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Production and preclinical evaluation of novel anti tuberculous antibiotic, transitmycin
1. Executive summary of the proposed research
According to WHO report, TB remains a leading cause of mortality worldwide in the 21 st century.
TB is a disease of poverty affecting mostly young adults in their most productive years (WHO report,
2010/2011). The worldwide problem caused by TB and the lack of new drugs in the market make it
imperative to develop novel drugs to fight efficiently preventing the rapid spread of drug resistant TB
strains. During the course of our anti TB drug discovery programme, a novel anti TB antibiotic,
Transitmycin was isolated from a marine Streptomyces sp R2 isolated from coral reef ecosystems of
Rameswaram, South India. Transitmycin showed promising activity against drug sensitive, MDR and XDR
M. tuberculosis isolates, some other bacterial pathogens and HIV. Further studies like production
improvement, drug formulations and preclinical studies will pave the way to bring this molecule at clinical
level. This proposal is aimed for the large scale production and preclinical evaluation of novel anti
tuberculous antibiotic, transitmycin.
2. Detailed description
2.1. Introduction
The incidence of infections caused by drug resistant bacteria continues to increase and remains a
serious threat to human health (Asolkar et al., 2010). Disease causing bacteria such as Mycobacterium
tuberculosis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa gradually develop
resistance to drugs. Out of all these the drug resistance developed by Mycobacterium tuberculosis against the
commonly used antibiotics is of major concern. Tuberculosis remains one among the leading causes of
infectious disease worldwide. One third of the world population is infected with Mycobacterium tuberculosis
and hence at risk of developing active TB (Boogoard et al., 2009).
The current first line TB regimen is more than 40 years old and consists primarily of rifampicin and
isoniazid. These antibiotics are effective in active drug susceptible TB, provided that patients complete the
course of treatment. However, there is a poor patients’ compliance due to the cost of drugs, adverse effects,
the long time required for completion of treatment (6-12 months) and the required number of drug doses.
Non-compliance has contributed to the emergence of multi drug resistant (MDR) and extensively drug
resistant (XDR) TB strains. MDR TB (strains resistant to isoniazid and rifampicin) often takes longer time to
treat with second line drugs. XDR-TB (MDR TB resistant to second line drugs including fluoroquinolones
and any one of the injectable drugs such as capreomycin, kanamycin and amikacin) is virtually incurable.
Furthermore, HIV/AIDS antiretroviral therapies are not always compatible with the current TB regimen
because of shared drug toxicities and drug interactions (Rivers and Mancera, 2008). In this context, there is
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an urgent need for developing novel antiTB drugs with less toxic side effects, improved pharmacokinetic
properties with extensive and potent activity against resistant strains and to reduce the total duration of
treatment (De Sousa, 2006).
Actinomycetes are the most economically valuable prokaryotes which are well known to produce
chemically diverse metabolites with wide range of biological activities. It has been estimated that about half
of the microbial bioactive metabolites notably antibiotics, antitumor agents, immuno suppressives and
enzyme inhibitors have been isolated from actinomycetes (Balagurunathan and Radhakrishnan, 2010).
Recently the rate of discovering new compounds from terrestrial actinomycetes has decreased but the rate of
re-isolation of known actinomycetes and antibiotics is on the increase. This has led researchers to explore
unique and extreme habitats such as marine environment for potentially new biosynthetic diversity. Marine
actinomycetes are the promising source for secondary metabolites (Lam, 2006). In the past 10 years, 659
marine bacterial compounds have been described in which 256 compounds have originated from
actinomycetes (Williams, 2008). From the discovery of streptomycin from Streptomyces griseus,
actinomycetes derived antibiotics are still in use for the treatment of tuberculosis. Due to the emergence of
MDR and XDR TB cases, search for novel antibiotics is still continuing.
During the course of our anti TB drug discovery, novel pigmented anti TB antibiotic, transitmycin
was isolated from Streptomyces sp R2 isolated from coral reef ecosystem of Rameswaram Coast, South
India. The main aim of this proposal is to improve the production of transitmycin and its preclinical
evaluation. The aim is also to formulate transitmycin for improving its stability and efficacy.
