Non-clinical studies in the process of new drug ...

Non-clinical studies in the process of new drugdevelopment – Part II: Good laboratory practice,metabolism, pharmacokinetics, safety and dose

translation to clinical studies

E.L. Andrade*, A.F. Bento*, J. Cavalli, S.K. Oliveira, R.C. Schwanke, J.M. Siqueira, C.S. Freitas,R. Marcon and J.B. Calixto

Centro de Inovacão e Ensaios Pré-clínicos, Florianópolis, SC, Brasil

Abstract

The process of drug development involves non-clinical and clinical studies. Non-clinical studies are conducted using differentprotocols including animal studies, which mostly follow the Good Laboratory Practice (GLP) regulations. During the early pre-clinical development process, also known as Go/No-Go decision, a drug candidate needs to pass through several steps, suchas determination of drug availability (studies on pharmacokinetics), absorption, distribution, metabolism and elimination (ADME)and preliminary studies that aim to investigate the candidate safety including genotoxicity, mutagenicity, safety pharmacologyand general toxicology. These preliminary studies generally do not need to comply with GLP regulations. These studies aim atinvestigating the drug safety to obtain the first information about its tolerability in different systems that are relevant for furtherdecisions. There are, however, other studies that should be performed according to GLP standards and are mandatory forthe safe exposure to humans, such as repeated dose toxicity, genotoxicity and safety pharmacology. These studies must beconducted before the Investigational New Drug (IND) application. The package of non-clinical studies should cover allinformation needed for the safe transposition of drugs from animals to humans, generally based on the non-observed adverseeffect level (NOAEL) obtained from general toxicity studies. After IND approval, other GLP experiments for the evaluation ofchronic toxicity, reproductive and developmental toxicity, carcinogenicity and genotoxicity, are carried out during the clinicalphase of development. However, the necessity of performing such studies depends on the new drug clinical application purpose.

Key words: Non-clinical studies; GLP studies; Safety; Pharmacokinetics; Toxicology

Introduction to Good Laboratory Practice:history and needs for implementation

The formal concept of ‘‘Good Laboratory Practice’’(GLP) was launched in the USA, during the 1970s, thanksto constant discussions about the robustness of the non-clinical safety data submitted to the FDA for New DrugApplications (NDA). At that time, inspections performed inthe laboratories, revealed:• Inadequate planning and flaws in studies execution;• Insufficient documentation of methods, results andeven fraudulent data (for example, the replacement ofdead animals during a study by other animals not properlytreated by the test article), which were not documented;• Use of hematological data from other studies as acontrol group;

• Exclusion of data concerning macroscopic observa-tions (necropsy);• Raw data changes in order to "adjust the results" forthe final report;

These observations and deficiencies became publicand, with the political effect of these claims, the FDA pub-lished the first proposals for regulation in 1976 and thefinal rules in 1979 (1). The GLP principles were the basisfor ensuring that the reports submitted to the FDA fully andreliably reflected the experimental work conducted. Forthe registration of pesticides, the US Environmental Protec-tion Agency (EPA) found similar problems with the qualityof the studies. Thus, the EPA published its own regulation

Correspondence: J.B. Calixto: <[email protected]>

*These authors contributed equally to this study.

Received June 21, 2016 | Accepted September 23, 2016

Braz J Med Biol Res | doi: 10.1590/1414-431X20165646

Brazilian Journal of Medical and Biological Research (2016) 49(12): e5646, http://dx.doi.org/10.1590/1414-431X20165646ISSN 1414-431X Review 1/19

mailto:[email protected]

http://dx.doi.org/10.1590/1414-431X20165646

draft in 1979 and 1980, and later, in 1983, the final ruleswere published in two separate parts – insecticides, fungi-cides and rodenticides (2) and control of toxic substances (3),reflecting their different legal bases.

In Europe, the OECD (Organization for EconomicCo-operation and Development) established a group ofexperts to formulate the first GLP principles. This was anattempt to:• Avoid non-tariff barriers to the marketing of chemicals;• Promote mutual acceptance (among member countries)of non-clinical safety data;• Eliminate the unnecessary duplication of experiments.

The initial proposals were subsequently adoptedby the OECD Council in 1981, through the "Decisionrelated to mutual acceptance of data in the assessment ofchemicals". The Data Mutual Acceptance (DMA), recom-mended by the OECD, states that "the data generated inthe testing of chemicals in an OECD member country,performed in accordance with the guidelines and GLPprinciples, should be accepted in other OECD membercountries to perform the evaluation and other uses relatedto the protection of man and the environment". In thefollowing years, several workshops were held in order toimprove the content and interpretation of the proposedprinciples. The result of these meetings was the publica-tion of a consensus or guideline (to support the experi-mental development). After 15 successful years, the GLPprinciples published by the OECD were reviewed bygroups of experts and adopted by the OECD Council in1997 (4). Internationally, adherence to the GLP principlesis a prerequisite for the mutual acceptance of data gener-ated in a study. Several OECD member countries haveincorporated the GLP principles in their legislation.

To facilitate mutual validation, in 1997 the OECDCouncil adopted the "Adherence of non-member countriesto the Council Acts related to the mutual acceptance ofdata in the assessment of chemicals", in which non-membercountries have the possibility of voluntarily adhering to theestablished standards and, following satisfactory imple-mentation, are allowed to be a part of the OECD program.This required the establishment of national control proce-dures, and according to which, the authorities of eachcountry should exchange information on the compliance ofinspected test facilities and also provide input on theprocedures for compliance control. In countries with noofficially recognized authorities, individual studies performedby the pharmaceutical industry, in which non-clinical safetydata are already developed under GLP standards, can bemonitored by foreign GLP inspectors (5).

Good Laboratory Practices in BrazilIn Brazil, the requirement for GLP began when the

Brazilian Institute of Environment and Renewable NaturalResources (IBAMA), by Decree No. 139 of December 21,1994, established that studies that aimed to assess thepotential environmental hazard of pesticides and other

chemicals, based on toxicological, ecotoxicological andphysical-chemical studies, for the registration and market-ing of these products in the country, should be performedby laboratories accredited by the National Institute ofMetrology, Quality and Technology (INMETRO), in accord-ance with the GLP principles (6). As a result, in 1995, thedocument "Principle of Good Laboratory Practice", withreference to the document published by the OECD calledSeries on Principles of GLP and compliance monitoring,was published by INMETRO. In 1996, the Decree No.139/94 was replaced by IBAMA Ordinance Decree No.84/96. The main idea, however, that laboratories perform-ing the tests for pesticide registration purposes should beaccredited by INMETRO, was maintained (7,8).

In 1997, INMETRO and IBAMA established togetherthe criteria for "accreditation", which was replaced in 2010by the term adopted until today: "recognition of compli-ance with the Principles of GLP", which is a confirmationby the Accreditation General Coordination (AGC) of atest facility adherence to the Principles of GLP and itsinclusion in the Brazilian Program of Conformity with GLP.Thus, in 1998, INMETRO and IBAMA started to createsuitable conditions for Brazil’s adhesion to the OECDrecommendations, allowing results generated in Brazilto be validated by OECD member countries. In 2009,by Decree No. 220, INMETRO appointed AGC as theBrazilian Compliance Monitoring Authority for the Princi-ples of GLP. In 2011, through AGC, Brazil obtained fulladherence to the OECD acts on mutual acceptance ofGLP data for the evaluation of chemicals, pesticides, theircomponents and related products. Brazil’s full adherence,in a straight vision, means that the results generated inBrazilian recognized laboratories could be automaticallyaccepted by other member countries. This was an impor-tant step for the inclusion of Brazil in the worldwidepharmaceutical scenario. For medicines regulation, GLPpreclinical studies are the initial part of a long and complexmultistep process, without which the release of chemicalsfor human use should definitely not be allowed.

In other words, Brazilian Test Facilities, recognized bythe AGC, started to have tests performed with substancesaccepted by OECD member and non-member countrieswith full adherence to Mutual Acceptance of Data (MAD).Other substances, such as pharmaceuticals, cosmetics,food and food additives, veterinary products, sanitizers,genetically modified organisms, among others, tested inAGC recognized test facilities in compliance with the GLPprinciples, are still not covered by the MAD system.Therefore, other countries have no obligation to accept thetests performed in Brazil with these substances, even ifrecognized by AGC (6).

Concepts in Good Laboratory PracticeThe concept given the term GLP can be considered an

example of concise and precise definition, which not onlydefines GLP as a quality program, but differentiates it from


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other systems. GLP restricts actions to organizational pro-cesses, where all steps related to non-regulated clinicaltrials should be developed, from conception and design tothe final stages (preparation of the final report andarchiving). Thus, GLP is defined as "a quality programrelated to organizational processes and conditions wherenon-clinical health and environmental safety are planned,performed, monitored, recorded, reported and archived."

