Comprehensive Notes on Drug Discovery and Development

Drug Discovery and Development

Main Stages

  • Drug discovery and drug development aim to achieve registration by regulatory authorities.
  • This allows the drug to be marketed legally for human use.
  • Next lesson: Importance of pharmacokinetic analysis (ADME processes).

Drug Discovery and Development Stages

  • Stages of a project aimed at producing a marketable drug to meet a particular medical need.

Drug Discovery Phase: Target Selection

  • Planning a project to discover a new drug: Where to start?
  • First, choose a new molecular target.
  • Drug targets are generally functional proteins (e.g., receptors, enzymes, transport proteins).
  • Historically, drug discovery programs were based on measuring complex responses in vivo, but this is now rare.
  • The first step is target identification.
  • Example: Breast cancer's estrogen sensitivity led to aromatase inhibitors (prevents estrogen synthesis).
  • In 2005, therapeutic drugs addressed 266 distinct human targets.
  • Many proteins that play a role in disease still lack cognate drugs.
  • These represent potential starting points for drug discovery.
  • Conventional biology, drawing on knowledge of disease mechanisms and chemical signaling pathways, coupled with genomic data, is the basis on which novel targets are most often chosen.
  • Genomics plays an increasing role by revealing new proteins involved in chemical signalling and new genes involved in disease.
  • Innovation in drug discovery is limited not by biology and primary pharmacology.
  • Other factors include unexpected adverse effects during clinical testing, the cost and complexity of drug discovery and development in relation to healthcare economics and increasing regulatory hurdles.

Drug Discovery Phase: Lead Finding

  • Next step after deciding on a biochemical target: find lead compounds.
  • Cloning of the target protein, using the human form because sequence variations among species are often associated with pharmacological differences.
  • Developing an assay system to measure, automatically, the functional activity of the target protein (e.g., binding assay).
  • High-throughput screening: automated testing of large numbers of chemical/biological compounds for a specific biological target, such as through binding assays.
  • Using combinatorial chemistry, which includes chemical synthetic methods that make it possible to prepare a large number (tens to thousands or even millions) of compounds in a single process, allows large families of related compounds to be made simultaneously.
  • Hits from the primary screen are used to prepare sets of homologues by combinatorial chemistry to establish critical structural features necessary for binding selectively to the target.
  • Several iterative cycles of synthesis and screening are usually needed to identify one or more lead compounds for the next stage.
  • Natural products (mainly from fungal and plant sources, used in the field of anti-infective, anticancer and immunosuppressant drug) as lead compounds.
  • The main disadvantage is that they are often complex molecules that are difficult to synthesise or modify by conventional synthetic chemistry, so that lead optimisation may be difficult and commercial production very expensive.

Drug Discovery Phase: Lead Optimization

  • Lead compounds found by random screening are the basis for this next stage.
  • The aim is to increase the potency of the compound on its target and to optimise it with respect to other characteristics, such as selectivity and pharmacokinetic properties.
  • Tests applied include a broader range of assays on different test systems, including studies to measure the activity and time course of the compounds in vivo, and checking for unwanted effects in animals, evidence of genotoxicity and usually for oral absorption.
  • The objective of the lead optimisation phase is to identify one or more drug candidates suitable for further development.

