The main questions that should be answered to facilitate the comprehension between the systemic exposure of a test article and the quantification of the absorbed fraction in the tissues are: i is the substance absorbed? Two protocols can be applied for toxicokinetic studies: a full protocol that aims to answer all aforementioned questions or a reduced protocol, in which only the main questions are answered to corroborate the interpretation of the toxicology findings. In the full toxicokinetic protocol other biological matrices should be collected besides blood, such as excrements urine and faeces , fat, muscles, liver, kidneys, possible target-organs and skin when the substance test is administrated by the dermal route.
The design of the study and the selection of the experimental protocol should be defined on a case-by-case basis; overall, they should be able to provide enough information to evaluate the risks and safety of the candidate substance 28 , Considering the importance of evaluating PK properties during the development of new drugs, the assays described in Table 1 are highly recommended.
It is recommended that the manufacturing process follows the Good Manufacturing Practices GMP in order to guarantee the quality, safety and efficacy of the pharmaceutical products and to ensure the manufacturing consistency and batch-to-batch reproducibility The recent advance in the development of new drugs has become a challenge for science, as the offer of new therapeutic approaches has required techniques that guarantee its safety in humans.
Non-clinical safety studies have been performed based on the experience and employment history in a specific animal species before safety tests have been performed in humans. Besides animal studies, several in vitro tests have been developed and validated for safety evaluation to discover the toxicological potential of substances.
However, these assays are sometimes complementary to the in vivo tests. The use of animals to evaluate the toxicity of compounds started in , when J. After this, Food and Drug Administration FDA scientists started to develop new methods, such as the ocular and cutaneous irritation in rabbits that were widely accepted and applied all over the world. In addition, the researchers of the National Institute of Cancer in the USA started to develop tests in mouse to predict the cancer-causing potential of new substances.
However, after and due to several births of children with limb deficiency caused by Thalidomide use during pregnancy, safety studies performed initially in animals were required. The IND application must contain the safety and efficacy data of the substance before the first human exposure see more details below At the end of , the OECD and the International Conference on Harmonization ICH published guidelines for toxicity in non-clinical tests for chemical and pharmaceutical substances, which are still recommended by the majority of the regulatory agencies.
Since its publication, new revisions and assays were implemented throughout the years, aimed at the promotion of more predictive and ethical tests that could reduce or even prevent the use of animals. These guidelines present the basis of how assays should be conducted, suggesting species to be used, duration of the assays, organs to be investigated and the analysis to be conducted, as well as which data should be presented in the final report.
Even so, these guidelines are still generating several doubts in the scientific and industrial communities as the assays are not presented in detail. The performance of non-clinical tests in sequence is an important factor in the development of a new medicine.
Despite of there is not a standard program to execute the assays, a well-designed planning helps avoiding several errors or unnecessary tests, besides saving time and financial resources. In Figure 1 , we suggest the non-clinical studies that should be performed during drug development Thus, in this section, we will discuss the main non-clinical safety tests that are required in the process of drug development, such as mutagenicity tests, acute, sub-chronic and chronic toxicity, developmental and reproductive toxicity, carcinogenicity, local tolerance and safety pharmacology.
In the initial phases of development, several promising selected substances follow exploratory safety test screening to assess possible toxic effects. The exploratory tests are normally performed in vitro , or with a reduced animal number and do not require conformity with GLP principles. For this reason, these studies present reduced costs in comparison to the GLP studies that are required in subsequent steps in the drug development process. The exploratory assays are essential for the initial decision making regarding the investment on a new substance, since it could provide relevant information, which directly affects the planning of non-clinical assays that will be performed.
One of the initial tests to evaluate the toxicity of a new substance is the preliminary Ames test. This test is performed to evaluate a possible genotoxicity effect and the detection of genetic alterations in organisms exposed to these substances.
The different genotoxicity tests detect potential genetic and chromosomal mutations in organisms. The Ames test is an in vitro mutation assay in bacteria and has the ability to detect any mutation promoted by the substance, enabling the reversion of the existent bacterial mutation and restoration of the functional bacterial capacity to synthesize an essential amino acid histidine. This test can either evaluate the mutation capacity of the substance or its metabolites.
The execution of this test is mandatory to most of the substances in the drug development process. Despite the test being regulated and required by the authorities, the preliminary assay is fundamental for the early detection of possible genotoxic effects of the test article.
