Compositions containing pepstatin and alginic acid or their salts, and uses thereof

Abstract

The present invention relates to aqueous ophthalmic compositions comprising pepstatin and alginic acid or a salt thereof, and the use of said compositions in methods for treating diseases or conditions of the ocular and / or periocular area associated with or resulting from the presence of pepsin in tears.

Description

[Technical Field]

[0001] The present invention relates to a composition, preferably for ophthalmic use, comprising pepstatin and alginic acid or a salt thereof, and optionally hyaluronic acid or a salt thereof, and to the use of said composition in a method for treating a disease or condition associated with or resulting from the presence of pepsin in extra-esophageal areas, preferably in tears, in a subject in need thereof, such as a subject with gastroesophageal reflux and / or reflux of gastric juices from the stomach into extra-esophageal areas (pharyngeal reflux or extra-esophageal reflux), for example, a disease or condition of the ocular and / or periocular area associated with or resulting from the presence of pepsin in tears. [Background technology]

[0002] Gastroesophageal reflux or gastroesophageal reflux disease (GERD for short) is an abnormal physiological condition characterized by the reflux (from the stomach into the esophagus) of gastric contents or acidic gastric juices (pH 1-2), including, for example, pepsin, hydrochloric acid, gastric juice, duodenal juice, and acidic gastric juice. Thus, in patients with GERD, reflux of gastric contents (even in small amounts) from the stomach into the esophagus irritates the esophageal epithelium, resulting in retrosternal burning and odynophagia, as well as increased dental caries (due to erosion of tooth enamel by gastric acid), postprandial retching, and a sour feeling in the upper esophagus and pharynx.

[0003] Gastroesophageal reflux is a very common condition in the population and is often associated with obesity, diabetes, conditions of increased gastric secretion, pregnancy, smoking, alcohol, and hiatal hernia.

[0004] In the absence of endoscopic lesions observable on gastroscopy, assuming that reflux of acidic contents does not cause esophagitis, gastric reflux is defined as nonerosive reflux or nonerosive reflux disease (NERD for short).

[0005] Laryngopharyngeal reflux (LPR for short) or laryngopharyngeal reflux disease, or extraesophageal reflux, are various terms used to identify conditions or disorders or diseases caused by the reflux of gastric contents from the stomach into areas outside the esophagus, which may be the upper respiratory tract, the eye (or eye), the periocular area (e.g., eyelid, conjunctiva, and lacrimal apparatus), and the ear system, as well as the Eustachian tube and middle ear.

[0006] In the context of the present invention, the periocular region is defined as the region around or around the eye (e.g., the eyelid, conjunctiva, and lacrimal apparatus). The eyelid is a skin membrane structure covering the eye. The conjunctiva is a mucous membrane covering the inner part of the eyeball and eyelid. The lacrimal apparatus is a collection of organs that allow the secretion of the tear film, including the lacrimal gland, and enable its outflow. The organs that allow its outflow include the lacrimal duct, which consists of the lacrimal punctum, lacrimal sac, and lacrimal channels or ducts or nasolacrimal duct.

[0007] Reflux of gastric contents or portions thereof (e.g., pepsin) from the stomach can cause inflammation and / or irritation at extraesophageal locations and can involve one or more of said extraesophageal areas, such as the upper respiratory tract, oral cavity, ear cavity, ocular (or eye) and / or periocular areas (e.g., eyelids, conjunctiva, and lacrimal apparatus).

[0008] The two diseases, GERD and LPR or extraesophageal reflux, may coexist or may manifest separately.

[0009] When GERD and LPR coexist, in addition to a disorder in the esophageal region, a patient with GERD may also have one or more disorders or diseases in the extraesophageal region.

[0010] When GERD and LPR do not coexist, subjects without GERD may experience discomfort or symptoms in one or more extraesophageal regions due to reflux of gastric contents from the stomach into these regions.

[0011] In view of the above, individuals with LPR (erosive or non-erosive) or LPR (extraesophageal reflux) may suffer from ocular surface disease (OSD), alternatively defined as, for example, lacrimal dysfunction syndrome (LDS for short), or dry eye syndrome. Indeed, the presence of pepsin (a component of gastric contents) has been detected in the tear secretions of subjects with gastroesophageal reflux.

[0012] Lacrimal Dysfunction Syndrome (LDS) is an eye disorder characterized by a reduction in the quantity and / or alteration of the quality of the tear film, whose primary function is to moisten and protect the front of the eye. The tear film performs the following important functions: i) Nutrition: Tears can ensure an adequate supply of oxygen and nutrients for the proper turnover of ocular surface cells; ii) Antibacterial: the presence of antibodies and enzymes in the tear film ensures a protective effect against external attacks; iii) cleaning; and iv) Lubrication Therefore, changes in tear film quantity / quality expose the eye to greater friction (determined by eyelid movement) and a greater risk of infection.Dyslacrimal syndrome is one of the most common ophthalmic disorders, with a high impact on middle-aged and elderly populations.

[0013] In the context of the present invention, the expressions "lacrimal dysfunction syndrome" (LDS) and "dry eye syndrome" are used interchangeably.

[0014] Another disease or condition that can occur in subjects with GERD, LPR, or extraesophageal reflux is inflammation of the eye or periocular area, particularly conjunctivitis, i.e., inflammation that affects the conjunctiva and results in the characteristic redness and bloodshot appearance of the eye. Conjunctivitis can occur in adults, children, and newborns alike.

