WO2023053144A1 - Composition synergique de malabaricone b et d'antibiotiques classiques contre des infections par staphylococcus aureus multirésistant aux médicaments - Google Patents

Composition synergique de malabaricone b et d'antibiotiques classiques contre des infections par staphylococcus aureus multirésistant aux médicaments Download PDF

Info

Publication number
WO2023053144A1
WO2023053144A1 PCT/IN2022/050875 IN2022050875W WO2023053144A1 WO 2023053144 A1 WO2023053144 A1 WO 2023053144A1 IN 2022050875 W IN2022050875 W IN 2022050875W WO 2023053144 A1 WO2023053144 A1 WO 2023053144A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
composition
malabaricone
aureus
gentamycin
Prior art date
Application number
PCT/IN2022/050875
Other languages
English (en)
Inventor
Radhakrishnan KOKKUVAYIL VASU
Neethu SIVADAS
Sidharth Chopra
Grace Kaul
Manjulika Shukla
Mathew DAN
Govind Murugan GOVINDAKURUP
Original Assignee
Council Of Scientific And Industrial Research An Indian Registered Body Incorporated Under The Regn. Of Soc. Act (Act Xxi Of 1860)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Council Of Scientific And Industrial Research An Indian Registered Body Incorporated Under The Regn. Of Soc. Act (Act Xxi Of 1860) filed Critical Council Of Scientific And Industrial Research An Indian Registered Body Incorporated Under The Regn. Of Soc. Act (Act Xxi Of 1860)
Publication of WO2023053144A1 publication Critical patent/WO2023053144A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

