In vivo -studies

Mari Madetoja, ERT

Made Consulting

mari.madetoja@madeconsulting.fi

Nobel Prizes

Since the beginning of the 20th Century, these prizes have charted the world's greatest medical advances. Of the 106 Nobel Prizes awarded

for Physiology or Medicine, 94 were dependent on research using animals. This includes every prize awarded for the past 30 years.

2016: Yoshinori Ohsumi - in vitro

2017: Jeffrey C. Hall, Michael Rosbash ja Michael W. Young - for their discoveries of molecular mechanisms controlling the circadian

Rhythm (Drosophila)

http://www.animalresearch.info/

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In vivo - statistics

• http://www.laaninhallitus.fi/lh/etela/hankkeet/ellapro/home.nsf/pages/BFD5CAFA94D8E7C7C225728A00475B11?opendocument

• 2016 - 105 615 operations – Academic studies

– Research purposes or

– Teaching

• 168 548 animals were housed but not operations were done (40% TG purposes)

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Contents

• Standards

– Toxicity

– PK

– Efficacy

• Different Animal Models

• Case: Cancer study

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Non-clinical guidelines

http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000083.jsp

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Compounds ‘‘drop out’’ of the process primarily for three reasons

1. Toxicity or (lack of) tolerance

2. (lack of) efficacy

3. (lack of) bioavailability of the active moiety in man -> pharmocokinetics

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©Gad.S.C._Drug Safety Evalution (2002)

Safety vs. efficacy vs.

pharmocokinetics

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Safety – non-clinical safety testing

• Safety pharmacology

• Acute (single dose) toxicity

• Subchronic/chronic (repeated dose) toxicity

• Genotoxicity

• Carcinogenicity

• Reproductive toxicity

• Special toxicity (immunotoxicity, local tolerance, environmental risk assessment, etc.).

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List of needed safety studies

• i. Acute toxicity study in rat (route to be finalized) • ii. Acute toxicity study in mice (route to be finalized) • iii. A 10-14 days dose range finding study in rats (non-GLP) • iv. A 10-14 days dose range finding study in non rodents (rabbits or dogs) (non-GLP) • v. Repeat dose 14 (minimum), 28 or 90 days toxicity study in rats • vi. Kinetic study • vii. Repeat dose 28 or 90 days toxicity study in non rodents (dog) • viii. Male fertility studies in rats • ix. In vitro genotoxicity studies:

– a. AMES test and/or – b. In vitro Chromosomal Aberration or – c. Mouse Lymphoma Assay

• x. Local toxicity Study (not sure, depends on route of administration) • xi. Allergenicity / hypersensitivity Study (if required):

– a. GPMT (Guinea Pigs Maximization Test) or – b. LLNA (Local Lymph Node Assay) in mouse – c. Buhler test

• xii. safetypharmacology studies – a. CV – b. CNS – c. respiratorysystem

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Results of a safety studies

• Toxicity limits

– No Observed Effects Level (NOEL)

– No Observed Adverse Effect Level (NOAEL)

– Lowest Observed Adverse Effect Level (LOAEL)

– Lowest Observed Effect Level (LOAEL)

• Target organs(s)

– Possible recovery

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Efficacy studies

• How to prove the efficacy

– In animal model

• Proper animal model???

– How to prove in vitro results in vivo?

• When in vitro efficacy results are not needed prove in vivo before clinical trials?

• Case by case

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Results of Efficacy studies

An idea about the therapeutic dose level

(dose have to be under toxic level - safety margin x10-50)

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Pharmacokinetics (PK) - ADME

• Absorption - the process of a substance entering the blood circulation.

• Distribution - the dispersion or dissemination of substances throughout the fluids and tissues of the body.

• Metabolization (or biotransformation, or inactivation) – the recognition by the organism that a foreign substance is present and the irreversible transformation of parent compounds into daughter metabolites.

• Excretion - the removal of the substances from the body. In rare cases, some drugs irreversibly accumulate in body tissue.

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Result of PK study

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Metabolities

The nonclinical characterization of metabolites with an identified cause for concern (e.g., a unique human metabolite) should be considered on a case-by-case basis

Usually the metabolites are well known (new drugs)

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NOTE

• You can not mix the efficacy study and toxicity/safety studies

– Two different study types

• Toxicity – over doses (GLP-study)

• Efficacy – therapeutic doses (non-GLP study)

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Quality of animal research

• The methodological quality of animal research in critical care: the public face of science (Annals of Intensive Care 2014, 4:26) (N=77) – Animal strain, sex, and weight or age were reported in

52 – 47 reported randomization – 31 reported some type of blinding – A sample size calculation was reported in 4 – Animal numbers were missing in the Methods section

in 16 – Extra animals used were mentioned in the Results

section in 31 (this number was unclear in 23)

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Carlijn R. Hooijmans, Marlies Leenaars and Merel Ritskes-Hoitinga ATLA 38, 167–182, 2010 :

A Gold Standard Publication Checklist to Improve the

Quality of Animal Studies, to Fully Integrate the Three Rs,

and to Make Systematic Reviews More Feasible

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Items from publication checklist

– Experimental design – Temperature – Ventilation – Humidity – Lighting – Bedding – Cage size/cage space/group size – Cage enrichment – Individual housing – Cage change – Handling/transport – Nutrition – Water – Blinding – Randomisation

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Experimental groups and controls — Quarantine and acclimatisation period after transportation to animal facility

— Species — Designation of strain (exact genetic code) — Origin and source of animals — Genetic background (outbred, inbred, F1 hybrid, mutant, transgenic, congenic, consomic, etc.) and generation — Definition of the experimental unit (individual animal/animals in one cage) — Number of animals per group (and possibly power and sample size calculations) — Sex — Age (at the beginning and the end of the experiment) — Weight (at the start of the experiment) — Microbiological status ● Conventional/specified pathogen-free (SPF)/gnotobiotic, germ-free ● Measures to protect microbiological status (for example, open-system, closed- system (SPF), individually ventilated cage racks, isolation unit)

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Experimental groups and controls (continued)

— Housing: Animal room ● Temperature ± range (regulated or not) ● Relative humidity ± range (regulated or not) ● Ventilation ● Over-pressure or under-pressure ● Air changes per hour ● Lighting ● Natural or artificial ● Number of hours light per 24 hours ● Light intensity ● Time when light is switched on ● Transitional decrease in light intensity ● Noise (music, etc.)

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Experimental groups and controls (continued)

— Water ● Type (analysis certificate available?)

● Pre-treatment (concentration of acidification or chlorination)

● Water schedule

● Quantity (ad libitum?)

● Frequency of water supply (in case of restriction)

● Frequency of change

● Bottles or automatic watering system

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Experimental groups and controls (continued)

— Housing: Cages ● Type and size ● Number of animals per cage (and if individually housed, why?) ● Bedding (reference; if not, type). Is batch analysis certificate available? Pre-treatment? ● Presence and type of cage-enrichment ● Frequency of cage change ● Frequency of handling

— Nutrition ● Type (natural-ingredient diets, chemically-defined diets or purified diets) ● Composition or batch number (if possible, use a reference) ● Pre-treatment ● Feeding regimes (ad libitum, meal feeding, restricted, etc.). If not ad libitum: ● Amount of food given ● Frequency and time of feeding

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The intervention

— Time schedule ● Day and time of intervention within experiment ● Time between intervention and sampling or processing

— Type of intervention — Description of operation techniques or other techniques and materials used — Dose and/or frequency of intervention (when applicable) — Administration route (enteral [oral or via the anus]/parenteral/trans-dermal) — Drugs and dose tested (product name, manufacturer, concentration) — Other products used (product name, manufacturer, concentration) — Method and time of sampling (blood, urine, etc.) — Anaesthesia (duration, type of drug and method) — Analgesia (type of drug and method) — Euthanasia (type of drug and method) — Description of general wellbeing of the animal during and at the end of the intervention and — in the case of compromised wellbeing — what relieving measures have been taken

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Different animal models

1. Induced (experimental) disease models

2. Spontaneous (mutant) disease models

3. Gene-modified disease models

1. Transgenic

2. Knock-outs

4. Chemically induced

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Induced (experimental) disease models

• Induced models are – healthy animals in which the condition to be

investigated is experimentally induced.

– often different from that of the corresponding disease in man. • The use of metrazol (pentylenetetrazol) as an animal

model of epilepsy

• Using cancer cells to cause tumors

• Occlusion of the middle cerebral artery as an animal model of ischemic stroke

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Spontaneous (mutant) disease models

• These models of human disease utilize naturally occurring genetical variants (mutants).

• Many hundreds of strains/stocks with inherited (peritty) disorders modeling similar conditions in humans have been characterized and conserved (eg. Diabetes models).

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Gene-modified disease models

• The rapid developments in genetic engineering and embryo manipulation technology during the past decade have made transgenic and knock-out disease models the perhaps most important category of animal disease models. A multitude of animal models for important diseases have been developed since this technology became available and the number of models seems to be increasing quickly.

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CURRENT EXAMPLES OF ANIMAL RESEARCH IN MEDICINE

• Addiction (monkeys) • Alzheimer’s Disease (mice) • Blindness [See also Amblyopia and transplanting retinal rod cells] (dogs, cats, mice) • Blood Thinners (rats, rabbits, monkeys) • Cystic Fibrosis (pigs) • Deep Brain Stimulation (monkeys) • Diabetes – Type I (mice) • Duchenne muscular dystrophy [Part 2] (mice) • Facial Transplants (monkeys) • Fragile X Syndrome (mice) • Heart Damage (rats) • High Cholesterol (mice, rats, rabbits, dogs, monkeys) • HIV/AIDS [Part 2] (monkeys, mice) • Huntington’s Disease (monkeys) • Human Papillomavirus (HPV) vaccine / Cervical Cancer (rabbits, cows, dogs) • Influenza [also see Swine Flu/Tamiflu] (mice) • Leber’s Congenital Amaurosis (dog) • Meningitis B (mice) • Multiple Sclerosis (mice) • Non-Hodgkin’s Lymphoma [and the treatment Rituximab] (mice, rats) • Paralysis [part 2] [and teaching animals to use paralysed limbs)(monkeys, rats) • Parkinson’s (macaque monkeys) • Polycystic Ovary Syndrome (monkeys) • Radiotherapy/Radiation Sickness (mice) • SARS (mice) • Skin cancer (flies) • Spinal Cord Injury (rats) • Stroke (rats) • TB Vaccine (mice) • Trachea Transplant (pigs, cows, dogs, rats, sheep) • Tuberculosis (Mice, Guinea Pigs) • Tumor Metastasis [and more] (mice) • X-ALD (mice)

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Terminology

• Genetically modified (GM) mouse – Transgenic – Knock out (poistogeeninen)

• Inbread – A strain is defined as inbred when it has been mated brother x sister

for 20 or more consecutive generations (F20) • A strain is congenic when it differs from a particular inbred strain at a single

locus as a result of backcrossing a particular allele into the inbred strain

• Outbread – Outbred stocks are most often used in research projects requiring

vigorous, economically priced animals, but in which considerations of specific genotype are of lesser importance

• Hybrid – F1 hybrids are the first generation of a cross between two inbred

strains

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Diabetes models

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Sampling

Species Reference weight (g) Blood volume

(ml/kg)$

Total blood volume

(TBV), normal adult

(ml)

Safe volume for

single bleed (ml)*

Bleed out volume

(ml)

Mouse 18 - 40 58.5 Male 1.5 - 2.4

Female 1.0 - 2.4 0.1 - 0.2

Male 0.8 - 1.4

Female 0.6 - 1.4

Rat 250 - 500 54 - 70 Male 29 - 33

Female 16 - 19

Male 2.9 - 3.3

Female 1.6 - 1.9

Male 13 - 15

Female 7.5 - 9

Hamster 85-150 78 Male 6.3 - 9.7

Female 7.1 - 11.2

Male 0.6 - 0.9

Female 0.7 - 1.1

Male 2.9 - 4.5

Female 3.3 - 5.2

Gerbil 55 - 100 66 - 78 Male 4.5 - 7

Female 3.8 - 6

Male 0.4 - 0.7

Female 0.4 - 0.6

Male 2.2 - 3.5

Female 1.9 - 2.9

Guinea pig 700 - 1200 69 - 75 Male 59 - 84

Female 48 - 63

Male 6 - 8

Female 5 - 6

Male 29 - 42

Female 24 - 31

Rabbit 1000 - 6000 57 - 65 58.5 - 585 5 - 50 31 - 310

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Animal producers

• Envigo (Harlan Research)

• Taconic

• The Jackson Laboratory

– Charles River Laboratories

• Janivier Labs

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CASE

Cancer study

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Test item = MC1500

Objective is to assess the an exosome-based drug delivery platform for a potent chemotherapeutic agent, paclitaxel (PTX), to treat Multidrug resistance (MDR) cancer.

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Task

• Plan an efficacy experiment

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What you have to think

1. Proper animal model

2. Samples – what are the end points?

3. Study period (one week, one month or?)

4. Control groups (positive and negative)

5. Dose

6. Dosing frequence

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Proper animal model

• Species

– Rat

– Mouse

– Dog

– Primates

• Strain

– Nude?

• Immunocompetent rodent models

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Samples – what are the end points?

• How you measure the efficacy?

• Tissues

– Analysis?

• Tumor size data during study?

• Blood samples

– Determination of the test item in the blood?

• Imaging (Pet jne..)

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Study period

• Tumor growth rate?

• When treatment begins

– Treatment

– Prevent

• Effect time of the drug

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Control groups

• Positive group

– Commercially available drug (if possible)

• Negative group

– No treatment or vehicle

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Dose and dosing frequence

• Dose level?

• How many dose levels?

– At least two

• Dose respose should be noticed

• Dosing frequence

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