2.2. Objectives:
 To produce transitmycin by solid state fermentation
 To improve the production yield of transitmycin through fermentation optimization
 To formulate transitmycin and test for bioactivity
 To evaluate transitmycin at preclinical level
2.3. Methodology:
Crude transitmycin will be produced from the marine Streptomyces sp R2 by adopting solid state
fermentation and extracted using ethyl acetate. Transitmycin will be purified by adopting Thin layer
chromatography and column chromatography methods. Structure and anti TB activity of transitmycin will be
confirmed by spectral analysis and Luciferase Reporter Phage (LRP) assay (Sivakumar et al., 2007;
Radhakrishnan et al., 2011).
Effect of critical medium components and culture conditions on transitmycin production will be
studied by adopting Response Surface Methodology (RSM) (Xiong et al., 2008). Factors which will be
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studied include carbon source, nitrogen source, minerals, pH, temperature and incubation period. Large scale
production of transitmycin will be carriedout using the optimized medium and culture conditions.
Formulations of transitmycin will be prepared by adding various along with pharmaceutically acceptable
additives, excipients and adjuvants. The composition will be formulated in various forms such as liquid,
solid, powder. The pharmaceutically acceptable additives and excipients will be selected from the group
comprising glycerol, lactic acid, poly ethylene glycol (PEG), salts such as KCl, cationic surfactants, anionic
surfactants and natural surfactants, lactose, sucrose, dextrose, sorbitol and mannitol; dextrins; polycarboxylic
acids, chitosan, vitamin C; polyethylene glycols, polyvinyl pyrrolidone, benzyl alcohol and polyvinylacetate.
The formulated transitmycin will be tested for anti TB activity by LRP assay.
In Phase I of this project, preclinical tests which are involving only the non animal study models will
be carriedout on transitmycin. Non-animal study models which will be used in this study may include
isolated organs, tissues, cells, cell components and microorganisms (Olejniczak et al., 2001). Tests which
will be carriedout include tests for mutagenicity, DNA damage & repair, mitotic recombination, etc.
2.4. Expected outcome:
 Conditions for improved transitmycin production will be optimized
 Formulations of transitmycin prepared and evaluated in this study may result in better stability and
efficacy
 Results of preclinical studies will pave the way for further clinical testing
3. Current Stage of development:
The microbial producer of Transitmycin, Streptomyces sp R2 to be used in this project was isolated
from sediment sample collected from coral reef ecosystem of Rameswaram Coast, South India. The active
compound, extracellular yellow pigment showing good anti TB and anti HIV activity was isolated from the
culture medium inoculated with the Streptomyces sp R2 through solvent extraction followed by bioassay
guided fractionation. Transitmycin also showed good activity against latent TB bacilli. Structure of the
active compound was determined based on their spectral properties. This compound is identified as novel
brominated compound, based on the published and patented literature search. This brominated compound is
named as Transitmycin. Patent application for this invention is filed through the IPR section of ICMR
[Patent application No: 247/DEL/2011 dated 2nd February 2011]
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4. Existing infrastructure and team
The Department of Bacteriology is one of the earlier formed departments of the institute and
developed several protocols and procedures for clinical trials on tuberculosis and has also
formulated the various treatment regimens for tuberculosis that are currently being followed at
National Level. The department had a major role in achieving milestones by providing laboratory
support like domiciliary treatment of tuberculosis, DOTS- provider facility for effective treatment,
short course chemotherapy, and population based study on the efficacy of BCG vaccine. The
department is functioning since 1956 till date and is accredited as Supra national reference
laboratory for tuberculosis by World Health Organisation. One of the major responsibilities of the
department is to provide active support for various controlled clinical trails being taken up by the
institute. The department has also initiated various research projects, both basic and operational, for
diagnosis and susceptibility testing of Mycobacterium tuberculosis. Evaluation of newer methods
and re-evaluation / optimization of existing methods followed in mycobacteriology is also being
continuously performed to achieve better diagnostic methods.
