EA047258B1 - COMPOSITION BASED ON POLYHEXAMETHYLENE BIGUANIDE FOR USE IN THE TREATMENT OF ACANTHAMOEBAL KERATITIS AND/OR FUNGAL INFECTIONS - Google Patents

Description

The present invention relates to a stable composition based on polyhexamethylene biguanide (PHMB) suitable for administration at the ophthalmic level, a method for its preparation and a corresponding dosage regimen effective for the treatment of acanthamoeba keratitis, and in particular for the destruction of cysts.

Acanthamoeba keratitis is an infection of the cornea, the clear tissue that lines the front of the eye. The infection is caused by Acanthamoeba, a microorganism commonly found in both streams and aquatic basins (lakes, oceans, and rivers), as well as in household tap water, swimming pools, hot tubs, soil, and air.

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

In general, Acanthamoeba has two phases of its life cycle: an active form (when the microorganism feeds and replicates) and a dormant form (when the microorganism defends itself from possible attacks by turning into a cyst). Cysts are the actual target for the development of effective anti-amoebic drugs.

Co-infection with bacterial species is common among patients with Acanthamoeba keratitis. In particular, Staphylococcus spp and Pseudomonas aeruginosa are the most common co-isolated bacterial pathogens [2, 3]. Particularly for the latter organism, an in vivo study suggests that the presence of bacteria is an important factor in the development and severity of acanthamoeba keratitis [4].

Acanthamoeba keratitis can be extremely painful, as the cornea is one of the most sensitive organs in the human body, with the highest density of pain receptors, and the products used to treat acanthamoeba keratitis can be quite difficult to tolerate and, in some cases, they also may irritate the surface of the cornea. Approximately 25% of cases of treatment of acanthamoeba keratitis even resulted in corneal transplantation. Without treatment, the disease progressed to blindness as a result of corneal vascularization and corneal scarring or perforation.

There are currently no approved drugs for the treatment of acanthamoeba keratitis. Biguanides and diamidines have shown efficacy against acanthamoeba keratitis in a number of clinical cases, although treatment regimens and concentrations of these drugs have been developed empirically.

Among the various options available, treatment with polyhexamethylene biguanide (PHMB) in an ophthalmic composition d at a concentration of 0.02% alone [5] or in combination with chlorhexidine 0.02%, propamidine 0.1% or dezomidine 0.1% is the most promising choice in the treatment of patients suffering from acanthamoeba keratitis.

Current treatments for acanthamoeba keratitis are lengthy and complex. They are started with hourly doses during the day, with no dosing at night, for approximately 1-2 weeks, then tapered according to response. Although every patient is different, those who are diagnosed early and receive adequate treatment immediately can typically expect 3 to 6 months of therapeutic treatment.

Additionally, repeated and long-term use of antibiotics and disinfectants such as those mentioned above significantly alters the composition of the conjunctival flora by increasing the percentage of pathogens such as Staphylococcus epidermidis to the detriment of the normal commensal flora, and this can have significant clinical consequences as Staphylococcus epidermidis is one of the main causes of eye diseases such as conjunctivitis, keratitis and endophthalmitis [6].

PHMB consists of n-time repeating biguanide units linked by hexamethylene chains, which create a cationic and amphipathic structure (Fig. 1).

PHMB-based drugs are a mixture of biguanide-based polymers with a molecular weight that can vary from 400 to 8000 amu. (atomic mass units) and has a variable degree of polymerization, where n can vary from 2 to 40.

The mechanism of action of PHMV has been the subject of various studies in in vitro experimental models. The SUV membrane is a system that mimics the chemistry of the human corneal epithelial cell membrane. When PHMB interacts with the SUV membrane bilayer, the interaction between phospholipids increases, water is displaced from the bilayer of acyl chains and polar heads, the region with acyl chains becomes less dynamic and selective, and PHMB is absorbed into the surface of the bilayer [7].

The cationic nature of PHMV allows it to interact with anionic polymers such as DNA. It has been indicated in the literature that PHMB interacts with DNA in aqueous solution, forming a complex between these two substances. The interaction involves electrostatic interaction between the cationic groups of the biguanide and the anionic phosphate groups of the deoxyribonucleotides of DNA in a molar ratio of approximately 1:1 [8].

The structure of Acanthamoeba, characterized by the exposure of DNA binding sites, provides easier access of PHMV to the protozoan DNA, facilitating the process of chromatin condensation and destruction

- 1 047258 of this microorganism, as has already been established for other bacteria such as Escherichia coli, Salmonella enteritidis and Staphylococcus aureus [9]. Although these studies are not specific to Acanthamoeba, they are useful to inventors in determining the optimal conditions under which PHMV binds to protozoan DNA and exhibits its best efficacy in phase e in the treatment of infectious keratitis.

In a previous clinical study I Papa V. et al. [10] it was shown that even higher concentrations of PHMB, amounting to 0.04, 0.06, 0.08%, are safe and well tolerated by healthy volunteers [8]. The pH and osmolarity of the composition, and even more so the molecular weight and polydispersity index of the PHMV polymer used, are not described. However, the concentration of PHMV alone is not sufficient to maximize the interaction of PHMV with Acanthamoeba DNA and treatment efficacy, as will be demonstrated below.

Patent application US 007/0140897 A1 [12] describes ophthalmic compositions based on active ingredients containing biguanide (including PHMB) at pH 4-6 for the treatment of acanthamoeba keratitis. However, the only compositions presented in the examples are based on alexidine, and not on PHMB. In any case, the significance of MW and PDI of biguanide-based polymers for interaction with pathogen DNA, or the specific concentrations of biguanide-based polymers used in combination with the above parameters, have not been described.

