1 Project Information 1 Project Title

- Equpment: standard laboratory glassware for organic synthesis and extraction, drying closets, stirrers, vacuum distillator.

- Gas chromatography, colon and thin-layer chromatography equipment.

- Spectrophotometer PerkinElmer LAMBDA 25 UV/Vis

- Elements analyzer ELEMENTAR VARIO LIII .

- Spectrometre IR Spectrum 100 FT-IR spectrophotometer (Perkin -Elmer) using the universal ATR sampling accessory.

- Spectrometre NMR "Bruker-Advance III" (400.13 and 100.61 MHz).
Institute of Phthisiopneumology
- System BACTEC MGIT 960 "Becton Dickinson", Germany - for detection and rapid test of M.tuberculosis sensitivity;

- Test-system for molecular genetics "GenoType®MTBDR plus" Hain - Lifescience, Germany - for quick test of M.tuberculosis sensitivity;

- System BD ProbeTecTM "Becton Dickinson"

- Fluorescent Microscopes Carl Ziess, Germania; Leica USA;

- Rooms for biological safity Class I and II ESCO biohazard safety cabinet, Esco Global

- Freezer -86 degrees Celsium, Binder GmbH, Germany

- Autoclave; SYSTEC MLV 5075, Systec GmbH, Germany

- Thermostate 37 degrees Celsius 400 l. Model BD 400. Binder GmbH, Germany

- Electronic balances, Adam Equipment Co Ltd., England

- Water distillators

- Centrifuges, pentru 30-60 tub, RCF 3000x - 8000x g, Eppendorf AG, Germany


2 Detailed Description of Work Plan
2.1 Introduction

What is the problem?

The problem with tuberculosis puts Moldova among the top five European countries with high rate of new infections. The current situation becomes more complicated because of high percentage (aprox. 25%) of MDRTB infections, with proportion of previously treated cases that became MDR-TB reaching 65.1%. [Zignol M. Bulletin of the WHO, 2012]

Zignol M. et al. Surveillance of anti-tuberculosis drug resistance in the world: an updated analysis, 2007–2010. Bulletin of the World Health Organization 2012; vol. 90, p. 111-119D. doi: 10.2471/BLT.11.092585

Thus, in the field of the micro- and nanoparticulate systems, a lyposomal system loaded with rifampicin is known. The system contains phosphatidile choline and cholesterol as main vehicles. In order to increase the specificity of the system accumulation in macrophages, it has been covered with macrophag-specific ligands – O-stearyl amylopectin and maleylated bovine serum albumin. [1]

The main disadvantages of this system are:

• 
  susceptibility to phospholipid oxidation, and as consequence, lower stability;
  
• 
  increased costs of the production technology;
  
• 
  short half-life time in the body;
  

The closest by composition known microparticulate systems are gelatin based microparticles covered with manosyl groups and loaded with isoniazid [8], and microspheres based on alginate-chitozan used for transport of different anti-tuberculosis drugs [9].

The main disadvantage of these systems is the fact that they only facilitate accumulation of drugs in the macrophages, but do not facilitate their penetration in the mycobacterial cell.

In the field of new anti-mycobacterial drugs, recently, many groups of researchers have proposed entities from different chemical classes: thiolactomycins [10], benzothiazones [11], substituted quinolinyl chalcones [12], cinnamic derivatives [13].

Development of new classes of anti-tuberculosis drugs is in permanent emergency since the resistance to older ones appears sooner than the new ones are registered.

The leading scientists in the field are:

1. 
   Makarov V - A. N. Bakh Institute of Biochemistry, Russian Academy of Science, Moscow, Russia
   
2. 
   Sharma M. - Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226001, India.
   
3. 
   Khuller G.K. - Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India
   
4. 
   Baltas M. - Université de Toulouse, Toulouse, France
   
5. 
   Tomioka H. - Department of Microbiology and Immunology, Shimane University School of Medicine, Izumo, Shimane, Japan
   
6. 
   Jordão A.K. - Universidade Federal Fluminense, Departamento de Química Orgânica, Instituto de Química, Outeiro de São João Baptista, Niterói, RJ, Brazil
   
