general guidelines for toxicopathology study
Post on 07-May-2015
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Presented
byDr. Rahul G. Kadam
Ph.D ScholarRoll NO. P1661
Toxicology is a branch of science that deals with toxins and poisons and their effects and treatment.
Toxicological screening is very important for the development of new drugs and for the extension of the therapeutic potential of existing molecules.
The US-FDA states that it is essential to screen new molecules for pharmacological activity and toxicity potential in animals (21CFR Part 314).
Toxicity tests are mostly used to examine specific adverse events or specific end points such as cancer, cardiotoxicity, and skin/eye irritation.
Toxicity testing also helps calculate the No Observed Adverse Effect Level (NOAEL) dose and is helpful for clinical trails.
Paracelsus (Father of Toxicology): determined specific chemicals responsible for the toxicity of plants and animals (dose-response relationship).
"All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy”
--Paracelsus
Mathieu Orfila, determined the relationship between poisons and their biological He is referred to as the father of modern toxicology.
Paracelsus (1493-1541)
Recent developments: after 1920
(introduced determine LD50 by USFDA )
Benefit –risk ratio can be calculated
Prediction of therapeutic index
Therapeutic index= Maximum tolerated dose Minimum curative dose
Smaller ratio, better safety of the drug
Pharmacological effects are same in man as in animals
Toxic effect in species will predict adverse effects in man
Giving high doses in animals improves predictability to man
Risk assessment can be made by comparison of toxic doses in test species with predicted therapeutic dose in man
PHASES OF DRUG DEVELOPMENT
(ANIMAL MAN)
PHASE III PHASE IVPHASE I
PHASE IPHASE IPRECLINICALPRECLINICAL PHASE II
Product Approval (NDA/MAA)
Patient studies
Entry to man(IND / CTA)
NoneNone
Healthy subjects
Safety andtolerability
Healthy subjects
Safety andtolerability
Genetic toxicity(in vivo)
Repeat dose toxicity testing
+Bioanalysis /
Toxicokinetics
Drug Metabolism
Reproductive Toxicity Testing(teratogenicity)
Genetic toxicity(in vivo)
Repeat dose toxicity testing
+Bioanalysis /
Toxicokinetics
Drug Metabolism
Reproductive Toxicity Testing(teratogenicity)
Patients
Small scale efficacy studies
Patients
Small scale efficacy studies
Patients
Large scalemulticentre
studies
Patients
Large scalemulticentre
studies
Chronic (long term) toxicity testing+
Bioanalysis / Toxicokinetics
Reproductive Toxicity Testing (fertility and pre/post natal)
Carcinogenicity studies
Drug Metabolism
Chronic (long term) toxicity testing+
Bioanalysis / Toxicokinetics
Reproductive Toxicity Testing (fertility and pre/post natal)
Carcinogenicity studies
Drug Metabolism
Patients
Large scalepost-marketing
studies
Patients
Large scalepost-marketing
studies
As requiredAs required
Genetic toxicity(in vitro)
Single / repeat dose
toxicity studies+
Bioanalysis / Toxicokinetics
Safety Pharmacology
Drug MetabolismLead
candidateIdentified
Clin
ical
Non
-clin
ical
MOLECULE
Studies should comply with GLPPerformed by trained and qualified staffUse of standardized and calibrated equipmentSOP’s followed in laboratory tasksAll documents should be preserved for minimum 5 years after marketing of the drug
OECD Guideline
EPA Guideline
FDA Guideline
GAITONDE Guideline
TOXICOKINETIC STUDIES
Generation of Pharmacokinetic data to access systemic exposure achieved in animals
Relation to dose level and the time course of toxicity study
To support choice of species & Treatment regimen
Design on clinical studies accordingly
Pharmacodynamic responses
Pharmacokinetic profile Species, sex, age of experimental animals
Susceptibility, sensitivity and reproducibility of test system
In vitro: Isolated organs, tissues cell-cultures
Mechanism of effect in vivo
Systemic toxicology studies
Single dose studies Repeated dose studies
Reproductive toxicology studies
Male fertility Female reproduction & Developmental studiesLocal toxicity studies
Hypersensitivity studies
Genotoxicity studies
Carcinogenicity studies
Preliminary Definitive
• Maximum Non Lethal dose(MNLD) determined
• MTD and MLD determined• Evaluate effects • Target organ of toxicity may be determined
a) SINGLE DOSE STUDIES/ ACUTE TOXICITY
METHOD