[0015] Often, disorders or conditions or diseases of extraesophageal regions (such as the ocular and periocular regions) are analyzed, diagnosed, and / or treated without consideration of their possible correlation with reflux of gastric contents (e.g., pepsin) from the stomach to extraesophageal regions.

[0016] As a result, many of the ocular and / or periocular area diseases or conditions (e.g., hypolacrimal syndrome (SDL), conjunctivitis, or ocular or periocular inflammation) are treated with currently available products (e.g., steroidal products) that do not specifically treat the underlying cause of the disease or condition and only provide temporary relief. For example, the commercial products do not effectively treat the presence of pepsin in the tears, eye, and / or periocular area.

[0017] BALESTRAZZI ALESSANDRA ET AL: "A new therapeutic approach for the Dry Eye Syndrome in patients with laryngopharyngeal reflux: first data" is a pilot study comparing two treatments in patients with dry eye syndrome and LPR. Patients were treated with Gastroftal eye drops and Gastroftal tablets or hyaluronic acid eye drops (Atlantis) alone for three months.

[0018] WO 2008 / 039984 A2 describes compounds that are inhibitors of cathepsin D (a member of the aspartyl protease subfamily) and pharmaceutical compositions containing said inhibitors. Such compounds have neurotrophic activity and are useful for the treatment and prevention of neuronal disorders (e.g., amyotrophic lateral sclerosis, multiple sclerosis, and muscular dystrophy). The pharmaceutical composition comprises alginic acid or a salt thereof.

[0019] US 4,339,439 A shows that co-administration of etintidine (a histamine H2 receptor antagonist) and pepstatin (a pepsin complexing agent) resulted in greater antiulcer activity in warm-blooded animals. Summary of the Invention

[0020] The technical problem addressed and solved by the present invention is to provide an ophthalmic, nasal or oral composition or mixture for the preventive and / or curative treatment of diseases or conditions of the extra-esophageal area, preferably diseases or conditions of the ocular and / or periocular area (such as lacrimal dysfunction syndrome (LDS), conjunctivitis, ocular or periocular inflammation, etc.), which are primarily caused by gastric reflux in the esophagus and / or extra-esophageal area and which are associated with or result from the presence of pepsin in said extra-esophageal area, preferably in tears.

[0021] Furthermore, the technical problem addressed and solved by the present invention is to provide an ophthalmic or nasal or buccal composition or mixture for use in said treatment methods that is stable, effective, easy to administer / apply, well tolerated and essentially free of side effects.

[0022] Through extensive and intensive research and development activities, the Applicant addresses and solves the above-mentioned technical problems by providing ophthalmic, nasal or oral compositions or mixtures comprising pepstatin and alginic acid or a salt thereof, and optionally hyaluronic acid or a salt thereof (in short, the compositions or mixtures of the present invention), as described below. Advantageously, the compositions and mixtures of the present invention are free of steroidal substances.

[0023] Besides being effective in the treatment of extraesophageal disorders, preferably the inflammatory ocular and / or periocular disorders mentioned, the ophthalmic compositions or mixtures of the invention based on pepstatin and alginic acid or salts thereof, optionally also based on hyaluronic acid or salts thereof, are stable, easy to administer / apply, well tolerated and essentially free of side effects.

[0024] In particular, the combination of pepstatin and alginic acid or a salt thereof acts effectively and synergistically to reduce and remove pepsin from tears, and this effectiveness is primarily due to the inhibitory action of pepstatin (even at low concentrations) and the simultaneous action of alginate as both a pepsin sequestering agent via nonspecific binding and as a lubricant.

[0025] Furthermore, the addition of an optional lubricant, preferably hyaluronic acid or a salt thereof, provides viscoelastic activity to the compositions of the present invention.

[0026] Additionally, the ophthalmic compositions or mixtures of the present invention are preferably free of steroidal substances.

[0027] Finally, the ophthalmic compositions of the present invention are easy to prepare and cost effective.

[0028] These and other objects, which will be apparent from the detailed description that follows, are achieved by the mixtures and compositions of the present invention by virtue of the technical characteristics claimed in the appended claims. [Brief explanation of the drawings]

[0029] [Figure 1] Absorption spectra of a blank without inhibitor (pepstatin) and a sample (pepsin); conditions: pepsin 0.02%, hemoglobin 2%, pH 1.5, 37°C [Figure 2] Absorption spectra of blank and pepsin samples in the presence of pepstatin (pepstatin: suspension in H2O) and comparison with the spectrum for free pepsin in the absence of pepstatin [Figure 3] Absorption spectra of blank (pepsin without pepstatin) and sample (pepsin + pepstatin) (pepstatin: dissolved in EtOH and diluted with water) [Figure 4]Pepsin activity spectrum in the presence of aqueous solution containing magnesium alginate; conditions: 0.02% pepsin, 2% hemoglobin, 1% aqueous solution of magnesium alginate; pH 1.5, 37°C [Figure 5] Absorption spectra of a blank (pepsin without pepstatin but with aqueous solution containing magnesium alginate) and a pepsin sample in the presence of aqueous solution containing magnesium alginate and pepstatin (pepstatin was dissolved in EtOH and then diluted with aqueous alginate). DETAILED DESCRIPTION OF THE INVENTION

[0030] A composition comprising: (i) A mixture M of active ingredients comprising or consisting of: (a) pepstatin or a pharmaceutically acceptable salt thereof, preferably an alkali metal or alkaline earth metal salt (e.g., magnesium, sodium, potassium, or calcium), and (b) alginic acid or a pharmaceutically acceptable salt thereof, preferably an alkali metal or alkaline earth metal alginate (e.g., magnesium, sodium, potassium, or calcium); form an object of the present invention, said composition may optionally comprise (ii) at least one additive and / or excipient of pharmaceutical or ophthalmic grade.

