In vitro studies - features, advantages and disadvantages. An integrated approach to the study of pharmacological substances in vitro, ex vivo, in vivo What is in vitro in vivo

- “in (on) a living organism”), that is, “inside a living organism” or “inside a cell”.

In science in vivo refers to conducting experiments on (or inside) living tissue in a living organism. This use of the term excludes the use of a part of a living organism (as is done in tests in vitro) or using a dead organism. Animal testing and clinical trials are forms of research in vivo.

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Excerpt characterizing In vivo

"No, now they will leave it, now they will be horrified at what they have done!" thought Pierre, aimlessly following the crowds of stretchers moving from the battlefield.
But the sun, covered with smoke, was still high, and ahead, and especially to the left of Semenovsky, something was seething in the smoke, and the rumble of shots, shooting and cannonade not only did not weaken, but intensified to the point of desperation, like a man who, straining himself, screams with all his might.

The main action of the Battle of Borodino took place in the space of a thousand sazhens between Borodino and the fleches of Bagration. (Outside this space, on the one hand, a demonstration by Uvarov's cavalry was made by the Russians in the middle of the day, on the other hand, beyond Utitsa, there was a clash between Poniatowski and Tuchkov; but these were two separate and weak actions in comparison with what happened in the middle of the battlefield. ) On the field between Borodino and the flushes, near the forest, in an open and visible stretch from both sides, the main action of the battle took place, in the simplest, most unsophisticated way.

Selezneva A.I. 1 , Kalatanova A.V. 2 , Afonkina O.V. 3

1 Candidate of Medical Sciences, Senior Researcher, 2 Junior Researcher, 3 Junior Researcher, St. Petersburg Institute of Pharmacy CJSC

INTEGRATED APPROACH TO THE STUDY OF PHARMACOLOGICAL SUBSTANCESIN VITRO, EX VIVO, IN VIVO

annotation

The article considers - effective planning and options for conducting experimental studies involving the optimal range of methods to establish possible directions of action of pharmacological substancesin vitro, ex vivo, in vivo. The ultimate goal of the integrated use of a battery of methods is to obtain reliable and sufficient experimental data, reduce the volume, cost and time of the study through the competent development of the design of the study and the use of data obtained at each stage.

Keywords: screening, preclinical studies, drugs, pharmacological substance, efficacy, safety, in vitro, ex vivo, in vivo.

Selezneva A.I. 1 , Kalatanova A.V. 2 , Afonkina O.V. 3

1 Candidate Medical Sciences, Senior Researcher, 2 Junior Researcher, 3 Junior researcher, “Institute of Pharmacy of Saint-Petersburg”

COMPLEX APPROACH TO STUDY PHARMACOLOGICAL AGENTS IN VITRO, EX VIVO, IN VIVO

Abstract

The article considers effective planning and options for experimental studies involving the optimum range of methods to identify possible areas of action of pharmacological agents in vitro, ex vivo, in vivo. The ultimate goal of the integrated use of a battery of methods to provide a reliable and sufficient in terms of experimental data, reducing the volume, cost and timing of the study by a competent study design and the use of data collected at each stage.

keywords: screening, pre-clinical studies, drugs, pharmacological agent, the efficiency, safety, in vitro, ex vivo, in vivo.

Successful study of the efficacy and safety of pharmacological substances directly depends on competent planning and development of study design. There are a large number of methods for both screening and volumetric assessment of the possible direction of action and toxic properties of pharmacological substances. These methods can be conditionally defined into three groups according to the methods of their implementation - methods in vitro, ex vivo, in vivo.

In vitro methods imply screening or volumetric evaluation of the efficacy and safety of pharmacological substances in model systems using reaction media, enzymes, cell lines, etc. Today, in the world scientific community, in vitro methods are very popular, both in terms of high innovation and in terms of humane treatment of animals. However, limiting studies of the efficacy and safety of pharmacological substances by in vitro methods is not advisable, since extrapolation of the results obtained to the whole organism is characterized by a high risk.

Ex vivo methods are, as a rule, isolated organs and tissues of living organisms. These methods are also widely known, and data from ex vivo studies tend to be more clinically relevant. However, as well as in vitro methods, the results of ex vivo studies cannot be the basis for starting clinical trials of a pharmacological substance.

