INVIVO (EXPERIMENTAL) PHARMACOLOGY

BY –RVS.CHAITANYA KOPPALA

ASSISTANT PROFESSOR,LPU

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PRECLINICAL PHARMACOLOGY

The preclinical pharmacology group provides support for drug discovery and drug development programs.

The group develops establishes and validates in vitro and in vivo pharmacokinetic and metabolic methods to screen and evaluate small molecules and peptides for their drug-like properties.

The group also establishes in vivo disease models to validate potential drug able targets and to evaluate therapeutic properties of preclinical drug candidates.

Applications:In vitro ADME:

Metabolic stability tests using human, mouse, rat, dog and monkey liver microsomesCytochrome P-450 inhibitionPlasma stability testPlasma protein binding testMetabolite identification

In vivo Pharmacokinetics (PK):Species - Mouse, RatRoute of administration – intravenous and oral

PK – Bio analysis:Method development for small molecules and peptidesPK parameter analysis including absolute bioavailability

In vivo pharmacology:Cancer•Mouse xenograft models, e.g., colon cancer; gastric cancer; lymphoma; myeloma; prostate cancer, ovarian cancer•Mouse orthotopic models, e.g., leukaemiaInfectious disease•Renal infection

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ANIMAL MODEL

• An animal model is defined as a specific combination of an animal species, challenge agent and route of exposure that produces a disease process or pathological condition that in multiple important aspects corresponds to the human disease or condition of interest.  

• In the context of animal model qualification, the model-defining natural history studies are the animal studies that establish the ranges of values of key parameters of the disease or condition that will be specified in the context of use for the qualified model and that will be used as measures of quality control and quality assurance when the model is replicated.

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Classification of animal models

1. EXPERIMENTAL : A model in which an experimenting induced conditions mimic a human disease

2. NEGATIVE : A model in which particular condition cant be produced , & is therefore studied to better understand the reason for the protective or resistant affects

3. SPONTANEOUS : A model in which the animal naturally develops a disease or some other conditions of interest

4. ALTERNATIVE MODEL is defined as technique that reduces or eliminates the need for live animals & there by prevents potential pain & distress in animals

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• Computer models and cell cultures are good for screening and are used frequently.

• Such models cannot replicate complicated interactions in the whole system.

• Final testing depends on studies in animals; sometimes it is required by law.

• Animal and non-animal models used in conjunction achieve the best answer.

Animal ModelsAnimal Models

There are three main types of animal models Homologous, Isomorphic and predictive.

Homologous - Animals have the same causes, symptoms and treatment options as would humans who have the same disease.

Isomorphic - Animals share the same symptoms and treatments.

Predictive - When the animals strictly display only the treatment characteristics of a disease

• An animal model is a living, non-human animal used during the research. Ex – Rats, Mice, Guinea pigs,

Rabbits, Monkeys etc.

• For the purpose of better understanding the disease without the added risk of causing harm to an actual

human being.

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In lab animal models :Species identity is known.There is a uniformity in gene chemistry.The parental history is known.Feed provided is designed and balanced. They are provided with good quality housing.Their hygiene ensured.They are almost free from disease.

Why Lab animals ? Why can’t we use wild ones ?

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Most animal models are Rodents Advantage :

• Their anatomy and physiology are comparable with that of human.

• Certain animals develop the same symptom comparable with human.

• By keeping the animals in a standard set up we can study the disease pattern, survival of causative germ outside the human body

• New drugs can be tested and developed.

• Easy to breed, handle them.

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Types of Animal models 1.SPONTANEOUS OR INHERITED ANIMAL

MODELS : (Naturally occurring animal disease or

conditions correspond to the same as in human)

Example: • Diabetes, • Hypertension,• Salmonella typhi infection.

2. INDUCED DISEASE (EXPERIMENTAL)ANIMAL MODELS:

Example:

• 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.

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Types of Animal models (Continued)

3. GENETICALLY MODIFIED ANIMAL MODELS Produced by the Insertion of foreign DNA

(transgenic) by micro- injection or removal or replacement of specific genes ( strong animal model for disease specific research)

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4.Negative model :

• Animals are resistant to a particular conditions.

(demonstrate a lack of reactivity to a particular stimulus).

Examples: • BALB/c mice are resistant for experimental

leprosy.• Gonococcal infection is not seen in rabbits

after an experimental treatment that induces the disease in other animals.

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What is animal testing?

