyme Prodrug Therapy
(GDEPT)
• An inactive prodrug can be activated to release ofAn inactive prodrug can be activated to release of
cytotoxic drug by an enzyme that has been delivered viacytotoxic drug by an enzyme that has been delivered via
gene to the tumor cell.gene to the tumor cell.
A gene encoding prodrug-activating enzyme is integratedA gene encoding prodrug-activating enzyme is integrated
into a genome of targeted tumor cells or viral vector underinto a genome of targeted tumor cells or viral vector under
the control of tumor-selective promoters.the control of tumor-selective promoters.
These cells, then express the enzyme that activates theThese cells, then express the enzyme that activates the
prodrug.prodrug.

55. 55
• Pro-prodrug (Double prodrugs)Pro-prodrug (Double prodrugs)
• Here the prodrug is further derivatised aHere the prodrug is further derivatised a
fashion such that two steps as enzymatically orfashion such that two steps as enzymatically or
chemically conversion lead to release activechemically conversion lead to release active
drug.drug.
CH C
NH2 O
NH2
S
CH3
CH3
C
O
O CH2 O C C
CH3
CH3
CH3
O
proprodrug
enzymatic clevage
S
CH3
CH3
C
O
OCH2OH
H
O
PRODRUG
N
S
CH3
CH3
O
H
Ampicillin ACTIVE

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Triple prodrug
Double prodrug
prodrug

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Conclusion
• Knowing “what happened for drugs in theKnowing “what happened for drugs in the
body and how drugs act” can help a chemistbody and how drugs act” can help a chemist
to design to more effective drugsto design to more effective drugs
• Prodrug strategy increases the success ofProdrug strategy increases the success of
Drug Discovery and Development process.Drug Discovery and Development process.

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References
• Adv. Drug Deliv. Rev. (2011),Adv. Drug Deliv. Rev. (2011),
• Advanced Drug Delivery Reviews 63 (2011) 3–23Advanced Drug Delivery Reviews 63 (2011) 3–23
• drugs From Discovery to Approval, 2009drugs From Discovery to Approval, 2009
• Evaluation of Drug Candidates for Preclinical Development, 2010Evaluation of Drug Candidates for Preclinical Development, 2010
• NNature reviewsature reviews,,drug discoverydrug discovery,, volume 7volume 7 ,,20082008,, 255255
• The AAPS JournalThe AAPS Journal, vol 10, no1,2008,92, vol 10, no1,2008,92
• Current Pharmaceutical Design, 2009, 15, 2236-2250Current Pharmaceutical Design, 2009, 15, 2236-2250
• Prog. Polym. Sci. 32 (2007) 933–961Prog. Polym. Sci. 32 (2007) 933–961
• Nature Reviews,drug discovery, volume 5 ,2006,147Nature Reviews,drug discovery, volume 5 ,2006,147
• Bioorg Med Chem. 2008 March 15; 16(6): 2764–2768.Bioorg Med Chem. 2008 March 15; 16(6): 2764–2768.
• Asian J. Research Chem. 2(2): April.-June, 2009Asian J. Research Chem. 2(2): April.-June, 2009
• Advanced Drug Delivery Reviews 26 (1997) 151–172Advanced Drug Delivery Reviews 26 (1997) 151–172
• Pharmacol Rev 56:53–102, 2004Pharmacol Rev 56:53–102, 2004

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Target Validation

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