Single dose tested in 2 rodent species 2 routes of administration Oral dosing of 2g/kg or 10 times of normal
human dose Observation for 14 days after dosing MNLD established Symptoms , signs reported Microscopic and Macroscopic evaluation
METHOD
Group of 20 animals of either sex dosed at MNLD 5 animals of each sex are observed for 48 hr and
conduct autopsy for early pathological changes Remaining 5 of each sex are observed for 14 days MTD and MLD established Signs of intoxication or recovery, changes in body
weight, pathological changes Complete macroscopic and microscopic examination Target organs can be identified
Two mammalian species(one should be non-rodent)
Long duration studies (30-180 days) Dose is dependent on dose-escalating studies Drug administered by clinical route Parameters monitored and recorded are:
Behavioral Physiological Biochemical Microscopic observations
b) REPEATED DOSE STUDIES/SUB-ACUTE OR CHRONIC TOXICITY
a) MALE FERTILITY
METHODOne rodent species(rat)
3 dose groups taken (each with 6 adult males),
1 control
Drug treatment by clinical route for 28-72 days
Mated with females in 1:2 ratio
Females getting pregnant should be examined
After 13 days of gestation
All male animals sacrificed
•Weights of testis, epididymus recorded & examined for their histology
•Sperms examined for motility & morphology
Segment I
19
Fertility and general reproductive performance study
Segment II Teratogenicity
Segment III Peri and post-natal study
Fertility and early embryonic development (rat) Embryo- foetal development (rat & rabbit)
Post natal development (rat) (post natal survival of offspring), growth parameters, vital senses, behavioral effects
b) FEMALE FETILITY
Drug administered to both males (28days) and females (14 days) before mating
Implantation Embryogenesis
Required when drug is administered by special route (other than oral) in humans
Study design: 2 species along with control used Dose dependent on dose escalating studies 3 dose levels
Dermal toxicity studies
Dermal photo-toxicity studies
Vaginal toxicity studies
Rectal tolerance studies
Rats & RabbitLocal signs (erythema, oedema), histological examination
Guinea pigUsed in treatment of leucodermaExamination of erythema & oedema formation
Rabbit or DogObservation of swelling, histopathology of vaginal wall
Rabbit or DogSigns of pain, blood or mucous, histology examination of rectal mucosa
Ocular toxicity studies
Parenteral drugs
Inhalation toxicity studies
Albino RabbitChanges in cornea ,Iris & aqueous humor, histological examination of eye
For intravenous/ intramuscular/ subcutaneous/ intra-dermal injectionSites of injection examined grossly and microscopically
One rodent and non rodent speciesAcute , sub-acute and chronic studies performedObservation of respiratory rateHistological examination of respiratory passages, lung tissue
Guinea Pig Maximization test
Local lymph node assay
Determination of Maximum non irritant or minimum irritant doseEvaluation of Erythema and oedema
Mice of one sex(either male or female)Drug treatment given on ear skinAuricular lymph node dissection after 5 daysIncrease in 3h-thymidine used for evaluation
To detect early tumorigenic effects in cases of chronic illness
In vitro tests:Test for gene mutation in BacteriaCytogenetic evaluation of chromosomal damage in mammalian cells
E.g.; Ames’s Salmonella Assay detects increased number of aberrations in metaphase chromosomesDNA strand breaks, DNA repair or recombination, Measurements of DNA adducts
In vivo tests:Chromosome damage in rodent hematopoietic cells
E.g.; Micronucleus Assay
Life-time Bioassays
Carcinogenicity studies are performed on:
Drug used for >6 months or frequent intermittent use for chronic diseases
Chemical structure of drug indicates carcinogenic potential
Therapeutic class of drugs which have produced positive carcinogenicity
Group sizes of 50 animals/sex at each of 3 dose levels
Control group is of double size
Record for onset of tumor development
Usually carried out for 24 months in rats and 18 months in mice (life span studies)
CONDUCT OF STUDY
EVALUATION OF RESULT
Incidence of cancers in control and test
Trend towards increasing incidence with increasing doses
Number of animals with single/multiple tumors
Macroscopic changes observed by autopsy
Histopathology of organs and tissues
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