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 Ophthalmic level This invention relates to a stable polyhexamethylene biguanide (PHMB)-based formulation suitable for administration, a method for preparing the same, and a related administration plan that is effective in treating Acanthamoeba keratitis, particularly in eradicating cysts. [Background technology]

[0002] Acanthamoeba keratitis is an infection of the cornea, the clear tissue that lines the anterior segment of the eye. This infection is commonly caused by Acanthamoeba, a genus of microorganisms found in both rivers and bodies of water (lakes, seas, and rivers), as well as in tap water, swimming pools, underwater massage baths, soil, and air. [Overview of the project]

[0003] Acanthamoeba keratitis is more common in contact lens wearers[1], but anyone with a corneal lesion can be at risk of developing this serious eye infection.

[0004] Generally, Acanthamoeba species have a two-phase life cycle: an active form (the form in which the microorganism feeds and replicates) and a dormant form (the form in which the microorganism protects itself from attacks that can occur through cyst formation). When developing effective anti-amebic drugs, cysts are the actual target.

[0005] Co-infection with bacterial species is common in patients with Acanthamoeba keratitis. Specifically, Staphylococcus species and Pseudomonas aeruginosa are the most commonly 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 very unpleasant and, in some cases, can irritate the surface of the cornea. Approximately 25% of treated cases of Acanthamoeba keratitis require a corneal transplant. If left untreated, the disease progresses to blindness as a result of corneal neovascularization and corneal scarring or perforation.

[0007] To date, there are no approved medications for the treatment of Acanthamoeba keratitis. While biguanides and diamidines have shown efficacy against Acanthamoeba keratitis in many clinical cases, the treatment regimens and concentrations of these medications have been developed empirically.

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

[0009] Current treatments for Acanthamoeba keratitis are long-term and challenging. These treatments begin with hourly doses during the day, avoiding nighttime administration, for approximately 1-2 weeks, and are then reduced depending on the patient's response. While it varies from patient to patient, those who receive an early diagnosis and prompt appropriate treatment can generally expect treatment to last 3-6 months.

[0010] Furthermore, repeated and prolonged use of antibiotics and disinfectants, such as those mentioned above, carries the risk of significantly altering the composition of the conjunctival microbiota by increasing the proportion of pathogens such as Staphylococcus epidermidis to a degree that is detrimental to the normal symbiotic bacterial flora. This can have significant clinical implications, as Staphylococcus epidermidis is one of the main causes of eye diseases such as conjunctivitis, keratitis, and endophthalmitis.[6]

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

[0012] PHMB-based formulations are mixtures of biguanide polymers having a molecular weight that can vary from 400 to 8,000 amu and a variable degree of polymerization in which n can range from 2 to 40.

[0013] The mechanism of action of PHMB has been the subject of various studies in in vitro experimental models. The SUV membrane is a system that mimics the chemical activity of the cell membrane of human corneal epithelium. When PHMB interacts with the bilayer of the SUV membrane, the cooperativity of the phospholipids increases, water is excluded from the bilayer consisting of acyl chains and polar heads, the acyl chain-containing region becomes less dynamic and selective, and PHMB is absorbed onto the surface of the bilayer [7].

[0014] The cationic nature of PHMB enables interaction with anionic macromolecules such as DNA. Literature has shown that PHMB interacts with DNA in aqueous solution, forming a complex between the two chemical species. This reaction involves electrostatic interactions between the cationic biguanide group and the anionic phosphate group of the DNA deoxyribonucleotide in a molar ratio of approximately 1:1 [8].

[0015] As has already been reported for other bacteria such as Escherichia coli, Salmonella enteritidis, and Staphylococcus aureus, the structure of Acanthamoeba, characterized by the exposure of its DNA binding site, facilitates PHMB's access to the protozoan's DNA, promoting the chromosome condensation and disruption processes of the microorganism [9]. Although these studies were not specific to Acanthamoeba, they were useful to the inventors in identifying the optimal conditions under which PHMB binds to the protozoan's DNA and exerts its best e-phase efficacy in the treatment of infectious keratitis.

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

[10] , even higher concentrations of PHMB—0.04%, 0.06%, and 0.08%—were shown to be safe and well-tolerated in healthy subjects [8]. The pH and osmolarity of the formulation were not described, nor were the molecular weight and polydispersion index of the PHMB polymer used. However, as will be discussed later, PHMB alone at these concentrations is not sufficient to maximize the interaction of PHMB with Acanthamoeba DNA and the efficacy of the treatment.

