RU2753511C2 - Composition of eye drops - Google Patents

Abstract

FIELD: medicine; ophthalmology.SUBSTANCE: group of inventions relates to the field of medicine, namely to ophthalmology, it is intended for the use when treating eye infections in animals. Ophthalmic solution contains at least 0.1 % wt./vol. of 2-amino-9-[[(1S,2R)-1,2-bis(hydroxymethyl)cyclopropyl]methyl]-1,9-dihydro-6H-purin-6-one and at least 15 % wt./vol. of cyclodextrin. A method for producing the specified solution is also presented, including following stages: a) a solution is obtained containing at least 15 % wt./vol. of cyclodextrin; b) 2-amino-9-[[(1S,2R)-1,2-bis(hydroxymethyl)-cyclopropyl]methyl]-1,9-dihydro-6H-purin-6-one is added to this solution; c) 2-amino-9-[[(1S,2R)-1,2-bis(hydroxymethyl)-cyclopropyl]methyl]-1,9-dihydro-6H-purin-6-one added at the stage b) is dissolved; and d) the stages b) and c) are repeated until the concentration of 2-amino-9-[[(1S,2R)-1,2-bis(hydroxymethyl)-cyclopropyl]methyl]-1,9-dihydro-6H-purin-6-one in the solution of at least 1 mg/ml is obtained.EFFECT: use of the group of inventions provides for a solution with improved stability, which allows one to re-use it at intervals of several hours during several days or weeks without precipitation formation, as well as good tolerance with healthy and infected eyes.14 cl, 9 ex

Description

Technology area

The present invention relates to an eye drop composition and its use for the diagnosis and treatment of eye infections in animals.

Prior art

Diagnosing eye infections in animals can be problematic. For example, diagnosis of feline herpes simplex virus type 1 (FHV-1) is usually based on clinical signs and PCR confirmation of viral DNA. However, this diagnosis is complicated by the possibility of false negative PCR results, and by the fact that positive PCR results can also reflect low prevalence of the virus and even latent infection. Therefore, it is often impossible to prove that the observed clinical signs are actually related to the presence of FHV-1 virus DNA. Accurate and prompt diagnosis is essential for effective treatment and the elimination of over-drug treatment.

Various antiviral compounds are known for the treatment of herpes virus infections. Compound 2-amino-9 - [[(1S, 2R) -1,2-bis (hydroxymethyl) -cyclopropyl] methyl] -1,9-dihydro-6H-purin-6-one (also known as “A-5021 ”) Is a potent inhibitor of herpes virus replication (Neyts et al., Antiviral Research 2001, 49, 115-120; Iwayama et al., Antimicr. Agents Chemother. 1998, 42, 1666-1670). For example, A-5021 has been reported to be a potent and selective antiviral agent against herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella zoster virus (VZV) in vivo. However, the medical and veterinary use of A-5021 is hampered by its low solubility. The solubility of A-5021 to pharmacologically active concentrations is highly dependent on pH, and obtaining a stable solution containing more than 1 mg / ml A-5021 (or A-5021 solutions with a concentration of 0.1% w / v) usually requires pH values either below 4 or above 10. Eye drop compositions having a pH value of about 7 and containing more than 1 mg / ml of A-5021 have been reported by Itahashi et al. (Cornea 2008, 27, 334-338), however, these compositions have poor stability and therefore cannot be used in practice. Therefore, the preparation of stable pharmaceutical formulations of dissolved A-5021 is not straightforward.

There is a need for improved methods for diagnosing viral infections and compositions that effectively treat (ocular) herpes infections that address at least one of the problems noted above.

The essence of the invention

The present invention discloses a method of dissolving A-5021 under isotonic and pH neutral conditions at concentrations of 1-10 mg / ml (0.1-1% w / v), which allows the use of A-5021 in stable liquid compositions such as eye drops. Such eye drops are especially useful in the treatment and diagnosis of eye infections in animals.

In a first aspect, the present invention relates to compositions comprising A-5021 at a concentration of at least 0.1% w / v that are stable. More specifically, the compositions are stable in that A-5021 does not precipitate, and more specifically, no precipitation is observed within 1-48 hours, preferably within 1 week, more preferably within 1 month, and most preferably within 3 or more than months after the preparation of the composition. More specifically, the compositions of the present invention include:

- at least 0.1% wt./about. A-5021 and

- at least 10% wt./about. cyclodextrin.

In particular embodiments of the invention, the composition is an aqueous solution. In some embodiments, the composition is an ophthalmic solution. In specific embodiments, the cyclodextrin used in the compositions of the present invention is hydroxypropyl beta cyclodextrin. In some embodiments, the composition further comprises at least 0.008% w / v. thiomersala.

In another aspect, the present invention relates to a composition as described above for use as a diagnostic and / or therapeutic composition.

In some embodiments, the compositions are for use in a method for treating an ocular herpes infection. In specific embodiments of the invention, the eye infection to be treated is a companion animal eye infection. In some embodiments, the companion animal is a feline. In specific embodiments, the ocular infection to be treated using the compositions of the present invention is an ocular infection in a feline. In specific embodiments of the invention, the methods of treatment provided comprise administering a composition of the invention to the eye two or three times a day for at least seven days.

In some embodiments, the compositions of the present invention are provided for use in a method for diagnosing an ocular herpes simplex infection. In specific embodiments, the eye infection is an eye infection in a companion animal. In specific embodiments, the companion animal is a feline. In specific embodiments of the invention, said diagnostic methods comprise administering a composition of the invention to the eye two or three times a day for one to seven days, and determining whether or not the clinical symptoms of an ocular infection have decreased, where a decrease in clinical symptoms indicates that the ocular the infection is an ocular herpes infection.