A study of the stability of compositions based on 0.02% PHMB with pH 4 in the work of Bouattour et al. 2018 [13] revealed the formation of degradation products (DP) after 90 days. The authors concluded that compositions based on PHMB cannot be stored for more than 60 days in EOS-LDPE (ethylene oxide sterilized low-density polyethylene) bottles at 25°C. The present inventors have identified a polyhexamethylene biguanide-based composition for ophthalmic administration that remains stable and effective at the highest active ingredient concentrations ranging from 0.04% (w/v) to 0.08%. (weight/volume), thanks to control of the molecular weight (MW) and polydispersity index (PDI) of the PHMB polymer, together with pH and osmolarity.

Research has shown that the use of PHMV with a high molecular weight and polydispersity index, together with a specific buffer system that maintains the pH of the PHMV composition 0.04%-0.08% (w/v) in the range of 5-6.5, synergistically creates optimal conditions for stability for at least 24 months and for the activity of the active ingredient.

For the reasons stated above, the PHMB molecule must actually be in a protonated form in order to exhibit its biocidal mechanism of action at its best. In its protonated, ionized form, PHMV penetrates Acanthamoeba-infected corneal epithelium and accumulates in the stroma due to electrostatic interaction with the negative charges of proteoglycans. It is here that PHMV exhibits its biocidal and cysticidal activity with a mechanism of action that, according to recent scientific research, is expressed through association with DNA through active interaction with the latter's phosphate groups. This connection blocks the process of amoeba DNA replication, causing the death of the pathogen [11]. In this sense, the effectiveness of the above-mentioned interaction between PHMV and DNA, and therefore the activity of PHMV itself, largely depends on the stability of the composition containing the active ingredient. In addition, administration of the composition of the invention in a precise stepwise dosing regimen to patients exposed to Acanthamoeba keratitis has proven to be particularly effective in terms of activity in completely killing Acanthamoeba cysts.

In this sense, since the composition according to the invention is used as monotherapy, it promotes compliance and accurate implementation of the therapeutic regimen by the patient, in particular due to the fact that there is no need for repeated instillation of various products during the day. In addition, given the selectivity and specificity of the product in relation to the target microorganism, a higher speed of action is provided with subsequent faster elimination of pathology, which prevents long periods of treatment over 3-6 months.

Again, compared to the multidrug therapeutic approach consisting of potent antiseptics and broad-spectrum antibiotics currently used, monotherapy (highly selective and specific) with 0.08% PHMB is clearly a safer option for preserving the diversity of the ocular microbiota during treatment and , therefore, to prevent the emergence of multidrug-resistant strains of Staphylococcus epidermidis.

Thus, the present invention relates to a liquid composition based on polyhexamethylene biguanide or its salts in a concentration ranging from 0.04 to 0.08%, containing a buffer system to maintain the pH in the range of 5-6.5 and an isotonic agent to maintain the osmolarity in the range 270-330 mOsm/kg, while the molecular weight of polyhexamethylene biguanide is in the range from 2300 to 6000 amu. and the polydispersity index of the polymer is in the range of 1.5 to 1.9, preferably 1.7 to 1.8, suitable for ophthalmic administration for use in the treatment of acanthamoeba keratitis or fungal infections.

Said fungal infections are preferably keratitis or keratomycosis,

- 2 047258 mediated by a pathogen selected from the group consisting of Candida albicans, Fusarium solani, Aspergillus niger, Aspergillus jumigatus, Aspergillus flavus and Cladosporium.

The above-mentioned fungal infections may also be present in combination with Acanthamoeba infection, particularly Fusarium solani and Cladosporium infections.

In an alternative embodiment of the invention, said treatment of infectious acanthamoeba keratitis is particularly effective in the case of co-infection with Pseudomonas aeuriginosa or Staphylococcus epidermis.

The therapeutic use of the 0.08% PHMB composition, which is active against Pseudomonas aeruginosa (data not shown, internal APE test study: ORPHAN DRUG - in vitro assay - 1305) actually appears to be highly effective in quickly and effectively eliminating the pathology in the case of the above coinfection. The 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.

In addition to the need to maintain a slightly acidic pH to ensure maximum protonation of the PHMV, another object of the present invention is to maintain the osmolarity of the composition at a value comparable to that of physiological tear fluid (which ranges from 290 to 320 mOsm/kg), preferably by adding an ionic isotonic agent selected from the group consisting of sodium chloride, potassium chloride, or non-ionic such as glycerin, mannitol, sorbitol; ionic buffer systems such as phosphate, citrate, bicarbonate or borate buffer, or non-ionic buffers such as trometamol, histidine, glycine, HEPES, which also contribute to osmoticity.

Osmotic pressure actually depends mainly on the ions present in the aqueous layer (Na ⁺ , K ⁺ , Cl ^- and HCO ₃ ).

According to preferred embodiments of the present invention, the isotonic agent is used in the following concentrations: NaCl (0.01-0.9%), KCl (0.01-1.19%), glycerol (0.01-2.6%), mannitol (0.01-5.1%) and sorbitol (0.01-5.5%).

In a specific embodiment of the polyhexamethylene biguanide liquid composition, the buffer system is an ionic buffer such as a phosphate, citrate, bicarbonate or borate buffer, or a non-ionic buffer such as trometamol, histidine, glycine, HEPES or a mixed system.

The buffer system preferably contains dibasic sodium phosphate dodecahydrate (Na ₂ HPO ₄ -12 H ₂ O) and monobasic sodium phosphate (NaH ₂ PO ₄ -H ₂ O). Dibasic sodium phosphate dodecahydrate is preferably used in a concentration in the range of 0.01-4.5% and monobasic sodium phosphate is used in a concentration in the range of 0.01-3.5%.

Alternatively, a citrate phosphate buffer containing dibasic phosphate dodecahydrate (Na ₂ HPO ₄ -12 H ₂ O) and citric acid (C ₆ H ₅ O ₇ -H ₂ O), or Tris/HCl, or boric acid can be used. acid/borate

If the composition of the invention is in the form of an ophthalmic gel or viscous solution, it is preferable to use a phosphate-free buffer, such as, for example, Tris buffer and/or boric acid or borates.

In a preferred embodiment, the pH of the liquid polyhexamethylene biguanide composition is 5.8.

