Synthesis of tetrazolium organic salts and their usage for application in histo-chemistry, cell biology, biochemistry and biotechnology. Submitted by Dr. Rupali M. Karai INCUBATOR: VENTURE CENTRE PROPOSAL CODE :- BIRAC/BIG/10685_ PI_Rupali

Synthesis of tetrazolium organic salts and their usage for application in histo -chemistry, cell biology, biochemistry and biotechnology . Submitted by Dr. Rupali M. Karai INCUBATOR: VENTURE CENTRE PROPOSAL CODE :- BIRAC/BIG/10685 _ PI_Rupali.

The proposed solution Technical Details (1/2) Cell proliferation and viability assays for cultured cells have been largely based on the reduction of colorless tetrazolium salts to colored formazans . Their principal advantages are avoiding the use of radioisotopes, together with easy and direct quantitative evaluation of viable cells. The applications and comparative results using the tetrazolium salt (TTC,MTT ) assay and other viability methods ( resazurin , neutral red, sulforhodamine B) have been described ( Skehan et al., 1990; Van Tonder et al., 2015; Da Luz et al., 2016). In this context sulfonated tetrazolium salts are useful reagents because of the water solubility of the colored formazan products. Formazan compounds were first described at the end of the 19th century but were rather overlooked until their potential as localization stains in living systems and redox viability probes were reported much later ( Mosmann , 1983; Carmichael et al., 1987 ). We plan to utilise local raw materials involving green chemistry to the best of our abilities and by replacing amyl alcohol with methanol and amyl nitrite with sodium nitrite. We also plan to reduce cost by recycling and reusing all the solvents and disposing the waste in the solid waste management facility. Slide no 2.

Technical Details (2/2) (Or Supporting Evidence) The structures of the tetrazolium ring and the corresponding formazan are shown in Fig. 1A and B respectively, and their properties and uses have been extensively described (Altman, 1976; Lillie, 1977, p. 227–228; Horobin , 1982; Seidler , 1992; Horobin and Kiernan, 2002, chapter 13). Different mono- and di- tetrazolium salts have been extensively used in histochemical applications ( Horobin and Kiernan, 2002, see chapter 13; Kiernan, 2015) but only mono- tetrazolium compounds are routinely employed for assessing cell viability MICROBIOLOGY,HISTOPATHOLOGY AND OTHER CHEMICAL STREAMS. The mono- tetrazolium salts TTC [ Triphenyl Tetrazolium Chloride ] ,3- (4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT, thiazolyl blue), and 5-cyano-2,3-di-(p- tolyl ) tetrazolium chloride (CTC) are used widely. Compared to MTT, which has three aromatic rings, the more efficient fluorescence of CTC is probably related to its more restricted molecular rotational freedom resulting from possessing only two aromatic rings ( Stockert and Blázquez -Castro, 2017, pp. 467–469). MTT was introduced for the histochemical detection of dehydrogenase activity by Pearse (1957). Neotetrazolium chloride (NTC), nitroblue tetrazolium chloride (NBT) and T etranitroblue tetrazolium chloride (TNBT) are widely used in histo chemical detection of dehydrogenase activity, and as redox indicators in combination with indigogenic methods ( Horobin and Kiernan, 2002, pp. 164–165 and 166 respectively; Van Noorden , 2010; Kiernan, 2015 ). Blue tetrazolium chloride (BTC) was developed and used for demonstration of enzymes in normal and neoplastic tissues ( Rutenburg et al., 1950), and as an indicator for seed germination and nanomolar detection of reducing sugars. We plan to focus on the widely used TTC [ T riphenyl Tetrazolium Chloride]. Slide no 3.

Project Team & Advisors What is the likely team composition and when it is likely to be completed? The main team members consists of myself and Dr. Murzban J. Karai . The additional staff requirements, we will appoint the persons’ in a month’s time. Dr M. J. Karai has behind him experience of 40 years. He is a visiting faculty to ICT and also on the board of numerous committees for the appointment of PhD scholars and their up gradation review panels. In the recent past he has been a member of the panel for the appointment of professors and asst. professors. H e is also a consultant to numerous chemical industries advising them on their process intensification and process developments. His mobile number is 9821245583 and email id is murzban.karai@gmail.com Slide no 4.