Drug Development Phase: Preclinical Development

  • Pre-clinical research involves evaluation of drug safety and efficacy in animal species that conclude to prospective human outcome.
  • The pre-clinical trials also have to acquire approval by corresponding regulatory authorities.
  • The regulatory authorities must ensure that trials are conducted in a safe and ethical way and would give approval for only those drugs which are confirmed to be safe and effective.
  • The aim of preclinical development is to satisfy all the requirements that have to be met before a new compound is deemed ready to be tested for the first time in humans.
  • Pharmacology deals with the pharmacokinetic and pharmacodynamic parameters of drug.
  • Pharmacokinetic studies are very important to make known the safety and efficacy parameters in terms of absorption, distribution, metabolism and excretion.
  • These studies give information on absorption rate for diverse routes of administration, which helps in selection of dosage form, distribution, rate of metabolism and elimination, which governs the half-life of the drug (the time it takes to reduce by half).
  • Half-life of the drug clarifies the safety outline of the drug which is obligatory for a drug to get approved by regulatory agencies.
  • The drug distribution mechanism elucidates the therapeutic effectiveness of the drug as it depends on the drugs bioavailability and its affinity.
  • Drug metabolism provides the probability of through phases of biotransformation process and formation of drug metabolites; it also helps in understanding the reactions as well as enzymes involved in biotransformation.
  • The pre-clinical trials can be conducted in two ways: PHARMACOLOGY
  • Toxicological studies of the drug can be performed by in-vitro and in-vivo test which evaluate the toxicological effects of the drug.
  • In-vitro studies can be performed to inspect the direct effects on cell proliferation and phenotype.
  • In-vivo studies can be performed for qualitative and quantitative determination of toxicological effects; as many drugs are species specific, it is essential to select appropriate animal species for toxicity study.
  • In-vivo studies to evaluate pharmacological and toxicological actions, including mode of action, are often used to support the basis of the proposed use of the product in clinical studies. TOXICOLOGY
The Ames Test
  • The Ames test is a simple and rapid test for drug mutagenicity, testing the ability of a drug to induce mutation in a cell.
  • Tests the ability of a molecule to produce changes in the DNA of a modified Salmonella typhimurium (a bacteria modified to maximize the probability of mutation).
  • The Salmonella typhimurium cannot synthesize histidine, requires it in the medium to be grown and live.
  • The culture containing the Ames bacteria will die if no mutation occurs.
  • S9 hepatic enzyme fraction is added to the medium to maximize the probability of formation of metabolites that would be seen in vivo with the compound, and the lipopolysaccharide coat of the bacterium is compromised to allow maximal entry of the compounds and the DNA repair capability of the bacterium is reduced.
  • If mutation occurs, it often confers the capability to synthesize histidine.
  • Therefore, if cells grow in histidine free media, it is likely that mutation has occurred.
Repeat Dosing Study
  • The overall aim is to assess organ toxicity and safety associated with repeat dosing to predict risk in humans.
  • Define different parameters such as:
    • NOAEL (No Observed Adverse Effect Level): the largest dose causing no observed toxicity or undesirable physiological effect
    • NOEL (No Observed Effect Level): the threshold for producing a pharmacologic or toxic effect
    • MTD (Maximum Tolerated Dose): refers to the largest dose that causes no obvious sign of ill health
  • Safety studies are done with a control group (vehicle only) and exposure to low doses (one to four-fold multiples of efficacious dose), mid doses (to determine dose responsiveness) and high doses (to Identify organ toxicity).
  • Endpoints for such studies are body weight and food consumption, ophthalmological effects, electrocardiography, clinical observations, mortality, organ weights and hepatic cytochrome P450 analyses.
  • Macroscopic and microscopic examination of tissue is done, this can be extensive as thousands of tissue samples are analyzed (in addition to thousands of samples for clinical pathology, hematology, blood coagulation, clinical chemistry and urinalysis).
The 3R Principles
  • 3R principles:
    • Developed over 50 years ago providing a framework for performing more humane animal research
    • Russel und Burch, "The Principles of Humane Experimental Technique", 1959
    • REPLACEMENT
      • Complete replacement of animal testing.
      • Alternatives to animal testing include in-vitro experiments, body on a chip, and in-silico experiments.
    • REDUCTION
      • Same quality of the result but lesser number of animals.
    • REFINEMENT
      • Reduction of the suffering of animals in the experiment and optimization of the conditions for housing and experimentation.
Choice of Methods
  • Replacement
    • Choice of methods (use of animals high > intermediate > none)
