Description:A FORMULATION TO INHIBIT THE GROWTH OF MULTIDRUG
RESISTANCE S. AUREUS.

FIELD OF INVENTION:

The present invention relates to a formulation to inhibit the growth of multidrug resistance S. aureus. More particularly, the formulation characterised with Montelukast as an inhibitor of efflux pumps in multidrug-resistant staphylococcus aureus that lowers the operating and capital costs associated with developing such drugs.

BACKGROUND ART:

In early 2017, the World Health Organization (WHO) made a notable contribution by publishing its first priority list of bacteria that are resistant to antibiotics. The list identified many infections that present substantial hazards to human well-being, with the objective of assisting in prioritizing research and advancing novel antibiotic therapies. Staphylococcus aureus (S. aureus) and its resistant variants, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), and vancomycin-resistant Staphylococcus aureus (VRSA), are classified as high priority pathogens. S. aureus is a Gram-positive bacteria frequently found on human skin and mucous membranes and linked to a range of human infections, such as bloodstream infections, infective endocarditis, infections of the skin, soft tissues, bone, joint. It also causes lung infections like pneumonia and empyema, gastrointestinal infections, meningitis, toxic shock syndrome, and urinary tract infections (PMID: 28722898). The management of staphylococcal infection has grown increasingly challenging in recent decades as a result of the emergence of different strains of MRSA and VRSA. MRSA is frequently present in both community and hospital-acquired infections, making beta-lactam antimicrobials ineffective for therapy. Vancomycin, once seen as a final option for treating MRSA, is currently facing challenges because to the rise of vancomycin-resistant strains such as VISA and VRSA. Conventional antibiotics that target cell wall production are not successful in treating these strains (PMID: 21867918).

Bacteria can acquire resistance to drugs through four primary mechanisms. First, they can modify the permeability of their cellular structure to prevent the entry of antibiotics. Furthermore, they can alter the specific targets that antibiotics typically target, making them ineffective. In addition, bacteria can generate enzymes that neutralize antibiotics, making them ineffective. Finally, bacteria can utilize efflux pumps to efflux out antibiotics from the cytoplasm, providing additional defense against the antibiotics. Multidrug resistance (MDR) in bacteria is mostly linked to increased efflux activity, whereas other mechanisms described are commonly connected with resistance to a single antibiotic (PMID: 31219077). Bacteria typically possess five predominant families of efflux pumps. The mentioned families are the small multidrug resistance (SMR) family, multidrug and toxin extrusion (MATE) family, major facilitator superfamily (MFS), resistance nodulation cell division (RND) family, and ATP binding cassette (ABC) family. Over 10 multidrug efflux pumps have been identified in S. aureus, located on either the chromosome or plasmids. The primary efflux pumps in S. aureus that play a role in multidrug resistance consist of MFS type pumps (NorA, NorB, NorC, MdeA, QacA/B, and Smr) and ABC type pumps (AbcA and Sav1866) (PMID: 23569469).

The increasing issue of antibiotic-resistant infections emphasises the need to explore alternative treatment options. Efflux pump inhibitors (EPIs) have the potential to significantly improve the efficacy of antibiotics by inhibiting the efflux of antibiotics from cells. The combination of EPIs and antibiotics shows great potential in addressing bacterial resistance resulting from overexpression of efflux pumps. An ideal EPI should have potent therapeutic effects without any toxic side effects and demonstrate a targeted approach towards prokaryotic efflux pumps while not possessing any antibacterial properties. However, EPIs identified till now have not been approved for clinical use due to their ineffectiveness, limited range of action, unfavorable drug behaviour in the body, or high levels of toxicity. Given the established pharmacokinetics and known adverse effects of FDA-approved drugs, evaluating them for EPI activity would be beneficial. If proven to be effective, they can undergo clinical trials and be manufactured more easily and at a lower cost (PMID: 35905077).

