FIELD OF INVENTION
[001] The present disclosure broadly relates to antimicrobial compounds and particularly refers to a composition comprising a combination of peptides displaying an enhanced antimicrobial activity.
BACKGROUND OF INVENTION
[002] Number of bacterial infections have been treated exclusively by antibiotics However, the antibiotics are unable to provide a desired effect in curtailing the growth of microorganisms due to spread of antibiotic resistance. Antibiotic resistance is defined as the ability of bacteria to survive a concentration of antibiotics that typically inhibits growth of the majority of other bacteria (Russell AD, J Pharm Pharmacology. 2000 Feb; 52(2):227-33). According to the World Health Organisation (WHO), the appearance of antibiotic resistance is classified as one of the biggest threat to human health (World Health Organization (2014), Antimicrobial resistance: global report on surveillance).
[003] The emergence of resistance to multiple antibiotics is common among the microorganisms such as Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis, etc. Among these microorganisms, Staphylococcus aureus (S. aureus) is a major human pathogen that causes a wide range of clinical infections. S. aureus is considered as the pathogen of great concern because of its intrinsic virulence, its ability to cause a diverse array of life-threatening infections, and its capacity to adapt to different environmental conditions. As rapidly as new antibiotics are introduced, Staphylococci have developed efficient mechanisms to neutralize them (Lowy FD J Clin Invest 2003 May 1; 111(9): 1265-1273).
[004] Conventionally, various antimicrobial actives including triclosan have been used for reducing the growth of bacteria. However, triclosan is associated with endocrine disruption and antimicrobial resistance (Foran CM, et alMar Environ Res. 2000 Jul-Dec;50(l-5): 153-6). Therefore, the efficacious use of triclosan is under global regulatory agency's scanner because of safety concern.

[005] Various efforts have been made to provide safe and efficacious broad-spectrum antimicrobial agents. For instance: US5607683A discloses an antimicrobial composition comprising a stabilizing acyclic polyether polymer, silver ion, a stabilizing anion, wherein the antimicrobial composition is useful for medical applications.
[006] US6107261A discloses an antibacterial composition comprising a phenolic antimicrobial agent; a surfactant selected from a group consisting of an anionic surfactant, a cationic surfactant, a non-ionic surfactant, an ampholytic surfactant, and mixtures thereof; a hydrotrope; a water-soluble hydric solvent; water, wherein the antimicrobial agent is present in an amount of at least 40% of saturation concentration, when measured at room temperature.
[007] Although a lot of efforts have been made to provide safe and efficacious antimicrobial agents, there still remains a need in art to provide a composition that provides an enhanced antimicrobial effect.
SUMMARY OF THE INVENTION
[008] In an aspect of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1.
[009] In another aspect of the present disclosure, there is provided a process for preparing a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent, to obtain the composition.
[0010] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and

appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0011] The following drawings form a part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may be
better understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[0012] Figure 1 illustrates the High-Performance Liquid Chromatography (HPLC)
profile of peptide 1 (SEQ ID NO: 1), in accordance with an embodiment of the present
disclosure.
[0013] Figure 2 illustrates the HPLC profile of peptide 2 (SEQ ID NO: 2), in
accordance with an embodiment of the present disclosure.
[0014] Figure 3 illustrates the HPLC profile of peptide 7 (SEQ ID NO: 3), in
accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. Definitions
[0016] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings

recognized and known to those of skill in the art, however, for convenience and
completeness, particular terms and their meanings are set forth below.
[0017] The articles "a", "an" and "the" are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0018] The terms "comprise" and "comprising" are used in the inclusive, open sense,
meaning that additional elements may be included. It is not intended to be construed
as "consists of only".
[0019] Throughout this specification, unless the context requires otherwise the word
"comprise", and variations such as "comprises" and "comprising", will be
understood to imply the inclusion of a stated element or step or group of element or
steps but not the exclusion of any other element or step or group of element or steps.
[0020] The term "including" is used to mean "including but not limited to".
"Including" and "including but not limited to" are used interchangeably.
[0021] For the purposes of the present document, the "antimicrobial efficacy" refers
to the ability of the composition to inhibit or kill the growth of microorganisms,
including but not limited to bacteria, viruses, fungi, parasites and combinations
thereof.
[0022] As used herein, peptide 1 indicates a peptide having an amino acid sequence
as said forth in SEQ ID NO: 1, or derivatives thereof.
[0023] Peptide 2 indicates a peptide having an amino acid sequence as said forth in
SEQ ID NO:2, or derivatives thereof.
[0024] The term "derivative thereof is intended to include any derivative of peptide,
or a combination of peptide with any derivative of peptide, or combinations of
peptides, wherein derivative including but not limited to salts, ethers, amide, acetyl,
myristoyl, palmitoyl, esters, PEGylated.
[0025] As used herein, antimicrobial peptides (AMPs) are expressed as first-line
host defences peptides exhibiting broad spectrum antimicrobial activity. AMPs are
short peptides consisting of amino acids in a range of 8 to 50. AMPs have low
molecular weight, wherein majority of AMPs are usually cationic in nature.
However, there are very few AMPs which are anionic in nature]