Definitions

  • the present invention relates to the utilization of a naturally abundant secondary metabolite in combination with conventional antibiotics in order to synergize its efficacy against multi drug resistant Staphylococcus infections.
  • the present invention particularly relates Malabaricone B in minimal dosage that can effectively synergize with the conventional antibiotic gentamycin. More particularly the present invention relates a composition of Malabaricone B with the conventional antibiotic gentamycin.
  • Herbal based drugs are of tremendous importance in treatment of various chronic diseases. Natural products -based antibiotics have long back history starting from the discovery of penicillin itself. Indeed, it has been amply documented that natural products are the progenitors of almost all the antibiotics in clinical utilization. Owing to their enormous potential, fewer side effects, and costeffectiveness herbal drugs gathered much attention among research. Antibiotic resistance amongst various strains of bacteria has currently emerged as a looming threat. In the case of infectious diseases, bacterial resistance over conventional antibiotics has become a challenging issue while the treatment. The scary numbers and emergence of several antibiotic-resistant superbugs in recent years demanded the development of new classes of effective therapeutic agents. In this context exploration of new antibacterial agents from natural resources having the ability to compete the multi-drug resistance has become more prominent.
  • the compounds including Myristic acid, a-monomyristylglyceride and Malabaricone C were isolated from nutmeg reported to have antimicrobial activity against upper airway respiratory tract bacteria, such as Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis.
  • the antibacterial activity of a series of fatty acids and a-monoacylglycerides with different carbon chain lengths against these bacteria was also measured and the result showed the high inhibitory activity of tridecanoic and tetradecanoic acids (Myristic acid), a- monododecanoylglyceride and a-monotetradecanoylglyceride (Tanaka, Yasuo.; Fukuda, Seiji.; Kikuzaki, Hiroe.; Nakatani, Nobuj. Antibacterial compounds from nutmeg against upper airway respiratory tract bacteria. ITE Letters on Batteries, New Technologies & Medicine (2000), 1(3), 412-417.).
  • Sen, et al reported the anti- promastigote activity of different extracts/fractions of M. malabarica and its constituent diarylnonanoids against Leishmania donovani promastigotes (MHOM/IN/83/AG83) using the MTS-PMS assay.
  • Preliminary screening revealed that methanol extract had potent leishmanicidal activity (ICso 31.O pg/mL).
  • Methanol extract afforded four diarylnonanoids (Malabaricones A-D, designated as Mai A, Mai B, Mai C and Mai D, respectively).
  • the IC50 values of Mai A-D were 16, 22, 27 and >50 pg/mL, respectively.
  • the result suggested that methanol extract of M.
  • malabarica especially its constituent compounds, Mai A and Mai B, have promising antileishmamal activity (Rupashree, Sen.; Baun, A. K.; Subrata, Chattopadhyay.; Mitali, Chatterjee. Antipromastigote activity of the malabaricones of Myristicamalabarica (Rampatri). Phytotherapy Research. 2007, 27(6), 592-5). Shafiei, et.al. described the antibacterial activities of ethyl acetate and ethanol extracts of flesh, seed, and mace of Myristica fragrans .
  • the bactericidal potential against three gram-positive cariogenic bacteria (Streptococcus mutans ATCC 25175, Streptococcus mitis ATCC 6249, and Streptococcus salivarius ATCC 13419) and three gram-negative periodontopathic bacteria (Aggregatibacter actinomycetemcomitans ATCC 29522, Porphyromonas gingivalis ATCC 33277, and Fusobacterium nucleatum ATCC 25586) were determined. Antibacterial activities of the extracts were tested by two-fold serial micro dilution, with minimum inhibitory concentrations (MIC).
  • MIC minimum inhibitory concentrations
  • MBC minimum bactericidal concentration
  • Sialidases are key virulence factors that remove sialic acid from host cell surface glycans, thus unmasking receptors to facilitate bacterial adherence and colonization. Isolation and characterization of novel inhibitors of the Streptococcus pneumoniae sialidases NanA, NanB, and NanC from Myristica fragrans seeds were reported. Of the isolated compounds Malabaricone C showed the most pneumococcal sialidases inhibition. These results suggested that Malabaricone C and neolignans could be potential agents for combating S.
  • JP 3886547B2 disclosed that a spice and an extract of a spice has wide range of antibacterial activities against alimentary toxicosis pathogens, especially clove, cinnamon or oregano or its extract has antibacterial activities against alimentary pathogens.
  • JPH11335274A disclosed the argingipain inhibitory activity of Malabaricone C isolated from nutmeg against gram-negative anaerobic bacteria Porphyromonas gingivalis.
  • WO1999056567 disclosed the antibacterial activity of different composition of nutmeg oleoresin isolated from M. fragrans.
  • KR100858196B1 has disclosed antibacterial activity of nutmeg extract measured as growth inhibition zone of Propionibacterium acnes.
  • US 20090192217 has disclosed the antibacterial activity of lignan compounds isolated from M. fragrans against the acne-causing bacteria, and acne treatment agents.
  • a primary objective of the present application to find a treatment of infectious diseases caused by the multi-drug resistance S. aureus (ATCC 29213 strain).
