Drug Action Lecture 1 Content

Fields that would not be studied without drug discovery knowledge

forensic pharmacology and toxicology

ex. arsenic trioxide for cancer treatment

Source of first medicinal drugs

• natural sources

• herbs, plants, seeds, roots, vines, barks, fungi

• only natural sources available for pharmaceuticals until mid-19th century

Examples of pharmacologically active substances derived from plants

• morphine from opium poppy

• nicotine from tobacco plant

• cannabinoids from cannabis leaves

• caffeine from tea and coffee

• cardiac glycosides (digoxin and digitoxin) from wooly foxglove

• quinine from cinchona tree

• salicylates from bark of white willow tree

Natural products and drug discovery

• natural products for medicine and health have been enormous

• important challenge to find biologically active compounds and develop these into new drugs

• popularity of natural products will continue because they are a matchless source of novel drug leads and inspiration for synthesis of non-natural molecules

• natural products provide important clues for identifying and developing synergistic drugs (neglected by research)

• rich historical record

When did the switch away from natural products to combinatorial chemistry occur?

1990s

might have led to current paucity of new drug candidates in development pipeline

Technological Changes in Drug Discovery

• Genomics, proteomics, metabolomics, bioinformatics, microbiome (target identification)

• transgenic and knockout animals (proof of concept)

• combinatorial chemistry (targeted-guide synthesis)

• structure-based and computer-aided design (X-ray/NMR)

• Automatic-high-throughput test models (u-HTS)

• data mining and systemic pharmacology (pharmacometrics)

• molecular phenotyping (omic-MolPAGE) and Biomarker

• ADMET profiles (HTS and in silico)

• AI system application

Drug Discovery Bottleneck

• “druggable” targets

• target validation

• poor ADMET and Efficacy

Disciplines Related to Drug Discovery

• long and complex process

• medicine (targeted-disease model-Medicinal chem)

• chemistry (novel method for new materials)

• biotechnology (target-lead validation/optimization)

• pharmacology (ADEMT-drug action)

• computer sciences (assistance in drug design)

Factors contributing to variation in drug response

• pharmacogenomics consideration

• age

• gender

• hepatic function

• immunological function

• diet

Modern Drug Discovery Procedures

• identification and validation of drug target (a “lead compound identification and optimization)

• assay development (how to test)

• therapeutic validation of a drug candidate (disease model selection, in vitro, in vivo)

• pre-clinical (toxicology, PK/PD)

• Clinical trials (investigational new drug (IND) and Phase I, II, III)

• Release of drug (New Drug Application, NDA)

• Follow up monitoring (FDA)

• Pharmacovigilance (PhV)

Drug Target Identification

• most often proteins

• ex. signaling transduction pathway as cancer treatment target

• nucleic acids also attractive targets for some diseases (viral infection, cancer)

• also ion channels, transporters

• target mechanisms: enzyme inhibitor - reversible or irreversible

• receptor

• nucleic acid

• ion channels

• transporters

• agonist or antagonist

• intercalcator (binder), modifier

• alkylating agent or substrate minic

• blockers or openers

• uptake inhibitors

Identification of Lead Compound and Optimization

• high-throughput-screening (HTS)

• rational design (structure-based design/targeted therapy)

High-Throughput-Screening

• normally use drug library

• natural product screening (50-70%)

• synthetic library (can be up to 1,000,000+ CMP)

• Combinatorial Chemistry

Rational Design

• structure-based design / targeted therapy

• enzyme/ligand targeted (Ras/EGFR)

• Targeted-guided synthesis (TGS)

• mAb (TNFa for arthritis, Lecanemab for AD)

• siRNA (treatment for Huntington disease)

• Vaccine (SPIKE protein for COVID-19, HBsAg for HBV)

Drug Design and Validation

• Rational design (structure-based screening)

• target validation

Rational design

• structure-based screening

• combines power of NMR spectroscopy, automatic docking, and X-ray crystallography

• provides means to apply structural info (NMR, modeling, X-ray) early in project to identify hits, select targets, and optimize the hits in terms of their affinities and specificities

Target validation

determination that molecular target is critically involved in disease process and potentially valuable point of invention for new drugs

Methods for Target Validation

• bio (macro) molecule may be involved in disease process, but to be a drug target it must be validated (shown to be critical in disease process)

• useful validation techniques:

  • gene knockout

  • RNA interference (RNAi)

  • Transgenic model (over expression)

  • CRISPR/Cas9 system

Gene knockout

does removal of the gene that encodes the target protein result in the death of a pathogen (disease-causing microbe)?

