Field of Invention:

The present invention relates to compositions comprising Sophorolipids, having anti-tubercular activity for potential use in the treatment of tuberculosis and multidrug resistant tuberculosis. The invention further relates to compositions comprising Sophorolipids and its compatibility with standard anti-tuberculosis drugs for potential use in treatment of tuberculosis and multi-drug resistant tuberculosis as a part of multi-drug regimen.

Background of the Invention:

Tuberculosis (TB), one of the oldest infectious disease known that continue to afflict millions of people each year. TB is caused by bacteria called Mycobacterium tuberculosis (Mtb). COVID-19 apart, TB is considered to be the leading cause of death due to communicable diseases. It is estimated that 10 million people were afflicted globally with TB in 2019 and 1.2 million people died of TB. India accounted for 26% of the world’s TB cases and reported 79000 deaths due to TB in the year 2019. COVID-19 has derailed the TB control efforts and threaten to reverse the progress made so far in reducing the global TB burden. Modeling studies estimate an increase of one million TB cases per year between 2020-2025 due to COVID-19. TB is a curable disease. A typical treatment regimen for tuberculosis consists of a combination of four drugs (rifampicin, isoniazid, ethambutol and pyrizinamide) as first-line therapy taken for six months. Moreover, a continuing threat to TB treatment and public health disease control is the presence of drug resistant tuberculosis, which is a serious clinical condition. Worldwide in 2019, close to half a million people developed rifampicin-resistant TB (RR-TB), of which 78% had multidrug-resistant TB (MDR-TB; resistant to first-line drugs rifampicin and isoniazid). India accounts for 27% of the drug resistant TB. There is a spectrum of severe drug resistant forms of TB ranging from MDR to pre-XDR and XDR, where XDR stands for extensive drug resistance, in which drug resistance extends to second-line drugs. The drug- resistant forms of TB have an intense and prolonged treatment with second-line drugs that can last up to 24 months. WHO’s 2020 update recommends three groups (Group A, B,C) of second-line drugs.

Despite available treatment options in place, there are several challenges associated with TB treatment. On a macro level, the cost of drugs, especially the newer ones, severe adverse drug reactions and treatment adherence are the inherent challenges. Lengthy, multi-drug regimens for treatment of TB have contributed to non-adherence to treatment by patients resulting in sub-optimal concentrations of the drugs systemically and hence catalyzing the emergence of drug resistance in the bacteria. There are hundreds of antibacterial drugs available, although only 18 drugs are useful for the treatment of TB. On a micro level, Mtb has a complex cell wall composition with a thick lipid cell envelope, which makes it difficult for drugs to penetrate to the target. Secondly, even some of the existing drugs in use have poor penetration in the TB lesions formed by the bacteria in the body leading to suboptimal effectiveness and accelerated development of drug resistance. Moreover, reports on resistance against newer or repurposed drugs like Bedaquiline, Delaminid, Clofazamine, etc. are already emerging, sometimes even without drug pressure or through cross-resistance.

Therefore, there remains a dire and continuous need in the art for the development of new compounds having anti-tuberculosis activity for use in treatment of tuberculosis and multi drug resistant tuberculosis.

In light of the above background, the present inventors have evaluated composition containing sophorolipids as a promising alternative for the treatment of Tuberculosis and tested its usability as a combination with existing TB drugs for potential use in managing tuberculosis and its severe forms like MDR and XDR TB. Objectives of the Invention

The main objective of the present invention is to provide composition comprising sophorolipids in effective anti-tubercular/inhibitory concentration, potentially useful for the treatment of drug sensitive and drug resistant forms of TB and a method for production of sophorolipid composition thereof.

A further objective is to evaluate the effectiveness of the sophorolipid composition against inhibition of tubercular bacteria residing in intracellular environment of the host cells.

A further objective is to provide sophorolipids concentration that does not negatively interact with known standard anti-TB drugs and hence possibly demonstrate its potential use for combination therapy.

Summary of the Invention

In line with the above objectives, the present invention provides composition comprising sophorolipids in effective anti-tubercular/inhibitory concentration, potentially useful for the treatment of drug sensitive and drug resistant forms of TB and method of production for compositions comprising sophorolipid and its effective inhibitory concentrations, potentially useful for the treatment of tuberculosis and multidrug resistant tuberculosis.