Furthermore, the in vitro micronucleus assay has emerged as one of the preferred methods for assessing chromosome damage. At this development level, the genotoxicity is restricted to in vitro tests; however, in the subsequent steps, other tests of genotoxicity are required, such as the in vivo micronucleus test.
The first in vivo toxicity study for a new substance is usually a dose range finding DRF study in rodents. For both scientific and welfare reasons, it is common practice to explore adverse effects in rodent species prior to non-rodent species. This increases the amount of information available for the design of non-rodent studies; for example, data from the initial rodent study can be used to set the starting dose, or allow specific monitoring of adverse effects in non-rodents.
MTD is defined as the highest dose tolerated in a toxicology study. The methodology is normally determined by parameters such as clinical signals, body weight changes, food consumption, morbidity and mortality. Besides that, several dose selection protocols also recommend the hematological and biochemical analysis execution, as well as the histopathological analysis of target organs to better determine the toxicity between the tested doses.
The acute or repeated-dose toxicity studies can be performed during the preliminary phase of the development process.
Although the MTD or dose escalation studies provide important information about drug toxicity, the repeated dose studies have more complete protocols, which consider the histopathology of a set of organs, more complex behavioral and clinical analysis, complete biochemical and hematologic analysis, ophthalmological analysis and groups for the evaluation of the side effects recovery after a treatment period. In the acute toxicity protocol, the effect of a single administration of three different doses is usually evaluated and the animals are observed for 14 days after treatment.
The OECD guidelines do not require the acute oral toxicity assay for pharmaceutical products, but some regulatory agencies suggest this assay. Also, according to the M3 guideline R2 34 , the acute toxicity is only recommended when there are no other studies about toxicity, such as MTD or dose escalation.
In this case, the acute toxicity studies can be limited and provide information about the administration routes and the doses to be administered. These data can be collected from non-GLP studies. However, the clinical treatment planning can only be supported by toxicological repeated dose studies performed in accordance to the GLP rules M3 R2 The short-term repeated-dose toxicity study is another protocol suggested during the exploratory phase.
The most indicated test is the repeated dose days oral toxicity study No. Since it evaluates the toxicity level of continuous administration, this test can provide more precise data, although it is more complex in comparison to the acute toxicity and MTD tests. This protocol is normally required for the first exposure of the substance in human Clinical phase I ; however, its execution will depend 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 be performed requires deep planning by the development team. Considering that the basis of defining which studies should be performed is related to the intended clinical use of the test article, the interaction between the non-clinical and clinical study teams is required.
Regulatory toxicology studies are mandatory in the drug development process and aim to evaluate the toxicity level of a substance using protocols that follow the guidelines recommended to conduct non-clinical studies of pharmaceutical products. In addition, it is important to emphasize that they have to be conducted in compliance with the GLP principles. After the preliminary toxicity studies, the GLP studies should be conducted in two animal species with the exception of mutagenicity tests.
The planning of these studies could be based on the data obtained by the exploratory studies of both efficacy and toxicity.
These findings could help to define doses, the duration of study, and any side effects that could require special attention. Some GLP toxicology studies are required before beginning clinical trials, but others could be conducted during different phases of the clinical trials; this will be discussed further in this section. Although there are no unique and standard plans for drug development, it is recommended to perform genotoxicity studies in vitro and in vivo as well as a study of dose selection and repeated-dose toxicity 28 days , before the first exposure of humans to the substance.
Usually, with these studies series, together with pharmacokinetic, efficacy, safety pharmacology and substance chemical characterization studies, it is possible to submit a dossier to the regulatory agencies to request permission to start the tests in humans.
It is important to emphasize that for toxicology studies following GLP principles, the test article should be in its final formulation, in other words, in the same formulation that will be used to treat individuals during the clinical studies, together with its complete chemical certificate of analysis. In addition, the route of administration should be, preferably, the same as that intended for human treatment.
In this step, the genotoxicity tests previously described in vitro Ames No. Thus, the in vivo micronucleus test No. In many cases, the genotoxicity assays, performed according to the GLP principles, are conducted before the repeated dose toxicity tests for decision-making reasons. However, it depends on the strategy programmed for each substance and on the obtained preliminary data.
Also, execution of the GLP genotoxicity test concomitant with initial repeated dose toxicity studies is common. An important decision during the planning of non-clinical studies is the duration of the repeated dose toxicity study that is normally based on the duration, therapeutic indication and planning of the clinical study.
