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Drug Information Association www.diahome.org 1

PRECLINICAL STUDIES

João B. Calixto

Center of Innovation and Preclinical Studies, Florianópolis, SC, Brazil.

DIA Meeting, São Paulo, October 20-22nd, 2013.


“Corpora Non Agunt NisiFixate’”

“A Substance will notwork unless it is bound”or

“A drug is efficaciousonly so long as it isbound to, andmodulating the actionof, its physiologicaltarget”

Source: Lancet 182: 445-451, 1913

1906, composto 606, Arsfenamina

(Salvarsan)

Paul Erlich (1854- 1915)

PreClinical Studies: Historical Perspectives


• At the end of the first world war it was recognized that closecollaboration between Pharmacologist, Physiologists, Physicians andMedicinal Chemistry was required to test new medicines.

• In the years between the first and second world war, the need offor careful testing of the mode of action and toxicology of new drugsin animal models prior testing in human was widely accepted byPhysicians.

• However, the died of at least 76 people in USA from poisoningwith a elixir of sulphanlamide containing 72% of dihetylene glycollead Ceiling and Cannon summarize the key principles for testingnew drugs which are accepted until today (Nuremberg Code).

• The voluntary consent of the human subject is absolutely essential.

• The experiment should be such as to yield fruitful results for the good of

society, unprocurable by other methods or means of study, and not random

and unnecessary in nature.

• The experiment should be so designed and based on the results of animal

experimentation and a knowledge of the natural history of the disease or

other problem under study that the anticipated results will justify the

performance of the experiment.

• The experiment should be so conducted as to avoid all unnecessary physical

and mental suffering and injury.

• No experiment should conducted where there is an a priori reason to believe

that death or disabling injury will occur; except, perhaps, in those

experiments where the experimental physicians also serve as subjects.

The Nuremberg Code - I



Preclinical Study: Why?

• May be effective in certain human disease-states(Pharmacology testing).

• Conducted to indicate that the drug may be testedsafely in humans (Toxicology testing).

• Fulfill Regulatory Requirement.


EXPLORATY (NON-GLP) TOXICOLOGY

In vitro screens, e.g.,

• In silco screens

• Mutagenicity

• Cytotoxicity

• Immunotoxicity

• Hepatotoxicity

• Embryotoxicity

Single and repeat dose rangefinding studies in 2 species

Timing of the Main Safety Assessment Studies DuringDrug Discovery and Development

REGULATORY (GLP) TOXICOLOGY

Safety pharmacology

Genotoxicity (in vitro and in vivo)

28-day repeat dose toxicity andrecovery in 2 species

3-12 month chronic toxicity in 2species

Reproductive toxicity in 1 specie,covering:

• Fertility and implantation

• Fetal development

• Pre- and postnatal effects

24-month carcinogenicity in 2species

Source: Drug Discovery and Development - Technology in transition, H.P.Rang; 230, 2006.

DISCOVERYPRECLINICAL

DEVELOPMENT

CLINICAL

PHASE I/II

CLINICAL

PHASE IV

IND NDA


Design of Pre-Clinical Studies

• Primary Pharmacology

• Secondary Pharmacology

• Toxicology

• Drug Kinetics (Toxicokinetics)

Preclinical Studies-I

Acute Toxicity• Objective: To determine Maximum Tolerated Dose (MTD) and

No Observable Effect Level (NOEL)

• Duration: Typically 14 days after single dose

• Animals Required: 2 species (rodent and non-rodent)

• Parameters:

Mortality | Clinical pathology | Gross necropsy | Weight change | Clinical observations

• Points to consider:

• Dose selection for repeat dose studies

• Choice of Species (Fialuridine)


Preclinical Studies - II

Sub Acute Toxicity•Objective: To determine toxicity after repeated administration

of the test material

•Duration: 14 – 28 days

•Animals Required: 2 species (rodent and non-rodent)

•Parameters:

Mortality | Clinical pathology | Urinalysis | Histology | Weight change | Clinical observation

•Points to consider:

Dosing regimen – similar to clinical

Recovery period

Durd Toxicokinetics | ImmunotoxicityDrug Information Association www.diahome.org 16

Preclinical Studies- III

Subchronic/Chronic Toxicity• Objective: In support of products used to treat chronic

conditions

• Duration: 30 days to 2 years

• Animals Required: 2 species (rodent and non-rodent)

• Parameters:

Mortality | Clinical pathology

Clinical obs | Behavioral Assessment

Histology | Weight change


Clinical Trials (EU)