GLP is based on three main figures:• Test Facility Management (TFM): the person(s) withthe authority and formal responsibility for the organizationand good functioning of the operational unit in accordancewith the principles of GLP. They are responsible, amongother functions, for the approval of all operational pro-cedures and the appointment of other figures in the GLP.The mention of management as the first pillar of theprinciples of GLP is not a coincidence. It is known that thequality of the program will only be successful if it is aninternal conviction of the TFM. It is not sufficient to makedeclarations of conformity that exalt the virtues of quality ifincorrect or non-validated information is transmitted toemployees, in regard to compliance with and adherence toGLP principles. This includes, for example, omitting whenprocedures are not accomplished, reducing investmentsrequired for compliance with the principles, and not attend-ing the quality unit requirements when the requestedchanges require greater investment, among others. Thecollaborators can conclude that only the appearance isimportant, rather than genuine compliance, which couldcompromise the entire system (for detailed TFM respon-sibilities, see section II of the NIT DICLA 035 (9)).• Quality Assurance Unit (QAU): the QAU is an internalsystem designed to ensure that the GLP principles aremet and that the studies conducted in the installation testare in accordance with these principles. For this purpose,it is a prerequisite that the QAU has independence. Anyactivity delegated to the QAU cannot compromise itsoperation and no member of the QAU can be involved inthe experimental development, unless they are monitoringfunctions. It is also essential that the person responsiblefor the QAU has direct access to different levels of manage-ment, in particular the TFM. It is the obligation of the personresponsible for the QAU to highlight any deviation or non-compliance with the principles of GLP in any part of thetest facility or in any procedure, so that corrective actionscan be established (for detailed information on the QAUfunctions, verify section II of the NIT DICLA 035 (9).• Study Director (SD): the SD is the only point of controlof a study and the only one who supports the study, sincethey are responsible for the study from the beginning tothe end. Thus, the SD ensures that scientific, regulatoryand administrative aspects of the study are completelycontrolled. The SD is usually the researcher responsiblefor the preparation and approval of the study plan, as wellas the data collection and/or its supervision, analysis,reporting and conclusions of the study. The SD has the

formal assignment of acting in accordance with the GLPprinciples, and must prioritize the scientific standard of thestudies related to the quality/efficacy of the experimentaldesign, evaluation and significance of the generateddata (for more details on the DE responsibilities, see thesection II of the NIT DICLA 035) (9).

Although not a requirement, the basic concepts of theGLP principles encourage the appropriate applicationof science, since the need to prepare a study plan withdetailed arguments about the reasons for performing thestudy, as well as producing the proposal, can certainlylead to a more rational execution of the study. An exampleto be mentioned among the complex relationships betweenthe GLP and science may be the determination of glucoselevels in biological samples. There are complex and highlyaccurate methods and also simple methods that can onlyidentify the presence or absence of glucose in a biologicalsample. Any method can be applied in accordance withthe principles of GLP (if performed according to the stan-dard operating procedure (SOP) and by allowing itsreproduction). However, it is clear that according to theaccuracy level required by the scientific proposal of thestudy, it is the regulatory agency’s role to reject any studywhich has methodologies that are unable to produce resultswith the required precision, reliability and reproducibility.

Thus, the GLP requirements are primarily implementedto ensure the quality and integrity of the data and are notdirectly related to scientific aspects, but to the applicationof its principles; however, the scientific aspects should betaken into consideration (10).

The regulatory authority can evaluate the data froma study in two ways: i) repeating all experiments, orii) rebuilding, step-by-step, all activities and circumstancesthat led to the outcome of the study. Although the first methodis the most reliable, it is impractical due to the high cost; inaddition, this often violates ethical principles, since it involvesrepeating previous studies and submitting more animals totoxicity studies. In turn, the second method, despite notproviding direct confirmation of the results, implies trust in thedata generated, simply because the planning and perfor-mance of the experiment, as well as the recording andreporting of data, can be traced and evaluated (if the workperformed in the test facility can be considered reliable) (10).

Primarily, the GLP has been developed to combatfraud in the generation and reporting of safety data.However, the goal of the GLP is much broader, as it is notonly a control mechanism allowing regulatory agencies tojudge the integrity of a study. The principles of the GLP aredesigned as a tool that also allows improvements in thestudy and data quality, by applying strict requirementsregarding documentation, providing the ability to rebuildany activity, and making the way back to its inception (10).

The GLP requirements are related to several issuesand are aimed at different organizational levels of a testfacility. Many of these requirements can be considered"common sense" and should be followed when working



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under the principles of GLP. Broadly, the requirements can besummarized in three points that are the central ideas in GLP:• Reproducibility: in general, this is the possibility of athird party rebuilding the full course of a safety study, evena long time after its completion, and even in the absenceof those who were actively involved in the conduct of theirstudy. This reconstruction capacity is the guarantee thatthe regulatory agency will need to prove that there wereno major faults in the conduct of the study; for example,that all animals received the correct dose of the test articleduring the entire duration of the study, the correct sampleswere collected and analyzed, and the compilation ofresults faithfully reflects the data collected. This providesassurance that the experiments were conducted asdescribed in the report submitted to the regulatory agency.• Responsibility: this is closely related to the first term.The documentation required to conduct a study, accordingto the principles of GLP, will report who did what andwho can be held responsible for likely errors. On the otherhand, if there are any questionable cases, it is also pos-sible to charge the correct person, if they are stillemployed in the test installation.• Awareness: the principles of GLP raise awareness ofbroad tasks, such as administrative work, which is aimedat the quality and transparency of studies conducted in thetest facility. The SDs perform the studies under theircontrol in an orderly manner, whilst also raising awarenessof achieving small routine tasks, which in theory can beconsidered dangerous by not requiring "double attention";if they are not archived, they may culminate in a failure toobserve significant effects.

All of these points require general principles to befollowed; e.g., that not only are records generated for eachactivity, event and/or condition, but that they are alsokept in an orderly manner to allow the full recovery of theinformation, whenever necessary.

Study of distribution, metabolism andpharmacokinetics (DMPK) of newsubstances

The main characteristics that determine the success ofa drug candidate are directly related to its kinetic proper-ties. In this context, through non-clinical studies that areplanned and properly executed, it is possible to char-acterize the pharmacokinetic profile of a substance aimingto establish an adequate dosage that enables patientadherence to treatment and the correct interpretation ofresults obtained from efficacy and safety studies. Mostly,the toxic effects of substances leading to the discontinua-tion of their development are associated with prolongedsystemic drug exposure, the formation of toxic/reactivemetabolites and/or possible drug-drug interactions (11).A number of drugs such as troglitazone, trovafloxacinand bromfenac (oral formulation) were withdrawn fromthe market due to the formation of reactive hepatotoxic

metabolites (12) and other drugs that are still on themarket such as acetaminophen and amiodarone, etc., cancause hepatotoxicity when used at high doses (12).

Considering the idea that the pharmacokinetic proper-ties of a substance are determinant to its success inclinical studies, the pharmaceutical industry has intro-duced DMPK (Drug Metabolism and Pharmacokinetic)studies in the early phases of new drug development.A recent study performed by four important pharmaceuticalcompanies (AstraZeneca, UK; Eli Lilly and Company,USA; GlaxoSmithKline, UK; and Pfizer, USA) showedthat, with the introduction of DMPK studies during the leadidentification phase, the pharmacokinetics represent only5% of the reasons for failure in clinical development (13).From the financial point of view, the discontinuity of aproject during the final phases generates losses of around90% of the total investment (14). Therefore, the DMPKstudies carried out during the non-clinical phases areextremely important, since they reduce the time and costsexpended with the development of new drugs. In addition,the DMPK performed during the early phases of drugdevelopment provide important information about thestructure of a molecule and possible modifications thatcan be made in order to optimize its DMPK properties.

Finally, data about the pharmacokinetic properties ofa new substance together with preliminary studies aboutits safety and efficacy are critical to take the decisionto continue or not (go/no-go decision) the developmentprocess of a new drug (15).

Factors that determine the pharmacokineticprofile of substances

Physical-chemical characteristics and physiologicalproperties

Several physical-chemical properties such as lipophil-icity, rate of dissolution, solubility, pKa and molecularweight, can directly interfere with the absorption, distribu-tion, metabolism and elimination of a substance (16,17).The lipophilicity and rate of dissolution are expressed asLogP (partition coefficient of non-ionic substance betweenthe hydrophilic and lipophilic phase in water/octanol system)and LogD (partition coefficient of ionized substances,normally weak acids and bases) (17). Substances withLogP45 are considered highly lipophilic and, althoughpresenting high membrane permeability, they also showlow solubility which hampers their absorption (16).

The solubility of a substance in different physiologicalconditions, as well as the lipophilic characteristic, also inter-feres with its absorption. One of the factors that directlyinfluences the solubility of a substance is its pKa(the negative base-10 logarithm of the acid dissociationconstant (Ka) of a solution). It defines the concentrationat which neutral and ionized species of a molecule areequally distributed in a specific pH (17). The knowledgerelated to the solubility and permeability of a substance is



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a very important aspect since it allows the prediction ofbioavailability after oral administration and its classifica-tion according to the Biopharmaceutical ClassificationSystem (BCS) (for details about BCS, see 18,19).

The absorption rate of a substance after oral admin-istration depends mainly on its capacity to cross theintestinal epithelium. Different in vitro methods are usedto determine the intestinal permeability of a substance,such as: i) human colon adenocarcinoma cells (Caco-2);ii) Madin-Darby canine kidney (MDCK) cells, and iii) theparallel artificial membrane permeability assay (PAMPA).However, Caco-2 cells are considered the gold-standardassay for permeability evaluation of substances that weredeveloped for the oral route of administration due to itsspontaneous differentiation process, which leads to theformation of an enterocyte monolayer with preserved mor-phological and functional characteristics (15,17). Besides thephysical-chemical properties, physiological factors asso-ciated with the binding affinity to plasma proteins and themetabolic stability of a substance are also important todetermine its DMPK properties (11,20).

The binding of a substance to plasma proteins, mainlyto albumin and acid a1-glycoprotein, occurs quicklyand reversibly until it establishes the kinetics equilibriumbetween the bound and unbound form. Only the unboundform is able to cross the capillary and reach the targetorgan. The concomitant administration of more than onesubstance could interfere with the binding affinity, produc-ing rather an exacerbated pharmacological effect or notproducing the desired therapeutic effect. The binding eval-uation of a substance to plasma proteins can be performedusing in vitro experiments by means of ultrafiltrationtechniques and equilibrium dialysis (11).

The metabolization process of a substance acts asa body’s defense system, leading to the modification offoreign substances (xenobiotics) by chemical processesto promote their elimination from the organism (20). Thebiotransformation studies allow evaluation of the meta-bolic stability level of a substance and the prediction ofpossible metabolites’ formation, which are more activethan the parent substance (prodrug), or even toxic meta-bolites; it also allows evaluation of whether the parentsubstance, which is often metabolically unstable, canreach the therapeutic concentration that is necessary toproduce the pharmacological effect (15,20).

The main organ in the body responsible for metabo-lization and detoxification of substances is the liver. It canalso occur in the lung, kidneys, gut and blood plasma.Drug-metabolizing enzymes of the system cytochromeP450 (CYP450), mainly CYP1A2, CYP2C9, CYP2C19,CYP2D6 and CYP3A4 are responsible for phase Ireactions (oxidation, reduction, hydrolysis, dealkylation,and deamination), which promote the conversion of alipophilic compound to more hydrosoluble metabolites,which are easily eliminated from the human and/or animalbody (15,20). CYP3A4 is the most abundant cytochrome

P450 isoform in human liver and has a broad substratespecificity. CYP3A4 is involved in the metabolism ofalmost 50% of all drugs available on the market (20). Todetermine the metabolic stability of a substance, bothin vitro and in vivo tests should be performed. The cellularsystems most often used for in vitro metabolic stabil-ity studies are: i) liver microsomes; ii) S9 fraction, andiii) culture of human hepatocytes for the determination ofhepatic clearance (15,20,21).

The identification of the main enzymes responsiblefor the metabolization of a substance in vitro generatesdetailed information about its metabolization process.It allows adequate guidance for the clinical study relatedto: i) substance interaction; ii) dose selection (to thosepatients with kidney or liver damage), and iii) toxic effectprediction (22–24). In parallel, the assay performed inmicrosomes that uses liver samples from humans andfrom other species should be conducted to evaluate pos-sible inter-species differences. Such results will indicatewhich species is more appropriate to execute the toxicitystudies, as well as to evaluate the possible involvementof the CYP3A4 and CYP2D6 enzymes in humans(15,20,21).

Several medicines were withdrawn from the marketdue to their interaction with other substances (mainlyrelated to the CYPs) for example: Seldanes (Terfenadine,Aventis Pharmaceuticals, USA), Posicors (Mibefradil,Roche, Switzerland), Propulsids (Cisapride, Janssen-Ortho,Canada), Lotronexs (Alosetron, Prometheus LaboratoriesInc., USA), Baycols (Cerivastatin, Bayer A.G., Germany)and Serzones (Nefazodone, Bristol-Myers Squibb, USA)(25). There are several examples of drugs that induceCYPs or, in other words, increase the metabolizationprocess, for example, barbiturates, rifampicin, omeprazoleand alcohol. On the other hand, drugs such as quinidine,ketoconazole and sulphaphenazole inhibit the CYPs reduc-ing the metabolization process (21–23). Besides theenzymes cited above, phase II enzymes (transferases),such as sulfotransferase, glucuronyl transferase andglutathione-S-transferase, enhance xenobiotic eliminationbased on the conjugation reactions. Other enzymes arealso involved in the chemical process of biotransforma-tion, such as alcohol dehydrogenase, aldehyde dehydro-genase, and NADPH-quinone oxidoreductase (22,23).Thus, the identification of enzymes responsible for theprocess of drug metabolization is recommended by reg-ulatory agencies during the discovery and developmentphases to evaluate possible drug-drug interactions (15,22).

Determination of pharmacokinetic properties in vivoThe in vivo pharmacokinetics assays allow the quanti-

tative evaluation of the time course of absorption, distri-bution, metabolism and elimination (ADME) of a newsubstance; this information is very useful for predicting thedesirable dosage and the appropriate posology protocol tobe used (15).



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Initially, during the non-clinical phase, performingexploratory pharmacokinetic studies is suggested. Thesestudies aim to support the pharmacology assays, the inter-pretation of toxicology and efficacy studies, and dosageselection and the compound/drug formulation optimiza-tion. During this phase, a reduced number of animals areused and blood samples are collected until 6 h afterthe administration of substances. A limited volume can becollected in accordance with the species and animalweight. For more details about the recommended bloodvolume to be collected, please see ‘‘Guidelines for Sur-vival Bleeding of Mice and Rats’’ developed by theNational Institutes of Health (26). It is recommended tofollow these limits since excess blood withdrawal caninterfere with the pharmacokinetics profile of the substance.

From initial pharmacokinetic (PK) screening, it ispossible to select substances that show adequate PKproperties and, after this, the selected substances aresubmitted to complete screening. The screening modelscurrently used are snapshot PK, rapid PK and full PK.However, the strategy choice depends on many factors,including materials and tools available, researchers’knowledge and definitions of the PK parameters to beanalyzed (27).

During the PK profile analysis of a substance, thefollowing parameters should be determined: i) area underthe curve (AUC); ii) maximum drug concentration in plasma;iii) time to reach the maximum concentration; iv) half-life;v) distribution volume; vi) clearance; and vii) bioavailability.

Desirable DMPK properties of a candidatesubstance intended for oral routeadministration

A substance intended to be used via the oral routeshould present some critical properties in DMPK studies:i) water solubility; ii) high permeability and low efflux inCaco-2 cells; iii) sufficient bioavailability to reach thedesirable plasma and organ to produce the pharmacolo-gical effect; iv) adequate half-life time to the intendedposology scheme in human; v) linear PK; vi) eliminationthat is not dependent on a single route or on a singlemetabolization enzyme, without forming active or reactivemetabolites in large amounts and without interactingwith metabolization enzymes in relevant concentrations;vii) acceptable safety margin (therapeutic index, preferablyhigher than 10 times); and viii) established PK-PD (pharma-codynamics) relation (15).

Toxicokinetic assay

The toxicokinetic assays are usually performed dur-ing toxicology studies and should be conducted in accord-ance with the GLP rules. The toxicokinetic assaysmeasure the systemic exposure of a substance in animals

and establish the relationship between the dose adminis-tered and the time course of the substance in the toxi-city studies. Indeed, the PK profile determination of asubstance following administration of multiple dosesenables the best interpretation of the toxicological findings.The toxicokinetic study also evaluates the potential of asubstance to accumulate in a specific organ and/or tissue.Thus, the data generated with these studies should con-tribute with the data obtained with the toxicology studiesin terms of interpretation of the toxicity tests and incomparison with the clinical data as part of the risk andsafety evaluation in humans. The toxicokinetic studies arepart of the non-clinical test battery recommended by theregulatory agencies (11,28).

The main questions that should be answered tofacilitate the comprehension between the systemic expo-sure of a test article and the quantification of the absorbedfraction in the tissues are: i) is the substance absorbed?;ii) what is the absorption rate?; iii) how is the substance(parent/metabolite) distributed within the body?; iv) is thesubstance metabolized?; v) if yes, in which organ/tissue,what is the rate of metabolization, and which metabolitesare formed?; vi) what is the route and rate of elimination?;and vii) what is the effect of the dose on absorption,distribution, metabolism, and elimination? (29).

Two protocols can be applied for toxicokinetic studies:a full protocol that aims to answer all aforementionedquestions or a reduced protocol, in which only the mainquestions are answered to corroborate the interpretationof the toxicology findings. In the full toxicokinetic protocolother biological matrices should be collected besidesblood, such as excrements (urine and faeces), fat, muscles,liver, kidneys, possible target-organs and skin (when thesubstance test is administrated by the dermal route).Moreover, if the substance in its parent form and/or itsmetabolites are volatile, additional animal groups shouldbe included for collecting excrements, carcasses and expiredair in order to determine the extension of absorption andthe route(s) of elimination of the substance. The design ofthe study and the selection of the experimental protocolshould be defined on a case-by-case basis; overall, theyshould be able to provide enough information to evaluatethe risks and safety of the candidate substance (28,29).

Considering the importance of evaluating PK proper-ties during the development of new drugs, the assaysdescribed in Table 1 are highly recommended.

Chemical characterization, manufacturingand manufacturing control

The chemistry, manufacturing and controls (CMC)of an active/final product are important for the adequateexecution of the non-clinical and clinical studies of a can-didate as well as for the correct interpretation and cor-relation between the results obtained in each phase of the



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discovery and development process. It is recommendedthat the manufacturing process follows the Good Manu-facturing Practices (GMP) in order to guarantee the quality,safety and efficacy of the pharmaceutical products and toensure the manufacturing consistency and batch-to-batchreproducibility (30).

Since CMC of an active substance/final product pro-vides important information that guarantees its identity aswell as its quality during the manufacturing process, theCMC submission to the application for product registrationrepresents one of the requirements of regulatory agen-cies. More details on the CMC of an active substance/finalproduct requested by regulatory agencies can be found inthe guideline M4Q (R1) (31).

Safety studies

Safety studies to evaluate toxicityThe recent advance in the development of new drugs

has become a challenge for science, as the offer of newtherapeutic approaches has required techniques thatguarantee its safety in humans. Non-clinical safety studieshave been performed based on the experience and employ-ment history in a specific animal species before safetytests have been performed in humans. Besides animalstudies, several in vitro tests have been developed andvalidated for safety evaluation to discover the toxicologicalpotential of substances. However, these assays are some-times complementary to the in vivo tests.

The use of animals to evaluate the toxicity of com-pounds started in 1920, when J.W. Trevan introduced the

Lethal Dose 50% (DL50) concept. After this, Food andDrug Administration (FDA) scientists started to developnew methods, such as the ocular and cutaneous irritationin rabbits that were widely accepted and applied all overthe world. In addition, the researchers of the NationalInstitute of Cancer in the USA started to develop tests inmouse to predict the cancer-causing potential of newsubstances. However, after 1960 and due to several birthsof children with limb deficiency caused by Thalidomideuse during pregnancy, safety studies performed initially inanimals were required. After those facts, the FDA requiredan Investigational New Drug (IND) application for all newsubstances that progress to clinical tests. The IND appli-cation must contain the safety and efficacy data of thesubstance before the first human exposure (see moredetails below) (32).

At the end of 1980, the OECD and the InternationalConference on Harmonization (ICH) published guidelinesfor toxicity in non-clinical tests for chemical and pharma-ceutical substances, which are still recommended by themajority of the regulatory agencies. Since its publication,new revisions and assays were implemented throughoutthe years, aimed at the promotion of more predictive andethical tests that could reduce or even prevent the use ofanimals.

These guidelines present the basis of how assaysshould be conducted, suggesting species to be used,duration of the assays, organs to be investigated and theanalysis to be conducted, as well as which data should bepresented in the final report. Even so, these guidelinesare still generating several doubts in the scientific and

Table 1. Recommended non-clinical assays of ADME/PK.

Non GPL

In vitro In vivo In vivo

1) Physical/chemical properties

[lipophilicity (log P/log D), solubility,chemical stability (pKa)]

2) Metabolic stability

3) Hepatic clearance4) Interaction between substances

(inhibition/induction of CYPs)

5) Physiological characteristics(plasma protein/tissue binding)

6) Permeability7) Plasmatic stability and total

blood/plasma partition

1) Pharmacokinetic profile

(concentration versus time)- Area under the curve- Cmax

- Tmax

- Distribution- Clearance

- Half-life time2) Biodisponibility bioavailability3) Linearity4) Metabolization

5) Routes of excretion

1) Toxicokinetic

- Pharmacokinetic profile(concentration versus time)

- Area under the curve

- Cmax

- Tmax

- Distribution

- Clearance- Half-life time

2) Biodisponibility3) Metabolization

4) Routes of excretion5) Quantification of biological fluids,organs, tissues, excrements and

expired air (when necessary)

ADME/PK: absorption, distribution, metabolism and elimination/pharmacokinetics; GLP: Good Laboratory Practices;Tmax: time-to-maximum; Cmax: maximum concentration. Source: adapted from (11).

GLP



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industrial communities as the assays are not presented indetail.

The performance of non-clinical tests in sequence isan important factor in the development of a new medicine.Despite of there is not a standard program to execute theassays, a well-designed planning helps avoiding severalerrors or unnecessary tests, besides saving time andfinancial resources. In Figure 1, we suggest the non-clinical studies that should be performed during drugdevelopment Thus, in this section, we will discuss themain non-clinical safety tests that are required in theprocess of drug development, such as mutagenicity tests,acute, sub-chronic and chronic toxicity, developmentaland reproductive toxicity, carcinogenicity, local toleranceand safety pharmacology.

Preliminary toxicology studies. In the initial phasesof development, several promising selected substancesfollow exploratory safety test screening to assess possibletoxic effects. The exploratory tests are normally performedin vitro, or with a reduced animal number and do notrequire conformity with GLP principles. For this reason,these studies present reduced costs in comparison to theGLP studies that are required in subsequent steps inthe drug development process. The exploratory assaysare essential for the initial decision making regarding theinvestment on a new substance, since it could providerelevant information, which directly affects the planning ofnon-clinical assays that will be performed.

One of the initial tests to evaluate the toxicity of anew substance is the preliminary Ames test. This test isperformed to evaluate a possible genotoxicity effect andthe detection of genetic alterations in organisms exposedto these substances. The different genotoxicity testsdetect potential genetic and chromosomal mutations inorganisms. The Ames test is an in vitro mutation assay inbacteria and has the ability to detect any mutationpromoted by the substance, enabling the reversion ofthe existent bacterial mutation and restoration of thefunctional bacterial capacity to synthesize an essentialamino acid (histidine). This test can either evaluate themutation capacity of the substance or its metabolites. Theexecution of this test is mandatory to most of the sub-stances in the drug development process. Despite the testbeing regulated and required by the authorities, thepreliminary assay is fundamental for the early detectionof possible genotoxic effects of the test article. Further-more, the in vitromicronucleus assay has emerged as oneof the preferred methods for assessing chromosomedamage. At this development level, the genotoxicity isrestricted to in vitro tests; however, in the subsequentsteps, other tests of genotoxicity are required, such as thein vivo micronucleus test.

The first in vivo toxicity study for a new substance isusually a dose range finding (DRF) study in rodents. Forboth scientific and welfare reasons, it is common prac-tice to explore adverse effects in rodent species prior to

Figure 1. Steps of non-clinical studies in drug development process. GLP: Good laboratory practice; IND: Investigational new drug;NDA: New drug application; ADME: absorption, distribution, metabolism and elimination.



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non-rodent species. This increases the amount of informa-tion available for the design of non-rodent studies; forexample, data from the initial rodent study can be usedto set the starting dose, or allow specific monitoring ofadverse effects in non-rodents. The Maximum ToleratedDose (MTD) test by dose escalation scheme is a commontest used for the dose selection in GLP toxicity studieswith a duration of 30 days. The MTD test allows theidentification of the dose at which target organ toxicity islikely to be observed, but without further study implicationsdue to the animals’ morbidity and mortality (33).

MTD is defined as the highest dose tolerated in atoxicology study. The methodology is normally determinedby parameters such as clinical signals, body weight changes,food consumption, morbidity and mortality. Besides that,several dose selection protocols also recommend thehematological and biochemical analysis execution, as wellas the histopathological analysis of target organs to betterdetermine the toxicity between the tested doses.

The acute or repeated-dose toxicity studies can beperformed during the preliminary phase of the develop-ment process. Although the MTD or dose escalationstudies provide important information about drug toxicity,the repeated dose studies have more complete protocols,which consider the histopathology of a set of organs, morecomplex behavioral and clinical analysis, complete bio-chemical and hematologic analysis, ophthalmologicalanalysis and groups for the evaluation of the side effectsrecovery after a treatment period. In the acute toxicityprotocol, the effect of a single administration of threedifferent doses is usually evaluated and the animals areobserved for 14 days after treatment. The OECD guide-lines do not require the acute oral toxicity assay forpharmaceutical products, but some regulatory agenciessuggest this assay. Also, according to the M3 guideline(R2) (34), the acute toxicity is only recommended whenthere are no other studies about toxicity, such as MTD ordose escalation. In this case, the acute toxicity studies canbe limited and provide information about the administra-tion routes and the doses to be administered. These datacan be collected from non-GLP studies. However, theclinical treatment planning can only be supported bytoxicological repeated dose studies performed in accord-ance to the GLP rules M3(R2) (34).

The short-term repeated-dose toxicity study is anotherprotocol suggested during the exploratory phase. Themost indicated test is the repeated dose 28-days oraltoxicity study No. 407 (35). Since it evaluates the toxicitylevel of continuous administration, this test can providemore precise data, although it is more complex in com-parison to the acute toxicity and MTD tests. This protocolis normally required for the first exposure of the substancein human (Clinical phase I); however, its execution willdepend on the objective of the clinical treatment regime,as its duration in humans is directly related to the non-clinical protocol. It is important to mention that deciding

which exploratory or regulated toxicology study will beperformed requires deep planning by the developmentteam. Considering that the basis of defining which studiesshould be performed is related to the intended clinical useof the test article, the interaction between the non-clinicaland clinical study teams is required.

Regulatory toxicology studies. Regulatory toxicologystudies are mandatory in the drug development processand aim to evaluate the toxicity level of a substance usingprotocols that follow the guidelines recommended to con-duct non-clinical studies of pharmaceutical products.In addition, it is important to emphasize that they have tobe conducted in compliance with the GLP principles. Afterthe preliminary toxicity studies, the GLP studies should beconducted in two animal species (with the exception ofmutagenicity tests). The planning of these studies couldbe based on the data obtained by the exploratory studiesof both efficacy and toxicity. These findings could help todefine doses, the duration of study, and any side effectsthat could require special attention. Some GLP toxicologystudies are required before beginning clinical trials, butothers could be conducted during different phases of theclinical trials; this will be discussed further in this section.

Although there are no unique and standard plans fordrug development, it is recommended to perform geno-toxicity studies (in vitro and in vivo) as well as a study ofdose selection and repeated-dose toxicity (28 days),before the first exposure of humans to the substance.Usually, with these studies series, together with pharma-cokinetic, efficacy, safety pharmacology and substancechemical characterization studies, it is possible to submit adossier to the regulatory agencies to request permissionto start the tests in humans. It is important to emphasizethat for toxicology studies following GLP principles, thetest article should be in its final formulation, in otherwords, in the same formulation that will be used to treatindividuals during the clinical studies, together with itscomplete chemical certificate of analysis. In addition, theroute of administration should be, preferably, the same asthat intended for human treatment. These requirementsare clearly described in the guidelines of non-clinicalstudies of the main regulatory agencies, such as the FDA,European Medicines Agency (EMA) and Agência Nacio-nal de Vigilância Sanitária (ANVISA, Brazil).

In this step, the genotoxicity tests previously described(in vitro Ames No. 471 (4) and in vitro micronucleusNo. 487 (36)) should be performed in accordance withthe GLP requirements, even when it has already undertakenexploratory studies. Thus, the in vivo micronucleus testNo. 474 (37) is also recommended, as it provides relevantgenotoxicity data of a substance involved in active pro-cesses, such as metabolization, pharmacokinetics andDNA repair, which are not totally detected by an in vitrosystem. This test evaluates micronucleus formation inerythrocyte samples from the bone marrow or from rodents’peripheral blood samples, allowing the identification of



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possible cytogenetic damage, resulting in micronucleusformation and chromosomal alterations. In many cases,the genotoxicity assays, performed according to the GLPprinciples, are conducted before the repeated dose tox-icity tests for decision-making reasons. However, it dependson the strategy programmed for each substance and onthe obtained preliminary data. Also, execution of the GLPgenotoxicity test concomitant with initial repeated dosetoxicity studies is common.

An important decision during the planning of non-clinical studies is the duration of the repeated dose toxicitystudy that is normally based on the duration, therapeuticindication and planning of the clinical study. Generally, theduration of toxicity studies conducted in two mammalianspecies (rodent and non-rodent) should be the same oreven longer than the studies in humans, but no more thanthe maximal time recommended by the M3(R2) guideline(34) for each species (see more details in Table 2). Thistable describes the recommended duration of repeated-dose toxicity studies to support the conduct of clinicaltrials. The relation between animal and human studies is avery important point in the drug development process,once the conduction and the choice of non-clinical studiesshould justify the time duration proposed for clinicaltreatment.

The repeated dose toxicity studies have guidelineswith a very well defined duration, such as the guidelinesNo. 407 (repeated dose 28-days oral toxicity study inrodents), No. 408 (repeated dose 90-days oral toxicitystudy in rodents), No. 410 (repeated dose 21/28-daysdermal toxicity study), No. 452 (chronic studies of toxicity),etc (4,35,36,38–40). One of the most used protocolsbefore the first exposure of substances in humans is therepeated dose for 28 days. As mentioned in the explor-atory studies section, this test aims to collect informationabout possible health risks using the repeated exposure toa substance, including its central effect, and that on theimmunological, endocrine and reproductive system.

Although this test is indicated for oral administration,other parenteral administration routes could be used ifwell justified and if they are similar to the clinical uses. Inaddition, the toxicity test following repeated doses of thesubstance could also be applied for 14 days when it isjustified by the short time of treatment in the clinical phase.Besides, it is highly recommended to add a recoverygroup to the study in order to observe possible toxiceffects recovery. The repeated dose toxicity study shouldbe performed in accordance with the GLP requirements.The obtained outcomes are fundamental for characteriz-ing the toxicity of the test article and provide a relationshipbetween the dose-response and toxicity data to determinethe no observed adverse effect level (NOAEL).

The toxicity data described in the guidelines sug-gested by the FDA comprise an important basis forthe IND application; however, it depends on the intendedapplication of each substance and can vary case-by-case.

After the authorization to start clinical studies, other non-clinical toxicity studies should be conducted; for example,sub-chronic and chronic studies. The toxicity evaluation isnormally classified in accordance with a chronologicalscale, such as the acute studies that are performed toverify the substance effect using single or repeated doseadministration for 24 h. Indeed, sub-acute studies arethose that comprise the toxic effects for 30 days, whereassub-chronic studies are defined by the toxic effect of asubstance between 30 and 90 days. Studies that aresuperior to 90 days are normally classified as chronic.However, this classification can be specific for somespecies; for example, chronic studies can be performed forsix months in rodents and 9 months in non-rodents (41).

The sub-chronic study (90 days) can be conducted inparallel with phase I clinical studies. This study is very similarto the toxicity study of 28 days, and the guidelines for bothrequire a daily treatment with at least three doses of thesubstance and the vehicle, together with clinical, biochem-ical, hematological, anatomical and histological analysis thatare detailed in each guideline. Despite standard measure-ments, some additional analyses could be included for theobservation of a particular effect of the substance, mainlywhen several toxic effects are described. These tests shouldbe conducted in accordance with the GLP principles; togetherwith the clinical data obtained from phase I, these can help todecide whether the study should continue or not to phase II.

The reproductive toxicology test occurs during theclinical studies, along with the teratogenic potential evaluation.The reproductive toxicology test is the most rigorous testapplied by the FDA and is a prerequisite for the approvalof new substances. In accordance with the guidelineS5(R2) (42), the drugs can affect the reproductive activity by:• Fertility and initial embryo-fetal development (implan-tation);• Embryo-fetal development or teratogenicity;• Pre- and post-birth development, including the mater-nal function.

Table 2. Recommended duration of repeated-dose toxicity studiesto support the conduct of clinical trials.

Maximum Duration ofClinical Trial

Recommended Minimum Durationof Repeated-Dose Toxicity Studies

to Support Clinical Trials

Rodents Non-rodents

Up to 2 weeks 2 weeks* 2 weeksBetween 2 weeks and

6 months

Same as clinical

trial

Same as clinical

trial46 months 6 months 9 months

*Clinical studies with lower duration than 14 days can be supportedby toxicity tests with same duration as the clinical study. Adaptedfrom M3(R2) Nonclinical Safety Studies for the Conduct of HumanClinical Trials and Marketing Authorization for Pharmaceuticals (33).



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The phase I study can often start in voluntary maleparticipants, even without reproduction/development data,as long as the substance has not shown evidence oftesticular damage in the studies of repeated doses of 2 to4 weeks’ duration (43). The requirement of reproductivetoxicology studies in the beginning of the clinical phase Imay differ in each country; however, it is quite often thatthese tests require clinical studies involving women offertile age. The fertility and implantation tests includemale (28 days) and female (14 days) treatments withthe substance before mating, and are characterized bythe semen analysis (counting and viability), number ofimplanted embryos and survival of the embryos at thesixth day of pregnancy. The embryonic and fetal testsare normally performed in two or three species (rats, mice,rabbits); the substance is administered to females in theinitial period of pregnancy (in rats, 6–16 days aftermating). In this case, the animals should be euthanizedbefore giving birth, aiming to count the embryo numberand observe abnormalities. In the pre- and post-develop-ment tests, females are treated during pregnancy andlactation, where the offspring can be observed accord-ing to the motor activity after lactation. In these cases,some pups are analyzed according to their abnormal-ities in different stages of development, even in adult-hood, to evaluate their sexual performance and theirsecond offspring (42,43). Despite some in vitro assaysof reproductive toxicity being routinely performed, theydo not provide enough data about teratogenic potentialin mammals and are not recognized and required by theauthorities (43).

The reproductive test battery is a requirement in thedrug development process for almost all regulatoryagencies; however, for herbal products, ANVISA sug-gests that these assays should not be performed. Thus,the following statement should be described in theproduct instructions: ‘‘it should not be used by pregnantwomen and nursing mothers since there are no studiesproviding its safety under these conditions’’ (44).

In addition to the general toxicology and reproduc-tive toxicity studies, the carcinogenicity test is usuallyrequired for drugs intended for continuous treatmentof 6 months or more. In these cases, carcinogenicitystudies should be carried out before the substances goto the market, but never before the beginning of clinicaltests. The carcinogenicity assay could be required incase of substances belonging to a known carcinogenicgroup or when chronic studies of toxicology presentconsistent evidence of the carcinogenic potential, oreven when there is evidence showing that the sub-stance or its metabolites are retained in the organism fora long period (43). Interestingly, in the absence of otherdata, substances with positive evidence in the geno-toxicity tests are considered carcinogenic to humansand are not submitted to long-lasting carcinogenicitytests. However, in case the substance has been used

for chronic treatment in humans, chronic tests (for about1 year) could be necessary to assess possible tumori-genic effects (45).

The carcinogenicity studies are normally carried outduring phase II and III of clinical development, usingonly a rodent species, especially rats. In addition, it isrecommended to perform other in vivo assays that canprovide additional information about the sensitivity ofthe carcinogenic substance, such as short duration testin transgenic mouse or carcinogenicity test of longduration in other rodent species (mouse). The carcino-genicity study of long duration in rats is usuallyconducted for at least 2 years of treatment with threeor four doses of the test article and the control.Generally, the lowest dose to be tested in these non-clinical studies is the maximal dose recommended inhumans, while the highest dose is the MTD obtainedin the previous safety studies. To perform the car-cinogenicity studies, it is necessary to include 50 to80 animals per group/gender. This means that theentire study needs around 600 to 800 animals beingtreated and evaluated for up 2 years. The ICH guide-lines (45–48) determine the rules to be followed in thesestudies, which require the performance of the studiesaccording to the GLP principles, with specific pathogen-free (SPF) animals and the histopathological analysiswith more than 50 tissue types being analyzed by aveterinary pathologist with experience in carcinogen-esis. The carcinogenicity test is one of the most difficultand expensive studies during the non-clinical develop-mental process.

Depending on the observed effects in the standardtoxicological studies, other tests could be required. Forexample, if the drug candidate induces alterations in theimmunological cells or in the lymphoid system tissues,immunogenicity studies could be necessary. Suchstudies are performed with substances that act bymodulating the immunological system or those causingalterations such as necrosis, apoptosis or interactions withcellular receptors shared by different tissues and non-target immunological cells (46). Some of these evi-dences could be obtained by hematological, biochem-istry and histopathological analysis obtained fromprevious toxicological studies. In these cases, assayssuch as the T-cell dependent antibody response (TDAR)test, immunophenotyping, natural killer cellular activity,etc. are recommended (48). Furthermore, for sub-stances previously known as immunogenic, the sensi-bility test could also be necessary. In addition, forsubstances that are administered topically, local toler-ance tests are required before the beginning of clinicalphase I, and could be part of other toxicology studies.This assay aims to evaluate the tolerance level of asubstance in different regions of the body with which itcould have contact. To perform these tests, the selection ofthe species depends on each assay type as well as the



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administration route, the dosage and also the exposuretime in accordance with the duration of the study to beconducted in humans. The local tolerance test can includethe administration route (dermal, parenteral, ocular, rectal,etc.) and tests of systemic toxicity. The guide CHMP/SWP/2145/2000 Rev. 1 (49) contains the detailed informationabout each test.

As previously described in this section, the toxicologytests are very important and require high responsibilityfrom the non-clinical and clinical teams. The availableamount of substances could require specific protocolsand requirements from the regulatory point of view. Forexample, the development of vaccines frequently doesnot require reproductive toxicity, mutagenicity or carcino-genicity tests. On the other hand, each substance hasparticular characteristics and is developed for the treat-ment of a specific and complex disease; for this reason,the development program should be analyzed case-by-case. Although the regulatory agencies suggest abasic battery of tests to be performed, it is fundamentalthat the developmental team anticipates possible addi-tional side effects to elaborate a complete clinic plan withimportant information and avoiding unnecessary studies.Thus, the previous and direct contact between the phar-maceutical industry and the regulatory agencies is highlyrecommended, aiming to establish the most appropriatetests for each drug candidate.

Safety pharmacologySafety pharmacology is a relatively new area in the

process of new drug discovery and development. It startedat the end of 1990 with a medical description of severecardiac side effects with the use of terfenadine (Seldanes,Marion Merrell Dow, USA). After thousands of medicalprescriptions, it was proven that terfenadine can causeTorsades de Pointes (TdP), which is a lethal cardiacsyndrome in healthy subjects caused when terfenadinewas used in high doses or in association with othermedicines (50). After this incident, the medication waswithdrawn from the market. So far, it was believed thatonly drugs used for cardiac indications could present thissevere side effect.

During the development phases of terfenadine, thetraditional non-clinical toxicological methods were used,which determined the toxicity of a substance in highdoses. However, by using these methods it was notpossible to detect the tendency of terfenadine to induceTdP. This problem could have been avoided if, during theroutine of the safety tests, a high-throughput screening(HTS) program using biomarkers to TdP had been used inthe initial discovery phases of the substance. However,this methodology was not part of the protocols for newdrug development. At that time, a specific area of drugdevelopment, named safety pharmacology, was createdin an attempt to identify the undesirable pharmaco-dynamic effects of drugs on physiological functions,

which are not identified in non-clinical toxicologicalstudies (51).

To determine the risk/benefit rate of a substance in thedevelopment phase is particularly difficult when rare, butpotentially lethal, side effects are a concern about the newdrug (51). In 2001 the ICH approved the guide S7A (52),which requires that the pharmaceutical industries per-form battery tests of safety pharmacology to determinepotentially undesirable pharmacodynamics effects of asubstance, mainly those related to the central nervoussystem (CNS), cardiovascular and respiratory systems aswell as implement supplementary tests to evaluate othersystems (53).

Currently, the tendency is that studies of safety phar-macology are not conducted only as a standard battery oftests recommended by the regulatory agencies, but also inan exploratory way in the initial phases of the develop-ment process. Thus, with the execution of in vitro, ex vivo,and in vivo preliminary tests of relatively low costs, it ispossible to detect early severe side effects allowing afast remodeling of the data and the reduction of problemsrelated to the safety of a new substance. In addition, suchstudies help with the decision about continuing the develop-ment phase or not. This initial phase is part of the processthat supports the selection and optimization of leadercandidate substances, in which usually it is not necessaryto follow the GLP requirements.

Preliminary studies of safety pharmacology. Mostof the problems that occur in developmental projects ofnew drugs or in the withdrawal from the market of anapproved new drug are usually associated to cardiovas-cular safety. Preliminary assays of cardiovascular safetypay special attention to the potential effect of a test articleon the cardiac conduction to assess as early as possiblewhether the drug candidate can induce a delay in therepolarization phase of the ventricular action potential(54). This phenomenon is often associated with the directblock or interruption in the maturation process of thepotassium channels hERG (alpha subunit Kv11.1) (55)that are channels of delayed rectification type rapidcodified by hERG gene type KCNH2 (56).

The relevance of the hERG channels in the cardiacelectric activity became evident after the demonstra-tion that genetic mutations in these channels have beenassociated with long QT syndrome (LQTS). LQTS is aproblem in the electric conduction of the myocardium thatalters the ventricular repolarization and, consequently,increases the vulnerability to the development of TdP-type ventricular arrhythmias and the chance of suddendeath (57).

Currently, it is well accepted that interference inventricular repolarization is reflected in the QT intervalincrease observed in the electrocardiogram, which is thetime required for the completion of both ventriculardepolarization and repolarization (58). The relationshipamong the hERG channels inhibition, non-clinical models



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of the QT interval evaluation, effects on the QT intervalin humans, and cardiac arrhythmia is well known anddescribed in the guidelines S7A and S7B (59), whichgive directions to the evaluation of risk of QT interval pro-longation (QT risk). Considering the relevance of theseevaluations, it is essential to perform screening for hERGchannel inhibition during the process of selection andoptimization of the molecule, using the HTS technique.In this context, all of the substances that cause anyinterference in the hERG channels are considered ofpotential risk to increase the QT interval. In cases whereinhibition of the hERG channels is persistent, the in silicomodeling is used in association with computationalchemistry to help in the medicinal chemistry to redirectthe molecule (60).

Besides the hERG assay, at the beginning of eachoptimization program, the leader substances should betraced in relation to their possible effects on other relevantcardiac ionic channels, such as the L-type calcium chan-nel (CaV1.2), sodium channel (NaV1.5) and the channel ofdelayed rectification type slow (Kv7.1, IKs) (61–63), sincethe activation or blocking of such channels can producepro-arrhythmic events. Other cardiac targets (a andb-adrenergic receptors) should be also evaluated at thisstage, as a routine investigation of the off-target effect (60).The cardiovascular safety in vitro studies can be supple-mented, if necessary, with more sophisticated assays, likethe extracellular action potential assay in human embryonicstem cell (ESC)-derived cardiomyocyte (64).

When leader substances are advanced in the optimiza-tion phase, cardiac effects could be tested using the heartperfusion test (Langendorff), which provides importantinformation about the electrophysiology, contractile activityand coronary blood flux. A discrete increase in the QTinterval could be detected in this model, which is the QTinterval prolongation predictive effect in human (60). Thus,the Langendorff test is considered a good method ofscreening to detect long QT interval in comparison to othermethods, such as telemetry in dogs, which fails to detectincreases lower than 10% (65). Still in the optimizationphase, the test articles could be evaluated on a scale ofintravenous doses in anesthetized rats to evaluate effectson the heart frequency and blood pressure. In theseevaluations, it is possible to observe dependent dosechanges, but further experiments should be performed,including mechanism of action studies or telemetry withawake animals. Also, test articles should be tested instudies with anesthetized dogs that provide additionalinformation about cardiac contractibility, cardiac debit andpulmonary vascular pressure (66). To evaluate possiblemechanisms of action, further experiments are oftenconducted, such as the action potential study in isolatedtissues and the study of isolated blood vessels (60). Thus,the global and integrated cardiovascular risk evaluationshould consider all of the results obtained in vitro, ex vivoand in vivo.

It is also important to mention that the preliminary safetypharmacology tests for small molecules should not berestricted to the cardiovascular system. Preliminary assaysshould also evaluate the effect of leader substances on theCNS, respiratory systems, as well as on other systems whennecessary. In the exploratory phase, the first in vivo testnormally executed is the Irwin test (67), which provides rapiddetection of the potential toxicity of the test article, the activedose scale and the main actions on the behavior andphysiological functions (68). In addition, if the substance isdesigned to treat CNS diseases or other physiologicalsystems, which have an action on the CNS, it should also betested in the preliminary studies to evaluate the abusepotential and addiction behavior, in accordance with the‘‘Non-clinical investigation of the dependence potential ofmedicinal products’’ (69). The tests to evaluate the suscep-tibility for abuse are also comprised in the ‘‘Abuse Potentialof Drugs’’ (70).

Regarding the respiratory safety determination, in silicotests are required to optimize the selected leader sub-stances. These substances are then crossed with a cellulartarget panel, which has many substances responsible forseveral respiratory side effects (e.g., contraction/relaxation ofthe smooth muscle or induction/inhibition of the mucusproduction). Also, biological assays are performed to identifythe activity of the substance on the respiratory system. Formore information about relevant cellular targets for respira-tory safety tests, please see (51). To confirm further possibleactions of the test article in those targets, which couldsuggest a relevant side effect, it is necessary to determinethe action profile, understanding whether it causes inhibition,activation or modulation (54). Indeed, the optimized leadersubstance can be treated in relation to its pulmonary venti-lation and the muscular tone of the respiratory tract usingrespiratory plethysmography in conscious rats and isolatedrat trachea, respectively (54).

GLP safety pharmacology studies. The second partof the safety pharmacology program includes a standardtest battery defined in guidelines S7A and S7B (52,59). Inthis phase, the decisions are not based on "excludingsubstances with potential side effects" but on "presentinga probably safe substance". Thus, there is a change in thedevelopment status, in which the regulatory authoritieshave to decide whether a substance will be evaluated inhumans or not.

The guideline S7A (52) describes three types of safetypharmacology studies: a core battery of tests, whichincludes assessments of vital physiological systems suchas CNS, cardiovascular and respiratory systems; supple-mental studies, which include more complex physiologicalsystems (gastrointestinal, renal, immune, etc.); and follow-up studies for core battery, which are more detailed anddirected to the characterization of specific adverse effectsobserved in the core battery. On the other hand, the ICHS7B guideline is particularly intended for evaluating the pro-arrhythmic risk of the candidate substance to new medicine.



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The essential assays summarized in this set of testsare performed before the phase I clinical trial, using thesame route of administration as conventional toxicologystudies (usually the same dose that will be used clinically).Assessments are generally conducted for a period of up to24 h after administration of the test article (51). The batteryof recommended tests should be performed in accordancewith GLP requirements. Moreover, for the posterior andsupplementary tests, there are no specific additional guide-lines, although its management should be as close aspossible to the GLP.

Importantly, there are conditions where the safetypharmacology studies are not required, such as local agents(dermal and ocular use) and cytotoxic agents for treatmentof patients with end-stage cancer (except cytotoxic agentswith novel mechanisms of action). For biotechnology-derivedproducts with highly specific binding to the target, it may besufficient to evaluate the safety pharmacology with toxicol-ogy studies and/or pharmacodynamic studies. On the otherhand, in biotechnology-derived products that represent anew therapeutic class, or do not have highly specific bindingto targets, an extensive safety pharmacology review shouldbe considered.

Battery tests of the cardiovascular system. The S7Aguideline (52) recommends the monitoring of generalcardiovascular parameters. In this context, heart rate,blood pressure (systolic, diastolic and average), ECGparameters and heart morphology are assessed, includingtests for the presence of cardiac arrhythmia. For this, thetelemetry technique in awake animals is used, which isusually in Latin square design or dose escalation, withcomplete wash out of the substance considering enoughtime required between the dosages. These studies oftenuse the same species as toxicology studies (49). Addi-tionally, S7A guideline (52) mentions that assessmentsof repolarization and conductance abnormalities shouldbe considered. These evaluations are described in moredetail in ICH S7B guideline, which is specific to the studyof the effect of substances on ventricular cardiac repo-larization to determine the pro-arrhythmic risk.

The strategy for the tests described in ICH S7Bguideline comprises the in vitro evaluation of the sub-stance activity on the hERG channels and in vivo on theQT interval. These assays are complementary tools;therefore, both should be conducted. The hERG assayis currently considered a model of choice for evaluation ofcardiac pro-arrhythmic risk. Although this assay, per-formed by binding techniques and automated technology(HTS assay), appears to be appropriate in the early stages(exploratory) of the safety studies, the manual hERGassay is advised for the cardiovascular tests battery.Despite being more laborious, the manual hERG assay isan indicator of function, as opposed to binding technique,which only measures the affinity of the test article to thereceptor. Additionally, this method more easily fits therequirements of GLP at this stage of development (71).

The results with the hERG assay cannot be a singlestandard in vitro test conducted for evaluating the pro-arrhythmic risk. Ventricular repolarization is a complexphysiological process, which cannot be summarized onlyin terms of hERG current activity. Agonists of calciumchannels, for example, are known agents capable ofprolonging the duration of action potential (DPA 90%) andpredispose to early and/or late post-depolarization afterdepolarization, which can lead to TdP (72). Cardiac riskrelated to this mechanism dependent on calcium cannotbe detected by the hERG assay. Furthermore, the use ofthe hERG assay can lead to incorrect conclusions oncardiac risk, as a partial inhibition of hERG current doesnot result in prolongation of the DPA-90 because of thecompensatory effects of other cardiac ion channels. Thus,to properly evaluate the integrated cardiac risk, furtherstudies should be considered, especially those designedto investigate the electrophysiological properties of thetest article, such as evaluation of AP duration usingdifferent pro-arrhythmic models (73).

Both the hERG assay and isolated Purkinje nervefibers are predictive tests, however, there is no in vitrotechnique that can completely reproduce the in vivo tests.Thus, as indicated in the regulatory guidelines, the in vivoapproach in awake animals monitored by telemetry is stillan essential component in assessing the pro-arrhythmicrisk. Therefore, both in vivo and in vitro tools should beapplied to maximize the chances of an accurate assess-ment of cardiac risk (74). As described in guideline ICHS7B, the set of results of these studies is part of theintegrated risk assessment and support the planning andinterpretation of subsequent clinical studies.

Battery tests of the CNS. The battery of safety tests onthe CNS is composed of simple tests using traditionaltechniques that can be performed quickly. These tests areoften carried out at the beginning of the discovery pro-cess of candidate substances to drugs as a form of drugscreening to eliminate those with CNS risks. Because ofits early application in the safety assessment process, suchstudies are conducted almost exclusively in rodents (68).Furthermore, neurological assessments can be performedin other species (e.g., in dogs, minipigs or monkeys) (75,76).These studies are generally performed blindly with10 animals per group (51). The functional observationbattery (FOB) (77) and Irwin test (67) can be used toevaluate the effects of a test article on the CNS via motoractivity parameters, behavioral changes, coordination, sen-sory and motor reflex and body temperature. Furtherstudies are related to assessment of the effects oncognitive function (potential for abuse, learning, memoryand attention) and brain function (electroencephalogram).Due to the complexity, there are no standard protocols andthere is also no requirement that these studies shouldfollow GLP principles (68).

Battery tests of the respiratory system. The battery ofthe safety respiratory system includes simple studies,



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mostly conducted independently of the toxicological studies,with a single administration or inhalation of the test article,conducted in accordance with GLP requirements (78).They are usually carried out in conscious rodents (in mostcases in rats) with eight animals per group given thegreater variability of respiratory parameters. Largeranimals, such as dogs and monkeys, can also be usedwhen necessary (e.g., if the target is absent in rodents orthe pharmacokinetic profile is not appropriate) (51). TheS7A guideline (52) suggests performing two series ofstudies: the test battery and the follow-up studies. The testbattery includes quantitative measurements of respiratoryrate, tidal volume and hemoglobin oxygen saturation (79).Follow-up studies are needed when there is suspicion ofside effects based on the pharmacological properties ofthe test or when the results of the test battery areindicative of side effects (78). In general, respiratory safetytests include evaluation of the "respiratory pump" efficiencyand gas exchange. The ventilatory pattern is evaluated bydirectly monitoring changes in lung volume and airflowgenerated by thoracic movements in conscious animals,using plethysmography. Head-out, dual chamber and wholebody plethysmography techniques are non-invasive meth-ods that are currently used to evaluate typical parameters ofrespiration including tidal volume, minute volume and mid-expiratory flow (EF50) (80).

If the core battery indicates, for example, flow limitationby a decrease in EF50 or a rapid shallow breathing pat-tern, the mechanical properties of the lung can be furtherevaluated functionally by invasive lung function tests orpulmonary manoeuvres in anesthetized animals usingtheir higher sensitivity and specificity. For the measure-ment of lung resistance and compliance, a pressure-sensitive catheter is inserted into the pleural cavity oresophagus for the measurement of pleural, airway, ortranspulmonary pressure (78).

Component interaction between safety pharmacologyevaluation and toxicology studies. There is a globaltendency to integrate some components of the safetypharmacology studies with toxicology evaluations (81).The development of non-invasive techniques such asECG monitoring together with respiration, temperatureand animal activity, using the external telemetry system,has contributed significantly to this practice (82). This mayenhance the overall strategy for risk assessment and hasadvantages such as increased sensitivity (e.g., increasedstatistical power) based on the relatively large number ofanimals used in toxicological studies, reduction of thenumber of animals needed for safety assessments (inaccordance with the guidelines of the NC3Rs), the inte-gration of safety pharmacology data with histopathologicaland hematological data and cost reduction (52).

As discussed above, currently the safety pharmacol-ogy studies are not restricted to running the standardbattery of tests designated by S7A and S7B guidelines(52,59) for regulatory submission. The teams involved are

committed to developing strategies for early assessmentof potential problems related to the safety of the candidatesubstances using different combinations of tests basedon scientific assessments and the particularity of eachsubstance, e.g., in a case-by-case basis. Due to integra-tion into the development process, these strategies notonly help in deciding whether to continue the project, butthey also guide the discovery teams. These actions leadto the identification of candidate substances with appro-priate safety profiles, thereby reducing attrition ratio andenabling a greater chance of success in development.

Dose transposition from animals to humans

Dose transposing between species involves the use oftools such as allometry, which allows estimation of thesafest starting maximum dose for clinical trials usually inhealthy volunteers. While the allometric method is ofgreat use in implementing doses, it is not applicable forendogenous hormones and proteins, as well as for thecalculation of the maximum dose allowed (83). Further-more, the physiological and biochemical differences betweenanimal species, such as drug metabolizing, enzymesexpression, carriers, among others, should be taken intoaccount; hence, in silico methods and PK/PD models arealso extensively used as support tools for transposingdoses for clinical trials (84,85).

One of the most common mistakes observed in non-clinical studies refers to the dose estimation for humanuse (Phase I trial) based upon studies carried out inanimals (efficacy and safety studies of a new substance).There is a misleading tendency for a linear transpositionbased on a simple conversion of dose calculation used insmall animals (mg/kg), extrapolated to a patient withan average weight of 60 kg. This math causes a hugedistortion, however, giving the feeling that the treatment ofa large animal would require an exorbitant amount of thetest article (86). For proper dose extrapolation from smallanimals to humans (first dose of the substance in humans)known as human equivalent dose (HED), it is fundamentalto consider an important parameter called body surfacearea (BSA). Table 3 shows the conversion of animal dosesto human equivalent doses based on BSA.

To determine whether HED is mandatory, the previousdetermination of NOAELs, which can be obtained in thesafety (animal toxicology) test, is used. The NOAEL param-eter represents the highest dose of tested article in animalspecie that does not produce significant adverse effects,as compared to the control group. Thus, to calculate theHED, the dose in animals (mg/kg) should be the NOAEL (87).

Investigational New Drug Application (IND)

Before starting clinical trials with a new substance,the FDA requires the sponsor/researcher to report on allof the non-clinical studies conducted with the candidate



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substance for the development of a new medicine as wellas the detailed plans for the clinical trials for such product(phases I, II, and III). IND is the mechanism by which theresearcher/sponsor informs the FDA about the necessaryrequirements to receive from the regulatory agency theauthorization to initiate the trials in humans (clinical trials).The sponsor/researcher has full responsibility for con-ducting the clinical studies. The details of the requiredcontent and format are described in detail in CFR title21 part 312 (88) and in the FDA ‘Guidance for Industry:Content and Format of Investigational New Drug Applica-tions (INDs) for Phase 1 Studies of Drugs, IncludingWell-Characterized, Therapeutic, Biotechnology-derivedProducts’ (34). Basically, the report for an IND requestcontains the following information:• Non-clinical studies results of the test article (in vitroand in vivo). The non-clinical test checklist may varyaccording with the product as well as with the clinical trial

duration. Furthermore, it must be demonstrated that non-clinical trials were performed in accordance with the GLPrequirements;• Complete chemical information about the new medi-cine candidate;• Detailed clinical protocols regarding the Phases I, II,and III experiments, in accordance with the Good ClinicalPractices (GCP) rules, as well as other non-clinical studiesto be conducted during the research phase in humans (89).

Once the IND application is submitted, the FDA informsthe investigator about the reception of documents andit has 30 days to review the data and approve or rejectthe request. According to the product under development,analysis of the IND request report is performed by thefollowing FDA centers: i) Center for Drug Evaluationand Research (CDER) and ii) Center for Devices andRadiological Health, and Center for Biologics Evaluationand Research (CBER) (90).

Conclusion and perspectives

In this review, we highlighted the most recent andrelevant aspects necessary to conduct non-clinical studiesto attend the guidelines to develop new drugs recom-mended by major regulatory agencies. Although greatefforts in recent years have been occurring to reduce, andperhaps in the future, ban the use of animals in the processof new drug development, several alternative methods arebeing adopted and recommended by the main internationalregulatory agencies. However, the use of animals in the newdrug development process is still required.

Based on this review, it is possible to conclude thatthere is no single recommended sequence for the achieve-ment of non-clinical studies during the process of newdrug development (Figure 1). Many of the studies may beperformed in parallel, and the sequence may vary widelydepending on the disease. The use of GLP standards isabsolutely necessary, especially for the evaluation ofsafety studies, and is a decisive factor for the acceptanceof non-clinical studies in other countries where GLP hasbeen recommended since 1970. Although Brazil adoptspractically the same procedures (guidelines) recom-mended by the FDA and EMA, few laboratories or nationalinstitutions can conduct non-clinical studies in accordancewith GLP requirements necessary for new drug registra-tion purposes. The lack of reproducibility and reliability ofnon-clinical studies has been a limiting factor in theprocess of new drugs development for some nationalpharmaceutical companies.

Therefore, the need for high quality standard animals,associated with well-designed protocols, qualified humanresources, use of positive and negative controls, blindexperiment execution, proper use of statistical analyses,among other aspects, are mandatory factors to obtainreliable and reproducible non-clinical results. Non-clinicalstudies should be strictly performed in accordance with

Table 3. Conversion of animal doses to human equivalent doses(HED) based on body surface area.

Species To convert animaldose mg/kg to

dose in mg/m2,

To convert animaldose in mg/kg to

HEDa in mg/kg,either:

multiply byKm factor

Divideanimaldose by

Multiplyanimaldose by

Human 37 – –Child (20 kg)b 25 – –Mouse 3 12.3 0.08

Hamster 5 7.4 0.13Rat 6 6.2 0.16Ferret 7 5.3 0.19

Guinea pig 8 4.6 0.22Rabbit 12 3.1 0.32Dog 20 1.8 0.54

PrimatesMonkeysc 12 3.1 0.32Marmoset 6 6.2 0.16

Squirrel monkey 7 5.3 0.19Baboon 20 1.8 0.54Micro-pig 27 1.4 0.73Mini-pig 35 1.1 0.95

aA 60-kg human is assumed. For species not listed or for weightsoutside the standard ranges, HED can be calculated from thefollowing formula: HED = animal dose in mg/kg � (animal weightin kg/human weight in kg)0.33. bThis Km value is provided forreference only since healthy children will rarely be volunteers forphase I trials. cFor example, cynomolgus, rhesus, and stumpail.Adopted from Guidance for Industry – Estimating the MaximumSafe Starting Dose in Initial Clinical Trials for Therapeutics inAdult (83).



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good institutional scientific practices and also employ-ing GLP requirements (indispensable for the requestand approval of a IND) in order to ensure the quality,

reproducibility and reliability of non-clinical data, whichwill support the early clinical studies contributing to thesuccessful development of a new drug.

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