Compositions for the prevention treatment of urinary tract infections comprising hyaluronic acid and diclofenac complexes

Abstract

Disclosed are instillable compositions in the form of aqueous solutions comprising hyaluronic acid or a salt thereof and a complex of diclofenac or a salt thereof with 2-hydroxypropyl-beta- cyclodextrin and use thereof in the prevention of urinary tract infections.

Description

[0001] “COMPOSITIONS FOR THE PREVENTION TREATMENT OF URINARY TRACT INFECTIONS COMPRISING HYALURONIC ACID AND DICLOFENAC COMPLEXES”

[0002] The present invention relates to compositions in the form of aqueous solutions comprising hyaluronic acid or a salt thereof and a complex of diclofenac or a salt thereof with 2-hydroxypropyl-beta-cyclodextrin for intravesical instillation and use thereof in the prevention of recurrent urinary tract infections.

[0003] STATE OF THE ART

[0004] Urinary tract infections (UTIs), defined microbiologically as the inflammatory response of the urothelium to microbial pathogens, are among the most common bacterial infections and, in 2019, there were an estimated 405 million cases of infection and 267,000 deaths worldwide due to urinary tract infections. About 25% of all antibiotic prescriptions are for urinary tract infections. Antibiotics are the first-line treatment for treatment and prevention of urinary tract infections of all types. However, rates of antimicrobial resistance are increasing, jeopardizing the effectiveness of antibiotics. Antimicrobial resistance is now one of the most substantial threats to patient safety worldwide.

[0005] Urinary tract infection with Escherichia coli is the most frequent cause of UTIs.

[0006] A study is reported in the literature in which it is concluded that diclofenac can have a direct antibacterial effect on strains of E.coli isolated from patients with urinary tract infection (Mazumdar et al., In vivo 20: 613-620, 2006).

[0007] However, in this context, there is a need to explore innovative approaches with respect to the direct antibacterial action of "non-antibiotic" compounds for managing the prevention of urinary tract infections.

[0008] US 2022 / 0273698 discloses the treatment of interstitial cystitis bladder pain syndrome comprising the sequential administration by intravesical instillation of a solution comprising a corticosteroid associated with a local anaesthetic or a non-steroidal anti-inflammatory and a solution of glycosaminoglycans such as hyaluronic acid or a salt thereof, heparin and chondroitin sulphate.

[0009] The local anaesthetic (lidocaine) and / or the non-steroidal anti-inflammatory (diclofenac) may be complexed with 2-hydroxypropyl-beta-cyclodextrin.

[0010] The complex of diclofenac or a salt thereof with 2-hydroxypropyl-beta-cyclodextrin diclofenac (HPpCD), described in EP 0658347, is used for the preparation of injectable forms for intramuscular, intravenous and subcutaneous administration.

[0011] DESCRIPTION OF THE INVENTION It has now been found that compositions in the form of instillable aqueous solutions comprising hyaluronic acid or a salt thereof (HA) or a complex of diclofenac or a salt thereof and 2 -hydroxypropyl-beta-cyclodextrin are particularly useful for the prevention of urinary tract infections towards the most common pathogens, such as for example Escherichia coli.

[0012] The invention therefore relates to compositions instillable in the form of aqueous solutions comprising hyaluronic acid or a salt thereof and a complex of diclofenac or a salt thereof and 2-hydroxypropyl-beta-cyclodextrin for use in the prevention of urinary tract infections.

[0013] The advantageous effects found for the compositions for use of the invention are greater than those obtainable by administering only diclofenac or the combination HA + uncomplexed diclofenac.

[0014] The complexation of diclofenac with 2-hydroxypropyl-beta-cyclodextrin (HPpCD) reduces bacterial colonization, thus allowing an effective prevention treatment of urinary infections even in the absence of a direct antibacterial effect. Although a potential direct antimicrobial activity of diclofenac on E. coli has been described (Mazumdar et al., In vivo 20: 613-620, 2006), the compositions of the invention did not unexpectedly demonstrate direct antibacterial effects, but were nevertheless surprisingly effective in reducing or counteracting bacterial colonization on the epithelia of the genitourinary system.