Drawbacks

The increasing issue of antibiotic-resistant infections emphasises the need to explore alternative treatment options. Efflux pump inhibitors (EPIs) have the potential to significantly improve the efficacy of antibiotics by inhibiting the efflux of antibiotics from cells. The combination of EPIs and antibiotics shows great potential in addressing bacterial resistance resulting from overexpression of efflux pumps. An ideal EPI should have potent therapeutic effects without any toxic side effects and demonstrate a targeted approach towards prokaryotic efflux pumps while not possessing any antibacterial properties. However, EPIs identified till now have not been approved for clinical use due to their ineffectiveness, limited range of action, unfavorable drug behaviour in the body, or high levels of toxicity.

In the prior art an application 2014WO-US21705 discloses use of levocetirizine and montelukast in the treatment of anaphylaxis. The embodiments described herein include methods and formulations for treating anaphylaxis and related acute allergic reactions. The methods and formulations include, but are not limited to, methods and formulations for delivering effective concentrations of levocetirizine and montelukast to a patient in need. The methods and formulations can comprise conventional and/or modified-release elements, providing for drug delivery to the patient.

In another prior art a PCT application WO2013/012199 discloses capsule formulation comprising montelukast and levocetirizine. The capsule formulation is disclosed for preventing or treating allergic rhinitis and asthma, which comprises two separate layers of: (1) a Montelukast layer comprising montelukast or a pharmaceutically acceptable salt thereof; and (2) a Levocetirizine layer comprising levocetirizine or a pharmaceutically acceptable salt thereof; and a method for the preparation thereof. The capsule formulation according to the present invention can completely separate two active ingredients, thereby minimizing the reactivity product stability against aging effects, and thus, can optimize the therapeutic effects.

In another prior art an US application US20230404992 A1 discloses topical formulations comprising montelukast and combinations with mussel adhesive proteins. There is provided topical pharmaceutical formulations comprising montelukast, or a pharmaceutical acceptable salt of solvate thereof, as well as combination products comprising (a) at least one montelukast, or a pharmaceutically combination products find particular utility in direct topical administration for the treatment of inflammation, of inflammatory disorders and/or of condition characterized by inflammation, including wounds, burns, psoriasis, acne and atopic dermatitis.

In yet another prior art a Patent specification 2015WO-GB541004 discloses new combination of pemirolast and montelukast. There is provided combination products comprising (a) pemirolast, or a pharmaceutically acceptable salt or solvate thereof; and (b) montelukast, or a pharmaceutically or solvate thereof. Such combination products5 find particular utility in asthma and related conditions

OBJECT OF INVENTION:

The principle object of the present invention is to identify efflux pump inhibitors of staphylococcus aureus from FDA-approved drug library.
Another object of the present invention is to make a formulation of efflux pump inhibitor and antibiotic combination for possible synergistic effect in treatment of multidrug-resistant staphylococcus aureus infection.
Yet another object of the present invention is to identify molecular mechanism of action of identified efflux pump inhibitor for efflux inhibition.

SUMMARY OF INVENTION:

This invention demonstrates the utilization of “montelukast” as an efflux pump inhibitor when combined with antibacterial agents, to effectively treat Multidrug-resistant staphylococcus aureus (MRSA). The study demonstrates the efficacy of “montelukast” in treating MRSA strains that have overexpressed efflux pumps. It also reveals that “montelukast” has synergistic effects when combined with fluoroquinolones (norfloxacin, moxifloxacin hydrochloride) and beta-lactams (cefotaxime sodium), resulting in enhanced cellular accumulation and reduced effective Minimum inhibitory concentration (MIC) in vitro. MRSA has proven to be quite challenging to manage in healthcare facilities due to its increased resistance to antibiotics, resulting in reduced cellular accumulation and higher effective concentration, primarily caused by the over expression of efflux pumps. The main efflux pumps involved in S. aureus resistance are norA, norB, and abcA. This study utilized an FDA-approved library to screen and identify efflux pump inhibitors. “Montelukast” was identified as one such inhibitor, demonstrating no antibacterial activity and having no impact on bacterial membrane potential or integrity. “Montelukast” significantly enhanced the efficacy of norfloxacin, moxifloxacin, and cefotaxime in killing MRSA, when formulated in the ratio of 1:4, 4:1, and 1:2 ratios respectively, both in vitro and in vivo with more than 4-fold reduction in MIC and more than 2 log reductions in bacterial burden, respectively. “Montelukast” demonstrated enhanced efficacy in killing MRSA when used in combination with moxifloxacin both in vitro and in vivo. It significantly reduced the bacterial load by 4.5 log in vivo and lowered the required concentration by 4-fold in vitro. Our study demonstrates that “montelukast” strongly interacts with MgrA, a master regulator of the efflux pumps in S. aureus, enhances MgrA's affinity for the norB promoter and effectively suppresses the expression of norB efflux pump. In light of the current challenges in treating MRSA and developing efflux pump inhibitors, utilising an FDA approved drug like “montelukast” could prove advantageous. This approach would streamline the process of inventing new efflux pump inhibitors, cut down on manufacturing cost, and minimize the need for higher antibiotic concentrations.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

