Drug discovery process Flashcards
Drug discovery stages
Stage 1 lead discovery - compounds with the desired activity are identified through screening of a large number of compounds.
Stage 2 lead optimization - optimising activity of lead compounds to identify the lead compound that will progress to clinical phase. End with demonstrating in vivo activity in animal models.
Stage 3 Submission of Investigational New Drug (IND) Application - allows progression of lead compound into human studies. This requires:
- Preclinical data incl. animal pharmacology and toxicology studies
- Chemical manufacturing information
- Clinical protocols describing how the compounds will be studies in humans
Phases of clinical trials
Phase 1 clinical trials aim to determine the safety and tolerability of the new drug in healthy volunteers.
- PK and PD monitored
- Safety endpoints using biomarkers
- Immunogenicity testing for biologics
Single Ascending Dose (SAD): the drug is given to a group of subjects and they are monitored. If the drug is OK then a second, higher dose is given to a different group of volunteers. This is repeated until intolerable side effects appear. This is used to determine the maximum tolerated dose (MTD).
Multiple Ascending Dose (MAD): same group of volunteers given cumulative additions of a low dose. This monitors safety, PK, PD and tolerability.
Phase 2 clinical trials use 100-300 patients to determine whether the candidate drug has the desired pharmacological effect in humans. Safety testing is also continued.
Phase 2A: determines therapeutic dose
Phase 2B: determines the efficacy of that dose of the candidate drug in a small population
Phase 3 clinical trials are usually randomised, blinded and carried out in multiple centres using 100s to 1000s of patients. Used to determine whether the candidate drug has the desired pharmacological effect in the relevant patient population, compared to placebo or the best current treatment. Safety testing still continues. New side effects may be identified as the population size increases.
At the end, you submit a New Drug Application for Marketing authorisation.
Phase 4 or post-marketing surveillance is monitoring of the drug in the wider population. This can help pick up rare adverse effects and the effects of a drug over a long period of time.
Drug targets
For something to be a good target, it must show disease association, be expressed selectively in the target organ and be druggable.
Druggable targets for low molecular weight (LMW) compounds include any protein with an active site or binding pocket.
Enzymes and GPCRs are the most common drug targets.
Target identification and validation
Target identification and validation links the target with the disease by determining if modulation of the target will help treat the disease and if it will lead to side effects based on the mechanism of action (on-target).
Target identification and validation requires an understanding of the disease as we need to identify targets that are the cause of the disease/symptoms or supportive of disease pathology (e.g. oppose symptoms like bronchodilators do).
This can be aided by bioinformatics, data from publications on disease pathology or genetic changes in the disease.
Functional validation of target involvement in disease is then performed using:
- Primary human cells using target knockouts or pharmacological modulators
- Animal models or human tissue experiments - e.g. transgenic animals with decreased/increased expression of the protein of interest to investigate phenotypic changes.
There are some issues with these validation methods including human vs animal differences and the existence of compensatory mechanisms.
- Compensatory mechanisms are not set up in knockdown animals, where the protein is not permanently inactivated but just silenced. These are therefore an alternative.
- Gene knockout is the complete elimination of genes from an organism. Gene knockdown is the reduction of the expression of a gene in an organism.
Lead identification
Following target identification and validation, we move on to lead identification of a molecule (or a series of molecules) that binds to the target to give the desired pharmacological effect.
Hit discovery requires the development of an assay capable of measuring target activity.
The assay should be simple but pharmacologically relevant, reproducible and reliable to identify a compound with the desired effect. The cost of the assay is an important consideration.
This is high-throughput screening.
Screening
High Throughput Screening (HTS)
- Screen of hundreds/thousands of compounds against a single target
Single, easy to measure readout e.g. calcium, reporter gene
Using a chemical library - a compilation of chemical compounds that may interact with your target. This required some knowledge of the structure of your target and possible points of interaction that can be exploited by molecules.
Virtual HTS can be performed using structures of chemicals in library to determine chances of interaction. Dependent on knowing the binding sites in the target.