[0026] For the purposes of the present document, the term "synergy" is used to describe the interaction or cooperation of two or more peptides to produce a combined effect greater than the sum of their separate effects. [0027] As used herein, aqua, glycerin, propylene glycol, ethylhexyl glycerin refers to the generally known components used in the art.
[0028] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
[0029] Unless defined 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0030] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein. [0031] To address the problem of pathogenic bacteria, various antibiotic therapies, antimicrobial actives and combinations thereof have been utilized. However, they have failed to provide the desired antimicrobial effect. The eradication of bacterial infections has been conventionally done by utilizing high concentrations and/or repeated administrations of the antibiotic drug. However, continuous use of antibiotic drugs at higher concentrations have increased the risk of selection of resistant population amongst the microorganisms. Alternatively, various antimicrobial agents have been deployed to eradicate many infectious diseases and to significantly improve our living environment. However, these antimicrobial

agents used as an alternative to antibiotics exhibit unfavourable properties such as
instability, haemolytic activity, high cost of production.
[0032] To overcome the afore-mentioned problems, the present disclosure provides
a safe and effective composition comprising a combination of peptides working at a
lower concentration range to provide the desired effect. The present disclosure
discloses a composition comprising a peptide 1 having an amino acid sequence as
set forth in SEQ ID NO: 1; and a peptide 2 having an amino acid sequence as set
forth in SEQ ID NO: 2, having a weight ratio in a range of 0.20:1-3:1. When the
composition interacts with the broad spectrum of microorganism, not limited to
bacteria, the composition facilitates and synergistically enhance the overall
antimicrobial efficacy against the microorganism. Due to the different structures
adopted by the peptide 1 and the peptide 2, both of them possess different
mechanisms of action for showing the antimicrobial effect against the
microorganisms.
[0033] Sequences:
[0034] SEQ ID NO: 1 depicts a peptide derived from C-terminal fragment of the
human cathelicidin antimicrobial peptide LL-37.
FKRIVQRIKDFLRNLV
[0035] SEQ ID NO: 2 depicts a hybrid peptide consisting of segment of human beta-
defensin-1 and humanized theta-defensin.
ACPIFTKIQGTYRGKAKRIGRRIC.
[0036] SEQ ID NO: 3 depicts a hybrid peptide.
FCPRRYKQIGTGLPGTKRIGRRIC
[0037] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID
NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as
set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the
peptide 2 has a weight ratio in a range of 0.20:1-3:1. In another embodiment of the
present disclosure, the peptide 1 and the peptide 2 has a weight ratio in a range of
0.25:1-2.75:1. In one another embodiment of the present disclosure, the peptide 1
and the peptide 2 has a weight ratio in a range of 0.25:1-2.5:1.

[0038] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.22:1-3:1.
[0039] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.25:1-2.5:1.
[0040] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, and wherein the peptide 1 has a weight percentage in a range of 0.002% to 0.017% with respect to the composition, and the peptide 2 has a weight percentage in a range of 0.002% to 0.017% with respect to the composition. In another embodiment of the present disclosure, the peptide 1 has a weight percentage in a range of 0.004% to 0.014% with respect to the composition, and the peptide 2 has a weight percentage in a range of 0.008% to 0.012%) with respect to the composition. In one another embodiment of the present disclosure, the peptide 1 has a weight percentage in a range of 0.008%> to 0.012%> with respect to the composition, and the peptide 2 has a weight percentage in a range of 0.004%) to 0.014%) with respect to the composition.
[0041] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.22:1-3:1, and wherein the peptide 1 has a weight percentage in a range of 0.002%> to 0.017%> with respect to the composition,

