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Mimicry of the Metabolism

Study applies biometric models to advance preclinical trials of natural products

The discovery of an active molecule derived from an element of Brazilian biodiversity may be the harbinger of a long journey fraught with uncertainties and risks to the production and commercialization of a new product. These uncertainties are inherent to the scientific activity itself, along with the risks involved in its commercial use. For this reason, developing an innovative drug is often a prolonged and costly activity. Statistics show that of the 300,000 compounds synthesized by industry, 20,000 (6.7%) are used in preclinical studies; of these, 200 (0.67%) reach clinical stage I; 40 (0.13%) clinical stage II; and 12 (0.004%) clinical phase III. Only eight of these (0.027%) are approved and in general one (0.003%) manages to obtain satisfactory market acceptance (Calixto, 2003)

In this respect, all efforts made to optimize these research and development (R&D) stages of biodiversity molecules are welcome. In the Biota/FAPESP Program, the group dedicated to these approaches is coordinated within BIOprospecTA, a subdivision of the Program that focuses primarily on biotechnology studies. In Brazil, research on identifying and isolating new active molecules of biodiversity is relatively well structured. However, the following stage (preclinical trials), which involves understanding the metabolism of these molecules, and the absorption and distribution of natural products, is one of the largest obstacles to research in the country.

Accordingly, the Thematic Project entitled Development of a platform for the study of in vitro and in vivo metabolism of natural products, a need for pre-clinical testing system was initiated in January 2010. The project, led by Dr. Norberto Peporine Lopes, from the Faculty of Pharmaceutical Sciences of Ribeirão Preto (USP), aimed to advance knowledge regarding the behavior of molecules with potential use in living organisms. To that end, the project was organized around a work platform that combines different analytical approaches to the selected molecules. “Several colleagues are seeking new active compounds and the fact that we fall under BIOprospecTA allows greater contact and the selection of targets for our studies”, explains Lopes.

The platform consists of a set of analyses that aim to reproduce the reactions of bioactive molecules in the human body, also known as biomimetic models. In these models, mass spectrometric techniques (for example, decomposition reactions in the gaseous phase of micromolecules ionized by electrospray) help construct maps of metabolites produced by the introduction of the molecule in the organism. After these exhaustive studies, in vivo tests are conducted using a small number of animals. “These models introduce the concept of extreme reduction and streamlining in the use of animals for research, such that their use raises more questions as opposed to doubts, as was the conventional practice”. Thus, these approaches allow interpreting the metabolism of natural products, essential in the initial phases of preclinical studies.

Over the course of four years the project involved ten institutions (USP/Ribeirão Preto and São Paulo, Unesp/Araraquara, UFOP, UEM, UFSE, UFPE, UFRGS, FIOCRUZ/Rio de Janeiro and the then business incubator called Lynchoflora ). Given its nature as a work platform, participation by researchers varied during the 4-year project, since the guiding element was the substance of the study and not the group itself. As a result, there was an intense flow of researchers, on average 20 from different fields, including product chemistry, pharmaceutics, pharmacognosis, pharmacology, pharmacokinetics, inorganic chemistry, organic and inorganic synthesis, and computational chemistry, acting in each stage of the product. Most were young researchers with up to five years of experience. “The preference for young researchers aimed at prioritizing the training of human resources to ensure the continuity of these new approaches”, explains Lopes.

From a scientific standpoint, the project resulted in approximately one hundred articles, one of which was on the cover of Angwandte Chemmie and in the Editor’s Choice section of Science Magazine.

More than 25 bioactive molecules were analyzed, 15 of which responded to some metabolic effect, and of these, two showed potential for industrial use. “One of the molecules studied is being patented in conjunction with FAPESP (São Paulo Research Foundation). Therefore, we believe that in the near future this patent could be in the negotiation phase in the Brazilian market, allowing us to secure partners for clinical studies” says Lopes. The aim is the development of a drug to treat American tegumentry leishmaniasis in a partnership between the University of São Paulo, the Biota/FAPESP Program and a PIPE/Fapesp project, coordinated by the Lynchoflora company, a spinoff of the Ribeirão Preto campus and FINEP.