Physiological/tissue screen
e.g. tissue/organ bath - looking at muscle contraction
Lead optimisation
Lead optimisation looks at:
- On target pharmacology: affinity, potency, selectivity and pharmacokinetics
- Secondary pharmacology and safety pharmacology
- Development of PK/PD relationships - interplay between the drug concentration in the body (PK) and the pharmacological effect (PD) - required for estimation of human dose
Refinement involves:
- Showing that the effect from those hit compounds is concentration-dependent
- Showing that hit compounds are interacting with the target
- Showing that hit compounds have a functional response, e.g. in tissues.
- Evaluating selectivity vs undesirable effects
- Start to develop structure-activity relationships - multiple compounds and medicinal chemists are involved
We can characterise the activity of hits using functional and binding assays, preferably including in vivo, full organism tests.
We might want to maker chemical structure changes to the hit compound to see if this improves potency/efficacy/solubility. Following changes, the compound has to be tested again.
The objective of lead optimisation is to identify a candidate molecule suitable for clinical studies.
We can start to look at PK using an in vitro ADME profile - e.g. use Caco cells as a model of intestinal epithelium to look at the ability to cross the epithelium (both directions).
In vitro ADME profile determines permeability across membranes, metabolic and chemical stability and effect on CYPs.
When identifying a clinical candidate consider activity, selectivity, PK profile including metabolism and secondary pharmacology, safety, stability , solubility and permeability.
In vitro absorption testing
The Parallel Artificial Membrane Permeability Assay (PAMPA) estimates passive, transcellular permeability.
A semipermeable lipophilic membrane on 96-well plates is used to mimic the cell membrane.
We measure how much of our compound passes from donor to acceptor wells.
The hydrophobic membrane has no transporter profile
It allows us to determine the apparent permeability, Papp, measured in cm/s.
Lipophilicity (LogP/LogD) plays a major role in passive diffusion. As LogD increases, PAMPA permeability also increases.
In vitro cell culture models which mimic the target site of interest can also be used in oral absorption studies.
Epithelial cell monolayers mimic the gut wall to model absorption. The most commonly used models are:
- Caco-2 human colon carcinoma cell line
- MDCK - Madin Darby Canine Kidney cell line expressing MDR1 (P-gp/ABCB1) and BCRP (ABCG2)
In vitro affinity for efflux transporters is assessed using ATPase assays and competitor assays (probe substrates).
Caco-2 permeability assays are conducted using permeable insert, sometimes called Transwell systems.
Caco-2 cells originate from a human colonic adenocarcinoma and differentiate to mimic the small intestine. They express a range of transporters and metabolic enzymes, similarly to the gut lumen.
For Caco-2 cells, we have to measure the permeability in both directions due to the presence of efflux transporters. This gives us an idea of what drugs act as substrates for the efflux transporter.
We have to determine the mechanism of drug transport:
- Paracellular passive
- Transcellular passive
- Active accumulation: Amino acids, dipeptide, glucose, OATPs
- Efflux: PGP, MRP2 but low BCRP
We also have to determine the affinity of the drug for active processes
Metabolism in the gut may also have to be considered. CYP3A4 and UGT are present, but in much lower concentrations than in the liver
MDCK cells can be used to assess the impact of transporters on permeability. As these are transfected cell lines, these can be transiently genetically modified to express only one transporter of interest.
In situ models, such as isolated perfused rat gut (IPRG) can also be used.
In vitro distribution testing
Transfected MDCK cell lines can be used to determine if a compound could have efflux-limited CNS penetration.
This could also be done for other tissues, e.g. using cells expressing polyamine transporters to measure potential accumulation into the lungs.
Protein binding measurement is also used. It is equilibrium dialysis with tissue homogenates.
Typically, plasma protein binding is measured in vitro using fresh plasma in filter devices or using dialysis
PPB determines the amount of free, unbound drug in circulation, which allows estimation of the amount of drug available to the target tissue.