• The term "animal testing" refers to procedures performed on living animals for purposes of research into basic biology and diseases, assessing the effectiveness of new medicinal products, and testing the human health and/or environmental safety of consumer and industry products such as cosmetics, household cleaners, food additives, pharmaceuticals and industrial/agro-chemicals.

• All procedures, even those classified as “mild,” have the potential to cause the animals physical as well as psychological distress and suffering.

• Often the procedures can cause a great deal of suffering. Most animals are killed at the end of an experiment, but some may be re-used in subsequent experiments.

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Can animal models of disease reliably inform human studies?

 

"The value of animal experiments for predicting the effectiveness of treatment strategies in clinical trials has remained controversial, mainly because of a recurrent failure of interventions apparently promising in animal models to translate to the clinic”

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EVIDENCES

Polio– Landsteiner and Popper proved it infectious; able to transmit disease to monkeys.

– Salk and Sabin developed their vaccine through work with chickens and monkeys.

Diabetes– Banting and Best showed importance of insulin in dogs.

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Infant Mortality– Studies in sheep and lambs led to use of steroids in treatment of respiratory distress

syndrome (formerly hyaline membrane disease), a major cause of death in premature infants.

– Advances in understanding and treatment of sudden infant death syndrome (SIDS) came from studies in rats, mice, dogs, and sheep

Cystic Fibrosis– A major killer of young adults.– Mouse models led to understanding role of chloride channels.– Genetic therapies on the horizon are an outgrowth of work in mice.

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High Blood Pressure (HBP)– Goldblatt linked HBP to kidney in rats, cats, and dogs; led to diuretics and angiotensin

converting enzyme inhibitors to treat high blood pressure.– Cushing linked HBP to brain in dogs; led to understanding sympathetic nervous

system influence on blood pressure and drugs to treat it.

Obesity– Major risk factor for diabetes mellitus, high blood pressure, heart attack, stroke and

certain cancers. – Epidemic in the United States: 64% of adults are overweight and 25% are obese.– Mouse models and Zucker obese rats shedding new light on causes of overeating,

importance of leptin receptors, and ways that obesity leads to disease.

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Bioterrorism– Smallpox vaccine from calves– “Two animal rule” – all vaccines must be tested for efficacy and safety in two animals

(typically rodent and non-human primate) before introduction in humans– Botulinum antitoxin tested in mice and non-human primates

AIDS– Numerous animal models in studies to understand the disease and how it attacks the

immune system.– Current anti-AIDS treatment developed in animal models have greatly extended life

expectancy and quality of life for AIDS victims.– AIDS vaccines being developed in monkeys.

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HISTORY

• The earliest references to animal testing are found in the writings of the Greeks in the 2nd and 4th centuries BCE.

• Aristotle and Erasistratus were among the first to perform experiments on living animals.

• Galen, a physician in 2nd-century Rome, dissected pigs and goats, and is known as the "father of vivisection".

• An Experiment on a Bird in an Air Pump, from 1768, by Joseph Wright

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• In the 1880s, Louis Pasteur convincingly demonstrated the germ theory of medicine by inducing anthrax in sheep.

• In the 1890s, Ivan Pavlov famously used dogs to describe classical conditioning.• Insulin was first isolated from dogs in 1922, and revolutionized the treatment

of diabetes.• On November 3, 1957, a Soviet dog, Laika, became the first of many animals to orbit the

earth.• In the 1970s, antibiotic treatments and vaccines for leprosy were developed using

armadillos, then given to humans.• The ability of humans to change the genetics of animals took a large step forwards in

1974 when Rudolf Jaenisch was able to produce the first transgenic mammal, by integrating DNA from the SV40 virus into the genome of mice.

• This genetic research progressed rapidly and, in 1996, Dolly the sheep was born, the first mammal to be cloned from an adult cell.

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STANDARDS• Specification for animal handling care• Treatment & transportation of experimental animals

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Indian Medical Organizations

To ensure uniform quality of clinical research by Good Clinical Practices throughout the country and to generate data for registration of new drugs before use in the Indian population.