[0015] The compositions for use of the invention preferably contain the complex of diclofenac sodium salt and 2-hydroxypropyl-beta-cyclodextrin in a concentration from 0.5 to 5 mg / mL of diclofenac or a salt thereof and from 2.0 to 25 mg / mL of 2-hydroxypropyl-beta-cyclodextrin (HPPCD).

[0016] The hyaluronic acid or a salt thereof used for the preparation may be of a high or a low molecular weight. Molecular weight Mw, determined by analytical techniques known to those skilled in the art such as gel permeation, is typically comprised between 1000 and 3000 kDa for high molecular weight and between 10 and 1000 kDa for low molecular weight.

[0017] The concentration of the hyaluronic acid or a salt thereof is preferably in the range of 0.7 to 1.5% w / v.

[0018] The compositions for use of the invention may also contain suitable buffering agents, for example phosphate buffer pH 7.4.

[0019] DETAILED DESCRIPTION OF THE INVENTION

[0020] The composition of the invention was evaluated in in vitro experimental models on reconstructed human epithelia in comparison with diclofenac and with the association of diclofenac and hyaluronic acid or a salt thereof.

[0021] The following samples were prepared as described below

[0022] Samples

[0023] Samples Description

[0024] 1 HA+compl D / HPPCD

[0025] 2 HA+D

[0026] 3 D

[0027] 4 (H-L HA)+D

[0028] 5 H-LHA + compl D / HPpCD

[0029]

[0030] HA=hyaluronic acid or a salt thereof

[0031] D = diclofenac sodium

[0032] HPpCD= hydroxypropyl-beta-cyclodextrin

[0033] Compl D / HPpCD= diclofenac sodium complex with hydroxypropyl-beta-cyclodextrin H-L HA= combination of hyaluronic acid or a high molecular weight salt thereof (Mw= 1000-3000 KDa) and hyaluronic acid or a low molecular weight salt thereof (Mw=10-1000 KDa)

[0034] Sample Preparation

[0035] • Sample 1 : HA +compl D / HPpCD solution

[0036] A 1% HA solution A (MW=1000-3000 kDa) is prepared. Such solution is obtained by solubilizing 0.516 g of HA in a final volume of 47 ml of phosphate buffered water pH 7.4, keeping the solution under mechanical stirring. The phosphate buffer consists of 0.746% dibasic sodium phosphate dihydrate, 0.119% sodium dihydrogen phosphate dihydrate and 0.404% NaCl. The solution A is then sterilized in an autoclave according to the technique known to the person skilled in the art.

[0037] Subsequently, a solution B consisting of the hydroxypropyl-beta-cyclodextrin: diclofenac sodium complex is prepared with the following amounts: 75 mg diclofenac sodium, 333 mg HPpCD, 0.18 mg Tween 20 in 1 ml water.

[0038] To solution A is added 3 ml of solution B, so as to obtain the final solution HA+compl D / HPPCD.

[0039] • Sample 2: HA+ D solution

[0040] A solution A is prepared as indicated in the preparation method of sample 1.

[0041] Subsequently, 225 mg of diclofenac sodium is solubilized in solution Awhile keeping the solution under mechanical stirring, bringing the final volume to 50 ml so as to obtain the final solution HA + D.

[0042] • Sample 3 : Solution D

[0043] The solution of only diclofenac sodium is prepared by solubilizing 225 mg of diclofenac sodium in an aqueous solution buffered with phosphate buffer pH 7.4, in a final volume of 50 ml, under mechanical stirring. The phosphate buffer consists of 0.746% dibasic sodium phosphate dihydrate, 0.119% sodium dihydrogen phosphate dihydrate and 0.404% NaCl.