  • Another object of the invention is to provide an efficient scaffold from natural origin for antibacterial drug development.
  • Another objective or the present application is to use Malabaricones; a naturally obtained phenylacylphenol from the species of Myristicaceae family endowed with excellent antibacterial activity and to synergize it with the conventional antibiotic gentamycin, to be used for the treatment of infectious diseases caused by the multi-drug resistance S. aureus (ATCC 29213 strain).
  • Yet another objective of the present application is to provide a synergistic combination of a naturally abundant secondary metabolites and conventional antibiotics to fight the challenges associated with the treatment of infectious diseases.
  • One other objective of the present application is to provide a synergistic composition of Malabaricone B and conventional antibiotics against multidrug resistant-staphylococcus aureus infections.
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 and
  • the ratio of Malabaricone B to gentamycin is 1:0.5.
  • the Malabaricone B a phenylacylphenol is extracted from fruit rinds of the species Myristica malabarica.
  • the MIC of the composition is 0.25 pg/mL.
  • the composition is effective against multidrug-resistant strains of staphylococci, selected from methicillin- sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA).
  • MSSA methicillin- sensitive S. aureus
  • MRSA methicillin-resistant S. aureus
  • VRSA vancomycin-resistant S. aureus
  • composition shows -92% reduction in biofilm, which is comparable to levofloxacin at 10X MIC.
  • the present invention also provides a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, synergistically effective amounts of Malabaricone B of Formula 1 and antibacterial drug selected from gentamycin
  • the pharmaceutically acceptable excipient is selected from the group consisting of gelatin, stearic acid, parabens and stabilizers like sorbitol and liquid non-volatile non-aqueous materials predominantly glycol such as propylene glycol.
  • Figure 1 illustrates the schematic representation of extraction and isolation procedure of the disclosed molecule in accordance with an embodiment of the present disclosure.
  • Figure 2 illustrates the time kill kinetics of the disclosed compound against S.aureus ATCC 29213 in accordance with an embodiment of the present disclosure.
  • Figure 3 illustrates the time kill kinetics of the disclosed compound in combination with gentamycin against S.aureus ATCC 29213 in accordance with an embodiment of the present disclosure.
  • Figure 4 illustrates the biofilm inhibition assay of the compound with standard drugs vancomycin and levofloxacin in accordance with an embodiment of the present disclosure.
  • Figure 5 illustrates the biofilm inhibition assay of the combination (Compound + Gentamycin) in accordance with an embodiment of the present disclosure.
  • Figure 6 illustrates the induced resistant mutant generation studies of the disclosed compound in accordance with an embodiment of the present disclosure.
  • Figure 7 illustrates the intracellular killing assay of the disclosed molecule against S. aureus ATCC 29213 in accordance with an embodiment of the present disclosure.
  • Figure 8 illustrates the in vivo animal efficacy of the disclosed molecule in neutropenic thigh infection model and /or skin infection model in accordance with an embodiment of the present disclosure.
  • the present invention provides a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 and antibacterial drug gentamycin.
  • MDR multidrug resistant
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 and antibacterial drug gentamycin wherein the ratio of Malabaricone B to gentamycin is 1:0.5.
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 a phenylacylphenol is extracted from the fruit rinds of the species Myristica malabarica.
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 wherein the MIC of the composition is 0.25 pg/mL.
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 is effective against multidrug-resistant strains of staphylococci, selected from methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA).
  • MSSA methicillin-sensitive S. aureus
  • MRSA methicillin-resistant S. aureus
  • VRSA vancomycin-resistant S. aureus
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 shows -92% reduction in biofilm, this was comparable to levofloxacin at 10X MIC.
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 further provides a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, synergistically effective amounts of Malabaricone B of Formula 1 and antibacterial drug selected from gentamycin
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 further provides the pharmaceutically acceptable excipient selected from the group consisting of gelatin, stearic acid, parabens and stabilizers like sorbitol and liquid non-volatile nonaqueous materials predominantly glycol such as propylene glycol.
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, Malabaricone B of Formula 1 and antibacterial drug gentamycin with a pharmaceutically acceptable excipient selected from the group consisting of gelatin, stearic acid, parabens and stabilizers like sorbitol and liquid non-volatile non-aqueous materials predominantly glycol such as propylene glycol.
  • composition comprises of a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, synergistically effective amounts of Malabaricone B of Formula 1