In vivo methods are classical for experimental pharmacology and represent studies on various species and lines of animals. In vivo methods provide reliable and sufficient results that can be successfully extrapolated to the clinic. There is a large amount of data on the anatomical, physiological, biochemical and other features of species and lines of experimental animals, which make it possible to establish the degree of relevance to humans and predict the results of clinical trials of pharmacological substances. However, despite the high informativeness of in vivo studies, the most successful approach to the development of study design can be provided by the results of in vitro and ex vivo studies. These methods also make it possible to significantly reduce the number of animals in the experiment, which is of key importance from the point of view of bioethics.

In this paper, possible options for a comprehensive assessment of the effectiveness of pharmacological substances using a battery of in vitro, ex vivo and in vivo methods are identified. The use of an integrated approach makes it possible to make the experimental study as informative and reliable as possible.

Comprehensive assessment of the effectiveness of pharmacological substancesin vitro, ex vivoandin vivo

For the study of the effectiveness of pharmacological substances, the screening of pharmacological activity, pilot studies and the study of mechanisms of action are of particular importance. So, new pharmacological substances can be synthesized or obtained from natural raw materials using various methods, a large number of stereoisomers or substances that differ in structure by one or more functional groups can be isolated. Conducting a full-fledged study of each of the candidates requires a lot of time, economic costs and the use of a large number of animals. The use of in vitro and ex vivo methods in most cases allows you to select the most promising candidates and reduce the amount of research.

An in vivo study provides volumetric data that are optimal for extrapolation to the clinic. The use of various models of diseases in animals, as well as the use of genetically modified species, contributes to the establishment of the mechanisms of pharmacological action, effective doses, the dynamics of the values of pathology markers during long-term course use, etc.

As an example, we present a comprehensive study of the effectiveness of pharmacological substance X, which potentially has antioxidant and cardioprotective properties, in the system of in vitro, ex vivo and in vivo methods.

The study design is presented in Table 1.

Table 1 - Design of a comprehensive study of the effectiveness of pharmacological substance X

At the first stage of the study of the mechanisms of action in vitro, it was found that the pharmacological substance X is characterized by a pronounced efficiency in relation to the hydroxyl radical and lipid peroxidation (Table 2).

Table 2 - The effectiveness of pharmacological substance X in in vitro studies

The effectiveness of the pharmacological substance X exceeded that of the reference drug Y.

It has been established that the presence of antioxidant properties of a pharmacological substance determines its cytoprotective properties. Numerous clinical and experimental studies have determined that oxidative stress plays a key role in the development of cardiovascular pathologies such as coronary artery disease, hypertension, atherosclerosis, coronary insufficiency and heart failure.

The results of in vitro studies made it possible to determine the main directions of experimental design ex vivo and in vivo, as well as to establish the possible mechanisms of action of the pharmacological substance.

The second stage of the research was to determine the cardioprotective properties of the pharmacological substance X in an ex vivo experiment conducted on an isolated heart using the Langendorff method. The study was carried out with the use of pharmacological substance X in three doses.

As a result of the second stage of the study, it was found that the values of pressure (LVP) and contraction velocity (dP/dt max) of the left ventricle against the background of ischemia followed by reperfusion of the isolated heart increase statistically significantly, which may indicate a positive inotropic effect of the drug (Fig. 1 ).

Rice. 1 - The effectiveness of pharmacological substance X in an ex vivo study.

The data obtained in the study in vitro and ex vivo give reason to assume the key mechanism of action and the pharmacological effect of the substance, and, therefore, to plan experiments in vivo.

Thus, since in vitro and ex vivo the pharmacological substance X was characterized by a pronounced cardioprotective activity, as well as an effect on the antioxidant system, models of cardiovascular pathologies, the pathogenesis of which is associated with oxidative stress and impaired myocardial contractility, were selected to study the specific activity in vivo. : acute myocardial infarction and arterial hypertension.

As a result of studies of the effectiveness of the pharmacological substance X in vivo on the model of acute myocardial infarction, the effect on the physiological and biochemical parameters of the simulated pathology was established (Table 3).

Table 3 - The effectiveness of the pharmacological substance X against the background of modeling acute myocardial infarction in rats, M ± m.

As a result of studies of the effectiveness of pharmacological substance X in vivo in spontaneously hypertensive rats, a pronounced decrease in systolic (SBP) and diastolic (DBP) blood pressure was observed both before the use of pharmacological substance X and 1 hour after (Table 4).