However, the results do not focus solely on the industrial use of molecules, but also on public health issues.  An example is the discovery of the action of caramboxin, a toxin in carambola (Averrhoa carambola), in the organism. Ingestion of fruit or juice by patients with kidney failure or acute kidney injury, or by diabetic individuals, may induce hiccups, vomiting, mental confusion, psychomotor agitation, prolonged seizures and even death.

Phase 2 is a new project Distribution and metabolism of natural and synthetic xenobiotics: from the comprehension of reactional process to tissue imaging generation that has just been approved by FAPESP within the Biota/FAPESP Program. This new phase will aim at increasing the number of molecules studied and incorporating new imaging tools into preclinical analyses in order to further improve biomimetic models. Moreover, the project has already distributed offspring throughout the country. Similar projects are being developed at Londrina State University (UEL) and the Federal University of Sergipe (UFSE). “The biomimetic models can be obtained commercially or from colleagues at the Faculty of Philosophy, Science and Arts of Riberão Preto (FFCLRP) of the University of São Paulo (USP) and the equipment, which is very expensive, can be shared, practically resolving the structural side of things. We are capable to conduct this type of analyses in the country, but we are not yet accustomed to work with different approaches simultaneously”, concludes Lopes.

Atividade citotoxica de staurosporinas em microorganismos marinhos-Brazilian Chemical Society (cedida pela SBQ)Understanding the development stages of a new drug[1]

The development process of a new drug, from the research stage to its introduction into the market, can be divided into four stages: basic research, development (preclinical and clinical trials), registration and post-commercialization.

Basic research attempts to identify promising new compounds for treating a particular disease. To that end, it is necessary to identify the stage in which the disease can be contained, in order to determine the target to be studied. This search can occur via several technological routes, such as chemical synthesis, prospection of natural compounds and biotechnology.

Molecule isolation is followed by preclinical trials, considered the R&D filter of new drugs. This stage involves laboratory (in vitro) and animal (in vivo) tests aimed at determining the safety and effectiveness of the new compound.

For safety, toxicity studies are carried out to determine the harmful effects of the drug on organ systems – especially the cardiovascular and reproductive systems – as well as genetic alterations. Effectiveness testing aims at observing the absorption, distribution, metabolization and excretion of the new compound and its degree of stability and purity.  It is important to emphasize the importance of this stage in drug development. Preclinical tests, particularly in animals, establish the margins of safety required for tests on human beings, that is, clinical trials.

Clinical trials, the most costly and time-consuming stage in the development process, consist of submitting the drug approved in the preclinical stage to safety and effectiveness testing in humans. Authorization to commercialize a drug can only be obtained based on clinical trials, which are subdivided into three phases according to their purpose:

  • Phase I: Aims to determine the tolerance/safety of a drug in a restricted number of healthy volunteers. To that end, subjects receive increasing doses of the drug.
  • Phase II: Tests are conducted to assess the therapeutic efficacy of the new compound in volunteers with the targeted pathology/disease, also in restricted numbers. The aim is to reach the optimal dose, that is, that which combines the best therapeutic effects and the lowest number of adverse reactions.
  • Phase III: Broadens therapeutic studies, with a large number of patients in order to determine the risks and benefits of the treatment. Comparative assessment is always performed, using another treatment as reference.

Following the positive results of these tests, it is possible to proceed to the third stage: registration of the new compound. Drug registration for the purposes of commercialization and use by the population is the responsibility of health regulatory agencies. To obtain a license, all information on the drug and its development phases must be entered on specific forms, which are submitted to these agencies for approval. In general, this process takes between one and two years. It is important to emphasize that the product must be registered in each of the countries where it will be commercialized.

The registration process is followed by post-commercialization, in which the effects and unexpected adverse reactions in users of the new drug must be monitored by the company and regulatory agencies. This stage is also known as pharmacovigilance or phase IV clinical trials.


By Paula Drummond de Castro

[1] Text adapted from Pieroni, J. P. et al. Terceirização da P&D de Medicamentos: panorama do setor de testes pré-clínicos no Brasil. BNDES Setorial, Rio de Janeiro, n. 29, p. 131-158, 2009