  • the present invention relates to Malabaricone, a naturally obtained phenylacylphenol from the species of myristicaceae family endowed with excellent antibacterial activity and synergize with the conventional antibiotic gentamycin, to be used for the treatment of infectious diseases caused by the multi-drug resistant S. aureus (ATCC 29213 strain).
  • the invention provides a synergistic combination of a naturally abundant secondary metabolites and conventional antibiotics to fight the challenges associated with the treatment of infectious diseases.
  • the invention provides an efficient scaffold from natural origin for antibacterial drug development.
  • aureus multi-drug resistant clinical isolates MRSA A, VRSA and VSSA
  • Enterococcus panel VSE and VRE
  • the invention discloses the important in- vitro and in -vivo screening techniques of the compound against MDR S. aureus clinical isolates.
  • the invention provides a highly active scaffold for the development of a cost -effective antibacterial drug for efficient management of infections of MDR strains of S. aureus which are generally resistance to existing antibiotic classes.
  • Vancomycin remains one of the first-line drugs for the treatment of MDR S. aureus infections however in recent years S. aureus isolates with complete resistance to vancomycin have emerged.
  • the antibacterial efficacy of the disclosed compound was comparable to vancomycin and in some aspects, it was more efficient than vancomycin. So, the invention in detail provides an alternative and efficient solution to address the issue related to antibiotic resistance which is of utmost importance.
  • the first aspect of the invention intends to disclose the isolation of Malabaricone B (NS-7) from the species M. malabarica.
  • Fruit rinds of Myristica malabarica (670 g) were collected on April 2017 from medicinal garden sites of Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI), Palode, Thiruvananthapuram District, Huawei, India.
  • JNTBGRI Jawaharlal Nehru Tropical Botanic Garden and Research Institute
  • the plant material was authenticated by Plant Genetic Resource Division KSCSTE-JNTBGRI and a voucher specimen [Myristica malabarica, Medicinal Garden site, Palode, Trivandrum, Huawei, April 2017, Govind, 83442 (TBGT)] was deposited in the herbarium of the institute. Dichloromethane extract of the dried material after successive column chromatographic separation technique afforded the desired compound.
  • Second aspect of the invention discloses the detailed in- vitro and in- vivo antibacterial screening of the compound Malabaricone B as well as its synergistic combination (Malabaricone B NS-7 + Gentamycin).
  • MDR multidrug resistant S. aureus infections
  • composition as disclosed herein, wherein the ratio of Malabaricone B to gentamycin is 1:0.5.
  • composition as disclosed herein, wherein Malabaricone B is a phenylacylphenol extracted from fruit rinds of the species Myristica malabarica.
  • compositions as disclosed herein wherein the MIC of the composition against the multidrug resistant (MDR) S. aureus is 0.25 pg/mL.
  • compositions as disclosed herein wherein the composition is effective against multidrug-resistant strains of staphylococci, selected from methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA) or vancomycin-resistant S. aureus (VRSA).
  • MSSA methicillin-sensitive S. aureus
  • MRSA methicillin-resistant S. aureus
  • VRSA vancomycin-resistant S. aureus
  • composition as disclosed herein, wherein the composition exhibits a concentration dependent bactericidal activity.
  • composition as disclosed herein, wherein the composition shows -92% reduction in biofilm, which was comparable to levofloxacin at 10X MIC.
  • a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, synergistically effective amounts of Malabaricone B of Formula 1 and antibacterial drug gentamycin
  • a synergistic antibacterial composition as disclosed herein, wherein the pharmaceutically acceptable excipient is selected from the group consisting of gelatin, stearic acid, parabens and stabilizers like sorbitol and liquid non-volatile non-aqueous propylene glycol.
  • a process for preparing a synergistic antibacterial composition for multidrug resistant (MDR) S. aureus infections comprising, mixing effective amounts of
  • Antibacterial susceptibility testing was carried out by broth micro dilution assay as per Clinical & Laboratory Standards Institute (CLSI) guidelines.
  • MIC Minimum inhibitory concentration
  • the results were summarized in Table 1.
  • Malabaricone B exhibited potent and selective activity against S. aureus ATCC 29213 among the various tested strains with an MIC value of 0.5 pg/ml and the compound was found to be inactive against other bacterial strains. Further activity studies of the compound were mainly focused on the ATCC 29213 strains of S. aureus.
  • MSSA methicillin-sensitive S. aureus
  • MRSA methicillin- resistant S. aureus
  • VRSA vancomycin-resistant S. aureus
  • Levofloxacin, Meropenem, Methicillin and Vancomycin were used as reference standards. In each strain two replicate tests were done (represented by I & II). Activity of the compound is found to be slightly higher than that of vancomycin against MRSA & VRSA panel.
  • VRSA vancomycin-resistant S. aureus
  • MIC of compound against Enterococcus panel including VSE & VRE was determined. MIC value of the disclosed compound is found to be slightly higher than that of vancomycin against Enterococcus panel which indicate that the compound is very active against this panel.
