[PHARM 5] Preclinical development

DRUG DEVELOPMENT

DRUG DEVELOPMENT

• The next step is involves transforming a compound from drug candidate to a product approved for marketing by the appropriate regulatory agencies.

chemical development

• check chemical stability under various conditions

• optimize and scale up synthesis and purification

• produce gmp batches as required for biological and clinical testing

pharmaceutical development

• develop and deliver formulations for biological testing

• develop dosage forms as required

• develop special delivery systems

drug metabolism/pharmacokinetics

• develop analytical techniques for measuring compound and metabolite concentrations in tissues

• develop major metabolites in relevant species, including man

• determine pk parameters

safety assessment

• safety pharmacology

• in vitro toxicology

• in vivo toxicology of various species

clinical development

• phase 1 (assessment of PK characteristics and side effects in normal volunteers)

• phase 2 (dose-finding and tests of efficacy in small groups of patients)

• phase 3 (definitive trials of efficacy in large groups of patients, including comparison with standard therapies)

early selection point

• Decision to take the drug candidate molecule into early (preclinical development)

decision to develop in man

• Entry of compound to Phase 1

full development decision point

• After phase 1 and phase 2a are completed

submission decision point

• Final decision to apply for registration

exploratory

• Aimed a giving a rough quantitative estimate of the toxicity of a compound when given acutely or repeatedly over 2 weeks; no need for GLP

regulatory

o Performed to GLP standards and comprise those that are required by regulatory agencies that will support approval of application

safety pharmacology

• This comprises of a series of protocol-driven studies aimed specifically at detecting possible undesirable or dangerous effects of exposure to drug in therapeutic doses

core battery safety test

1. performed on all compounds intended for systemic use

follow up and supplementary test

1. performed if on battery test, results mechanisms need to be determined, or if there is a drug-drug interaction.

exploratory toxicology studies

• Studies conducted in rodents and non-rodents · Single dose with widely-spaced doses (10,100, 1000 mg/kg) over a 14-day observation.

• Usually 4 or 5 doses will be tested.

• Dose escalation protocol may be used in which animals treated are administered with increasing doses until signs of toxicity appears or until a dose of 2000mg/kg.

• Animals are killed and autopsied to determine target organ toxicity.

• Followed by multiple-dose ranging studies in which the drug is given twice daily for 2 weeks.

• Limitation: INSUFFICIENT for supporting first human evaluation

genotoxicity

• defined as in vitro and in vivo tests designed to detect compounds that induce genetic damage by various mechanisms

• Enable hazard identification with respect of damage to DNA and its fixation

Ames test

a. An increase in the number of surviving colonies of S. typhimirium in the absence of histidine denotes mutagenicity.

chronic toxicology studies

• The objective of these studies is to look for toxicities after repetitive dosing of the compound when a steady state is reached.

experimental design for chronic toxicity studies

• Must be performed under GLP conditions

• One rodent plus one non-rodent plus additional species

• Separate male and female groups

• Average study requires 200 or more rats, 60 dogs

• Urine and blood sampling done regularly

• Killed after several months, then autopsied or histologic examination.

carcinogenicity testing reproductive or developmental toxicity studies

special tests

carcinogenicity testing

• Normally required before a drug can be marketed especially if the compound class belongs to a known class of carcinogens or has chemical features associated with carcinogenicity

• Chronic studies show evidence of precancerous changes Compound or metabolites are retained in tissues

reproductive/developmental toxicology studies

• The requirement for reproductive toxicology data as a prelude to clinical trials differs from country to country.

• General rule: Clinical trials involving women should be preceded by relevant toxicological data.

segment 1 test

• fertility and implantation involving treating both males (28 days) and females (14 days) with drug prior to mating

segment 2 test

• for embryonic and fetal development, drug being given to female during initial gestational stage.

segment 3

• for pre and perinatal development; dosing female rats with drug throughout gestation and lactation.

LD50

o Dose required to kill 50% of a group of experimental animals o Has been largely abandoned as a useful measure of toxicity, and no longer needs to be measured for new compounds

no toxic effect level

o Largest dose in the most sensitive species in a toxicology study of a given duration which produced no observed toxic effect

no observed adverse effect level

o Largest dose causing neither observed tissue toxicity nor undesirable physiological effects such as sedation, seizures, or weight loss

maximum tolerated dose

o Usually applies to long-term studies and represents the largest dose tested that caused no obvious signs of ill-health

no observed effect level

o Represents the threshold for producing any observed pharmacological effect or toxic effect

drug repositioning

• Process of uncovering new indication of approved/failed/abandoned compounds. · More practical, less time-consuming and less costly. · 3 approaches:

computational

• machine-learning algorithms

experimental

· - provide clear evidences between drug and diseases

mixed approaches

• combination of two

aspirin celecoxib dapoxetine duloxetine fingolimod ketoconazole minoxidil raloxifene rituximab sildenafil thalidomide topiramate zidovudine

Examples of drugs successfully repositioned A cute dog doesnt fight kittens, makes rr shoot to the zky HJAHDAHDAHHAHAHAHAHAHAHAHA

conventional/experimental computational mixed approaches

3 approaches in repositioning

molecular modeling

• This refers to a data-driven science branch with many algorithms and databases being created or adapted as a response to new data forms (Xia, 2017)

• It uses high performance computing used in interpolating laboratory experiments and theory (Aminpour et al., 2019)

high performance computing

used in interpolating lab experiments

structure-based design

• Structure is KNOWN

• 2D/3D conformation

• Can be elucidated

• It is a multi-disciplinary and iterative process that is well-established in research institution and pharmaceutical industry.