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2) LABORATORY ANIMAL HUSBANDRY

• Housing• Animal environment• Food , water & bedding• Sanitation• Identification & record keeping• Emergency , weekend & holiday care

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3) Veterinary care

• Preventative medicine• Surveillance , diagnosis , treatment & control of diseases• Anesthesia & analgesia• Surgery & post surgical care• Euthanasia

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CRITERIA`S TO BE CONSIDERED FOR SELECTION OF LIVE ANIMAL EXPERIMENT

• Procedure should yield results beneficial to well being• The species & no of animals should be appropriate for

experimental purpose• The proposed procedures shouldn’t unnecessarily duplicate

previous experiment• Appropriate analgesics , anesthetics & tranquilizing drugs to be

used to minimize pain & discomfort

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• Methods of euthanasia• The individuals performing the experimental procedure &

caring for the animals need to be properly trained

In general procedures that causes minimal pain or discomfort to humans & place the animals in minimum distress are considered ACCEPTABLE

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FOOD & WATER REQUIREMENTSSPECIES DAILY FOOD REQUIREMENT DAILY WATER REQUIREMENT

Mouse 3 – 6 g 3 – 7 ml

Rat 10 – 20 g 20 – 30 ml

Hamster 7 – 15 g 7 – 15 ml

Guinea pig 20 – 30 g 12 15 ml / 100 g

Rabbits 75 – 100 g 80 – 100 ml / kg

Cat 100 – 225 g 100 – 200 ml

Dog 250 – 1200 g 100 – 400 ml / day

Primate 40 g / kg 350 – 1000 ml

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FASTING• Like humans , animals are often fasted in preparation for blood

collection

Non human primates , G pig : 18 – 24 hrsMice : 4 – 6 hrs

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ANESTHESIA & ANALGESIA• Investigators using live animals must employ appropriate anesthetic ,

analgesic , sedative agents necessary to control pain & distress , unless use of such agents would interfere with specific objectives of the research

• Most of the PAIN RELIEVING AGENTS are controlled substances & must be ensured that drug supplies are adequately protected & inventoried in accordance with the requirement of the statute

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EUTHANASIA

•Methods of euthanizing laboratory animals are chosen to induce rapid unconsciousness and humane death without pain or distress.• The methods that are preferred are those published by councils of veterinarians. •The animal can be made to inhale a gas, such as carbon monoxide and carbon dioxide, by being placed in a chamber, or by use of a face mask, with or without prior sedation or anesthesia

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• PHYSICAL EUTHANASIA : cervical dislocation & decapitation recommended only when scientifically justified Cervical dislocation is considered humane for poultry , mice , rats weighing less than 200 g & rabbit weighing less than 1 kg

• VERIFICATION OF DEATH : Irregardless of specific euthanasia methods used , it is imperative that death be verified by examining the animal for cessation of vital signs

CPCSEA Guidelines

• GOAL•VETERINARY CARE •ANIMAL PROCUREMENT•QUARANTINE, STABILISATION AND SEPERATION•ANIMAL CARE AND TECHNICAL PERSONNEL •PERSONAL HYGIENE•ANIMAL EXPERIMENTATION INVOLVING HAZARDOUS AGENTS

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CPCSEA Guidelines

• MULTIPLE SURGICAL PROCEDURES ON SINGLE ANIMAL•DURATION OF EXPERIMENTS•PHYSICAL RESTRAINTS•CAGING OR HOUSING SYSTEM•FOOD,BEDDING AND WATER•SANITATION AND CLEANLINESS•WASTE DISPOSAL AND PEST CONTROL•RECORD KEEPING•SOP’s

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CPCSEA GuidelinesAnaesthesia And Euthanasia :-

•Painful procedure should be carried under anesthesia only.•Anesthesia is given for full duration•Animal is prepared for anesthesia by keeping them fasting overnight and by using pre anesthetics.•Commonly used anaesthetics : pentobarbitone, ketamine, urethane etc.•Euthanasia :- events where an animal is required to be sacrificed on termination of an experiment or for other ethical reasons.

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Light / Temperature controlLight / Temperature control Rodents are nocturnal

the optimum light is 400 Lux is allowed (Rodents are photophobic)

21-23 ºC has to be maintained

Animal colonies are usually Air-conditioned and continuous supply of electric current is ensured (UPS).

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Essential Environmental Essential Environmental conditionsconditionsMacro environment

Temperature control

Hygrometer Cage distribution Rack fabricationLight source

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ANIMAL HANDLINGANIMAL HANDLING

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Techniques:

• Sexing

•Behavioral studies

•Oral feeding

•Intraperitoneal injection

•Subcutaneous injection

•Intradermal injection

•Intramuscular injection

•Intravenous injection

• Different blood collection techniques.

Handling / Restraining

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•Sexing Rat- the distance between the anal and genital orifices is greater in the male (left) compared to the female (right).

