Title: Preclinical Testing of Drugs
1Preclinical Testing of Drugs
- Ali Said Faqi, D.V.M., Ph.D., D.A.B.T
- MPI Research, Inc.
- Mattawan, Michigan
2Drug 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.
3Factors 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).
4Preclinical 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).
5PK/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.
6PK/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.
7Genotoxicity
- 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 -
8Genotoxicity- 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.
9Genotoxicity- 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
10Reproductive 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.
11Spontaneous 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
12Skeletal Evaluation Using CT-Scan
13Animal 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.
14Chronic 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.
15Age
- 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.
16Beagle 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.
17Minipigs 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
18Comparison 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.
19Comparison 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.
20Study 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
21Study 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.
22Clinical 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
23Clinical 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.
24Distinguishing 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.
25Carcinogenicity 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).
26Maximum 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.
27Common 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.
28Carcinogenicity 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.
29Carcinogenicity 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.
30Endpoints
- 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.
31Transgenic 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