• This is used when THE STRUCTURE OF THE TARGET IS KNOWN/AVAILABLE.

• Drugs discovered: Zanamivir, Nelfinavir, Aleglitazar

• Target Identification

• Modeling and visualization of macromolecular structure

• Binding site prediction and analysis

• Molecular docking

• Structure-based virtual screening

• Validation of molecular docking

aleglitazar nelfinavir zanamivir

drugs discovered through structure based design

zinc/zinc20

a. Freely available database of commercially available compounds for molecular docking

PubChem

a. Database of small chemical molecules and their biologic activities

ChemSpider

a. All of the chemical structures of compounds

ChemBL

a. Small molecule database contain information of ADMET and binding of huge number of bioactive substances

Drug Bank

a. Comprehensive database with information about the drug, their target, and other useful information

homology modeling

• 3D images of similar protein

• Template structure

• Assumption: Two identical structures adopt similar 3D structure.

RasMol, PyMol, Chimera

• Visualize macromolecules in atomic levels

• · These are visualization tools that play a significant role in viewing and analyzing the predicted and experimentally-determined 3D structures of macromolecules at the atomic level.

PyMol

• widely used cross-platform molecular graphics tool for 3D visualization of proteins, small molecules, nucleic acids, electron densities and surface trajectories

RasMol

• provides rotation of molecule interactively by viewing molecules in various forms.

PDB databases

• Contain fully reviewed and experimentally validated information of protein structure

• Focused on amino acids and residues

• Residue + candidate

• Single worldwide archive of structural data of biologic macromolecules. It includes data obtained from X-ray crystallography and NMR submitted by biologists and biochemist

• Text based query

• Sequence based query

molecular docking

• If the structure of the ligand will fit in receptor binding site

• Strength of binding

blind docking

• Do not know where it will bind

• A: utilize other binding site

• Time consuming

flexible rigid

2 types of docking

flexible docking

• Ligand and receptor sidechain are kept flexible as well as the binding energy

• For induced-fit docking, main chain is moved to integrate the conformational changes of receptor upon ligand binding.

• Yields better approach than conventional docking

• Disadvantage: may yield poor docking scores due to incorrect ligand binding.

rigid docking

• Internal geometry of ligand and receptor is kept fixed during docking simulation.

• Successful lead finding: aspartic protease in HIV yielded a candidate inhibitor with higher potency using the DOCK program

structure based virtual screening

• Involves docking and screening of a compound database against the drug target, followed by scoring based on binding of free energy.

• Ligands are ranked according to their affinity to the target, and the most promising compounds are shown at the top of the list. SBVS methods require that the 3D structure of the target protein be known so that the interactions between the target and each chemical compound can be predicted in silico (Liu et al., 2018).

pose selection docking a decoy set

2 methods of validating molecular docking

pose selection

§ Docking programs are used to re-dock a compound with known conformation and orientation into the target site (Hevener et al., 2019).

docking a decoy set

o inactive or suspected inactive compounds that have been seeded against the target with compounds having known activity (Hevener et al., 2019 and Jain, 2008).

AutoDock

a. Predicts the affinity of binding and poses of small molecules to a 3D structure of the target protein

AutoDock Vina

a. Virtual screening and molecular docking b. Updated version

Glide Schrodinger

Molecular modeling

Hex

a. Docking simulation

MoE

a. Molecular operating environment b. Docking simulations

Ligand based drug design

• It is an alternative protocol and plays a tremendous role when the structure of the target protein is unknown or cannot be predicted by available modeling methods.

• It uses statistical methods to correlate the activity of the ligand to structural information.

• Pharmacophore mapping

• QSAR (Quantitative Structure-Activity Relationship)

pharmacophore mapping

• abstract description of molecular features necessary for molecular recognition of a ligand by a macromolecule.

• features can be used as a query for searching and retrieving potential leads from chemical compound databases.

hydrophobic centroids aromatic rings hydrogen bond donors/acceptors, cations and anions

Examples of pharmacophoric features

quantitative structure activity related

• Predicts the biological activity of chemical compounds in drug designing.

• It uses mathematical tools to find the relationship between structure and biologic activity.

• In here, a QSAR model is built.

2D QSAR

o Uses 2D properties descriptors: steric, electrostatic, hydrophobicity and geometric behavior to interpret biologic activity.

3D QSAR

o Used to overcome limitation of 2D QSAR wherein it is unable to determine correlation between physicochemical properties and 3D spatial arrangement.

2D 3D multidimensional

QSAR models

Computer based field analysis

• Based on the concept that biological activity of a small molecule depends on the molecular field.

• Uses Lennard Jones and Coulombic potential to calculate steric and electrostatic fields.

• Affected by Van der waals

Comparative Molecular Similarity Indices analysis

o More precise and accurate because algorithm is affected by Van der waals.

highest occupied molecuar orbital

o Energy of small molecules that can donate electron during formation of complex

lowest occupied molecular orbital

o Ability of molecules to take electron from associated protein

Lumo gap

difference between HOMO AND LUMO

Lipinski's rule, polar surface area, polarizability, refractivity, van der waals forces

ADMET Prediction and analysis

molecular dynamics simulation

• It is a computational study of biomolecules.

• It can predict actual interaction of the ligand at the atomic level.

1981

captopril

dorzolamide

1995

indinavir ritonavir saquinavir

1996

tirobifan

1998

oseltamivir zanamivir

1999

aliskiren rallegravir

2007