In a preferred embodiment of the polyhexamethylene biguanide liquid composition of the present invention, the concentration of PHMB is selected from the group 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).

The polyhexamethylene biguanide liquid composition of the present invention suitable for ophthalmic administration is a sterile solution which may be in the form of eye drops.

Alternatively, in the presence of a viscosity increasing agent (eg, xanthan gum, gellan gum, polyvinyl alcohol, hyaluronic acid, sodium hyaluronate, cellulose derivatives such as carboxymethylcellulose or hydroxypropylcellulose), the polyhexamethylene biguanide composition of the present invention may be in the form of an ophthalmic gel or viscous solution.

According to a further preferred embodiment of the invention, the liquid composition based on polyhexamethylene biguanide of the present invention further contains a penetration enhancer, which can be selected from Tween 80 and benzalkonium chloride.

In addition, according to a preferred embodiment, the liquid polyhexamethylene biguanide composition for use according to the invention may further contain 0.02% chlorhexidine, 0.1% propamidine, 0.1% dezomidine in combination with PHMB. When PHMV is administered in combination with a second active agent, administration may be sequential, concurrent, or separate,

- 3 047258 that is, in the same composition of eye drops or in separate compositions.

The present invention also relates to a liquid composition based on polyhexamethylene biguanide according to the invention for the treatment of Acanthamoeba keratitis by administering stepped dosages of 16 drops per day for 5 days, 8 drops per day for 7 days, 6 drops per day for 7 days and 4 drops per day until clinical normalization. Clinical normalization was 88% at an average of 4 months. This stepped dosing differs from the intensive dosing previously described in the Phase I study by Papa et al. [10], since it is more effectively involved in the elimination of cysts and, consequently, in the clinical normalization of acanthamoeba keratitis.

The present invention also relates to a method for preparing a liquid polyhexamethylene biguanide composition according to the invention, which includes the following steps:

(1) adding a buffer system at a concentration ranging from 0.01% to 4.5% (w/v) and an isotonic agent at a concentration ranging from 0.01% to 5.5% (w/v) to the purified water at easy and continuous mixing; it is possible to add a viscosity enhancer selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose and hydroxypropylcellulose;

(2) adding polyhexamethylene biguanide having a molecular weight in the range of 2300-6000 amu. and a polydispersity index in the range of 1.5-1.9, preferably 1.7-1.8, at a concentration in the range of 0.04% to 0.08% (w/v) with gentle stirring;

(3) bringing to the desired volume by adding purified water;

(4) sterilization by filtration or heat.

In case of a viscous solution, addition of a viscosity enhancer selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose and hydroxypropylcellulose must also be performed after step (1). In this case, in the presence of non-filterable polymers, heat sterilization is used instead of filtration sterilization.

The solution is then distributed into single-dose containers or vials, which are then sealed. Single-dose containers can be made of low-density polyethylene (LDPE) and each package can contain 5 units of one dose.

In a specific embodiment of the method for preparing a liquid composition based on polyhexamethylene biguanide, the buffer system for maintaining the pH in the range of 5-6.5 is an ionic buffer such as phosphate, citrate, bicarbonate, Tris buffer, borate or non-ionic buffer such as trometamol, histidine , glycine, HEPES, or mixed system. The buffer system is preferably selected from the group consisting of dibasic sodium phosphate dodecahydrate/monosodium phosphate, dibasic sodium phosphate dodecahydrate/citric acid, Tris/HCl or boric acid/borate.

According to a preferred embodiment, the buffer system is based on dibasic sodium phosphate dodecahydrate (Na ₂ HPO ₄ -12 H ₂ O) and monobasic sodium phosphate (NaH ₂ PO ₄ -H ₂ O). Dibasic sodium phosphate dodecahydrate is preferably used in a concentration in the range of 0.01-4.5% (w/v) and monobasic sodium phosphate is used in a concentration in the range of 0.01-3.5% (w/v).

The isotonic agent to maintain osmolarity in the range of 270-330 mOsm/kg is preferably sodium chloride.

The process for preparing the composition was also developed to ensure that both the original molecular weight and the polydispersity index of the active ingredient PHMB are maintained in the final product in the range of 2300-6000 amu. and 1.5-1.9, preferably 1.7-1.8 respectively. These parameters were found to be responsible for the effectiveness of the final product.

Finally, the present invention relates to therapeutic ophthalmic lenses loaded or derivatized with a liquid composition of the invention based on polyhexamethylene biguanide for controlled release of the active ingredient for the treatment of acanthamoeba keratitis or co-infection with Pseudomonas aeuriginosa. This embodiment provides continuous release of the composition over an extended period of time and has the added benefit of patient compliance and adherence.

The present invention will be described below for illustrative but non-limiting purposes, according to a preferred embodiment with particular reference to the accompanying drawings, wherein:

in fig. 1 shows the structure of the hexamethylene biguanide repeating unit in PHMB;

in fig. 2 shows a flow diagram of a method for producing compositions based on PHMV;

in fig. 3 shows a chromatogram obtained by gel filtration chromatography of PHMB in API;

in fig. 4 shows a chromatogram obtained by gel filtration chromatography of PHMB in the finished product; from comparison with the chromatogram in Fig. 2, relating to raw materials, it is obvious that the production method is able to maintain unchanged the molecular weight distribution of the raw materials until the final product;

- 4 047258 in Fig. Figure 5 shows a comparison of stability data for a composition based on 0.08% PHMB at pH 7.4 and pH 5.8;

in fig. Figure 6 shows a comparison of PHMV-DNA binding as a function of molecular weight. From the direction of the graph it can be seen that when using high molecular weight PHMV, the polymer-DNA bond is more efficient since one or more nucleic acid molecules are involved. A high-molecular-weight polymer is capable of forming cross-bridges between different nucleic acid molecules, forming a network of connections (line with circles). On the contrary, the line with squares reflects the establishment of a linear type bond between PHMB and DNA, where the polymer, characterized by a low molecular weight, does not exhibit the ability to form cross-bridge bonds, but forms a bond in which only one nucleic acid molecule is involved;

in fig. 7(A) shows the results of a stability study of 0.08% PHMV formulations using batches of PHMV with different molecular weights (MW) and polydispersity indices (PDI), expressed as % recovery (w/v) of PHMV at T0 and after 12 months (T12 );

in fig. 7(B) shows the results of a stability study of a 0.08% PHMV composition using a batch of PHMV characterized by MW = 2641 amu. and PDI = 1.6. The study was carried out under three different temperature conditions: long-term stability 25°C, RH 40%; intermediate stability 30°C, RH 65%; accelerated stability 40°C RH 25%. Stability results expressed as % recovery (wt/vol) of PHMV at T0 and after 3 months (T3) are shown.

To better illustrate the invention, the following non-limiting examples are presented, where various PHMV-based compositions were tested and compared, assessing the effect on the stability of certain parameters of the composition, such as pH, osmolarity, molecular weight and polydispersity index of the active ingredient PHMV.

Example 1. Method for producing compositions based on PHMV.

The method for preparing the composition according to the invention is illustrated in the diagram in FIG. 2.

Dibasic sodium phosphate dodecahydrate (Na ₂ HPO ₄ -12 H ₂ O), monosubstituted sodium phosphate (NaH = PO ₄ -H ₂ O) and sodium chloride are added sequentially and dissolved in a certain volume of purified water with continuous gentle stirring.

The mixing step is critical to maintaining the original molecular weight and polydispersity index of the PHMV and must be performed without foaming or swirling in the solution. Preferably, a stirrer or wheel is used that is immersed in the solution for mixing. Finally, PHMV (0.08%) is added.

The solution is brought to volume (100 ml) by adding a sufficient amount of purified water and with constant stirring. Then the pH and appearance of the solution are controlled.

Sterilization is performed by filtration through a 0.2 micron filter. The nature of the filter membrane and the dynamics (pressure, flow) are chosen in such a way as to prevent the product from being forced through the membrane in order to avoid depolymerization or breaking of the polymer chain.

After filtration, the integrity of the 0.2 µm filter is monitored using an electronic control system.

Finally, the sterile solution is used to fill single-dose containers using blow/fill/seal technology under aseptic conditions.

This automated technology allows the vial to be filled and sealed under aseptic conditions with a solution volume ranging from 0.1 to 1000 cm ³ .

The PHMV solution appears as a clear, colorless or pale yellow liquid.

At the end of the preparation process, the finished product is characterized for its main components, including any impurities, using suitable chromatographic methods.

A comparison of the chromatogram of PHMV in API (Fig. 3) and the chromatogram of PHMV after the composition preparation process (Fig. 4) shows that the molecular weight distribution of PHMV does not change.

Example 2. Comparative studies of the stability of compositions based on RNMV.

The resulting PHMV-based compositions are sterile solutions, the relevant physicochemical parameters of which are pH and osmolarity.

As described in detail, the choice of a slightly acidic pH is due to the need to obtain a stable composition, which cannot be obtained at a pH close to neutral.

For this purpose, various compositions based on 0.08% PHMB were developed at different pH values (5.8; 6.2; 7.4) with subsequent testing of their stability at 25, 40 and 60°C.

To obtain a composition based on 0.08% PHMB, pH 5.8, the following ingredients were weighed:

- 5 047258

Table 1

Ingredient Function % (mass/volume) NaH ₂ PO ₄ H ₂ O buffer 0.827 g Na ₂ HPO ₄ 12H ₂ O buffer 0.240 g NaCl isotonic agent 0.520 g RNMV active ingredient 0.08 g Sterile water solvent bring to 100 ml

In table 2 below shows the physicochemical characteristics of the final composition at pH 5.8 and during the phases of the process of its preparation.

table 2

rn Osmolarity (mOsm/kg) UV spectrum of the active agent Recovery (UV quantitative) Buffer solution (before adding RNMV) - 289 - - Buffer with PHMV (before sterilization) 5.8 290 corresponds - Composition sterilized by filtration 5.8 297 corresponds 100.4

To prepare the 0.08% PHMB, pH 6.2 composition, the following ingredients were weighed in accordance with the same process described above.

Table 3

Ingredient Function % (mass/volume) NaH ₂ PO ₄ H ₂ O buffer 0.735 g Na ₂ HPO ₄ 12H ₂ O buffer 0.480 g NaCl isotonic agent 0.510 g RNMV active ingredient 0.08 g Sterile water solvent bring to 100 ml

In table Table 4 shows the physicochemical characteristics of the final composition at pH 6.2 and at the stages of the method for its preparation.

Table 4

rn Osmolarity (mOsm/kg) UV spectrum of the active agent Recovery (UV quantitative) Buffer solution 6.05 291 - - (before adding PHMV) Buffer with PHMV (before sterilization) 6.07 295 corresponds - Composition sterilized by filtration 6.20 287 corresponds 100.9

To obtain a composition based on 0.08% PHMB, pH 7.4, the following ingredients were weighed.

- 6 047258

Table 5

Ingredient Function % (mass/volume) NaH ₂ PO ₄ H ₂ O buffer 1.91 g Na ₂ HPO ₄ 12Η ₂ Ο buffer 0.19 g NaCl isotonic agent 0.44 g RNMV active ingredient 0.08 g Sterile water solvent bring to 100 ml

In table Figure 6 shows the physicochemical characteristics of the final composition at pH 7.4 and at the stages of the method for its preparation.

Table 6

pH Osmolarity (mOsm/kg) UV spectrum of the active agent Buffer solution (Before adding PHMB) - - - Buffer with PHMV (before sterilization) 7.45 286 corresponds Composition sterilized by filtration 7.45 286 corresponds Composition sterilized in an autoclave 7.33 295 corresponds Filtered composition after 2 weeks at 25°C 7.36 293 corresponds

The stability of the 0.08% PHMB formulation in a single dose container was then compared at pH 5.8 and 7.4 at 25°C (long term conditions), 40°C (accelerated conditions) and 60°C (stress conditions). ). The results of the stability analysis are shown below.

