Biological Screening of Drug Activities PDF

Biological Screening of Drug Activities Dr. Maha Abdollah Lecture 4 Anti-cancer Bioassays part II (in vivo)  IDENTIFY DRUG TARGET WHO PRIORITY MEDICINE Which pharmacological...

Biological Screening of Drug Activities Dr. Maha Abdollah Lecture 4 Anti-cancer Bioassays part II (in vivo)  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  Hallmarks of Cancer What are the main properties of cancer cells?  Accelerator = oncogene Definition of Cancer Brakes = Tumour suppressor gene ✓ A term for diseases in which abnormal cells divide without control and can invade nearby tissues. ✓ If we imagine a normal cell is like a car, its movement (division) is governed by the gas (oncogenes) and the brakes (tumour suppressor genes) then a cancer cells is like a car which lost its brakes leading to a speeding car down a cliff (i.e. uncontrollable cell division)!  What is the Main Therapeutic Effect of Anticancer Agents? Cancer arise form a single malignant cell, the therapeutic goal of cancer chemotherapy may require killing all actively growing tumour cells. Antineoplastics or Anticancer drugs are the drugs that prevent or inhibit the proliferation of neoplasms. Achievement of a therapeutic effect by combination therapy.  Then, how to design a bioassay to test the effect of a new anti-cancer drug? Generally speaking we are looking for drugs or treatments that inhibit cell proliferation Reduce tumour growth & prolong survival in vivo  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  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  Why mice are the most commonly used animals for anti-cancer drug screening? 1. Mice are small, easy to handle. 2. Short generation time & accelerated lifespan 3. manageable costs, space, and time. 4. Striking similarity to humans in anatomy, physiology, and genetics. 5. Over 95% of the mouse genome is similar to humans. Many of the genes responsible for complex diseases are shared between mice and humans. 6. Mouse genome can be directly manipulated.  Types of Animal Models (In General) 1. Spontaneous or inherited 2. Induced (experimental) 3. Genetically modified  Types of Animal Models Used in Cancer 1. Transplantable 2. Carcinogen Induced 3. Genetically modified 4. In vivo hollow fiber assay  Types of Cancer Animal Models 1-Transplantable There are 2 types of transplantation models: Allograft and Xenograft Allograft transplantation models (Syngeneic models) Transplantation between two genetically identical individuals of the same species. Because theTransplantation cancer tissues andModels the recipient share ancestry, the transplant : Cell line based allografts is not rejected by the host's immune system. e.g. transplantation of murine cancer cells into a mouse of the same strain from which the cells have been derived.  Types of Cancer Animal Models 1-Transplantable ansplantation There are 2Models types of :transplantation Human Tumor Xenografts models: Athymic “nude”mice developed in 1960’s Xenograft transplantation models Mutation in nu gene on chromosome 11 Phenotype: retarded growth,between Transplantation low fertility, two genetically different individuals of different species. This no fur, immunocompromised method involves actual human cancer cells or solid tumors which are transplanted into a – Lack thymus gland, T-cell immunity host mouse. The host mice are special, that they have impaired immune systems and the First human tumor xenograft of colon adenocarcinoma by Rygaard foreign cells will not be rejected by the host. & Poulson, 1969 14 Evaluation of anticancer agents - Dr. Anup Thorat 42  Xenograft Transplantable Models ✓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 immunocompromised mice and their inability to reject xenografts (tissues from other species) and therefore they became a very valuable research tools for implantation of human tumours. ✓Major advantage: Because the cancer carries human genetic material it may be more representative of the properties and mutations of the human cancer. ✓Major disadvantage: Because of the changes to the host immune system, it may not mimic the situation in actual patients. It is costly to maintain these animals due to their compromised immunity  Xenograft Sites Xenograft Sites Subcutaneous tumor (NCI method of choice) with IP drug administration Intraperitoneal Intracranial Intrasplenic S.C model in nude mice Renal subcapsule Site-specific (orthotopic) organ inoculation  Xenograft Study Endpoints Toxicity Endpoints: – Drug related death – Net animal weight loss Efficacy Endpoints: – Tumor weight change – Treated/control survival ratio – Tumor growth assay (corrected for tumor doubling time)  Types of Cancer Animal Models 2- Carcinogen Induced The disease is experimentally created through chemical injections of a carcinogen. Carcinogen: a substance which can cause cancer either by inhaltion, ingestion or skin contact. Studies invoking cancerous changes in mouse models help us to understand many of the environmental assaults that we face, and will aid in the development of effective treatments for resulting cancers. However, the main disadvantage of these models is they can take a very long time to develop  Types of Cancer Animal Models 2- Carcinogen Induced Cancer site Cancer Type Species Carcinogen Colon Adenocarcinomas Rat AOM (azoxymethane) Prostate Adenocarcinomas Rat MNU (methylnitrosourea) Esophagus Squamous cell Rat NMBA(N-nitroso- carcinoma methylbenzylamine) Breast Adenocarcinoma Mice NMU (N-Nitroso-N- methylurea)  Types of Cancer Animal Models Carcinogen-Induced 2- Carcinogen Induced (PROS & CONS) Advantages Disadvantages – Mimics initiation steps of – Health hazard to some cancer investigator – Can study early events – Variability of disease – Used to id predisposing progression conditions – Can require large animal – Can study prevention numbers – Penetrance (all animals may not get disease)  Types of Animal Models 3- Genetically Engineered Transgenic models are produced by the insertion of foreign DNA by micro- injection (transgenic or knock in) OR removal/replacement of specific genes (knockout). The genes manipulated with this technique could be: ✓Oncogenes (KI) ✓Tumor suppressor genes (KO) ✓Growth factors (KI) ✓Cell cycle regulators (KO/KI) https://emice.nci.nih.gov/  Types of Animal Models 4- In vivo Hollow Fiber Assay ✓The hollow fiber assay (HFA) is a fast in vivo assay to determine the cytotoxic effect of drugs, as well as their pharmacodynamic effects on human tumor cell lines grown in hollow fibers that are implanted subcutaneously or intraperitoneally in mice or rats. ✓The HFA has been optimized at the National Cancer Institute (NCI) and is a unique in vivo model for drug discovery 22 In Vivo Hollow Fiber Assay Semipermeable biocompatible fibers made of polyvinylidene fluoride (PVDF) filled with cancer cells are implanted i.p or s.c. usually in mice but other species can be used One mouse can support the growth of up to twelve cancer cell lines, and therefore enables to test more than one cell line simultaneously in one animal. The pores in the fiber allow soluble bioactive agents (e.g., proteins, such as growth factors produced by tumor cells or host) to bypass the fiber, but tumor cell to host cell contact is not possible.  IN VIVO HOLLOW FIBER ASSAY (CONT.) Can be transplanted in both immunocompromised and in immunocompetent mouse strains, since the immune cells of the host can not infiltrate into the hollow fiber After treatment, fibers are removed and active cell proliferation and cellular characteristics are analyzed in vitro, including DNA damage induction, cell death induction (Apoptosis), protein expression levels and cell morphology. In the HFA the anti-cancer drug activity is defined as a reduction of the number of viable cells (e.g. using MTT assay).  NCI Hollow Fiber Assay The NCI currently uses a panel of 12 tumor cell lines for routine hollow fiber screening of anticancer drug activities. It is used as the initial in vivo assessment to determine potential activity of agents that have reproducible activity in the in vitro anticancer drug screen  Pros of NCI Hollow Fiber Assay 1. Provide info about drug efficacy and saves time (procedure takes less than 2 weeks)& materials (e.g., test compounds). Unlike high costs of large-scale animal testing using xenograft tumor models. 2. The HFA will prevent testing inactive compounds in a xenograft model in case the experimental agents exhibit only minimal anti-tumor activity. 3. Cancer treatments that appear promising in vitro are often less effective in solid tumors because of the proliferative and microenvironmental heterogeneity that develops in these tumors during their growth in a three-dimensional structure; cells in a hollow fiber also grow in a three-dimensional structure.  Pros of Hollow Fiber Assay (cont.) 4. The pharmacokinetic behavior & potential biotransformation of the drug is taken into account. 5. HFA is excellently suited to perform initial in vivo experiments to study combinations, because the compounds will have a dynamic in vivo interaction.  Cons of Hollow Fiber Assay 1. The HFA was not developed to replace the classic xenograft model. 2. Complex interactions which occur when the transplanted tumor cells are growing in and interacting with the host’s tissue can not be analyzed. 3. Cells can not metastasize to secondary organs of the animal, since the fiber prevents cellular migration. 4. Although angiogenesis in the tumor will not be induced when these fibers are used, neovascularization occurs to supply the fiber with nutrients. This is possibly induced by growth factors that are released by the tumor cells in the fiber.  Summary-Hollow Fiber Assay ✓Therefore, the hollow fiber assay can be used perfectly as a preliminary tool to asses the capacity of a compound to reach tumor cells growing in two distinct physiologic compartments (s.c. or i.p.) ✓ Useful to asses whether the drug can reach pharmacologically active concentrations in the tumor cells.  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, which can occur spontaneously or can be chemically or virally induced. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line. Can you give an example of immortal cells? Cancer cells (will be explained in more details next lecture) Do you know who are these women? A woman who revolutionized medicine and biomedical research without even knowing it! Oprah Winfery playing Deborah Oprah Winfery Henrietta Lacks Lacks  The Immortal Life of Henrietta Lacks A non-fiction (true story) book written by Rebecca Skloot, a science journalist Her name was Henrietta Lacks, but scientists know her as HeLa. She was a poor black tobacco farmer whose cells— taken without her knowledge in 1951—became one of the most important tools in medicine, vital for developing the polio vaccine, cloning, gene mapping, in vitro fertilization, and more. Henrietta’s cells have been bought and sold by the billions, yet she remains virtually unknown, and her family can’t afford health insurance. The story timeline And the ethical debate continues……..  Overview Design a flyer, leaflet, comic, HISTORY OF HELA flash cards, poster, CELLS video, etc. →Henrietta’s story PROJECT Present a research paper that used Each group will have 15 minutes RESAERCH PAPER HeLa cells in drug screening  Project Description Divide yourselves into 5 groups (7-8 students per group) You will be asked to do a 10-15 minutes group presentation and all the students will have to talk during the presentation and all the students will be asked questions The project will include 2 main sections: 1. A brief history about HeLa cells and the story behind them 2. Pick a recent research paper in which HeLa cells were used and present it  1- A Brief History about HeLa cells & the story Behind Them Design a flyer, leaflet, comic, cards, poster, video, etc. (whatever you want) to tell Henrietta’s story There are a lot of resources that tell the story of the HeLa cells for example: The book: The Immortal Life of Henrietta Lacks The website of the book author: http://rebeccaskloot.com/the-immortal- life/ The movie (same name as the book) The internet! Be creative and find more resources!!  PICK A SCIENTIFIC DISCOVERY IN WHICH HELA CELLS WERE USED AND BRIEFLY TALK ABOUT IT For example I picked nanotechnology Find information about how HeLa cells helped in the development of the field of nanotechnology  2- PICK A RECENT RESEARCH PAPER IN THE SELECTED FIELD AND PRESENT IT Try to find a paper which is short and easy to read Look for recent papers between 2000 and 2018 You will understand in more details how to find and read a paper in the tutorial Make sure you understand the paper as you will be asked questions…. For instance I found this paper…  Project Marking Criteria Mark Assessment Criteria -Good flow of information from one section to the next Presentation 5 -Good command of English Skills -Clear and easy to follow and understand Knowledge -The student was able to answer all the questions coherently and 5 understanding -The students show good understanding of the topics covered in of the topic the presentation -Creativity and innovation in presenting the information 5 Originality -Going beyond the recommended resources Total marks (15 marks) The project accounts for 15% of the total module marks