Preclinical Studies- IV

Carcinogenicity• Objective: To evaluate the tumorigenic potential in animals

and risk to humans

• Duration: 12 months or more

• Species: Mouse or Rat

• Parameters: Tumor development | Clinical pathology | Clinicalobservations and assessment


Pharmacology, Pharmacokinetic or Toxicology (mechanistic in vitro and invivo) data

Structure-activity relationships

Compound accumulation over long-term use

Continuous use in humans for 6 months +Drug Information Association www.diahome.org 18

Pre-Clinical Studies: Choice of Animal Species

• Rats, Mice, Rabbit, Guinea pig, Dogs, Pigs and Monkeysare the most commonly used animals in preclinicalstudies;

• Ideally , the species of choice should have the samepharmacokinetic profile as in humans, however, thisinformation is either incomplete or missing;

• Under such circumstance, select the most sensitivespecies for evaluating the safety of the substance;


Pharmacokinetics Modeling: Objectives

• Predict levels of test article in the body (absorbed,internal, target organ dose).

• Predict levels of test article or metabolites in excreta(biomarkers of internal dose).

• Predict dosages associated with a given level in thebody or excreta (dose reconstruction).


Absorption, Distribution, Metabolism and Elimination (ADME)

Advantages performingADME testing early in theprocess, before a moleculegets into development:

• Improvement of

Pharmacokinetic (PK)

properties.

• Saves time and costs

associated with

progressing an unsuitable

drug candidate through the pipeline.

The ADME properties thatare routinely measuredare:

• Solubility

• Permeability

• Plasma protein binding

• Movement across

transporters

• Metabolism by liver

enzymes


Safety Pharmacology [SP]-I

Cardiovascular system SP Evaluation:

• CORE:

– Hemodynamics (blood pressure, heart rate)

– Autonomic function (Cardiovascular Challenge)

– Electrophysiology (EKG in dog).

• NON-CORE

– QT Prolongation


Safety Pharmacology [SP]- II

Respiratory System [SP] Evaluation

• Respiratory functions

– Measurement of rate and relative tidal volume in conscious animals.

• Pulmonary Function

– Measurement of rate, tidal volume and lung resistance in anaesthetized animals.


Safety Pharmacology [SP]- III

Central Nervous System SP Evaluation

• Functional Observation Battery (Irvin Test)

• Learning and memory

– Measurement of learning ability and cognitive function in rats.

Secondary Organ System SP evaluation

• Renal System

• GI System

• Immune System

• Others Drug Information Association www.diahome.org 34

Why Study the hERG Channel and/or the Cardiac Action Potential?

• Acquired or congenital long QT syndromes may cause cardiacarrhythmias and sudden death in otherwise young and healthypersons.

• Six non-anti-arrhythmic drugs produced acquired long QTsyndrome and were withdrawn from the market.– Seldane® (terfenadine - antihistamine)– Hismanal® (astemizole - antihistamine)– Propulsid® (cisapride - prokinetic)– Serlect® (sertindole -antipsychotic)– Raxar® (grepafloxin - antibiotic)– Zagam® (sparfloxicin - antibiotic)

• Multiple classes/pharmacophores implicated• Observed effects primarily associated with block of the hERG

channel• Episodes exceedingly rare, but sometimes lethal


Genotoxicity• Minimum of 2 in vitro Assays:

– Bacterial mutation Assay

• Ames test (point mutations) – Chromosomal Aberration

• Mammalian cells in vitro or in vivo

• Dominant Lethal Tests in Rodents

OR– Mouse Lymphoma Assay

• If either of them positive or for Phase II– Micronucleus Assay (In vivo Assay).– Others Assays: Unscheduled DNA Synthesis

• Not needed for Biologics


Local Tolerance

• Dermal (skin) Irritation/Sensitization

• Eye Irritation

• Phototoxicity– The potential for sunlight (or other light frequencies)

to transform a drug or a metabolite is:

– a useful tool for activating some drugs and

–A cause for a significant adverse effects for others (Quinolone antibiotics).



*Figures originally published in French francs have been converted into Euros. Costs refer to 2000-2001.

Source: Nature Reviews/ Drug Discovery, 3: 523, 2004.