Generally, the duration of toxicity studies conducted in two mammalian species rodent and non-rodent should be the same or even longer than the studies in humans, but no more than the maximal time recommended by the M3 R2 guideline 34 for each species see more details in Table 2. This table describes the recommended duration of repeated-dose toxicity studies to support the conduct of clinical trials. The relation between animal and human studies is a very important point in the drug development process, once the conduction and the choice of non-clinical studies should justify the time duration proposed for clinical treatment.
The repeated dose toxicity studies have guidelines with a very well defined duration, such as the guidelines No. One of the most used protocols before the first exposure of substances in humans is the repeated dose for 28 days. As mentioned in the exploratory studies section, this test aims to collect information about possible health risks using the repeated exposure to a substance, including its central effect, and that on the immunological, endocrine and reproductive system.
Although this test is indicated for oral administration, other parenteral administration routes could be used if well justified and if they are similar to the clinical uses. In addition, the toxicity test following repeated doses of the substance could also be applied for 14 days when it is justified by the short time of treatment in the clinical phase.
Besides, it is highly recommended to add a recovery group to the study in order to observe possible toxic effects recovery. The repeated dose toxicity study should be performed in accordance with the GLP requirements. The obtained outcomes are fundamental for characterizing the toxicity of the test article and provide a relationship between the dose-response and toxicity data to determine the no observed adverse effect level NOAEL.
The toxicity data described in the guidelines suggested by the FDA comprise an important basis for the IND application; however, it depends on the intended application 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 is normally classified in accordance with a chronological scale, such as the acute studies that are performed to verify the substance effect using single or repeated dose administration for 24 h.
Indeed, sub-acute studies are those that comprise the toxic effects for 30 days, whereas sub-chronic studies are defined by the toxic effect of a substance between 30 and 90 days.
Studies that are superior to 90 days are normally classified as chronic. However, this classification can be specific for some species; for example, chronic studies can be performed for six months in rodents and 9 months in non-rodents The sub-chronic study 90 days can be conducted in parallel with phase I clinical studies.
This study is very similar to the toxicity study of 28 days, and the guidelines for both require a daily treatment with at least three doses of the substance and the vehicle, together with clinical, biochemical, hematological, anatomical and histological analysis that are detailed in each guideline.
Despite standard measurements, some additional analyses could be included for the observation of a particular effect of the substance, mainly when several toxic effects are described. These tests should be conducted in accordance with the GLP principles; together with the clinical data obtained from phase I, these can help to decide whether the study should continue or not to phase II.
The reproductive toxicology test occurs during the clinical studies, along with the teratogenic potential evaluation. The reproductive toxicology test is the most rigorous test applied by the FDA and is a prerequisite for the approval of new substances.
In accordance with the guideline S5 R2 42 , the drugs can affect the reproductive activity by:. The requirement of reproductive toxicology studies in the beginning of the clinical phase I may differ in each country; however, it is quite often that these tests require clinical studies involving women of fertile age.
The fertility and implantation tests include male 28 days and female 14 days treatments with the substance before mating, and are characterized by the semen analysis counting and viability , number of implanted embryos and survival of the embryos at the sixth day of pregnancy.
The embryonic and fetal tests are normally performed in two or three species rats, mice, rabbits ; the substance is administered to females in the initial period of pregnancy in rats, 6—16 days after mating.
In this case, the animals should be euthanized before giving birth, aiming to count the embryo number and observe abnormalities. In the pre- and post-development tests, females are treated during pregnancy and lactation, where the offspring can be observed according to the motor activity after lactation. In these cases, some pups are analyzed according to their abnormalities in different stages of development, even in adulthood, to evaluate their sexual performance and their second offspring 42 , Despite some in vitro assays of reproductive toxicity being routinely performed, they do not provide enough data about teratogenic potential in mammals and are not recognized and required by the authorities The reproductive test battery is a requirement in the drug development process for almost all regulatory agencies; however, for herbal products, ANVISA suggests that these assays should not be performed.
In addition to the general toxicology and reproductive toxicity studies, the carcinogenicity test is usually required for drugs intended for continuous treatment of 6 months or more.
In these cases, carcinogenicity studies should be carried out before the substances go to the market, but never before the beginning of clinical tests. The carcinogenicity assay could be required in case of substances belonging to a known carcinogenic group or when chronic studies of toxicology present consistent evidence of the carcinogenic potential, or even when there is evidence showing that the substance or its metabolites are retained in the organism for a long period Interestingly, in the absence of other data, substances with positive evidence in the genotoxicity tests are considered carcinogenic to humans and are not submitted to long-lasting carcinogenicity tests.
