Preclinical pharmacology cum Invivo pharamcology

1. 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.

3. Applications:
In vitro ADME:
Metabolic stability tests using human, mouse, rat, dog and monkey liver microsomes
Cytochrome P-450 inhibition
Plasma stability test
Plasma protein binding test
Metabolite identification
In vivo Pharmacokinetics (PK):
Species - Mouse, Rat
Route of administration – intravenous and oral
PK – Bio analysis:
Method development for small molecules and peptides
PK 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., leukaemia
Infectious 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.

8. 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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9. 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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10. 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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11. 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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12. 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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13. 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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3 R ` S

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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 REQUIREMENTS
SPECIES 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 hrs
Mice : 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

34. 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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35. 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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36. CPCSEA Guidelines
Anaesthesia 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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37. 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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38. Essential Environmental conditionsEssential Environmental conditions
Macro environment
Temperature control
Hygrometer
Cage distribution
Rack fabrication
Light source
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39. 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.

40. Handling / Restraining
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41. •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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42. Tools for oral feeding in rodentsTools for oral feeding in rodents
Ball-tipped oral feeding needle with syringe
Leather glove
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43. 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.

44. Tools for injection :Tools for injection :
75% alcohol cotton ball for surface disinfection
Medium size towel for restraining the rat
25G 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.

48. 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 µl per site to avoid tissue trauma.

49. 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.

51. 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. 51
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

53. 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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54. 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
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Collection of blood from tail vein

55. 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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56. 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 sinus
Medial 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

60. Drug safety testing
1) Acute Toxicity
2) Subacute Toxicity
3) Chronic Toxicity
Other Toxicities –
1) Reproductive Toxicology
2) Mutagenicity
3) Carcinogenicity
4) Primary Irritation Testing
5) Antigenicity
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61. Statistical Methods
They 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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62. Major classes of Screening
1) ANS – Cholinergic / anticholinergic , adrenergic / antiadrenergic ,
Ganglion blocking / stimulating
2) 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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63. Drugs Acting on CNS
1) 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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64. 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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65. 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.,. 17

66. 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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67. 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.
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68. 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 20

69. Other ACTIVITIES
1) 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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70. Statistical Methods
They 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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71. THANK YOU