WO2023047421A1 - Plant-based compositions for inhibiting multi-drug resistance (mdr) and potentiating antibiotics in bacterial pathogens - Google Patents

Abstract

Plant-based compositions for inhibiting multi-drug resistance (MDR) and potentiating antibiotics in bacterial pathogens The present invention is related to a composition comprising plant-based multi-drug resistance inhibitors (MDRi) for potentiating antibiotics against clinically relevant gram-negative and gram-positive pathogens. Further, the present invention provides a pharmaceutical formulation comprising the plant-based MDRi along with pharmaceutically acceptable excipients. Also, the present invention provides method of preparing the composition comprising MDRi. Method of treatment and uses of the composition comprising MDRi has also been provided in the present disclosure.

Description

Plant-based compositions for inhibiting multi-drug resistance (MDR) and potentiating antibiotics in bacterial pathogens

FIELD OF INVENTION

This invention generally relates to drug science. Specifically, the present invention is a formulation of plant-based extracts that potentiate antibiotics to effectively combat clinically relevant multi-drug resistant (MDR) gram-negative and gram-positive bacterial pathogens. The present invention provides a pharmaceutical active formulation of plant-based extracts blended with pharmaceutically acceptable excipients. Also, the present invention provides the method of preparing the plant-based formulation. The method of treatment and uses of the formulation for potentiation of antibiotics is provided in the present disclosure.

BACKGROUND AND PRIOR ART

There has been a dramatic increase in the number of bacterial pathogens with MDR to antibacterial agents in the recent years. The World Health Organization (WHO), Center for Disease Control and Prevention (CDC) in United States and European Center for Disease Prevention and Control (ECDC) consider MDR bacteria to be a major public health concern.

According to a recent UN report in June 2019, antimicrobial resistance is projected to be the leading cause of death by 2050, resulting in 10 million deaths annually. Traditionally, antibiotics such as penicillin, tetracycline, erythromycin, vancomycin are used to treat several infectious bacterial diseases in humans. These medicines become ineffective against the infectious pathogens as the pathogens develop resistance. The pathogens effectively develop mechanisms such as blocking permeation of entry, modifying through enzymes, and efflux pump mechanism etc. against the antibiotics to acquire the drug resistance. The efflux pump mechanism takes the form of a complex protein that spans the cell wall. This protein is programmed to identify an antibiotic and systematically pump it from inside to outside the cell wall of the pathogens. This is called a drug resistance pump. As new antibiotics are developed, the bacterial drug resistance pumps also succeed in ejecting the new drugs, which may be chemically very different from old ones. This ability to eject different antibiotics constitutes the MDR pump. There are five classes of pumps identified so far. These pumps can extrude both first and second-line drugs such as isoniazid, ethambutol, fluoroquinolones, and aminoglycosides.

Plants can produce their own compounds (antimicrobial) to counteract infections on their own. Defense molecules, and not new antibiotics, can disrupt the mechanism of resistance. A potentiating compound can be an enzyme modifier (like P-lactamase inhibitors) or can block the bacterial membrane protein that pumps antibiotics out (MDR or efflux pump). By their action, the potentiators increase the potency of the weak antibiotic by several folds. These compounds are called multi-drug resistance inhibitors (MDRi). MDRi can increase the lifespan of several antibiotics. Many such compounds described in literature are active against gram-positive bacteria, particularly Staphylococcus aureus. It is perhaps the gram-negative species such as Pseudomonas, Escherichia and Acinetobacter that will be the most problematic bacteria to treat.

Beta-lactamases (BLs) are a diverse class of enzymes produced by pathogenic bacteria that break open the beta-lactam ring, inactivating the beta-lactam antibiotic. P-lactamase production is among the most clinically relevant mechanisms of resistance for gram-negative bacterial pathogens.

Based on molecular homology, they are classified as follows: Classes A, C, and D have a serine residue at the active site, whereas class B enzymes have zinc at the active site i.e., metallo-beta- lactamases (MBLs). Class A includes extended- spectrum BLs (ESBLs) and Klebsiella pneumoniae carbapenemases (KPCs), class B includes the MBLs (NDM, IMP, and VIM), class C includes AmpC, and class D includes oxacillinases (OXAs) (World Health Organization, 2020).