Stability data:

0.08% PHMB composition, pH 5.8 at 25°C ± 2°C and relative humidity 40% ± 5 (long-term conditions).

Table 7

Week Limits

Analyzes Unit measurements Min Max 0 13 26 39 52 65 104 Appearance Transparent, without foreign particles rel.* COOT. COOT. COOT. COOT. COOT. COOT. Color Colorless COOT. COOT. COOT. COOT. COOT. COOT. COOT. pH 5.3 6.3 5.7 5.6 5.8 5.8 5.8 5.8 5.8 Osmolarity mOsm/kg 270 330 290 274 281 276 293 277 276 Bigu anid (HPLC) Complies with standard COOT. COOT. COOT. COOT. COOT. COOT. COOT Bigu anid test (UV) %L.S. 90 But 97.6 97.8 99.7 96.2 98.8 92.5 94.6 Impurities A % a.s. 5.0 0.8 1.0 1.0 1.8 0.8 1.0 0.9 Impurities B+ WITH % a.s. 5.0 0.9 0.9 1.2 2.4 1.1 1.4 1.1 Loss of water % 5.0 0.0 0.1 0.1 0.1 0.1 0.2 Sterile ness Complies with European Pharmacopoeia Sterile Sterile

* correspond: corresponding.

0.08% PHMB composition, pH 5.8 at 40°C ± 2°C and relative humidity 25% (accelerated).

- 7 047258

Table 8

Limits weeks Analysis Unit measurements Min. Max. 0 13 26 Appearance Transparent, free of foreign particles resp. resp. resp. Color Colorless resp. resp. resp. pH 5.3 6.3 5.7 5.6 5.8 Osmolarity mOsm/kg 270 330 290 274 276 Bigu anid (HPLC) Complies with standard resp. resp. Biguanide test (UV) %L.S. 90 110 97.6 96.7 99.2 Impurities A % a.s. 5.0 0.8 1.0 0.9 Impur. B+ C % a.s. 5.0 0.9 1.0 1.3 Loss of water % 5.0 0.1 0.2 Sterility Complies with European Pharmacopoeia Sterile

* correspond: corresponds

0.08% PHMB composition, pH 7.4 at 60°C ± 2°C and 20% relative humidity (stress conditions).

_______________________________________Table 9, Week Limits

Analysis Unit measurements Min. Max 0 1 2 Appearance Transparent, without foreign particles resp. ratio resp. Color Colorless resp. ratio resp. pH 7.0 7.8 7.3 7.2 7.3 Osmolarity mOsm/kg 270 330 276 294 299 Biguanide (ABS ratio 235/220nm) 1.5 1.4 1.3 Sterility Complies with European Pharmacopoeia Sterile Sterile

* correspond: corresponding.

0.08% PHMB composition, pH 5.8 at 60°C ± 2°C and relative humidity 20% (stress conditions).

Table 10

Week Limits

Analysis Unit measurements Min. Max. 0 1 2 Appearance Transparent, free of foreign particles resp. resp. resp. Color Colorless resp. resp. resp. pH 5.3 6.3 5.8 5.6 5.7 Osmolarity mOsm/kg 270 330 289 296 302 Biguanide (ABS ratio 235/220nm) 1.4 1.5 1.5 Sterility Complies with European Pharmacopoeia Sterile Sterile

- 8 047258 * cor.: corresponding.

The results presented in Fig. 5 show that the stability of the PHMB-based composition obtained by maintaining the pH in the range of 5-6.5 is not achieved at a pH close to neutral (pH 7.4).

Example 3. Study of the RNMV-DNA connection.

A PHMV-DNA association study was performed to evaluate the equivalence of low molecular weight PHMV and high molecular weight PHMV.

The ability of PHMV to bind to DNA can be considered independent of the specific type of DNA, as indicated in the literature [8], while the binding ability and efficiency of binding to DNA depends on the molecular weight and polydispersity index of the PHMV polymer. When the polymer has a molecular weight in the range of 2300 to 6000 amu. and a polydispersity index in the range of 1.5–1.9, DNA binding is more efficient, probably because the polymer length is more suitable for the formation of cross-links involving multiple nucleic acid molecules, according to the mechanism proposed by Allen et al. [8]. In contrast, when the polymer is of low molecular weight, the bond will be linear and involve a single nucleic acid molecule.

Then a comparative study was carried out with different samples of PHMB polymers to evaluate the ability to bind to nucleic acid molecules. PHMV (sample A) was degraded by acid and high temperature treatment for 5 hours to obtain low molecular weight PHMV polymers.

PHMV (sample B) did not receive any type of treatment and had a molecular weight that fell within the stated range.

The ability of both samples to bind nucleic acid molecules was then assessed. The experimental method proposed here is based on the interaction between an aqueous solution of PHMB and an aqueous solution of DNA. The amount of PHMB-DNA complex was measured as the difference between the initial amount of both substances in solution and the residual amount after the end of the reaction. The amount of both substances in solution is monitored using spectrophotometric measurements at 2 different wavelengths, 260 nm for DNA and 235 nm for PHMB, respectively.

In conclusion, our data demonstrate an increased ability of high molecular weight PHMV to interact with DNA by cross-bridging multiple nucleic acid molecules and, therefore, greater efficacy in the treatment of infectious acanthamoeba keratitis or coinfection with Pseudomonas aeruginosa. The type of bond formed between the PHMB polymer and the DNA is much more efficient, as shown in the graph of FIG. 6.

Example 4. Study of stability depending on molecular weight and polydispersity index PHMV.

A comparative study was performed on three different starting batches based on PHMV, characterized by different molecular weights and polydispersity indices (PDI):

batch L-17GR185627, MW = 2517 amu, PDI = 1.73, batch F-693/LU/101, MW = 1170 amu, PDI = 1.93;

batch F-693/LU/118, MW = 440 amu, PDI = 2.14.