[0044] • Sample 4: Solution (H-L HA) + D:

[0045] A hyaluronic acid solution C or a high and low molecular weight salt thereof is prepared at the final percentage of 0.85%. Such a solution is obtained by solubilizing 0.439 g of hyaluronic acid or a high weight salt thereof (MW=1000-3000 kDa) and 0.448 g of hyaluronic acid or a low molecular weight salt thereof (MW=10-1000 kDa) in an aqueous solution buffered with phosphate buffer pH 7.4, in a final volume of 47 ml. The phosphate buffer consists of 0.746% dibasic sodium phosphate dihydrate, 0.119% sodium dihydrogen phosphate dihydrate and 0.404% NaCl. Solution C is then sterilized in an autoclave according to known techniques.

[0046] Subsequently, 225 mg of diclofenac sodium are added to solution C under mechanical stirring, bringing the final volume to 50 ml so as to obtain solution (H-L HA) + D.

[0047] • Sample 5: H-LHA+ compl D / HPpCD solution:

[0048] A solution C is prepared as indicated in the preparation method of sample 4.

[0049] Subsequently, a solution B is prepared as indicated in the preparation method of sample 1. To solution C is added 3 ml of solution B, so as to obtain the final solution H-LHA +compl D / HPPCD.

[0050] METHODS

[0051] In vitro reconstructed human epithelial models are the closest in morphology (multilayered epithelium), biochemical and physiological properties to human tissues in vivo and currently represent the most promising alternative to animals, ex vivo explants and submerged cell monolayers for in vitro evaluation of safety and efficacy of topically applied products (Gordon S. et al., Altex 32(4), 2015).

[0052] Therefore, reconstructed human bladder epithelium (RHBE / S / 13) that was infected (colonized) with Escherichia coh (DSM1103) suspended in reconstituted simulated urine was used to induce an infection. The selected E.coli strain is recognized as responsible for uropathogenic infections and recurrent cystitis.

[0053] Epithelial barrier integrity (TEER) and permeability testing were performed with Lucifer Yellow (LY) to evaluate the ability or not of the samples to preserve epithelial barrier integrity and permeability when compromised by E. coli.

[0054] The following RHBE were used:

[0055] RHBEs not colonized and not treated

[0056] RHBEs colonized

[0057] RHBEs colonized and treated.

[0058] A suspension of E.coli (104-105CFU / mL) is initially prepared in a simulated urine solution, which is used to colonize the RHBEs when necessary.

[0059] The composition of the simulated urine is as follows: 3.55 g of urea, 0.750 g of creatinine, 0.500 g of ammonium citrate, 2 g of sodium chloride, 0.4125 g of potassium chloride, 0.125 g of potassium bisulphate, 0.050 g of magnesium sulphate, 0.4375 g of monobasic potassium phosphate, and 0.125 g of potassium bicarbonate were dissolved in 125 mL of ultrapure water. Ultrapure water was added up to reaching a final volume of 250 mL. It was used within 24 hours after preparation.

[0060] The RHBEs are prepared as follows:

[0061] Not colonized and not treated RHBEs: are kept in their culture medium without antibiotic.

[0062] Colonized RHBEs, maintained in their culture medium without antibiotic were infected for 2 hours with 30 pl of E.coli suspension. Excess bacteria were removed after colonization.

[0063] Colonized and treated RHBEs, maintained in their culture medium without antibiotic, prior to being colonized as reported for colonized RHBEs, were treated for 4 h with 50 pl of each sample under evaluation.

[0064] All the RHBEs were incubated at 37°C, 5% CO2 in saturated humidity. The TEER and LY% analyses are performed after 16 hours of incubation. Both analyses were performed in duplicate.

[0065] COUNT OF VIABLE ESCHERICHIA COLI IN THE HOMOGENIZED TISSUE FRACTIONS.