RNA interference

• involves double-stranded ribonucleic acid (dsRNA) interfering with the expression of genes with sequences complementary to the dsRNA

• results in a reduction of the production of the protein (target) in question

Transgenic model

• over expression

• over expression of a certain protein to accelerate or slow down disease progress

CRISPR/Cas9

gene editing

Approach to select a lead small molecule as potential drug candidate for EGFR target

• high-throughput-screening (HTS) from synthetic/combinatory/natural product compound library

• rational design to target EGFR (ex. synthesis of peptide-mimics on key binding site “alpha helix”)

• fragment-based design and approach (ex. set up EGFR as bait protein for target-guided synthesis to discovery active, lead compound bigger than original two inactive fragments)

Disease models for lead molecule validation

• cancer

• cardiovascular disease

• diabetes

• infection disease (HIV, Hepatitis, TB, Influenza, SARS, SARS-Cov2)

• Neurogenerative diseases (AD, PD)

• Genetic disease (Huntington, ALS)

• Others (obesity, autism, psychological disease)

Assay Development

• in vitro Enzymatic Assay (FTase, Kinase, P450)

• In vitro Ligand binding assay (125I-PDGF/R)

• Protein-Protein/Peptide interaction (FP, ELISA, FRET, AlphaScreen, Luc-Assay, ChIP)

• Cell cycle analysis (FACS)

• Western Blot Analysis (SDS-PAGE, 2-D gel)

• Microarray, Proteomics, Metabolomics, NGS analysis

• loss-of-function (knock-down)/gain-of-function (knock-in)

• Cell-based Assay (MTT, Apoptosis, Migration, Proliferation, Transformation, Toxicity, GFP-tagged)

• In vivo mouse model (nude, diabetes, transgenic, ko models)

• PK/PD model

Clinical trials are designed to

determine safety and tolerance in man (MTD), pharmacokinetics (PK/PD); bioavailability for range of doses; determine pharmacological profiles

Main phases of pre-clinical and clinical trials

• pre-clinical: animal studies, submission of “investigational new drug” application to gov (FDA)

• Phase I: test of normal, healthy human volunteers (MTD)

• Phase II: evaluate the safety and efficacy of a drug in patients

• Phase III: test on large patient number study to establish efficacy vs. a placebo or comparator compound

• Phase IV: long-term surveillance/monitoring of adverse reactions

Combinatorial chemistry

• aim is the generation of large numbers of compounds very quickly

• combinatorial: relating to or involving combinations

• impacts between lead discovery and lead optimization

Lipinski’s rule of five

• 4 parameters that define the drug-like of potential drug candidates based on analysis of existing drug molecules

• got name from cut-off values for each of these parameters of which all have values of 5 or multiple of 5

• Rule states that poor absorption or permeation is more likely when:

  • compound has more than 5 H-bond donors (some of OHs and NHs)

  • more than 10 H-bond acceptors (sum of Ns and Os)

  • MW over 500

  • LogP is over 5 (or MLogP is over 4.15)

• useful guide in drug design

Prodrug

• drug which is given (taken) in an inactive form

• once administered, prodrug is metabolized by body into the biologically active compound

Prodrug strategies

• used to overcome variety of problems

• alter solubility

  • may assist in achieving desired formulation

• improve membrane permeability and drug bioavailability

  • absorption: crossing hydrophobic cell membrane

  • if drug is too polar, may not pass membrane

  • if drug is too non-polar, may not come back out of membrane

• slow release of active agent

  • if drug eliminated from body quickly, effective dosage cannot be sustained

  • slow release of active agent by controlled release from a prodrug allows a more controlled dosage of the active being released into the body

• masking drug toxicity or side effects

  • many anticancer agents are cytotoxic, but it is the cancerous cells only which we want to kill

  • masking toxicity can be achieved by pro-drugging and the active agent is accumulates preferentially in the tumor (due to leaky vasculature)

AUC

• area under the concentration vs time curve after the administration of an extravascular dose

• function of the fraction of drug dose that enters the systemic circulation and clearance

• after IV and extravascular doses, AUC can determine bioavailability for extravascular dose

Cytochrome P450

• enzyme

• research developed considerably over past 20 years, well-established understanding