7. 
   Miller M.J. - Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, USA.
   

1. 
   Vyas S. P., Kannan, M. E., Jain, S. et al. Design of liposomal aerosol for improved delivery of rifampicin to alveolar macrophages. International Journal of Pharmaceutics. 2004, 269, 37–49;
   
2. 
   Pandey, R., Sharma, S. & Khuller, G. K. Nebulization of liposome encapsulated antitubercular drugs in guinea pigs. International Journal of Antimicrobial Agents. 2004, 24, 93–4;
   
3. 
   Sharma R., Saxena, D., Dwivedi, A. K. et al. Inhalable microparticles containing drug combinations to target alveolar macrophages for treatment of pulmonary tuberculosis. Pharmaceutical Research. 2001, 18, 1405–10;
   
4. 
   Edwards D., Fiegel J., Sung J. Particles for treatment of pulmonary infection. Patent WO2007/011396. Published: 25.01.2007
   
5. 
   Shim C. K., Yang R. Pharmaceutical composition for lung targeting. Patent KR20080043333 20080509. Published: 09.05.2008
   
6. 
   Ahmad Z., Pandey R., Sharma S., Khuller G.K. Alginate nanoparticles as antituberculosis drug carriers: formulation development, pharmacokintecs and therapeutic potential. The Indian Journal of Chest Diseases&Allied Sciences. 2006, vol. 48., p. 171–176;
   
7. 
   Tiwari S., Chaturvedi A.P., Tripathi Y.B., Mishra B. Macrophage-specific targeting of isoniazid through mannosylated gelatin microspheres. AAPS PharmSciTech, 2011, 12(3), 900-8;
   
8. 
   Saraogi G.K., Sharma B., Joshi B., Gupta P., Gupta U.D., Jain N.K., Agrawal G.P. Mannosylated gelatin nanoparticles bearing isoniazid for effective management of tuberculosis. J. Drug Target. 2011, 19(3):219-27;
   
9. 
   Pandey R., Khuller G.K. Chemotherapeutic potential of alginate-chitosan microspheres as antitubercular drug carriers. Journal of Antimicrobial Chemotherapy. 2004, vol. 53, p. 635 – 640;
   
10. 
    Kamal, A., Azeeza, S., Malik, M.S. et al.: Efforts towards the development of new antitubercular agents: potential for thiolactomycin based compounds. J. Pharm. Pharm. Sci. 11, 56(s)-80(s) (2008)
    
11. 
    Makarov, V., Manina, J., Mikusova, K. et al.: Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science. 5928, 801-804 (2009)
    
12. 
    Sharma, M., Chaturvedi, V., Manju, Y.K. et al.: Substituted quinolinyl chalcones and quinolinyl pyrimidines as a new class of anti-infective agents. Eur. J. Med. Chem. 44, 2081-2091 (2009)
    
13. 
    De, P., Yoya, G.K., Bedos-Belval, F., Constant P. et al.: Design, synthesis and biological evaluation of new cinnamic derivatives as antituberculosis agents. J. Med. Chem. 54, 1449-1461 (2011)
    

Different biotechnological and pharmaceutical companies are looking for the new developments in the field of treatment of tuberculosis. One of the companies is Evonik Industries AG from Essen, Germany. The company has expertise for feasible studies, formulation and product development, drug delivery formulation analysis, scale-up, clinical lot preparation, clinical trial manufacturing, and commercial manufacturing (aseptic manufacturing and terminal sterilization). They work with virtually all drug classes, including small molecules, peptides, proteins, nucleic acids, and cell fragments. Evonik’s patented microparticle technologies includes microparticles for treating or preventing intracellular infections such as tuberculosis and HIV. 

Another company in the field is Innate Immunotherapeutics Limited from Auckland, New Zealand. This is a public unlisted biotech company that has designed and manufactured a unique immunomodulator microparticle technology. This technology can be used to induce the human immune system to fight certain cancers and infections, or turn off certain immune mechanisms which lead to autoimmune diseases such as Multiple Sclerosis. The same technology can be used in the design of better vaccines to potentially treat or prevent diseases such as influenza, cancer, malaria, or tuberculosis.