Figure 1 illustrates screening of Montelukast as an efflux pump inhibitor (EPI) in accordance with the present invention;

Figure 2 illustrates In vitro efficacy of montelukast as an efflux pump inhibitor having no cytotoxic effect in accordance with the present invention;

Figure 3 illustrates Montelukast enhances efficacy of antibiotics both in vitro and in vivo in accordance with the present invention;

Figure 4 illustrates Montelukast regulation of efflux pump gene expression and interaction with MgrA in accordance with the present invention;

Figure 5 illustrates Montelukast increases dimerisation and phosphorylation of MgrA in accordance with the present invention.

DETAILED DESCRIPTION:

As shown in Figure 1 there is illustrated screening of Montelukast as an efflux pump inhibitor (EPI). Panel A displays a heat map illustrating the results of screening FDA-approved drugs for their potential to inhibit efflux pumps using an EtBr accumulation assay. In this assay, higher fluorescence intensity indicates a greater accumulation of EtBr within the cell. Black boxes represent potential drugs with efflux inhibitory properties, characterized by a fluorescence intensity increase above 30%. Panel B shows a bar plot of concentration dependent increase of EtBr accumulation by montelukast. Panel C shows bar plot of montelukast reducing EtBr efflux at 6 μg/ml (10 μM), Chlorpromazine (CPZ) a known efflux pump inhibitor was used at ½ MIC concentration as a positive control

Identify efflux pump inhibitors of staphylococcus aureus from FDA-approved drug library.

An ethidium bromide (EtBr) accumulation assay was used to identify efflux pump inhibitors of S. aureus. A library of 774 FDA-approved drugs was evaluated to determine if they had the potential to inhibit efflux pumps. Drugs that exhibited more than 30% increase of EtBr fluorescence than control were classified as potential efflux pump inhibitors (EPIs). The screening identified several drugs that exhibited efflux inhibition properties, as shown in Figure 1A. These drugs were then refined using an exclusion criterion, which eliminated drugs with intrinsic fluorescence intensity, known antibacterial properties, and known toxicity towards mammalian cells from the list of potential efflux pump inhibitors. Montelukast was determined to be the most effective inhibitor of the efflux pump, as it resulted in the highest accumulation of EtBr compared to other identified potential EPIs. Montelukast demonstrated a concentration dependent increase in EtBr accumulation, indicating a dose-dependent effect on efflux inhibition (Figure 1B). Furthermore, montelukast demonstrated reduction in EtBr efflux at 6 μg/ml (10 μM), as depicted in Figure 1C.

