Preclinical Testing of Drugs

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Preclinical Testing of Drugs

• Ali Said Faqi, D.V.M., Ph.D., D.A.B.T
• MPI Research, Inc.
• Mattawan, Michigan

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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.

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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).

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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).

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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.

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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.

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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

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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.

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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

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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.

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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

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Skeletal Evaluation Using CT-Scan
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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.

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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.

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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.

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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.

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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

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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.

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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.

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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

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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.

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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

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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.

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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.

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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).

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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.

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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.

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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.

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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.

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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.

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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