PPB also influences drug clearances as only the unbound, free drug is available for clearance by the liver and kidneys.
The ratio is measured at equilibrium.
In vitro elimination testing
HEK or MDCK cell lines can be used to measure hepatic elimination.
Primary cell cultures, such as primary proximal tubule cells, can also be grown.
Kidney slices are also used, but these require organ donation.
Isolated perfused kidneys are also used. Intact organ structure is required and this method is expensive and has a short viability of <2h.
Most enzymes involved in metabolism are found in microsomes. These are prepared from cellular smooth endoplasmic reticulum by differential centrifugation.
Microsomes contain all membrane-bound proteins of the cell, such as cytochromes P450s and UDP-glucuronosyltransferases, but cofactors need to be added to initiate metabolism.
Hepatocyte preparations can also be used. These contain all metabolising enzymes of the liver (microsomal and cytosolic).
They are used for assessing metabolic stability and metabolite identification and ideal for assessing species differences in metabolism.
However, there can be some loss of function seen in vitro, such as loss of proliferation and CYP function, but this can be reversed to some extent by de-repressing transcription factors and adding antioxidants.
Intrinsic clearance can be calculated from these assays.
For drug metabolism studies, the in vitro metabolism data can be ‘scaled’ to represent clearance in the human liver. This is in vitro-in vivo extrapolation (IVIVE).
Pharmacokinetic-pharmacodynamic relationships
Pharmacokinetic-pharmacodynamic relationships compare the drug effect to drug exposure over time in vivo. This is required for an estimation of the clinical dose.
The dose and AUC can be used to calculate other PK parameters. This needs to be carried out in animals to predict the potential PK profile in humans and determine FIH dose.
The concentration-response curve is used to determine pharmacodynamics.
Preclinical evaluation
Preclinical evaluation is part of lead optimization and involves safety and toxicology profiling.
The target dose in humans is determined using animal to human dose prediction.
Pharmacokinetics and efficacy (pharmacodynamics) are also evaluated.
The maximum safe dose is determined and the therapeutic index predicted.
Testing for off-target effects is done to determine target selectivity. This involves determining secondary pharmacology and looking at the drug’s effects at similar targets.
We must look at both exaggerated pharmacology and off-target side effects.
Safety pharmacology involves exploratory tests using a single dose, while toxicology looks at chronic dosing.
In vitro screens cannot pick up toxic effects caused by metabolites, and often effects in one organ lead to toxicity elsewhere, which can only be seen in vivo.
Immunogenicity may also only be identified in human subjects.
Exploratory testing is used to establish a dose range.
This can use a single dose to measure drug absorption and duration of action.
Different doses are used in different animals - single ascending dose.
Cumulative dosing can also be carried out - e.g. increasing the dose every 2 days until a toxic effect is observed. This is a multiple ascending dose study.
This gives an indication of a safe dose and the duration of action of the drug.
It also enables human equivalent dose (HED) calculation.
Both of these are required for regulatory approval
Toxicological testing
Toxicological testing determines the toxic effects of drugs at various doses with acute or chronic exposure.
We can test for genotoxicity such as mutagenicity.
We also test carcinogenesis and teratogenesis, which can only be detected by long-term animal studies. Carcinogenicity testing is carried out for drugs expected to be used for 6 months or more or for special concerns, e.g. for a compound in a known class of carcinogens. These are not necessary if mutagenicity has already been detected.
The assay can be modified to include a microsomal activation system.
Non-genotoxic carcinogens, such as those acting via histone modification, may be missed.
We can test for chromosomal abnormalities using CHO cells grown in the presence of our compound.
Tissue-specific toxicity needs to be tested for. Long-term dosing is performed to see if the drug accumulates in any organs or tissues.
Chronic toxicology testing requires one rodent and one non-rodent species, using both male and female animals.
Blood and urine samples are taken and respiratory, cardiovascular and GI effects are monitored.
Autopsies are performed to get as much date out of each animal as possible.
Testing also includes PK measurements for extrapolation to human dosing.