As shown in Figure 2 there is illustrated in vitro efficacy of montelukast as an efflux pump inhibitor having no cytotoxic effect: Panel A shows time-kill curve of montelukast at 6 μg/ml (10 μM) having no effect on S. aureus growth. Panel B shows microscopy images of Live-Dead analysis (Syto9/PI staining) of montelukast at 6 μg/ml (10 μM) by having no effect on bacterial membrane integrity, 70% ethanol was used as dead control. Montelukast effect on S. aureus membrane potential was studied by using membrane potential sensitive dye DiOC2. Panel C shows the flow cytometry analysis of montelukast at 6 μg/ml (10 μM) having no effect on bacterial membrane potential, CCCP (Carbonyl cyanide 3-chlorophenylhydrazone) a known membrane depolarizer was used at 5 μM concentration as a positive control. Panel D shows the cell viability curve of montelukast having no cytotoxic effect on mammalian cell lines (HEK293T: Human Kidney epithelial cell line and A549: Human lung cancer cell line) up to 40 μM concentration with more than 75% viable cells.

Furthermore, montelukast was found to lack antibacterial properties (Figure 2A), have no impact on bacterial membrane integrity (Figure 2B), membrane potential (Figure 2C), and have no cytotoxicity for mammalian cell lines (Figure 2D). These findings establishes montelukast as a promising efflux pump inhibitor.

Identify antibiotic and efflux pump inhibitor combination for possible synergistic effect in treating drug-resistant staphylococcus aureus infection.

Figure 3: Montelukast enhances efficacy of antibiotics both in vitro and in vivo: Panel A shows in vitro synergistic effect of montelukast in combination with antibiotics. The synergy was determined by taking into consideration of the Fractional Inhibitory Concentration (FIC) value wherein FIC lesser than 0.5 (i.e., more than 4-fold reduction in MIC) was considered synergistic, FIC between 0.5 & 1 was considered additive, FIC between 1 & 4 as indifferent and FIC greater than 4 as antagonistic. The FIC calculation is given in Panel B where the minimum inhibitory concentration (MIC) of antibiotics and montelukast was determined both individually and in combination.

Subsequently, the FIC was calculated using the equation: FIC = FICA + FICB. FICA = MIC of A in combination/ MIC of A alone and FICB = MIC of B in combination/ MIC of B alone, where A is antibiotics and B is montelukast. Panel C shows the photographs of infected and treated skin areas of mice right flank region on day 3 (start of treatment) and day 8 (day of sacrifice). Panel D shows bacterial burden in the skin tissues in control (without infection), MRSA mu50 infected, antibiotics and montelukast treated mice determined on day 8 by CFU count method where PBS: Phosphate buffered saline, pH 7.4, Control: No infection or treatment, Mont: Montelukast, Nor: Norfloxacin, Mox: Moxifloxacin and Cefo: Cefotaxime.

The study investigated the potential of using montelukast in conjunction with antibiotics to enhance the efficacy of antibiotics in treating MRSA. This was done in vitro by checkerboard synergy experiment and in vivo by synergy study in a mouse skin infection model. The study primarily evaluated three antibiotics: norfloxacin and moxifloxacin hydrochloride, which are both fluoroquinolones, and cefotaxime sodium, which is a beta-lactam antibiotic. The three antibiotics are selected based on their status as substrates being effluxed by overexpressed efflux pumps of MRSA, namely norA, norB, and abcA. We selected these three efflux pumps because they are the most common pumps that cause multidrug resistance in S. aureus. The in vitro synergy study showed that montelukast exhibited synergistic effects when combined with norfloxacin and cefotaxime against both sensitive (MSSA) and resistant (MRSA) S. aureus strains, with a Fractional Inhibitory Concentration (FIC) of less than 0.5 i.e., 4-fold reduction in minimum inhibitory concentration (MIC) (Figure 3A & 3B). However, moxifloxacin was found to effective synergistically in combination with montelukast only in MRSA with FIC 0.16 (Figure 3A & 3B). In addition, when studied the effect of antibiotics and montelukast on the treatment of MRSA infection in an in vivo mouse model of skin infection, it was found that the separate administration of antibiotic and montelukast was not effective in treating MRSA infection, resulting in a reduction of less than 1 log in colony forming units (cfu) (Figure 3D). On the other hand, the combination of montelukast with moxifloxacin, norfloxacin, and cefotaxime, there was a significant decrease in bacterial burden by 4.5 log cfu, 3.4 log cfu, and 2.1 log cfu respectively (Figure 3D). Overall, montelukast increased the efficacy of moxifloxacin in treating MRSA infection both in vitro (4-fold reduction in MIC, Figure 3B) and in vivo (4.5 log reduction in MRSA cfu, Figure 3D).