In another aspect, the invention provides compositions comprising effective amounts of Sophorolipids along with other standard anti-tuberculosis drugs for potential use in treatment of tuberculosis and multi-drug resistant tuberculosis as a part of multi-drug regimen.

Description of drawings:

Figure 1 depicts Layout of 96-well microtiter plate for setting up REMA assay with 7 different concentrations of the 2 batches of sophorolipid composition and 1 Mycobacterium tuberculosis (Mtb)strain. Figure 2 depicts Representative image of DS- and MDR-Mtb colonies observed on 7H11 plates after 4 weeks of incubation (NC- no drug, PC- lpg/ml Rif, 42.1- batch of sophorolipid composition at 1% concentration)

Figure 3 SEM micrographs of a: Control (untreated) DS- Mtb cells; b & c: sophorolipid composition treated DS-Mtb exposed for 6 hours (b), and 2 days(c) ; d: Control (untreated) MDR-Mtb cells; e a & f: sophorolipid composition treated MDR-Mtb exposed for 6 hours (d) and 2 dyas (f); Marked areas were digitally zoomed and shown as inset

Figure 4 SEM micrograph of sophorolipid composition treated MDR-Mtb exposed for 2 days (at 5000X magnification); Arrow indicates cell fragmentation Figure 5 Representative image of Growth reduction of intracellular DS-Mtb exposed to 0.005%, 0.01%, 0.025% of sophorolipid composition and positive control (Rif lpg/ml). The bars depict the average CFU/ml with standard deviation for the error bar. Each concentration was tested in triplicates and average is reported

Figure 6 Representative image of Growth reduction of H37Rv with time, on exposure to 0.005%, 0.01%, and 0.025% of sophorolipid composition and positive control (Rif 1 pg/ml)

Figure 7 depicts Synergy Checkerboard Assay

Description of structure:

The figure below shows the structure of sophorolipid mixture produced by Candida bombicola

Lactonic Sophorolipid Acidic Sophorolipid

Detailed Description of the Invention

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

Unless specified otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, to which this invention belongs. To describe the invention, certain terms are defined herein specifically as follows.

Unless stated to the contrary, any of the words, “including”, “includes”, “comprising”, and comprises” mean “including without limitation” and shall not be construed to limit any general statement that it follows to the specific or similar items.

As used herein in the specification, the term “effective inhibitory amount” or “effective inhibitory concentrations” means and include an amount which is sufficient to prevent growth of tuberculosis bacteria in vitro.

Sophorolipids are an emerging class of biocompounds with amphiphilic naturenaturally produced from Candida bombicola as a mixture of lactonic and acidic sophorosides of 17-U-hydroxydecanoic acid with a typical lactone to acid ratio of 7:3. They are biodegradable low-foaming surfactants with high detergency and hardness-tolerance properties. The sophorolipid composition provided in the present invention comprise a residue of sophorose, the disaccharide consisting of two glucose residues linked by the b-1,2' bond, and fatty acid as an aglycone. The sophorolipid is a fermentation product of vegetable oils rich in oleic acid group and the sugar molecule. Accordingly, in an embodiment, the invention provides a process for preparation of sophorolipid composition which process comprises; a) fermenting a composition comprising oils or a fatty acid substrate of oleic (C18: l), linoleic (C18:2) and a-linolenic (C18:3) acids; a nitrogen source and salts using a yeast strain Candida bombicola AATCC 22214 by maintaining pH at 3.2 to 6.5 under aeriation; and b) extracting sophorolipid from fermented broth using suitable organic solvents.

In an embodiment, the carbon source is selected from glucose, dextrose, com starch liqueur and sucrose with a concentration of 10-100 g/1.

In an embodiment, the nitrogen source may include yeast extract, ammonium sulphate, urea, and peptone at a concentration of 1-10 g/1.

In an embodiment, the salts can be selected from the group consisting of potassium diphosphate, potassium mono-phosphate, calcium chloride, and magnesium sulphate at a concentration of 0.1-5 g/1.