[0066] At the end of the incubation period, the tissues were collected for the viable counts of the homogenized tissue. The tissues were separated from the plastic insert with a scalpel, placed in a vial containing 0.5 mL of sterile saline and treated with the Minilisis homogenizer (3 cycles of 30 sec, minimum power). Residual bacterial viable counts (CFU) were determined on tissue homogenates (adherent). The vital counts of the homogenates were then monitored on NUTRIENT plates by means of the diffusion plate method using 10-fold dilutions in sterile saline and sending 100 pL for each dilution; all plates were incubated at 37°C under aerobic conditions for 24-48 hours.

[0067] The following formula was then used to calculate CFU / mL:

[0068] CFU / ml = (number of colonies x dilution factor) / plate volume (mL).

[0069] All results were then converted to CFU / tissue and expressed as logarithmic values. TEER (Trans-Epithelial-Electrical-Resistance) ANALYSIS

[0070] TEER is the measurement of the movement of ions through the paracellular pathway regulated by the polarized surfaces of the plasma membrane and the tight cell-cell junctions that together prevent the movement of solutes and ions across epithelia. TEER is an indirect assessment of the stability of tight junctions and consequently is a direct measure of barrier functionality in epithelial tissue: it reflects the overall strength of the barrier linked to both epithelial structure and thickness. Maintaining the stability and electrical resistance of an epithelium is critical for essential physiological processes; therefore, significant changes in TEER may represent an early expression of cellular damage.

[0071] The trans-epithelial electrical resistance is expressed in Ohm*cm2. The lower the TEER value, the more the tissue is subjected to damage and therefore to an increase in the permeability of the tight junctions. In treated tissues, if the sample has a protective effect, after infection it is observed that the TEER value remains higher than in the untreated colonized tissues.

[0072] LY ANALYSIS (LUCIFER YELLOW)

[0073] Lucifer Yellow (LY) is a fluorescent dye impermeable to the cell membrane, used as a probe to study paracellular permeability in tissues. In cases where the cellular junctions are intact, the LY has a very low permeability, however, if the junctions between the cells are damaged, the spread of the LY increases. In this case, the probe was used to verify the integrity of the cellular junctions in the tissues in the presence of the analysed samples.

[0074] LY diffusion was assessed by quantifying changes in fluorescence levels between the apical and basolateral compartment of the tissue. Specifically, the flow % of LY is evaluated. High levels of LY % indicate increased damage to cellular junctions.

[0075] The calculation to obtain the LY % is as follows:

[0076] LY Flow % = (RFU BL / RFU AP t=0) x 100

[0077] (BL=basolateral; APt=0=apical. Mean RFU of LY 500 pM)

[0078] RFU= fluorescence measurement

[0079] MUCOADHESION METHOD Mucoadhesion was assessed using the rheological synergism method.

[0080] Such method is based on the use of a viscometer that determines the variation of the viscosity (expressed in mPa.s) of a mixture containing the polymer and the mucin (component glycoprotein of the mucus) with respect to the sum of the individual contributions due to the polymer and the mucin. (Hassan EE et al., Pharmaceutical Res., Vol 7, No. 5, 491-495, 1990).

[0081] This variation, called rheological synergism, is calculated according to the following formula:

[0082] A = rj (mixture)- [ (polymer) + rj (mucin)]

[0083] Where: Arp = rheological synergism

[0084] rj (mixture) = viscosity of the polymer-mucin mixture

[0085] rj (polymer) = viscosity of the polymer solution

[0086] rj (mucin) = viscosity of the mucin solution

[0087] To make the data comparable, the relative rheological synergism is used:

[0088] relative rj = A I rj +1

[0089] where rj = rj (polymer) + rj (mucin).

[0090] In the presence of interactions between polymer and mucin, the parameter assumes values greater than 1.

[0091] RESULTS COUNT OF VIABLE ESCHERICHIA COLI CELLS IN HOMOGENIZED TISSUE FRACTIONS.