Identify molecular mechanism of action of identified efflux pump inhibitor for efflux inhibition.

As shown in Figure 4 there is illustrated montelukast regulation of efflux pump gene expression and interaction with MgrA. Panel A shows the bar plot of montelukast affecting the gene expression of efflux pumps and their regulators at 6 μg/ml (10 μM). 16s rRNA was employed as an internal control. Panel B shows the bar plot of relative expression of pknB to rsbU in absence and presence of montelukast at 6 μg/ml (10 μM). Panel C shows the circular dichroism spectra and relative secondary structure content of purified MgrA in absence and presence of 6 μg/ml (10 μM) montelukast. Panel D shows with increasing montelukast concentration the interaction of montelukast with purified MgrA increases as measured form relative tryptophan intensity by fluorimeter. Panel E shows montelukast binding to MgrA in 1:1 stoichiometry as determined by Job's plot. Panel F shows the comparison of binding affinities of MgrA with different efflux pump gene promoters in absence and presence of 6 μg/ml (10 μM) montelukast.

To understand how montelukast inhibits the activity of efflux pumps, we conducted a study to examine the impact of montelukast on the gene expression of these efflux pumps and their regulators. This was done through quantitative real-time PCR analysis. The gene expression study (Figure 4A) showed that montelukast treatment did not have any impact on the expression of norA (0.2-fold) and abcA (0.4-fold) in MRSA. On the other hand, the treatment of montelukast resulted in a significant reduction in norB expression, decreasing it by 3.9-fold. Furthermore, the use of montelukast resulted in a significant reduction in the gene expression of mgrA by 1.9-fold. It appears that montelukast could inhibit the norB efflux pump by reducing the expression of mgrA, which is responsible for activating norB in unphosphorylated conditions. Furthermore, we analysed the expression of pknB, a serine/threonine kinase that phosphorylates MgrA, and rsbU, a serine/threonine phosphatase that dephosphorylates MgrA. Both genes showed a decrease in expression after montelukast treatment. On the other hand, when comparing the ratio of pknB to rsbU, it was found that montelukast treatment led to a 1-fold increase compared to the untreated control (Figure 4B), suggesting that pknB has a significant role. This suggests that montelukast treatment may positively affect the phosphorylation of MgrA, which in turn can result in stronger repression of norB gene expression as phosphorylated MgrA acts as a direct repressor of norB efflux pump expression.

Phosphorylated MgrA binds directly to the norB promoter and exhibits direct repression action. Therefore, in order to verify if montelukast undergoes phosphorylation and if it enhances the binding capability of MgrA to the norB promoter, we cloned and purified the MgrA protein. The secondary structure content and stability of purified MgrA were evaluated by circular dichroism spectroscopy, which showed that MgrA is in native confirmation with no alteration in its secondary structure (Figure 4C). Furthermore, Fluorimetry was used to investigate any potential interaction of MgrA and montelukast. The fluorimetry analysis revealed a strong interaction between MgrA and montelukast, with a binding affinity (Kd) of approximately 28.5 ± 4 nM (Figure 4D). Additionally, the binding stoichiometry of MgrA and montelukast was evaluated using Job's continuous variation approach, revealing a 1:1 binding stoichiometry between MgrA and montelukast (Figure 4E).
Since, MgrA regulates efflux pump gene expression by directly interacting with the promoter regions, we evaluated the effect of MgrA binding to efflux pump gene promoters in absence and presence of 6 μg/ml (10 μM) montelukast by electrophoretic mobility shift assay (EMSA). The EMSA analysis (Figure 4F) revealed that in absence of montelukast MgrA binds to all the efflux pump gene promoters having highest affinity for norB promoter (Kd ~ 4.3 ± 1.1 μM) followed by norA promoter (Kd ~ 10.6 ± 3.4 μM), abcA promoter (Kd ~ 11.3 ± 4 μM) and mgrA promoter (Kd ~ 15.5 ± 2.7 μM). However, in presence of montelukast MgrA showed strong binding to norB promoter (Kd ~ 1.7 ± 0.4 μM) and mgrA promoter (Kd ~ 5.8 ± 1.7 μM) having non-significant effect on norA promoter (Kd ~ 9.2 ± 4.2 μM) and abcA promoter (Kd ~ 14.5 ± 7 μM).

