Use of phenytoin as a new tool for cross-linking eye collagen, ophthalmic compos itions containing it and related preparation methods

Abstract

Phenytoin and / or derivatives thereof are effective, alone or with Riboflavin, in a method of topically treating keratoconus and axial myopia, which includes contacting it with collagen and / or ocular structures to improve the strength of corneal and sclerocorneal collagen. Different formulations containing both Phenytoin sodium salt and Fosphenytoin are identified and tested, and a plurality of products containing them are described, in the form of inclusion complexes, gels and eye drops, developed to obtain and improve the solubilization of Phenytoin itself, to be administered to obtain the cross-linking of eye collagen and counteract the weakness thereof in diseases such as keratoconus and axial myopia, as well as the related preparation methods.

Description

[0001] USE OF PHENYTOIN AS A NEW TOOL FOR CROSS-LINKING EYE COLLAGEN, OPHTHALMIC COMPOSITIONS CONTAINING IT AND RELATED PREPARATION METHODS

[0002] DESCRIPTION

[0003] The present invention relates to a method of cross- linking the collagen present in a collagenous tissue, which includes contacting the collagenous tissue with an amount of an agent releasing phenytoin or derivatives thereof such as fosphenytoin (FS) and inclusion complexes thereof, effective in cross-linking collagen.

[0004] The invention further relates to a method of inhibiting the loss of structural integrity of a collagenous tissue during transplantation-related transport which includes contacting the collagenous tissue with an amount of an agent releasing phenytoin or a derivative thereof and inclusion complexes thereof, effective for inhibiting the loss of structural integrity of said collagenous tissue.

[0005] The present invention also provides a composition for ophthalmic administration comprising an agent releasing Phenytoin Fosphenytoin and inclusion complexes thereof, together with ophthalmically suitable carriers or excipients.

[0006] Lastly, this invention provides a method of altering the refractive power of a cornea which includes contacting the cornea with an agent releasing phenytoin or fosphenytoin and inclusion complexes thereof so as to perform cross-linking in the cornea and thus alter the refractive power of the cornea itself.

[0007] SCOPE OF THE INVENTION The Applicants, starting from the fact that it has been reported in the literature that phenytoin has various positive effects on the wound healing process and is potentially useful in treating corneal injuries and assuming that the direct action of a biological cofactor could be involved, have carried out experimental research aimed at verifying that phenytoin sodium (and also the prodrug thereof, phenytoin phosphate) was able to exert a direct collagen protection action.

[0008] The results obtained confirmed that phenytoin sodium (or the phosphate derivative thereof) can be used electively alone, or even in association with Riboflavin, to improve collagen strength (structure) in Keratoconus, while taking advantage of the positive effects of said molecule on the wound healing process, in a sort of dual positive strategy.

[0009] This led the same Applicants to experiment with the use thereof in both treating keratoconus and in progressive myopia, since in both such diseases, keratoconus and progressive myopia, Phenytoin and derivatives thereof, alone or with Riboflavin, has demonstrated surprising therapeutic efficacy against the abnormalities of growth and alteration of corneal cells, keratinocytes, which are no longer capable of ensuring an adequate architecture of the cornea in the presence of such diseases.

[0010] BACKGROUND ART

[0011] The cornea is a transparent convex barrier which serves to keep the structure of the eye intact and focus light on the retina. The cornea derives the structural strength, shape, and integrity thereof from corneal collagen. The strength of the intertwined collagen filaments is due to the covalent crossed bonds established between and within the collagen filaments and between the collagen and glycoproteins in the matrix.

[0012] KERATOCONUS is a degenerative disease of the cornea linked to corneal collagen weakness, where the biomechanical strength of the corneal collagen is reduced by a series of conditions which lead to a progressive deformation of the cornea itself. It is assumed that it is caused by genetic, environmental factors or hormonal dysfunctions.

[0013] AXIAL MYOPIA is instead a disease in which there is an alteration of eye collagen and more specifically structural alterations of scleral collagen, in which an alteration of the structure of the collagen forming the scleral shell is demonstrated (1-6).

[0014] The human sclera mainly consists of type 1 collagen (up to 50-70%) which is distributed between the equator and the posterior pole region of the eyeball (7-8). Throughout the myopia progression period, the thinning of existing fiber bundles is accompanied by an active change in the diameter distribution of the collagen fibers. Therefore, the sclera, especially the outer layers, contains small collagen fibers, the average diameter of which is less than 60-70 mm.