  • Compound was screened to determine the synergy between FDA approved antibiotics commonly used for the treatment of MDR staphylococcus infections which include Ceftazidime, Gentamycin, Levofloxacin, Linezolid, Meropenem, Minocycline, Rifampicin and Vancomycin. Checkerboard method was used to determine synergy between compound and the antibiotics. According to the recommendations of CLSI, serial two-fold dilutions of each drug to at least double the MIC were freshly prepared prior to testing. MIC of the drug alone and the combination with gentamycin (1:0.5) was determined.
  • XFIC fractional inhibitory concentrations
  • XFIC FIC A + FIC B
  • FIC A is the MIC of drug A in the combination/MIC of drug A alone
  • FIC B is the MIC of drug B in the combination/MIC of drug B alone.
  • the combination was considered synergistic when the XFIC ⁇ 0.5, indifferent when the XFIC > 0.5 to 4, and antagonistic when the XFIC > 4.
  • Obtained results from the synergy studies were summarized in Table 8.
  • Compound showed potent antibacterial efficacy with an MIC value in the range of 1-2 against S. aureus ATCC 29213 strains.
  • compound synergized with gentamycin showed an enhanced activity against S.
  • aureus ATCC 29213 strains Compound exhibited an FIC value of 0.265 with gentamycin indicating synergistic interaction. The result was impressive and indicates antibacterial potential of the disclosed compound can be effectively utilized in combination with existing drugs to achieve enhanced activity for the treatment of MDR staphylococcus infections.
  • Microbial biofilm has a primary role in the pathogenesis of diseases and in the attachment of multicellular organisms to a fouled surface and is responsible for antimicrobial resistance.
  • Compound was tested along with drug to determine the anti-biofilm activity.
  • FIG 4 illustrates the biofilm inhibition assay of the compound with standard drugs vancomycin and levofloxacin.
  • Activity of Malabaricone B (NS- 7) was found to comparable with standard drug vancomycin in biofilm inhibition assay.
  • FIG 5 illustrates the biofilm inhibition assay of the combination (Compound + gentamycin). The results showed that at IX MIC caused -92% reduction in biofilm, which was comparable to levofloxacin 10X. It proved that the combination in lower concentration was found to be more effective in inhibiting the bacterial biofilm formation than the individual drugs at higher concentration. So, the conditions of severe S. aureus infections can be treated with this particular combination more efficiently.
  • the propensity of bacterial resistance over the disclosed compound was evaluated using serial exposure of the compound against bacteria. Resistance studies with the disclosed compound and levofloxacin against S. aureus ATCC 29213 after the number of serial passages indicated that the compound is active against the gram -positive bacteria and the bacteria have developed no resistance over the compound. FIG 6 illustrates the induced resistant mutant generation studies of the disclosed compound. The propensity to generate resistance in compound was lower as compared to levofloxacin.
  • Intracellular killing assay of the compound was performed to find out its ability to kill the intracellular S. aureus ATCC 29213. Vancomycin and levofloxacin were used as the positive control. Macrophages are placed in a culture with the bacteria.
  • FIG 7 illustrates the intracellular killing assay of the disclosed molecule against S. aureus ATCC 29213. After 30 minutes the remaining bacteria were killed with a standard antibiotic and the phagocytes werestained and examined for number of bacteria thathave killed. Compound was found to be effective against bacteria inside macrophages. 14. In vitro post antibiotic effect (PAE) of the compound
  • Post-antibiotic effect was a well-established pharmacodynamics parameter that reflects an arrested bacterial growth following the removal of the active antibacterial agent from the growth medium.
  • the clinical relevance of the PAE is probably most important when designing dosage regimens. It allows determining the dosing schedule of antibiotics.
  • Post antibiotic effect of the compound at different concentration against S. aureus 29213 was carried out. The results revealed that the compound suppress the bacterial growth in the administration of less dosage as compared to levofloxacin and vancomycin. Short exposure was needed for better activity. TABLE ll In vitro post antibiotic effect of the compound in different concentration
  • FIG 8 illustrates the in- vivo animal efficacy of the disclosed molecule in neutropenic thigh infection model and /or skin infection model.
  • Compound exhibited potent in- vitro antimicrobial activity against drug-resistant isolates, its efficacy in in- vivo was then determined in a murine neutropenic thigh infection model. The results showed that effectiveness of compound in reducing the bacterial load in infected mice was comparable to that of vancomycin.
  • the in- vivo efficacy data of NS-7 shows good animal efficacy at 50 mg/kg as compared to vancomycin at 25 mg/kg.
  • EXAMPLE-2 Isolation and characterization of Malabaricone B from the DCM extract of the rind.
  • the compound was screened for its antibacterial potential against ESKAP pathogen panel utilizing CLSI guidelines.
  • the panel consists of E. coli, S. aureus, K. pneumonia, P. aeruginosa and A. baumannii.
  • the bacterial cultures were inoculated in Muller-Hinton broth.
  • Optical density (OD) of the cultures were measured at the wavelength of 600 nm followed by dilution for -104 cfu/mL.