[0017] U.S. Patent Application Publication 2007 / 0140897A1

[12] describes an ophthalmic formulation based on an active ingredient containing a pH 4-6 biguanide (including PHMB) for the treatment of Acanthamoeba keratitis. However, the only formulation exemplified is alexidine-based, not PHMB-based. In any case, the relevance of the MW and PDI of the biguanide polymer in its interaction with the pathogen's DNA, nor the specific concentrations of the biguanide polymer used in combination with the above parameters, are not described.

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

[13] of a formulation based on 0.02% PHMB at pH 4 revealed that degradation products (BPs) had already formed after 90 days. These authors concluded that PHMB-based formulations cannot be stored for more than 60 days in EOS-LDPE bottles at 25°C. The authors of this invention have now found that by adjusting the molecular weight (MW) and polydispersity index (PDI) of the PHMB polymer, along with pH and osmolality, stability and efficacy can be maintained at the highest active ingredient concentration in the range of 0.04%(w / v) to 0.08%(w / v). Ophthalmic administration We are identifying polyhexamethylene biguanide-based formulations for use.

[0019] Studies have shown that using high molecular weight, high polydispersity index PHMB in conjunction 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 for at least 24 months and for the activity of the active ingredient. [Modes for carrying out the invention]

[0020] For the reasons described above, in reality, for the PHMB molecule to optimally exert its biocidal mechanism of action, it must be in its protonated form. In its ionized form after protonation, PHMB passes through the epithelium of the cornea infected with Acanthamoeba and accumulates in the corneal stroma by electrostatic interaction with the negative charge of proteoglycans. In this corneal stroma, according to the latest scientific research, PHMB exhibits its biocidal and cystsicidal activity through a mechanism of action that is expressed through binding to DNA through extensive interaction with the phosphate groups of DNA. This binding blocks the replication process of amoeba DNA, causing the death of the pathogen

[11] . Thus, the effectiveness of the interaction between PHMB and DNA described above, and hence the activity of PHMB itself, is greatly affected by the stability of the formulation containing the active ingredient. Furthermore, administration of the formulation of the present invention in a strict stepwise dosing schedule in patients with Acanthamoeba keratitis has been found to be particularly useful from the perspective of the activity against complete eradication of Acanthamoeba cysts.

[0021] In this sense, since the formulation object of the present invention is a monotherapy, it is particularly possible to avoid repeatedly infusing various products during the day, which is advantageous for patient compliance and adherence to the treatment regimen. Furthermore, considering the selectivity and specificity of the product against target microorganisms, greater rapidity of action is ensured, and as a result, the pathological condition is recovered earlier, avoiding a long treatment period of 3 to 6 months.

[0022] Repeatedly, compared with multi-drug therapy composed of currently used powerful antiseptics and broad-spectrum antibiotics, monotherapy with 0.08% PHMB (highly selective and specific) is indeed 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] Therefore, the present invention provides a buffer system for maintaining the pH within the range of 5 to 6.5, and an isotonic agent for maintaining the osmolarity within the range of 270 to 330 mOsm / Kg, and a polyhexamethylene biguanide-based solution or its salt with a concentration in the range of 0.04% to 0.08%. The molecular weight of the polyhexamethylene biguanide is in the range of 2,300 to 6,000 amu, the polydispersity index of the polymer is in the range of 1.5 to 1.9, preferably in the range of 1.7 to 1.8, and it is for use in the treatment of acanthamoeba keratitis or fungal infections. Ophthalmic administration It relates to a polyhexamethylene biguanide-based solution or its salt suitable for

[0024] The fungal infection is preferably keratitis or corneal mycosis 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 coexist with Acanthamoeba infections, especially Fusarium solani infections and Cladosporium infections.

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

[0027] Therapeutic use of 0.08% PHMB preparations active against Pseudomonas aeruginosa (data not described, internal APE test study: orphan drug-in vitro assay-1305) has demonstrated high performance in achieving rapid and effective recovery from the aforementioned co-infections. Polyhexamethylene biguanides may take the form of inorganic or organic salts selected from the group consisting of chlorides, bromides, sulfates, phosphates, mesylates, formates, citrates, or maleates.