Costs of Conventional Preclinical Toxicology Studies

Test Cost ranges*

• Acute toxicity (rodents) €3,900-4,600

• Subacute toxicity (four weeks in rats-dogs) €143,000-183,000

• Subacute toxicity (four weeks in monkeys) €125,000

• Subacute toxicity (thirteen weeks in rats-dogs) €213,000-305,000

• Subacute toxicity (thirteen weeks in monkeys) €190,000

• Chronic toxicity (twenty-six weeks in rats and thrity-nine weeks in dogs)

€366,000-488,000

• Chronic toxicity (thirty-nine weeks in monkeys) €290,000

• Mutagenic potential (three basic tests) €25,000-69,000

• Carcinogenic potential (mice or rats) €1,124,000-2,287,000

• Effect on reproductive performance €313,000-458,000

• Complete budget €2,290,000-6,000,000 or more

Allocation of R&D investments by function(%)



Center of Innovation and Preclinical Studies - CIEnP


Main Building

5.300 m2 of constructed area, divided into:

• Basement: parking and support area

• Rodent facility: 600 m2 for the creation/maintenance of rodents standard SPF

with international certification

• Research Labs: About 20 laboratories with high level equipment to perform

research

• Office and Administration

• Auditorium: to host smaller events ~ 120 people

• Business incubator: 1.000 m2 for development of new ideas and companies

• Staff of about 60-80 people, including 15-20 PhDs

Non-rodent animal facility

Secondary building with about 2.000 m2 only for maintenance of non rodent animals

(nonhuman primates, dogs, rabbit, guinea pig, pigs among others).

Center of Innovation and Preclinical Studies

Center of Innovation and Preclinical Studies: MAIN OBJECTIVES


• To conduct preclinical studies: pharmacodynamics, safetypharmacology, pharmacokinetics and toxicology (rodents andnon-rodents), acting in accordance with the guidelines of goodlaboratory practices;

• To support the research, development and innovation to thepharmaceutical companies, supporting drug development;

• To integrate the national network of drug development,cooperating with companies, institutions and technologicalmechanisms / agents to promote innovation;

• To contribute to the establishment of national competence andthe generation of patents in Brazil and abroad.


Depending on national legal situations, the GLP requirements for non-clinical laboratorystudies conducted to evaluate drug safety cover the following classes of studies :• Single dose toxicity• Repeated dose toxicity (sub-acute and chronic)• Reproductive toxicity (fertility, embryo-foetal toxicity and teratogenicity, peri-/post-natal toxicity)• Mutagenic potential• Carcinogenic potential• Toxicokinetics (pharmacokinetic studies which provide systemic exposure data for theabove studies)• Pharmacodynamic studies designed to test the potential for adverse effects (Safetypharmacology)• Local tolerance studies, including phototoxicity, irritation and sensitisation studies, ortesting for suspected addictive and/or withdrawal effects of drugs.

WHAT IS GLP?

Good Laboratory Practice is defined as a quality system concerned with theorganisational process and the conditions under which non-clinical health andenvironmental safety studies are planned, performed, monitored, recorded, archived andreported.


Standard Operating Procedures - CIEnP

SOP’s area Number

• Toxicology 10

• Toxicology support 25

• Animal house 32

• GLP general procedures 10

• Equipments (just those involved with toxicology and animal house)

37

• Organizational (just those involved directly with GLP)

39

• RENAMA 8

• TOTAL 161


Softwares for regulatory compliance -CIEnP

The Pristima® Suite is designed as a fully integrated preclinicaldata management system that ensures seamless interoperabilitythroughout the full preclinical lifecycle process, helps streamlinereporting and submission processes through the delivery of real-time statistical analysis, promotes quality data management at alltimes through business rules, security and audit trails, andsupports an unlimited number of simultaneous studies and users.


Softwares for regulatory compliance-CIEnP

Pristima® VM delivers extensivecapabilities for Vivarium Managementand Veterinary Care. This uniquesolution delivers key features thateffectively bridge the gap between thebusiness, animal management andscientific aspects of research absent inmany oftoday’s preclinical solutions. Pristima®VM’s rich graphical user interface anddetailed reports and visualizationsdeliver the most comprehensive animalfacility solution.


Softwares for regulatory compliance

The eQCM document management module provides full document control overdifferent types of file formats, keeping a detailed history of all changes.eQCM's document management provides cycle control over the creation,approval, modification and retirement of obsolete versions of documents.Automated workflows ensure that documents are routed through all applicablereviewers and authorized in a controlled manner that complies with all regulateddocument management requirements, including the collection of electronicsignatures for specified workflow actions. Document tasks are sent to acentralized workload and notifications to the user’s e-mail facilitating the timelycompletion of critical document activities.

Sapiens Parque – Florianópolis, SC



www.cienp.org.br


Center of Innovation and Preclinical Studies

http://memoria.cnpq.br/index.htm

http://memoria.cnpq.br/index.htm

João Batista Calixto,

PhD.

Managing Director : CIEnP

[email protected]

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