Chronic toxicity leading to irreversible damage, such as nerve or cardiovascular damage, would limit the marketability of the drug.
Reproduction/development testing requires treating both males and females prior to mating and testing fertility. Embryo testing involves treating females during the first stages of pregnancy and looking at embryo development to test for teratogenicity. The same can also be done for dosing during lactation.
What is considered an acceptable side effect depends on the condition being treated.
It is important to remember that there may be species differences in toxicological responses.
Drug-drug interactions
Drug-drug interactions can occur due to effects on drug metabolism, especially due to inhibition of CYP enzymes.
Induction of drug metabolism can also occur. We need to look at whether drug metabolism in cultured hepatocytes increases.
For example, ketoconazole inhibits CYP3A4 and causes terfeneadine cardiotoxicity.
CYP enzymes can be induced by: Phenytoin (VGSC blocker for epilepsy), Barbiturates, Corticosteroids and Rifampicin (CYP3A4)
Marketing Authorisation Application Requirements
The components of a marketing authorisation application are:
- Module 1 - Administrative, regional or national information e.g., application form, proposed summary of product characteristics (SmPC), labelling, package leaflet, expert signature pages, environmental risk assessment (ERA), pharmacovigilance system and risk management plan (RMP).
- Module 2 - High level summaries - the Quality Overall Summary (QOS), the Non-clinical Overview/Summaries, and the Clinical Overview/Summaries.
- Module 3 - Chemical, Pharmaceutical and Biological documentation (quality and manufacturing)
- Module 4 - Toxicological and Pharmacological test data (non-clinical) performed on the active substance and drug product
- Module 5 - Clinical trials data on the drug product
The European Medicines Agency (EMA) tends to look at info in module 2 (high level summaries) and then explore any difficulties in more detail in the other sections.
The Food and Drug Administration (FDA) start at the detail in modules 3 and 4
Modules 2,3,4,5 are submitted as a Common Technical Dossier (CTD)
Drug registration procedures (EMA)
Application types are:
- New medicinal product (new active substance) - this is a more stringent review
- Generic medicinal products, “hybrid” medicinal products and similar biological medicinal products - you have to prove bioequivalence to an already approved drug product
- Well-established medicinal use (WEU) for the product, supported by bibliographic literature
- New, fixed combination of active substances in a medicinal product
- Informed consent from a marketing authorisation holder for an authorised medicinal product – licence exists but applicant wants a duplicate licence
Centralised procedure (CP) – one application and one assessment coordinated by the European Medicines Agency (EMA) leads to EU-wide approval.
- Compulsory for drugs with a new active substance for HIV/AIDS, cancer, diabetes, neurodegenerative diseases, immunological dysfunctions, viral diseases, biotech and advanced therapy medicines, and orphan medicines.
- Optional for other products – eligibility request is required.
- Single trade name across EU
Decentralised procedure (DCP) – multiple applications, one assessment by a national agency. Subsequent approval granted by other nations based on mutual recognition.
Mutual recognition procedure (MRP) – existing MA approval in one country. Apply for approval in other countries and MA granted based on mutual recognition.
National procedure (NP) – single MAA submitted to one national agency, assessment and approval carried out by that agency
UK drug approval post-brexit
Post-brexit, the UK is administered by the Medicines and Healthcare products Regulatory Agency (MHRA).
Existing EU MAs were converted into Great Britain (GB) MAs effective from 1 January 2021.
Existing EU MAs remain valid in Northern Ireland
A new reliance procedure is available to obtain a GB MA that relies on a recent EU approval.
The rolling review for novel medicines is a new route for MAA. Modules of the CTD are incrementally submitted for pre-assessment by the MHRA.
This allows a rolling review - identify problems earlier.
Post Marketing Surveillance
Post marketing surveillance is part of pharmacovigilance.
This involves monitoring of side effects/adverse drug reactions.
The yellow card scheme is used in the UK.
This allows us to monitor drug effects in subpopulations and identify potential rare side effects and long-term effects over the lifetime of a population.