The failure to find a potent broad spectrum antibacterial from plants may be because plants use different chemical strategies for controlling infections. Berberine, a plant alkaloid from the Berberis species, increases membrane permeability and intercalates to DNA, thereby killing the pathogen. Due to the presence of MDR pumps in the pathogen, this compound is readily extruded, rendering it ineffective. Berberis possesses another compound, 5-alpha methoxyhydnocarpin, that blocks the MDR pumps of gram-positive bacteria wherein the minimum inhibitory concentration potency of berberine increased to 1000-fold (Stermitz et al., 2000). The efflux of compounds is controlled by efflux pumps coded by genes that are found in chromosomes or plasmids. Multiple types of efflux pumps can be seen in a single organism itself and there are five families of efflux pump proteins discovered. These pumps are chromosomally coded, and the categories are resistance nodulation division (RND) family, major facilitator superfamily (MFS), small multi-drug resistance (SMR) family, multi-drug and toxic compound extrusion (MATE) family and ATP binding cassette (ABC). Comparative genomic studies revealed high degrees of homology between the pump genes (>70%) and amino acid sequences (>80%) of the pump proteins of one such family both within a species and across different species (Piddock, 2006). Each family contains several pumping mechanisms, but these pumps are similar in structure and function. The RND family contains at least four different pumps that expel antibiotics of beta lactam, fluoroquinolones, tetracycline, chloramphenicol etc. and is found in Pseudomonas aeruginosa and Escherichia coli (Srikumar et al., 1998). The homology between the pumps of both species is very high (Nikaido, 1998). MATE MDR efflux pumps have been described in various bacteria that include P. aeruginosa, S. aureus, Haemophilus influenzae, Vibrio cholera etc. Resistance to multi-drug substances in Lactococcus lactis through ABC transporters was reported by van Veen et al. (1996). A study in Saccharomyces cerevisiae revealed the involvement of at least 23 proteins in the MFS of the MDR mechanism (Sa-Correia and Tenreiro, 2002). A great majority of the identified SMR family members are of bacterial origin, although the first archaeal SMR transporter was characterized in 2003 (Ninio and Schuldiner, 2003).

Nosocomial infections in intensive care units of hospitals constitute many of the global MDR cases and this grows day by day in the absence of effective potentiators of antibiotic drugs. A few secondary metabolites from plants have been found to have minimal antibacterial activities but no compound has been identified so far in potentiating any antibiotics.

Considering the above, there remains a need for the provision of novel and effective plant-based solution for P-lactamase and multi-drug resistance_inhibitors through potentiating antibiotics. The present invention aims to provide a formulation of plant-based MDRi for potentiating antibiotics against clinically relevant gram-negative and gram-positive pathogens.

SUMMARY OF THE INVENTION

The present invention is related to a formulation of plant-based MDRi for potentiating antibiotics against clinically relevant gram-negative and gram-positive pathogens. The said plant-based MDRi are extracted from the plants selected from the group consisting of Nigella sativa, Terminalia bellirica, Syzygium aromaticum, Drosera species, Plumbago species, Camellia sinensis and Emblica officinalis. The antibiotics are selected from Amikacin (AMK) from Aminoglycoside group, Azithromycin (AZM) from Macrolide group, Ciprofloxacin (CIP) from Quinolone group, Cotrimoxazole (COT) from combination of two drugs, sulfamethoxazole and trimethoprim, Ceftriaxone (CTR) from Cephalosporin (P-lactam) group and Imipenem IMP from Carbapenem (P-lactam) group.

Further, the present invention provides a pharmaceutical formulation comprising the plant-based MDRi along with pharmaceutically acceptable excipients. Also, the present invention provides method of preparing the formulation comprising MDRi. The method of treatment and uses of the formulation comprising MDRi has also been provided in the present disclosure. BRIEF DESCRIPTION OF FIGURES AND DRAWINGS

The accompanying drawings illustrate some of the embodiments of the present invention and, along with the descriptions, explain the invention. These drawings have been provided by way of illustration and not by way of limitation. The components in the drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of the aspects of the embodiments.

Figure 1 illustrates antibiotic discs with Acinetobacter baumannii.

Figure 2 illustrates plant formulation + antibiotic discs with A. baumannii.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is now described with reference to the tables/figures and specific embodiments, including the best mode contemplated by the inventors for carrying out the invention. This description is not meant to be construed in a limiting sense, as various alternate embodiments of the invention will become apparent to persons skilled in the art, upon reference to the description of the invention. It is therefore contemplated that such alternative embodiments form part of the present invention.

Unless defined otherwise, 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. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Some of the terms are defined briefly here below; the definitions should not be construed in a limiting sense.

The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

The term “plurality” as used herein is defined as “one, or more than one”. Accordingly, the terms “one”, “at least one” would all fall under the definition of “plurality”.

Whether or not a certain feature or element was limited to being used only once, either way it may still be referred to as “one or more features” or “one or more elements” or “at least one feature” or “at least one element”. As used herein, the term "pharmaceutically acceptable carrier vehicle/or excipient" refers to a carrier medium or excipient which does not interfere with the effectiveness of the biological activity of the active ingredients, and which is not toxic to the host at the concentrations at which it is administered. This term includes diluents, binders, fillers, colors, flavors, solvents, polymers, disintegrants, dispersion media, coatings, isotonic agents, absorption delaying agents, and the like. The use of such terms and media for the formulation of pharmaceutically active substances is well-known in the art (for example, "Remington's Pharmaceutical Sciences", E. W. Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

In an aspect of the present invention, a formulation comprising a plant-based MDRi has been provided, wherein said plant-based MDRi are extracted from the plants selected from the group consisting of Nigella sativa, Terminalia bellirica, Syzygium aromaticum, Drosera species, Plumbago species, Camellia sinensis and Emblica officinalis; and wherein said formulation potentiates antibiotics against clinically relevant gram-negative and gram-positive pathogens.

In an embodiment, the ratio between the plant-based formulation and antibiotics ranges from 0.01 : 1 to 1:1.

In another embodiment, said plant-based formulation is quantified with the following concentration of biomarkers: thymoquinone from Nigella sativa in an amount of 0.3%;

95% pure Curcuminoids; a minimum of 50% of tannin from Terminalia bellirica;

10% eugenol from Syzygium aromaticum; naphthoquinone from Drosera species in an amount of 1%; plumbagin from Plumbago species in an amount of 4% ; tannin from Camellia sinensis in an amount of 1%; and ascorbic acid from Emblica officinalis in an amount of 0.4%.

In another embodiment, said antibiotics are selected from Amikacin (AMK) from Aminoglycoside group, Azithromycin (AZM) from Macrolide group, Ciprofloxacin (CIP) from Quinolone group, Cotrimoxazole (COT) from combination of two drugs, sulfamethoxazole and trimethoprim, Ceftriaxone (CTR) from Cephalosporin (0- lactam) group and Imipenem IMP from Carbapenem (0- lactam) group.

In an embodiment, said antibiotics are in concentration range of 10-50pg. In another embodiment, said gram-positive pathogens are selected from Enterococcus faecalis and Staphylococcus aureus.

In another embodiment, said gram-negative pathogens are selected from Acinetobacter baumanii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.

In an embodiment, said formulation can be formulated in the form of liquid or as solid powders or granules, for oral administration.

In another embodiment, said formulation is a sustained release formulation, e.g., for oral administration or for parenteral administration.

In an embodiment, said formulation can be formulated as fixed dose combination or can be given along with the antibiotic as a concurrent, simultaneous, or concomitant administration.

In a second aspect of the present invention a pharmaceutical formulation has been provided which comprises the formulation as claimed in claim 1 and at least one or more pharmaceutical carriers/vehicles/excipients, wherein, when said pharmaceutical formulation is administered to a subject suffering from or susceptible to a microbial infection, the plant-based MDRi efficiently minimizes the ejection of the antibiotic from the outside of the cell wall thereby suppresses antibiotic resistance.

In a third aspect of the present invention a method of preparing the formulation of the present invention has been provided, wherein said method comprises the steps of: extracting plants individually with organic solvent or aqueous or hydro-solvent mixtures under 70°C and concentrated under vacuum to get the desired concentration of active constituents; drying the obtained extracts under vacuum with reduced temperatures ranging from 40-45°C to remove any solvent residue; and blending the dried extract in different combinations, followed by testing the active constituents for efficacy.

In a fourth aspect of the present invention, a method of treating microbial infections by administering an effective amount of pharmaceutical formulation of the present invention in a subject has been provided.

In a fifth aspect of the present invention, use of the formulation of the present invention for treating diseases caused by gram-negative and gram-positive pathogens has been provided. In accordance with the above, the present inventors have screened several plant materials to identify and develop effective MDRi that can efficiently minimize the ejection of the antibiotic from the outside of the cell wall and further potentiate the antibiotic during the course of the treatment.

These MDRi can work as potentiator, efflux pump inhibitor, enzyme modifying inhibitor and can increase membrane permeation in the bacterial pathogens to make the respective antibiotic more effective in culminating the pathogens.

The present disclosure with reference to the following accompanying examples describes the present invention. However, those of skill in the art should, considering the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. It is understood that the examples are provided for the purpose of illustrating the invention only and are not intended to limit the scope of the invention in any way.

EXAMPLES:

Example 1: PLANT-BASED FORMULATION PREPARATION

The following plants are individually extracted with organic solvent or aqueous or hydro-solvent mixtures at 70°C for 4 hours to get the desired concentration of active constituents. The extracts are dried under vacuum with reduced temperatures ranging from 40-45°C to remove any solvent residue and then blended in different combinations and tested for the efficacy.

The raw materials used in the formulation contain the following with percentage of active constituents:

• Nigella sativa (thymoquinone 0.3%)

• Curcuminoids (95% pure)

• Terminalia bellirica (tannin >50%)

• Syzygium aromaticum (eugenol >10%)

• Drosera species (naphthoquinone - 1%)

• Plumbago species (plumbagin - 4%)

• Camellia sinensis (tannin - 1%)

• Emblica officinalis (ascorbic acid - 0.4%)

Species of Drosera not limited to D. peltata, D. indica, D. burmannii, and species of Plumbago not limited to P. zeylanica, P. indica, P. auriculata that are available in India.

There are 250 species of Drosera available globally, and the prominent ones are listed below with their availability in countries:

• Drosera rotundifolia (Australia and South Asia)

• Drosera peltata (India)

• Drosera indica (India)

• Drosera burmannii (Australia and South Asia)

There are many species of the Plumbago genus, and the prominent ones are listed below with their availability in countries:

• Plumbago auriculata Lam. (South Africa, introduced in India)

• Plumbago europaea L. (Mediterranean and Central Asia)

• Plumbago indica L. (Southeast Asia, Indonesia, the Philippines, and southern China)

• Plumbago pulchella Boiss. (Mexico) Plumbago wissii Friedr. (Namibia)

Plumbago zeylanica L. (Australia, Sri Lanka, and India)

Example 2: MULTI-DRUG RESISTANCE STUDY RESULTS

Two studies were conducted to determine the efficacy of the formulation and the results of a combination that show the highest efficacy are given below.

Code of Antibiotic/ Antibiotic discs (6mm) used:

AMK - Amikacin - Aminoglycoside group - 30pg

AZM - Azithromycin - Macrolide group - 30pg

CIP - Ciprofloxacin - Quinolone group - lOpg

COT - Cotrimoxazole - Combination of two drugs, sulfamethoxazole and trimethoprim - 25 pg

CTR - Ceftriaxone - Cephalosporin (P-lactam) group - 10 and 30pg

IMP - Imipenem - Carbapenem (P-lactam) group - lOpg

MDR pathogens used:

Acinetobacter baumanii - Gram-negative

Enterococcus faecalis - Gram-positive

Escherichia coli - Gram-negative

Klebsiella pneumoniae - Gram-negative

Pseudomonas aeruginosa - Gram-negative

Staphylococcus aureus - Gram-positive

2.1: Study 1 - Disc Diffusion Assay

The plant-based formulation is subjected to a disc diffusion assay with and without antibiotics and the results are tabulated in Table 1 below.

+ - Mild efficacy; ++ - Moderate efficacy; +++ - High efficacy

PF - Plant formulation; ND - Not done Table 1:

• Acinetobacter baumannii - Highly effective alone and in combination with all antibiotics

• Enterococcus faecalis - Mildly effective alone, highly effective with Amikacin and moderately effective with Azithromycin, Ceftriaxone, Ciprofloxacin, Cotrimoxazole and Imipenem

• Escherichia coli - Mildly effective

• Klebsiella pneumoniae - Not effective alone, highly effective with Azithromycin, moderately effective with Amikacin, mildly effective with other antibiotics except Ciprofloxacin

• Pseudomonas aeruginosa - Not effective alone, highly effective with Azithromycin and Imipenem, moderately effective with Amikacin, mildly effective with Ceftriaxone,

Ciprofloxacin and Cotrimoxazole

• Staphylococcus aureus - Highly effective alone and with Azithromycin

2.2: Study 2 - MIC (Minimum Inhibitory Concentration) Determination

The MIC of antibiotics of the respective clinical pathogens are listed in the table 2 below:

Table 2:

The MIC of the formulation alone and in combination with antibiotics is in the table 3 below:

Table 3

Claims

We Claim:

1. A formulation comprising plant-based multi-drug resistance inhibitors (MDRis), wherein said plant-based MDRi are extracted from the plants selected from the group consisting of Nigella sativa, Terminalia bellirica, Syzygium aromaticum, Drosera species, Plumbago species, Camellia sinensis and Emblica officinalis; and wherein said formulation potentiates antibiotics against clinically relevant gram-negative and grampositive pathogens.

2. The formulation as claimed in claim 1, wherein said formulation with the quantified biomarkers are present in the following concentration: thymoquinone from Nigella sativa in an amount of 0.3%;

95% pure curcuminoids; a minimum of 50% of tannin from Terminalia bellirica;

10% eugenol from Syzygium aromaticum; naphthoquinone from Drosera species in an amount of 1%; plumbagin from Plumbago species in an amount of 4% ; tannin from Camellia sinensis in an amount of 1%; and ascorbic acid from Emblica officinalis in an amount of 0.4%.

3. The formulation as claimed in claim 1, wherein said antibiotics are selected from

Amikacin from the aminoglycoside group, Azithromycin from the macrolide group, Ciprofloxacin from the quinolone group, Cotrimoxazole from a combination of sulfamethoxazole and trimethoprim, Ceftriaxone from the cephalosporin (P-lactam) group and Imipenem from the Carbapenem (P-lactam) group.

4. The formulation as claimed in claim 3, wherein said antibiotics are in a concentration range of 10-50pg.

5. The formulation as claimed in claim 1, wherein said gram-positive pathogens are selected from Enterococcus faecalis and Staphylococcus aureus.

6. The formulation as claimed in claim 1, wherein said gram-negative pathogens are selected from Acinetobacter baumanii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.

7. The formulation as claimed in claim 1, wherein said formulation can be formulated in the form of liquid or as solid powders or granules, for oral administration.

8. The formulation as claimed in claim 1, wherein said formulation is a sustained release formulation, e.g., for oral administration or for parenteral administration.

9. The formulation as claimed in claim 1, wherein said formulation can be formulated as fixed dose combination or can be given along with the antibiotic as a concurrent, simultaneous, or concomitant administration.

10. A pharmaceutical composition comprising the formulation as claimed in claim 1 and at least one or more pharmaceutical carriers/vehicles/excipients, wherein, when said pharmaceutical composition is administered to a subject suffering from or susceptible to a microbial infection, the plant-based formulation of multi-drug resistance inhibitors either efficiently minimizes the ejection of the antibiotic from the outside of the cell wall thereby suppresses antibiotic resistance or potentiation of antibiotics to surpass enzyme modification by the pathogens.

11. A method of preparing the formulation as claimed in claim 1 , wherein said method comprises the steps of: extracting plants individually with organic solvent or aqueous or hydro-solvent mixtures at 70°C for 4 hours to get the desired concentration of active constituents; drying the obtained extracts under vacuum with reduced temperatures ranging from 40-45°C to remove any solvent residue; and blending the dried extract in different combinations, followed by testing the active constituents for efficacy.

12. A method of treating microbial infections by administering an effective amount of pharmaceutical formulation as claimed in claim 11 in a subject.

13. Use of the formulation as claimed in claim 1, for treating diseases caused by gramnegative and gram-positive pathogens.