    • animals > organ systems > perfused organs > complex of cells/tissue in vitro > single cells > cellular component
  • Refinement
    • Choice of methods (avoidance of pain, suffering, harm)
    • No anaesthesia/analgesia (break-off criteria established)
    • Anaesthesia and analgesia as well as wound care
  • Reduction
    • Entire experiment with anaesthesia w/o reawakening (final experiment)
    • e.g. by:
    • Limitation of the number of experimental animals to a minimum (planning of experiments using statistic methods)
    • Avoiding repetition of animal experiments
    • Repeated use of experimental animals (§ 18 TierSchVersV)
Problems Concerning Animal Testing
  • Problems concerning animal testing
    • One prevalent problem is that animals are bred for health while drugs are usually targeted toward unhealthy individuals
    • This has led to proposals that more safety testing should be done in animal models of disease (i.e., rat models of type II diabetes, heterozygous p53 knock-out mice for chemically induced carcinogenicity, lipopolysaccharide-induced inflammation and heterozygous superoxide dismutase-knockout mice to unveil mitochondrial toxicity).
    • Another important difference is in the relative exposure to populations of varying size.
    • Specifically, even in the most rigorous safety testing studies, the number of animals exposed to a drug is very much smaller than the numbers of humans that will be exposed to the drug as it becomes widely available to the greater human population (perhaps 1000-2000 animals exposed as opposed to 1,000,000 humans).
    • Another problem is that animals are bred for homogeneity to yield consistent experimental results, while the human population is much more diverse (60% of human genes may be alternatively spliced).
    • There are proposals to utilize human tissue to a greater extent in safety testing in an effort to obviate differences due to species. Thus, human blood vessel endothelial cells, hepatocytes and human kidney proximal cells have been used in safety studies for drug testing.
    • Schematic diagram of increasing population exposure to a drug as it moves from discovery to development, and eventually to the marketplace in the general population. Shown in the box are four drugs found to produce serious toxic effects only when released to the greater population
Problems Concerning Animal Model
  • Problems concerning animal model
    • Ideally, an animal model should resemble the human disease in the following ways:
    • 1. similar pathophysiological phenotype (face validity)
    • 2. similar causation (construct validity)
    • 3. similar response to treatment (predictive validity)
    • Although models for many important disorders (e.g., epilepsy, diabetes, hypertension and gastric ulceration) based on knowledge of the physiology of the condition, are available, and have been used successfully to produce new drugs, however their success in predicting therapeutic efficacy is far from perfect
    • 1. Many diseases, particularly in psychiatry, are defined by phenomena in humans that are difficult or impossible to observe in animals, which rules out face validity
    • 2. The cause of many human diseases is complex or unknown; to achieve construct validity for many degenerative diseases, we need to model the upstream (causative) factors rather than the downstream (symptomatic) features of the disease, although the latter are the basis of most of the simple physiological models
    • 3. Relying on response to treatment as a test of predictive validity carries the risk that drugs acting by novel mechanisms could be missed, because the model will have been selected on the basis of its responsiveness to known drugs.
Genetic and Transgenic Animal Models
  • Genetic animal model
    • By selective breeding, it is possible to obtain pure animal strains with characteristics closely resembling certain human diseases; genetic models of this kind include spontaneously hypertensive rats, genetically obese mice, epilepsy-prone dogs and mice, rats with deficient vasopressin secretion, and many other examples; in many cases, the gene responsible have not been identified
  • Transgenic animal model
    • Generated through manipulation of the germline, these animal models replicate human disease and are expected to be predictive of therapeutic drug effects in humans; this technology can be used in many different ways, for example:
    • to inactivate individual genes, or mutate them to pathological forms
    • to introduce new (e.g. human) genes
    • to overexpress genes by inserting additional copies
    • to allow gene expression to be controlled by the experimenter
    • Examples:
    • transgenic mice that overexpress mutated forms of the proteins which are important in the pathogenesis of Alzheimer’s disease. When they are a few months old, these mice develop pathological lesions and cognitive changes resembling Alzheimer’s disease, and provide very useful models with which to test possible new therapeutic approaches
    • transgenic mice with mutations in tumour suppressor genes and oncogenes are widely used as models for human cancers
    • However, also transgenic mice can be misleading in relation to human disease (example: gene responsible of causing cystic fibrosis)
GLP Standards
  • GLP standards are not usually adopted until projects get beyond the discovery phase