In an embodiment, oils or the fatty acid substrate of Cl 8: 1 in a concentration of 40-80% was preferred as a substrate for the production of sophorolipid. The oils may be selected from the group consisting of oleic oil, soya oil, rice bran oil, sunflower oil, mauha oil, cotton seed oil, or fatty acid with a concentration of 10- 150 g/1. The substrate feeding is carried out during the process batch wise, at time intervals of approximately 6 to 24 hours.

In an embodiment, the fermentation pH is maintained during the entire cycle, which is around 140-180 log hrs, at about 3.2 to 6.5 at the start, preferably at 3.2 to 3.5.

In an embodiment, the fermentation temperature is an appropriate temperature at which the yeast can grow and produce a higher yield of sophorolipid and is usually 20 to 35°C, preferably 23 to 27°C. In an embodiment, the speed of aeration and agitation were maintained at 0.1 to 2.0 wm and 100 to 300 rpm respectively. Aeration and agitation are required to be performed to provide sufficient dissolved oxygen conditions in the culture.

The method of production of sophorolipid includes a further step to extract sophorolipid from fermented broth. After fermentation, the entire broth was allowed to stand for sedimentation of the cell biomass. The cell biomass was then removed by decanting and a viscous layer of sophorolipid was isolated which is used for further extraction and purification. In an alternate process variant, the sophorolipid was extracted by centrifugation or microfiltration.

Suitable solvents that can be used for the extraction and purification of viscous solution of sophorolipids can be selected from the group consisting of Water, Ethyl Acetate, Methanol, Ethanol, Hexane or combinations thereof or any other suitable organic solvent. The organic solvent layer can be recovered by decanting to obtain purified sophorolipid. The remaining solvent in the product was recovered by vacuum distillation. The final yield of the sophorolid obtained was calculated in g/1.

The purified Sophorolipid goes through further testing to check the quality of the products, including HPLC, critical micelle concentration (CMC), surface tension, emulsification index, specific gravity, viscosity, and stability of the product. Sophorolipid is a glycolipid in which long -chain hydroxy fatty acid and sophorose (sugar) are combined, and when produced by Starmerella bombicola, it is a mixture of lactone form and acid form, which is confirmed by HPLC.

In another embodiment, the present invention provides a composition, for inhibiting growth of Mycobacterium tuberculosis (Mtb) and multi-drug Resistant tuberculosis, comprising Sophorolipid in an effective concentration range of 0.025% to 5%; wherein, the sophorolipid is characterized by a 50% ± 5 % Lactonic and 50% ± 5 % acidic Sophorolipid content.

In another embodiment, the Sophorolipid composition is further characterized by critical micelle concentration of 40 mg/L, Interfacial tension at 1.37 mN/m and Surface tension at 35-36 mN/m.

In yet another embodiment, the invention provides compositions for inhibiting growth of Mycobacterium tuberculosis (Mtb) and multi-drug Resistant tuberculosis, comprising Sophorolipid in an effective concentration range of 0.025% to 5%; optionally formulated with suitable pharmaceutical excipients or carriers selected from the group consisting of diluents, binders, lubricants, fillers, disintegrants, glidants, pH adjusting agents, polymers, coloring agents, etc.

This optimum combination of lactonic and acidic functionalities formed in the sophorolipids, according to the process of the present invention imparts the anti- mycobacterial activity to the compositions.

In an embodiment, the sophorolipid composition, thus manufactured by the present invention, was evaluated for its capability to inhibit growth of Mycobacterium tuberculosis (Mtb) in vitro.

According to another embodiment, the study of mechanism of action in producing the anti-mycobacterial activity, has been shown to cause changes or ruptures in the cellular membrane and the consequent death of the pathogen. Additionally, based on some literature studies the Sophorolipids of the present invention have been shown to act as an active stabilizer and safety carrier in microemulsion drug delivery formulations and hence may help in improving site-specific bioavailability of conventional TB drugs. Therefore, the sophorolipid composition prepared according to the invention are a promising alternative to boost the efficacy of existing TB drugs thereby managing tuberculosis and its severe forms like MDR and XDR TB.