[0031] Pepstatin (also called pepstatin A) is an aspartyl protease inhibitor. It is a hexapeptide containing the amino acid statin (Sta, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic) acid, with the sequence isovaleryl-valyl-valyl-statyl-alanyl-statin (Iva-Val-Val-Sta-Ala-Sta) (e.g., CAS number: 26305-03-3). Pepstatin is a molecule produced by actinomycetes. At neutral pH (buffered), pepstatin is in its acidic form and can be dissolved in alcohol (e.g., ethanol) and then diluted with water. Alternatively, in the salt form, pepstatin can be dissolved in water at neutral pH. Preferably, for the preparation of the mixtures or compositions of the present invention comprising (a), (b) and optionally (c) and / or other components described in the present invention, pepstatin is used as such, i.e., as pepstatin not in the form of a salt.

[0032] The alginic acid contained in the mixture or composition of the present invention, together with (a) and, optionally, (c) and / or other components described herein, is preferably alginic acid (e.g., CAS No. 9005-32-7) having an average molecular weight comprised within the range of about 50 kDalton (kDa) to about 800 kDa; preferably about 100 kDa to about 600 kDa; more preferably about 200 kDa to about 400 kDa, e.g., about 240 kDa (atomic mass units).

[0033] Advantageously, said (b) alginic acid or salt thereof is obtained from seaweed.

[0034] In a preferred embodiment, the mixture of active ingredients M contained in the composition of the present invention comprises or consists of: (a) pepstatin or a salt thereof, (b) alginic acid or a salt thereof, preferably an alkali metal or alkaline earth metal salt (e.g., magnesium, sodium, potassium, or calcium), more preferably magnesium alginate, and (c) a lubricant, preferably hyaluronic acid or a pharmaceutically acceptable salt thereof, more preferably an alkali metal or alkaline earth metal hyaluronate (e.g., magnesium, sodium, potassium, or calcium), even more preferably sodium hyaluronate.

[0035] The lubricant (c), which may optionally be included in the mixture M of the composition of the invention together with (a) and (b) according to any one of the embodiments of the invention, may be selected from hyaluronic acid or a salt thereof, carboxymethylcellulose, hypromellose, xanthan gum, polyvinylpyrrolidone (PVP), polyvinyl alcohol, and mixtures thereof; preferably hyaluronic acid or a salt thereof.

[0036] The hyaluronic acid or salt thereof (e.g., sodium hyaluronate) contained in the mixture or composition of the present invention, together with (a) and (b) and, where appropriate, other components described herein, is preferably a linear or branched hyaluronic acid or salt thereof having an average molecular weight within the range of about 200 kDa to about 5,000 kDa; preferably about 1,000 kDa to about 3,000 kDa; more preferably about 1,500 kDa to about 2,000 kDa.

[0037] According to a preferred embodiment, the compositions of the present invention comprise or consist of: (a) pepstatin; (b) an alkali metal or alkaline earth metal alginate, preferably magnesium alginate; (c) an alkali metal or alkaline earth metal hyaluronate, preferably sodium hyaluronate, and ophthalmic grade additives and / or additives.

[0038] According to a more preferred embodiment, the compositions of the present invention comprise or consist of (a) pepstatin, (b) magnesium alginate, (c) sodium hyaluronate, and ophthalmic grade additives and / or additives.

[0039] According to a further preferred embodiment, the compositions of the present invention comprise or consist of (a) pepstatin, (b) magnesium alginate derived from alginic acid obtained from seaweed, having an average molecular weight of about 200 kDa to about 400 kDa, e.g., about 240 kDa, (c) sodium hyaluronate having an average molecular weight of about 1,500 kDa to about 2,000 kDa, and ophthalmic grade additives and / or additives.

[0040] In addition to (a) pepstatin, (b) alginic acid or a salt thereof, and, where appropriate, (c) a lubricant, preferably hyaluronic acid or a salt thereof, the mixture M contained in the composition of the present invention together with additives may further comprise at least one further active ingredient (d) selected from the group consisting of plant extracts (e.g., extracts of green tea (Camellia sinensis)), amino acids, vitamins A, B, C, D and / or E, and mixtures thereof.

[0041] The compositions of the present invention comprising said mixture M comprising (a), (b), and optionally (c) may comprise at least one acceptable pharmaceutical or food grade additive and / or excipient, i.e., a substance without therapeutic activity that is suitable for pharmaceutical or food use. In the context of the present invention, acceptable additives and / or excipients for pharmaceutical or food use include all auxiliary substances known to those skilled in the art for preparing compositions in semi-solid or liquid form, such as diluents, solvents (such as water), solubilizers, acidifiers, thickeners, sweeteners, lubricants, surfactants, preservatives, stabilizers, pH-stabilizing buffers, and mixtures thereof.

[0042] The compositions and mixtures M of the present invention comprise (a), (b), and optionally (c) according to any one of the described embodiments, and preferably do not comprise a steroid compound.