However, in case the substance has been used for chronic treatment in humans, chronic tests for about 1 year could be necessary to assess possible tumorigenic effects The carcinogenicity studies are normally carried out during phase II and III of clinical development, using only a rodent species, especially rats.
In addition, it is recommended to perform other in vivo assays that can provide additional information about the sensitivity of the carcinogenic substance, such as short duration test in transgenic mouse or carcinogenicity test of long duration in other rodent species mouse. The carcinogenicity study of long duration in rats is usually conducted for at least 2 years of treatment with three or 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 in humans, while the highest dose is the MTD obtained in the previous safety studies. This means that the entire study needs around to animals being treated and evaluated for up 2 years. The ICH guidelines 45 — 48 determine the rules to be followed in these studies, which require the performance of the studies according to the GLP principles, with specific pathogen-free SPF animals and the histopathological analysis with more than 50 tissue types being analyzed by a veterinary pathologist with experience in carcinogenesis.
The carcinogenicity test is one of the most difficult and expensive studies during the non-clinical developmental process. Depending on the observed effects in the standard toxicological studies, other tests could be required. For example, if the drug candidate induces alterations in the immunological cells or in the lymphoid system tissues, immunogenicity studies could be necessary.
Such studies are performed with substances that act by modulating the immunological system or those causing alterations such as necrosis, apoptosis or interactions with cellular receptors shared by different tissues and non-target immunological cells Some of these evidences could be obtained by hematological, biochemistry and histopathological analysis obtained from previous toxicological studies. In these cases, assays such as the T-cell dependent antibody response TDAR test, immunophenotyping, natural killer cellular activity, etc.
Furthermore, for substances previously known as immunogenic, the sensibility test could also be necessary. In addition, for substances that are administered topically, local tolerance tests are required before the beginning of clinical phase I, and could be part of other toxicology studies. This assay aims to evaluate the tolerance level of a substance in different regions of the body with which it could have contact. To perform these tests, the selection of the species depends on each assay type as well as the administration route, the dosage and also the exposure time in accordance with the duration of the study to be conducted in humans.
The local tolerance test can include the administration route dermal, parenteral, ocular, rectal, etc. As previously described in this section, the toxicology tests are very important and require high responsibility from the non-clinical and clinical teams.
The available amount of substances could require specific protocols and requirements from the regulatory point of view. For example, the development of vaccines frequently does not require reproductive toxicity, mutagenicity or carcinogenicity tests.
On the other hand, each substance has particular characteristics and is developed for the treatment of a specific and complex disease; for this reason, the development program should be analyzed case-by-case. Phase I trials Researchers test an experimental drug or treatment in a small group of people for the first time. Phase II trials The experimental drug or treatment is given to a larger group of people to see if it is effective and to further evaluate its safety.
Phase III trials The experimental study drug or treatment is given to large groups of people. Researchers confirm its effectiveness, monitor side effects, compare it to commonly used treatments, and collect information that will allow the experimental drug or treatment to be used safely. Examples of other kinds of clinical research Many people believe that all clinical research involves testing of new medications or devices.
As such, certain personalities and individual motivations may be more suited for a client-focused career while other psychologists may thrive in policy studies. Resource: Top 25 Doctoral Programs in Psychology. The career track for non clinical psychologists is research and academia-oriented. This means that core courses will include research design, statistical methods and science-based writing. However, a doctorate in philosophy will boost your career options especially in the highly competitive field of applied or organizational research.
In this field, you can expect to find work in various administrative positions in elementary, middle and secondary schools. Teaching positions in post-secondary schools are always open to along with behavioral and social research fellowships.
Importance of Non-Clinical Science. By Editorial Team March 17, Share to Facebook Share to Twitter email print page Bookmark for later. The regulatory process The search for successful therapeutic interventions to treat PD and improve the quality of life for people living with PD involves a long, difficult, and expensive process. Types of preclinical research There are two types of preclinical research that are performed on potential therapies: In vitro — studies performed in a test tube, culture dish, or other mediums outside of a living organism In vivo — studies performed in a living organism, usually done in animal models 1 In vivo studies are critical for toxicology, researching the potential side effects of a new drug or therapy.
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