SEXING

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Tools for oral feeding in Tools for oral feeding in rodentsrodents

Ball-tipped oral feeding needle with syringeLeather glove

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ROUTES OF ADMINISTRATION

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Oral gavage-Restrain the animal by the scruff.

-Place the tip or ball of the needle into the animal’s mouth.

-Slide the tip gently past the back of the tongue.

-The needle should slide easily down the esophagus, if properly placed. DO NOT FORCE

-If any resistance is met, remove the needle and reinsert.

-Do not aspirate.

-Once the needle is properly placed, administer the material.

Tools for injection :Tools for injection :

75% alcohol cotton ball for surface disinfectionMedium size towel for restraining the rat25G 5/8” needle with 1cc syringe for injection

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Intraperitoneal Injection :•Restrain the mouse by the scruff method. Expose

the ventral side of the animal, tilting the head

down at a slight angle.

•Swab the site with 70% ethanol.

•The sterile needle should be placed, bevel up, in

the lower right or left quadrant of the animal’s

abdomen.

•Insert the needle at a 30° angle.

•Aspirate to ensure proper placement and inject

the material.

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Intravenous injection•Warm the animal under a heat lamp or disposable

handwarmers (not exceeding 85 - 90° F).

•Place the animal in a restraint device and stabilize the

tail between the thumb and forefinger.

• Swab the tail with 70% ethanol.

•Attempt the injection starting at the middle or slightly

distal part of the tail.

• With the tail under tension, insert the needle, bevel up,

approximately parallel to the vein and insert the needle

at least 3 mm into the vein.

•DO NOT ASPIRATE, as it will cause the vein to collapse.

•Inject the material in a slow, fluid motion.

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Subcutaneous Injection •Restrain the animal by the scruff method. Use

your thumb and forefinger to make a tent of

skin over the scruff.

•Swab the area with 70% ethanol.

•Insert the needle, bevel up, at the base of the

tent.

•The needle should be inserted parallel to the

skin and should be directed toward the

posterior of the animal.

•Aspirate to ensure proper placement and

inject the material.

Intradermal Injection 

A properly performed intradermal injection will result in a small, round skin welt. 

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•In order to perform intradermal injections, the

animal should be anesthetized.

•Shave or pluck an injection site on the back of

the animal to remove the hair.

•Swab the site with 70% ethanol.

•Insert the needle into the skin, bevel up,

holding the needle nearly parallel to the plane

of the skin.

•Do not aspirate.

•Inject the material.

•The volume of the injection should be limited

to 50 l per site to avoid tissue trauma.

Insert the needle, bevel up, into the caudal thigh at a 45° angle.

Insert the needle into the caudal thigh, bevel up. 

Method I: Method II:Intramuscular injection

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Method III:

• Identify the caudal thigh muscles, swab the area with 70% ethanol, aspirates and inject the material. 

Method IV:

•Gently pull the foot of the leg to be injected through the restrainer and locate the caudal thigh muscle. 

Let the rat relax on the top of the lid.Stretch the body of the rat by pulling up it’s tail and then cover the rat with a towel by your left hand.

The injection site should be in the lower left quadrant of the abdomen because vital organs are absent from this area

Only the tip of the needle should penetrate the abdominal wall to prevent injection into the intestines.

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Overview of intraperitoneal injection

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Methods for blood collection :

•Collection of blood from tail vein

•Collection of blood from cardiac puncture

•Retro-orbital Sinus Blood Collection 

Tools for collection of blood from tail vein

• 75% alcohol cotton ball for surface disinfection• 27G1/2” needle with 1 ml syringe for blood

withdrawal• A vial for blood collection

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Optimal site of blood withdrawal is around the distal one-third of the tail since this part of tail gives better visualization of the veins

Disinfect the tail with 75% alcoholic cotton ball

Create negative pressure inside the syringe

Pull back the plugger by the ring finger to withdraw blood from the tail vein 54

Collection of blood from tail vein

Tools for collection of blood from cardiac puncture

• 75% alcohol cotton ball for surface disinfection• 24G needle with 10cc syringe for blood withdraw• 15 cc centrifuge tube for blood collection

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Disinfect the left thorax with 75% alcoholic cotton ball

Search for the point of maximum heart beat

Insert the needle straight on the selected point and withdraw blood by your left hand

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Collection of blood from cardiac puncture

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Retro-orbital Sinus Blood Collection 

•The retro-orbital sinus is the site located behind the eye at

the medial or lateral canthus, just underneath the

conjunctival membrane.