[0015] This higher number of small collagen fibers has been shown to explain the lower tensile strength of the tissue, facilitating the expansion of the sclera.

[0016] The Applicants, starting from the need to strengthen the collagen structure which has been thoroughly studied, have re-evaluated Phenytoin, an old molecule, belonging to a class of drugs called anticonvulsants which act by decreasing the abnormal electrical activity of the brain: this primary activity is also accompanied by positive effects on the wound healing process.

[0017] BACKGROUND ART

[0018] In the literature, clinical studies related to phenytoin indicate several main features: decreased inflammatory response, increased fibrous proliferation, increased collagen content and increased formation of new blood vessels.

[0019] See Bibliography: no. 16-22.

[0020] Furthermore, in recent years, phenytoin has been shown to be useful in treating corneal injuries. 1% isotonic phenytoin eye drops are described.

[0021] Bazin’s work (23) demonstrated that phenytoin can promote cross-linking in both normal skin and granulomatous tissue in rats. Wounds treated with phenytoin showed a significant increase in collagen deposition and neovascularization, which resulted in an increase in the tensile strength of the wound and accelerated healing of both open and closed wounds.

[0022] Vernillo et al.(24) (Vernillo AT, Ramamurthy NS, Lee HM, Rifkin Br: The effect of phenytoin on collagenase and gelatinase activities in UMR 106-01 rat osteoblastic osteosarcoma cells. Matrix 10(l):27-32, 1990) demonstrated that phenytoin can inhibit bone resorption, in UMR 106-01 rat osteoblastic osteosarcoma cells, through the action thereof on the transcription, synthesis and / or secretion of collagenolytic enzymes, collagenase and gelatinase.

[0023] WO 2015 / 138786 A1 (UNIV COLUMBIA (US); PAIK DAVID CHOOHYUN [US] ET AL.) September 17, 2015 discloses the use of agents releasing formaldehyde for use in treating keratoconus. These agents induce the cross-linking of corneal and scleral tissue by releasing formaldehyde and thus cross-linking collagen and improving tissue rigidity. In particular, hydantoin and derivatives thereof are proposed as agents releasing formaldehyde. The ophthalmic compositions described can be in the form of drops.

[0024] EP 1700616 A1 (UNIV MIGUEL HERNANDEZ DE ELCHE [ES] of September 3, 2006 describes compounds for treating dry eye caused by photorefractive surgery. Agents which block the electrical activity of damaged nerve endings in the neuroma have been found for this condition, in particular after procedures such as excimer laser surgery.

[0025] In particular, the blocking agents can be DPH. Ophthalmic compositions comprising 0.001-0.1% DPH are described.

[0026] WO 2021 / 108022 A1 (DISPERSOL TECHNOLOGIES LLC [US] June 3, 2021 describes pharmaceutical formulations comprising complexes of a pharmaceutically active ingredient (API) with a cyclic oligomer. These inclusion complexes are formed to enhance drug properties, such as oral bioavailability. In particular, HPBCD can form water-soluble inclusion complexes with several drugs, such as fosphenytoin. Active pharmaceutical ingredients (APIs) could be used to treat an eye disorder.

[0027] AMINIFARD MOHAMMAD NAEIM ET AL., in the article "Evaluation of Topical Phenytoin Eye drop 1% Therapeutic Effects on Corneal Alkali Burns in Rabbit Model" in IRANIAN JOURNAL OF OPHTHALMOLOGY, Vol. 24, no. 3 January 1, 2012, (pages 33-38) studies the impact of topical 1% phenytoin on healing corneal epithelial defects in a rabbit model. The topical phenytoin significantly reduces the wound surface areas with respect to the controls. Phenytoin can be useful in treating corneal injuries. A 1% isotonic phenytoin eye drops described.

[0028] WO 2006 / 013084 Al (ROYAL COLLEGE OF SURGEONS IE [IE]; KELLY JOHN [IE] ET AL: of February 9, 2006 concerns topical formulations containing phenytoin for wound healing. The formulations are in the form of a gel with a carbomer as gelling agent, in particular Carbopol or as a stabilized matrix with cyclodextrin such as HPBCD, as a complexing agent. Phenytoin is present in an amount between 0.5-5.0% by weight and is used in the form of the sodium salt thereof or as fosphenytoin. The use of a Carbomer results in a stable and homogeneous dispersion of phenytoin at a pH value close to physiological value and is described as particularly advantageous .