Table 4 - Change in blood pressure during the course use of pharmacological substance X

Note - * p ‹ 0.05 compared to the control group

Thus, as a result of using a comprehensive assessment in vitro, ex vivo and in vivo, the high efficiency of the new pharmacological substance X was established and possible mechanisms of action were determined. The cardiotonic and cardioprotective effect of the drug was established on the isolated heart model using the Langendorff method and in the modeling of acute experimental myocardial infarction in vivo. When using the new drug in spontaneously hypertensive animals, a persistent decrease in blood pressure was observed, as well as a decrease in the initial pressure figures by the end of the course of treatment. It was found that a key role in the realization of the pharmacological effects of the pharmacological substance X is played by its antioxidant activity, which was confirmed in studies of antiradical and reducing ability in vitro.

The use of in vitro and ex vivo methods made it possible to significantly reduce the volume of experimental animals, since, based on their results, effective doses of pharmacological substance X and the most suitable experimental models were selected.

Literature

Menshchikova E.B. Oxidative stress: Pathological conditions and diseases. - Novosibirsk: ARTA, 2008. 284 p.;
Toropova Ya.G. Perfusion of an isolated heart by the Langendorff and Nilli method: possibilities of application in scientific research / Ya.G. Toropova, N.Yu. Osyaev, R.A. Mukhamadiyarov // Translational medicine. - 2014. No. 4 - S. 34-39.

References

Russell W.M.S., Burch, R.L. The Principles of Humane Experimental Technique. – London: Methuen & Co. 238pp.;
Directive 2010/63/EU of the European Parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes // Official Journal of the European Union. 2010. P. 33 - 79;
Humane Science in the 21st Century: abstracts of the 9th World Congress, Prague, 2014. Volume 3, No. 1. 336 pp.;
Mathers J. Antioxidant and cytoprotective responses to redox stress // Biochem Soc Symp.- Vol. 71. – P.157-176;
Addabbo F. Mitochondria and Reactive Oxygen Species / F. Addabbo, M. Montagnani, M.S. Goligorsky // Hypertension. – 2009. 53. – P. 885-892;
Men'shhikova E.B. Okislitel'nyj stress: Patologicheskie sostojanija i zabolevanija. - Novosibirsk: ARTA, 2008. 284 s;
Toropova Ja.G. Perfuzija izolirovannogo serdca metodom Langendorf i Nilli: vozmozhnosti primenenija v nauchnyh issledovanijah / Ja.G. Toropova, N.Ju. Osjaev, R.A. Muhamadijarov // Transljacionnaja medicina. - 2014. No. 4 - S. 34-39.

Research is conducted on microorganisms, cells, or biological molecules outside of their normal biological context. They are colloquially referred to as test-tube experiments. These studies in biology and its sub-disciplines have traditionally been carried out in test tubes, flasks, petri dishes, etc., and since the beginning of molecular biology have included methods, the so-called omics. Studies that use components of an organism isolated from their normal biological environment are more detailed and more convenient than analyzes with whole organisms. In contrast, in vivo studies are conducted on animals, including humans and whole plants.

Examples

Examples of in vitro studies: isolation, growth and identification of cells derived from multicellular organisms (cell culture or tissue culture); subcellular components (mitochondria or ribosomes); cellular or subcellular extracts (eg wheat germ or reticulocyte extracts); purified molecules such as proteins, DNA or RNA); and industrial production of antibiotics and pharmaceuticals. Viruses that replicate only in living cells are studied in the laboratory in cell or tissue culture, and many zoovirologists refer to this as in vitro work to distinguish it from in vivo work on whole animals.

The polymerase chain reaction is a method for the selective replication of specific DNA and RNA sequences in a test tube.
Protein purification is the isolation of a specific protein from a complex mixture, which in many cases is obtained from homogenized cells or tissues.
In vitro fertilization is carried out in a cup using sperm and an egg. The fertilized embryo(s) are then implanted into the uterus of the expectant mother.
In vitro diagnostics is a wide range of medical and veterinary laboratory tests. They are necessary for diagnosing diseases and monitoring the clinical condition of patients, and the material is blood samples, cells or other tissues of the patient.