[0043] According to an aspect of the present invention, the mixture or composition of the present invention is not formulated for oral use.

[0044] The ophthalmic compositions of the present invention, comprising (a), (b) and, optionally, (c) (according to any one of the described embodiments), may be in liquid form (e.g., water-based eye drops), and exhibit high stability (preferably ≧24 months), homogeneity and no formation of sediment in liquid form, preferably water- or oil-based eye drops, or semi-solid form, preferably ointment, salve, gel or cream.

[0045] Compositions of the invention in liquid form, for example in the form of eye drops, may contain pepstatin in a molar concentration (M) comprised within the range of 0.1 nM (0.0001 μM) to 50 μM; preferably 0.01 μM to 5 μM; more preferably 0.5 μM to 2 μM (e.g., about 1 μM).

[0046] According to an embodiment of the present invention, the composition of the present invention may comprise the following in the following weight percentages (%) based on the total weight of the composition: (a) pepstatin, as such, 0.0000001% (1x10 -8 %) to 0.001% (1x10 -3 %), preferably 0.000001% (1x10 -6 %) to 0.0003% (3x10 -4 %), more preferably 0.00003% (3 x 10 -5 %) to 0.0001% (1x10 -4%); (b) alginic acid or a salt thereof, preferably magnesium alginate, 0.01% to 10%, preferably 0.05% to 2%, more preferably 0.05% to 0.4%, for example 0.2%; and, optionally, (c) a lubricant, preferably hyaluronic acid or a salt thereof, more preferably sodium hyaluronate, 0.01% to 10%; preferably 0.05% to 2%; more preferably 0.05% to 0.30%, for example 0.15%.

[0047] In the context of the present invention, the expression "composition" is used to designate a pharmaceutical composition or a medical device composition according to the European regulations on medical devices [EU 2017 / 745-(MDR), Directive 93 / 42 / EEC-(MDD)].

[0048] The compositions and mixtures M of the invention, for use as medicaments, comprising (a), (b) and, where appropriate, (c) and / or additives / excipients according to any one of the described embodiments, form the subject of the present invention.

[0049] The compositions and mixtures M of the invention, comprising (a), (b) and, where appropriate, (c) and / or additives / additives according to any one of the described embodiments, for the preventive and / or curative treatment of disorders or diseases of the ocular and / or periocular area, preferably inflammatory disorders or diseases, such as, for example, lacrimal dysfunction syndrome (LDS), conjunctivitis, ocular or periocular inflammation (e.g., blepharitis, keratitis, uveitis), primarily associated with or resulting from the presence of pepsin in the tears of a subject in need of treatment, form the subject of the present invention.

[0050] The presence of pepsin in tears is plausibly due to gastric reflux in the esophageal and / or extraesophageal regions.

[0051] The presence of pepsin in tears may be determined through conventional diagnostic methods suitable for detecting pepsin in fluids and known to those skilled in the art.

[0052] The presence of pepsin in tears is manifested in subjects with gastric reflux and / or diseases or conditions associated with said gastric reflux.

[0053] The disease or condition associated with gastric reflux is selected from the group comprising or consisting of gastroesophageal reflux disease (GERD), laryngopharyngeal reflux disease (RFL) or extraesophageal reflux, esophagitis (acute or chronic inflammation of the esophageal mucosa), esophageal ulcer, de-epithelialization of the esophageal mucosa, acid reflux, heartburn, gastric bloating, epigastric pain, dyspepsia, nausea, chronic cough, bronchospasm, sore throat, laryngitis, pharyngeal or hypopharyngeal bolus, heartburn, dysphonia, nasopharyngeal inflammation, and all disorders primarily caused by or caused in conjunction with gastric reflux.

[0054] The diseases or conditions associated with gastric reflux, including diseases or conditions of the ocular and / or periocular area, preferably inflammatory diseases or conditions (e.g., dysfunctional lacrimal gland syndrome (SDL), conjunctivitis, ocular or periocular inflammation (e.g., blepharitis, keratitis, uveitis), etc.), may be present in the absence of a diagnosis of gastroesophageal reflux disease (GERD), i.e., even in subjects who do not have GERD.

[0055] Furthermore, the present invention further relates to a method for producing the mixture or composition of the present invention, preferably for ophthalmic use (in short, the method of the present invention), which comprises step 1) dissolving pepstatin in a suitable solvent (e.g., an alcoholic solvent, ethanol or ethyl alcohol, isopropyl alcohol, glycerin, propylene glycol, sorbitol 20 or Tween® 20 (a mixture of partial triesters of sorbitol and its mono- and dianhydrides, and stearic acid), ethoxylated hydrogenated castor oil 40 molar (Peg-40 hydrogenated castor oil); preferably an alcoholic solvent, more preferably ethanol) at neutral pH (obtained using a buffer; preferably a borate buffer) to obtain a pepstatin alcoholic solution, preferably having a concentration within the range of 0.01 mg / ml to 1 mg / ml, preferably between 0.1 mg / ml and 1 mg / ml, for example 1 mg / ml. Step 1) is followed by step 2) of diluting the pepstatin alcohol solution with an aqueous solution containing an alginate, preferably an alkali metal or alkaline earth metal alginate, more preferably magnesium alginate.

[0056] Pepstatin at neutral pH is insoluble in water and in the prior art, pepstatin is used in the form of an aqueous suspension.