[0029] LEE SIANG YIN ET AL: in the article: "LIPID MICROEMULSION-BASED HYDROGELS FOR EFFECTIVE TOPICAL DELIVERY OF PHENYTOIN" INTERNATIONAL JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES", VOL. 8, NO. 11 OF October 28, 2016 page 240, proposes developing lipid microemulsion hydrogels as topical drug carriers for phenytoin. The oil-in-water / O / W lipid emulsions were obtained from different oils, including soybean oil, and loaded with phenytoin. A microemulsion containing soybean oil showed the highest potential for phenytoin release .

[0030] In light of the above, the inventors carried out experimental research aimed at verifying that phenytoin sodium (and also the prodrug thereof, phenytoin phosphate) was able to exert a direct collagen protection action, also verifying which was the most suitable topical formulation for the release of phenytoin on the corneal epithelium.

[0031] REEVALUATION OF PHENYTOIN AS A UV CROSS-LINKING INDUCER

[0032] The Applicants verified the assumption that Phenytoin sodium could be a photosensitizer considering the peculiar chemical structure of Phenytoin (5,5— diphenyl-2,4-imidazolidinedione ) containing two Phenolic Rings, and for the knowledge of such a molecule therein, as a photosensitizing drug. They performed a number of experiments, reported in detail below, which demonstrate that Phenytoin (sodium salt) could be, at least in small part, a producer of superoxide Anions when irradiated by UV light, as amply demonstrated for riboflavin (and the prodrug derivative thereof, riboflavin phosphate).

[0033] With such results, thoroughly discussed below, the Applicants assumed that Phenytoin could be used, alone or with Riboflavin, to enhance collagen strength in Keratoconus, while taking advantage of the positive effects of the molecule on the wound healing process, in a sort of dual positive strategy.

[0034] This approach arises from the critical examination of the techniques already known in the literature to obtain the cross-linking of collagen. The techniques are: chemical methods, such as glutaraldehyde, formaldehyde, glyceraldehyde, ribose, glucose and beta- nitro aliphatic alcohols, genipin introduced in the cornea;

[0035] - physical methods such as dehydration heat treatment 7 (drying water from collagen), thermal heating and UVC or gamma radiation;

[0036] All these methods cannot be used on human collagen because they are toxic. photochemical method, focused mainly, if not exclusively, on the use of Riboflavin which, activated at 366 nm, produces ROS causing the cross-linking of collagen. This method has been widely used on humans for the treatment of keratoconus and validated by extensive literature .

[0037] Such a method, although less dangerous than the others, has critical peculiarities related to the use of UV Light: in fact, irradiated riboflavin exclusively carries out the role of superoxide producer

[0038] - (Reactive Oxygen Substances): the activity thereof is linked to the chemical reaction and does not involve other mechanisms such as the induction of enzymatic activity or the stimulation of cells.

[0039] Although widely applied with good results, the photochemical method has some room for further improvement as demonstrated by various scientific works (ref. 25, 26).

[0040] The Applicants thus decided to verify the ability of phenytoin sodium salt to produce ROS, using the well- known procedure applied to riboflavin and the derivative thereof, phenytoin phosphate.

[0041] Simply put, Phenytoin solutions were irradiated at 366 nm for 15 minutes, in a small Irradiation chamber provided with WOOD'S light lamps (366nm) with incident light intensity 633 μW / cm2.

[0042] The production of Superoxide anion is detected by reduction of NBT (Nitro blue Tetrazolium salt) transformed into Formazan Blue detected at 560 nm.

[0043] The concentration of Superoxide Anion can be calculated considering that 1 mole of Reduced NBT (Formazan) at 560 nm has a molar absorption of 15000 (1 μM absorbs 0.015) and that 10 μM of Reduced NBT are produced by 50 μM of Superoxide. (Ref. 27).

[0044] Phenytoin is related to barbiturates in chemical structure, phenobarbital: but it has a ring with five members. The chemical name is sodium 5,5-diphenyl-2,4-imidazolidinedione, having the following structural formula: The chemical-physical profile of the molecules clarifies that:

[0045] Phenytoin and Phenytoin sodium are profoundly different, specifically considering water solubility.