and the peptide 2 has a weight percentage in a range of 0.002% to 0.017% with respect to the composition.
[0042] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.25:1-2.5:1, and wherein the peptide 1 has a weight percentage in a range of 0.002% to 0.017% with respect to the composition, and the peptide 2 has a weight percentage in a range of 0.002% to 0.017% with respect to the composition.
[0043] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof; and (c) at least one excipient selected from a group consisting of aqua, glycerin, propylene glycol, ethylhexyl glycerine, and combinations thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1.
[0044] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof; and (c) at least one excipient selected from a group consisting of aqua, glycerin, propylene glycol, ethylhexyl glycerine, and combinations thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.22:1-3:1.
[0045] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof; and (c) at least one excipient selected from a group consisting of aqua, glycerin, propylene glycol, ethylhexyl glycerine, and combinations thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.25:1-2.5:1.

[0046] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising:(a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent, to obtain the composition.
[0047] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising:(a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent selected from a group consisting of water, ethyl alcohol, 2-propanol, 1-propanol, and combinations thereof; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent, to obtain the composition. In another embodiment of the present disclosure, the at least one solvent is water. [0048] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising:(a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent is done at a temperature in a range of 20°C to 40°C, to obtain the composition. In another embodiment of the present disclosure, contacting the peptide 1, the peptide 2, and the at least one solvent is done at a temperature in a range of 25°C to 40°C. In one another embodiment of the present disclosure, contacting the peptide 1, the peptide 2, and the at least one solvent is done at a temperature in a range of 26°C to 38°C.

[0049] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising:(a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent, to obtain the composition, and wherein the composition has a pH in a range of 4.5 to 7.5. In another embodiment of the present disclosure, the composition has a pH in a range of 5.0 to 7.0.
[0050] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising:(a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent is done at a temperature in a range of 20°C to 40°C, to obtain the composition, and wherein the composition has a pH in a range of 4.5 to 7.5. [0051] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising:(a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent selected from a group consisting of water, ethyl alcohol, 2-propanol, 1-propanol, and combinations thereof; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent, is done at a temperature in a range of 20°C to 40°C, to obtain the composition, and wherein the composition has apHin a range of 4.5 to 7.5.

[0052] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising:(a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.22:1-3:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent, to obtain the composition.
[0053] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising:(a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.25:1-2.5:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent, to obtain the composition.
[0054] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising:(a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, said process comprising: (i) obtaining a peptide 1; (ii) obtaining a peptide 2; (iii) obtaining at least one solvent; and (iv) contacting the peptide 1, the peptide 2, and the at least one solvent, to obtain the composition, and wherein the peptide 1 has a weight percentage in a range of 0.002% to 0.017% with respect to the composition, and the peptide 2 has a weight percentage in a range of 0.002% to 0.017% with respect to the composition.
[0055] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the

peptide 2 has a weight ratio in a range of 0.20:1-3:1, and wherein the composition is part of at least one substance selected from a group consisting of antibacterial hand wash, disinfectants, antibacterial hand sanitizers, soaps, antibacterial wipes, antibacterial spray, antibacterial hand-cream, antibacterial lotion, and combinations thereof.
[0056] In an embodiment of the present disclosure, there is provided a composition comprising: (a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and (b) a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1, wherein the composition enhances antimicrobial efficacy, and wherein the composition is effective in reducing the growth of at least microorganism selected from a group of bacteria, viruses, fungi, parasites, and combinations thereof. In another embodiment of the present disclosure, the composition is effective in reducing the growth of Gram-positive bacteria. In one another embodiment of the present disclosure, the composition is effective in reducing the growth of Staphylococcus aureus. (S. aureus)
[0057] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the peptide 1 is derived from C-terminal fragment of the human cathelicidin antimicrobial peptide LL-37, and the peptide 2 is a hybrid peptide consisting of segment of human beta-defensin 1 and domain of theta-defensin.
[0058] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.
EXAMPLES
[0059] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as

commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply. The working and non-working examples as depicted in the forthcoming sections highlight the criticality of the working percentages of different components in achieving the synergistic composition of the present disclosure. It is further specified that the peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and the peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, in a range of 0.20:1-3:1, is critical to achieve the synergistic composition in providing an enhanced antimicrobial efficacy. The replacement of any of the components specified above with any other component substantially affects the desired result. Also, deviating from the disclosed ranges does not provide the desired effect.
Materials and Methods
[0060] a) The components - peptide 1 and peptide 2 used for arriving at the final
composition of the present disclosure were custom synthesized from Genscript INC,
USA & USV Private Limited, Mumbai. Peptide 1 (SEQ ID NO: 1) is C-terminal
fragment of LL-37, human cathelicidin antimicrobial peptide. Peptide 1 is 16 amino
acids in length.
[0061] Peptide 2 (SEQ ID NO: 2) is a hybrid peptide composed of human beta-
defensin-1 and humanized theta-defensin, wherein peptide 2 is 24 amino acids in
length. Both peptide 1 and peptide 2 are cationic in nature. The physiochemical
parameters of the afore-mentioned peptides shown in Table 1.

[0062] The HPLC analysis and purification of the peptide 1, peptide 2, and peptide 7 was carried out separately. It was observed that the peptide 1, peptide 2 and peptide 7 were >95% pure. The purity of the peptide 1, peptide 2, and peptide 7 was calculated on the basis of HPLC profile as depicted in Figure 1, Figure 2, and Figure 3.
[0063] b) Different media chemicals such as tryptone soya broth (TSB), tryptone soya agar (TSA) were procured from HiMedia laboratories, Mumbai, India and Mueller-Hinton broth (MH broth) was procured from Sigma Chemicals, Merck. [0064] c) For the purpose of evaluating bactericidal activity of the composition, strain of Gram-positive bacteria, i.e. Staphylococcus aureus (ATCC 6538) was used. [0065] The culture of the bacteria was maintained on Tryptone soya agar (TSA) and was sub-cultured for every two weeks. The glycerol stock was used for maintaining and storing the bacterial culture at -80°C for further use.
EXAMPLE 1
Preparation of the composition having combination of peptide 1 and peptide 2 [0066] The composition of the present disclosure was prepared by obtaining 9.8mg of peptide 1 and 13.13mg of peptide 2. The peptide 1 and peptide 2 was contacted with lOOmL of water at a temperature in a range of 20°C to 40°C, to obtain the composition, wherein the composition has a pH in a range of 4.5 to 7.5. The composition was further aliquoted and stored at -20 °C till further use.

EXAMPLE 2
Preparation of neutralizer and neutralizer solution
a) Neutralizer
[0067] For the purpose of testing the antimicrobial activity of the composition of the present disclosure, neutralizer was prepared by a) obtaining 30g/L of polysorbate 80, 30g/L of saponin and 3g/L of lecithin; b) obtaining 250mM of potassium phosphate buffer having a pH of 7.2; and c) contacting polysorbate80, saponin, lecithin and potassium phosphate buffer, to obtain the neutralizer.
b) Neutralizer solution
[0068] The neutralizer solution was prepared by utilizing sodium phosphate buffer having a pH of 7.4.
[0069] lOOmM of sodium phosphate buffer was prepared by a) obtaining 3.1 gram of NaH2P04.H20 and 10.9 gram of Na2HP04; b) obtaining 1 liter of water; c) contacting NaFfePO^fbO, Na2HP04; and water, to obtain the first mixture; d) final pH of the first mixture was adjusted with NaOH to 7.4.
EXAMPLE 3
Evaluation of the antimicrobial efficacy of the composition
[0070] The contact kill experiment was used to study the antimicrobial activity and/or bactericidal efficacy of the peptide 1, peptide 2, and combinations thereof against a bacterial strain, herein referred to as Staphylococcus aureus ATCC 6538. In the contact kill experiment, dilution-neutralization method was deployed to test the antimicrobial efficacy of the peptide 1, peptide 2, and combinations thereof. The cells of the bacteria were exposed to different concentrations of peptide 1, peptide 2, and combinations thereof for about 1 minute +5 seconds. The control contained no peptide and was treated in identical conditions.
[0071] The contact kill experiment was conducted in temperature bath, wherein the temperature was set at 20°C (Eppendorf Thermomixer comfort). The single colony of bacterium was picked from the TSA plate and grown in MH broth at 37°C.

[0072] The absorbance of bacterial suspension was measured in Thermo Multiskan at 600nm using cuvette (BRAND, Polystyrene 2.5 mL volume cuvette). [0073] Bacterial suspension of about 108 CFU/ml was prepared Different concentrations of each peptide and combinations were prepared in water. Bacterial suspension and peptides were equilibrated at a temperature of 20°C for about 10 minutes.
[0074] For the purpose of the present disclosure, the contact kill experiment was carried out by the following steps: a) 50uL of S. aureus suspension was obtained; b) different concentrations of each peptide and peptide combinations were obtained; c) 50uL of lOOmM of sodium buffer having a pH of 7.4 was obtained; d) different concentration of peptides was contacted with 50uL of lOOmM of sodium buffer, to obtain a first mixture; e) the first mixture was contacted with 50uL of S. aureus suspension for about 1 minute and vortexed, to obtain a second mixture; f) after contact time of 1 minute, 20uL aliquot of second mixture was immediately diluted into 96-well plate containing 180uL of neutralizer solution and left for about 5 minutes; g) the serially diluted aliquots were spotted on TSA plates; h) the TSA plates were incubated at a temperature of 37°C for about 24 hours; and i) the colonies in the plate were counted and fraction of surviving bacteria was calculated. [0075] Activity of the peptides was evaluated by logio reduction factor that was calculated as the difference between logarithms of CFU/ mL before and after exposure to tested concentrations of peptides.
EXAMPLE 4
Antimicrobial efficacy exhibited by the peptide 1 (SEQ ID NO: 1) and peptide
2 (SEQ ID NO: 2)
[0076] The contact kill experiment (as described in example 3) was carried out by
utilizing different concentrations of peptide 1, peptide 2, and combinations thereof.
After 1 minute of contact time, the antimicrobial efficacy of these peptides was tested
individually and in combinations thereof, against Staphylococcus aureus (S. aureus)
ATCC 6538. Table 2 shows the antimicrobial efficacy of peptide 1 represented by
Pepl and peptide 2 represented by Pep 2, and combinations thereof against the

growth of S. aureus. The antimicrobial efficacy is demonstrated by log reduction value(s).
[0077] It can be observed from Table 2, that peptide 1 and peptide 2 when tested individually against S. aureus, displayed increased reduction in the growth of bacteria (S. aureus). The reduction in the growth of bacteria was increased with increasing concentration of the afore-mentioned peptides.
[0078] Further, for the purpose of the present disclosure, when peptide 1 and peptide were allowed to interact at different ratios to form different combinations (peptide 1: peptide 2), i.e. 1(1:1), II (0.5:1), 111(0.25:1), IV (2:1), V (1:1), VI (0.5:1), VII (4:1), VIII (2:1), and IX (1:1). It was observed that the combinations I, II, III, IV, V, VI,

VIII, and IX exhibited synergy in antimicrobial efficacy as compared to the antimicrobial efficacy exhibited by additive effect of individual concentrations of peptide 1 and peptide 2.
[0079] However, it was observed that combination VII did not show any synergy in antimicrobial efficacy. The combined effect of the combination VII showed a log reduction of 3.7 that was less than the log reduction (3.7+0.1=3.8) exhibited by additive effect of the constituents present in said combination. [0080] Therefore, it can be inferred from Table 2 that the combinations I, II, III, IV, V, VI, VIII, and IX present in the disclosed weight ratio range (0.20:1-3:1), exhibiting synergy in antimicrobial efficacy were considered as the working examples of the present disclosure, whereas combination VII (having weight ratio of 4:1) present outside the disclosed weight ratio range that did not show any synergy in terms of log reduction, was considered as the non-working example of the present disclosure.
EXAMPLE 5
Non-Working Examples of the present disclosure
[0081] To test the antimicrobial efficacy, peptide 7 was used in place of peptide 1.
Peptide 7 represented by SEQ ID NO: 3 is a hybrid peptide, having amino acid
sequence "FCPRRYKQIGTGLPGTKRIGRRIC". It is 24 amino acids in length and
has a charge of 7.
[0082] Contact kill experiments were carried out with peptide 2 and peptide 7. The
peptide 2 and peptide 7 were tested at individual concentrations and in combinations
(peptide 2: peptide 7) X, XI, XII, XIII, XIV, and XV against S. aureus for 1 minute,
as given in Table 3

[0083] Referring to Table 3, it can be observed that the peptide 2 and peptide 7 when present in combinations such as X, XI, XII, XIII, XIV, and XV, did not show synergy in antimicrobial efficacy as compared to the antimicrobial efficacy exhibited by tested individual concentrations of peptide 2 and peptide 7. For instance: 12uM of peptide 2 and 48uM of peptide 7 when present individually, showed a log reduction of 0.2 each. However, 2uM of peptide 2 and 48uM of peptide 7 when present in combination X, log reduction of the combination X was 0.4, which was equal to log reduction of said peptides present individually. Therefore, no significant synergy was observed in antimicrobial efficacy of combination X.
[0084] It can be inferred that as none of the combinations of peptide 2 and peptide 7 showed any synergy in antimicrobial efficacy against S. aureus. Therefore, combinations of peptide 2 and peptide 7 were considered as the non-working examples of the present disclosure. Hence, it can be inferred that mere combinations of any peptide do not show any synergistic result, on the other hand, a combination of peptide 1 and peptide 2 within a weight ratio range as disclosed in the present disclosure displays a synergistic effect.
[0085] Overall, the combination of peptide 1 and 2 in the disclosed weight ratio range of 0.20:1-3:1 is essential to have a synergistic composition in providing an enhanced antimicrobial efficacy. Replacing even a single component with another component does not provide the desired effect. Also, deviating from the disclosed weight ranges does not exhibit the desired result.

Advantages of the present disclosure
[0086] The present disclosure discloses a composition comprising: a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof; and a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.20:1-3:1. The composition provides a synergistic antimicrobial effect and broad spectrum activity. Also, the composition works synergistically at lower concentrations to provide antimicrobial efficacy. Therefore, present disclosure disclosed a cost-effective production of the composition. The composition as disclosed herein can be used as an alternative for antibiotics or can be used in conjunction with antibiotics, providing a promising therapy for combating the growth of multiple drug resistant microorganism. Also, the composition can be exploited by incorporating the same in at least one substance selected from a group consisting of antibacterial hand wash, disinfectants, antibacterial hand sanitizers, soaps, antibacterial wipes, antibacterial spray, antibacterial hand-cream, antibacterial lotion, and combinations thereof. Further, as peptides are the active components of the composition, it can be regarded as safe to use as there are no adverse effected associated with it.

I/We Claim:
1. A composition comprising:
a) a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1,
or derivatives thereof; and b)a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2,
or derivatives thereof,
wherein the peptide 1 and the peptide 2 has a weight ratio in a range of
0.20:1-3:1.
2. The composition as claimed in claim 1, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.22:1-3:1.
3. The composition as claimed in claim 1, wherein the peptide 1 and the peptide 2 has a weight ratio in a range of 0.25:1-2.5:1.
4. The composition as claimed in claim 1, wherein the peptide 1 has a weight percentage in a range of 0.002% to 0.017% with respect to the composition, and the peptide 2 has a weight percentage in a range of 0.002% to 0.017% with respect to the composition.
5. The composition as claimed in claim 1, wherein the composition further
comprises at least one excipient selected from a group consisting of aqua,
glycerin, propylene glycol, ethylhexyl glycerine, and combinations thereof.
6. A process for preparing the composition as claimed in claim 1, said process
comprising:
a) obtaining a peptide 1 having an amino acid sequence as set forth in SEQ ID NO: 1, or derivatives thereof;
b) obtaining a peptide 2 having an amino acid sequence as set forth in SEQ ID NO: 2, or derivatives thereof;
c) obtaining at least one solvent; and
d) contacting the peptide 1, the peptide 2, and the at least one solvent, to obtain the composition.
7. The process as claimed in claim 6, wherein the at least one solvent is selected
from a group consisting of water, ethyl alcohol, 2-propanol, 1-propanol, and
combinations thereof.

8. The process as claimed in claim 6, wherein contacting the peptide 1, the peptide 2, and the at least one solvent is done at a temperature in a range of 20°C to 40°C.
9. The composition as claimed in claim 1, wherein the composition has a pH in a range of 4.5 to 7.5.
10. The composition as claimed in claim 1, wherein the composition is part of at least one substance selected from a group consisting of antibacterial hand wash, disinfectants, antibacterial hand sanitizers, soaps, antibacterial wipes, antibacterial spray, antibacterial hand-cream, antibacterial lotion, and combinations thereof.
11. A composition as claimed in any one of the claims 1-5, wherein the composition enhances antimicrobial efficacy.