Preclinical study. Assist.Prof. Witthawat Wiriyarat Faculty of Veterinary Science, Mahidol University

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1 Preclinical study Assist.Prof. Witthawat Wiriyarat Faculty of Veterinary Science, Mahidol University

2 Overviews Principle of animal use Biological activity/pharmacodynamics Animal species and model selection Number/gender of animals Administration/dose selection

3 History of the Use of Animal Models in Biomedical Science Erasistratus of Alexandra ( BC) carried out the research on the functions of the heart and respiratory systems in pigs. The first textbooks on anatomy by Galen ( ) were based on dissections on pigs and apes but not on human cadavers (which was forbidden by religious and legal authorities)

4 Louis Pasteur ( ) used animals in the validation of the experimental method in microbiology Pasteur investigated anthrax and rabies pathogenesis in animal models Pasteur further enhanced the concept of vaccination of dogs against rabies virus and finally tested his theory of vaccination on Joseph Meister, who had been bitten by a rabid dog

5 Koch s postulate Robert Koch in Germany introduced the concept of specificity into medicine and the germ theory of disease

6 History of the Use of Animal Models in Biomedical Science In the 20 th century, cardio-pulmonary resuscitation, the discipline of immunology and translational research on organ transplantation were all primarily developed through the use of animal models. Today, animal models are used in virtually every field of biomedical research

7 On November 3, 1957, a Russian dog, Laika, became the first animal to orbit the earth

8 WHAT IS AN ANIMAL MODELS? An animal model in which normative biology or behavior can be studied, or in which a spontaneous or induced pathological process can be investigated, and in which the phenomenon in one or more respects resembles the same phenomenon in humans or other species of animal. (US.NRC) Source of photo:

9 Why do we need animals for research & education? Animal models are used to: Help researchers understand the fundamental ways in which diseases affect living tissue. Develop and test treatments for illness or injury Develop new methods and approaches to the cure and alleviation of disease and disability Train future scientists and physicians.

10 Can Results from Animal Studies Really Be Applied to Humans? There are many similarities between animals and man. Examples include: Immune function in mice Cardiovascular function in dogs Animals provide index of safety. Nuremberg Code mandates that animal studies precede and support human studies. Declaration of Helsinki mandates that medical research on humans must be supported by preceding animal research. Nearly all medical advances of the past century started with research in animals.

11 Animal models Invertebrate models are very useful in the fields of neurobiology, genetics and development and notable examples of invertebrates use for such purposes include the C. elegans and Drosophila.

12 Animal models Vertebrate models are responsible for many advances in biology and medicine and are extremely important in translational research. This includes the use of both small animal models (e.g. mice, rats, rabbits) and large animal models (e.g. dogs, pigs, monkeys)

13 Broad areas of animal model are used in biomedical research include; 1. Pharmaceutical research including the development of biological products 2. Toxicology testing 3. Development and testing of new medical devices 4. Surgical research 5. Pathophysiological research

14 Broad areas of animal model are used Generally, two or more species (one rodent, one non-rodent) are tested because a drug may affect one species differently from another Besides treatment efficacy, animal models are also used to determine the pharmacokinetics

15 Ethical concern for animal using Three-Rs concept Replacement Reduction Refinement

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17 CLASSIFICATION OF DISEASE MODELS 1. Induced (experimental) disease models 2. Spontaneous (genetic) disease models 3. Transgenic disease models 4. Negative disease models 5. Orphan disease models

18 Relevant animal model for nonclinical evaluation of vaccines An animal that develops an immune response similar to the expected human response after vaccination. It is acknowledged that species specific differences in immune responses are likely. Ideally, the animal species chosen should be sensitive to the pathogenic organism or toxin under consideration. (WHO guidelines on nonclinical evaluation of vaccines)

19 Use of Animal Models in Vaccine Development Animal models can be used in a variety of ways to study Disease pathogenesis, route of infection and transmission of diseases Characterization of host immune response to natural infection and vaccination Mechanisms of protection following vaccination, infection or treatment of disease

20 Use of Animal Models in Vaccine Development Vaccine immunogenicity and potency testing Demonstrate safety (Tox studies) Development for new method for vaccine delivery and formulation Development for novel vaccination concept such as in utero or maternal immunization Enable selection of a protective dosage for humans

21 Biological activity/ pharmacodynamics Vaccine biological activity was measured by potency test but do not necessarily reflect the mechanism of protection in humans. Classical challenge studies in animals immunized with the vaccine under consideration have been developed into routine potency assays e.g. for diphtheria and tetanus toxoids. Where no suitable animal challenge model exists, potency is often based on measurement of immune responses, usually serological (e.g. influenza and hepatitis B vaccines).

22 Biological activity/ pharmacodynamics A pharmacodynamic study for a vaccine product is generally conducted to evaluate the immunogenicity and may also extend to include the pharmacology of an adjuvant. Immunogenicity data derived from appropriate animal models may guide selection of the doses, schedules and routes of administration to be evaluated in clinical trials.

23 Biological activity/ pharmacodynamics Nonclinical studies should be designed to assess relevant immune responses including functional immune response leading to protection It should be recognized that animal models frequently fail to predict immunogenicity and efficacy in humans.

24 Animal species and model selection Ideally, for vaccine safety studies, the selected species should fulfil several criteria: The selected species should develop an immune response following immunization (humoral and/or cell mediated) that is similar to the expected response in humans after vaccination.

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26 Animal species and model selection The selected species should be susceptible to the pathogen, reflecting the course of infection in man (and in the case of live attenuated vaccine strains, permitting evaluation of viremia/bacteremia). Two or more species may be necessary to characterize the product, for example where the mechanism of protection induced by the vaccine is not well understood In general, the use of outbred animals is recommended.

27 Considerations for Animal Model Selection Species Susceptibility to pathogen Similarity to human response Display similar characteristics to human disease and pathogenesis Immune response Route and volume of administration Endpoints of study Manipulations required Cost Facility space and required biosafety containment level Availability of sufficient animals Availability of reagents for immunological analyses

28 Animal models for influenza virus

29 Summary of signs of disease and pathology present in the different animal models for influenza virus Margine I and Krammer F, Pathogens, 2014

30 Animal immunodeficiency retrovirus

31 HIV/AIDS animal models Chimpanzees Macaques/SIV or SHIV SCID-hu mouse

32 Specific animal models AG129 mice for DENV

33 Number/gender of animals The size of the treatment group depends on the animal model chosen. The number of animals used in studies using nonhuman primates would be expected to be less than that in studies that used rodents For small animal models, e.g. rats and mice, it is recommended that approximately 10 males + 10 females per group be studied. In general, the approximate age at the start of the study for rodents is 6 8 weeks, and for rabbits, 3 4 months. Source: WHO

34 Number/gender of animals For adjuvanted vaccine, for mice and rat, it is recommended that at least 10 animals/sex/group be used for the necropsy at the end of the treatment interval, and at least 5 animals/sex/group be used for the necropsy at the end of the recovery period. For rabbits, it is recommended that at least five animals/sex/group/time interval be used.

35 Group of animals The study design should include several group of animals: Negative control group(s) Active control groups (e.g. vaccine formulation without antigen) Treatment group

36 Administration/dose selection The toxicity study should be performed using a dose that maximizes exposure of the animal to the candidate vaccine and the immune response induced for example, peak antibody response. If feasible, the highest dose (in absolute terms) to be used in the proposed clinical trial should be evaluated in the animal model.

37 Administration/dose selection However, the dose is sometimes limited by the total volume that can be administered in a single injection, and guidelines on animal welfare should be followed. Alternatively, a dose that exceeds the human dose on a mg/kg (HED) basis and that induces an immune response in the animal model may be used.

38 Administration/dose selection The number of doses administered to the test animals should be equal to or more than the number of doses proposed in humans. The dosing interval used in the toxicity study may be shorter (e.g. an interval of 2 3 weeks) than the proposed interval in clinical trials in humans.

39 Forster R, 2012

40 Route of administration The route of administration should correspond to that intended for use in the human clinical trials. Traditional routes are by intramuscular, subcutaneous or intradermal routes. There is also current interest in alternative routes of administration for vaccines including mucosal routes (such as nasal, rectal or vaginal), oral and topical administration, and these routes can generally be achieved without difficulty in laboratory animals.

41 Single dose toxicity study Provide preliminary tolerability and safety data for a new vaccine Useful information in order to evaluate the acute actions of a vaccine These studies are generally performed in rodents, and typically the study design will be that of a rodent acute toxicity study with administration of the FHD or greater. Forster R, 2012

42 Local tolerance The purpose of local tolerance studies is to evaluate tissue reactions at the site of administration by gross observation and by histopathology. If there are marked reactions, follow-up studies may examine the persistence of material (vaccine antigen and/or adjuvant) at the injection site and in draining lymph nodes can usually be made during the repeat-dose toxicity study or stand-alone study can be set up for more detailed investigations Forster R, 2012

43 Repeated dose study The treatment regime used in the nonclinical study should follow the proposed clinical regime In order to provide confidence in the safety of the dosing schedule, the number of administrations in the toxicity study should exceed the number planned for human administration The full human dose should be tested exactly as it will be given in the proposed clinical use, in the same formulation and at the same volume.

44 Monitoring of the pharmacodynamic response during the repeat dose toxicity study is a valuable element of the study; confirm the relevance of the animal model permits identification of non-responders correlation of the toxic actions with the measured immune response

45 Special Considerations Adjuvants Additives (Excipients and preservatives) Vaccine formulation and delivery device Alternate routes of administration particular types of vaccines

46 Example 1. Forster R, 20

47 Example 2. Forster R, 201

48 Forster R, 201

49 Example 3. Wiriyarat W, 2008

50 Wiriyarat W, 2008

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52 Wiriyarat W, 2008

53 Reference WHO guidelines on nonclinical evaluation of vaccines, WHO Technical Report Series, No. 927, Forster R. Study designs for the nonclinical safety testing of new vaccine products. Journal of pharmacological and toxicological methods. 2012;66(1):1-7.

54 Thank you