Market size The size of the market is huge to the tune of 3 cr and growing about 30% annually by the day. These compounds have been utilised extensively in the biochemistry field and their consumption will grow by leaps and bounds in the future too. These compounds have n annual demand of 1.5 to 2 tons. Even at a conservative price tag of INR 30,000/- to 32,000/- per kg and 40% net profit the ROI is very attractive. Competitive Landscape Today Today n o company is manufacturing all these compounds. An internet search throws up re-packers and traders masquerading as manufacturers. On a worldwide scale there are only three manufacturers. We have developed the manufacturing process form basic raw materials and thus enable us to market these compounds at a reasonable cost and of enhanced purity. In next 5 years In next 5 years too we envisage a tremendous potential growth in the demand of TTC and other Tetrazoliums . We are confident in the flexibility of our process and pricing to ward off any serious threat to our sales. Slide no 5.

Define Differentiation of Proposed Solution AS HAS BEEN MENTIONED EARLIER IN DEVELOPING THE MANUFACTURING PROCESSESS WHICH SUPPORT US TO MARKET THE COMPOUNDS AT A RESONABLE COST AND OF ENHANCED QUALITY. THE PROJECT IS TOTALLY FEASIBLE AS THE MARKET IS SET AND THE SAMPLES WHICH WERE SUPPLIED TO POTENTIAL BUYERS HAVE BEEN RECEIVED POSITIVELY.AT END OF THE 16 MONTHS PROJECT TIMELINE WE WILL BE POSITIVELY READY WITH A COMMERCIALLY VIABLE PRODUCT. Mention what is the Value Proposition THE MARKET VALUE IS OF 3.0 cr AND GROWING BY 30% ANNUALLY.THE PROFIT MARGINS HOVER IN THE RANGE OF 40 TO 50%. Slide no 6.

When do you plan to set up your startup? THE BASIC FACILITY IS IN PLACE AND WILL BE MADE OPERATIONAL SOON.ALL THE MANUFACTURING LICENSES FROM MIDC,MSEB,MPCB HAVE BEEN OBTAINED. Have you discussed about your solution with Target Customers? How many >100,>50,>25,>10,>5, None? What is the feedback? YES THE SAME HAS BEEN DISCUSSED WITH NUMEROUS PARTIES AND SAMPLES HAVE BEEN SUPPLIED TO THEM AND HAVE RECEIVED THEIR APPROVAL.AT THE MOMENT THE TARGET CUSTOMERS ARE 6. Have you discussed your solution with business mentor? What is the feedback? Na Slide no 7.

Projected Time to Hit the Market 3-5 yr Road Map THE FIRST TWO YEARS WILL BE UTILISED IN SUPPLYING THE LOCAL MARKET . THE NEXT TWO YEARS WILL BE FOCUSSING ON TAPPING THE EXPORTS MARKET AND BY THE FIFTH YEAR STABILISING THE PRODUCTION. Business Plan Target Customer The target customers have been tapped and their feedback is positive. Once production commences, we will satisfy their total requirements. Revenue Model We will be following a zero debt model. We will recycle the earnings from the sales and undertake further production. We plan to purchase in bulk raw materials to reduce cost. We will also be tapping investors as the scale of operation balloons. Our product is unique and a long drawn one. We shall market it at a reasonable price tag and shall outdo potential competitors in the future too. Development of many such biomedical compounds and commercial scale up of same has been envisaged in the future. Our SWOT analysis is very positive. SWOT ANALYSIS STRENGTH OUR STRENGTH LIES IN THE R&D CAPABILITY WE HAVE ASSEMBLED OVER THE PAST MANY YEARS. WE HAVE GARNERED EXPERIENCE IN SYNTHETIC PROCESSES AND ANALYTICAL R&D. WEAKNESS OUR MAIN WEAKNESS IS THE FINANCIAL CONSTRAINTS. OPPORTUNITES MASSIVE OPPORTUNITIES AWAIT SME IN THE FIELD OF SPECIALITY CHEMICALS MANUFACTURING.MORESO WHEN TRADITIONAL CHINESE SPACE IS BEING VACATED. THREATS WE CAN FACE COMPETITION IF THE CHINESE COMPANIES REDUCE COST, BUT WE HAVE CALCUALTED THE RISK AND ARE READY TO FACE IT. slide no 8.

Backup Slides THE SYNTHETIC ROUTE A] SYNTHESIS OF HYDRAZONE BY REACTION OF BENZALDEHYDE AND PHENYL HYDRAZINE B] SYNTHESIS OF DIAZOTISED ANILINE BY USE OF SODIUM NITRITE AND HYDROCLORIC ACID C] SYNTHESIS OF THE FORMAZAN BY CONDENSATION OF DIAZOTISED ANILINE AND THE HYDRAZONE D] OXIDATION OF THE FORMAZAN TO GIVE THE TETRAZOLIUM SALT E] PURIFICATION. USES OF 2,3,5-Triphenyl Tetrazolium Chloride TTC is used to differentiate between metabolically active and inactive tissues . [enzymes important in the oxidation of the organic compounds and thus cellular metabolism] while it remains in its unreacted state in areas of necrosis since these enzymes have either denatured or degraded. HISTO-CHEMICAL TTC has been employed in autopsy pathology to identify myocardial infractions. Healthy viable heart muscles will stain deep red from cardiac lactate dehydrogenase while areas potential infractions will be paler. TTC detects ischemic injury as early as 30 minutes after coronary occlusion in rats and all hearts with within 3 to 6 hrs. Experiments in the use of TTC for cerebral infractions are also on. TTC is also used for counting for viable microbial contaminants in pharmaceuticals and cosmetics.in presence of antibiotics TTC is also used for biochemical experiments to indicate cellular respiration. Wallhauser has employed TTC to detect inhibition of bacterial growth while testing antibiotics in body fluids. Similarly a method was developed by using TTC as an indicator for inhibition of test organisms in presence of antibiotics substance in milk. Ref Wallhauser :- Naturwissenschaften 38, 237,1951. TTC IS BEING USED AS A CHROMOGENIC MARKER . The white compound is reduced to red TPF [1,3,5-Triphenyl formazan ] in living tissues due to the activity various hydrozenases Slide no 9.

Work plan (Gantt chart). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 X X X X X X X X X X X X X X X X X X.

Milestones. Duration (months) Milestone targets 3 Setting up the team, Ordering of the e quipment and Installation of the equipment. 6 Testing of the equipment and commencement of the pilot scale commercial batches. 9 Completion of the pilot scale commercial batches. 12 Evaluation of the product manufactured in the pilot scale batches and Commencement of the commercial scale production. 15 Evaluation of the product manufactured in the commercial scale production. 18 Closure and preparation of the final report..

Budget. Heads Amount (Rs lakh ) Basis Equipment About 15 L For the ordering of the HPLC and UV-VIS spectrophotometer Consumables About 4 L Consumables to be utilized for batch Man power About 15 L For appointment of support staff Incubation services 3.5L For the organization providing the same Travel 0.5 L For marketing and promotion of the product. External services 10 L For any engineering or production related consultancy Contingency 1.6 L Safety net for any unexpected expenditure..

Novelty NOVELTY AS HAS BEEN MENTIONED EARLIER IN DEVELOPING THE MANUFACTURING PROCESSESS WHICH SUPPORT US TO MARKET THE COMPOUNDS AT A RESONABLE COST AND OF ENHANCED QUALITY . FURTHERMORE THE MANUFACTURING PROCESS HAS BEEN STANDERDISED USING BASIC RAW MATERIALS,SO WE WILL NOT BE DEPNDANT ON OTHERS FOR THE INTERMEDIATES. THE PROJECT IS TOTALLY FEASIBLE AS THE MARKET IS SET AND THE SAMPLES WHICH WERE SUPPLIED TO POTENTIAL BUYERS HAVE BEEN RECEIVED POSITIVELY . THE NOVELTY LIES IN THE USE OF NON-TOXIC AND CHEAP RAW MATERIALS NAMELY ,METHANOL,CHLOROFORM,BENZALDEHYDE,SODIUM NITRITE ETC. Feasibility The initial feasibility study by supplying the commercial samples have thrown up encouraging results. We have been inundated with additional orders and we look forward to sustain the same. IP Strategy NA Slide no 13.

Reviewer’s concerns. Concerns raised by TEP Comments of applicant addressing concerns The solution addressing a niche market and the business plan needs to be strengthened. The business plan has been elaborated further as per reviewer’s request The applicants claims that they have sold 1-5 kg, it would have been helpful if the comparison data with the existing competitor is shown experimentally. We have incorporated the requested information by outlining our experimental layout..

Funds Received So far (BIRAC & Non BIRAC) NONE.