Accordingly, in one preferred embodiment, the present invention provides compositions comprising effective inhibitory amounts of sophorolipids for potential use in treatment of tuberculosis and multidrug resistant tuberculosis.

According to another preferred embodiment the present invention provides efficacy of the compositions comprising sophorolipids that changes or ruptures the Mtb outer membrane causing the death of the bacteria.

According to another preferred embodiment, the present invention provides evaluation of the capability of sophorolipids to inhibit tubercular bacteria residing inside host cells.

According to another preferred embodiment, the invention provides compatibility of compositions comprising effective inhibitory amount of Sophorolipids along with other standard anti-tuberculosis drugs for potential use in multi-drug regimen for treatment of tuberculosis and multi -drug resistant tuberculosis.

The standard anti-tuberculosis drugs according to the invention are selected from the group consisting of rifampicin (Rif), isoniazid (Inh), ethambutol, Pyrazinamide, Moxifloxacin(Mfx).

In another embodiment, the compositions of the present invention comprising Sophorolipids in effective inhibitory amounts either alone or in combination with other standard anti-tuberculosis drugs, can be formulated with one or more suitable pharmaceutical excipients or carriers.

In a further embodiment, the sophorolipid composition provided according to the present invention shows anti-tubercular activity against both drug-sensitive and drug-resistant forms of tuberculosis bacteria as demonstrated by REMA assay and Agar proportion method in vitro. According to this experiment, the concentrations of sophorolipid composition ranging from 0.05% to 5% were tested against a laboratory strain that is drug sensitive, three different drug sensitive clinical isolates and three different drug resistant clinical isolates. Clinical isolates were obtained from tuberculosis patients. Some strains showed effectiveness even at the concentration of 0.05%. But overall, for all drug sensitive and MDR strains, effective concentration range was 0.5%-5% on solid conditions and 0.5%-l% in liquid conditions.

In a further embodiment, sophorolipid composition shows intracellular inhibition of tubercular bacteria as demonstrated by in vitro human THP-1 monocyte cell line derived macrophage infection model. A sophorolipid composition concentration of 0.005 to 0.025% showed dose dependent inhibition in macrophages infected with drug-sensitive strain of tuberculosis, with 0.025% concentration showed similar inhibition as standard anti-tubercular drug rifampicin.

According to another embodiment, the sophorolipid composition does not have negative interactions with sentinel anti-TB drugs used in the treatment of drug- sensitive and drug-resistant strains like Rifampicin, Isoniazid and Moxifloxacin, as shown by Resazurin Drugs Combination Microtiter Assay (REDCA).

Those of ordinary skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments or examples disclosed herein, but is intended to cover modifications within the objectives and scope of the present invention as defined in the specification. The presented examples illustrate the invention, but they should not be considered to limit the scope of the invention in any way. Examples

Example 1:

Sophorolipid composition Production method:

The sophorolipid composition prepared according to the present invention is a composition with typical properties of the production process. The sophorolipid composition production was performed using the yeast strain Candida bombicola AATCC 22214, also reclassified as a Starmerella bombicola. The fed batch fermentation was carried out using the seed inoculum which was prepared in a 100-liter fermenter, whereas the pilot batch was carried out in a 1000L fermenter. The composition for both fermenters is as follows: one of the carbon sources includes glucose, dextrose, com starch liqueur and sucrose with a concentration of 10-100 g/1. The nitrogen source may include yeast extract, ammonium sulphate, urea, and peptone at a concentration of 1-10 g/1. The different salts, such as potassium diphosphate, potassium mono-phosphate, calcium chloride, and magnesium sulphate are used at a concentration of 0.1-5 g/1 concentrations. One of the important compositions of media is the substrate for the production of sophorolipid, which may be vegetable oil or fatty acid obtained from vegetable oil. The substrate is selected on the basis of fatty acid composition, including fatty acids C18:l, C18:2 and C18:3. Those oils or fatty acids that include fatty acid Cl 8: 1 composition of 40-80% was selected for the production. These oils may be oleic oil, soya oil, rice bran oil, sunflower oil, mauha oil, cotton seed oil, or fatty acid with a concentration of 10-150 g/1. Substrate feeding carried out batch wise, at time intervals of approximately 6 to 24 hours.