The Drug Development Phase: Clinical Development

  • Clinical trials are an important and highly specialised form of biological assay, designed specifically to measure therapeutic efficacy and detect adverse effects
Principles and Organisation of Clinical Trials
  • A clinical trial aims to compare the response of a test group of patients receiving a new treatment (A) with that of a control group receiving an existing ‘standard’ treatment (B) controlled clinical trials; the use of controls is crucial in clinical trials; usually, the controls are provided by a separate group of patients from those receiving the test treatment, but sometimes a crossover design is possible in which the same patients are switched from test to control treatment or vice versa, and the results compared randomisation is essential to avoid bias in assigning individual patients to test or control groups concern arises over the ethics of assigning patients at random to particular treatment groups (or to no treatment) informed consent declaration of Helsinski
  • The clinical trial does not normally give any information about potency or the form of the dose-response curve, but merely compares the response produced by two or more stipulated therapeutic regimens; however, the organisation of clinical trials, with controls against bias, is immeasurably more complicated, time-consuming and expensive than that of any laboratory-based assay.
Avoidance of Bias
  • Two main strategies that aim to minimise bias in clinical trials: Randomisation
    • If two treatments, A and B, are being compared on a series of selected patients, the simplest form of randomisation is to allocate each patient to A or B by reference to a series of random numbers
    • One difficulty, particularly if the groups are small, is that the two groups may turn out to be ill-matched with respect to characteristics such as age, sex or disease severity stratified randomisation avoids the difficulty by dividing the subjects into age, sex, severity, or other categories, random allocation to A or B being used within each category. It is possible to treat two or more characteristics of the trial population in this way, but the number of strata can quickly become large, and the process is self-defeating when the number of subjects in each becomes too small. As well as avoiding error resulting from imbalance of groups assigned to A and B, stratification can also allow more sophisticated conclusions to be reached; B might, for example, prove to be better than A in a particular group of patients even if it is not significantly better overall
Double-Blind Technique
  • Double-blind tecnique
    • neither subject nor investigator is aware at the time of the assessment which treatment is being used it is not always possible, however. A dietary regimen, for example, can seldom be disguised; with drugs, pharmacological effects may reveal to patients what they are taking and predispose them to report accordingly In general, however, the double-blind procedure, with precautions if necessary to disguise such clues as the taste or appearance of the two drugs, is used whenever possible
The Size of the Sample
  • The size of the sample, clinical trial should involve the minimum number of subjects, and much statistical thought has gone into the problem of deciding in advance how many subjects will be required to produce a useful result the results of a trial cannot be absolutely conclusive, because it is based on a sample of patients, and there is always a chance that the sample was atypical of the population from which it came Two types of erroneous conclusion are possible, referred to as type I and type II errors a type I error occurs if the results show a difference between A and B when none actually exists (false positive) so from it depends the statistical significance of the result; a type II error occurs when someone fails to detect a real difference between A and B (false negative) and from it depends the statistical power of the result.
    • a trial may give a significant result before the planned number of patients have been enrolled, so it is common for interim analyses to be carried out at intervals
    • If this analysis gives conclusive result, or if it shows that continuation is unlikely to give a conclusive result, the trial can be terminated, thus reducing the number of subjects tested in planning clinical trials, it is necessary to decide the purpose of the trial in advance, and to define the outcome measures accordingly
  • Meta-analysis is a procedure that make possible, by the use of statistical techniques, to combine the data obtained in several individual trials (provided each has been conducted according to a randomised design) in order to gain greater power and significance; the Cochrane Collaboration; the Cochrane Library
Key Aspects of Clinical Trials
  • A clinical trial is a special type of bioassay done to compare the clinical efficacy of a new drug or procedure with that of a known drug or procedure (or a placebo).
  • At its simplest, the aim is a straight comparison of unknown (A) with standard (B) at a single-dose level.
    • The result may be: 'B better than A', 'B worse than A', or 'No difference detected'. Efficacy, not potency, is compared.
    • To avoid bias, clinical trials should be:
    • controlled (comparison of A with B, rather than study of A alone)
    • randomised (assignment of subjects to A or B on a random basis)
    • double-blind (neither subject nor assessor knows whether A or B is being used).
    • Type I errors (concluding that A is better than B when the difference is actually due to chance) and type II errors (concluding that A is not different from B because a real difference has escaped detection) can occur; the likelihood of either kind of error decreases as the sample size and number of end-point events is increased.
    • Interim analysis of data, carried out by an independent group, may be used as a basis for terminating a trial prematurely if the data are already conclusive, or if a clear result is unlikely to be reached.
    • All experiments on human subjects require approval by an independent ethical committee.
    • Clinical trials require very careful planning and execution, and are inevitably expensive.
    • Meta-analysis is a statistical technique used to pool the data from several independent trials.
Good Clinical Practice
  • The conduct of trials has to comply with an elaborate code known as Good Clinical Practice, covering every detail of the patient group, data collection methods, recording of information, statistical analysis and documentation
  • At the end of phase III, the drug will be submitted to the relevant regulatory authority for licensing; the dossier required for this is a massive and detailed compilation of preclinical and clinical data. Evaluation by the regulatory authority normally takes a year or more, and further delays often arise when aspects of the submission have to be clarified or more data are required

Pharmacovigilance

  • Pharmacovigilance is the science and activities relating to the detection, assessment, understanding and prevention of adverse effects or any other medicine-related problem
  • The European Medicines Agency (EMA) coordinates the European Union (EU) pharmacovigilance system and operates services and processes to support pharmacovigilance in the EU
  • Before a medicine is authorised for use, evidence of its safety and efficacy is limited to the results from clinical trials, where patients are selected carefully and followed up very closely under controlled conditions. This means that at the time of a medicine's authorisation, it has been tested in a relatively small number of selected patients for a limited length of time
  • After authorisation the medicine may be used in a large number of patients, for a long period of time and with other medicines Certain side effects may emerge in such circumstances; it is therefore essential that the safety of all medicines is monitored throughout their use in healthcare practice
  • EU law therefore requires each marketing authorisation holder, national competent authority and EMA to operate a pharmacovigilance system. The overall EU pharmacovigilance system operates through cooperation between the EU Member States, EMA and the European Commission. In some Member States, regional centres are in place under the coordination of the national competent authority