The department has its various sections in three floors including the important components
like BSL Level 3 facility of 2400 sq ft area and other laboratory facility of 7300 sq ft. Major fund
providers for the departmental activities are ICMR, WHO- USAID, DST, and DBT. The
department has the strength with human resource at various cadres:
1. Permanent staff: 2 scientific staff, 35 staff in the technical cadre
2. Ad- hoc project: 3 Consultants, 2 staff of research cadre, 13 staff in the technical cadre
3. Students (Doctoral & post doctoral studies) – 7
Phase I
I. Deliverables:

Development of optimized medium for the increased production of transitmycin

Transitmycin formulations with better stability and efficacy may be developed

Suitability of transitmycin for further evaluation at phase II of this project may be determined based
on the results of preclinical testing
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II. Strategy:
Marine Streptomyces sp R2
[Transitmycin (Tr) producer]
I. Production of Tr
II.Optimization of Tr Production
[solid state fermentation]
[Response Surface Methodology]
Crude Tr preparation
Effect of C, N & minerals, pH
[using ethyl acetate]
Temp & incubation period, etc
Tr purification
Quantification of crude Tr
[chromatography & bioassay]
& testing for Anti TB activity
Transitmycin
III. Tr Formulation
IV. Preclinical studies on Tr
[+Excipients, Adjuvants, additives]
Non-animal Models
Testing for stability
[Organs, cells, tissues, microbes, cell components]
Testing for anti TB activity
Time Frame
April
2012
to
June 2012
July 2012
to
September
2012
I. Tr Production (PU)
*
*
II. Tr Production optimization
(IIT, NIRT)
*
*
Objectives
Duration
October
January
2012
2013
to
to
December
March
2012
2013
April 2013
to
June 2013
July 2013
to Sep
2013
*
*
*
III. Tr Formulation (IIT, NIRT)
*
*
IV. Tr preclinical Trial (PU, IIT,
NIRT
Data compilation
*
*
*
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PU – Periyar University, Salem; IIT – Indian Institute of Technology, Madras; NIRT – National Institute for
Research in Tuberculosis
Budget Breakup
(A) Non-Recurring
Expenditure
Equipment
13,00,000
(B) Recurring
(i) Manpower for 18 months
(ii) Consumables/ Contingencies (and for
Scientist ’C’
9,68,665
2 JRF/SRF
7,48,800
16,00,000
outsourcing certain procedures)
(iv) Travel
1,00,000
(C)Overhead charges
3,41,746
Grand Total (A+B+C)
50,59,211
Since this project involves three Institutes viz., Periyar University (PU), Salem; Indian Institute of Technology,
Madras (IIT); National Institute for Research in Tuberculosis (NIRT), Chennai split up budget will be
submitted once the project is sanctioned.
Phase II Strategy:
In drug development, pre-clinical development is a stage of research that begins before clinical trials
(testing in humans) can begin, during which important feasibility, iterative testing and drug safety data is
collected. The main goals of pre-clinical studies (also named preclinical studies and nonclinical studies) are
to determine a product's ultimate safety profile. For instance, drugs may undergo pharmacodynamics (PD),
pharmacokinetics (PK), ADME, and toxicity testing through animal testing. This data allows researchers to
allometrically estimate a safe starting dose of the drug for clinical trials in humans. Most pre-clinical studies
must adhere to Good Laboratory Practices (GLP) in ICH Guidelines to be acceptable for submission to
regulatory agencies such as the Food & Drug Administration. Toxicity studies of Tr include organs targeted
by that drug, as well as any long-term carcinogenic effects or toxic effects on mammalian reproduction. The
information collected from these studies is vital so that safe human testing can begin. Typically, in drug
development studies animal testing involves two species. Depending on drugs functional groups, it may be
metabolized in similar or different ways between species, which will affect both efficacy and toxicology.