The fermentation pH is maintained during the entire cycle, which is around 140- 180 log hrs, at 3.2 to 3.5, preferably about 3.2 to 6.5 at the start. The fermentation temperature is an appropriate temperature at which the yeast can grow and produce a higher yield of sophorolipid and is usually 20 to 35°C, preferably 23 to 27°C. The speeds of aeration and agitation were maintained at 0.1 to 2.0 vvm and 100 to 300 rpm respectively. Aeration and agitation are required to be performed to provide sufficient dissolved oxygen conditions in the culture. The method of production also includes a further step to extract sophorolipid from fermented broth. After fermentation, the entire broth was allowed to stand for sedimentation of the cell biomass. The cell biomass was then removed by decanting and a viscous layer of sophorolipid was used for further extraction and purification. These steps can be omitted in a process variant by using centrifugation or microfiltration. Water and organic solvents such as Ethyl Acetate, Methanol, Ethanol, Hexane, or any other suitable organic solvent were used for the extraction and purification of the sophorolipid in the remaining viscous solution. The organic solvent layer was recovered by decanting to obtain purified sophorolipid. The remaining solvent in the product was recovered by vacuum distillation. The final yield was calculated in g/1. In the above-state of conditions, a yield of 251.59 g/1 is achieved on the basis of the oven drying method.

The purified Sophorolipid goes through further testing to check the quality of the products, including HPLC, critical micelle concentration (CMC), surface tension, emulsification index, specific gravity, viscosity, and stability of the product. Sophorolipid is a glycolipid in which long -chain hydroxy fatty acid and sophorose (sugar) are combined, and when produced by Starmerella bombicola, it is a mixture of lactone form and acid form, which is confirmed by HPLC.

Example 2:

Composition of Sophorolipid:

The sophorolipid is a glycolipid and is a combination comprising of Lactonic and Acidic functionality. Lactonic part is the ester formed by linkage of acid to alcohol part of sugar and is a closed ring. The optimum combination of lactonic and acidic formed in the sophorolipid composition imparts the antimicrobial and preservative character. Generally, it is sold and can be provided as aq. Solution and the active content is adjusted as per the end application and the form needed. Based on the fermentation cycle the lactonic and acidic ratio could be tailored. Accordingly, the sophorolipid composition prepared according to the process of the present invention is characterized by a typical content of 50% ± 5 % Lactonic and 50% ± 5 % acidic Sophorolipid content, respectively.

The Surface properties were measured using Kyowa DY300 tensiometer and standard SOPS. The CMC of the Sophorolipid composition made above was measured at 40 mg/L, Interfacial tension using Heptane-water standard method was 1.37 mN/m and Surface tension was 35-36 mN/m.

Example 3:

Antimycobacterial activity of sophorolipid composition against human pathogen (Mtb) by REMA assay and Agar proportion method

1. Resazurin Microtiter Plate Assay:

The REMA assay involved the addition of micro-quantities of a bacterial isolate and the drug to be tested in the wells of a 96-well plate (Thermo Fisher Scientific, Roskilde, Denmark). After incubation, the growth of the culture was checked by the addition of a redox dye- Resazurin (Sigma, MO, USA). In presence of live bacteria, Resazurin undergoes an oxidation-reduction reaction to give Resorufin, resulting in a colour change from blue to pink. The REMA assay was performed with a standard laboratory strain (H37Rv) and three different Drug sensitive (DS)- and Multi-drug resistant (MDR)-Mtb clinical isolates.

Sophorolipid compositions were tested for a range of concentrations- 0.05%, 0.1%, 0.5%, 1%, 2%, 3% and 5% and compared with standard drug Rifampicin (Rif) (Sigma, MO, USA) at a concentration of lpg/ml as control. DS strains used were sensitive to Rif and hence did not show growth in the assay, while MDR strains were resistant to Rif and hence showed growth in the assay. The cell density of Mtb cultures was adjusted to 1 McFarland (0.295 OD600nm), a final dilution of 1:20 was used in the assay. The plate was set up as shown in Figure 1 by mixing appropriate amount of Sophorolipid composition and culture suspension. Plates were incubated at 37°C for 7 days. After completion of the incubation period, 30 mΐ of 0.01% w/v resazurin sodium solution was added to each well and the plates were incubated again at 37°C for 24 hours. The plates were then observed for colour change from blue to pink/colourless, which indicated growth (Table 1). The lowest drug concentration that prevented colour change was taken as the upper limit for the minimum inhibitory concentration (MIC) range.

