1 Project Information 1 Project Title

Brevicarine group alkaloids will be extracted from Carex brevicollis DC according to the method described in [Macaev F. et al. Moldovan patent №4009 from 15.07.2008, Macaev F. et al.Moldovan patent application №2011 0013 from 02.04.2011]

Synthesis of the heterocyclic compounds will be realized based on the available alkaloids from the β-carbolin derivatives group and substituted hydrazides of the an anthanilic, 3-amine as well as 4-amine benzoic acids. The main step of these syntheses is cyclization of linear derivatives will be realized by comprising both alternative approaches (TMTD/ DMF or CS₂/KOH/EtOH). Thus, due to the presence of two highly reactive functional groups in the 5-aryl-2-thio-1,3,4-oxadiazoles molecules, reaction of alkylation with –SH group participation and bonding of the nucleophil reagents, including brevicarine and brevicolin alkaloids, a wide number of substances will be obtained with different pharmacophores. On the other hand, as a consequence of antranilic acid hidrazide transformation into 3-amino-2-chinazolin-4-(3H)-one, a new group of compounds with a wide range of active centers (-SH, -CO, NH, -NH₂, -N=) will be obtained. These will serve as starting point for the synthesis of a large number of traptamine derivatives. According to this method, reactions of alkylation, cuaternization, recyclisation, and condensation will be applied.
Nano-encapsulation of the substance into cyclodextrins

Cyclodextrins form host-guest inclusion nanocomplexes with the substances in water solution if the latest possess smaller polarity than water. There are 4 main routes to obtain nanocomplexes and related systems for nanocomplexes formation prove.

Equimolar physical mixtures (p.m.) of the substances and cyclodextrins are prepared by homogeneous blending in the agate mortar of exactly weighed amounts.

Kneaded products are prepared from physical mixtures by adding small volumes of bi-distilled water during the 60 minutes of kneading in the agate mortar. After the last portion of water had been added, the obtained homogeneous slurry is kneaded to dry.

Equimolar amounts of the substances and the cyclodextrins are dissolved in bi-distilled water. The resulting mixture is stirred at 35 °C for 2 h. After the equilibration of 24 hours the clear solutions are coevaporated at 40 °C.

Equimolar amounts of a substance and a cyclodextrin are dissolved in water and mixed for 2 hours at 35 °C. After the equilibration period of 24 hours the clear solutions are frozen and subsequently lyophilized in a freeze-dryer of Alpha 1-2 LD type.

FT_IR spectra are very rich in information. The complex formation can be detected by band shifts or change in band appearance. FT-IR spectra will obtained using the KBr pellet technique. They will collected in the 4000 to 350 cm^-1 spectral range with the resolution of 2 cm^-1.

DSC analysis will be performed with a Schimadzu DSC-60. Weighed samples (2.0 – 2.2 mg) will be scanned in aluminium pans at a scan rate of 10 °C min^-1 over a temperature range of 30 – 300 °C.

Study of the anti-tuberculosis activity will be performed in vitro on sensible and resistant strains of Mycobacterium tuberculosis. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) will be determined first for the pure substances, then for the encapsulated complexes.

Toxicity tests of the substances that will show the highest anti-tuberculosis activity will be performed on the animal models (mice). To determine the acute toxicity the lethal dose test (LD₅₀) will be applied. Subchronic toxicity tests will be employed to determine toxicity likely to arise from repeated exposures of several weeks to several months. Detailed clinical observations and pathology examinations will be conducted. Basic parameters of these tests are: species – mice, age – young adults, number – 10 of each sex, dosage – three dose levels plus a control group, include the toxic dose level plus NOAEL, observation period – 90 days.
2.9 Sustainability Planning

Which “Market” will we study?
After the end of the project, the scientific activities of the team can be continued under two main directions: financial support from donors interested in the research in field of new types of tuberculosis treatment and in partnership with the companies already working in the field.

Starting with the first direction, all the main available programs supporting the study of tuberculosis treatment will be taken in consideration for submitting the follow-up projects. These main programs and donors include: FP7/Horizon2020 (specific programme Cooperation, theme Health); US NIAID, NIH; Bill and Melinda Gates Foundation (Global Health Program, Priority Tuberculosis); the Global Fund to Fight AIDS, Tuberculosis, and Malaria; United States Agency for International Development (USAID); Institute Pasteur; German Federal Ministry of Education and Research (BMBF); World Health Organization (WHO). Besides the main governmental and non-governmental donors, there is a group of big pharmaceutical companies funding the research in the field of new drugs and formulations for anti-tuberculosis treatment: Otsuka Pharmaceutical, AstraZeneca, Novartis, Sequella, Pfizer, Sandoz.

Main donors are concentrated in the developed countries as EU, USA, and Japan. For the first phase, we will concentrate on the European donors, being more accessible, especially after Moldova's association to the FP7, starting the January 2012. Concerning these donors, we will have to find more information about the new requirements of the future programmes under Health theme FP7/Horizon2020, as well we experience lack of information of other national European donors funding the collaborative research with other countries in the field of tuberculosis: EU member states and non-member countries (Norway, Switzerland), as well as some major EU-close partners, as Turkey, Russia, and Israel.