For each batch, a 0.08% PHMB composition was obtained, characterized by pH = 5.8, osmolarity 0.280 mOsm/kg.

The stability of the PHMV composition was then assessed at T0 and after 12 months (T=12) at a temperature of 25°C±2°C, RH 40±5%. The following parameters were assessed:

appearance, extraction% (weight/volume) of the active ingredient, the results are shown in table. 11 and in the graph of FIG. 7A.

Table 11

The consignment MW (Yes) PDI Testing Months T=0 T= 12 L-17GR185627 2517 1.73 Appearance Acceptable Acceptable Recovery % PHMV (UV) 101.5 97.1 F-693/LU/101 1170 1.93 Appearance Acceptable Unacceptable Recovery % PHMV (UV) 100 35.8 F-693/LU/118 440 2.14 Appearance Acceptable Unacceptable Recovery % PHMV (UV) 100 12.2

The stability study confirms that the most stable 0.08% PHMV composition is the one characterized by the highest molecular weight PHMV, i.e. lot L-9 047258

17GR185627, MW (molecular weight) = 2517 amu, pH = 5.8 and with a PDI value of 1.73. No significant degradation products (DPs) were observed throughout the stability study period.

Solutions of 0.08% PHMV, characterized by low molecular weight (batch F-693/LU/101, MW = 1170 amu - batch F-693/LU/118, MW = 440 amu) , indicate a reduced % recovery of PHMV after 12 months and, therefore, instability of the composition and reduced effectiveness.

Thus, it is obvious that the parameters of molecular weight and polydispersity index PHMV, together with the pH of the solution, make a synergistic contribution to providing a stable and effective composition.

In addition, stability studies were carried out on solutions of 0.08% PHMB, at pH 5.8 and osmolality 0.290 mOsm/kg, characterized by MW = 2641 amu. and PDI = 1.6. The study was carried out under three different temperature conditions: Long-term stability 25°C, RH 40%; Intermediate stability 30°C, RH 65%; Accelerated stability 40°C RH 25%. Stability results are shown below and in FIG. 7(B):

Table 12

Conditions Test Months T=0 t=z 25°C, RH 40% Appearance Acceptable Acceptable % PHMV recovery 103.7 103.1 (UV) 30°C, RH 65%; Appearance Acceptable Acceptable Recovery % PHMV (UV) 103.7 101.7 40°С RH 25% Appearance Acceptable Acceptable Recovery % PHMV (UV) 103.7 105.3

Stability test results indicate that the product is stable under all temperature conditions tested, as the % recovery of PHMV is within the specified limits, 90-110% (w/v). Other chemical-physical parameters tested (data not shown), such as pH, osmolality and quantitation of extracted impurities, remained stable and within specified limits under all three climate conditions tested.

Example 5: Clinical study.

A randomized, multicenter, double-blind, parallel-group, phase 3 study to evaluate the efficacy, safety, and tolerability of the 0.08% PHMB composition of the invention compared with conventional combination therapy of 0.02% PHMB + 0.1% propamidine in adult men is detailed below. and women suffering from acanthamoeba keratitis.

This study was designed as a superiority study in accordance with EMA (European Medicines Agency) requirements (CPMP/EWP/482/99). The study includes a screening visit for inclusion, a treatment period including short outpatient visits, and follow-up visits.

A total of 130 people with Acanthamoeba keratitis were assigned to the following two treatment groups in a 1:1 ratio.

Group 1: 0.08% PHMB + placebo.

Group 2: 0.02% RNMV + 0.1% combination therapy.

Patients.

This study was conducted on men and women suffering from Acanthamoeba keratitis who were at least 12 years of age.

Target.

The main objective of the study is to compare the clinical normalization rate (CRR) at 12 months after randomization (CRR_12) between 0.08% PHMB + placebo and combination therapy 0.02% PHMB + 0.1% propamidine, assessing the difference between CRR_12 in terms of degree uncertainties, and testing the therapeutic superiority or lack of superiority of monotherapy with 0.08% PHMV.

Another purpose of the study is to obtain information on the safety of the ophthalmic composition based on 0.08% PHMB according to the invention.

Hypothesis.

The primary hypothesis tested is that the CRR12 of subjects receiving monotherapy with the 0.08% PHMV formulation is greater or non-inferior, below the still acceptable limit (Δ), relative

- 10 047258 specifically CRR12 combination therapy with 0.02% PHMB + 0.1% propamidine administered in accordance with the treatment protocol indicated below.

Secondary hypotheses are:

adverse effects associated with toxicity are lower with 0.08% PHMV monotherapy compared with combination therapy;

cure time is lower in subjects receiving 0.08% PHMV monotherapy compared to combination therapy.

Clinical normalization obtained within 12 months of treatment initiation is 67%, as reported in a retrospective study by the sponsor (Study 038/SI).

Concomitant and previous treatment

Subjects are authorized to take or have taken the following medications:

Antibiotics: Topical use of moxifloxacin is approved for the treatment of concomitant bacterial infections. However, it is not approved as a prophylactic antibiotic for the treatment of patients with corneal ulcers; PHMV is already a broad-spectrum antibacterial agent and no additional antibacterial agent is needed. Topical moxifloxacin is not approved as a prophylactic antibiotic in patients with corneal ulcers; PHMB is a good broad-spectrum antibacterial agent and does not require an additional antibacterial agent.

Antiviral and antifungal drugs: The use of these drugs during the study is not permitted. Any use of these drugs should be discontinued at the start of the study.

Anti-inflammatory drugs: For subjects receiving steroid therapy at study entry (who are already taking topical steroids, such as for misdiagnosis of HSV keratitis or as adjuvant treatment for bacterial keratitis), the following options exist.