Databases

  • PubMed is a free resource supporting the search and retrieval of biomedical and life sciences literature (article/review) with the aim of improving health–both globally and personally
  • The PubMed database contains more than 35 million citations and abstracts of biomedical literature. It does not include full text journal articles; however, links to the full text are often present when available from other sources, such as the publisher's website or PubMed Central (PMC)
  • Available to the public online since 1996, PubMed was developed and is maintained by the National Center for Biotechnology Information (NCBI), at the U.S. National Library of Medicine (NLM), located at the National Institutes of Health (NIH)
Minimizing Results Obtained
  • How we can minimize the results obtained?
    • Keywords
    • Boolean Operators
    • Parentheses or brackets
    • Tags
Boolean Operators and Parentheses or Brackets
  • Boolean Operators are words that connect search terms or key words together to broaden or narrow the results retrieved. In library research they are often used with the library's research databases or the library catalog.
  • The three Boolean operators are AND, OR, and NOT.
    • AND narrows your search results by limiting your results to those that contain both words connected with AND.
    • OR expands your search results by including results that contain one word, the other word, or both words.
    • NOT narrows your search results by limiting your results to those that contain the word you designate before NOT, but not the word after NOT.
  • Venn Diagram Visualization:
    • AND: Retrieves results with Peanut Butter and Jelly.
    • OR: Retrieves results with peanut butter, with jelly, and with both.
    • NOT: Retrieves results with peanut butter, and exclude those with jelly or PB with jelly.
  • Some database searches allow you to combine Boolean operators and parentheses or brackets to enhance your search further. Information in these parentheses or brackets is read first. Then the information outside the parentheses and brackets is applied to the search.
  • For example:
    • (college OR university) AND athletics will search for all articles containing "college athletics" or "university athletics" as if you had searched for college AND athletics and then done a second search for university AND athletics, saving you valuable time if you wanted to search for both of these synonyms.
    • Often times these databases will have an Advanced Search feature that may make this process easier to understand and enter. Different databases may treat parentheses and the order of Boolean Operators differently, so if you plan to use them extensively in a particular search, check the database's help features or consult a librarian if you are confused or not receiving the results you believe you should be receiving.
Tags
  • Commonly Used Field Tags:
    • Author [AU]
    • Date of Publication [DP]
    • Journal [JO]
    • Pagination [PG]
    • Volume [VI]
    • Issue [IP]
    • MeSH Terms [MH]
    • Language [LA]
    • Title [TI]
    • Title/Abstract [TIAB]
Text Availability
  • Common Text Availability Filters:
    • Abstract
    • Free full text
    • Full text
ClinicalTrials.gov

ClinicalTrials.gov is a database of privately and publicly funded clinical studies conducted around the world and was developed and is maintained by the National Center for Biotechnology Information (NCBI), at the U.S. National Library of Medicine (NLM), located at the National Institutes of Health (NIH)

Searching ClinicalTrials.gov
  • Search parameters (all fields optional)
    • Status : Recruiting and not yet recruiting studies, all studies
    • Condition or disease (For example: breast cancer): Obesity
    • Other terms (For example: NCT number, drug name, investigator name) : oleoylethanolamide
    • Country
Example Clinical Trials Listed
  • Example information provided for listed trials:
    • Status
    • Study Title
    • Conditions
    • Interventions
    • Locations
  • Study Design
    • Study Type: Interventional (Clinical Trial)
    • Estimated Enrollment: 120 participants
    • Allocation: Randomized
    • Intervention Model: Parallel Assignment
    • Intervention Model Description: We are testing the hypotheses that high fat diet consumers will show the greatest benefit from supplementation with oleoylethanolamide (OEA) in terms of weight loss maintenance, because OEA targets brain adaptations that are related to high fat diet.
    • Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
    • Primary Purpose: Basic Science
    • Official Title: Targeting the Gut-brain Axis to Facilitate Weight Loss in High Fat Diet Consumers
    • Actual Study Start Date: April 6, 2021
    • Estimated Primary Completion Date: February 26, 2026
    • Estimated Study Completion Date: February 26, 2026