[0046] In fact, Phenytoin is insoluble in water so it is more lipophilic than hydrophilic, thus it should have more permeability on the membranes of the eyes.

[0047] Phenytoin sodium salt is instead partially soluble in water and due to the hydrophilic behavior thereof is less permeable to ocular membranes.

[0048] It is also necessary to bear in mind that phenytoin sodium salt is soluble in water but the resulting pH is above 10, a specification so peculiar that it forms a major limitation to the use of such a molecule on the ocular surface, even for short times of 10-20 minutes.

[0049] As for Riboflavin, the chemical structure of

[0050] RIBOFLAVIN is shown here below:

[0051] RIBOFLAVIN-5'-PHOSPHATE SODIUM SALT DIHYDRATE The results after 15 minutes of irradiation of Phenytoin sodium salt and Riboflavin phosphate are here compared . RESULTS

[0052] 15 MINUTES OF IRRADIATION

[0053] EXAMPLE 1

[0054] A) PHENYTOIN Sodium salt

[0055] IRRADIATION EXPERIMENT

[0056] B) RIBOFLAVIN PHOSPHATE

[0057] IRRADIATION EXPERIMENT Conclusion

[0058] The simple experiments described above show how the sodium salt of phenytoin, when irradiated at 366 nm, with an intensity of 633 μW / cm2, can produce superoxide anion: such behavior can, at least in part, be explained by the fact that the chemical structure could be a photo sensitizing agent as previously assumed by the Applicants .

[0059] - However, the ability of the sodium salt of phenytoin to produce superoxide anion is very poor and far lower than that of riboflavin phosphate, respectively 13.79 μM / min / mM (Phenytoin sodium salt) and 292 μM / min / Mm (riboflavin phosphate).

[0060] Such a preliminary result, if it defines a new possible profile of Phenytoin, did not totally satisfy the Applicants, since the weak capacity to produce Superoxide exerted by Phenytoin does not justify / explain the several biological / biochemical effects of this molecule especially in vivo (cells or tissue).

[0061] Furthermore, although it does not require the presence of an external biological cofactor, it still requires the presence of a specific radiation at 366 nm because without the presence of light at 366nm there can be no effect: riboflavin itself produces superoxide anion due to the effect of light.

[0062] REEVALUATION OF PHENYTOIN: DIRECT INTERACTION WITH THE STRUCTURE OF COLLAGEN

[0063] In light of the above, the Applicants turned their attention to verifying the possible interaction between phenytoin and COLLAGEN, a polymer which is notoriously involved in general in the wound healing process, and more specifically, as assumed by the Applicants, in healing keratoconus.

[0064] To this end, they therefore decided to incubate collagen and phenytoin sodium salt (solubilized by a mixture of propylene glycol / ethanol / water (40 / 10 / 50 v / v)) together.

[0065] - 1st Experiment

[0066] 0.66 ml of aqueous solution containing 666 mcg of collagen was added to 10 mg of phenytoin sodium salt solubilized in 1 ml of mixture and incubated for 4 / 6 hours

[0067] To avoid the presence of the organic solvent used to solubilize the sodium salt of phenytoin, the Applicants decided to carry out the same experiment / incubation with fosphenytoin which is freely soluble in water.

[0068] - 2nd Experiment

[0069] 0.66 ml of aqueous solution containing 666 mcg of collagen was added to 10 mg of phenytoin phosphate completely solubilized in 1 ml of water and incubated for 4 / 6 hours

[0070] The sample after incubation was analyzed by Mass Spectrometry: this technique is used to analyze proteins in the proteomic field, to differentiate proteins, and to identify possible cross-linking with high specificity and sensitivity (28). The technique mainly consists of Ion Source, Injection System, HPLC system, Informatics Apparatus .

[0071] BRIEF DESCRIPTION OF THE ANALYTICAL PROCEDURE

[0072] 1) The protein (in our case collagen) is hydrolyzed overnight by trypsin (high potency hydrolytic enzyme)

[0073] 2) The obtained Hydrolytic Products are analyzed with ionization mass spectrometry (Electro Spray Ionization - (ESI),

[0074] 3) This treatment produces further protein fragments with their own positive charge identified by an HPLC system,

[0075] 4) Each fragment, with its own molecular weight and charge (m / z), is identified by a Peak having its own Retention Time (rt),

[0076] 5) All this data is collected and quantified by a powerful DATA Processor Computer Apparatus.