The phosphorylation of MgrA in S. aureus typically happens when it is in a dimeric state. Hence, we performed a western blot analysis to quantify the proportions of dimeric MgrA, monomeric MgrA, and phosphorylated MgrA in the absence and presence of 6 μg/ml (10 μM) montelukast. This was achieved by treating the MgrA protein with cell lysate, as shown in Figure 5A & 5B. The analysis revealed that the proportions of dimeric MgrA and phosphorylated MgrA were elevated in MgrA treated with cell lysate, as compared to untreated MgrA protein. In addition, the presence of montelukast further elevated the levels of phosphorylated MgrA, suggesting that montelukast treatment promotes the phosphorylation of MgrA.

The study demonstrates that montelukast is an inhibitor of efflux pumps in S. aureus. Additionally, montelukast synergistically with moxifloxacin, enhancing moxifloxacin's effectiveness in treating MRSA infection. Montelukast enhanced the effectiveness of moxifloxacin by inhibiting the norB efflux pump, which effluxed out moxifloxacin. Ultimately, the study demonstrates that montelukast administration increases the phosphorylation of MgrA, resulting in an enhanced affinity of MgrA for the norB promoter. This, in turn, leads to stronger binding of MgrA to the promoter, resulting in increased suppression of the norB efflux pump gene.

As shown in Figure 5 there is illustrated montelukast increase dimerization and phosphorylation of MgrA: Panel A shows western blot of increase in dimeric MgrA and phosphorylated MgrA proportion in bacterial cell lysate (20 μl) treatment of purified MgrA in presence of montelukast (40 μM) (lane 4) in comparison to untreated purified MgrA in absence (lane 1) and presence of cell lysate (lane 3). Lane 2 shows only bacterial cell lysate control. Panel B shows bar plot of relative percentage integrated density showing proportion of monomeric, dimeric and phosphorylated MgrA in different conditions. The relative percentage integrated density was measured from western blot using ImageJ software. Mont: Montelukast.

Inventive step

• A formulation was characterised to inhibit the growth of multidrug resistance S. aureus.

• Montelukast was characterised as an inhibitor of efflux pumps in multidrug-resistant staphylococcus aureus.

• Montelukast was found to improve the efficacy of norfloxacin, moxifloxacin, and cefotaxime in killing MRSA, when formulated in the ratio of 1:4, 4:1, and 1:2 ratios respectively in treating multidrug-resistant staphylococcus aureus infection, both in vitro and in vivo.

• Montelukast was found to strongly interact with MgrA, increase MgrA phosphorylation and MgrA’s affinity for the norB promoter, repressing norB expression and making moxifloxacin more effective in treating multidrug-resistant staphylococcal infection.
Advantages:
So far, no efflux pump inhibitor has been cleared for clinical usage. The current research found montelukast, an efflux pump inhibitor that lowers the operating and capital costs associated with developing such drugs, by screening an FDA-approved drug library. Additionally, it explains montelukast, generally used for the treatment of asthma, having no antibacterial properties, enhances the effectiveness of moxifloxacin in treating MRSA when taken in combination, in both in vitro and in vivo. Montelukast is an FDA-authorized medicine with well-established pharmacokinetic and pharmacodynamics features. This ensures that there are no concerns regarding adverse drug reactions or high levels of toxicity in the body.

While the present disclosure has been described with reference to certain embodiments and exemplary embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the present disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present disclosure without departing from its scope.
, Claims: