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Preclinical Testing of Drugs

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Title: Preclinical Testing of Drugs


1
Preclinical Testing of Drugs
  • Ali Said Faqi, D.V.M., Ph.D., D.A.B.T
  • MPI Research, Inc.
  • Mattawan, Michigan

2
Drug Development Process
  • Discovery and synthesis.
  • Preclinical development (chemical testing,
    biological testing, pharmacology, toxicology,
    safety, etc.).
  • Clinical development (phases I-III).
  • Regulatory review, marketing approval.
  • Market launch.
  • Post-marketing development.

3
Factors Affecting the Type of Preclinical Testing
  • Chemical structure (analogous to known
    compounds).
  • Proposed human indication.
  • Target population.
  • Method of administration.
  • Duration of administration (Acute, Chronic).

4
Preclinical Support for Phase II-III Clinical
Trials
  • Phase II-III
  • Completed battery of genotoxicity.
  • Completed battery of reproductive toxicity.
  • Extended repeated dose toxicity.
  • Extended Pharmacokinetic studies.
  • Phase III
  • Chronic toxicity (rodent non rodent).
  • Carcinogenicity.
  • Supplemental studies (special safety concerns as
    alerted).

5
PK/TK Studies Objectives
  • Primary
  • Determine the systemic exposure achieved in
    animals and its relationship to dose level and
    the time course of toxicity study.
  • Secondary
  • Relate the exposure achieved in toxicity studies
    in toxicological findings and contribute to the
    assessment of the relevance of these findings to
    clinical safety.

6
PK/TK Studies Objectives (cont.)
  • Secondary
  • Support the choice of species and treatment
    regimen in nonclinical toxicity studies.
  • Provide information which, in conjunction with
    the toxicity findings contributes to the design
    of subsequent nonclinical toxicity studies.

7
Genotoxicity
  • Genetic toxicology testing focuses on the process
    of mutagenesis, which includes
  • Induction of DNA damage
  • Gene mutations and
  • Chromosomal aberrations
  • Minimum of 2 in vitro Assays
  • Bacterial mutation Assay
  • Ames test (point mutations)-
  • This assay uses mutant bacterial strains which
    have lost the ability to synthesize a specific
    amino acid.
  • If TA affects bacterial DNA, some bacterial cells
    regain their ability to synthesize the amino acid
    through reverse-mutation

8
Genotoxicity- continue
  • Chromosomal Aberration
  • The assay examines the cytogenetic effect of a TA
    and the Chinese Hamster Lung Cell line is
    commonly used.
  • OR
  • Mouse Lymphoma Assay
  • Can be used as replacement of in vitro
    Chromosomal aberration. It detects not only
    clastogenicity, but also gene mutation.

9
Genotoxicity- continue
  • If either of them are positive or for Phase II
  • Micronucleus Assay (In vivo Assay).
  • It detects immature erythrocytes with
    micronuclei.
  • The increase in micronuclei formations indicates
    chromosomal damage or abnormal mitotic
    activities.
  • Others Assays Unscheduled DNA Synthesis (UDS)
  • UDS assay measures the ability of a test article
    to induce DNA lesions by measuring the increase
    in DNA repair.
  • Not needed for Biologics

10
Reproductive Toxicity Testing
  • Three segments or ICH guidelines
  • Segment I or (ICH 4.1.1) General Fertility
    Reproductive Performance.
  • Segment II or ICH 4.1.3 Embryo-Fetal
    Development Study (birth defects).
  • Segment III or ICH 4.1.2 Peri- and Postnatal
    Study including maternal function.

11
Spontaneous Malformation in Different Species
  • Species Range () of malformation
  • Mouse 1 - 18.6
  • Rat 0.02- 1.9
  • Rabbit 0.7- 6.3
  • Dog 0.2- 1.9
  • Rhesus Monkey 0.1- 0.4
  • Cyno Monkey 0.4
  • Human 0.14 -13.8

12
Skeletal Evaluation Using CT-Scan
13
Animal Species
  • Rats, Mice, Dogs, Pigs and Monkeys are the most
    commonly used animals in preclinical studies.
  • Ideally , the species of choice should have the
    same pharmacokinetic profile as in humans,
    however, this information is either incomplete or
    missing,
  • Under such circumstance, select the most
    sensitive species for evaluating the safety of
    the substance.

14
Chronic Toxicity Study
  • Species 2 ( a rodent and a non-rodent).
  • In rodents chronic studies are usually for 6
    months to 2 years.
  • In non-rodents chronic studies are usually for 1
    year but may be longer.
  • The length of exposure is somewhat dependent on
    the intended period of use in humans.

15
Age
  • Younger and still growing animals are preferred
    at the initiation of a sub-chronic study.
  • When mice or rats are selected, they should be
    about six weeks and not more than eight weeks.
  • When dog is chosen treatment should begin at 4-6
    months but no more than 9 months.

16
Beagle Dogs in Safety Assessment
  • Advantages
  • Medium size,
  • moderate length of hair coat,
  • ease of handling and
  • adaptability to living in group housing
  • Disadvantages
  • Variation in body weight and size
  • Loud
  • Greater test material requirements
  • Availability
  • Exercise and special housing requirements.
  • Dogs have natural tendency to vomit.

17
Minipigs in Toxicity Studies
  • In Europe pigs are used in pharmaceutical studies
    in place of dogs and primates.
  • Main advantage
  • Similarity to human in
  • Cardiovascular anatomy and physiology
  • GI and digestion
  • Renal system
  • Immune system
  • Human skin
  • Thickness and permeability
  • Pigmentation

18
Comparison between Rodent and Non- Rodent Study
Design
  • Number of Animals
  • 4 to 11 times as many rodents are used in
    toxicity studies as non-rodents
  • Differences in Study Activities
  • Blood Collection
  • In rodent studies (satellite animals) are usually
    used.
  • Non-rodents blood samples from the main study
    animals without compromising their health status.
  • Dosing
  • Dietary is mostly used in rat studies. Capsule
    dosing is probably the most appropriate route of
    oral administration in dogs and gavage for
    monkeys.

19
Comparison between Rodent and Non- Rodent Study
Design
  • Handling of Animals
  • Rodents are relatively easy to work with. In
    contrast, primates are often difficult to handle
    because of their size, strength, emotionality and
    aggressiveness.
  • Behavioral Evaluation
  • Behavioral assessment of non-rodents is generally
    difficult. Difficulties associated with their
    larger size, handling, manipulation and their
    greater awareness of and reactivity to the
    experimenter.

20
Study Activities in a General Toxicity Study
  • Daily observations Pretreatment and twice daily
    during the study.
  • This consists of a home-cage observation with
    notation of clinical signs indicative of poor
    health.
  • Physical Examinations Pretreatment and after
    dosing during weeks 2 and 4
  • Physical examination involves evaluation of gait,
    mobility, and reflexes as well as the examination
    of the head (eyes, ears, mouth, teeth, etc.).
  • ECG Pretreatment and after dosing during weeks 2
    and 4

21
Study Activities- cont.
  • Ophthalmic Examinations Pretreatment and during
    Week 4.
  • Body Weight Pretreatment, Weekly and prior to
    scheduled necropsy.
  • Food consumption Pretreatment and Weekly
  • Dogs do not spill much of their feed.
  • Blood and urine collections Pretreatment, and
    during weeks 2 and 4.
  • Serial blood can be easily collected in dogs
    (Jugular).
  • Due to the difficulty in obtaining sufficient
    volume of urine in dogs over short period, urine
    is usually collected overnight.
  • Pharmacokinetics On Days 1 and 28.

22
Clinical Chemistry
  • Hepatocellular leakage enzymes
  • ALT, AST, SDH, LDH
  • Cholestatic enzymes
  • AP, GGT
  • Liver function tests
  • Total bilirubin, direct bilirubin, indirect
    bilirubin, bile acids, ammonia
  • Pancreatic parameters
  • Amylase, lipase
  • Lipid parameters
  • Cholesterol, triglycerides
  • Muscle parameters
  • Creatine kinase (CK), AST, ALT, LDH

23
Clinical Chemistry
  • Calcium, potassium Often influenced by kidney
    function, kidney parameters should be evaluated
    concurrently.
  • Kidney Parameters Urea nitrogen, Creatinine
  • Urine specific gravity should be evaluated
    concurrently when evaluating renal function.
  • Kidney function affect proteins, minerals,
    electrolytes, acid-base balance and hematopoietic
    parameters.

24
Distinguishing Factor Between Chronic and
Carcinogenicity Study
  • The chronic toxicity studies are designed to
    define no effect levels and the potential
    systemic toxicity,
  • Whereas, the oncogenicity study is designed to
    measure potential for induction of tumors.

25
Carcinogenicity Studies
  • Group size
  • The minimum group size is 50/sex- some use up to
    80/sex
  • Number of control groups
  • Two control groups are favored
  • Criteria for dose selection
  • Most regulatory guidelines require the highest
    dose administered be the estimated maximum
    tolerable dose (MTD).
  • This is generally derived from sub-chronic
    studies (90-days).

26
Maximum Tolerable Dose
  • Maximum tolerable dose (MTD) has been defined by
    some regulatory agencies as the dose that
    suppresses body weight gain slightly (e.g., 10)
    in a 90-day sub-chronic study.
  • However, it may also be considered parameters
    other than weight, such as physiological and
    pharmacokinetic data and urinary metabolite
    profiles, as indicator of an appropriate MTD.

27
Common Shortfalls in Study Design
  • Using the wrong animal model.
  • Using the wrong route or dosing regimen.
  • Using the wrong vehicle or formulation of test
    material.
  • Using the wrong dose level.

28
Carcinogenicity Studies-Cont.
  • Study Duration
  • The duration is two years for both rats and mice.
  • Strains used
  • Sprague-Dawley rats
  • CD1-mice
  • Route of Administration
  • Two most common routes are diet and gavage.
  • Drinking water, dermal application or injections
    may be used.
  • Survival
  • Reduced survival in a carcinogenicity study may
    or may not be drug related.
  • No unanimity among the regulatory agencies as to
    the minimum survival required to produce a valid
    carcinogenicity study.

29
Carcinogenicity Studies-Cont.
  • The FDA Red Book II Draft guideline does not
    recommend early termination due to decreased
    survival.
  • The EMEA guidelines differ in that they suggest
    termination of the study when survival in the
    control groups reaches 20,
  • While Japanese guidelines suggest termination at
    25 survival in the control or low dose group.
  • These provisions do not request termination of
    the study when drug-related mortality may be
    present only in the high dose group.

30
Endpoints
  • A carcinogenicity study is more focused than a
    chronic study- few endpoints are evaluated.
  • Pathology- limited to neoplastic and
    preneoplastic tissues
  • Only Pathology is considered in details.
  • Body weight- to ensure that there is no excessive
    toxicity to invalidate the study.
  • Survival- key to determine when to terminate the
    study.
  • Food consumption- measured to ensure that the
    dietary administration doses are accurate.

31
Transgenic Mouse Models
  • The objective is to use them as an alternative to
    the traditional lifetime mouse bioassay.
  • FDA would accept validated transgenic mouse
    models.
  • Four transgenic mouse models have been broadly
    evaluated.
  • P53/- Mouse Model
  • Sensitive for some mutagenic carcinogens, such as
    benzene. This is the most popular model in the
    USA.
  • A six month duration has been proven to be
    insufficient.
  • The XPA -/- Mouse model
  • The model is sensitive to both UV and genotoxic
    carcinogens and also to some non genotoxic
    carcinogens
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