Table 1: Interpretation of results for REMA assay

Results:

Table 2 represents the results obtained with Sophorolipid composition batch BPS 42.1. Both DS- and MDR-Mtb strains, showed no colour change at concentrations 0.1%, 0.5% and 1%, indicating bacterial killing by Sophorolipid composition at these concentrations. Hence, the effective range for this batch was from 0.1% to 1%, and the MIC was determined to be 0.1%.

It was noted that MDR strains, resistant to Rif, showed growth in the presence of Rifampicin control, but were susceptible to Sophorolipid composition (Table 2 &

3)·

Table 2: Growth observed for Mtb strains for Sophorolipid composition/Biogod batch 42.1 by REMA

2. Agar Proportion Method (APM):

The APM relies on a colony-count approach, where the drug/composition to be tested was added in the solid agar growth medium (Middlebrook 7H11 agar medium (Difco) and the bacterial cultures were spread onto, or spotted on the agar plates. After incubation for 3-4 weeks, the colony-forming units (CFU) on the drug-containing plates were counted and compared to a ‘no-drug’ control plate in order to determine the inhibitory effect on the bacteria. APM was carried out for the standard laboratory strain H37Rv and the three different DS and MDR clinical Mtb isolates. Sophorolipid composition was tested for concentrations- 0.05%, 0.1%, 0.5%, 1%, 2%, 3% and 5%.

Results:

Table 3 and Figure 2 represent combined results for all Mtb strains tested with Sophorolipid composition batch BPS 42.1. As can be seen, there was no growth of Mtb (DS and MDR) on the Sophorolipid composition plates even at the lowest concentration of the drug. This indicates that Sophorolipid composition batch 42.1 showed an anti-mycobacterial effect from 0.05% to 5%, with an MIC of 0.05%.

Table 3: Growth observed with DS- and MDR-Mtb strains for Sophorolipid composition/Biogod batch 42.1 by APM (‘+’ indicates growth, indicates No growth)

Example 4:

Scanning electron micrographs of Sophorolipid composition treated DS and MDR-Mtb strains:

Scanning electron micrographs (SEM) were undertaken to show the effect of sophorolipid composition (BG) on cell morphology of DS and MDR-Mtb strains. DS and MDR-Mtb strains were treated with 0.5% sophorolipid composition and the cells were harvested at 0 hours (control/untreated cells), 6 hours and 2 days for SEM studies. The cells were fixed in 2% glutaraldehyde (Sigma) in 0.1M Sodium cocodylate buffer (Sigma) at room temperatures for 20 mins in dark. A 10m1 smears were prepared on a glass coverslip coated with 0.01% Poly-L-Lysine (Sigma), dehydrated in a series of increasing ethanol concentration, sputter coated with palladium and imaged. Imaging was carried out in Environmental Scanning Electron Microscope (FEI Quanta 200 with tungsten electron source). Images were recorded at 3nm resolution, under high vacuum at three different magnifications- 1000X, 5000X and 10000X.

Results:

When compared to control untreated cells (Figure 3a, 3d), it was observed that Sophorolipid composition treated bacteria after 6 hours exposure had a wrinkled appearance (Figure 3b, 3e). The morphology of the cells was rough compared to the smooth outer surface of untreated Mtb. Though the cells appeared to have a similar size after treatment, the shape was distorted from the normal rod-like form of the bacteria. At 2 days post-exposure to Sophorolipid composition, cell damage and degradation was more evident (Figure 3c, 3f), and there was an accumulation of cell debris in both DS and MDR strains. The MDR-Mtb had a smoother cell surface compared to the DS strain at 6 hours post sophorolipid composition exposure (Figure 3b, 3e), however, after 2 days of treatment, clear fragmentation of the MDR cells was observed as shown in Figure 4.