[0077] The flow of the total procedure can be summarized as follows: the protein sample treated with Phenytoin (block 1) is compared with the reference standard = intact collagen (block II). The digestion of the treated proteins is analyzed (block III) and compared with the analysis of intact proteins (block IV).

[0078] The fragments of each sample are identified by the following parameters: m / zmed m / z mean (mass / charge ratio), m / zmin, m / zmax, rtmed Mean retention time, rtmin Minimum retention time, rtmax Maximum retention time, n peaks peaks Number, KO peaks detected by the algorithm R (XCMS) in the Sample (Standard Collagen), WT peaks detected by the algorithm R (XCMS) in the Control (Treated Collagen).

[0079] - The results obtained following the procedure and analysis, briefly described above, are summarized in the following TABLE: 734.5744573 327.305 8078.510633 7595.894275 232068.8671 589.1224101 323.7 7597.04436 11179.124780 1646316.5136

[0080] The Table shows that all the Peaks belonging to the Standard Collagen are much more intense / stronger than the Peaks belonging to the Treated Collagen Samples, (Drug A, Drug B) at least 8 / 10 times: in short, the collagen peaks incubated with both Phenytoin and Fosphenytoin are practically nil.

[0081] - Such a phenomenon could be explained by two main assumptions :

[0082] 1) by a profound conformational change of COLLAGEN, which makes the treated collagen samples resistant to the hydrolytic effect of trypsin.

[0083] Trypsin exerts the strong hydrolytic action thereof precisely using the molecules of Lysine and Arginine present in the collagen structure, therefore the results obtained allow the Applicants to assume that the Lysine and Arginine residues are involved in the modification / alteration of the Collagen treated by Phenytoin sodium salt and Fosphenytoin.

[0084] Such a result is new and never been described in the literature and is certainly independent of the presence of a biological cofactor.

[0085] 2) The fact that collagen in the presence of Phenytoin sodium salt and Fosphenytoin is virtually unhydrolyzed by Trypsin could be explained by a specific inhibition of the enzyme Trypsin, a serine protease which hydrolyzes proteins on the carboxyl side of the amino acids lysine and arginine. On the other hand, Trypsin belongs to the large family of Metalloproteases which are very important in the development of ocular disorders involving a weakening of collagen, such as keratoconus or axial myopia (references a, b, c.).

[0086] Such a result is unexpected, not described in the literature and is also certainly independent of the presence of a biological cofactor. CONCLUSIONS

[0087] The Applicants, considering the results obtained, and experimentally verifying the reproducibility thereof, have developed different formulations containing both Phenytoin sodium salt and Fosphenytoin, which could be used in the treatment of Keratoconus and Axial Myopia.

[0088] Therefore, a plurality of products containing Phenytoin in the form of micro emulsions, inclusion complexes, gels and eye drops are identified and described below, capable of obtaining and improving the solubilization of Phenytoin itself, to be administered to obtain the cross-linking of ocular collagen and counteract the weakness thereof in diseases such as Keratoconus and Axial Myopia which are the subject of the present industrial patent. The methods for obtaining them are also provided.

[0089] PREPARATIONS CONTAINING PHENYTOIN FOR CROSS-LINKING EYE

[0090] COLLAGEN

[0091] A) MICROEMULSIONS

[0092] Two formulations are provided:

[0093] 1) Phenytoin micro emulsion 0.252%

[0094] 2) Phenytoin micro emulsion 0.504%

[0095] 1) PHENYTOIN MICROEMULSION 0.252% Phenytoin Emulsion Formulation 0.252%

[0096] First step:

[0097] Solubilization of Phospholipids S75, Phenytoin, 5 Triglycerides and Vitamin E TPGS

[0098] The weighed raw materials are transferred into a 10-liter evaporation Flask. At this point, the Flask is placed on a Rotavapor and heated until it reaches a temperature of 50°C (by immersing the latter in a tank 10 of water heated to a temperature of 50°C). During the heating step, the flask is gently rotated for at least 12 hours. The complete dispersion of the Phenytoin dispersed in the phospholipids and triglycerides is obtained.

[0099] 15 Second Step: Tris buffer preparation with EDTA and Vitamin E TPGS

[0100] 1 kg of Tris buffer is prepared with EDTA and Vitamin E TPGS inside a 25-liter food-grade plastic bucket.

[0101] Third step:

[0102] Hydration of phospholipid powder and mixing

[0103] The Phospholipid Powder is hydrated with 1 kg of Tris buffer with EDTA and Vitamin E TPGS prepared in the step. The mixing occurs in a 3-liter plastic container using the Silverson AX5 vertical homogenizer.

[0104] A whitish Macro Emulsion is obtained.

[0105] Fourth step:

[0106] Microemulsion Preparation by High Pressure Homogenization

[0107] The macro emulsion is extruded with a high pressure extruder (900 bar) 5 times. A clear product is obtained. Fifth step:

[0108] Addition of Hypromellose

[0109] After allowing the microemulsion to cool, Hypromellose (1.5g) is added thereto, which is suitably mixed using the Silverson AX5.(vertical).

[0110] Sixth step: Sterilization by filtration

[0111] Using a Sartorius Flexboy Sterile Bag (3 L) connected to a filtering system, consisting of a pre- filter (0.8-0.2 pm) and a 0.45-0.2 pm sterilizing filter (indissolubly connected to the bag), the microemulsion is filtered (sterilized) at room temperature .

[0112] 2) PHENYTOIN MICROEMULSION 0.504%

[0113] Phenytoin Emulsion Formulation 0.504%

[0114] First step: Solubilization of Phospholipids S75, Phenytoin, Triglycerides and Vitamin E TPGS

[0115] The weighed raw materials are transferred into a 5- liter evaporation Flask. At this point, the Flask is placed on a Rotavapor and heated until it reaches a temperature of 50°C (by immersing the latter in a tank of water heated to a temperature of 50°C). During the heating step, the flask is gently rotated for at least 12 hours. The complete dispersion of the Phenytoin dispersed in the phospholipids and triglycerides is obtained.

[0116] Second Step: Tris buffer preparation with EDTA and Vitamin E TPGS

[0117] 1 kg of Tris buffer is prepared with EDTA and Vitamin E TPGS inside a 25-liter food-grade plastic bucket. Third step:

[0118] Hydration of phospholipid powder and mixing

[0119] The Phospholipid Powder is hydrated with 1 kg of Tris buffer with EDTA and Vitamin E TPGS prepared in step 2. The mixing occurs in a 3-liter plastic container using the Silverson AX5 vertical homogenizer.

[0120] A whitish Macro Emulsion is obtained.

[0121] Fourth step

[0122] MICROEMULSION Preparation BY High Pressure Homogenization

[0123] The macro emulsion is extruded with a high pressure extruder (900 bar) 5 times. A clear product is obtained.

[0124] Fifth step

[0125] Addition of Hypromellose

[0126] After allowing the microemulsion to cool, Hypromellose (1.5g) is added thereto, which is suitably mixed using the Silverson AX5.(vertical).

[0127] Sixth step

[0128] Sterilization by filtration

[0129] Using a Sartorius Flexboy Sterile Bag (3 L) connected to a filtering system, consisting of a pre- filter (0.8-0.2 pm) and a 0.45-0.2 pm sterilizing filter (indissolubly connected to the bag), the microemulsion is filtered (sterilized) at room temperature.

[0130] The above formulations indicate a concentration range between 10 mM 0.252% and 20 mM, 0.504%, but it is implicit that the reported Micro emulsion preparation technique is capable of producing such a product at a concentration of active substances lower than that indicated of 0.504% 20 and 0.252% mM, for example, of 1 mM equal to 0.0252%, by those skilled in the art, putting common laboratory knowledge into practice. B) PHENYTOIN HYDROXYPROPYL-B-CYCLODEXTRIN COMPLEX

[0131] Different types of cyclodextrins have been studied for complexing with Phenytoin to obtain the solubilization thereof.

[0132] Following the experiments carried out, the Applicants obtained the complexation between hydroxypropylated 3-cyclodextrins and phenytoin and an improvement in solubility was observed.

[0133] The following was used:

[0134] 2-Hydroxypropyl-β-cyclodextrin

[0135] CAS Number: 128446-35-5

[0136] Molar Mass 1541.54 g / mol

[0137] Formula :C63H12O42

[0138] PREPARATION OF PHENYTOIN HPBCD INCLUSION COMPLEX

[0139] The following procedure was carried out:

[0140] 252 mg of Phenytoin (1) mM were dissolved in 500 ml of ethanol by magnetic stirring overnight.