There is a plenty of companies specialiazing in microparticles production for different medical purposes that might be interested in partnering for new production. One of the examples is Phosphorex Inc from Hopkinton, MA, USA. They can encapsulate any molecule, e.g. a drug (small or large molecule), or a dye (fluorescent or non-fluorescent) into micropsheres and nanospheres. They also are specialized in coating biomolecules (e.g., protein, peptide, oligonucleotide or antibody) onto the surface of microspheres and nanospheres.

Also, PolyMicrospheres, Division of Vasmo, Inc from Indianapolis, IN, USA works in the field. PolyMicrospheres and Advanced Nanotechnologies, Divisions of Vasmo, Inc., are leaders in developing Microparticle and Nanoparticle-based Drug Delivery Systems with embedded chemotherapeutics and other pharmaceutical compounds for the controlled-release delivery of drugs to affected tissues over an extended period. The portfolio of their technologies includes: controlled-release technology, extended-release delivery, targeted and site-specific delivery, drug stabilization and solubilization, micro-encapsulation and matrix technologies, emulsification and solvent evaporation, emulsification and solvent extraction, complex coacervation, protein gelation, spray drying.

The market description will be given in the section 1.19. Other companies that are specializing in the field of pulmonary drug delivery will be investigated in the market research during the implementation of the project.

• 
  new active substances in the core with proved or highly potential anti-tuberculosis activity. The substances will be synthesized or extracted from the local medicinal plants resources.
  
• 
  encapsulation of the substances in cyclodextrins as main encapsulating agents that will also serve as promotors of the substances penetration in the mycobacterial cell.
  
• 
  loading of the nano-encapsulated drugs into the alginate-chitosan microparticles that will help them to penetrate into macrophages infected with mycobacteria.
  

• 
  obtaining of new nano-encapsulated entities with potential antituberculosis activity: brevicarin and brevicolin, analogues of traptamin alkaloid from the derivatives of 1,3,4-oxadiazoles an 3-amino-2-chinazolin-4-(3H)-ons;
  
• 
  determination of anti-mycobacterial activity of the substances and their nanocomplexes through in vitro studies on the M.tuberculosis;
  
• 
  evaluation of toxicity of the substances and their nanocomplexes on laboratory animals
  

• 
  Synthesis and extraction of substances with anti-tuberculosis activity
  
• 
  Nano-encapsulation of the substances
  
• 
  Biological evaluation of the activity and toxicity of he substances and their encapsulated forms.
  

This should improve the treatment of tuberculosis, by:

• 
  reducing the time of treatment
  
• 
  reducing the dosage of the drugs
  
• 
  reducing the frequency of the drugs’ administration
  
• 
  reducing thus side effects, especially on liver
  
• 
  increasing thus patient compliance with the regimen of treatment
  

1. 
   Brzostek A., Pawelczyk J., Rumijowska-Galewicz A., Dziadek B., Dziadek J. Mycobacterium tuberculosis is able to accumulate and utilize cholesterol. Journal of Bacteriology. 2009, vol. 191, no. 21, p. 6584 – 6591;
   
2. 
   Donova M.N., Nikolayeva V.M., Dovbnya D.V. et al. Methyl-b-cyclodextrin alters growth, activity and cell envelope features of sterol-transforming. Mycobacteria. Microbiology. 2007, vol. 153, p. 1981 – 1992;
   

Prof. Macaev has successfully participated in 10 international projects as coordinator (INTAS projects Ref. № 2000-0711 and Ref. № 2006-8064, MD-US projects CRDF/MC2-3007 and CRDF/MERL-7031, MD-Russia project - Ref. № 06.21CRF, MD-Belarus project Ref. № 05.15BF, MD-Ukraine project Ref. № 10.820.09.01/UA, MD-Germany project Ref. № 09.820.05.08 GF, The Royal Society International Joint Project Ref. № JP090309), as well as member of a group (SCOPES, Ref. Nr.110823).

Dr. Pogrebnoi has successfully participated in 4 international projects as member of a group in INTAS project Ref. № 2000-0711, MD-US project CRDF/MC2-3007, SCOPES, Ref. Nr.110823, MD-Ukraine project Ref. № 10.820.09.01/UA.