  • Working stock solutions of concentration 2.56 mg/ml were prepared for different antibiotics using DMSO from Stock solution of 10 mg/ml.
  • Working stock solutions were diluted further for differential dilutions of 64-0.5 pg/ml.
  • a cytotoxicity assay was performed as per published protocols.
  • Vero African green monkey kidney cells were seeded in a 96-well plate and grown for 24 hours at 37°C in a humidified atmosphere of 5% CO2 and 95% air in DMEM medium supplemented with 10% fetal bovine serum, 1.5 g Sodium bicarbonate/L, 100 pg/mL of Penicillin and 10 pg/mL of Streptomycin. The next day different concentrations of the compound were added in the media in triplicate.
  • MTT ((3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide)] was added to a final concentration of 1 mg/mL for 2 hrs.
  • the media was removed by gently inverting the plate.
  • DMSO was added to the wells to solubilize the formazan crystals and absorbance was read at 595 nm in a plate reader. The absorbance of treated vs. untreated cells were compared. The % Survival vs. Concentration of compound was plotted to determine the CC50. The selectivity index is calculated as CC50/MIC.
  • Doxorubicin was used as positive control and each experiment was repeated in triplicate.
  • the combination was considered synergistic when the XFIC ⁇ 0.5, indifferent when the XFIC > 0.5 to 4, and antagonistic when the XFIC > 4.
  • aureus ATCC 29213 strains was cultured overnight in TSB supplemented with 1% glucose on rotary shaker at 37°C, 180 rpm. The culture was diluted in the ratio 1:100 and diluted culture (0.2 mL/well) was transferred into 96 well flat bottom tissue culture plates. The plates were covered with adhesive lid to maintain low oxygen conditions thereby increase biofilm formation and were incubated for 48 hrs. at 37°C. Subsequently, the media was decanted, plates were rinsed gently thrice with lx PBS (pH 7.2) and the planktonic bacteria was removed. The plates were refilled with TSB containing different concentrations of drug/drug combination and incubated for 24 hrs. at 37°C.
  • Post drug treatment media was once again decanted, and wells were rinsed thrice with PBS.
  • the plates were incubated at 60°C for 1 hr. for biofilm fixation and stained by 0.06% crystal violet for 10 mins.
  • Wells were rinsed with PBS and biofilm bound crystal violet was eluted by 30% acetic acid (0.2 mL each) and quantified by measuring the absorbance at 600 nm.
  • serial passaging was initiated by harvesting bacterial cells growing at the highest concentration of the compound with one-half of the MIC and inoculating into fresh media. This inoculum was subjected to another MIC assay. After 24 hrs. of incubation period, the cells growing in the highest concentration of the compound from the previous passage were once again harvested and assayed for the MIC. The process was repeated for 35 passages. The MIC value of the compound was plotted against the number of passages and the fold increase in MIC was determined.
  • PAE post antibiotic effect
  • EXAMPLE- 10 In vivo efficacy in neutropenic thigh infection model and /or skin infection model
  • mice of same age and same weight were grouped into 3 animals/per cage and will be marked on the tail.
  • Mice were injected IP with 1 mL syringe with 26G needles containing 100 pL of Cyclophosphamide at a dose of 150 mg/Kg and 100 mg/kg, 24 hr. and 1 hr. respectively before the introduction of infection to induce neutropenia.
  • Mid log phase bacteria (10 7 -10 9 ) in PBS was injected into the thigh IM. After 3 & 6 hours post bacterial infection, the test compound -200 pL (200 mg/kg) per mice was administered IP. Untreated control animal was sacrificed after 2 hours of the inoculation of bacteria to confirm establishment of infection. Following the sacrifice, thigh will be removed, homogenized individually in 5 ml of PBS, serially diluted and plated on the Mueller- Hinton agar plates for CFU count.
  • composition of Malabaricone B and gentamycin was prepared by mixing the compound and the drug in the ratio 1:0.5 along with pharmaceutical excipients selected from agents like gelatin, stearic acid, parabens and stabilizers like sorbitol and liquid non-volatile non-aqueous materials predominantly glycol such as propylene glycol.
  • the main advantages of the present invention include the development of a novel antibacterial agent from natural origin against MDR-SA infection.
  • Clinical relevance of the disclosed molecule is an added advantage in terms of less dosage administration and short exposure time for excellent activity compared to existing antibiotics. Side effects and further health complications associated with conventional antibiotics can be reduced using a naturally derived scaffold.
  • Synergy with the existing antibiotic Gentamycin and the enhanced activity of the disclosed molecule is found to solve the problem of antimicrobial resistance over different class of antibiotics.
  • the disclosed compound as well as the combination (NS-7 + Gentamycin) exhibits excellent activity against MDR strains of S. aureus and Enterococcus panel which is interesting and suggest the potential utility of the compound to introduce a new mechanism of action to cure the multidrug resistant S. aureus infections. Effective utilization of a naturally abundant molecule for the antibacterial drug development provides a cost-effective technique.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Communicable Diseases (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

