Biological Screening of Drug Activities Lecture 2

Biological screening of Drug Activities Dr. Maha Abdollah Lecture 2  What is Biological screening of drug activities? What is drug screening? ✓It is the process by which a compound is proven to be either pharmacologically active or inert. ✓It is...

Biological screening of Drug Activities Dr. Maha Abdollah Lecture 2  What is Biological screening of drug activities? What is drug screening? ✓It is the process by which a compound is proven to be either pharmacologically active or inert. ✓It is carried out to determine any possible pharmacological activity the tested compound might have (qualitative analysis)  Drug Development process oWhy haven’t we cured cancer, Alzheimer's, dementia, diabetes… yet? oHow to overcome the emerging problem of antibiotic resistance? Where does this module fit in the Drug Development process? From Rang and Dale’s Pharmacology, 8th Edition page 719  Steps of Pre-Clinical Studies File for approval as an Investigational New Drug (IND) 5 4 Establish Effective and Toxic Doses Screen the Drug in the Assay 3 Develop a Bioassay 2 Indentify a Drug Target 1 Let’s watch the video….. The drug development process and clinical Trial Phases Summary STEPS OF PRE-CLINICAL STUDIES File for approval as an Investigational New Drug (IND) 5 4 Establish Effective and Toxic Doses Screen the Drug in the Assay 3 Develop a Bioassay 2 Indentify a Drug Target 1 Regulatory approval Clinical Phase 4 Post marketing surveillance Pharmacovigilance  IDENTIFY DRUG TARGET WHO PRIORITY MEDICINE Which pharmacological classes are in desperate need for new drugs? Gap 1 Antimicrobials and Treatment(s) exist but will Influenza pandemic soon become ineffective Gap 2 WHO Priority Treatment(s) exist but the pharmaceutical delivery CVS, Cancer, HIV/AIDS, Medicine and mechanism or formulation is Depression Pharmaceutical gaps not appropriate for the target population Gap 3 Acute Stroke, Treatment does not exist OR Osteoarthritis, Alzheimer's is not sufficiently effective. and dementia  Disease models How to mimic a biological system? MOLECULAR LEVEL Molecular level Cellular level (in vitro) Organ/tissue level (ex-vivo) Organism level (in vivo) Why are these terms in italic? Because they are in Latin In vivo = in a living body Ex vivo = outside the living body In vitro = in glass  MOLECULAR ASSAYS Directed at a single subcellular target: to measure/analyze DNA, RNA, gene expression, protein level, protein-protein interactions, etc. Examples: PCR Gel electrophoresis Western Blot Southern Blot Sequencing ELISA (Enzyme-linked immunosorbent assay) Can be divided into proteomics and genomics These topics have been covered before in the Biochemistry module. (Please revise)  CELLULAR ASSAYS (IN VITRO) MOLECULAR LEVEL Cell culture refers to the removal of cells from their normal multi-cellular organism and their subsequent growth in a favorable artificial environment to maintain their viability  How are cell lines derived? The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation. Favourable artificial environment  Culture conditions-I Culture conditions vary widely for each cell type, but the favorable artificial environment in which the cells are cultured invariably consists of a suitable vessel containing the cell culture media. Cell culture media is is a solution rich in essential nutrients that can help maintain the growth of cells outside their normal environment they consist of: ✓a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals) ✓growth factors ✓hormones ✓gases (O2, CO2) ✓In a regulated physico-chemical environment (pH, tonicity &temprature)  The properties of Culture Medium Isotonic. Buffers to maintain the pH at7.4 Salts and trace elements. Amino acids and vitamins. Energy source (carbohydrates) as glucose. Fatty acids and lipids ❖The culture medium should be supplemented immediately before use by: ✓fetal bovine serum (FBS): a complex mix of albumin, growth factors and lipids (the most important component of medium) ✓antibiotics such as penicillin and streptomycin to prevent any infection  Culture conditions-II Suitable vessel: Sterile single-use disposable plastic tissue culture flasks are usually treated to provide a hydrophilic surface to facilitate attachment of anchorage dependent cells. Most cells are anchorage-dependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture).  Can cells live forever in culture? SENESCENCE VS. IMMORTALITY Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as senescence; these cell lines are known as finite. However, some cell lines become immortal through a process called transformation. When a finite cell line undergoes transformation; it acquires the ability to divide indefinitely → so it becomes a continuous cell line. Can you give an example of immortal cells? Cancer cells (will be explained in more details next lecture)  CELL LINEs Transformation Finite cell lines Continuous cell lines ✓Limited life span ✓Grow indefinitely ✓Go through a limited number of ✓Grows either in monolayer or in cell generation suspension ✓Properties: ✓Properties: o Contact inhibition o Absence of contact inhibition o Density limitation o No density limitation ✓Less growth rate ✓High growth rate ✓Doubling time (24-92hrs.) ✓Doubling time (12-24hrs.)  Physiological assays MOLECULAR LEVEL Organ/tissue level (ex-vivo) Organism level (in vivo)  EX VIVO ASSAYS These techniques employ an isolated tissue or organ of a recommended living system to study the effect of tested samples in suitable physiological conditions within the stipulated time of organ survival outside the body. e.g. Use of any isolated organ from animals (heart, kidney, muscle, uterus..etc.) in a glassware to study the effect of test compound within the period of its survival outside the living body with provision of only oxygen, glucose and isotonic salts to maintain cell & cell organelles integrity.  IN VIVO ASSAYS These techniques aim to study the biological effect or response of the test compound under screening in a living system directly. e.g. rodents, rabbits, frogs, monkeys, dogs etc. Most commonly used are rodents ANIMAL MODELS  TYPES OF ANIMAL MODELS 1. Spontaneous or inherited 2. Induced (experimental) 3. Genetically modified  TYPES OF ANIMAL MODELS 1-SPONTANEOUS Naturally occurring animal disease or conditions correspond to the same as in human Spontaneous models are genetic variants, which mimic the human condition. The variance occurs naturally through mutation and not by experimental induction. Example: ✓Nude mice (nu): a strain with a spontaneous genetic mutation that causes a deteriorated or absent thymus, resulting in an inhibited immune system due to a greatly reduced number of T cells. It also leads to loss of hair (see picture). ✓This leads to their inability to reject xenografts (tissues from other species) and therefore they became a very valuable research tools for implantation of human tumours.  TYPES OF ANIMAL MODELS 2- INDUCED (EXPERIMENTAL) The disease is experimentally created either through surgical modifications, chemical injections or change of diet. Examples: Myocardial infarction is induced by coronary artery surgical ligation Streptozotocin induced diabetes in rats Atherosclerosis induced by feeding with high fat diet Experimentally produced cancer by injecting aromatic amines or hydrocarbons like DMBA (Dimethyl benz anthracene) organ specific carcinogen. It serves as a tumor initiator in murine models. Rotenone induced Parkinson's disease  TYPES OF ANIMAL MODELS 3- GENETICALLY MODIFIED Transgenic models are produced by the insertion of foreign DNA by micro- injection (knock in) OR removal/replacement of specific genes (knockout). They are strong animal model for disease specific research KO Transgenic mice normal expressing green Knock out (KO) of normal hair gene fluorescent protein (GFP), which glows green under blue light.  Biological variability In order to fulfill the requirements for a good bioassay; experimental design must aim to reduce biological variability Biological variability: each measured biological parameter is liable to variation when re-measured.  Individual Variation Species Variation Biological variations from Biological variations due animal to animal within to species difference the same species What causes biological variability? Diurnal Variation Seasonal Variation Biological variations from Biological variations from day time to night time one season to another  How to reduce biological variability? 1. Selection of animals 2. Replication 3. Use of controls and reference standards 4. Bias counteraction (randomization & blinding)  1- SELECTION OF ANIMALS Studies done in vitro should be done on the same cell line extracted from the same species of animals. e.g. can’t test the test compound on a human cell line and the standard on a murine cell line and compare the results. Studies done in vivo on animals should be of the same species, age, weight, sex..etc.  2- REPLICATION The effect of the drug on a given parameter should be repeated several times and the mean value (average) is taken to reduce the variability and error. (↑No.----> ↓ variability & error ) 3 to 6 wells per treatment (in vitro) or 6-30 animals/ group (in vivo) within the same experiment. The entire experiment should also be repeated at least 3 times.  3- USE OF CONTROLS AND REFERENCE STANDARD Aim: to reduce the biological variability due to changes in procedure, time or conditions. There are international standards which are used for comparison and their activity is constant.  4- BIAS COUNTERACTION Research bias, also called experimenter bias, is a process where the scientists performing the research influence the results, in order to portray a certain outcome. Two approaches could be done to reduce bias a) Randomization b) Blinding  4- BIAS COUNTERACTION A) RANDOMIZATION A method based on chance alone by which study participants (humans, animals, etc.) are randomly assigned to a treatment group. Randomization minimizes the differences among groups by equally distributing subjects with particular characteristics among all the trial arms (test vs. control or standard).  4- BIAS COUNTERACTION B) BLINDING (MASKING) A blind or blinded-experiment is an experiment in which information about the test is masked (kept) from the participant and/or the researcher, to reduce or eliminate bias. ✓Randomization reduce selection bias ✓Blinding reduce observation bias