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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

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FDA Definition of a DrugAny chemical agent which effects any biological processAny chemical agent which effects any biological process“ “ An active ingredient that is intended to furnish pharmacological activity or An active ingredient that is intended to furnish pharmacological activity or

other direct effect in the diagnosis, cure, mitigation, treatment, or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or to affect the structure of any function of the prevention of a disease, or to affect the structure of any function of the human body, but does not include intermediates used in the synthesis of human body, but does not include intermediates used in the synthesis of such ingredient.”such ingredient.”

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5,000–10,000Screened

5,000–10,000Screened

250Enter Preclinical

Testing

250Enter Preclinical

Testing

5Enter

Clinical Testing

5Enter

Clinical Testing

1Approved by

the FDA

1Approved by

the FDA

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12

10

8

6

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2

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Net Cost: $802 million invested over 15 years Source: 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 carried Most research is carried

out on diseases which out 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, or Drug Target = specific macromolecule, or

biological system, which the drug will biological 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

HN

HO

CH3

O

O

HN

O

CH3

O CH3

O

CH3

O

CH3O

HN

HO

CH3

codeineheroin

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 a test In an artificial environment, as in a test

tube or culture mediatube or culture media• In vivoIn vivo:: In the living body, referring to tests In the living body, referring to tests

conducted in living animalsconducted in living animals• Ex vivoEx vivo:: Usually refers to doing the test on a Usually 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, mutagenicityAnimal 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 are Most metabolic products are less pharmacologically activeless pharmacologically active• Close relationshipClose relationship between the between the biotransformation of drugsbiotransformation of drugs

and and normal biochemical processesnormal biochemical processes occurring in the body: occurring in the body:• Metabolism of drugs involves many pathways associated with the Metabolism of drugs involves many pathways associated with the

synthesis of endogenous substrates such as steroid hormones, cholesterol synthesis of endogenous substrates such as steroid hormones, cholesterol

and bile acids and bile acids • Many of the enzymes involved in drug metabolism are principally Many 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 the These enzymes metabolize drugs only because the drugs resemble the drugs 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 by Convert parent compound into a more polar (=hydrophilic) metabolite by adding adding or unmasking functional groupsor 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 aqueous solubility to further increase aqueous solubility• 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 success To increase success • To increase bioavailabilityTo increase bioavailability• To pass liver metabolism “First pass To 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 uptakeProdrug can be a choice

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What is the Prodrug?• Prodrug - a pharmacologically inactive compound that is Prodrug - a pharmacologically inactive compound that is

converted to an active drug by a metabolic biotransformation converted 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 for conversion occurs as soon as the desired goal for designing the prodrug is achieved.designing the prodrug is achieved.

• Prodrugs currently constitute Prodrugs currently constitute 5%5% of known drugs and a larger percentage of of 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 SolubilityImprove patient acceptability (decrease pain on injection)•improve chemical stability•minimize toxicity and side effects

•Improved Permeability and BioavailabilityAlter or improve absorption.

•Prolonged Half-LifeAlter biodistributionAlter metabolismeliminationTissue-Targeted Delivery

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characteristics of prodrugs• It should not have intrinsic pharmacological It should not have intrinsic pharmacological

activity.activity.• The prodrug must be readily transported to The prodrug must be readily transported to

site of action.site of action.• The prodrug must be selectively cleaved to The 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 active drug It does not contain a temporary linkage between the active drug & a carrier moiety but designed from a molecular modification & a carrier moiety but designed from a molecular modification of the active principle itself.of the active principle itself.

It is a compound that is converted to active drug by a Metabolic It is a compound that is converted to active drug by a Metabolic biotransformation.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

CH3O

OH

CH3OO

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 to Contain 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 labile inactive 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 group group attached to the drugsattached to the drugsTripartite- Tripartite- Carrier group is attached via Carrier group is attached via linker to druglinker to drugMutual ProdrugsMutual Prodrugs- - Two drugs linked Two drugs linked togethertogether

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• Mutual prodrugMutual prodrug • In such type of prodrug two pharmacologically In such type of prodrug two pharmacologically

active agents are coupled to form a single active 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 Aspirin Eg.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 prodrugAntibody-drug conjugatePeptide–drug conjugate ...• Membrane transporter-associated prodrug • Polymeric prodrug PEG–drug conjugate HPMA–drug conjugate PLGA- –drug conjugate • Enzyme cleavable prodrugAntibody-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 are where optimal therapeutic benefit of a drug is obtained & unwanted effect are minimized.minimized.

• It is desirable for It 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 and The 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 Drug One important parameter in prodrugs for site specificity is the Drug

therapeutic index.therapeutic index. A drug after its absorption into systemic circulation gets distributed A drug after its absorption into systemic circulation gets distributed into into

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 effect The 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 get If 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 approach To 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 Barrier In a brain targeting, delivery of drug is limited by Blood Brain Barrier (BBB).(BBB).

The Blood Brain Barrier can allows The 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 molecules But the larger, more water soluble and ionic molecules do notdo not readily cross readily cross BBB.BBB.

The BBB allows only lipophilic molecules to enter brain, on this basis The 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 cancertherapeutics

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

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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.

IN

I

O NH

O

C O 2 H

C O 2 H

IN

O H

I

carboxypeptidaseG2Carboxypeptidase G2

Nitrogen mustard as a Glutamic acid conjugate Activated Nitrogen mustard

L-Glu + CO2+

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Gene-Directed Enzyme Prodrug Therapy (GDEPT)

• An inactive prodrug can be activated to release of An inactive prodrug can be activated to release of cytotoxic drug by an enzyme that has been delivered via cytotoxic 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 integrated A gene encoding prodrug-activating enzyme is integrated into a genome of targeted tumor cells or viral vector under into 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 the These cells, then express the enzyme that activates the prodrug.prodrug.

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• Pro-prodrug (Double prodrugs)Pro-prodrug (Double prodrugs)• Here the prodrug is further derivatised a Here the prodrug is further derivatised a

fashion such that two steps as enzymatically or fashion such that two steps as enzymatically or chemically conversion lead to release active chemically conversion lead to release active drug.drug.

CH C

NH2 O

NH2S

CH3

CH3

C

O

O CH2 O C C

CH3

CH3

CH3O

proprodrugenzymatic clevage

S

CH3

CH3

CO

OCH2OH

H

O

PRODRUG

N

S

CH3

CH3

O

H

Ampicillin ACTIVE

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Triple prodrug

Double prodrugprodrug

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Conclusion• Knowing “what happened for drugs in the Knowing “what happened for drugs in the

body and how drugs act” can help a chemist body and how drugs act” can help a chemist to design to more effective drugs to design to more effective drugs

• Prodrug strategy increases the success of Prodrug 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, Evaluation of Drug Candidates for Preclinical Development,

2010 2010 • NNature reviewsature reviews,,drug discoverydrug discovery,, volume 7 volume 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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? ?? ?

? ? ?? ? ?

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Target Validation

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