In another aspect, the present invention relates to methods for preparing stable ophthalmic solutions containing A-5021. In particular, the methods include the stages at which:

a) get a solution containing at least 10% wt./about. cyclodextrin;

b) A-5021 is added to this solution;

c) dissolve the A-5021 added in step b);

d) repeating steps b) and c) until the concentration of A-5021 in solution is at least 1 mg / ml;

e) if necessary, add at least 0.08 mg / ml thiomersal.

In certain embodiments of the invention, the cyclodextrin in step a) is hydroxypropyl-beta-cyclodextrin.

Detailed description of the invention

The present invention will be described with reference to specific embodiments, but the present invention is limited only by the claims and not by these embodiments. Any reference numerals in the claims should not be construed as limiting their scope.

As used herein, the singular includes both the singular and the plural, unless the context clearly indicates otherwise.

The terms “comprising,” “comprises,” and “consists of,” as used herein, are synonymous with the terms “including,” “includes,” or “comprising,” “contains,” and are inclusive or open-ended and do not exclude additional, not listed components. elements or steps of the method. The terms "comprising", "comprises" and "consists of" when referring to the recited components, elements or process steps also include embodiments that "consist of" the recited recited components, elements or process steps.

In addition, the terms first, second, third, etc. in the description and in the claims are used to distinguish between similar elements and not necessarily to describe sequential or chronological order, unless otherwise indicated. It should be understood that the terms used in this manner are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein are capable of operating in sequences other than those described or illustrated herein.

Values that are used in relation to the measurand, such as parameter, quantity, time duration, etc., are intended to cover deviations of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1 % or less, even more preferably +/- 0.1% or less, of the indicated value, insofar as such deviations are suitable for the described invention. It should be understood that each meaning as used herein is also specifically and preferably disclosed.

The disclosure of numerical ranges with end values includes all numbers and sub-ranges included in the respective ranges, as well as the indicated end values.

All documents cited in this description are included in this description by reference in their entirety.

Unless otherwise indicated, all terms used in the disclosure of an invention, including technical and scientific terms, have the meaning commonly understood by one of ordinary skill in the art to which this invention relates. By way of additional guidance, definitions of terms used in the description are included for a better understanding of the present invention. The terms and definitions used in this document are provided solely to assist in understanding the invention.

The term "w / v" as used herein means weight / volume. If a solution is stated to contain a certain compound at a concentration of x% w / v, this means that 1 liter of solution contains 10 g of the compound.

The term "sulfo", used alone or as part of another substituent, means a —SO ₃ H group or a salt thereof.

Reference in this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one variation" or "in variation" in various places throughout this specification do not necessarily refer to the same variation. In addition, particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to those skilled in the art from this description, in one or more embodiments. In addition, while some of the embodiments described herein include some features but not other features included in other embodiments, combinations of features of various embodiments are within the scope of the present invention and form various embodiments as those skilled in the art will appreciate. in this area. For example, in the appended claims, any of the features of the claimed embodiments may be used in any combination.

The present invention provides liquid compositions containing compound A-5021 (i.e. 2-amino-9 - [[(1S, 2R) -1,2-bis (hydroxymethyl) cyclopropyl] methyl] -1,9-dihydro- 6H-purin-6-one) represented by formula (I):

Unlike other nucleoside analogs, compound A-5021 is poorly soluble in water. Although, given the structural similarities between A-5021 and the known herpes drug penciclovir (ie, 2-amino-9- [4-hydroxy-3- (hydroxymethyl) butyl] -3H-purin-6-one), it is likely that it can be formulated as a sodium salt hydrate composition, such a composition is highly alkaline and therefore incompatible with applications where a physiological composition is required.

Thus, specific applications of A-5021 were difficult due to the impossibility of obtaining a sufficiently high concentration of the active ingredient in a physiologically compatible liquid composition.

For example, a suitable eye drop composition should have a neutral pH (ie, pH 7) or near neutral pH. The inventors have found that only cyclodextrins such as hydroxypropyl beta cyclodextrin (Kleptose® HPB) are suitable for physiological liquid compositions A-5021, since only the use of such agents results in a solution with a concentration of 0.1% to 1% mass./about., which is well tolerated by healthy eyes and eyes infected with the herpes virus (see example A1). In addition, the composition must be stable so that it can be reused at intervals of several hours, preferably over several days or weeks, without precipitation in the composition.

Accordingly, in a first aspect, the present invention relates to a composition comprising

- at least 0.1% wt./about. A-5021; and

- cyclodextrin.

Obtaining a physiologically acceptable solution of the active ingredient at a concentration of 0.1% wt./about. or more allows it to be formulated in various forms, such as aqueous solutions (which can be applied as sprays or drops), aqueous gels, and the like.

In certain embodiments of the invention, the composition is an ophthalmic solution.

In certain embodiments of the invention, the composition further comprises one or more solvents, such as water. In some embodiments, the composition is an aqueous solution. The term "aqueous" as used herein means that more than 50 volume percent of the solvent is water.

In certain embodiments of the invention, the composition according to the present invention consists of a solvent of at least 0.1% w / v. A-5021 and cyclodextrin. In certain embodiments, the implementation of the composition according to the present invention consists of at least 0.1% wt./about. A-5021 and cyclodextrin.

Concentration A-5021 0.1% w / v provides a minimal sufficient therapeutic effect of the composition for treating herpes virus infections in domestic animals or other species of animals larger than rodents. However, in certain embodiments of the invention, a higher concentration of A-5021 can lead to an increase in the therapeutic effect of the composition. Accordingly, in certain embodiments, the composition comprises at least 0.10%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% , 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95% or 1% A-5021 (percentages are% w / v).

The use of cyclodextrin makes it possible to obtain stable compositions containing A-5021 at a concentration of at least 0.1% w / v, for example 0.2% w / v. or 0.5% w / v, which are well tolerated when used as directed when physiologically acceptable solutions are required, such as administration to the eye and / or wound. A higher concentration of cyclodextrin usually produces a higher concentration of A-5021. In certain embodiments of the invention, the composition comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95% or 99% cyclodextrin (percentages are% w / v).

Cyclodextrin may include one or more of beta-cyclodextrin, gamma-cyclodextrin, or alpha-cyclodextrin. In certain embodiments, the cyclodextrin comprises beta-cyclodextrin. In other embodiments, the cyclodextrin comprises a modified or substituted cyclodextrin. For example, cyclodextrin may contain one or more substituents selected from hydroxypropyl (eg 2-hydroxypropyl), sulfobutyl, and methyl. In certain embodiments, the cyclodextrin is hydroxypropyl (beta) cyclodextrin (eg, Kleptose® HPB), more particularly 2-hydroxypropyl (beta) cyclodextrin. Hydroxypropyl beta-cyclodextrin significantly increases the solubility of A-5021 and is well tolerated when administered to the eye. In certain embodiments of the invention, the cyclodextrin comprises methyl cyclodextrin, for example, randomly methylated beta cyclodextrin. In certain embodiments, the cyclodextrin is sulfobutylether-cyclodextrin, eg, sulfobutylether-beta-cyclodextrin (eg Captisol®).

In certain embodiments, the compositions of the present invention further comprise one or more preservatives. Indeed, multi-dose pharmaceutical compositions usually contain preservatives, which allows them to be used repeatedly within a day and / or several days after opening the package. However, such preservatives should not interfere with the physiological tolerance of the composition, while ensuring the stability of the composition over an extended period of time.

The present inventors have found that thiomersal is most effective in preservative efficacy trials without causing adverse effects. Accordingly, in certain embodiments of the invention, the composition of the present invention further comprises thiomersal (i.e. (2-carboxylatophenyl) sulfanylethylmercury sodium; also known as thimerosal), which is represented by a compound of formula (II):

The authors of the present invention found that thiomersal at a concentration of 0.005% wt./about. or more (for example 0008%, 0.01% or 0.02% w / v) is especially effective for antimicrobial preservation (see example a1). Accordingly, in certain embodiments of the invention, the composition of the present invention comprises at least 0005, 0008, 0.01, 0.015, or 0.02% w / v. thiomersala.

In certain embodiments of the invention, the composition comprises at least 0.01% w / v. thiomersal and at least 0.2% wt./about. A-5021. The maximum allowable concentration of thiomersal is regulated in various countries. Thus, the concentration of thiomersal is preferably not more than 0.02% w / v, for example 0.01, 0.015, 0.018, or 0.02% w / v.

In order to be able to use the compositions according to the present invention in cases where strict physiological tolerance is required, for example, in ophthalmic applications, the pH of the composition must be within the range of ocular comfort. Accordingly, the pH of the compositions according to the present invention is preferably in the range between 5 and 8.5, in particular between 6 and 8 and in particular between 6.4 and 7.8, most preferably between 6.6 and 7.4. In certain embodiments, the compositions of the present invention may further comprise one or more buffering agents such as a phosphate buffer, eg sodium phosphate. In certain embodiments, the compositions of the present invention may further comprise one or more pH adjusters such as sodium hydroxide, hydrochloric acid, or combinations thereof.

The inventors of the present invention have found that the use of cyclodextrin does not require the use of solubilizing agents with elevated pH.

In addition, the physiological tolerability of the compositions according to the present invention assumes minimal difference from local osmotic pressure. In certain embodiments, when the compositions are intended for ophthalmic use, the compositions preferably have an osmotic pressure or osmolarity that is equal to or nearly equal to those of the tear fluid (i.e., about 300 mOsm / L), preferably 200 to 400 mOsm / L. In some embodiments, the compositions of the present invention may further comprise one or more tonicity adjusting agents, eg, selected from the group consisting of dextrose, glycerol, mannitol, potassium chloride, sodium chloride, and phosphate buffers. Tonicity agents can be used to alter the osmotic pressure or osmolarity of the composition.

One skilled in the art will further appreciate that the compositions of the present invention are preferably sterile and free of exogenous particles.

Thus, as indicated above, the compositions according to the present invention may contain, in addition to the active ingredient A-5021 and cyclodextrin, one or more preservatives and / or buffering agents. In certain embodiments of the invention, the compositions of the present invention consist essentially of an active ingredient and a cyclodextrin and an aqueous solution such as a buffer. In certain embodiments of the invention, the compositions do not contain an elevated pH solubilizing agent.

The present inventors have found that the compositions of the present invention are particularly useful for the diagnosis and treatment of herpes infections. Accordingly, in a further aspect, the present invention relates to a composition as described above for use in medicine, in particular for use as a therapeutic and / or diagnostic composition.

In certain embodiments, the present invention provides a composition as described above for use in a method for treating a herpes infection. In certain embodiments, the herpes infection is an ocular herpes infection. Ophthalmic herpesvirus infections have been reported in a variety of animals, including cats (e.g. examples b1, b2 and b3 below), dogs (e.g. Ledbetter et al., Veterinary Microbiology 2009, 138, 98-105), and horses (e.g. Kershaw et al., Virus Research 2001, 80, 93-99). Accordingly, in certain embodiments of the invention, the infection is an infection in an animal, for example, selected from the feline, canine, and equine family.

In certain embodiments of the invention, the compositions are provided for treating an infection in a companion animal, in particular in a feline, and even more particularly in a feline. Thus, in certain embodiments of the invention, the infection is a feline herpes virus infection. The present inventors have found that the compositions of the present invention are very effective in treating ocular herpes infections in companion animals such as felines such as cats.

In addition, the inventors have found that the treatment of herpes eye infections using the compositions of the present invention is particularly successful when the composition is administered to the (infected) eye two or three times a day. The optimal frequency of use may depend on factors such as the severity of the infection and the concentration of A-5021 in the composition. In certain embodiments of the invention, compositions containing about 0.5% wt./about. A-5021 is administered twice a day. In certain embodiments, lower concentration compositions, for example about 0.2% w / v, are administered three times a day. In certain embodiments of the invention, the composition is administered to the (infected) eye two or three times a day for at least one, two, three or four weeks. In certain embodiments, treatment is continued for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the clinical symptoms resolve.

Compositions according to the invention for use in the treatment of ocular infections are generally administered directly to the eye, in particular applied to the cornea. However, other means of administration are also contemplated, such as intraocular injection.

Feline herpes virus is one of the most common infectious diseases in cats. Accordingly, the present invention provides therapeutic compositions for use in the treatment of feline ocular infections. In certain embodiments of the invention, the treatment regimen is as described above. Thus, in specific embodiments, the treatment regimen is a composition containing from 0.1 to 1% w / v. A-5021, includes two or three injections per day. Typically, eye infections are characterized by one or more symptoms, such as ocular discharge, conjunctivitis, keratitis, dendritic ulcers, geographic ulcers, corneal sequestration, corneal edema, vascularization, blindness, eosinophilic conjunctivitis, eosinophilic keratitis, stromal keratitis, stromal keratitis. Eye symptoms are usually preceded by a history of respiratory symptoms.

In addition, the present inventors have found that the compositions of the present invention are extremely well tolerated by animals suffering from clinical symptoms of an ocular infection that increase sensitivity in the eyes, such as eye discharge, conjunctivitis, keratitis, dendritic ulcers, geographic ulcers, corneal sequestration, corneal edema, vascularization, blindness, eosinophilic conjunctivitis, eosinophilic keratitis, stromal keratitis, uveitis, dry eyes, or other clinical symptoms. Thus, in specific embodiments, the compositions of the present invention are intended for use in the treatment of ocular infections characterized by one or more of the symptoms described above.

In certain embodiments, the compositions of the present invention are used to treat ocular infections that have been diagnosed as herpes virus infections. For example, dendritic and geographic ulcers (feline ulcerative keratitis) are considered a pathognomonic clinical symptom of FHV-1. Thus, in certain embodiments of the invention, the compositions are used to treat an ocular infection in cats suffering from a dendritic or geographic ulcer.

In addition, as described above, the presence of a herpes infection can be determined virologically. Thus, in certain embodiments of the invention, the compositions are used to treat an ocular infection in a feline diagnosed with feline herpes simplex virus type 1 (FHV-1) infection.

In accordance with the foregoing, the present invention relates to the use of compositions as described above for the manufacture of a medicament for the treatment of an ocular infection, more particularly an ocular herpes infection, as described herein. In addition, the present invention relates to methods of treating an ocular infection comprising administering a composition as described above to an (infected) eye, in particular two or three times a day as described above.

It has been observed that treatment of ocular herpes virus infections with the compositions of the present invention results in a significant improvement in clinical symptoms within one week and even after one or two days. In view of this surprising efficacy, the compositions of the present invention are additionally useful in methods for diagnosing eye infections, more particularly eye infections in felines. More specifically, the inventors have found that the compositions of the present invention can be used as a diagnostic agent for confirming or excluding feline herpes virus type 1 (FHV-1) as a cause of feline eye infections.

Indeed, ocular herpesvirus infections typically result in a variety of different and often nonspecific clinical symptoms, including ocular discharge, conjunctivitis, keratitis, corneal sequestration, eosinophilic conjunctivitis, eosinophilic keratitis, stromal keratitis, uveitis, and dry eyes. In the case of feline eye disease, other viral, bacterial and fungal pathogens can cause similar clinical disease. More specifically, it has been described that the cause of one or more of these clinical ocular symptoms may be: feline calcivirus, feline immunodeficiency virus, feline leukemia virus, Chlamydophila felis, Mycoplasma spp. (M. felis and M. gatae) and even immune-mediated diseases.

Consequently, the clinical diagnosis of FHV-1 is challenging and laboratory confirmation is often required. However, most virological methods for detecting FHV-1 are relatively insensitive and often a source of false negative and false positive results. Currently, polymerase chain reaction (PCR) is considered the most sensitive method for detecting FHV-1 DNA in eye tissues. However, the results of PCR tests are proving inconsistent, with the result that many veterinary ophthalmologists abandon PCR testing as a diagnostic tool and rely only on the medical history and examination of the patient.

Because A-5021 is a highly specific herpes drug that is ineffective against bacterial and fungal infections, and other viruses other than herpes viruses (e.g. FHV-1) that can cause similar symptoms (e.g. feline calcivirus, leukemia virus feline and feline immunodeficiency virus), the rapid response to treatment with the composition of the present invention allows it to be used as a diagnostic agent, more particularly as a diagnostic therapeutic agent. Therefore, in the case of eye infections in felines of unknown etiology, the response to treatment with the composition according to the present invention makes it possible to diagnose a first-line eye infection, in particular to determine whether the infection is herpetic or non-herpetic.

Accordingly, the present invention further relates to compositions as described above for use in a method for diagnosing an ocular infection, in particular an ocular infection in a companion animal. More specifically, the diagnostic methods contemplated herein include the steps of a) administering a composition according to the present invention having a composition and dosage as described above, and b) determining, over a period of time, whether the clinical symptoms have improved or not, where improvement in clinical symptoms is an indicator of the presence of herpes virus infection. In additional specific embodiments, the methods of the present invention comprise administering a composition of the invention daily and determining for 1 to 7 days, for example, within 1-2 days, within 2-4 days, or within 5-7 days, whether there is an improvement. clinical symptoms of infection. In certain embodiments of the invention, taking into account the effectiveness of the treatment, the methods include determining whether there is improvement within 1-4 or even 1-2 days.

In a further aspect, the present invention relates to methods for preparing a soluble and stable composition A-5021 at a concentration of at least 1 mg / ml. Indeed, as described above, the present inventors have identified methods for preparing stable solutions with an increased concentration of this active ingredient that are compatible with direct administration to the eye and / or wounded tissue. In particular, the compositions according to the present invention are stable in that they do not precipitate, which allows reuse of the same dose of the substance over several days to several weeks.

In particular, the methods of the present invention include the steps of:

a) obtaining a solution containing at least 10% wt./about. cyclodextrin, for example hydroxypropyl beta-cyclodextrin;

b) adding to this solution A-5021;

c) dissolving the A-5021 added in step b);

d) repeating steps b) and c) until a concentration of A-5021 in solution is at least 1 mg / ml;

e) if necessary, adding at least 0.08 mg / ml thiomarsal.

In certain embodiments of the invention, the solution obtained in step a) is an aqueous solution. It should be borne in mind that repeating steps b) and c) is only necessary if the concentration of 1 mg / ml has not been reached after the first execution of steps b) and c). In certain embodiments of the invention, A-5021 is added in several stages, for example in 2-5 stages.

The dissolution step A-5021 is generally carried out by mixing, for example using a vortex. In certain embodiments of the invention, step c) comprises heating the mixture obtained in step b), preferably to a temperature between 45 and 60 ° C. The skilled person will understand that this step can be carried out without the need for specific action, especially when a limited amount of the active ingredient is added.

As indicated above, the present inventors have found that the use of cyclodextrin does not require the use of solubilizing agents at elevated pH. Thus, in certain embodiments of the invention, the methods of the invention do not include the addition of a solvent having a pH greater than 7.9.

The following non-limiting examples illustrate the present invention.

Examples of

The following examples are provided for the purpose of illustrating the present invention and are in no way intended to limit the scope of the present invention, and should not be interpreted in any way as limiting the scope of the present invention.

a) Dissolution of A-5021 to pharmacologically active concentrations

Example A1 - Dissolution tests

To determine the solubility of A-5021 (i.e. 2-amino-9 - [[(1S, 2R) -1,2-bis (hydroxymethyl) cyclopropyl] methyl] -1,9-dihydro-6H-purine-6- she) first, the first series of tests of 33 experiments with suspension was carried out. In these experiments, 33 different systems were examined (differing in terms of buffers, co-solvents, solubilizing agents). The following 6 buffers were used: HCl buffer (pH 2), phosphate buffer (pH 6.8 and 7.4), tris (hydroxymethyl) aminoethane buffer (pH 9), sodium carbonate / sodium bicarbonate (pH 10.8) and potassium dihydrogen phosphate (pH 12). Dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), N-methylpyrrolidone (NMP), ethanol (EtOH), and propylene glycol at various concentrations were used as co-solvents. Solubilizing agents used were propylene glycol, glycerin, solutol HS 15, polysorbate 80, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (Kleptose® HPB), Cremophor EL, Cremophor RH 60, polyethylene glycol (PEG) 300, PEG-400 cyclodextrin (e.g. Captisol®). Solubility was determined by filtering the suspensions after 24 hours equilibration. The concentration of A-5021 in the filtrates was analyzed by high performance liquid chromatography (HPLC), from which the corresponding solubility was calculated.

It was found that the addition of an organic cosolvent to solutions buffered to pH 12 did not improve solubility. However, the addition of solubilizing agents such as hydroxypropyl-β-cyclodextrin, Captisol® and propylene glycol (used at up to 80% v / v) to buffer systems above pH 11 did improve solubility. The highest solubility of A-5021 (8.64 mg / ml) was obtained in 80 vol% propylene glycol in 20 vol% buffer (pH 11.3).

Based on these results, 10 additional solubility experiments were performed with different pH values to determine the effect of pH on the solubility profile of A-5021. Were considered two different buffering systems, co-solvents and solubilizing agents with a focus on the systems that gave the best results described above. The solubility was determined again by filtering the suspensions after 24 hours of equilibration. The concentration of A-5021 in the filtrates was analyzed by HPLC, from which the corresponding solubility was calculated. In general, no higher solubility values were found than described above. When using an acidic pH (i.e. pH 2.8) compared to the previously used pH 11.3, the solubility at room temperature of A-5021 decreases slightly from 8.64 mg / ml to 8.5 mg / ml. Close to neutral pH of the buffer system (80% v / v propylene glycol and 20% v / v phosphate buffer at pH 7.4), which is of interest for the development of ophthalmic solutions (taking into account the topical application of A-5021 for the eyes infected with the herpes virus), it was possible to dissolve A-5021 at a concentration of 8.18 mg / ml. When 2-hydroxypropyl beta-cyclodextrin (HPBC) was used as a solubilizing agent instead of propylene glycol, the solubility of A-5021 decreased compared to propylene glycol buffer systems. When using 60% vol./about. HCl buffer and 40% w / v. HPBC solubility of A-5021 was 6.76 mg / ml and 4.91 mg / ml at pH 2.2 and pH 7, respectively. A further decrease in the solubility of A-5021 was noted when captisol® (30% w / v captisol® and 70% w / v buffer) was used as the solubilizing agent. With a further increase in the amount of propylene glycol to 5.7-33.34% v / v. in a system that already contains 33.33-40% wt./about./about. HPBC and 33.34-54.3% v / v. buffer, a decrease in the solubility of A-5021 was observed (at pH 7.0-7.4, the solubility of A-5021 was 3.28-4.05 mg / ml).

Based on the above results and in order to obtain composition A-5021 with pharmacologically active concentrations (preferably about 0.5 mg / ml), it was decided to investigate additional solubilizing agents and / or to maintain high amounts of propylene glycol at neutral pH. A total of 28 vehicle candidates were tested to dissolve A-5021 at a concentration of 5 mg / ml at neutral pH: propylene glycol at a concentration of 20% to 80% w / v, glycofurol at a concentration of 5% to 50% w / vol., glycerin in a concentration from 15% to 50% w / v., 15% w / v. glycerin + 15 wt./about. glycofurol, 25% w / v propylene glycol + 25% w / v glycofurol, propylene glycol (range: 15% -60% w / v) + glycerin (range: 5% -15% w / v), 25% w / v. Kleptose® HPB + 25% propylene glycol, Cremophorus RH 40, Cremophorus EL, solutol HS 15, 40-50% Kleptose® HPB, miglyol 812N, liquid paraffin, polyvinylpyrrolidone K12.

Only four carriers allowed the dissolution of A-5021 at a concentration of 5 mg / ml (0.5% w / v) at a pH close to neutral (pH in the range of 6.98 - 7.11). These carriers were as follows (% w / v in water):

(i) 80% w / v. propylene glycol;

(ii) 50% w / v. Kleptose® HPB;

(iii) 60% w / v. propylene glycol + 15% w / v glycerin;

(iv) 60% w / v. propylene glycol + 10% w / v glycerin.

The osmotic pressure of the propylene glycol contained in the composition / solution showed hypertonicity (high osmolarity, non-physiological). Values from 6925 mOsm to 11725 mOsm were observed, indicating the need to check local tolerance.

When tested in the eyes of healthy rabbits, mice and cats, local tolerance to solutions containing propylene glycol was good. However, when ophthalmic solutions containing propylene glycol were instilled into the eyes of mice and cats infected with the herpes virus, severe intolerance was observed. Observed clinical manifestations of local intolerance immediately after dosing included the following: eye closure / squinting (in cats and mice), vocalization (in cats and mice), drooling (in cats), scraping movements (in cats), eye scratching (in cats) and head shaking (in cats and mice). The severity of symptoms ranged from mild to severe. Symptoms were similar in animals treated with carriers (i), (iii) or (iv) (propylene glycol solutions with or without glycerin, not containing A-5021, and propylene glycol solutions with or without glycerol containing A- 5021), indicating that the intolerance was not directly related to the presence of A-5021. Therefore, Kleptose® HPB (hydroxypropyl-beta-cyclodextrin) is the only solubilizing agent that is suitable for the preparation of ophthalmic solutions containing A-5021 at a concentration of 0.1% to 1% w / v, for example 0.2 % up to 0.5% wt./about; The pH of the solution containing Kleptose® HPB at a concentration of 0.2% to 0.5% w / v was 6.0-8.0 with an osmotic pressure of about 350 mOsm.

Since the Kleptose® HPB formulation is a multifunctional ophthalmic solution for eyes infected with herpes, the Kleptose® HPB formulation must be tested for efficacy against microbes. For this purpose, a series of nine formulations based on Kleptose® HPB with various antimicrobial preservatives were tested:

- no preservative;

- 0.01% benzalkonium chloride (BAK);

- 0.02% BAK;

- 0.01% BAK + 0.01% ethylenediaminetetraacetic acid (EDTA);

- 0.008% thiomersal;

- 0.01% thiomersal;

- 0.02% thiomersal;

- 1.5% benzyl alcohol;

- 0.15% methyl para-hydroxybenzoate (PHBM) + 0.05% propyl para-hydroxybenzoate (PHBP).

The effectiveness of preservatives has been tested (criteria for effectiveness are based on the criteria of the European Pharmacopoeia). The compositions were infected with S. aureus, P. aeruginosa, C. albicans, A. brasiliensis (i.e. yeast, mold and bacteria) and tested at various time points after infection (after 6 hours, 24 hours, 7 days, 14 days and 28 days). Formulation A-5021 based on Kleptose® HPB without preservative did not meet EP criteria A or B, which justifies the inclusion of an antimicrobial preservative. Compositions A-5021 based on Kleptose® HPB with 0.01% - 0.02% BAK or 1.5% benzyl alcohol, or 0.15% PHBM + 0.05% PHBP did not meet criteria A or B. Composition A- 5021 based on Kleptose® HPB with 0.01% BAK + 0.01% EDTA did not meet criteria A or B. Thus, of the nine formulations tested, only three formulations containing thiomersal (0.008% w / v, 0 , 01% w / v and 0.02% w / v) were determined to be sufficiently effective for A-5021 solutions based on Kleptose® HPB (0.2% and 0.5% w / v. ). A high concentration of Kleptose® HPB (i.e. hydroxypropyl beta-cyclodextrin) may be responsible for inhibiting other preservatives.

Thus, the following compositions A-5021 remained as ophthalmic products for treating eye diseases in pets infected with herpes, since these compositions are compatible with topical instillation in the eye: 0.2-0.5% w / v. A-5021 at 20-50% w / v Kleptose® HPB using 0008-0.02% w / v thiomersal as an antimicrobial preservative (adding water to the required volume).

Example a2 - Composition with a concentration of A-5021 0.5% w / v (composition A)

A total of 5 mg / ml A-5021 was dissolved in a 50% w / v solution. Kleptose® HPB in water, then stirred and heated to a temperature between 45 and 60 ° C. Thereafter, no crystallization was observed for several days after storage at room temperature and at 2 ° C to 8 ° C. The composition had an osmotic pressure of 350 mOsm and a pH of 7.03.

Thiomersal was added as a preservative at a concentration of 0.01% w / v. The effectiveness of the preservative was tested in accordance with the general method of the European Pharmacopoeia. Sampling was performed 6 hours, 7 days, 14 days and 28 days after infection. There was a 3.2 log ₁₀ decrease in S. aureus and a 1.5 log ₁₀ decrease in P. aeruginosa 6 hours after infection. After this recovery, S. aureus and P. aeruginosa were not observed. No recovery of S. aureus and P. aeruginosa was observed at any of the sampling points (7 days, 14 days and 28 days after infection).

Evaluated the stability of the composition containing 0.5% wt./about. A-5021 and 0.01% w / v. thiomersal in 50% wt./about. Kleptose® HPB, using an accelerated stability test in which the composition was held for 3 months at 40 ° C and 75% relative humidity. No decrease was observed with respect to theoretical A-5021 content. No signs of degradation products were observed above the 0.3% cut-off value. The thiomersal content was considered acceptable in relation to the differences in the methods of analysis and manufacture.

Example a3 - Composition with a concentration of A-5021 0.2% w / v (composition B)

A total of 2 mg / ml A-5021 was dissolved in 20% w / v. Kleptose® HPB (added water to volume), then mixed and heated to a temperature between 45 and 55 ° C. Thiomersal was added at a concentration of 0.01% w / v. The composition had a pH of 6.4. The effectiveness of the preservative was tested in accordance with the general method of the European Pharmacopoeia. A decrease of 2.1 log ₁₀ in S. aureus was noted at 6 hours after infection. No recovery of S. aureus was observed after this (last sampling was 7 days after infection). There was a 1.7 log ₁₀ decrease in P. aeruginosa 6 hours after infection. No recovery of P. aeruginosa was observed after this (last sampling was 7 days after infection).

There was no recovery of C. albicans or A. brasiliensis at 7 days, 14 days and 28 days after infection. The testing of the efficacy of the preservatives was stopped on the 7th day after infection, since the therapeutic efficacy of the composition was already demonstrated on the 7th day.

Evaluated the stability of a solution containing 0.2% wt./about. A-5021, 20% w / v Kleptose® HPB and 0.01% w / v. thiomersal using an accelerated stability test in which the composition was kept for 1 month at 40 ° C and 75% relative humidity. No decrease was observed with respect to theoretical A-5021 content. No signs of degradation products were observed above the 0.3% cut-off value.

b) Treatment of herpes infections

For the treatment of various cats with clinical signs caused by acute FHV-1 infection, ophthalmic solutions of compositions A and B were used as described above.

Example b1 - treatment 3 times a day with a composition containing 0.5% wt./about. A-5021

A 15-year-old Chartreuse cat with a history of FHV-1 eye herpes (with recurrences) was presented to a veterinary ophthalmologist after a new phase of acute herpes infection. The right eye was affected and various dendritic ulcers were noted (by fluorescein staining and imaging in cobalt blue light). The cat was treated three times a day with Composition A as described above. After 10 days of treatment, the cat's eye was seen again by a veterinary ophthalmologist. The right eye was completely open. Although the fluorescein test showed two small remaining epithelial branches, the ulcers were almost completely healed. In addition, the treatment was very well tolerated as the treatment did not cause pain, discomfort or irritation. The treatment was continued for 10 days, after which the cat recovered completely.

Example b2 - Treatment 3 times a day with a composition containing 0.2% w / v. A-5021

A litter of two 5 week old European Shorthair kittens (one female and one male) were infected with FHV-1. FHV-1 is a common disorder in newborn kittens, resulting in respiratory illness, fever, sneezing or coughing, nasal discharge, and often symblepharon (i.e., partial or complete adhesion of the ocular conjunctiva of the eyelids to the bulbar conjunctiva of the eyeball). At birth, the entire litter of kittens is infected to some extent.

Two kittens were presented to a veterinary ophthalmologist; PCR (polymerase chain reaction) confirmed a positive result for FHV-1 on conjunctival swabs. The left eye of the male kitten showed signs of typical FHV-1-induced conjunctivitis. There were also suspicions of dendritic ulcers, but their confirmation was difficult due to conjunctival edema.

The cat was even more seriously infected, there was corneal edema in the left eye and a severe symblepharon in the right eye, from the third eyelid, covering the entire right eye. Both kittens were treated three times daily with ophthalmic solution of composition B as described above. After seven days of eye instillations, the kitten was completely healed, and the kitten was no longer infected, as confirmed by a negative PCR result for FHV-1 (conjunctival swabs).

Example b3 - Treatment with a composition containing 0.2% w / v. A-5021, twice a day

A 6 week old European Shorthair male kitten was presented to a veterinary ophthalmologist with respiratory symptoms. The left eye remained closed, there was corneal edema and small blood vessels that formed a small corneal granuloma. The kitten was diagnosed with the herpes virus. The kitten was treated twice daily with ophthalmic composition B as described above, and its condition improved within 7 days of treatment: the left eye was more open, the corneal granuloma decreased and the blood vessels were retracted. Some residual edema remained. The treatment was continued at the same frequency twice a day until the kitten was completely healed.

c) Diagnosis and treatment of herpes infections

Example c1

A 1 year old European Shorthair cat was presented to a veterinary ophthalmologist with the following clinical signs: sneezing and severe conjunctivitis in the right eye. The cat was diagnosed with interstitial keratitis. No corneal or dendritic ulcers were observed, so a definitive clinical diagnosis of FHV-1 could not be made. However, it was suggested that FHV-1 might be the cause, and conjunctival swabs were taken for FHV-1 PCR analysis. FHV-1 PCR results were negative. Therefore, a virologic diagnosis of FHV-1 could not be made. Treatment was started with ophthalmic composition A as described above; no other drugs or corticosteroids were administered. There was a marked improvement in the first week of treatment. After two weeks of treatment, the cornea and anterior chamber of the eye were clearly visible. The inflammatory response, which was observed at the first dose, regressed. Thus, the diagnosis of FHV-1 disease can be based on successful treatment with ophthalmic solution A-5021 with marked improvement during the first week of treatment. This was confirmed by further successful treatment and clinical improvement noted 16 days after the start of treatment. The cat was examined again 103 days after the treatment, and it was found that the cornea was calm and had no sequelae of the disease.

Example c2

A 4-year-old European Shorthair cat was presented to a veterinary ophthalmologist with the following clinical signs: the right eye is closed with the presence of several interstitial corneal vessels. The cat was diagnosed with interstitial keratitis, which has a fundamentally vascular reaction. No corneal or dendritic ulcers were observed in this adult cat, so a definitive clinical diagnosis of FHV-1 could not be made. Conjunctival swabs were taken for FHV-1 PCR analysis. FHV-1 PCR results were negative. Therefore, a virologic diagnosis of FHV-1 could not be made. Pretreatment was initiated with ophthalmic composition A as described above; no other drugs or corticosteroids were administered. There was a marked improvement in the first week of treatment. After three weeks of treatment, the cat felt much better, the eye was clearer, and the corneal vessels significantly regressed. Thus, the diagnosis of FHV-1 disease was based on successful treatment with ophthalmic solution A-5021 with marked improvement during the first week of treatment. The diagnosis was confirmed by further successful treatment and clinical improvement noted 21 days after the start of treatment.

Example c3

A total of 20 field cats of different breeds and ages with different ocular manifestations were selected, the main cause of which was considered FHV-1 (according to the diploma of the European College of Veterinary Ophthalmology), eye swabs were taken for PCR analysis for FHV-1, cats were treated with various compositions A-5021 (at a concentration of 0.2% and 0.5%). The treatment was carried out topically by instilling eye drops into the affected eyes with a frequency of 2 or 3 times a day. Positive PCR results for FHV-1 were obtained in 60% of these cases (12 of 20 cats), negative results for FHV-1 were obtained in 25% of cases, and in 15% of cases no PCR results were obtained (for example, due to with DNA extraction error). Of the FHV-1 PCR negative cases, all cats responded quickly to treatment with significant clinical improvement already seen in the first week of treatment (false negative based on PCR). In contrast, one cat that tested positive for FHV-1 DNA based on PCR did not respond to treatment (false positive based on PCR and clinical diagnosis).

Therefore, herpes specific drug A-5021, formulated as an ophthalmic product, is a rapid diagnostic drug for herpes infected eyes.

Claims (20)

1. Ophthalmic solution containing: at least 0.1% wt./about. 2-amino-9 - [[(1S, 2R) -1,2-bis (hydroxymethyl) cyclopropyl] methyl] -1,9-dihydro-6H-purin-6-one and at least 15% wt./about. cyclodextrin. 2. The ophthalmic solution according to claim 1, which is an aqueous solution. 3. An ophthalmic solution according to claim 1 or 2, wherein the cyclodextrin is hydroxypropyl-beta-cyclodextrin. 4. Ophthalmic solution according to any one of paragraphs. 1-3, additionally containing at least 0.008% wt./about. thiomersala. 5. Ophthalmic solution according to any one of paragraphs. 1-4 for use in a method for treating ocular herpes infection. 6. The ophthalmic solution of claim 5, wherein the eye infection is an eye infection of a companion animal. 7. The ophthalmic solution according to claim 6, wherein the pet is a feline. 8. Ophthalmic solution according to any one of paragraphs. 5-7, wherein the method comprises administering the composition to the eye two or three times a day for at least seven days. 9. Ophthalmic solution according to any one of paragraphs. 1-4 for use in a method for diagnosing ocular herpes infection. 10. The ophthalmic solution according to claim 8 or 9, wherein the eye infection is an eye infection of a companion animal. 11. The ophthalmic solution of claim 10, wherein the companion animal is a feline. 12. A method for producing an ophthalmic solution according to claim 1, including the stages at which: a) get a solution containing at least 15% wt./about. cyclodextrin; b) to this solution is added 2-amino-9 - [[(1S, 2R) -1,2-bis (hydroxymethyl) cyclopropyl] methyl] -1,9-dihydro-6H-purin-6-one; c) dissolve 2-amino-9 - [[(1S, 2R) -1,2-bis (hydroxymethyl) -cyclopropyl] methyl] -1,9-dihydro-6H-purin-6-one added in step b) ; and d) repeat steps b) and c) until a concentration of 2-amino-9 - [[(1S, 2R) -1,2-bis (hydroxymethyl) cyclopropyl] methyl] -1,9-dihydro-6H- purin-6-one at least 1 mg / ml. 13. The method of claim 12, wherein the cyclodextrin is hydroxypropyl-beta-cyclodextrin. 14. The method of claim 13, further comprising step e) adding at least 0.008 mg / ml thiomersal.