Drug Discovery and Development: Prodrug strategy

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

FDA Definition of a Drug:

3 FDA Definition of a Drug Any chemical agent which effects any biological process “ An active ingredient that is intended to furnish pharmacological activity 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 human body, but does not include intermediates used in the synthesis of such ingredient.”

R&D Is Risky & Costly:

4 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 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 Approval Additional post-market testing Preclinical: laboratory & animal tests Compound Success Rates by Stage R&D Is Risky & Costly

Choosing a Disease:

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

Identifying a Drug Target:

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

Proposing new drug (Drug Discovery):

7 Proposing new drug (Drug Discovery) Drug source and selection approaches Irrational approach Rational approach Antisense Approach RNAi Approach Biologics Gene Therapy . . .

In silico methods:

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

Choosing the Bioassay:

9 Choosing the Bioassay Definitions: In vitro : In an artificial environment, as in a test tube or culture media In vivo : In the living body, referring to tests conducted in living animals Ex vivo : Usually refers to doing the test on a tissue taken from a living organism.

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10 What is Pharmacology ? Pharmacology Pharmacokinetics Pharmacodynamics What the body does to drug What the drug does to body Toxicology, carcinogenicity, mutagenicity Animal Tests, In Vitro Assays, and In Silico Methods Formulations and Delivery Systems

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11 Pharmacokinetics and Pharmacodynamics

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12 What the body does to the drug - A bsorption - D istribution M etabolism E xcretion Pharmacokinetics Bioavailablity <1 Bioavailablity =1

Distribution:

13 Distribution Passive diffusion Facilitated diffusion Active transport

Drug Metabolism:

14 Drug Metabolism Most metabolic products are less pharmacologically active Close relationship between the biotransformation of drugs and normal biochemical processes occurring in the body: Metabolism of drugs involves many pathways associated with the synthesis of endogenous substrates such as steroid hormones, cholesterol and bile acids Many of the enzymes involved in drug metabolism are principally designed for the metabolism of endogenous compounds These enzymes metabolize drugs only because the drugs resemble the natural compound

Phases of Drug Metabolism:

15 Phases of Drug Metabolism Phase I Reactions Convert parent compound into a more polar (=hydrophilic) metabolite by adding or unmasking functional groups (-OH, -SH, -NH 2 , -COOH, etc.) Often these metabolites are inactive May be sufficiently polar to be excreted readily Phase II Reactions Conjugation with endogenous substrate to further increase aqueous solubility Conjugation with glucuronide , sulfate, acetate, amino acid Liver is principal site of drug metabolism: Other sites include the gut, lungs, skin and kidneys For orally administered compounds, there is the “First Pass Effect” Intestinal metabolism Liver metabolism Enterohepatic recycling Gut microorganisms - glucuronidases

Drug Metabolism - Phase I:

16 Drug Metabolism - Phase I Phase I Reactions Oxidation Reduction Hydrolytic cleavage Alkylation (Methylation) Dealkylation Ring cyclization N-carboxylation Dimerization Transamidation Isomerization Decarboxylation

Drug Metabolism - Phase II :

17 Drug Metabolism - Phase II Conjugation reactions Glucuronidation by UDP-Glucuronosyltransferase: (on -OH, -COOH, -NH 2 , -SH groups) Sulfation by Sulfotransferase: (on -NH2, -SO 2 NH 2 , -OH groups) Acetylation by acetyltransferase: (on -NH 2 , -SO 2 NH 2 , -OH groups) Amino acid conjugation (on -COOH groups) Glutathione conjugation by Glutathione-S-transferase: (to epoxides or organic halides) Fatty acid conjugation (on -OH groups) Condensation reactions

Excretion :

18 Excretion

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19 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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21 IND Review Process (when Preclinical research finished)

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22 NDA Review Process

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23 To increase success To increase bioavailability To pass liver metabolism “First pass effect” To increase Half Life To increase the target to non-target uptake Prodrug can be a choice

What is the Prodrug?:

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

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25 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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27 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

characteristics of prodrugs:

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

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Classification of Prodrugs:

31 Classification of Prodrugs 1- Carrier linked prodrug 2- Bioprecursor

2-Bioprecursor prodrugs:

32 2-Bioprecursor prodrugs It does not contain a temporary linkage between the active drug & a carrier moiety but designed from a molecular modification of the active principle itself. It is a compound that is converted to active drug by a Metabolic biotransformation. Types of activation- Oxida tion (most common method) Reduction Phosphorylation (For antiviral agents)

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33 Oxidation Example – Nabumetone, which is a Non-steroidal anti-inflammatory prodrug used in arthritis

1- Carrier linked prodrug:

34 1- Carrier linked prodrug Contain a group that can be easily removed enzymatically to reveal the true drugs Ideally the group removed is pharmacologically inactive and nontoxic while the connecting bond must be labile for efficient activation in vivo Bipartite- Composed of one carrier group attached to the drugs Tripartite- Carrier group is attached via linker to drug Mutual Prodrugs - Two drugs linked together

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35 Mutual prodrug In such type of prodrug two pharmacologically active agents are coupled to form a single molecule which acts as carrier for others. Eg.benorylate is a mutual prodrug of Aspirin & Paracetamol.

Carrier linked prodrug(bi & tri partite):

36 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) :

39 Prodrugs for Site Specificity (targeted therapy) 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 minimized. It is desirable for highly toxic compound such as employed in a cancer. 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. One important parameter in prodrugs for site specificity is the Drug therapeutic index. A drug after its absorption into systemic circulation gets distributed into target as well as non-target site. The distribution to non-targeted tissue may leads to undesirable toxic effect and also insufficient concentration to the target site. If the target is too long and take more time for distribution the drug may get eliminated without reaching such a site. To minimize such a problems in a targeted drug delivery prodrug approach has been used.

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40 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

Targeting to brain:

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

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Targeting to Tumor:

43 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.

Criteria for Success With Enzyme-Prodrug Therapies:

44 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.

Antibody targeted drugs as cancer therapeutics:

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

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46 Antibody targeted drugs as cancer therapeutics- continued

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47 pH-labile Hydrazone linkers

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49 PSA labile spacer

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Antibody-Directed Enzyme Prodrug Therapy (ADEPT):

51 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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52 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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53 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. Carboxypeptidase G2 Nitrogen mustard as a Glutamic acid conjugate Activated Nitrogen mustard L -Glu + CO2 +

Gene-Directed Enzyme Prodrug Therapy (GDEPT) :

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

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55 Pro-prodrug (Double prodrugs) Here the prodrug is further derivatised a fashion such that two steps as enzymatically or chemically conversion lead to release active drug.

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56 Triple prodrug Double prodrug prodrug

Conclusion:

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

References:

58 References Adv. Drug Deliv. Rev. (2011), Advanced Drug Delivery Reviews 63 (2011) 3–23 drugs From Discovery to Approval, 2009 Evaluation of Drug Candidates for Preclinical Development, 2010 Nature reviews,drug discovery, volume 7 ,2008, 255 The AAPS Journal , vol 10, no1,2008,92 Current Pharmaceutical Design, 2009, 15, 2236-2250 Prog. Polym. Sci. 32 (2007) 933–961 Nature Reviews,drug discovery, volume 5 ,2006,147 Bioorg Med Chem. 2008 March 15; 16(6): 2764–2768. Asian J. Research Chem. 2(2): April.-June, 2009 Advanced Drug Delivery Reviews 26 (1997) 151–172 Pharmacol Rev 56:53–102, 2004

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Target Validation:

60 Target Validation

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