• important in metabolism and safety testing

• decrease adverse effects of drugs through biotransformation and bioactivation

• unresolved: orphan P450s, ligand cooperativity, kinetic complexity, prediction of metabolism, overall contribution of bioactivation to drug idiosyncratic problems, extrapolation of animal tests to humans, contribution of genetic variation

Phase I Clinical Trial

• tests for safety

• small number of healthy volunteers (10-100)

• given range of doses (maximum-tolerant dosage/MTD) and monitored for signs for toxicity

Phase II Clinical Trial

• tests for safety, efficacy, dosage

• small number of persons (~500)

• controlled and double-blind

Phase III clinical trial

• tests for safety, efficacy, side effects

• large population (1000s)

• detect side effects in small percentage of patients

• double-blind, controlled

Phase IV

• post marketing evaluation

• long term safety

ADR

• adverse drug reaction

• describes harm associated with the use of given medications at a normal dosage

• may occur following a single dose or prolonged administration of a drug or result from the interaction of two or more drugs

• different from side effect: side effects may be beneficial

• study of ADRs: pharmacovigilance (PhV)

Common ADR

• Abortion, miscarriage, or uterine hemorrhage

• Addiction

• Birth defects, Bleeding of intestine, Cardiovascular disease

• Deafness and kidney failure death, following sedation

• dementia

• depression or hepatic injury

• diabetes

• diarrhea

• erectile dysfunction

• fever

ADE

• adverse drug event

• any adverse change in health or side effect that occurs in a person who participates in a clinical trial while the patient is receiving the treatment or within specified period afterward

• ADEs in patients participating in clinical trials must be reported to local institutional review board (IRB) and the study sponsor

• “serious” adverse events must be reported to FDA immediately

  • death, hospitalization, life-threatening events, cancer, fetal exposure

• minor adverse events documented in annual summary send to regulatory authority

• sponsor collects ADE reports from local researchers and notifies all participating sides of ADEs at other sites

PhV

• Pharmacovigilance

• pharmacological science relating to the detection, assessment, understanding, and prevention of adverse effects, particularly long term and short term side effects of medicines

• science of collecting, monitoring, researching, assessing, and evaluating info from healthcare providers and patients on the adverse effects of medications, biological products, herbalism, and traditional meds with view to

  • identify new info about hazards associated with meds

  • preventing harm to patients

• process of collection begins in phase I of clinical trial and continues after approval

• around the world (FDA, WHO)

Pharmacoepidemiology

• study of the utilization and effects of drugs in large numbers of people

• provides estimate of the probability of beneficial effects of a drug in a population and the probability of adverse effects

• bridge science spanning clinical pharmacology and epidemiology

Drug Discovery Steps

1. Target Identification

2. Target Validation

3. Lead Compound Identification

4. Lead Optimization

5. Preclinical Testing

6. Clinical Trials

~200 enter preclinical testing after 7-10 years and 600M to 800M dollars

~5 enter clinical testing after 6-12 years and 1.2 to 1.4 billion dollars

Drug Development Stages and Timeline

1. Drug discovery (5-6 years)

2. Phase I clinical trials (3-6 months)

3. Phase II clinical trials (1-2 years)

4. Phase III clinical trials (1-4 years)

5. Drug review

6. FDA approval

7. Post-market monitoring

1 approved by FDA/EMA after 1-2 years and ~50M dollars

Alzheimer’s Disease

• no cure, no effective drug can stop or slow down

• annual economic impact: $290 billion in 2019

• only one antibody for AD approved since 2003

• cause is still chicken and egg argument

• group of disease with similar neurological symptoms, can be grouped into different subtypes

• age is most significant risk factor; aging impairs immune system

• majority of AD cases can be defined as being an immunological disease (immunesenescence) with neurological symptoms

Therapeutics against AD

• proteinase inhibitors: acetylcholinesterase inhibitor

• beta secretase inhibitor, gamma secretase inhibitor

• small molecules that inhibit aggregation

• vaccine against amyloid beta and tau

• antibodies against ABeta or Tau as therapies

• anti-inflammation agents (COX1 and COX2 inhibitors)

Cell Therapeutic for Alzheimer’s Disease

• target: aggregated ABeta

• Method: antigen sensitized DCs as vaccine

• Model: Human APPswe transgenic mouse model

• Molecules: Different ABeta peptides with mutation

• Lead screening: PWT, PDM, PFM, PFDM, E22W, G24V

• Assay development: ELISA, Flow cytometry

• Toxicology: dose escalation

• Application: IND, Phase, I, II, III, IV

• project started in 2005 and will submit IND app this month