In the second phase, we will try to reach out the USA and Global donors, first finding their names. We will have to learn about their conditions of participation, their willingness to fund our research, their openness to receiving proposals from foreign scientists.

For the companies working in the field, main stakeholders in the field of treatment of tuberculosis are also situated in the developed countries, the same as main donors. Besides, the big companies listed above, there are many SME working in the field that could be interested in future partnership with them.

Their main niche in the market includes pulmonary drug delivery for the treatment of different lung infections, nano- and microparticulate drug delivery,

We need to gather the following information about these companies: names of companies, main contacts, level of their interest in our research results, their willingness to pay for your technology, license our innovation, or contract us to solve their problems, their openness to working with foreign scientists.
What is our specific market research objective?
The market research objectives that we will address during the lifetime of our project are divided in 2 main directions according to the above mentioned information: gathering information about main donors that can fund our research, and partners, companies that are willing to cooperate with us.

Concerning the first direction the specific objectives will be:

• 
  search for EU and non-EU European national programmes funding collaborative research on tuberculosis;
  
• 
  search for USA and global programmes funding collaborative research on tuberculosis;
  
• 
  raising team awareness of FP7/Horizon 2020 and other programmes future actions on tuberculosis through participation in the info-days and seminars organized by local NCPs and the European Commission;
  
• 
  enhancing knowledge and understanding of FP7/Horizon2020 and other funding programmes conditions of participation, eligibility, application procedures, and project writing tips through participation in trainings, seminars, and info-days organized by local NCPs and the European Commission;
  
• 
  working with potential partners on elaboration and submission of new projects.
  

• 
  search for EU and non-EU companies interested in new drug based treatments of tuberculosis;
  
• 
  determining the companies with interest in our research results and most advantageous conditions of collaboration;
  

• 
  meeting new potential partners for FP7/Horizon2020 and other programmes tuberculosis projects through participation in the thematic conferences, symposia, brokerage and partnering events;
  
• 
  improving our visibility for potential partners and programme committees through creating and maintaining the group webpage in 3 languages: English, Romanian, and Russian; creating group profile on Europe Enterprise Network, on CORDIS; creating single-page presentation for national partners catalogues that are distributed internationally.
  

As well, we will gather and translate in 3 languages all the information about the group and its activities. This information will be used to complete the group web page and the group partner search profiles and activities presentations.

By the end of Quarter 2, we will have selected 3 funding organizations with the eligibility criteria, funding opportunities most favorable for our team. As well, we will have visited 2 events from the list of events for partnering opportunities, there we will try to meet the representatives of the companies selected in the previous quarter. The group members will have attended at least 4 trainings, seminars, and info-days on funding opportunities, project writing, application procedures organized locally, or through webinars.

We will have contacted the previously elaborated list of companies that might be interested in the results of our research concerning our possible collaboration. For the beginning, we will propose to establish the collaboration under a funding of external donors determined in the list above, unless other possibilities are proposed by the company.

Moreover, we will have created and distributed to potential partners single-page presentations of the groups in English, as well as group profiles on CORDIS, EEN, Innovative Medicines Platform.

By the end of Quarter 3, we will have developed and launched the group’s web-page. We will have selected 3 companies for further collaboration under different conditions: external funding, their private funding, mixed funding external/private, depending on their preferences and our agreements. We will have visited 4 more trainings, seminars, and info-days on funding opportunities, project writing, application procedures, project submission organized locally, or through webinars.

By the end of Quarter 4, with the companies selected previously, we will have created a consortium including other partners as well that will submit a project on anti-tuberculosis drugs to one of the 3 funding organizations we will determine by the end of 2^nd Quarter.

What will result?
The main result of our marketing search is going to be an agreement with a company/group of companies to continue development of the new anti-tuberculosis drugs based on the entities developed under this project.

In the case if our assumptions proved correct, when the project results are positive and we manage to find a company interested in their follow-on development and marketing.

In the case if we do not get positive results and no company will be interested to continue research with other entities, we will come with a proposal to submit a common project to one of the funding programs we will have found.

Thus, by the end of the Quarter 4 we will have a consortium created either for collaborative follow-on development of the anti-tuberculosis drug, or for new research under external funding.

Definite stages of the project implementation are represented in matrix diagram in Table 1.

Table 2 graphically displays timing and duration of the stages involved, as well as interdependence of the stages.
4. Personnel commitments

Table 3 displays personnel commitments for the project implementation.

Tables 4 , 5, and 6 display the equipment, materials, services, and other direct costs to be purchased for the project.

Table 7 displays the costs necessary for travel outside and within country of residence.

Tables 8 and 9 display aggregate financial information and define the first advanced payment.

Table 10 contains detailed information about each individual person involved.