[0028] In addition to the need to maintain a slightly acidic pH so that PHMB is in its maximally protonated state, a further objective of the present invention is to maintain the molar osmolarity of the formulation at a value equivalent to that of tears (ranging in the range of 290-320 mOsm / Kg) by adding, preferably, an ionic isotonic agent selected from the group consisting of sodium chloride and potassium chloride, or a nonionic isotonic agent such as glycerol, mannitol, or sorbitol, which also contributes to osmotic pressure; an ionic buffer system such as phosphate buffer, citrate buffer, bicarbonate buffer, or borate buffer; or a nonionic buffer such as trometamol, histidine, glycine, or HEPES.

[0029] Osmotic pressure is actually the amount of ions (Na) present in the aqueous layer. + , K + Cl - It primarily relies on , and HCO3.

[0030] According to a preferred embodiment of the present invention, the isotonic 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 the polyhexamethylene biguanide-based liquid formulation, the buffer system is an ionic buffer such as phosphate buffer, citrate buffer, bicarbonate buffer, or borate buffer, or a nonionic buffer such as trometamol, histidine, glycine, HEPES, or a mixed system.

[0032] The buffer system preferably contains disodium phosphate dodecahydrate (Na2HPO4·12H2O) and sodium dihydrogen phosphate (NaH2PO4·H2O). 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, a 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 When the solution is in the form of a gel or viscous solution, it is preferable to use, for example, Tris buffer and / or a phosphate-free buffer such as boric acid or a borate.

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

[0036] In preferred embodiments 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), and is preferably 0.08% (w / v).

[0037] Ophthalmic administration The polyhexamethylene biguanide-based liquid formulation of the present invention, which is suitable for use, is a sterile solution that can be made into eye drops.

[0038] Alternatively, in the presence of a thickening agent (for example, xanthan gum, gellan gum, polyvinyl alcohol, hyaluronic acid, sodium hyaluronate, carboxymethylcellulose, or hydroxypropylcellulose or other cellulose derivatives), the polyhexamethylene biguanide-based formulation of the present invention may be used. ophthalmological It can be in the form of a gel or a viscous solution.

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

[0040] Furthermore, according to a preferred embodiment, the polyhexamethylene biguanide-based solution for use of the present invention may further contain, in combination with PHMB, 0.02% chlorhexidine, 0.1% propamidine, and 0.1% desomeidine. When PHMB is administered in combination with the 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 for the treatment of Acanthamoeba keratitis by administering a stepwise dose 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. Clinical recovery was 88% over an average of 4 months. This stepwise dose differs from the intensified dose previously reported in a Phase I trial conducted by Papa et al.

[10] because it more effectively intervenes in cyst eradication and, consequently, clinical recovery from Acanthamoeba keratitis.

[0042] The present invention further relates to a method for preparing a liquid formulation based on the polyhexamethylene biguanide of the present invention, comprising the following steps: (i) Adding the buffer system in a concentration of 0.01% to 4.5% (w / v) and the isotonic agent in a concentration of 0.01% to 5.5% (w / v) to purified water while gently and continuously stirring; optionally adding a thickener selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose, and hydroxypropylcellulose; (ii) Add a 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) while gently stirring; (iii) The process of adjusting the final volume by adding purified water; (iv) A process of sterilization by filtration or heat.

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

[0044] This solution is then dispensed into single-dose containers or vials and sealed. These single-dose containers may be made of low-density polyethylene (LDPE), and each package can contain 5 single-dose units.

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

[0046] In a preferred embodiment, the buffer system is based on disodium phosphate dodecahydrate (Na2HPO4·12H2O) and sodium dihydrogen phosphate (NaH2PO4·H2O). Disodium phosphate dodecahydrate is preferably used at a concentration in the range of 0.01% to 4.5% (w / v), and sodium dihydrogen phosphate is used at a concentration in the range of 0.01% to 3.5% (w / v).

[0047] The isotonic agent used to maintain the molar osmotic pressure concentration within the range of 270 to 330 mOsm / Kg is preferably sodium chloride.

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

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

[0050] The present invention will be described below, for illustrative purposes only and without limitation, based on preferred embodiments, with particular reference to the accompanying drawings. [Brief explanation of the drawing]

[0051] [Figure 1]Figure 1 shows the structure of the hexamethylene biguanide repeat unit in PHMB; [Figure 2] Figure 2 shows a flowchart of the preparation method for PHMB-based formulations; [Figure 3] Figure 3 shows the chromatogram obtained by gel permeation chromatography of PHMB in the API. [Figure 4] Figure 4 shows the chromatogram obtained by gel permeation chromatography of PHMB in the final product; a comparison with the chromatogram of the raw material in Figure 2 clearly demonstrates how this manufacturing method can maintain the molecular weight distribution of the original raw material up to the final product. [Figure 5] Figure 5 shows a comparison of stability data for formulations based on 0.08% PHMB at pH 7.4 and pH 5.8. [Figure 6] Figure 6 shows a comparison of PHMB-DNA binding with respect to molecular weight. From the trend in this graph, it can be understood that using PHMB with a high molecular weight results in more effective polymer-DNA binding because it includes one or more nucleic acid molecules. High molecular weight polymers can form crosslinks between different nucleic acid molecules, creating a binding network (lines containing circles). Conversely, lines containing squares indicate the establishment of linear binding between PHMB and DNA. Polymers characterized by lower molecular weights do not demonstrate suitability for crosslink formation, but they do establish bindings containing 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 index (PDI), expressed as the recovery rate (w / v) of PHMB at T0 and 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=2,641amu and PDI=1.6. This study was conducted under three different temperature conditions: long-term stability (25°C, RH40%); intermediate stability (30°C, RH65%); and accelerated stability (40°C, RH25%). The stability results are shown as the w / v recovery rate of PHMB at T0 and 3 months (T3). [Examples]

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

[0053] (Example 1): Method for preparing a PHMB-based formulation A flowchart in Figure 2 shows the method for preparing the formulation of the present invention.

[0054] Disodium phosphate dodecahydrate (Na2HPO4·12H2O), sodium dihydrogen phosphate (NaH2PO4·H2O), and sodium chloride are added sequentially, and dissolved in a large amount of purified water while gently stirring.

[0055] This stirring step is important for maintaining the initial molecular weight and polydispersity index of PHMB and must be performed in a way that does not create bubbles or vortices in the solution. It is preferable to use a stirring bar or impeller that is submerged in the solution for stirring. Finally, add PHMB (0.08%).

[0056] Add a sufficient amount of purified water while continuously stirring to bring the solution to its final volume (100 ml). Next, adjust the pH and appearance of the solution.

[0057] Sterilization is performed by filtration with a 0.2 μM filter. To avoid depolymerization or cleavage of polymer chains, the properties and dynamics (pressure, flow rate) of the filter membrane are selected to prevent the product from forcibly passing through the membrane.

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

[0059] Finally, using this sterile solution, single-use containers are filled under sterile conditions using batch-fill-form-and-fill (B / F / S) technology.

[0060] This automated technology allows for a range of 0.1 to 1,000 cm. 3 Vials can be filled and sealed under sterile conditions with solution volumes within this range.

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

[0062] At the end of the manufacturing process, the final product is characterized by its main components, including any impurities, using a preferred chromatography method.

[0063] A comparison of the PHMB chromatogram in the API (Figure 3) and the PHMB chromatogram after formulation preparation (Figure 4) shows that the molecular weight distribution of PHMB remains unchanged.

[0064] (Example 2): Comparative study on the stability of PHMB-based formulations The manufactured PHMB-based formulation is a sterile solution in which pH and molar osmolality are relevant physicochemical parameters.

[0065] As detailed, the selection of a slightly acidic pH was based on the need to obtain a suitable formulation that could not be obtained at a pH near neutral.

[0066] For this purpose, various formulations based on 0.08% PHMB at different pH values ​​(5.8, 6.2, and 7.4) were developed, and their stability at 25°C, 40°C, and 60°C was subsequently verified. To prepare a 0.08% PHMB-based formulation at pH 5.8, the following components 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 formulation based on 0.08% PHMB at pH 6.2, the following components were weighed according to the same method as described 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 formulation based on 0.08% PHMB at pH 7.4, the following components 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] Next, a comparison was made regarding the stability of 0.08% PHMB-based formulations in single-dose containers at pH 5.8 and pH 7.4 under 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 preparation, pH 5.8, 25℃±2℃, 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 PHMB-based formulations obtained by maintaining the pH within the range of 5 to 6.5 is not achieved at pH levels around neutral (pH 7.4).

[0086] (Example 3): Study of PHMB-DNA binding To evaluate the equivalence between low-molecular-weight PHMB and high-molecular-weight PHMB, we conducted studies on PHMB-DNA binding.

[0087] The DNA-binding ability of PHMB can be considered independent of the specific DNA species studied, as shown in reference [8], although the DNA-binding ability and effectiveness depend on the molecular weight and polydispersity index of the PHMB polymer. Binding to DNA is more effective 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, likely because the length of the polymer is more suitable for the formation of 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 binding becomes linear and involves a single nucleic acid molecule.

[0088] Next, to evaluate the binding ability to nucleic acid molecules, a comparative study was conducted using different PHMB polymer samples. PHMB (Sample A) was decomposed by acid and high-temperature treatment for 5 hours to obtain a low molecular weight PHMB polymer. PHMB (Sample B) underwent no type of treatment and is 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 herein is based on the interaction between aqueous PHMB and aqueous DNA solutions. The content of the PHMB-DNA complex was measured as the difference between the initial content of both substances in the solution and the residual amount after the reaction was complete. The content of both substances in the solution was monitored by spectrophotometric measurements at two different wavelengths: 260 nm for DNA and 235 nm for PHMB.

[0090] In conclusion, data show that the ability of high molecular weight PHMB to interact with DNA is increased through the formation of crosslinks with multiple nucleic acid molecules, resulting in greater efficacy in treating infectious Acanthamoeba keratitis and co-infections with Pseudomonas aeruginosa. As shown in the graph in Figure 6, the type of binding formed between the PHMB polymer and DNA is significantly more effective.

[0091] (Example 4): Stability study related to molecular weight and polydispersity index of PHMB A comparative study was conducted on three different PHMB-based initiation batches characterized by the following different molecular weights and polydispersity index (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] A 0.08% PHMB formulation was prepared for each lot, characterized by a pH of 5.8 and a molar osmolarity of 0.280 osmol / kg. The stability of the PHMB formulation was then evaluated at T0 and 12 months (T=12) under conditions of 25°C ± 2°C and 40 ± 5% relative humidity (RH). The parameters considered were as follows: - Appearance - Recovery rate of active ingredient (w / v)

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

[0094] [Table 11]

[0095] This stability study confirmed that the most stable 0.08% PHMB formulation is batch L-17GR185627, which features the highest molecular weight PHMB, i.e., MW=2517amu, pH=5.8, and PDI value 1.73. No significant degradation products (BP) were observed during the stability study.

[0096] In 0.08% PHMB solutions characterized by low molecular weight (batch F-693 / LU / 101, MW=1170amu - batch F-693 / LU / 118, MW=440amu), a decrease in PHMB recovery rate after 12 months was observed, indicating instability and reduced efficacy of the formulation.

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

[0098] Furthermore, a stability study was conducted on a 0.08% PHMB solution with a pH of 5.8, an osmolality of 0.290 osmol / kg, characterized by MW=2641amu and PDI=1.6. This study was performed under three different temperature conditions: long-term stability (25°C, RH40%); intermediate stability (30°C, RH65%); and accelerated stability (40°C, RH25%). These stability results are shown below and in Figure 7(B).

[0099] [Table 12]

[0100] The stability test results showed that the PHMB recovery rate remained within the specified limit of 90–110% (w / v), indicating that the product is stable under all the temperature conditions tested. Other chemophysical parameters tested (data not provided), such as pH, osmolality, and impurity recovery assays, also maintained stability and remained within their specified limits under all three climatic conditions tested.

[0101] (Example 5): Clinical trial A randomized, multicenter, double-blind, parallel-group Phase 3 trial 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 + 0.1% propamidine in adult male and female subjects with Acanthamoeba keratitis is described in detail below.

[0102] This study is intended as a superiority study based on EMA requirements (CPMP / EWP / 482 / 99). The study consists of a screening visit for selection and a treatment period including short-term outpatient visits and follow-up visits.

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

[0104] patient: This study was conducted on male and female subjects aged 12 years or older who had Acanthamoeba keratitis.

[0105] the purpose: The primary objective of this study is to compare the Clinical Resolution Rate (CRR) (CRR_12) at 12 months after randomization between 0.08% PHMB + placebo and 0.02% PHMB + 0.1% propamidine combination therapy, evaluate the difference in CRR_12 in relation to uncertainty, and 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 ophthalmic formulation based on 0.08% PHMB of the present invention.

[0107] hypothesis: The primary hypothesis to be tested is that, when administered according to the treatment protocol described below, the CRR12 of subjects treated with 0.08% PHMB monotherapy will be higher, not lower, or at least within acceptable limits (Δ) compared to the CRR12 of subjects treated with 0.02% PHMB + 0.1% propamidine combination therapy.

[0108] The second hypothesis is as follows: - Undesirable toxicity-related effects are less frequent with 0.08% PHMB monotherapy compared to combination therapy; - Healing time was shorter in subjects who received 0.08% PHMB monotherapy compared to those who received combination therapy.

[0109] As shown in the sponsor's retrospective study (Study 038 / SI), 67% of patients achieved clinical recovery within 12 months of the start of treatment.

[0110] Combination therapy and pretreatment The subjects have been observed to have taken or have a history of taking the following medications:

[0111] (antibiotics): Topical moxifloxacin is approved for the treatment of associated 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 antibiotics are 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 antibiotics are needed for this purpose.

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

[0113] (Anti-inflammatory drugs): For subjects who are receiving steroid treatment at the start of the study (for example, those already using topical steroids for a misdiagnosis of 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 approved in this study. Patients using other topical steroids at the start of the study will need to adjust their administration frequency. Diclofenac is the only oral NSAID approved in this study and will be added in 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 will need to discontinue treatment after randomization. c. Subjects who were not using topical steroids at the time of enrollment in the study may initiate topical steroids during the study along with an oral NSAID (recommended diclofenac; 75 mg to 150 mg / day, divided into 2 to 3 doses), as specified in the protocol.

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

[0115] dose The dosages used in clinical efficacy trials are as follows: The treatment involves a gradual dose regimen: 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, treat only one eye (the right eye unless there is a difference in severity, such as the more susceptible eye requiring treatment) according to the prescribed dosage and consider it for testing. Treat the other eye 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 clinical recovery rate over 12 months. Patients were divided into those who received prior steroid treatment and those who did not.

[0120] [Table 14]

[0121] result: Primary efficacy: Clinical recovery rate over 12 months Clinical trial results clearly show that 0.08% PHMB monotherapy is not inferior to 0.02% PHMB + 0.1% propamidine combination therapy in terms of clinical recovery rate (CRR_12) at 12 months. * The CRR_12 for 0.08% PHMB monotherapy corresponds to 87.1%. * The CRR_12 of 0.02% PHMB + 0.1% propamidine corresponds to 89.5%.

[0122] Monotherapy is always preferred over combination therapy, and the results show no statistically significant difference.

[0123] The percentage of subjects who had been previously treated with corticosteroid-based therapy is shown below: - 22.6% in the case of 0.08% PHMB monotherapy - 10% in the case of 0.02% PHMB + 0.1% propamidine combination therapy

[0124] The CRR12 clinical recovery rates of patients who had received pre-steroid treatment and those who had not are shown below: * The CRR_12 of 0.08% PHMB monotherapy corresponds to 91.4% * The CRR_12 of 0.02% PHMB + 0.1% propamidine corresponds to 87.8%

[0125] Since the CRR_12 in combination therapies carried out in normal clinical practice is about 63 - 67%, the CRR_12 obtained with monotherapy is unexpected in both cases. This clinical trial, therefore, shows that monotherapy under the described formulation conditions (MW, PDI, and PHMB polymer concentration 0.04% - 0.008%, pH and osmolality) is not only equivalent 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 efficacy: healing time) The healing time related to the treatment is: * 0.08% PHMB group: 138.3 days * (range 32 - 365 days) <00000​​​​​​​​​The 0.08% PHMB group showed a wider range of healing times. Generally, monotherapy is associated with longer healing times than combination therapy, but unexpectedly, the lower limit of the healing time range for monotherapy was 32 days. These results were not statistically significant.

[0128] Table 15 shows the results regarding the time-to-care period for patients who received treatment.

[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). <Note> The embodiments of the present invention include the following: <Section 1> A polyhexamethylene biguanide-based liquid formulation 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 the pH in the range of 5 to 6.5, and an isotonic agent for maintaining the molar 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 formulation for use as described in <Section 1>, for the treatment of co-infections caused by the genus Acanthamoeba and Pseudomonas aeruginosa or Staphylococcus epidermis. <Section 3> A polyhexamethylene biguanide-based solution for use as described in <Item 1> or <Item 2>, wherein the buffer system is selected from the group including 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 buffers. <Section 4> A polyhexamethylene biguanide-based liquid preparation for use as described in 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. <Section 5> A polyhexamethylene biguanide-based liquid preparation for use according to any one of items 1 to 4, wherein the isotonic agent is sodium chloride or potassium chloride. <Section 6> A liquid preparation based on polyhexamethylene biguanide for use according to any one of items 1 to 5, wherein the polydispersity index of the polymer is in the range of 1.7 to 1.8. <Section 7> A polyhexamethylene biguanide-based liquid preparation for use as described in any one of items 1 to 6, having a pH of 5.8. <Section 8> A liquid preparation based on polyhexamethylene biguanide for use as described in any one of items <1> to <7>, in the form of an eye wash, eye drops, ophthalmic gel, or viscous solution. <Section 9> A polyhexamethylene biguanide-based liquid preparation for use as described in <Item 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. <Section 10> A polyhexamethylene biguanide-based liquid formulation for use according to any one of items 1 to 9, further comprising a penetration enhancer selected from Tween 80 and benzalkonium chloride. <Section 11> A polyhexamethylene biguanide-based liquid formulation for use as described in any one of items 1 to 10, further comprising 0.02% chlorhexidine, 0.1% propamidine, or 0.1% desomeidine. <Section 12> A polyhexamethylene biguanide-based solution for use as described in any one of items <1> to <11>, administered ophthalmally in a stepwise dose 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 polyhexamethylene biguanide-based liquid preparation according to any one of items <1> to <12>, comprising the following steps: (i) Adding the buffer system in a concentration of 0.01% to 4.5% (w / v) and the isotonic agent in a concentration of 0.01% to 5.5% (w / v) to purified water while gently and continuously stirring; optionally adding a thickener selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose, and hydroxypropylcellulose; (ii) Add a 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) while gently stirring; (iii) The process of adjusting the final volume by adding purified water; (iv) A process of sterilization by filtration or heat. <Section 14> A method for preparing a polyhexamethylene biguanide-based liquid preparation according to item 13, wherein the buffer system is selected from the group including 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 buffers. <Section 15> A method for preparing a polyhexamethylene biguanide-based liquid preparation 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. <Section 16> A method for preparing a polyhexamethylene biguanide-based liquid preparation according to any one of items 13 to 15, wherein the isotonic agent is sodium chloride or potassium chloride. <Section 17> An eye lens loaded with a polyhexamethylene biguanide-based solution described in any one of items <1> to <11> for controlled release of the active ingredient for use in the treatment of Acanthamoeba keratitis, co-infection with Acanthamoeba and Pseudomonas aeruginosa, or fungal infections.

Claims

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 the pH in the range of 5 to 6.5, and an isotonic agent for maintaining the molar osmotic pressure concentration (osmotic pressure) 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 polyhexamethylene biguanide is in the range of 1.5 to 1.

9.

2. A polyhexamethylene biguanide-based liquid formulation for use according to claim 1, for the treatment of co-infections caused by the genus Acanthamoeba and Pseudomonas aeruginosa or Staphylococcus epidermidis.

3. A polyhexamethylene biguanide-based liquid preparation for use according to claim 1 or 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 mixtures thereof.

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

6. A liquid preparation based on polyhexamethylene biguanide for use according to any one of claims 1 to 5, wherein the polydispersity index of the polyhexamethylene biguanide is in the range of 1.7 to 1.

8.

7. A liquid preparation based on polyhexamethylene biguanide for use according to any one of claims 1 to 6, wherein the pH is 5.

8.

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

9. A polyhexamethylene biguanide-based liquid preparation 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 liquid formulation for use according to any one of claims 1 to 9, further comprising a penetration enhancer selected from Tween 80 (registered trademark) and benzalkonium chloride.

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

12. A polyhexamethylene biguanide-based solution for use according to any one of claims 1 to 11, administered ophthalmally in a stepwise dose 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 preparation according to any one of claims 1 to 12, comprising the following steps: (i) Adding the buffer system in a concentration of 0.01% to 4.5% (w / v) and the isotonic agent in a concentration of 0.01% to 5.5% (w / v) to purified water while gently and continuously stirring; optionally adding a thickener selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose, and hydroxypropylcellulose; (ii) Add a 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) while gently stirring; (iii) The process of adjusting the final volume by adding purified water; (iv) A process of sterilization by filtration or heat.

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

8.

15. A method for preparing a polyhexamethylene biguanide-based liquid preparation 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 mixtures thereof.

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

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

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