Background of the invention The present invention refers to the pharmaceutical field since it provides a composition comprising resveratrol and polymyxins for use as antibacterial agent against polymyxin-resistant Gram-negative pathogens, for the treatment of urinary tract infections (UTI), for use for intestinal decontamination, for use for the treatment of bacterial intestinal infectious disease and for the treatment of lower respiratory tract infections (LRTI) .

State of the art

Resveratrol (3, 5, 4 ' -trihydroxy-trans-stilbene) is a stilbenoid found in numerous plants that has been investigated for potential therapeutic effects in various areas including cancer (Pavan AR, Silva GD, Jornada DH, Chiba DE, Fernandes GF, Man Chin C, Dos Santos JL. 2016. Unraveling the Anticancer effect of curcumin and resveratrol. Nutrients. 8:628), neurodegenerative (Moussa C, Hebron M, Huang X, Ahn J, Rissman RA, Aisen PS, Turner RS . 2017. Resveratrol regulates neuro-inflammation and induces adaptive immunity in Alzheimer's disease. J Neuroinflammation . 14:1) and cardiovascular disorders (Shah A, Quon A, Morton JS, Davidge ST. 2017. Postnatal resveratrol supplementation improves cardiovascular function in male and female intrauterine growth restricted offspring. Physiol Rep .5:2) . Resveratrol is also known to have antiviral (Abba Y, Hassim H, Hamzah H, Noordin MM. 2015. Antiviral activity of resveratrol against human and animal viruses. Adv Virol. http://dx.doi.org/10.1155/2015/184241) and antibacterial activity, for example against Gram-negative bacteria such as Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa , Providencia stuartii , and Enterobacter cloacae, in combination with antibiotics such as chloramphenicol, kanamycin, streptomycin (Seukep JA, Sandjo LP, Ngadjui BT, Kuete V. 2016. Antibacterial and antibiotic-resistance modifying activity of the extracts and compounds from Nauclea pobeguinii against Gram-negative multi-drug resistant phenotypes. BMC Complement Altern Med. 16:193) .

Colistin consists of a cationic cyclic decapeptide linked to a fatty acid chain through an -amide linkage, with a molecular weight of 1750 Da, having bactericidal activity against Gram-negative aerobic bacilli, including Acinetobacter species, P. aeruginosa, Klebsiella species, Enterobacter species, Escherichia coli, Salmonella species, Shigella species, Citrobacter species, Yersinia pseudotuberculosis, Morganella morganii , and Haemophilus influenzae . Unfortunately, Burkholderia cepacia, Proteus species, Providencia species, Serratia species, Edwardsiella species, and Brucella species are all resistant to colistin. Synergistic activity of colistin has been reported with chloramphenicol (Wei WJ, Yang HF. 2017. Synergy against extensively drug-resistant Acinetobacter baumannii in vitro by two old antibiotics: colistin and chloramphenicol. Int J Antimicrob Agents. doi:

10.1016/j . ijantimicag.2016.11.031) , carbapenems, fosfomycin, sulbactam, rifampicin (Justin R. Lenhard, Roger L. Nation, Brian T. Tsuji. 2016. Synergistic combinations of polymyxins. International Journal of Antimicrobial Agents. 48:607-613) .

The increasing prevalence of infections caused by multidrug resistant (MDR) bacterial pathogens has dramatically limited the options for effective antibiotic treatments, especially in the hospital setting. The recent emergence and dissemination of extensively drug resistant (XDR) Gram-negative pathogens including colistin only-susceptible Pseudomonas aeruginosa , carbapenem-resistant Acinetobacter baumannii , and carbapenem- resistant Enterobacteriaceae, has revamped the interest for colistin as one of the few classes of drugs that retain activity against these pathogens (Falagas ME, Kasiakou SK. 2005. Colistin: the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin Infect Dis. 40:1333) . Unfortunately, the increased use of colistin in the clinical practice, both in human and veterinary medicine, and its massive use in animal husbandry, for example as a growth promoter, has led to an increase in colistin resistance among clinically relevant Gram-negative bacteria (Kaye KS, Pogue JM, Tran TB, Nation RL, Li J. 2016. Agents of last resort: polymyxin resistance. Infect Dis Clin North Am. 30:391) .

International application n. W02012/ 129499 discloses a topical antibacterial formulation comprising resveratrol and polymyxin B. The synergistic effect of polymyxins in combination with other antibiotics against resistant strains of the gram negative Acinobacter baumannii is known in the art (Wentao Ni, Xiaodi Shao, Xiuzhen Di, Junchang Cui, Rui Wang, Youning Liu, 2015, In vitro synergy of polymyxins with other antibiotics for Acinetobacter baumannii: A Systematic review and meta-analysis, Int. J. Antimicrobial Agents. 45:8-18) .

Korean Patent n. KR20160044768 discloses the synergistic effect of polymyxin B in combination with ciclopirox on drug resistance gram negative bacteria.

Chinese patent n. CN104127487 discloses the synergistic effect of resveratrol in combination with tetramethylpyrazine for curing digestive tract diseases in animals.

It is also known in the art the effect of resveratrol alone as antibacterial against gram positive bacteria (Paulo L, Ferreira S, Gallardo E, Queiroz JA, Domingues F, 2010, Antibacterial activity and effect of resveratrol on human pathogenic bacteria, W J Microbiol Biotechnol. 26:1533-1538) .

The objective technical problem is to restore antibacterial efficacy of colistin against colistin-resistant Gram-negative pathogens by providing an alternative synergistic combination for the treatment of bacterial infections associated to multi-drug resistant (MDR) Gram-negative pathogens .

The same inventors unexpectedly found that, despite the lack of any significant intrinsic antimicrobial activity, resveratrol exhibited a strong synergistic effect with colistin against polymyxin-resistant Gram- negative pathogens .

Object of the invention

With reference also to the attached claims, object of the present invention is a pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for the treatment of urinary tract infections (UTI) .

Another object of the present invention is a pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for intestinal decontamination.

Another object of the present invention is a pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for the treatment of bacterial intestinal infectious disease.

Another object of the present invention is a pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for the treatment of lower respiratory tract infections (LRTI) .

Detailed description of the invention Within the meaning of the present invention resveratrol means 3,5,4'- trihydroxy-trans-stilbene .

Within the meaning of the present invention polymyxin means antibiotic with a chemical structure of a cyclic peptide with a hydrophobic tail; polymyxins include polymyxin B and polymyxin E, known as colistin, pharmaceutically acceptable salts thereof, or a mixture thereof.

Within the meaning of the present invention multidrug resistant bacterial pathogens means any bacteria which are resistant to multiple antimicrobial drugs and in particular antibiotics, in particular Gram- negative species.

Within the meaning of the present invention polymyxin-resistant Gram- negative pathogens means any bacterial being resistant to polymyxin in particular Gram-negative bacteria such as E. coli, S. maltophilia , E. cloacae, K. pneumoniae, A. baumannii, and P. aeruginosa.

Within the meaning of the present invention a pharmaceutically active amount of an active ingredient means the amount of active ingredient giving the desired pharmaceutical effect without side effects.

Within the meaning of the present invention and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients means any substance formulated with the active ingredient for long-term stabilization, for therapeutic enhancement, facilitating drug absorption, reducing viscosity, enhancing solubility, facilitate stability, such as antiadherents , binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, vehicles, which can be chosen by the person skilled in the pharmaceutical field in view of the common skills.

Within the meaning of the present invention urinary tract infections (UTI) means infections affecting parts of the urinary tract, such as infections of the lower urinary, bladder infection, cystitis and infections of upper urinary tract, kidney infection, pyelonephritis. Within the meaning of the present invention lower respiratory tract infections (LRTI) means infections affecting parts of lower respiratory tract, such as infections of trachea, infections of primary bronchi, and lungs infections .

The above lower respiratory tract infections (LRTI) may be associated to cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary diseases .

Within the meaning of the present invention intestinal decontamination means to eliminate or reduce the amount of target bacteria carbapenem- resistant Enterobacteriaceae that are also colistin-resistant (CRE COL- R) , which are present as colonizers in the intestine of patients who are at high risk of developing infection, because of neutropenia, surgery, previous recurrent CRE COL-R infections or multiple comorbidities.

Within the meaning of the present invention bacterial intestinal infectious disease means infection of the gastrointestinal tract including gastroenteritis, diarrhea, dysentery, enterocolitis.

With reference also to the attached claims, object of the present invention is a pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for the treatment of urinary tract infections (UTI) .

Another object of the present invention is a pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for intestinal decontamination.

Another obj ect of the present invention is a pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for the treatment of bacterial intestinal infectious disease.

Another object of the present invention is a pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for the treatment of lower respiratory tract infections (LRTI) .

Preferably in the composition the amount of resveratrol is equal or lower than 5 grams .

More preferably in the composition the amount of resveratrol is comprised between 2.5 grams and 5 grams .

Even more preferably in the composition the amount of resveratrol is 5 grams .

Preferably in the composition the amount of polymyxin is comprised between 125 milligrams and 450 milligrams.

More preferably in the composition the amount of polymyxin is 150 milligrams .

Preferably in the composition polymyxin is colistin. Preferably polymyxin is colistin sulphate.

Preferably the composition is formulated to be administered orally or by inhalation .

More preferably the pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for the treatment of urinary tract infections (UTI) or for use for intestinal decontamination or for the treatment of bacterial intestinal infectious disease is formulated to be administered orally. More preferably the pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for the treatment of lower respiratory tract infections (LRTI) disease is formulated to be administered by inhalation.

Preferably lower respiratory tract infections (LRTI) are selected from the group consisting of infections of trachea, infections of primary bronchi and lungs infections .

More preferably the above lower respiratory tract infections (LRTI) are selected from the group consisting of lower respiratory tract infections (LRTI) associated to cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary diseases.

In a preferred embodiment of the present invention the pharmaceutical composition comprising pharmaceutically active amounts of at least one polymyxin and pharmaceutically active amount of resveratrol and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients for use for the treatment of urinary tract infections (UTI) or for use for intestinal decontamination or for the treatment of bacterial intestinal infectious disease, comprises 5 grams of resveratrol and 150 milligrams of colistin sulphate and pharmaceutically acceptable additives and/or pharmaceutically acceptable excipients and is for oral administration .

Examples

The in vitro activity of resveratrol alone and in combination with colistin against a collection of clinically relevant Gram-negative pathogens of different species was tested.

A panel of polymyxin-resistant strains of various clinically-relevant Gram-negative species was included in the study, as shown in table 1, wherein MDR means resistant to carbapenems (imipenem, meropenem, ertapenem) , β-lactamase inhibitors combinations (amoxicillm-clavulanate, piperacillin-tazobactam) , expanded-spectrum cephalosporins (cefotaxime, ceftazidime, cefepime) and fluoroquinolones. COL-R means colistin resistant and Nd means not determined.

Table 1

Colistin sulfate and resveratrol were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France) . For susceptibility testing, each agent was freshly prepared according to the CLSI guidelines (CLSI 2015) in the appropriate solvent. Resveratrol was dissolved in DMSO . Minimal Inhibitory Concentrations (MICs) were determined by reference broth microdilution method following CLSI procedures (CLSI 2015) , and interpreted accordingly to the EUCAST breakpoints version 7.0

(www.eucast.org) .

The antimicrobial activity of resveratrol in combination with colistin was investigated by the checkerboard method, using cation-adjusted Mueller-Hinton broth (MHB, Becton, Dickinson and Company, France) in 96- well microtiter plates (Tascini C, Tagliaferri E, Giani T, Leonildi A, Flammini S, Casini B, Lewis R, Ferranti S, Rossolini GM, Menichetti F. 2013. Synergistic activity of colistin plus rifampin against colistin- resistant KPC-producing Klebsiella pneumoniae . Antimicrob Agents Chemother. 57:3990) . Each well was inoculated with 50 μΐ of a suspension of 5 x 105 CFU/ml of cells in a final volume of 100 μΐ . Inocula were prepared by direct suspension in MHB of bacteria grown overnight on Mueller-Hinton agar (MHA, Oxoid Ltd, United Kingdom) plates. The bacteria were then incubated for 16-20 h at 35°C ± 2°C. The total fractional inhibitory concentration (∑FIC) for each combination was calculated according to EUCAST document E.Def 1.2 (European Committee for Antimicrobial Susceptibility Testing. 2000. Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. EUCAST definitive document E.Def 1.2. EUCAST, Basel, Switzerland) as follows: ∑FIC equals FIC of agent A plus FIC of agent B, where the FIC of agent A or B is the MIC of agent A or B in the presence of resveratrol divided by the MIC of agent A or B alone. Synergistic effect was defined when the value of ∑FIC was lower or equal to 0.5. Controls with DMSO at the highest concentration were always included to rule out a potential synergistic effect of DMSO used for resveratrol solubilization.

The above experiments revealed that resveratrol did not show any inhibitory effect at concentrations up to 500 μg/ml with any of the tested strains. In checkerboard assays, the combination of resveratrol plus colistin showed a notable dose-dependent synergistic effect against polymyxin- resistant strains of naturally susceptible species such as Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli, Stenotrophomonas maltophilia, P. aeruginosa and A. baumannii, as shown in table 2, wherein a asterisk indicates a value of ∑FIC ≤0.5.

Table 2

Colistin MICs for all polymyxin-resistant strains could be reduced below the breakpoint for susceptibility (2 μg/ml) in most cases with resveratrol concentrations of 128 μg/ml (Table 2) except for E. coli FI- 4451 in which the colistin resistance was mcr-1 plasmid-mediated. Controls with DMSO at the highest concentration tested (2.5%) confirmed that the observed effects were not related with the presence of DMSO.