•This method is intended for survival blood collection.

•Not more than 10% of the blood volume should be removed

at one sampling.

•A topical ophthalmic anesthetic must be used prior to

performing this procedure.

•With a gentle rotating motion, insert the heparinized or

non-heparinized micro-hematocrit capillary tube through

the sinus membrane.

•Continue rotating the tube at the back of the orbit until

blood flows.

•Collect the appropriate volume of blood.

Retro orbital sinusMedial canthus

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Retro-orbital Sinus Blood Collection

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They include:

-Plethysmometer

-Plus Maze

-T-Maze

-Pole-climbing apparatus

-Jumping box

-Eddy’s hot plate

-Electro-convulsometer etc.,.

Equipments

Drug safety testing 1) Acute Toxicity

2) Subacute Toxicity3) Chronic Toxicity

Other Toxicities –1) Reproductive Toxicology2) Mutagenicity3) Carcinogenicity4) Primary Irritation Testing5) Antigenicity

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Statistical MethodsThey include:-Collection of data and calculation of means.

-Experimental designs

-Errors.

-Variables.

-Analysis of Variance

-Tests of Hypothesis.-Statistical tests Ex : t- Test, F - Test etc.,.

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Major classes of Screening1) ANS – Cholinergic / anticholinergic , adrenergic / antiadrenergic , Ganglion blocking / stimulating2) Peripheral CNS – Neuromuscular blocking agents, Sedatives, anaesthetics.3) Cardiovascular – Antihypertensive, antianginal, cardiotonics.4) Metabolic – Antihyperlipedemics, antidiabetics, diuretics.5) Immunopharmacological - Antiallergic, anti-inflammatory, immunostimulants.6) Gastro-intestinal Antimicrobials – Anti-ulcer, gastrokinetic , antibacterials .7) Chemotherapeutic – Antineoplastics.

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Drugs Acting on CNS1) Muscle Co-ordination:-Grip strength in Mice

-Rota Rod Testing in Mice.

2) Hypnotic Activity:

-Potentiation of Hexobarbitone-induced sleeping time.

-Experimentally induced insomnia in rats

3) Others : Anti-epileptic, anxiolytics etc.,.

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Drugs Acting on Peripheral Nervous System

1) Local Anaesthetic Activity:-Conduction Anaesthesia in the -Sciatic nerve of Frog and rats

-Conduction Anaesthesia on the -Mouse tail

-Corneal Anaesthesia in Rabbits

2) Neuromuscular blocking activity:

-Rabbit head drop method

-Isolated Phrenic Nerve Diaphragm Preparation

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Drugs Acting on Cardio Vascular System

1) Anti-hypertensive Activity:-Acute renal hypertension in Rats

-Chronic renal hypertension in Rats

-DOCA- Salt induced hypertension

2) Anti-arrhythmic Activity:-Chemically induced arrhythmias.-Electrically induced arrhythmias.

-Mechanically induced arrhthmias.

3) Others : Cardioprotectives, Cardiotonics etc.,.

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Drugs Acting on Respiratory System

1) Bronchodilator and anti-asthmatic activity:-Spasmolytic activity on Guinea Pigs Isolated Tracheal Chain and Lung Strip.

-Bronchial Perfusion of the Isolated lung.

2) Anti-tussive and Expectorant Activity:-Citric acid Inhalation Induced Cough in Guinea Pigs.-Acute study of Mucus secretion In Rabbits

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Analgesics, Anti-inflammatory and Anti-pyretic Activity

1) Inflammatory actions:-Carrageenin-induced Paw Oedema

-UV Erythema in Guinea Pigs.

2) Pyretic activities:

-Antipyretic testing in Rats.

-Antipyretic testing in Rabbits. 19

3) Analgesic activities:Chemically Induced Nociception-Writhing tests.

-Formalin test in Rats.

Electrical Stimulation Methods-Electrical stimulation of the Tail-Flinch-jump test in Mice

Other methods – Tail-flick Test, - Tail Immersion Test

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Other ACTIVITIES1) They include actions on :-Urinary tract.-GI system.-Liver.-Anti-diabetic activity.-Anti-fertility.-Actions on Eye.-Anti-stressor and anti-oxidant activities.

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Statistical MethodsThey include:-Collection of data and calculation of means.

-Experimental designs

-Errors.

-Variables.

-Analysis of Variance

-Tests of Hypothesis.-Statistical tests Ex : t- Test, F - Test etc.,. 22

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