In vitro testing is used to characterize the specific adsorption, distribution, metabolism and excretion (ARME) of drugs or common chemicals within a living organism. For example, experiments with Caco-2 cells will help assess the absorption of compounds through the mucosa of the gastrointestinal tract. It is possible to determine the division of connections between organs to study distribution mechanisms. Suspension or plate culture of primary hepatocytes or hepatocyte-like cell lines (HepG2, HepaRG) can be used to study and quantify chemical metabolism. These parameters of the ARME processes can then be integrated into so-called "physiologically based pharmacokinetic models" or PMPOs.

Video about in vitro studies

Benefits in vitro

In vitro studies allow for a species-specific, simpler, more convenient and more detailed analysis than whole-body analysis. Just as whole animal studies are increasingly replacing human trials, in vitro studies are replacing whole animal studies.

Simplicity

Living organisms are extremely complex functional systems formed by tens of thousands of genes, protein and RNA molecules, inorganic ions of small organic compounds and complexes. The environment in which they are located is spatially organized by membranes, and in multicellular organisms organs and systems are responsible for this. These myriad components interact with each other and with the environment to process food, remove waste, move components to the right place, and respond to signaling molecules, light, other organisms, heat, sound, taste, balance, and touch.

This complexity makes it difficult to determine the relationships between individual components, as well as the study of basic biological functions. In vitro work simplifies the system under study, so the researcher can focus on a small number of components.

For example, the identification of immune system proteins (such as antibodies) and the mechanism by which they recognize and bind to foreign antigens would remain unclear. However, the extended use of in vitro work has made it possible to isolate proteins, identify the cells and genes that produce them, and study the physical features of interaction with antigens. In addition, it was possible to determine how this interaction leads to cellular signals that activate other components of the immune system.

Species specificity

Another advantage of in vitro methods is that human cells can be studied without "extrapolation" from the cellular response of the experimental animal.

Convenience, automation

In vitro methods can be miniaturized and automated, providing high throughput screening methods for testing molecules in toxicology or pharmacology.

Flaws

The main disadvantage of experimental in vitro studies is that it is difficult to extrapolate the results of the work back to the biology of the intact organism. To avoid overinterpretation of the results, in vitro researchers must be careful. This can lead to erroneous conclusions about the biology of the organism and system.

For example, scientists developing a new viral drug to treat infection with a pathogenic virus (eg, HIV-1) may conclude that the potential drug serves to prevent viral replication in vitro (usually in cell culture). However, before a drug can be used in a clinical setting, it will need to undergo a series of in vivo trials to determine its safety and efficacy in intact organisms (usually successively in small animals, primates and humans). In general, most drug candidates that are effective in vitro are not effective in vivo due to problems with drug delivery to diseased tissues, toxicity to important body parts that were not reflected in the original in vitro studies, or other problems. .

Extrapolation from in vitro to in vivo (IVIVE)

Results obtained from in vitro experiments cannot usually be transformed to predict the response of the whole body in vivo. Therefore, it is extremely important to develop a consistent and reliable extrapolation procedure from in vitro to in vivo results. In general, two decisions were made:

Increasing complexity of in vitro systems for tissue reproduction and interaction between them (as in "man on a chip" systems).
Use of mathematical modeling to numerically simulate the behavior of a complex system, where in vitro data provide model parameter values.

The two approaches are not incompatible: improved in vitro systems will provide more accurate data for mathematical models. On the other hand, more and more sophisticated in vitro experiments are gathering more and more numerous, complex and promising data for integration. Here we need mathematical models, such as in systems biology.

Extrapolation in pharmacology

In pharmacology, IVIVE studies can be used to approximate pharmacokinetics (PK) or pharmacodynamics (PD). Since the time and intensity of exposure to a given target depend on the time of concentration of the course of a potential drug (related molecule or metabolites) into the target site, the sensitivity of tissues and organs in vivo may be completely different or even the opposite of that observed on cultured cells in vitro. . This indicates that extrapolation effects observed in vitro require a quantitative in vivo PK model. It is generally accepted that physiologically based PK models (BFMs) play a central role in extrapolation.

In the case of early effects or effects without intercellular communication, it is assumed that the same concentration of cellular exposure causes the same effects in qualitative and quantitative terms, in vitro and in vivo. In this situation, it is sufficient to develop a simple PD model of the in vitro dose-response relationship and transpose without change to predict effects in vivo.