Example 5:

In vitro Sophorolipid composition intracellular Mtb inhibition assay using CFU enumeration method:

Mtb is an intracellular pathogen and is known to infect and reside in macrophages during infection in humans. Macrophages derived from THP-1 cells; a human derived monocyte cell-line is a well-established in vitro model for tuberculosis infection and can serve as a good model for evaluating potential in vivo effectiveness of new drugs. This is carried out by measuring the rate of Mtb growth inside macrophages with and without drug exposure using CFU enumeration method. Here we used THP-1 cells infected with drug sensitive stain H37Rv for evaluating Sophorolipid composition’s ability to inhibit intracellular growth of Mtb. THP-1 cells were seeded on to 96 wells and differentiated to macrophages by treating with 20 ng/ml of phorbol 12-myristate 13 -acetate (Sigma) for 48 hours. Initial analysis showed that the Sophorolipid composition concentrations of upto 0.025% was non-toxic to macrophages and inhibited the DS strain used (MIC of this strain 0.025% sophorolipid composition), and hence was selected for intracellular inhibition assay. Macrophages were infected with Mtb in the ratio of 1:1 and incubated for 2 hours. After 2 hours, the excess bacteria was washed and the cells in triplicate wells were treated with 0.005% Sophorolipid composition or 0.01% Sophorolipid composition or 0.025% Sophorolipid composition or 1 ug/ml Rifampicin (positive control) or basal media (no drug control; NC). Cells were incubated for 2 days or 5 days, lysed with 0.01% SDS and number of surviving Mtb were counted by CFU method on 7H11- agar plates as described above. The intracellular Mtb inhibition ability was determined by calculating the CFU/ml (N) at times 0 hours, 2 days and 5 days, the “Growth Rate (GR)” of drug-treated and untreated conditions at 2 and 5 days, and “normalized growth rate inhibition” at time t in the presence of drug at concentration c.

The “Growth Rate (GR)” was calculated using the following formula:

The “normalized growth rate inhibition” was calculated by the below formula fc(c,t)

GR inhibition = 2 ^k — 1

Where, k(c,t) is the growth rate of drug-treated cells and k(0) is the growth rate of untreated control cells (4). GR value inhibition value of, a. 1 indicates no growth inhibition b. 0 tol indicates partial growth inhibition c. 0 indicates complete growth inhibition

Results:

Tables 4 and 5 illustrate the comparison of CFU/ml and GR inhibition values for different concentrations of Sophorolipid composition (BG) and PC (Rif lpg/ml). Figures 4 and 5 illustrates representative experiment in graphical format showing growth reduction of Mtb exposed to Sophorolipid composition and Rifampicin compared to untreated control (No drug control). Results show that there was a concentration-dependent inhibition of intracellular growth of the Mtb strain tested, when the infected macrophages were treated with various concentrations (0.005%, 0.01%, and 0.025%) of Sophorolipid composition for 2 days and 5 days. Importantly results show that inhibition by 0.025% Sophorolipid composition was comparable to standard anti-TB drug Rifampicin at lpg/ml concentration, demonstrating that Sophorolipid composition can inhibit intracellular bacteria. Table-4: Observed CFU/ml for different concentrations of Sophorolipid composition and Rifampicin

Table-5: Comparison of GR inhibition value for different concentrations of

Sophorolipid composition with Rifampicin

Example 6:

Resazurin Drugs Combination Microtiter Assay (REDCA):

The interactions between two drugs can be synergistic, additive, indifferent, or antagonistic. Such interactions can be studied by checkerboard assays (Figure 3) where the effect of drug combinations is compared with its individual activity. Synergism is defined when the effect between two test drugs is greater than the sum of the effects of the individual drugs. An additive effect is seen when the combined effect is equal to the sum of the effect of each agent alone. Indifference is when no effect is observed with both drugs combined. In antagonism, one drug can reduce the effectiveness of the other drug in action. The comparison is represented as the Fractional Inhibitory Concentration (FIC) index which is calculated using the following formula, where A and B are the MIC of each antibiotic in combination (in a single well), and MIC _A and MICB are the MIC of each drug individually.

