Polyhexamethylene biguanide-based formulations for use in the treatment of acanthamoeba keratitis and / or fungal infections

Abstract

The present invention relates to stable formulations based on polyhexamethylene biguanide (PHMB) suitable for administration at ophthalmic levels, methods for their preparation, and related dosing regimens effective in the treatment of Acanthamoeba keratitis, particularly effective in eradicating cysts.

Description

[Technical Field]

[0001] The present invention provides Ophthalmic grade The present invention relates to stable formulations based on polyhexamethylene biguanide (PHMB) suitable for administration in the ophthalmic solution, methods for their preparation, and related dosing regimens effective in the treatment of Acanthamoeba keratitis, particularly effective in eradicating cysts. [Background technology]

[0002] Acanthamoeba keratitis is an infection of the cornea, the transparent tissue lining the front of the eye. The infection is caused by Acanthamoeba, a microorganism commonly found in both water bodies (lakes, oceans, and rivers), as well as household tap water, swimming pools, hydromassage bathtubs, soil, and air. Summary of the Invention

[0003] Acanthamoeba keratitis is more common in contact lens wearers [1], but anyone with corneal disease can be susceptible to developing this serious eye infection.

[0004] Generally, Acanthamoeba has a two-phase life cycle: an active form (in which the organism feeds and replicates) and a dormant form (in which the organism protects itself from potential attacks by encysting). The cysts are the actual target when developing effective anti-amoebic drugs.

[0005] Co-infection with bacterial species is common in patients with Acanthamoeba keratitis. Specifically, Staphylococcus spp. and Pseudomonas aeruginosa are the most commonly co-isolated pathogenic microorganisms. [2,3] In particular, in vivo studies have suggested that the presence of the latter microorganism is an essential factor in the development and severity of Acanthamoeba keratitis. [4]

[0006] Because the cornea has the highest density of pain receptors and is one of the most sensitive organs in the human body, Acanthamoeba keratitis can be extremely painful. Products used to treat Acanthamoeba keratitis can be extremely intolerable and, in some cases, can irritate the corneal surface. Approximately 25% of treated cases of Acanthamoeba keratitis result in corneal transplantation. If untreated, the disease progresses to blindness as a result of corneal neovascularization and scarring or perforation.

[0007] To date, no drugs have been approved for the treatment of Acanthamoeba keratitis. Biguanides and diamidines have shown efficacy against Acanthamoeba keratitis in many clinical cases, but the treatment regimens and concentrations of these drugs have been empirically developed.

[0008] Among the various options available, treatment with polyhexamethylene biguanide (PHMB) in the form of a 0.02% ophthalmic preparation alone [5] or in combination with 0.02% chlorhexidine, 0.1% propamidine, or 0.1% desomeidine is the most promising option for treating patients with Acanthamoeba keratitis.

[0009] Current treatments for Acanthamoeba keratitis are lengthy and difficult. These treatments begin with hourly doses during the day, avoiding nighttime use, for approximately 1-2 weeks, then are tapered based on response. While each patient is different, those who receive an early diagnosis and begin appropriate treatment promptly can generally expect to receive therapeutic treatment for 3-6 months.

[0010] Furthermore, repeated and prolonged use of antibiotics and disinfectants, such as those mentioned above, risks significantly altering the composition of the conjunctival microbiota by increasing the proportion of pathogens, such as Staphylococcus epidermidis, to the detriment of the normal commensal microbiota. This could have important clinical implications, as Staphylococcus epidermidis is one of the main causes of ocular diseases such as conjunctivitis, keratitis, and endophthalmitis [6].

[0011] PHMB consists of several repeating (n) biguanide units linked by hexamethylene chains, forming a cationic and amphiphilic structure (Figure 1).

[0012] PHMB-based formulations are mixtures of biguanide polymers with molecular weights that can vary from 400 to 8,000 amu and variable degrees of polymerization, where n can range from 2 to 40.

[0013] The mechanism of action of PHMB has been the subject of extensive research in in vitro experimental models. SUV membranes are systems that mimic the chemistry of human corneal epithelial cell membranes. When PHMB interacts with the SUV membrane bilayer, it increases phospholipid cooperativity, expels water from the bilayer consisting of acyl chains and polar head groups, reduces the dynamics and selectivity of the acyl chain-containing regions, and allows PHMB to be absorbed to the bilayer surface [7].

[0014] The cationic nature of PHMB allows it to interact with anionic macromolecules such as DNA. It has been shown in the literature that PHMB interacts with DNA in aqueous solution, resulting in the formation of a complex between these two species. This reaction involves electrostatic interactions between the cationic biguanide groups and the anionic phosphate groups of DNA deoxyribonucleotides in a molar ratio of approximately 1:1 [8].

[0015] As previously reported for other bacteria, such as Escherichia coli, Salmonella enteritidis, and Staphylococcus aureus, the structure of Acanthamoeba, characterized by exposed DNA-binding sites, facilitates PHMB access to the protozoan DNA, promoting the chromosome condensation and destruction process.[9] Although these studies were not specific to Acanthamoeba, they helped us identify the optimal conditions for PHMB to bind to the protozoan DNA and exert its maximum e-phase efficacy in treating infectious keratitis.

[0016] A previous Phase I clinical trial by Papa V et al.

[10] showed that even higher PHMB concentrations of 0.04%, 0.06%, and 0.08% were safe and well-tolerated in healthy subjects. [8] The pH and osmolarity of the formulations were not described, nor were the molecular weight and polydispersion index of the PHMB polymer used. However, as discussed below, PHMB alone at those concentrations is not sufficient to maximize its interaction with Acanthamoeba DNA and therapeutic efficacy.

[0017] US Patent Application Publication No. 2007 / 0140897A1

[12] describes ophthalmic formulations based on biguanides (including PHMB) with a pH of 4-6 for the treatment of Acanthamoeba keratitis. However, the only formulation exemplified is alexidine-based, not PHMB-based. In any case, neither the relevance of the MW and PDI of biguanide polymers in their interaction with pathogen DNA, nor the specific use concentrations of biguanide polymers in combination with these parameters, are described.

[0018] A stability study by Bouattour et al. 2018

[13] of a 0.02% PHMB-based formulation at pH 4 revealed the formation of degradation products (BPs) already after 90 days. The authors concluded that PHMB-based formulations cannot be stored for more than 60 days in EOS-LDPE bottles at 25 °C. The present authors have now demonstrated that by adjusting the molecular weight (MW) and polydispersity index (PDI) of the PHMB polymer, along with the pH and osmolarity, they are able to maintain stability and efficacy at the highest active ingredient concentrations in the range of 0.04% (w / v) to 0.08% (w / v). Ophthalmic administration The patent identifies polyhexamethylene biguanide-based formulations for

[0019] Studies have shown that the use of high molecular weight, high polydispersity index PHMB, along with a specific buffering system that maintains the pH of 0.04%-0.08% (w / v) PHMB formulations within the range of 5-6.5, synergistically provides optimal conditions for stability and activity of the active ingredient for up to at least 24 months. DETAILED DESCRIPTION OF THE INVENTION

[0020] For these reasons, the PHMB molecule must be in its protonated form to best exert its biocidal mechanism of action. In its protonated, ionized form, PHMB penetrates the corneal epithelium infected with Acanthamoeba and accumulates in the corneal stroma through electrostatic interactions with the negative charges of proteoglycans. In the stroma, PHMB exerts its biocidal and cysticidal activity, according to recent scientific research, through extensive interactions with DNA phosphate groups. This binding blocks the amoeba's DNA replication process, resulting in the death of the pathogen.

[11] Thus, the effectiveness of this interaction between PHMB and DNA, and thus the activity of PHMB itself, is significantly affected by the stability of the formulation containing the active ingredient. Furthermore, administration of the formulations of the present invention in a strict stepwise regimen in patients with Acanthamoeba keratitis has been found to be particularly useful in terms of their activity towards complete eradication of Acanthamoeba cysts.

[0021] In this sense, the formulation object of the present invention, being a monotherapy, favors patient compliance and adherence to the treatment regimen, since it avoids repeated infusions of various products, especially during the day. Furthermore, given the selectivity and specificity of the product against the target microorganism, a greater rapidity of action is ensured, resulting in a faster remission of the pathology and avoiding long treatment periods of 3 to 6 months.

[0022] Again, compared with currently used multi-drug therapies consisting of strong antiseptics and broad-spectrum antibiotics, monotherapy with 0.08% PHMB (highly selective and specific) certainly represents a safer option in preserving the diversity of the ocular microbiota during treatment and thus avoiding the emergence of multi-resistant Staphylococcus epidermidis strains.

[0023] Accordingly, the present invention provides a solution or salt thereof based on polyhexamethylene biguanide at a concentration ranging from 0.04% to 0.08%, which comprises a buffer system for maintaining a pH within the range of 5 to 6.5 and an isotonicity agent for maintaining an osmolarity within the range of 270 to 330 mOsm / Kg, wherein the molecular weight of the polyhexamethylene biguanide is in the range of 2,300 to 6,000 amu, and the polydispersity index of the polymer is in the range of 1.5 to 1.9, preferably 1.7 to 1.8, for use in the treatment of Acanthamoeba keratitis or fungal infections. Ophthalmic administration The present invention relates to a liquid preparation based on polyhexamethylene biguanide or a salt thereof, which is suitable for the following:

[0024] The fungal infection is preferably keratitis or keratomycosis mediated by a pathogen selected from the group consisting of Candida albicans, Fusarium solani, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Cladosporium.

[0025] The above fungal infections may also coexist with Acanthamoeba infections, particularly Fusarium solani and Cladosporium infections.

[0026] In another embodiment of the present invention, the treatment of infectious Acanthamoeba keratitis is particularly effective in cases of co-infection with Pseudomonas aeuriginosa or Staphylococcus epidermis.

[0027] Therapeutic use of a 0.08% PHMB formulation active against Pseudomonas aeruginosa (data not shown, Internal APE Test Study: Orphan Drug - In Vitro Assay - 1305) has demonstrated its high performance in rapidly and effectively reversing the pathology of the above co-infections. Polyhexamethylene biguanide may be in the form of an inorganic or organic salt selected from the group consisting of chloride, bromide, sulfate, phosphate, mesylate, formate, citrate, or maleate.

[0028] In addition to the need to maintain a slightly acidic pH so that PHMB is in a maximally protonated state, a further object of the present invention is to maintain the osmolarity of the formulation at a value equivalent to the physiological value of tear fluid (which ranges from 290 to 320 mOsm / Kg), preferably by the addition of an ionic tonicity agent selected from the group consisting of sodium chloride, potassium chloride, or a non-ionic tonicity agent such as glycerol, mannitol, or sorbitol, which also contribute to osmolality; an ionic buffer system such as phosphate buffer, citrate buffer, bicarbonate buffer, or borate buffer, or a non-ionic buffer such as trometamol, histidine, glycine, or HEPES.

[0029] Osmotic pressure is actually caused by the ions (Na + , K. + , Cl - , and HCO3).

[0030] According to a preferred embodiment of the present invention, the tonicity agents are used in the following concentrations: NaCl (0.01% to 0.9%), KCl (0.01% to 1.19%), glycerol (0.01% to 2.6%), mannitol (0.01% to 5.1%), and sorbitol (0.01% to 5.5%).

[0031] In certain embodiments of polyhexamethylene biguanide-based liquid formulations, the buffer system is an ionic buffer such as phosphate buffer, citrate buffer, bicarbonate buffer, or borate buffer, or a non-ionic buffer such as trometamol, histidine, glycine, HEPES, or a mixed system.

[0032] The buffer system preferably comprises disodium phosphate dodecahydrate (NaHPO·12H O) and sodium dihydrogen phosphate (NaHPO·H O). Disodium phosphate dodecahydrate is preferably used at a concentration in the range of 0.01% to 4.5%, and sodium dihydrogen phosphate is preferably used at a concentration in the range of 0.01% to 3.5%.

[0033] Alternatively, citrate phosphate buffer containing disodium phosphate dodecahydrate (Na2HPO4·12H2O) and citric acid (C6H5O7·H2O), or Tris / HCl or boric acid / borate may be used.

[0034] The formulation of the present invention ophthalmological If in the form of a gel or viscous solution, it is preferred to use phosphate-free buffers such as, for example, Tris buffers and / or boric acid or borate salts.

[0035] According to a preferred embodiment of the polyhexamethylene biguanide based solution, the pH is 5.8.

[0036] In a preferred embodiment of the polyhexamethylene biguanide-based liquid formulation of the present invention, the concentration of PHMB is selected from the group consisting of 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), and 0.08% (w / v), preferably 0.08% (w / v).

[0037] Ophthalmic administration The polyhexamethylene biguanide-based solutions of the present invention suitable for use are sterile solutions that can be in the form of eye drops.

[0038] Alternatively, the polyhexamethylene biguanide-based formulations of the present invention in the presence of a thickening agent (e.g., xanthan gum, gellan gum, polyvinyl alcohol, hyaluronic acid, sodium hyaluronate, cellulose derivatives such as carboxymethylcellulose or hydroxypropylcellulose) may be ophthalmological It may be in the form of a gel or a viscous solution.

[0039] According to a further preferred embodiment of the present invention, the polyhexamethylene biguanide-based solution of the present invention further comprises a penetration enhancer which may be selected from Tween 80 and benzalkonium chloride.

[0040] Also, according to a preferred embodiment, the polyhexamethylene biguanide-based solutions for use in the present invention may further comprise 0.02% chlorhexidine, 0.1% propamidine, or 0.1% desomedine in combination with PHMB. When PHMB is administered in combination with a second active agent, the administration may be carried out sequentially, simultaneously, or separately, i.e., in the form of the same eye drops or separate formulations.

[0041] The present invention further relates to a polyhexamethylene biguanide-based solution of the present invention for the treatment of Acanthamoeba keratitis by administering escalating doses of 16 drops / day for 5 days, 8 drops / day for 7 days, 6 drops / day for 7 days, and 4 drops / day until clinical recovery, which averaged 88% over a 4-month period. This escalating dose differs from the intensified dose previously reported in a Phase I study by Papa et al.

[10] because it more effectively intervenes in eradicating cysts and thus in clinical recovery from Acanthamoeba keratitis.

[0042] The present invention further relates to a method for preparing a polyhexamethylene biguanide-based liquid formulation of the present invention, comprising the steps of: (i) adding, under gentle and continuous stirring, to purified water, the buffer system at a concentration ranging from 0.01% to 4.5% (w / v) and the tonicity agent at a concentration ranging from 0.01% to 5.5% (w / v); and optionally, adding a viscosity-increasing agent selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose, and hydroxypropylcellulose; (ii) adding polyhexamethylene biguanide having a molecular weight in the range of 2,300 to 6,000 amu and a polydispersity index in the range of 1.5 to 1.9, preferably 1.7 to 1.8, at a concentration in the range of 0.04% to 0.08% (w / v) with gentle stirring; (iii) bringing to final volume by adding purified water; (iv) Sterilizing by filtration or heat.

[0043] In the case of viscous solutions, the addition of a thickening agent selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose, and hydroxypropylcellulose should also be added after step (i), in which case heat sterilization is used in the presence of a non-filterable polymer instead of sterilization by filtration.

[0044] This solution is then dispensed into unit dose containers or vials which are then sealed. The unit dose containers may be made from low density polyethylene (LDPE) and each package may contain 5 single dose units.

[0045] In a particular embodiment of the method for preparing a polyhexamethylene biguanide-based liquid formulation, the buffer system for maintaining a pH in the range of 5 to 6.5 is an ionic buffer such as phosphate buffer, citrate buffer, bicarbonate buffer, Tris buffer, borate buffer, or a nonionic buffer such as trometamol, histidine, glycine, HEPES, or a mixed system, preferably selected from the group consisting of disodium phosphate dodecahydrate / sodium dihydrogen phosphate, disodium phosphate dodecahydrate / citric acid, Tris / HCl, or boric acid / borate salts.

[0046] According to a preferred embodiment, the buffer system is based on disodium phosphate dodecahydrate (NaHPO·12H O) and sodium dihydrogen phosphate (NaHPO·H O). Disodium phosphate dodecahydrate is preferably used at a concentration in the range of 0.01% to 4.5% (w / v), and sodium dihydrogen phosphate at a concentration in the range of 0.01% to 3.5% (w / v).

[0047] The tonicity agent for maintaining the osmolarity within the range of 270 to 330 mOsm / Kg is preferably sodium chloride.

[0048] The preparation method of the formulation has also been developed to ensure that both the initial molecular weight and polydispersity index of the active ingredient PHMB are maintained in the final product within the ranges of 2,300-6,000 amu and 1.5-1.9, preferably 1.7-1.8, respectively, as these parameters have been shown to be related to the efficacy of the final product.

[0049] Finally, the present invention relates to a therapeutic eye lens loaded with or derivatized with the polyhexamethylene biguanide-based liquid formulation of the present invention for the controlled release of the active ingredient for the treatment of Acanthamoeba keratitis or Pseudomonas aeuriginosa co-infection. This embodiment allows for continuous release of the formulation over an extended period of time, further benefiting patient compliance and adherence to treatment.

[0050] The invention will now be described by way of example and not by way of limitation on preferred embodiments with particular reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0051] [Figure 1]Figure 1 shows the structure of the hexamethylene biguanide repeat unit in PHMB; [Figure 2] Figure 2 shows the flow chart of the preparation of PHMB-based formulations; [Figure 3] FIG. 3 shows the chromatogram obtained by gel permeation chromatography of PHMB in API. [Figure 4] FIG. 4 shows the chromatogram obtained by gel permeation chromatography of PHMB in the final product; a comparison with the chromatogram in FIG. 2 for the raw material makes it clear how this manufacturing process is able to maintain the molecular weight distribution of the original raw material all the way to the final product. [Figure 5] Figure 5 shows a comparison of stability data for 0.08% PHMB-based formulations at pH 7.4 and pH 5.8. [Figure 6] Figure 6 shows a comparison of PHMB-DNA binding with molecular weight. From the trend in this graph, it can be seen that using PHMB with a higher molecular weight results in more effective polymer-DNA binding due to the inclusion of one or more nucleic acid molecules. High-molecular-weight polymers are able to form crosslinks between different nucleic acid molecules, forming a network of bonds (lines containing circles). Conversely, lines containing squares indicate the establishment of linear bonds between PHMB and DNA, while polymers characterized by lower molecular weights are unable to form crosslinks but establish bonds involving only one nucleic acid molecule. [Figure 7A] Figure 7(A) shows the results of a stability study of 0.08% PHMB formulations using PHMB batches with different molecular weights (MW) and polydispersity indices (PDI), expressed as the percent recovery of PHMB (w / v) at TO and after 12 months (T12). [Figure 7B]Figure 7(B) shows the results of a stability study of a 0.08% PHMB formulation using a PHMB batch characterized by MW = 2,641 amu and PDI = 1.6. The study was conducted under three different temperature conditions: long-term stability (25°C, RH 40%); intermediate stability (30°C, RH 65%); and accelerated stability (40°C, RH 25%). Stability results, expressed as PHMB recovery (w / v) at T0 and after 3 months (T3), are shown. [Example]

[0052] The following non-limiting examples are provided to better illustrate the present invention, in which various PHMB-based formulations are tested and compared to evaluate the effect on stability of several formulation parameters, such as pH, osmolarity, molecular weight and polydispersity index of the active ingredient PHMB.

[0053] Example 1: Preparation of PHMB-based formulations A method for preparing the formulations of the present invention is shown in the flow diagram of FIG.

[0054] Add disodium phosphate dodecahydrate (Na2HPO4·12H2O), sodium dihydrogen phosphate (NaH2PO4·H2O), and sodium chloride in that order and dissolve in a large amount of purified water while continuing to stir gently.

[0055] This stirring step is important to maintain the initial molecular weight and polydispersity index of PHMB and must be performed without forming bubbles or vortexes in the solution. It is preferable to use a stir bar or impeller that can be submerged in the solution for stirring. Finally, PHMB (0.08%) is added.

[0056] The solution is brought to a final volume (100 ml) by adding sufficient purified water with continuous stirring. The pH and appearance of the solution are then adjusted.

[0057] Sterilization is achieved by filtration through a 0.2 μM filter. The properties and dynamics (pressure, flow rate) of the filter membrane are chosen to prevent the product from being forced through the membrane, to avoid depolymerization or scission of the polymer chains.

[0058] After filtration, the integrity of the 0.2 μm filter is regulated by an electronic control system.

[0059] Finally, the sterile solution is used to fill unit dose containers under aseptic conditions using form-fill-seal (B / F / S) technology.

[0060] This automated technique allows for measurements of 0.1 to 1,000 cm 3 The vials can be filled and sealed under aseptic conditions with solution volumes ranging from 0.1 to 100 mg / L.

[0061] The PHMB solution appears as a clear, colorless or pale yellow liquid.

[0062] At the end of the manufacturing process, the final product is characterized in its main components, including any impurities, by suitable chromatographic methods.

[0063] Comparison of the PHMB chromatogram at API (Figure 3) with that after formulation (Figure 4) shows that the molecular weight distribution of PHMB remains unchanged.

[0064] Example 2: Comparative Stability Study of PHMB-Based Formulations The manufactured PHMB-based formulations are sterile solutions with pH and osmolarity being the relevant physicochemical parameters.

[0065] As detailed above, the choice of a slightly acidic pH is dictated by the need to obtain a suitable formulation that cannot be obtained at a near-neutral pH.

[0066] For this purpose, various formulations based on 0.08% PHMB at different pH values ​​(5.8, 6.2, 7.4) were developed and then their stability was examined at 25° C., 40° C., and 60° C. To prepare a formulation based on 0.08% PHMB at pH 5.8, the following ingredients were weighed:

[0067] [Table 1]

[0068] Table 2 below shows the physicochemical characteristics of the final formulation at pH 5.8 and at each stage of its preparation process.

[0069] [Table 2]

[0070] To prepare a 0.08% PHMB-based formulation at pH 6.2, the following ingredients were weighed according to the same method as above:

[0071] [Table 3]

[0072] Table 4 below shows the physicochemical characteristics of the final formulation at pH 6.2 and at each stage of its preparation process.

[0073] [Table 4]

[0074] To prepare a 0.08% PHMB-based formulation at pH 7.4, the following ingredients were weighed:

[0075] [Table 5]

[0076] Table 6 below shows the physicochemical characteristics of the final formulation at pH 7.4 and at each stage of its preparation process.

[0077] [Table 6]

[0078] A comparison was then conducted on the stability of 0.08% PHMB-based formulations in single-dose containers at pH 5.8 and pH 7.4 at 25°C (long-term), 40°C (accelerated), and 60°C (stress conditions).

[0079] The results of the stability analysis are shown below:

[0080] (Stability data) 0.08% PHMB formulation, pH 5.8, 25°C ± 2°C, relative humidity 40% ± 5 (long-term)

[0081] [Table 7]

[0082] [Table 8]

[0083] [Table 9]

[0084] [Table 10]

[0085] The results shown in Figure 5 indicate that the stability of the PHMB-based formulation obtained by maintaining the pH within the range of 5–6.5 is not achieved at pHs around neutral (pH 7.4).

[0086] Example 3: PHMB-DNA binding studies To assess the equivalence between low and high molecular weight PHMB, PHMB-DNA binding studies were performed.

[0087] While the DNA binding capacity of PHMB can be considered independent of the specific DNA species studied, as shown in [8], its DNA binding capacity and effectiveness depend on the molecular weight and polydispersity index of the PHMB polymer. When the polymer has a molecular weight in the range of 2,300–6,000 amu and a polydispersity index in the range of 1.5–1.9, DNA binding is more effective, presumably because the polymer length is more suitable for forming crosslinks involving multiple nucleic acid molecules according to the mechanism proposed by Allen et al. [8]. Conversely, when the polymer is characterized by a low molecular weight, the bonds are linear and involve a single nucleic acid molecule.

[0088] Next, a comparative study was conducted using different PHMB polymer samples to evaluate their binding ability to nucleic acid molecules. PHMB (Sample A) was degraded by acid and high temperature treatment for 5 hours to obtain a low molecular weight PHMB polymer. PHMB (Sample B) was not subjected to any type of treatment and was characterized by a molecular weight within the claimed range.

[0089] Next, the ability of both samples to bind to nucleic acid molecules was evaluated. The experimental method proposed here is based on the interaction between an aqueous solution of PHMB and an aqueous solution of DNA. The content of PHMB-DNA complexes was measured as the difference between the initial content of both species in solution and the amount remaining after the reaction was completed. The content of both species in solution was monitored spectrophotometrically at two different wavelengths: 260 nm for DNA and 235 nm for PHMB.

[0090] In conclusion, the data indicate that the increased ability of high molecular weight PHMB to interact with DNA through the formation of cross-links with multiple nucleic acid molecules results in greater efficacy in treating infectious Acanthamoeba keratitis and Pseudomonas aeruginosa co-infection. As shown in the graph in Figure 6, the type of bond formed between the PHMB polymer and DNA is much more effective.

[0091] Example 4: Stability studies related to molecular weight and polydispersity index of PHMB A comparative study was carried out on three different PHMB-based starting batches characterized by different molecular weights and polydispersity indices (PDI): Batch L-17GR185627, MW=2517amu, PDI=1.73, · Batch F-693 / LU / 101, MW=1170amu, PDI=1.93; · Batch F-693 / LU / 118, MW=440amu, PDI=2.14.

[0092] For each lot, a 0.08% PHMB formulation was prepared, characterized by a pH of 5.8 and an osmolarity of 0.280 osmol / Kg. The stability of the PHMB formulation was then evaluated at T0 and after 12 months (T=12) under conditions of 25°C ± 2°C and 40 ± 5% relative humidity (RH). The following parameters were considered: -Appearance -Recovery rate of active ingredient (w / v)

[0093] These results are shown in Table 11 below and in the graph of Figure 7A.

[0094] [Table 11]

[0095] This stability study confirms that the most stable 0.08% PHMB formulation is the one characterized by the highest molecular weight PHMB, i.e., batch L-17GR185627, with MW = 2517 amu, pH = 5.8, and a PDI value of 1.73. No significant degradation products (BPs) were observed during the stability study.

[0096] The 0.08% PHMB solution (Batch F-693 / LU / 101, MW=1170 amu - Batch F-693 / LU / 118, MW=440 amu), characterized by its low molecular weight, showed a decrease in PHMB recovery after 12 months, indicating formulation instability and reduced efficacy.

[0097] Thus, it is clear that the parameters of molecular weight and polydispersity index of PHMB, along with the pH of the solution, contribute synergistically to providing a stable and effective formulation.

[0098] Additionally, stability studies were conducted on a 0.08% PHMB solution with a pH of 5.8 and an osmolality of 0.290 osmol / Kg, characterized by a MW of 2641 amu and a PDI of 1.6. The studies were conducted under three different temperature conditions: long-term stability (25°C, 40% RH); intermediate stability (30°C, 65% RH); and accelerated stability (40°C, 25% RH). These stability results are shown below and in Figure 7(B).

[0099] [Table 12]

[0100] The results of the stability study indicate that the product is stable under all temperature conditions studied, with PHMB recoveries falling within the specified limits of 90-110% (w / v). Other chemical and physical parameters studied (data not shown), such as pH, osmolality, and impurity recovery assays, also remained stable and within the specified limits under all three climatic conditions tested.

[0101] Example 5: Clinical Trial A randomized, multicenter, double-blind, parallel-group Phase 3 study to evaluate the efficacy, safety, and tolerability of the 0.08% PHMB formulation of the present invention compared to the conventional combination therapy of 0.02% PHMB plus 0.1% propamidine in male and female adult subjects with Acanthamoeba keratitis is described in detail below.

[0102] The study is intended as a superiority study in accordance with EMA requirements (CPMP / EWP / 482 / 99) and will consist of a screening visit for inclusion and a treatment period including short outpatient visits and follow-up visits.

[0103] A total of 130 subjects with Acanthamoeba keratitis were assigned in a 1:1 ratio to one of two treatment groups: Group 1: 0.08% PHMB + placebo Group 2: 0.02% PHMB + 0.1% combination therapy.

[0104] patient: The study was conducted in male and female subjects aged 12 years and older with Acanthamoeba keratitis.

[0105] the purpose: The primary objective of this study was to compare the Clinical Resolution Rate (CRR) (CRR_12) 12 months after randomization between the combination therapy of 0.08% PHMB + placebo and 0.02% PHMB + 0.1% propamidine, to evaluate the difference between CRR_12 in terms of uncertainty, and to examine the therapeutic superiority or non-inferiority of 0.08% PHMB monotherapy.

[0106] A further objective of this study is to obtain information regarding the safety of the 0.08% PHMB-based ophthalmic formulations of the present invention.

[0107] hypothesis: The primary hypothesis to be tested is that the CRR12 in subjects treated with 0.08% PHMB formulation monotherapy will be higher, non-inferior, or acceptably lower (Δ) than the CRR12 of 0.02% PHMB + 0.1% propamidine combination therapy when administered according to the treatment protocol described below.

[0108] The second hypothesis is: -Toxic-related undesirable effects are less with 0.08% PHMB monotherapy compared with combination therapy; - Healing time will be less in subjects receiving 0.08% PHMB monotherapy compared to combination therapy.

[0109] As shown in a sponsored retrospective study (Study 038 / SI), clinical recovery achieved within 12 months of treatment initiation was 67%.

[0110] Concomitant and prior treatment Subjects are taking or have taken the following medications:

[0111] (antibiotics): Topical moxifloxacin is approved for the treatment of concomitant bacterial infections. However, topical moxifloxacin is not approved as a prophylactic antibiotic for treating patients with corneal ulcers because PHMB is already a broad-spectrum antibiotic and no additional antibiotic is needed. Topical moxifloxacin is not approved for use as a prophylactic antibiotic in patients with corneal ulcers because PHMB is a good broad-spectrum antibiotic and no additional antibiotic is needed.

[0112] (Antiviral and antifungal drugs): Use of these medications is not permitted during the study. Any intake of these medications at the start of the study must be discontinued.

[0113] (Anti-inflammatory): For subjects who are on steroid therapy at study entry (e.g., already using topical steroids for misdiagnosed HSV keratitis or as adjunctive treatment for bacterial keratitis), the following options are available: a. Discontinuation, maintenance, or reduction of steroid administration. Dexamethasone (0.1% or 0.15%) is the only topical steroid permitted in this study. Patients using other topical steroids at the start of the study will need to change the frequency of administration. Diclofenac is the only oral NSAID permitted in this study, and will be added at an appropriate single dose (75 mg to 150 mg / day, divided into 2 to 3 doses). b. Subjects using NSAIDs or cyclosporine at the start of the clinical trial must discontinue treatment after randomization. c. Subjects who were not using topical steroids at study entry may be initiated on topical steroids along with an oral NSAID (recommended diclofenac; 75 mg to 150 mg / day, divided into 2 to 3 doses) during the study, as specified in the protocol.

[0114] Other topical treatments allowed: lubricants, mydriatics (cyclopentolate, homatropine, or atropine), and antiglaucoma medications.

[0115] dose The doses used in clinical efficacy studies are as follows: Administration of escalating doses: 16 drops / day for 5 days, 8 drops / day for 7 days, 6 drops / day for 7 days, and 4 drops / day until clinical recovery.

[0116] Treatment Allocation: Protocol for bilateral disease: If both eyes are affected, only one of the two (the right eye unless there is a difference in severity, such as requiring treatment of the more compromised eye) will be treated according to the therapeutic dose and considered for the study. The other eye will be treated according to standard practice.

[0117] Table 13 below summarizes the demographics and distribution of patients participating in the study.

[0118] [Table 13]

[0119] Table 14 shows the 12-month clinical recovery rate, with patients divided into those with and without prior steroid treatment.

[0120] [Table 14]

[0121] result: Primary efficacy: 12-month clinical recovery rate The results of the clinical trial demonstrated that 0.08% PHMB monotherapy was non-inferior to 0.02% PHMB + 0.1% propamidine combination therapy in terms of clinical recovery rate (CRR_12) at 12 months. * The CRR_12 of 0.08% PHMB monotherapy was 87.1%. * The CRR_12 of 0.02% PHMB + 0.1% propamidine was 89.5%.

[0122] Monotherapy was always preferable to combination therapy, and the results did not show a statistically significant difference.

[0123] The percentage of subjects previously treated with corticosteroid-based therapy is shown below: -0.08% vs. 22.6% for PHMB monotherapy -10% for 0.02% PHMB + 0.1% propamidine combination therapy

[0124] The CRR12 clinical recovery rates for patients with and without prior steroid treatment are shown below: * The CRR_12 of 0.08% PHMB monotherapy was 91.4%. * The CRR_12 of 0.02% PHMB + 0.1% propamidine was 87.8%.

[0125] The CRR_12 obtained with monotherapy is unexpected in both cases, since the CRR_12 obtained with combination therapy in routine clinical practice is approximately 63-67%. This clinical trial therefore demonstrates that monotherapy under the described formulation conditions (MW, PDI, and PHMB polymer concentration 0.04%-0.008%, pH, and osmolality) is not only comparable to combination therapy, but in both cases the results obtained are better than those reported in previous clinical practice (retrospective study 083 / SI).

[0126] (Secondary benefit: healing time) Treatment-related healing times are: * 0.08%PHMB group: 138.3 days * (Range: 32 to 365 days) * 0.02% PHMB + propamidine 0.1% group: 117.1 * days (range 54-214 days) * Average

[0127] The 0.08% PHMB treatment showed a wider healing time range. Generally, longer healing times are associated with monotherapy versus combination therapy, but unexpectedly, the lower end of the healing time range for monotherapy showed recovery at 32 days. These results were not statistically significant.

[0128] Table 15 shows the results for time-to-care for treated patients.

[0129] [Table 15]

[0130] References [1] Kilvington S, Larkin DF. Acanthamoeba adherence to contact lenses and removal by cleaning agents. Eye1990;4:589-593. [2] Sharma R., et al.Coinfection with Acanthamoeba and Pseudomonas in contact lens-associated keratitis. Optom Vis Sci. 2013; 90(2): e53-5 [3] Singh A., et al. Acanthamoeba Keratitis Versus Mixed Acanthamoeba and Bacterial Keratitis: Comparison of Clinical and Microbiological Profiles. Cornea. 2020; 39:1112-1116. [4] Nakagawa H., et al.Number of Bacteria and Time of Coincubation With Bacteria Required for the Development of Acanthamoeba Keratitis. Cornea. 2017; 36:353-357. [5] Larkin D. F., etal., Treatment of Acanthamoeba keratitis with polyhexamethylene biguanide,Ophthalmology, 99 (1992) 185. [6] Dave S.B., Changes in ocular flora in eyes exposed to ophthalmic antibiotics. Ophthalmology. 2013;120:937-41. [7] Ian J. et al.,Effects of Polyhexamethylene Biguanide and Polyquaternium-1on PhospholipidBilayer Structure and Dynamics, J. Phys.Chem. B 2015, 119,10531-10542. [8] M. J. Allen, et al.White Cooperativity in the binding of the cationic biocide polyhexamethylene biguanide to nucleic acids. Biochemicaland Biophysical Research Communications 318 (2004) 397-404. [9] K. Chindera, et al. The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes. ScientificReports 6:23121.

[10] Papa V. et al. Ocular safety and tolerability of high dose PHMB (Polyhexanide) in healthy volunteers. ARVO,2017.

[11] Sowlati-Hashjin S., et al. Insights into the Polyhexamethylene Biguanide (PHMB) Mechanism of Action on Bacterial Membrane and DNA: A Molecular Dynamics Study. J Phys Chem B. 2020;124:4487-4497.

[12] Domanda di brevetto US2007 / 0140897 A1.

[13] Y. Bouattour et al. Stability of an ophtalmic formulation of polyhexamethylene biguanide in gamma-sterilized and ethylene oxide sterilized low density polypropylene multidose eyedroppers. PEERJ, vol. 6 pag. e4549(2018). <Additional Notes> Aspects of the present invention include the following. <Section 1> 1. A polyhexamethylene biguanide-based solution suitable for ophthalmic administration for use in the treatment of Acanthamoeba keratitis and / or fungal infections, comprising polyhexamethylene biguanide at a concentration in the range of 0.04% to 0.08% (w / v), a buffer system for maintaining a pH in the range of 5 to 6.5, and a tonicity agent for maintaining an osmolarity in the range of 270 to 330 mOsm / kg, wherein the molecular weight of the polyhexamethylene biguanide is in the range of 2,300 to 6,000 amu, and the polydispersity index of the polymer is in the range of 1.5 to 1.9. <Section 2> A polyhexamethylene biguanide-based liquid preparation for use as described in <Item 1> for the treatment of simultaneous infections caused by Acanthamoeba and Pseudomonas aeruginosa or Staphylococcus epidermis. <Section 3> A polyhexamethylene biguanide-based liquid formulation for use according to <Item 1> or <Item 2>, wherein the buffer system is selected from the group consisting of phosphate buffer, citrate buffer, bicarbonate buffer, borate buffer, Tris buffer, glycerol buffer, mannitol buffer, sorbitol buffer, trometamol buffer, histidine buffer, glycine buffer, HEPES buffer, and mixed systems. <Section 4> The polyhexamethylene biguanide-based liquid preparation for use according to <Item 3>, wherein the buffer system is selected from the group consisting of disodium phosphate dodecahydrate / sodium dihydrogen phosphate, disodium phosphate dodecahydrate / citric acid, Tris / HCl, and boric acid / borate salts. <Section 5> The polyhexamethylene biguanide-based liquid preparation for use according to any one of <Item 1> to <Item 4>, wherein the tonicity agent is sodium chloride or potassium chloride. <Section 6> A liquid preparation based on polyhexamethylene biguanide for use according to any one of <Item 1> to <Item 5>, wherein the polydispersity index of the polymer is within the range of 1.7 to 1.8. <Section 7> A polyhexamethylene biguanide-based liquid preparation for use according to any one of <Item 1> to <Item 6>, having a pH of 5.8. <Section 8> A polyhexamethylene biguanide-based liquid preparation for use according to any one of <Item 1> to <Item 7>, which is in the form of an eyewash, eye drops, ophthalmic gel, or viscous solution. <Section 9> A polyhexamethylene biguanide-based liquid preparation for use according to <Item 8>, in the form of an ophthalmic gel or viscous solution, further comprising a thickener selected from the group consisting of xanthan gum, gellan gum, polyvinyl alcohol, hyaluronic acid, sodium hyaluronate, carboxymethylcellulose, and hydroxypropylcellulose. <Section 10> A polyhexamethylene biguanide-based liquid preparation for use according to any one of <Item 1> to <Item 9>, further comprising a penetration enhancer selected from Tween 80 and benzalkonium chloride. <Section 11> A polyhexamethylene biguanide-based liquid preparation for use according to any one of <Item 1> to <Item 10>, further comprising 0.02% chlorhexidine, 0.1% propamidine, or 0.1% desomedine. <Section 12> A polyhexamethylene biguanide-based solution for use according to any one of <Item 1> to <Item 11>, administered ophthalmically in graded doses of 16 drops / day for 5 days, 8 drops / day for 7 days, 6 drops / day for 7 days, and 4 drops / day until clinical recovery. <Section 13> A method for preparing a liquid formulation based on polyhexamethylene biguanide according to any one of <Item 1> to <Item 12>, comprising the following steps: (i) adding, under gentle and continuous stirring, to purified water, the buffer system at a concentration ranging from 0.01% to 4.5% (w / v) and the tonicity agent at a concentration ranging from 0.01% to 5.5% (w / v); and optionally, adding a viscosity-increasing agent selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose, and hydroxypropylcellulose; (ii) adding polyhexamethylene biguanide having a molecular weight in the range of 2,300 to 6,000 amu and a polydispersity index in the range of 1.5 to 1.9, preferably 1.7 to 1.8, at a concentration in the range of 0.04% to 0.08% (w / v) with gentle stirring; (iii) bringing to final volume by adding purified water; (iv) Sterilizing by filtration or heat. <Section 14> Item 13. The method for preparing a polyhexamethylene biguanide-based liquid formulation according to <Item 13>, wherein the buffer system is selected from the group consisting of phosphate buffer, citrate buffer, bicarbonate buffer, borate buffer, Tris buffer, glycerol buffer, mannitol buffer, sorbitol buffer, trometamol buffer, histidine buffer, glycine buffer, HEPES buffer, and a mixed system. <Section 15> Item 14. A method for preparing a polyhexamethylene biguanide-based liquid formulation according to item 14, wherein the buffer system is selected from the group consisting of disodium phosphate dodecahydrate / sodium dihydrogen phosphate, disodium phosphate dodecahydrate / citric acid, Tris / HCl, and boric acid / borate salts. <Section 16> The method for preparing a polyhexamethylene biguanide-based liquid preparation according to any one of <Item 13> to <Item 15>, wherein the isotonicity agent is sodium chloride or potassium chloride. <Section 17> An eye lens loaded with the polyhexamethylene biguanide-based liquid formulation according to any one of Items 1 to 11, for controlled release of an active ingredient for use in the treatment of Acanthamoeba keratitis, simultaneous infection with Acanthamoeba and Pseudomonas aeruginosa, or fungal infection.

Claims

1. 1. A polyhexamethylene biguanide-based liquid formulation for ophthalmic administration for use in the treatment of acanthamoeba keratitis and / or fungal infections, comprising polyhexamethylene biguanide at a concentration in the range of 0.04% to 0.08% (w / v), a buffer system for maintaining a pH in the range of 5 to 6.5, and a tonicity agent for maintaining an osmolality in the range of 270 to 330 mOsm / kg, wherein the molecular weight of the polyhexamethylene biguanide is in the range of 2,300 to 6,000 amu, and the polyhexamethylene biguanide has a polydispersity index in the range of 1.5 to 1.

9.

2. 2. A polyhexamethylene biguanide-based solution for use according to claim 1 for the treatment of co-infections with Acanthamoeba and Pseudomonas aeruginosa or Staphylococcus epidermis.

3. 3. A polyhexamethylene biguanide-based liquid formulation for use according to claim 1 or claim 2, wherein the buffer system is selected from the group comprising phosphate buffer, citrate buffer, bicarbonate buffer, borate buffer, Tris buffer, glycerol buffer, mannitol buffer, sorbitol buffer, trometamol buffer, histidine buffer, glycine buffer, HEPES buffer, and mixed systems.

4. 4. The polyhexamethylene biguanide-based liquid formulation for use according to claim 3, wherein the buffer system is selected from the group consisting of disodium phosphate dodecahydrate / sodium dihydrogen phosphate, disodium phosphate dodecahydrate / citric acid, Tris / HCl, and boric acid / borate.

5. A polyhexamethylene biguanide-based solution for use according to any one of claims 1 to 4, wherein the tonicity agent is sodium chloride or potassium chloride.

6. A polyhexamethylene biguanide-based liquid formulation for use according to any one of claims 1 to 5, wherein the polyhexamethylene biguanide has a polydispersity index in the range of 1.7 to 1.

8.

7. A polyhexamethylene biguanide-based liquid formulation for use according to any one of claims 1 to 6, having a pH of 5.

8.

8. A polyhexamethylene biguanide-based solution for use according to any one of claims 1 to 7, in the form of an eyewash, eye drops, ophthalmic gel or viscous solution.

9. 9. The polyhexamethylene biguanide-based solution for use according to claim 8, in the form of an ophthalmic gel or viscous solution, further comprising a thickening agent selected from the group consisting of xanthan gum, gellan gum, polyvinyl alcohol, hyaluronic acid, sodium hyaluronate, carboxymethylcellulose, and hydroxypropylcellulose.

10. A polyhexamethylene biguanide-based solution for use according to any one of claims 1 to 9, further comprising a penetration enhancer selected from Tween 80 and benzalkonium chloride.

11. A polyhexamethylene biguanide-based solution for use according to any one of claims 1 to 10, further comprising 0.02% chlorhexidine, 0.1% propamidine or 0.1% desomedine.

12. 12. A polyhexamethylene biguanide-based solution for use according to any one of claims 1 to 11 by ophthalmic administration in graded doses of 16 drops / day for 5 days, 8 drops / day for 7 days, 6 drops / day for 7 days, and 4 drops / day until clinical recovery.

13. A method for preparing a polyhexamethylene biguanide-based liquid formulation according to any one of claims 1 to 12, comprising the steps of: (i) adding, under gentle and continuous stirring, to purified water, the buffer system at a concentration ranging from 0.01% to 4.5% (w / v) and the tonicity agent at a concentration ranging from 0.01% to 5.5% (w / v); optionally, adding a viscosity increasing agent selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose, and hydroxypropylcellulose; (ii) adding polyhexamethylene biguanide having a molecular weight in the range of 2,300 to 6,000 amu and a polydispersity index in the range of 1.5 to 1.9 at a concentration in the range of 0.04% to 0.08% (w / v) with gentle stirring; (iii) bringing to final volume by adding purified water; (iv) Sterilizing by filtration or heat.

14. A method for preparing a polyhexamethylene biguanide-based liquid formulation according to claim 13, wherein the polyhexamethylene biguanide has a polydispersity index in the range of 1.7 to 1.

8.

15. 15. A method for preparing a polyhexamethylene biguanide-based liquid formulation according to claim 13 or claim 14, wherein the buffer system is selected from the group comprising phosphate buffer, citrate buffer, bicarbonate buffer, borate buffer, Tris buffer, glycerol buffer, mannitol buffer, sorbitol buffer, trometamol buffer, histidine buffer, glycine buffer, HEPES buffer, and mixed systems.

16. 16. The method for preparing a polyhexamethylene biguanide-based liquid formulation according to claim 15, wherein the buffer system is selected from the group consisting of disodium phosphate dodecahydrate / sodium dihydrogen phosphate, disodium phosphate dodecahydrate / citric acid, Tris / HCl, and boric acid / borate.

17. A method for preparing a polyhexamethylene biguanide-based liquid formulation according to any one of claims 13 to 16, wherein the tonicity agent is sodium chloride or potassium chloride.

18. 12. An eye lens loaded with the polyhexamethylene biguanide-based solution of any one of claims 1 to 11 for controlled release of an active ingredient for use in the treatment of Acanthamoeba keratitis, co-infection with Acanthamoeba and Pseudomonas aeruginosa, or fungal infections.

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