[0057] Conversely, the method of the present invention allows pepstatin to be dissolved in an aqueous phase without the use of solvents that are potentially toxic and unsuitable for ophthalmic use.

[0058] For clarity, to achieve the purposes of the present invention, the components (or active ingredients) (a), (b), and optionally (c) of the mixture or composition of the present invention may be administered simultaneously or separately (preferably with a time interval of 5 to 30 minutes), and may be administered in any order. Preferably, the active ingredients are administered to a subject simultaneously, and even more preferably, in a single composition for a more rapid effect and ease of administration. When the active ingredients of the present invention are administered in a single composition, said single composition corresponds to the composition of the present invention.

[0059] In the context of the present invention, the term "subject" is used to refer to a human or animal subject, preferably a mammal (e.g., a pet such as a dog, cat, horse, sheep, or cow). Preferably, the compositions of the present invention are used in a method of treating a human subject.

[0060] Unless otherwise specified, a reference to a composition or mixture or the like containing an amount of a component "falling within the range of x to y" is used to indicate that the range endpoints are included, even if not expressly stated, and that the component may be present in the composition or mixture or the like in all amounts within the range.

[0061] Unless otherwise specified, the content of an ingredient in a composition or mixture refers to the weight percent (%) of the ingredient relative to said total weight of said composition or mixture.

[0062] Unless otherwise specified, a statement that a composition, mixture, or other "comprises" one or more components means that other components may be present in addition to the components specified, and a statement that a composition, mixture, or other "consists of" specified components means that the presence of other components not specified is excluded.

[0063] In the context of the present invention, the expression "method of treatment" is used to indicate an intervention in a subject in need of treatment characterized by administering to the subject a therapeutically effective amount of a composition or mixture of the present invention to eliminate, reduce / reduce or prevent a disease or condition and its symptoms or disorders.

[0064] The phrase "therapeutically effective amount" refers to that amount of active compound that elicits a biological or medicinal response in a tissue, system, mammal, or human, as determined and defined by an individual, researcher, veterinarian, physician, or other clinician or health care professional. [Example]

[0065] Examples of ophthalmic compositions in liquid form according to the present invention are reported in Tables 1, 2, 3 and 4. (solution buffered to approximately pH 7.6±0.1: 7.4-7.8±0.1). [Table 1] [Table 2] [Table 3] [Table 4]

[0066] Experimental Part I. Method The method used to measure the enzymatic activity of pepsin alone or with an inhibitor (pepstatin) was a modification of the Anson method [Anson et al., J. Gen. Physiol. 1931, 16, 59], which uses denatured hemoglobin as a substrate. Enzyme activity was determined after precipitation of the unhydrolyzed substrate with trichloroacetic acid. Enzyme activity was determined spectrophotometrically from the concentration of soluble peptides liberated by proteolysis. Reactions studied: (Hemoglobin + H2O) + Pepsin → Oligopeptide

[0067] II. Reagents: Hydrochloric acid 12M Human hemoglobin (Sigma-Aldrich) Pepsin (BDH; 1 Anson unit per gram): enzyme Trichloroacetic acid solution 6.1N (Sigma-Aldrich) Pepstatin A: an inhibitor (or inhibitor)

[0068] III. Conditions T = 37 °C, pH = 1.5, A 280nm , light path 1 cm, reaction time 10 min. Pepsin activity is maximum at pH 1.5, where the experiment was conducted.

[0069] Hemoglobin 2%, Pepsin 0.02%, Inhibitor 0.01%

[0070] IV. Preparation Substrate: Weigh 250 mg of human hemoglobin into a 10 mL flask, bring the volume up with distilled water, and stir in a 37°C water bath for 10 minutes to obtain a 2.5% solution. Pass the solution through a filter paper to remove any insoluble residue. Take 8 mL of the filtrate and increase the volume to 10 mL with acidified water to obtain a 2% (w / V) solution with a final pH of 1.5.

[0071] Enzyme: Weigh 5 mg of pepsin into a 5 mL flask and make up the volume with acidified water (pH = 1.5).

[0072] Enzyme combined with inhibitor: Weigh 5 mg of pepsin into a 5 mL flask. Separately prepare a 0.5 mg / mL solution or suspension of inhibitor (pH = 1.5) (check the pH with a pH meter and, if necessary, adjust the pH with 12 M hydrochloric acid so that the total volume remains constant). Add the 0.5 mg / mL solution or suspension of inhibitor to the flask containing pepsin, and continue stirring with a magnet for 10 minutes before proceeding with the experiment.

[0073] Trichloroacetic acid: Dilute 6.1N trichloroacetic acid solution 20 times with distilled water to obtain a 5% (w / V) solution.

[0074] V. Experimental Procedure 0.25 mL of substrate (hemoglobin) is pipetted into a suitable vial and almost completely immersed in a water bath adjusted to 37°C. This is stirred with a magnet for 10 minutes at 37°C. 50 microliters of enzyme solution (pepsin) or enzyme combined with inhibitors (pepsin + pepstatin A) are then added and the system is incubated for exactly 10 minutes. After this time, 0.5 mL of 5% trichloroacetic acid solution is added, followed by leaving it for another 5 minutes.

[0075] Using a graduated pipette, add exactly 3 mL of distilled water (pH = 1.5) to the vial. Stir the solution and filter it using a 0.45 μm syringe filter. Record the absorption spectrum (absorbance (A) is always less than 1) from 300 nm to 250 nm using a spectrophotometer, with the absorbance value at 280 nm as the reference.

[0076] Blank: 0.25 mL of substrate (hemoglobin) is pipetted into a suitable vial and almost completely immersed in a water bath adjusted to 37°C. This is stirred with a magnet for 10 minutes. 50 microliters of a 0.5 mg / mL inhibitor solution (without enzyme) is then added and the system is incubated for exactly 10 minutes. After this time, 0.5 mL of a 5% trichloroacetic acid solution is added, followed by leaving it for another 5 minutes.

[0077] Fifty microliters of a 1 mg / mL enzyme solution (without inhibitors) is introduced. Using a graduated pipette, exactly 3 mL of distilled water (pH = 1.5) is added to the vial. The solution is then vortexed and filtered using a 0.45 μm syringe filter. The absorbance at wavelengths from 300 nm to 250 nm is recorded using a spectrophotometer, with the absorbance value at 280 nm as the reference.

[0078] VI.Calculation Each experiment was repeated at least twice. The final absorbance value (A) at 280 nm was the average of the absorbance values ​​obtained (uncertainty in the values ​​was always much less than 10%). A, ΔA, is the mean of the ΔA X =A 280サンプル(物質X) -A280ブランク(物質X) and ΔAo=A 280サンプル(遊離ペプシン) -A 280ブランク(遊離ペプシン) (in the absence of inhibitors) and is related to pepsin inhibitors. The calculated ΔA is proportional to the amount of soluble aromatic amino acid residues liberated into solution by the proteolytic action of pepsin alone or in the presence of an inhibitor (pepstatin A).

[0079] Figure 1 shows the absorption spectra of the blank and sample (reaction time = 10 min) in the absence of any inhibitor. o =0.40

[0080] The maximum absorbance difference determined between the sample and blank at 280 nm was 0.34, which is taken as the proteolytic action of the pepsin enzyme on the substrate within 10 minutes in the absence of any inhibitor under these experimental conditions.

[0081] The residual activity in the presence of substance X was calculated based on the following formula: formula 1 Residual activity=(ΔA X / ΔA o )x100

[0082] VII. Results and Discussion

[0083] VII.A. Pepstatin [Table 5]

[0084] The concentration of the substance (0.01%) was chosen to observe the inhibition of pepsin and to minimize the light scattering caused by the inhibitor. The Anson method used allows for the standardization of the absorbance increase for each substance with respect to the scattering that has the same effect on both the blank and the sample. In fact, the absorbance difference at 280 nm was measured independently of the scattering phenomenon.

[0085] Pepstatin showed a complete reduction in residual activity (ΔA X =0).

[0086] Although pepstatin is insoluble in HO, a suspension made in the laboratory was completely effective for its intended purpose (ΔA X =0).

[0087] Figure 2 shows the absorption spectra of the blank and sample in the presence of 0.01% pepstatin (in HO, suspension; the absorbance of the blank is greater due to the pepstatin suspension) (reaction time = 10 min), ΔA o = 0.004, and the spectrum ΔA for free pepsin in the absence of inhibitors X =0.340.

[0088] Pepstatin was completely dissolved in ethanol (EtOH) at a concentration of 1 mg / mL. Such a solution could be diluted with water. Pepstatin maintained its inhibitory activity and remained soluble when diluted from EtOH with water. This was demonstrated by the fact that pepstatin maintained its inhibitory activity and that no scattering effect was observed with respect to the blank (the aggregation index of the molecule was not determined in this case).

[0089] Next, the activity of pepsin in the presence of 1 μM pepstatin was measured under the same test conditions as the previous study. Pepstatin was added to 2% hemoglobin, followed by the addition of pepsin. From the above perspective, we investigated whether the binding of the enzyme to the inhibitor was immediate or slow on the experimental timescale (t = 10 min) (kinetic study). A 1 μM pepstatin / 2% hemoglobin solution was prepared, and after adding the enzyme, various activities were measured. The experimental results are shown in Figure 3.

[0090] FIG. 3 shows the absorption spectra of the blank (pepsin without pepstatin) and sample at pH=1.5 in the presence of 1 μM pepstatin (dissolved in EtOH (1 mg / mL) and diluted with HO) (reaction time=10 min).

[0091] Figure 3 directly shows that at a concentration of 1 μM, activity is completely reduced. From a kinetic standpoint, if activity is essentially zero, this is clearly due to immediate binding of the enzyme and inhibitor, given that the enzyme has no time to exert its proteolytic effect when the inhibitor is already present in 2% hemoglobin.

[0092] VII.B. Magnesium Alginate As a post-experimental procedure, a sterile aqueous solution containing 0.2% w / w magnesium alginate (shortly alginate solution) was added to the hemoglobin solution.

[0093] The selected concentration of the alginate solution in hemoglobin was 1%. This concentration prevented diffusion and prevented complete loss of activity. In hemoglobin, the alginate solution inhibited pepsin, reducing its activity by 60% relative to free pepsin at pH 1.5 (Figure 4). Figure 4 shows the reduction in pepsin activity in the presence of a 1% aqueous magnesium alginate solution at pH 1.5.

[0094] VII.C. Pepstatin + Magnesium Alginate To test the synergistic effect of pepstatin (a composition according to the invention) in combination with magnesium alginate on overall pepsin inhibition, a hemoglobin solution containing 1% of the 0.2% w / w magnesium alginate-containing aqueous solution (see paragraph VII.B.) in which pepstatin was dissolved at 1 μM was prepared at pH 1.5 (the amount of alginate-containing solution added to the hemoglobin was such that the desired final pepstatin concentration was obtained), and various activities were measured after addition of the enzyme (pepsin). The experimental results are shown in Figure 5.

[0095] Dilution of a 1 mg / mL pepstatin solution in ethanol with aqueous magnesium alginate does not impair the solubility and inhibitory capacity of pepstatin.

[0096] Figure 5 shows the absorption spectra of a blank (pepsin without pepstatin but with 1% hemoglobin solution containing magnesium alginate) and a sample (reaction time = 10 min) at pH = 1.5 in the presence of 1% magnesium alginate solution and 100 nM pepstatin (pepstatin was dissolved in EtOH (1 mg / mL) and then diluted with aqueous magnesium alginate solution).

[0097] The synergistic effect of pepstatin together with magnesium alginate was such that a pepstatin concentration of 100 nM (an order of magnitude lower than pepstatin in water) was able to completely eliminate pepsin activity.

[0098] VIII. Conclusion Pepsin can be found in the tears of patients with gastroesophageal reflux. This enzyme, if still active, can damage the eye due to its proteolytic action. Alginate inhibits pepsin noncompetitively and in a dose-dependent manner. Pepstatin A, on the other hand, inhibits pepsin at picomolar concentrations. The pepstatin-magnesium alginate binary system has a synergistic inhibitory effect, completely inactivating the pepsin enzyme at picomolar concentrations, while simultaneously sequestering the enzyme through nonspecific binding by magnesium alginate, which exerts a lubricating effect. This binary system therefore treats and prevents eye damage caused by pepsin.

[0099] Pepstatin is insoluble in water and, in fact, is always used as a suspension, capable of inhibiting pepsin in any case. The applicant has discovered a method for dissolving such inhibitors in water without the use of toxic solvents. An ethanolic solution of pepstatin can be diluted with water. It maintains its inhibitory activity and remains soluble even after dilution. Pepstatin dissolved in water in this manner is capable of stoichiometrically inhibiting pepsin. There is no suspension or precipitation in the solution. The binding of pepsin in water is immediate on a human timescale, unless the pepsin added to hemoglobin during the experiment is able to exert its proteolytic action in the presence of an inhibitor.

[0100] IX. Manufacturing method The steps for preparing a water-based liquid form of a composition containing pepstatin and magnesium alginate (composition according to the present invention, Table 6) are described below.

[0101] Step 1: Measure the water and pour it into a beaker with a magnetic stir bar. Start stirring and heat the water to 50°C. Add buffer salts Ingredients: Demineralized water, disodium tetraborate, boric acid

[0102] Step 2: Gradually add the ingredients in the order listed (1. Sodium Chloride, 2. Magnesium Alginate) and wait for complete dissolution. The resulting solution is left stirring for 30 minutes.

[0103] Filtration step 1: Filter the resulting solution through a 0.2 μm PES filter

[0104] Step 3: After the first filtration, measure the water and pour it into a beaker. Start stirring and heat the water to 50°C. Add an ethanol solution of pepstatin (e.g., at a concentration of 1 mg / ml) The resulting solution is left stirring for 30 minutes. Pepstatin: Isovaleryl-L-valyl-L-valyl-statyl-L-alanyl-statin or Isovaleryl-L-valyl-L-valyl-4-(S)-amino-3-(S)-hydroxy-6-methyl-heptanoyl-2-alanyl-4-(S)-amino-3-(S)-hydroxy-6-methyl-heptanoic acid

[0105] Filtration step 2: Filter the resulting solution through a 0.2 μm PES filter [Table 6]

[0106] Physical properties of the compositions in Table 6: pH about 7.53; Osmolality: Approximately 304 mOsm Kg -1 ; Viscosity approximately 1.309cSt; Density approximately 1.003g mL -1

[0107] Measurement methods for the physical quantities described: Osmolality: Osmometer Viscosity: Ostwald viscometer (measured at room temperature) pH: pH meter Density: Pycnometer

[0108] Expected amount of pepstatin in the composition: 1 μM or less Pepstatin is soluble in ethanol up to 1 mg / mL.

[0109] An embodiment E(n) of the present invention is shown below.

[0110] E1. An aqueous ophthalmic composition comprising: (i) a mixture M comprising or consisting of: (a) pepstatin or a pharmaceutically acceptable salt thereof; and (b) alginic acid, or a pharmaceutically acceptable salt thereof; (ii) at least one ophthalmic grade additive and / or excipient;

[0111] E2. The composition of E1, wherein the mixture M further comprises (c) hyaluronic acid or a pharmaceutically acceptable salt thereof.

[0112] E3. The above (i) mixture (M) is (a) Pepstatin; (b) an alkali metal or alkaline earth metal alginate, preferably magnesium alginate; and (c) hyaluronates of alkali metals or alkaline earth metals, preferably sodium hyaluronate; The composition of E2, comprising, or consisting of:

[0113] E4. The composition of any one of E1 to E3, wherein the composition is formulated for ophthalmic use; preferably formulated in an ophthalmic liquid form, more preferably eye drops or water-based eye drops; or formulated in an ophthalmic semi-solid form, preferably an ointment, salve, gel, or cream.

[0114] E5. The composition of any one of E1 to E4 for use as a medicament.

[0115] E6. A composition described in any one of E1 to E4 for the preventative and / or definitive treatment of a disease or condition of the ocular and / or periocular area associated with or resulting from the presence of pepsin in tears in a subject in need of treatment.

[0116] E7. The composition of E6, wherein the disease or condition of the eye and / or periocular area is selected from the group comprising or consisting of lacrimal dysfunction syndrome (LDS) or dry eye syndrome, conjunctivitis, conjunctivitis of the cornea, ocular inflammation, periocular inflammation, blepharitis, keratitis, and uveitis; preferably selected from the group comprising or consisting of lacrimal dysfunction syndrome (LDS) or dry eye syndrome, conjunctivitis, keratitis, ocular inflammation, and periocular inflammation.

[0117] E8. The composition of E6 or E7, wherein the composition is for the preventative and / or curative treatment of a disease or condition of the ocular and / or periocular area in (i) a subject with gastric reflux from the stomach to the esophagus and / or extraesophageal area, or (ii) a subject with a disease or condition associated with or resulting from said gastric reflux.

[0118] E9. The composition of E8, wherein the disease or symptom associated with or resulting from gastric reflux from the stomach to the esophagus and / or extraesophageal regions is selected from the group comprising or consisting of gastroesophageal reflux disease (GERD), erosive or non-erosive laryngopharyngeal reflux (LPR) or extraesophageal reflux, esophagitis, esophageal ulcer, de-epithelialization of the esophageal mucosa, gastric reflux, heartburn, gastric bloating, epigastric pain, dyspepsia, nausea, chronic cough, bronchospasm, sore throat, laryngitis, pharyngeal or hypopharyngeal bolus, heartburn, dysphonia, and nasopharyngeal inflammation.

[0119] E10. A method of making the composition of any one of E1 to E4, said method comprising: Step 1) Dissolve pepstatin in a solvent of neutral pH, preferably an alcoholic solvent or ethanol, to obtain an alcohol solution of pepstatin. Next Step 2) Diluting the alcoholic solution of pepstatin into an aqueous solution containing an alginate, preferably an alkali metal or alkaline earth metal alginate, more preferably magnesium alginate. A method comprising:

Claims

1. An aqueous ophthalmic composition comprising: (i) a mixture M comprising, or alternatively consisting of: (a) pepstatin or a pharmaceutically acceptable salt thereof; and (b) alginic acid, or a pharmaceutically acceptable salt thereof; (ii) at least one ophthalmic grade additive and / or additive;

2. The composition of claim 1 , wherein the mixture M further comprises (c) hyaluronic acid or a pharmaceutically acceptable salt thereof.

3. The (i) mixture (M) is (a) pepstatin; (b) an alkali metal or alkaline earth metal alginate, preferably magnesium alginate; and (c) an alkali metal or alkaline earth metal hyaluronate, preferably sodium hyaluronate; 3. The composition of claim 2, comprising or consisting of:

4. 4. The composition of any one of claims 1 to 3, wherein the composition is formulated for ophthalmic use; preferably in an ophthalmic liquid form, more preferably eye drops or water-based eye drops; or in an ophthalmic semi-solid form, preferably an ointment, salve, gel or cream.

5. A composition according to any one of claims 1 to 4 for use as a medicament.

6. A composition according to any one of claims 1 to 4 for the preventive and / or curative treatment of diseases or conditions of the ocular and / or periocular area associated with or resulting from the presence of pepsin in the tears of a subject in need of treatment.

7. 7. The composition of claim 6, wherein the disease or condition of the eye and / or periocular area is selected from the group comprising or consisting of lacrimal dysfunction syndrome (LDS) or dry eye syndrome, conjunctivitis, conjunctivitis of the cornea, ocular inflammation, periocular inflammation, blepharitis, keratitis, and uveitis; preferably selected from the group comprising or consisting of lacrimal dysfunction syndrome (LDS) or dry eye syndrome, conjunctivitis, keratitis, ocular inflammation, and periocular inflammation.

8. The composition of claim 6 or 7, wherein the composition is for the preventive and / or curative treatment of diseases or symptoms of the ocular and / or periocular area in (i) a subject with gastric reflux from the stomach to the esophagus and / or extraesophageal area, or (ii) a subject with a disease or symptom associated with or resulting from said gastric reflux.

9. 9. The composition of claim 8, wherein the disease or symptom associated with or resulting from gastric reflux from the stomach to the esophagus and / or extraesophageal region is selected from the group comprising or consisting of gastroesophageal reflux disease (GERD), erosive or non-erosive laryngopharyngeal reflux (LPR) or extraesophageal reflux, esophagitis, esophageal ulcer, de-epithelialization of the esophageal mucosa, gastric reflux, heartburn, gastric bloating, epigastric pain, dyspepsia, nausea, chronic cough, bronchospasm, sore throat, laryngitis, pharyngeal irritation or hypopharyngeal bolus, heartburn, dysphonia, and nasopharyngeal inflammation.

10. 5. A method for producing a composition according to any one of claims 1 to 4, said method comprising: Step 1) Dissolving pepstatin in a solvent of neutral pH, preferably an alcoholic solvent or ethanol, to obtain an alcoholic solution of pepstatin. Next Step 2) Diluting the alcoholic solution of pepstatin into an aqueous solution containing an alginate, preferably an alkali metal or alkaline earth metal alginate, more preferably magnesium alginate. A method comprising:

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