The FIC Index value of

1. <0.5 indicates synergistic effect

2. 0.5-4 indicates additive effect or indifference

3. >4 indicates antagonistic effect

Here we used a resazurin based checkerboard assay called resazurin drugs combination microtiter assay(REDCA) for evaluating the synergistic activity of Sophorolipid compositions (BG) with first-line anti-TB drugs, Rif and Inh for DS strains, and in addition, second-line drug Moxifloxacin (Mfx) for MDR strains. MDR strains are sensitive to Mfx. The assay was performed with three different clinical isolates of both DS- and MDR-Mtb strains. A range of concentrations of the drugs and Sophorolipid was decided based on MIC values known for each strain. Two-fold dilutions of drugs were made aseptically using Middlebrook 7H9 broth (Difco) and the assay was performed. The range tested is mentioned in the footnote of Tables 4 and 5. In each plate, Sophorolipid concentrations were added along the columns, and Rif/Inh/Mfx were added along the rows. Sterility controls and no drug controls were maintained. Plates were incubated at 37°C for 7 days. After the incubation period, 30m1 of 0.01% w/v Resazurin sodium (Sigma) solution was added to each well and plates were incubated at 37°C for 24h-48h in dark. The plates were then observed for colour change from blue to pink/colourless, which indicated growth. No colour change indicates inhibition of growth and killing of bacteria. MIC was determined for each drug alone and in combination with Sophorolipid. Based on the MIC values, the FIC Index for each drug combination was calculated and the type of drug interaction was deduced. Results:

Table 4 represents the results obtained from the three DS strains. As can be seen, a decrease in MIC of Rif and Inh was observed in the presence of Sophorolipid composition, indicating a synergistic or additive effect of Sophorolipid with standard anti-TB drugs. The FIC values calculated for Sophorolipid and Rif were between 0.5 and 4, suggesting an additive effect of the two drugs for the given strains. Since MIC of Inh was beyond the range of concentrations tested, the FIC value was calculated considering the next higher concentration. A similar additive effect was seen for Sophorolipid and Inh with two of the three DS strains.

For MDR strains, the second-line drug Moxifloxacin was tested along with Sophorolipid composition for synergistic action. In addition, Rif and Inh, although ineffective, were also checked to see whether there is a decrease in MIC in presence of Sophorolipid composition. Since MIC for these drugs was greater than the highest standard concentration, the FIC value was calculated by considering the next 2-fold higher concentration. As shown in Table 6 and Table 7, an additive effect was observed for all the tested anti-TB drugs with sophorolipid composition for one clinical isolate (DR3). However, except for Rif + sophorolipid composition combination for DR2 strain (FIC value=l, Additive effect), there was no effect (Indifference) observed for any drug combination in the other strains.

Table 6: Observed MIC values, FIC Index and drug combination effect observed for DS strains

Footnote: Concentrations tested: DS1- sophorolipid composition - 0-1% v/v; Rif 0-2 pg/ml. Inh-0-0.2 pg/ml; DS2- sophorolipid composition - 0-1% v/v; Rif 0- 0.12 pg/ml. Inh-0-0.1 pg/ml; DS-3- sophorolipid composition - 0-1% v/v; Rif 0- 0.5 pg/ml. Inh-0-0.05 pg/ml

Table 7: Observed MIC values, FIC Index and drug combination effect observed for MDR strains Footnote: Concentrations tested: DR1 and DR2- sophorolipid composition - 0-1% v/v; Rif 0-4 pg/ml. Inh-0-1.6 pg/ml. Mfx- 0-0.5 pg/ml ; DR-3- sophorolipid composition - 0-1% v/v; Rif 0-4 pg/ml. Inh-0-1.6 pg/ml. Mfx- 0-0.25 pg/ml

Conclusion for REDCA:

REDCA checkerboard assay established an additive effect between sophorolipid composition and, Rif & Inh for drug-sensitive strains. This suggests that sophorolipid composition do not negatively interact (antagonize) with first-line anti-TB drugs tested and maybe used in combination. For drug-resistant strains, indifference was observed and similar to additive effect suggest sophorolipid composition did not negatively interact with the tested drugs and thus can be used alone or may be used in combination.