La présente invention concerne une combinaison synergique d'un nouvel agent antibactérien malabaricone B dérivé naturellement de formule 1 à partir de l'espèce de Myristica malabarica et de la gentamycine antibiotique classique approuvée par la FDA pour l'activité améliorée contre les superbactéries résistantes aux antimicrobiens. La présente invention concerne également le criblage détaillé in vitro et in vivo du composé dans diverses souches bactériennes à Gram positif et à Gram négatif, y compris la MDR et explique bien l'efficacité antibactérienne du composé correspondant. Des études in vivo montrent le fait que le composé est aussi efficace que le médicament existant vancomycine. La pertinence clinique de la molécule décrite est un avantage ajouté en termes d'administration de dose inférieure et de temps d'exposition court pour une excellente activité par comparaison avec des antibiotiques existants. En particulier, la présente invention concerne un nouveau médicament tête de liste efficace dérivé naturellement pour traiter des infections MDR.
PCT/IN2022/050875 2021-10-01 2022-09-30 Composition synergique de malabaricone b et d'antibiotiques classiques contre des infections par staphylococcus aureus multirésistant aux médicaments WO2023053144A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202111044825 2021-10-01
IN202111044825 2021-10-01

Publications (1)

Publication Number Publication Date
WO2023053144A1 true WO2023053144A1 (fr) 2023-04-06

Family

ID=85780529

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2022/050875 WO2023053144A1 (fr) 2021-10-01 2022-09-30 Composition synergique de malabaricone b et d'antibiotiques classiques contre des infections par staphylococcus aureus multirésistant aux médicaments

Country Status (1)