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Milestones and time frame [for 5 years]
Milestones
Time duration[in years]
Acute and Repeated-dose toxicity
2012-2013
Adverse effects on male and female fertility/
Embryotoxicity/postnatal toxicity
2013-2014
Genotoxicity and Tumorigenicity
2014-2015
Sensitization/immunosuppression
2015-2016
Local and other particular adverse events
2016-2017
Estimated budget
Manpower (for 5 years)
Expenditure
Scientist C-6 nos
14520000
JRF-6
3735000
SRF-6
4200000
Consumables/ Contingencies
8000000
Outsourcing
3500000
Equipments
9800000
Overhead charges
3395500
Total
47150500
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IP Strategy
The antibiotic is not licensed to any pharma company.
 Provide intellectual incentives for partner.
 Avoid harsh or inappropriate acquisitiveness.
 Listen, and welcome new ideas or approaches.
 Be patient, and expect to walk before running.
 Explicitly define (and quantify) any dissatisfactions.
 Do not assume anything about the partner.
 Find the right balance of parallel and serial actions.
 Meet the partner team and maximize face-to-face communications.
 Be aware that sometimes it really is best to let partners do it their way.
 Be honest and aware of your own strengths and weaknesses.
 Understand your partner’s culture and personality.
 Adapt your communication style to the partner’s personality.
 Define roles and metrics of success clearly and explicitly.
Market Overview and Business Model
 Understand the phases of drug discovery, from building a screening cascade to clinical trials
Learn how to build the target product and candidate profiles for a drug
 Discover the key stakeholders- ICMR
 Explore the legal and regulatory framework that applies to drug discovery-DCI
 Understand the economics behind drug discovery-Pharma industry BD
 Consider where the industry will head in the future for marketing the formulated drug-Pharma
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References:
1. Rivers, E.C., and R.L. Mancera. 2008. New antituberculosis drugs in clinical trials with novel
mechanisms of action. Drug Discovery Today. 13(23); 1090-1098.
2. Boogaard, J., G.S. Kibiki., E.R. Kisanga., M.J. Bocice, and R.E. Aarnoutse. 2009. New drugs
against tuberculosis: problems, progress and evaluation of agents in clinical development.
Antimicrobial Agents and Chemotherapy. 53(3); 849-862.
3. Asolkar, R.N., T.N. Kirkland., P.R. Jensen, and W. Finical. 2010. Arenimycin, an antibiotic
effective against rifampin- and methicillin- resistant Staphylococcus aureus from the marine
actinomycete Salinispora arenicola. The Journal of Antibiotics. 63; 37 – 39.
4. Tuberculosis: World Health Organization Report. 2010/2011.
5. Balagurunathan R and M. Radhakrishnan. 2010. Biotechnological, genetic engineering and
nanotechnological potential of actinomycetes. In: Industrial Exploitation of Microorganisms
Editor(s): D.K. Maheshwari, R.C. Dubey, R. Saravanamurthu R.C. Dubey. I.K. International
Publishing House Pvt.Ltd, New Delhi Pp.436
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tuberculosis (MDR-TB) based on natural products. Fitoterapia. 80: 453-460.
7. Lam, K.S. 2006. Discovery of novel metabolites from marine actinomycetes. Curr Opin Microbiol.,
9; 245–251.
8. Williams, P.G. 2008. Panning the chemical gold: marine bacterial as a source of therapeutics. Trends
in Biotechnol., 27; 45-52.
9. Sivakumar, P.M., S.P. Seenivasan, V. Kumar, and M. Doble. 2007. Synthesis, antimycobacterial
activity evaluation, and QSAR studies of chalcone derivatives. Bioorg Med Chem Lett., 17:1695–
1700.
10. Radhakrishnan, M., R. Balagurunathan, N. SelvaKumar, Mukesh Doble and Vanaja Kumar. 2011.
Bioprospecting of marine derived actinomycetes with special reference to antimycobacterial activity.
Indian Journal of Geo-Marine Sciences, 40(3); 407-410
11. Xiong, Z.Q., X.R. Tu, and G.Q., Tu. 2008. Optimization of medium composition for actinomycin
X2 production by Streptomyces sp. JAU4234 using Response Surface Methodology. 35; 729-734.
12. Oleijniczak, K., P. Gunzel, and R. Boss. 2001. Preclinical testing strategies. Drug Information
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