[0092] E.COLI VITAL COUNT HOMOGENIZED TISSUE ANALYSIS CFU / ml Log (CFU / tissue)

[0093] RHBE not colonized and not < 1.00 X 101NA

[0094] treated

[0095] RHBE colonized 5.70 X 1065.2

[0096] RHBE colonized and treated 6.00 X 1065.3

[0097] with Sample 1

[0098] RHBE colonized and treated 4.73 X 1065.2

[0099] with Sample 5

[0100]

[0101] The results show that the composition of the invention has no direct antimicrobial effect.

[0102] TEER AND LY% ANALYSIS Samples Description TEER LY% Negative RHBE tissue not treated and

[0103] control (NC) not colonized with E.coli 55.4 4.57

[0104] Positive control RHBE tissue treated and colonized

[0105] (CNZ) with E.coli 32.8 66.74

[0106] 1 HA+compl D / HPPCD 58.4 2.37

[0107] 2 HA+D 40.1 3.23

[0108] 3 D 41.7 16.1

[0109] 4 (H-L HA)+D 34.4 10.04

[0110] 5 H-L HA + compl D / HPpCD 54.9 2.62

[0111]

[0112] The results obtained from the TEER and LY% analyses of the colonized and treated RHBEs show that the composition of the invention, while lacking direct antimicrobial effect, unexpectedly improves the barrier effect of the urothelium itself towards the colonization of E.coli (Figure 1). The results also show that the composition of the invention is less mucoadhesive than HA alone (Figure 2).

[0113] The results obtained demonstrate that the combination of HA and diclofenac complexed in HPpCD (Sample 1) shows an increase in the barrier effect of the urothelium and a reduction in permeability towards Escherichia coli greater than diclofenac alone or the combination HA +diclofenac; therefore, the complexation of diclofenac with HPpCD, although not having a direct antimicrobial effect, reduces the bacterial colonization of the urothelium.

[0114] In fact, see the comparison between Sample 1 and Sample 2 where the latter differs due to the absence of HPpCD. Likewise, this effect also occurs with mixtures of high and low molecular weight HA, see Sample 5 vs Sample 4, thus resulting in a peculiarity of the diclofenac HPpCD complex in the hyaluronic acid system or a salt thereof in which it is solubilized.

[0115] The effect is also unexpected because the combination of HA with the diclofenac / 2-hydroxypropyl beta cyclodextrin (D / HPpCD) complex is less mucoadhesive than HA alone.

[0116] The protective effect obtained by the invention is superior to that obtained with aqueous solution of diclofenac alone, see Sample 3 vs Sample 1 and Sample 5.

Claims

CLAIMS1. Instillable compositions in the form of aqueous solutions comprising hyaluronic acid or a salt thereof and a complex of diclofenac or a salt thereof and hydroxypropyl-beta-cyclodextrin for use in the prevention treatment of urinary tract infections.

2. Compositions for use according to claim 1 comprising the complex of diclofenac sodium and hydroxypropyl-beta-cyclodextrin.

3. Compositions for use according to claim 1 or 2, wherein the complex of diclofenac or a salt thereof and hydroxypropyl-beta-cyclodextrin comprises 0.5 to 5 mg / mL of diclofenac or a salt thereof and 2.0 to 25 mg / mL of hydroxypropyl-beta-cyclodextrin.

4. Compositions for use according to one of claims 1 to 3, wherein the concentration of the hyaluronic acid or a salt thereof is in the range of 0.7 to 1.5% w / v.

5. Compositions for use according to one of claims 1 to 4, wherein the hyaluronic acid or a salt thereof used for the preparation of the composition has an average molecular weight Mw comprised between 1000 and 3000 kDa.

6. Compositions for use according to one of claims 1 to 5, wherein the hyaluronic acid or a salt thereof used for the preparation of the composition comprises a high average molecular weight fraction Mw comprised between 1000-3000 kDa and a low average molecular weight fraction Mw comprised between 10-1000 kDa.

7. Compositions for use according to one of claims 1 to 6 comprising buffering agents.

8. Compositions for use according to one of claims 1 to 7, wherein the infections are sustained by Escherichia coli.

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