Country Link
WO (1) WO2023053144A1 (fr)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHAKRABORTY MOUTOSHI, AFRIN TAMANNA, MUNSHI SAURAB KISHORE: "Microbiological quality and antimicrobial potential of extracts of different spices", FOOD RESEARCH, vol. 4, no. 2, 14 October 2019 (2019-10-14), pages 375 - 379, XP093056193, ISSN: 2550-2166, DOI: 10.26656/fr.2017.4(2).303 *
DATABASE TKDL ANONYMOUS : "JĒtĪphalĒdilepaŠ (01), knowledge knows since 200 years", XP093056194 *
DATABASE TKDL ANONYMOUS : "Upadažšaspho°evalepaŠ, knowledge knows since 200 Years", XP093056197 *
THAKARE RITESH; SHUKLA MANJULIKA; KAUL GRACE; DASGUPTA ARUNAVA; CHOPRA SIDHARTH: "Repurposing disulfiram for treatment ofStaphylococcus aureusinfections", INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS, ELSEVIER, AMSTERDAM, NL, vol. 53, no. 6, 1 January 1900 (1900-01-01), AMSTERDAM, NL , pages 709 - 715, XP085697183, ISSN: 0924-8579, DOI: 10.1016/j.ijantimicag.2019.03.024 *

Similar Documents

Publication Publication Date Title
KR102443442B1 (ko) 천연 보존제 및 항미생물제, 및 이들의 조성물
Mazumder et al. Antibacterial potentiality of Mesua ferrea Linn. flowers
Ebere Okwu et al. A novel antimicrobial phenanthrene alkaloid from Bryopyllum pinnatum
Yi et al. Synthesis and antibacterial activities of novel pleuromutilin derivatives with a substituted pyrimidine moiety
Mazlan et al. Synergistic antibacterial activity of mangiferin with antibiotics against Staphylococcus aureus
Kwon et al. Antimicrobial and immunomodulatory effects of Aloe vera peel extract
JPH01500993A (ja) ジスコルハブジン化合物およびそれらの使用方法
Lee et al. Antibacterial and synergistic activity of prenylated chalcone isolated from the roots of Sophora flavescens
Yuan et al. Antibacterial compounds and other constituents of Evernia divaricata (L.) Ach
CN111184866B (zh) 细菌感染的组合治疗
Alimirzaee et al. 1‐methyl malate from Berberis integerrima fruits enhances the antibacterial activity of ampicillin against Staphylococcus aureus
WO2023053144A1 (fr) Composition synergique de malabaricone b et d'antibiotiques classiques contre des infections par staphylococcus aureus multirésistant aux médicaments
CN116617361A (zh) 百里香酚在制备mcr-1酶抑制剂中的应用
Khan et al. In vitro anti-pseudomonal potential of Juglans regia and Otostegia limbata leaves extract against planktonic and biofilm form of Pseudomonas aeruginosa
KR101501346B1 (ko) 왕머루포도나무 뿌리 추출물 및 이의 분획물을 유효성분으로 포함하는 반코마이신 내성 장구균 감염증 예방 또는 치료용 조성물
UGUR et al. Antimicrobial effects of propolis extracts on Escherichia coli and Staphylococcus aureus strains resistant to various antibiotics and some microorganisms
CN114716384A (zh) 一种含有3,4-二氢嘧啶或嘧啶侧链的截短侧耳素类衍生物及其制备与应用
Alkhafaji et al. Antimicrobial Activity of Momordica cochinchinensis Seeds and Seeds Aril Extract
WO2011063615A1 (fr) Amides macrocycliques, compositions pharmaceutiques, leurs procédés de préparation et leurs utilisations
EP3906251A1 (fr) Peptides thiazolyle pour le traitement d'infections mycobactériennes non tuberculeuses
KR20120079455A (ko) 폴리사이클릭 펩타이드 화합물을 포함하는 항균용 조성물 및 이의 생산방법
Ahmed et al. Andrographis paniculata (Burm. f) Wall. ex Ness: a potent antibacterial plant
KR101828023B1 (ko) 시계꽃 추출물을 함유하는 항균용 조성물
Veerappan et al. EVALUATION OF ANTIMICROBIAL POTENTIALS OF Cardiospermum halicacabum Linn.
US20100092581A1 (en) Antibacterial compositions comprising an extract from arceuthobium

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22875348

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE