Drug Discovery & Development

DR. KARUN KUMAR

JUNIOR RESIDENT – II

DEPT. OF PHARMACOLOGY

INTRODUCTIONCreation of a new drug involves :-

1. Drug discovery (Research) :- Identification of a

potential therapeutic target Selection of a single

molecule for testing in humans.

2. Drug development (Development) :- Preclinical

studies that support initial clinical trials through

approval of the drug by regulatory authorities.

3. Commercialization (Marketing) :- Product

Therapeutic application Sales

Drug discovery process

Process by which pharmaceutical, biotechnology,

academic & Govt. laboratories identify or screen

compounds to find potentially active therap. agents

Multidisciplinary nature of drug discovery

Drug discovery process

Stages of drug discovery

Target identification

• TargetMolecular recognition site to which drug binds

• Target may be• Protein molecule

• A receptor

• Enzyme

• Transport molecule

• Ion channel

• Tubulin

• Immunophilin

Therapeutic drug targets

Strategies to find new drug targets

1. Conventional strategies

a) Analysis of pathophysiology

b) Analysis of MOA of existing drugs

2. New strategies

a) Disease genes

b) Disease-modifying genes

Disease genes

• Abl-kinase Molecular target for Imatinib

• Secretase inhibitors Anti-Alzheimer drugs

• Most promising field Cancer therapies

• Identifying d/s gene Valuable pointers to drug

targets

Disease modifying genes

• Most important category

• Approaches used :-

1. Gene expression profiling

2. Gene knockout studies

Gene expression profiling

Principle Development of any disease phenotype

involves changes in gene expression in cells & tissues

involved

Method DNA microarray (‘gene chips’)

DNA microarray

Cluster analysis of gene expression experiment (Dendrogram)

Gene knockout screening

• Generation of transgenic ‘gene knockout’ strains of mice

• Examples

1. Inactiv. Of gene for ACE/ATR ↓ B.P.

2. Elim. Of gene encoding GABAA Irrit. In mice

3. Cathepsin K Osteoporosis

4. Melanocortin receptors Obesity

• Flatworm & zebrafish Speed up the process

Target validation• Experimental approach by which a “potential” drug

target can be tested & given further credibility

• Main approach• Pharmacological mGluR for pain

• Genetic • Antisense oligonuceotides

• RNA interference

• Transgenic animals

Phase D0 ‘Target identification’ (Research with intent of identifying a therapeutic target)

Phase D1 + D2 ‘Lead identification’ (Initial screening to discover chemical template for lead optimization).Tests include HTS,LTS(in vitro & in vivo)

Phase D3 ‘Lead optimization’ (Optimiz. & biol. Testing of compound).Outcome Best in potency & selectivity, as well as ADME properties

Phase D4 Completion of studies to allow 1st

application in man

Antisense Oligonucleotides

Stretches of RNA complementary to gene of interest

↓

Bind to cellular mRNA (Prevent its translation)

↓

Inhibits expression of specific genes

↓

Role of ‘specific genes’ in disease phenotype can be determined

RNA interference

Short lengths of dsRNA (siRNAs)

↓

Activate RISC (RNA induced silencing complex)

↓

Destroys corresponding fmRNA in the cell

↓

Gene silenced

Transgenic animals

Important definitions• Screening Testing many compounds in assays

relevant to the disease in question

• Hit A compound that passes such a ‘screen’

• Primary hit Compound giving positive result in a

screening assay

• Confirmed hit Compound is confirmed as

positive when assay is repeated

• Validated hit Confirmed hit that shows selective

activity

• Lead compound A new chemical entity that could be

developed in a new drug by optimizing beneficial

effects & minimizing S/E

Should comply with Lipinski’s rule of 5 :-

• Mol. Wt. < 500 Da cLogP < 5

• No. of H bond donors <5 Sum of no. of Ns & Os <10

• Drug candidate End result of lead optimization (A

compound judged suitable for precl. & cl. Develop.)

• Development compound Drug candidate that has

been accepted for further development

Screening1. Selectivity screening In vitro tests (If compound

is selective to merit further investigation)

2. Pharmacological profiling

• In vitro profiling Study on isolated tissues has been

the mainstay

• In vivo profiling Imaging technologies used

• MRI

• USG

• PET

• X-ray densitometry tomography

Animal models of disease

1. Acute physiological & pharmacological models

• Seizures induced by electrical stimulation of the

brain Epilepsy

• Histamine induced bronchoconstriction Asthma

• Eddy’s hot plate test Pain

• Injection of LPS & cytokines Septic shock

• Elevated plus maze test Anxiolysis

2. Chronic physiological or pharmacological models

• Use of Alloxan / Streptozotocin Type I DM

• Inducing brain ischemia Stroke

• Inducing coronary ischemia IHD

• “Kindling” Epilepsy

• Self-admin. of opiates/nicotine Drug dependence

• Cholesterol fed rabbits Hypercholesterolemia &

atherosclerosis

• Immunization with myelin basic protein MS

• Admin. Of MPTP Parkinson’s disease

• Transplant. Of malignant cells into immunodef.

Animals Progressive tumors (Cancer)

3. Genetic models

1. SHR

2. Seizure-prone dogs

3. Rats insensitive to ADH

4. Obese mice

1. CF

2. DMD

3. Leptin gene (mutated in ob/ob mice)

5. Alzheimer’s disease (APP)

Validity criteria (Willner 1984)

1. Face validity Accuracy with which model

reproduces the phenomena char. human d/s

2. Construct validity Extent to which etiology of

human disease is reflected in model

3. Predictive validity Extent to which

manipulation (drug t/t) is predictive of effects in

the human disorder

Identification of hits

1. Compound centered approach Traditional

Compound is identified

↓

Biological profile is explored

↓

If compd. displays desirable pharmacologic activity

↓

Compound is refined & developed further

2. Target centered approach

Putative drug target (receptor/enzyme) is identified

↓

Researchers search for compounds which interact with target (agonist/antagonist/modulator)

Search maybe:

1. Systematic Uses info. About structure of target as a starting point

2. Shotgun approach All compounds in a large library of substances are tested in a high-speed automated assay

High-throughput screening

• Simplest target centered approach

• Uses a target based assay & robotic automation to

test thousands of compounds in a few days time

• 2 critical aspects :-

1. A large library of compounds must be available

for screening

2. Assay (Simple/sophisticated) that leads to rapid

identification of true hits must be developed

Lead identification

Library is “run through” the assay (96/384-well plate)

↓

‘Primary hits’ are examined more closely

↓

Further screening is done (To eliminate false positives & false negatives)

↓

Leads are advanced in ‘lead optimization’ process

Lead optimization

Physical, chemical, biological & pharmacological

properties of promising lead molecules (that

appear to interact with the target in a desirable

way) are

Characterized & refined with the ultimate goal

Of selecting a single molecule to enter into

Clinical testing & formal drug development

Factors causing termination 1. Failure to demonstrate efficacy in a rigorous animal

model of human disease

2. Low bioavailability

3. Extensive/complex metabolism Potentially

dangerous reactive metabolites

4. Toxic effects in preliminary animal toxicology studies

5. In vitro evidence that molecule may damage DNA

6. Extremely difficult chemical synthesis

Drug development

• All activities involved in transforming a compound from

drug candidate Product approved for marketing by

appropriate regulatory authorities

• Falls in 3 main parts :-

1. Technical development Ensuring quality of end-

product

2. Investigative studies Safety & efficacy

3. Managerial functions Co-ordination,

documentation & liaison with regulatory authorities

Stages of drug developmentStage No. of yrs.

Synthesis/isolation of compound 1-2

Preclinical studies (Scr.,eval.,pk. & short term toxicity test. in animals)

2-4

Scrutiny & grant of permission for clinical trials 0.25-0.5

Pharmaceutical formulation, standardization ofchemical/biological/immuno-assay of the compound

0.5-1

Clinical studies: Phase I,II,III trials; Long term animal toxicity testing

3-10

Review & grant of marketing permission 0.5-2

Post-marketing surveillance No fixed duration

Phases of Precl. & Clinical Development

Phase Primary goal Dose Patient monitorNumber of

participants

Preclinical

Testing of drug in non-human subjects

UnrestrictedA graduate level researcher-Ph.D.

In vitro & in vivo animal models

Phase 0Oral bioavailability & half-life of drug

SubtherapeuticClinical researcher

10 people

Phase ITesting of drug on healthy volunteers (safety)

Subtherapeuticwith asc. Doses

Clinical researcher

20-100 people

Phase IITesting of drug on patients (efficacy & tolerability)

Therapeutic doseClinical researcher

100-300 people

Phase IIITesting of drug on pts.(confirm efficacy)

Therapeutic doseClinical research.& Physician

1000-2000 people

Phase IVPost-marketing surveillance (A/E, D-D I)

Therapeutic dose PhysicianAnyone seeking treatment

Phase V Translational research No dosing None All reported use

DeMets D., Friedman L. and Furberg C.(2010).Fundamentals of Clinical Trials. Springer 4th EditionGoodman and Gilman's The Pharmacological Basis of Therapeutics, (2011) 12th Edition

Preclinical development

Aim Meet all requirements before a new compound is

deemed ready to be tested for the 1st time in humans

Work falls into :-

1. Safety pharmacology Testing to check that drug

does not produce hazardous acute effects

2. Preliminary toxicological testing Eliminate

genotoxicity & determine maximum non-toxic dose of

drug (given daily for 28 days & tested in 2 species)

1. Pharmacokinetic testing (ADME studies) in laboratory animals

2. Chemical & pharmaceutical dev

3. Pharmacokinetic testing (ADME studies) in

laboratory animals

4. Chemical & pharmaceutical development

Assess feasibility of large scale synthesis &

purification

5. Assess stability of compound under various

conditions

6. Develop a formulation suitable for clinical studies

• Work done in accordance with GLP

IND

• New drug ready to be studied in humans a

Notice of Claimed Investigational Exemption for a

New Drug (IND) must be filed with the FDA

• It includes :-

1. Info. on the composition and source of the drug

2. Chemical and manufacturing information

• Includes :-

1. Info. on the composition and source of the drug

2. Chemical and manufacturing information

3. All data from animal studies

4. Proposed plans for clinical trials

5. Names and credentials of physicians who will

conduct the clinical trials

6. A compilation of the key data relevant to study of

the drug in humans that has been made available

to investigators and their institutional review

boards

Phase 0 (Microdosing study)

• Developed by FDA & EMA as “cost-cutting” tools

Very low doses (1/100th of estimated human dose or

max. of 100 µg total dose of candidate drug) are

administered to healthy volunteers

↓

Pharmacok worked out using AMS with radiolabelled

drug/LC-MS to measure ultra low drug levels

• Subpharm. dose No toxic / therapeutic eff. But

yields human pharmacokinetic information

• Elaborate animal studies & costly phase I human

trials could be avoided for candidate drugs

• Useful in more precise selection of doses for phase

I study

• They are promising & most regulatory authorities

are willing to allow & consider them

Phase I• Studies carried out Phase I clinics (All vital func. Are

measured & emergency/resuscitative facilities avail.)

• Clinical investigators Clinical pharmacologists

• Trial subjects Healthy volunteers (25-100)

• Objectives :-

1. Check for safety (Drug affects CV, hepatic or renal

functions adversely)

2. Check for tolerability (Drug produces unpleasant

symptoms like headache, nausea & vomiting)

1. 3.

2. 3.

3. Determine whether humans & animals show

significant pharmacokinetic differences

4. Determine a safe clinical dosage range in humans

(Common rule Begin with 1/5th or 1/10th of

MTD in animals & calc. it for 70 kg body wt.)

5. Determine the pharmacokinetics of the drug in

humans (Whether deficiency in drug effect is due

to lack of absorption / faster elimination)

6. Detect any predictable toxicity

Pharmacokinetic parameters

1. Cmax Peak drug &/or metabolite concentration

2. Tmax Time to peak drug &/or metabolite conc.

3. AUC0-∞ Area under conc.-time curve e.p. to inf.

4. AUC0-T AUC calc. to a specific time point T

5. T1/2 Time taken for level of drug to dec. by 1/2

6. VD Volume of distr.

7. CL Clearance

8. MRTMean residence time (Avg. time a drug molecule rem. In body after rapid i.m. injection)

Dosing & blood sampling schedule for phase I studies

Phase II

• Drug studied for 1st time in pts. with target disease

• Main purpose Gather evidence that drug has

effects suggested by preclinical trials

• End points :-

1. Definitive end point (Measures drug effect

directly Pain relief [analgesic])

2. Surrogate end point (Predictive of the definitive

end point Reduction in tumor size[anticancer])

Phase IIa

• “Proof of concept”/ “Proof of claims”

• Preliminary evidence of efficacy & safety

• Up to 200 pts. are studied Potential therapeutic

benefits & side effects are observed

• Establishment of dose range for more definitive

therapeutic trials in phase IIb

• Study design Single blind (Subject unaware if he

is taking placebo/positive control/new drug)

Phase IIb

• Dose-finding studies

• To confirm efficacy with statistical significance

• Determine the optimal dose & dosing regimen

• Large no. of patients used (200-400)

• Study design Double blind (3rd party holds the

code identifying each medication & this code is not

deciphered until all clinical data has been coll.)

Phase III• Large scale, multicentered randomized double blind

trials to further establish safety & efficacy

• Made using “Double-blind cross over” designs to

minimize errors

• NDA After completion of phase III trials,

sponsors file a “New Drug Application” with the

drug control authorities of that country

• NDS If documentation is acceptable & in

compliance, drug enters market with NDS

Double blind Cross Over DesignPt. grp. (randomized)

Week 1 Week 2 Week 3

I Standard drug

Placebo New drug

II Placebo New drug Standard drug

III New drug Standard drug

Placebo

Phase IV

• Surveillance phase during the post marketing

clinical use of the drug

• Used to discover

• Relatively rare side effects

• Previously unknown drug interaction

• Previously unknown therapeutic use detected by a

chance discovery

Phase V

• Aims to bridge the gap between basic and clinical research1

• Encompasses laboratory studies, clinical demands, public health and health management, policies and economics1

• Crucial in the evolution of contemporary biomedical science1

• It is used to signify the integration of a new clinical treatment into widespread public health practice2

1-Translational research: from benchside to bedside.N C Keramaris, N K Kanakaris, C Tzioupis, G Kontakis, P V GiannoudisAcademic Department of Trauma and Orthopaedics, Leeds Teaching Hospitals, University of Leeds, Great George Street, Leeds LS1 3EX, UK.Injury (Impact Factor: 1.93). 07/2008; 39(6):643-50. DOI: 10.1016/j.injury.2008.01.051 Source: PubMed

2-Margaret A. Rogers (June 2009). "What are the phases of intervention research?". American Speech-Language-Hearing Association. Retrieved Jan 8, 2013.

Preclinical safety assessment

1. Exploratory toxicology

• Rough estimate of toxicity (2 weeks)

• Provides an indication of main organs & physiological

systems involved

2. Regulatory toxicology (GLP)

• Reqd. by regulatory authorities/EC before compound

can be given for 1st time to humans

• Studies reqd. to support an application for marketing

approval

Timing of safety assessment

Genotoxicity

• Mutagenicity Chemical alteration of DNA

sufficient to cause abnormal gene expression in the

affected cell & its offspring

• Chromosomal damage

• End results Carcinogenesis & Teratogenicity

(detected by long-term animal studies)

Toxicokinetics

“Generation of pharmacokinetic data, either as an

integral component in the conduct of non-clinical

toxicity studies, or in specially designed supportive

studies, in order to assess systemic exposure” (ICH

Guideline S3A)

• Pharmacokinetics applied to toxicological studies

Toxicity measures

1. NTEL Largest dose in most sensitive species

2. LD50 Estimated dose reqd. to kill 50% expt. An.

3. NOAEL Largest dose causing neither tissue

toxicity nor undesirable physiological effects

4. MTD Largest dose tested causing no obvious

signs of ill-health

5. NOEL Threshold for producing any observed

pharmacological or toxic effect

THANK YOU