A. Interrupting, maintaining or reducing the dose of steroids. Dexamethasone (0.1% or 0.15%) is the only topical steroid allowed in this study. Patients using other topical steroids at study entry should adjust the frequency of their administration. Diclofenac is the only oral NSAID (non-steroidal anti-inflammatory drug) approved in a clinical trial and is given in appropriate doses (75 mg - 150 mg per day, divided into 2-3 doses).

b. Subjects using an NSAID or cyclosporine at the start of the clinical trial should discontinue treatment after randomization.

V. Subjects not using topical steroids at study entry may begin taking topical steroids along with an oral NSAID (diclofenac recommended; 75 mg to 150 mg daily, divided into 2 to 3 doses) during the study as specified in the protocol.

Other approved local therapeutic treatments: lubricants, mydriatics (cyclopentolate, homatropine or atropine) and antiglaucoma drugs.

Dosage

The dosage used in the clinical efficacy study is as follows:

administration in a stepwise dosage of 16 drops per day for 5 days, 8 drops per day for 7 days, 6 drops per day for 7 days and 4 drops per day until clinical normalization.

Assignment of treatment type.

Protocol for bilateral pathology: If both eyes are affected, then only one of them (the right one, unless there is a difference in severity requiring treatment of the more affected eye) will be treated according to the treatment dosage and evaluated in the study. The other eye will be treated according to standard clinical practice.

In table 13 The demographics and distribution of patients participating in the study are presented below.

- 11 047258

Table 13

RNMV 0.08% + placebo RNMV 0.02%+ 0.1% propamidine Total Screened 69 66 135 Not screened 0 0 0 Randomized 69 66 135 Did not receive study drug ¹ 0 1 (1.5%) 1 (0.7%) Safety Analysis Kit ¹ 69 (100%) 65 (98.5%) 134 (99.3%) Completing Study ² 58 (84.1%) 57 (87.7%) 115 (85.8%) Premature withdrawal from study ² I (15.9%) 7 (10.8%) 18 (13.4%)

Did not receive study drug

means 0 1 (100.0%) 1 (100.0%) Complete set of analyzes ¹ 66 (95.7%) 61 (92.4%) 127 (94.1%) Completing Study ² 56 (84.8%) 53 (86.9%) 109 (85.8%) Premature withdrawal from study ² 10(15.2%) 7(11.5%) 17 (13.4%) DX not confirmed Lack of primary 3 (100%) 4 (80%) 7 (87.5%) performance evaluation 0 1 (20%) 1 (12.5%) Analysis protocol set ¹ 62 (89.9%) 57 (86.4%) 119(88.1%) Completing Study ² 54 (87.1%) 50 (87.7%) 104 (87.4%) Premature withdrawal from study ² 8 (12.9%) 6 (10.5%) 14(11.8%) Maximum PD 4(57.1%) 4 (44.4%) 8 (50%) Not FAS (full set of analyses) 3 (42.9%) 5 (55.6%) 8 (50%)

¹ Percentages based on the number of patients randomized in each treatment group.

² Percentages based on the number of subjects included in the analysis set.

In table Figure 14 shows the level of 12-month clinical normalization. Patients were divided into those with and without prior steroid treatment.

- 12 047258

Table 14

12-month clinical normalization rate RNMV 0.08% PHMV h placebo (N=66) RNMV -0.02%+ 0.1% General Propamidine Quantity (N=61) (N=127) Patients* *s Without normalization 7 (22.6%) 2 (10%) 9 (17.6%) previous steroid therapy Normalization 24 (77.4%) 18 (90%) 42 (82.4%) Patients without Without normalization 3 (8.6%) 5 (12.2%) 8 (10.5%) previous steroid therapy Normalization 32 (91.4%) 36 (87.8%) 68 (89.5%)

*Use of any of these drugs prior to therapy:

Corticosteroids and anti-infective drugs in combination, corticosteroids.

Percentages are based on the number of subjects in each subgroup and treatment.

Results.

Primary effectiveness: 12-month clinical normalization rate.

The results of the clinical trial revealed no superiority of PHMB 0.08% monotherapy compared to PHMB 0.02% + propamidine 0.1% combination therapy in terms of clinical normalization rate at 12 months (CRR_12).

* CRR_12 for 0.08% PHMV monotherapy is 87.1%.

* CRR_12 for 0.02% PHMV + propamidine 0.1% is 89.5%.

Monotherapy is always preferred over combination therapy and the results do not show statistically significant differences.

The percentage of subjects previously treated with corticosteroid-based therapy is shown below:

22.6% for 0.08% RNMV monotherapy;

10% for 0.02% RNMV + propamidine 0.1% combination therapy.

The clinical normalization rate of CRR_12 for patients with and without prior steroid treatment is shown below:

* CRR_12 for monotherapy 0.08% RNMV is 91.4%;

* CRR_12 for 0.02% PHMV + propamidine 0.1% is 87.8%.

The CRR_12 obtained with monotherapy in both cases is unexpected, since combination therapy used in routine clinical practice produces a CRR_12 of approximately 63-67%. Thus, the clinical study not only demonstrates that monotherapy under the described composition conditions (MW, PDI and 0.04%-0.008% PHMB polymer concentration, pH and osmolarity) is equivalent to combination therapy, but in both cases the results obtained are better than previously described in clinical practice (retrospective study 083/SI).

Secondary effectiveness: treatment time.

The treatment time associated with the therapy is:

* 0.08% PHMV group: 138.3 days* (range 32-365 days);

* 0.02% PHMV group + propamidine 0.1%: 117.1* days (range 54-214 days).

* average value.

0.08% PHMV shows treatment time in a wider range. Longer treatment times are generally associated with monotherapy compared to combination therapy, but unexpectedly the lower end of the treatment time range for monotherapy shows normalization at day 32. The results show no statistical difference.

In table 15 shows the results for the treatment time of patients.

- 13 047258 ____________________________________________________________Table 15

RNMV 0.02% +

RNMV 0.1%

0.08%+ placebo Propamidine

Treatment time (days) (N=56) (N=54)

Visit at the end of the study

n/nmiss 56/0 54/0 Average (DS) 138.3 (67.3) 117.1 (47) Average 125 112 Q1,Q3 93, 152 85, 151 Min., Max. 32, 365 54.214 p-value for difference between groups 0.0934

treatments n/nmiss = number of subjects with estimated/missing data.

Q1 = first quartile, Q3 = third quartile, SD = standard deviation. ^-Mann-Whitney test.

BIBLIOGRAPHY

[1] Kilvington S, Larkin DF. Acanthamoeba adherence to contact lenses and removal by cleaning agents. Eye 1990;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., et al., 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-lon Phospholipid Bilayer Structure and Dynamics, J. Phys. Chern. In 2015, 119, 10531-10542.

[8] M. J. Allen, et al. White Cooperativity in the binding of the cationic biocide polyhexamethylene biguanide to nucleic acids. Biochemistry and Biophysical Research Communications 318 (2004) 397-404.

[9] K. Chindera, et al. The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes. Scientific Reports 6:23121.

[10] Papa V. et al. Ocular safety and tolerance 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 Chern B 2020;124:4487-4497.

[12] Domanda di brevetto US2007/0140897 Al.

[13] Y. Bouattour et al. Stability of an ophthalmic formulation of polyhexamethylene biguanide in gamma-sterilized and ethylene oxide sterilized low density polypropylene multidose eyedroppers. PEERJ, vol. 6 pages e4549 (2018).

Claims (4)

1. A liquid composition based on polyhexamethylene biguanide containing polyhexamethylene biguanide in a concentration in the range of 0.04%-0.08% (w/v), a buffer system to maintain the pH in the range of 5-6.5, an isotonic agent to maintain the osmolarity in the range of 270 -330 mOsm/kg, characterized by a molecular weight of polyhexamethylene biguanide in the range of 2300-6000 amu. and a polymer polydispersity index in the range of 1.5-1.9, for ophthalmic administration for the treatment of acanthamoeba keratitis and/or fungal infections. 2. Liquid composition based on polyhexamethylene biguanide according to claim 1 for the treatment of coinfections caused by Acanthamoeba and Pseudomonas aeruginos or Staphylococcus epidermis. 3. Liquid composition based on polyhexamethylene biguanide according to claims 1, 2, where the buffer system is selected from the group containing phosphate, nitrate, bicarbonate, borate, Tris (tris(hydroxymethyl)aminomethane), glycerol, mannitol, sorbitol, trometamol, histidine, glycine , HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer or mixed system. 4. The liquid composition based on polyhexamethylene biguanide according to claim 3, wherein said buffer system is selected from the group consisting of dibasic sodium phosphate dodecahydrate/monosodium phosphate, dibasic sodium phosphate dodecahydrate/citric acid, Tris/HCl or boric acid/borate. 5. Liquid composition based on polyhexamethylene biguanide according to any one of claims 1 to 4, where the isotonic agent is sodium chloride or potassium chloride. 6. Liquid composition based on polyhexamethylene biguanide according to any one of claims 1-5, characterized by a polymer polydispersity index in the range of 1.7-1.8. 7. Liquid composition based on polyhexamethylene biguanide according to any one of claims 1-6, having a pH of 5.8. 8. Liquid composition based on polyhexamethylene biguanide according to any one of claims 1-7 in the form of collyria, eye drops, ophthalmic gel or viscous solutions. 9. The liquid composition based on polyhexamethylene biguanide according to claim 8 in the form of an ophthalmic gel or a viscous solution, further comprising a viscosity enhancer selected from the group consisting of xanthan gum, gellan gum, polyvinyl alcohol, hyaluronic acid, sodium hyaluronate, carboxymethylcellulose and hydroxypropylcellulose. 10. Liquid composition based on polyhexamethylene biguanide according to any one of claims 1 to 9, additionally containing a penetration enhancer selected from Tween 80 and benzalkonium chloride. 11. Liquid composition based on polyhexamethylene biguanide according to any one of claims 1-10, additionally containing 0.02% chlorhexidine, 0.1% propamidine or 0.1% dezomidine. 12. A method for producing a liquid composition based on polyhexamethylene biguanide according to any one of claims 1-11, including the following stages: (1) adding a buffer system at a concentration ranging from 0.01 to 4.5% (w/v) and an isotonic agent at a concentration ranging from 0.01 to 5.5% (w/v) to purified water under mild and continuous stirring; it is possible to add a viscosity enhancer selected from sodium hyaluronate, xanthan gum, polyvinyl alcohol, carboxymethylcellulose and hydroxypropylcellulose; (2) adding polyhexamethylene biguanide having a molecular weight in the range of 2300-6000 amu. and a polydispersity index in the range of 1.5-1.9, at a concentration in the range of 0.04%-0.08% (w/v) with gentle stirring; (3) bringing to the required volume by adding purified water; (4) sterilization by filtration or heat. 13. The method according to claim 12, where the polydispersity index of the polymer is in the range of 1.7-1.8. 14. A method for producing a liquid composition based on polyhexamethylene biguanide according to claim 12, where the specified buffer system is selected from the group consisting of phosphate, citrate, bicarbonate, borate, Tris, glycerol, mannitol, sorbitol, trometamol, histidine, glycine, HEPES buffer or mixed systems. 15. The method of producing a liquid composition based on polyhexamethylene biguanide according to claim 14, where the specified buffer system is selected from the group consisting of dibasic sodium phosphate dodecahydrate/monobasic sodium phosphate, dibasic sodium phosphate dodecahydrate/citric acid, Tris/HCl or boric acid/borate. 16. A method for producing a liquid composition based on polyhexamethylene biguanide according to any one of claims 12 to 15, where the isotonic agent is sodium chloride or potassium chloride. 17. An ophthalmic lens loaded with a liquid polyhexamethylene biguanide composition according to any one of claims 1 to 11 for controlled release of the active ingredient for use in the treatment of Acanthamoeba keratitis, Acanthamoeba and Pseudomonas aeruginosa co-infections or fungal infections.