[0141] 1.54 g of hydroxypropyl-β-cyclodextrin (1mM) were added and dissolved at 70°C by rotavapor for at least 8 hours.

[0142] Mixing and rotavapor heating were repeated twice. A clear solution was obtained.

[0143] The Phenytoin solution included in hydroxypropyl- β-cyclodextrin was lyophilized and after lyophilization a soft powder was obtained.

[0144] Two different formulations of Phenytoin HPBCD COMPLEX eye drops were then prepared as follows:

[0145] METHOD FOR PREPARING 0.252% PHENYTOIN HPBCD COMPLEX EYE

[0146] DROPS 1.792 g of soft powder was solubilized with 100 ml of distilled water and other ingredients were added to obtain the following eye drops formula: 0.252% Phenytoin HPβCD 1.54% COMPLEX

[0147] METHOD FOR PREPARING 0.504% PHENYTOIN HPBCD COMPLEX EYE DROPS 3.584 g of soft powder was solubilized with 100 ml of distilled water and other ingredients were added to obtain the following eye drops formulation.

[0148] 0.504% Phenytoin HPβCD 3.08% COMPLEX

[0149] The formulations above indicate a range of concentrations between 10 mM and 20 mM, but it is implicit that the reported preparation technique of 0.504% HPBCD Complex eye drops is capable of producing such a product at a concentration of active substances lower than that indicated of 0.504% 20 and 0.252% 10 mM, for example of 1 mNM equal to 0.0252%, by those skilled in the art, putting common laboratory knowledge into practice.

[0150] The inclusion in beta-cyclodextrins of the association Phenytoin + Riboflavin was also then tested due to the insolubility thereof in water. PHENYTOIN + RIBOFLAVIN ASSOCIATION BOTH INCLUDED IN BETA-CYCLODEXTRINS

[0151] A molar ratio of: 10 mm phenytoin - 1 mm riboflavin was chosen. This ratio was chosen to emphasize the effect of phenytoin with respect to base riboflavin, but obviously does not exclude other ratios in a range of 10mm phenytoin and 10mm riboflavin.

[0152] The:

[0153] PREPARATION OF PHENYTOIN (10MM) EYE DROPS IN

[0154] BETACYCLODEXTRINS + RIBOFLAVIN (0.1MM) IN CYCLODEXTRINS, was obtained in three stages as follows: 1) The:

[0155] PREPARATION OF A PHENYTOIN HPBCD INCLUSION COMPLEX is carried out as follows: a) 0.252 mg of Phenytoin (1mM) is dissolved in 500 ml of ethanol by magnetic stirring overnight; b) 1.54 g of hydroxypropyl-β-cyclodextrin (1mM) are added and dissolved at 70°C by rotavapor for at least 8 hours; c) Mixing and heating in a rotavapor is repeated twice, until a clear solution is obtained; d) the Phenytoin solution included in hydroxypropyl-β-cyclodextrin thus obtained is lyophilized, obtaining a soft dry powder.(1.792 g).

[0156] 2) The preparation of a:

[0157] RIBOFLAVIN HPBCD INCLUSION COMPLEX is carried out as follows: a) 37.6 mg of RIBOFLAVIN (O.1mM) are dissolved in 500 ml of ethanol by magnetic stirring overnight; b) 154.0 mg of hydroxypropyl-β-cyclodextrin (O.1mM) were added and dissolved at 70°C by rotavapor for at least 8 hours; c) Mixing and heating in a rotavapor was repeated twice, until a clear solution was obtained (191.6 mg); d) the RIBOFLAVIN solution included in hydroxypropyl-β-cyclodextrin thus obtained is lyophilized, obtaining a soft dry powder.

[0158] At this point, we move on to the

[0159] 3) PREPARATION OF TWO-COMPONENT (BIMODAL) EYE DROPS 1.792 g of dry soft powder OF PHENYTOIN IN BETA-

[0160] CYCLODEXTRINS AND 0.191 g of dry soft powder of RIBOFLAVIN IN BETA-CYCLODEXTRINS are solubilized with 100 ml of distilled water and other ingredients are added to obtain the following eye drops formula: 10mM Phenytoin in HPβCD COMPLEX + 1mM Riboflavin in HPβCD COMPLEX

[0161] We then went on to prepare: C) CLEAR CARBOPOL GELS PROCEDURES FOR PREPARING A 0.252% PHENYTOIN GEL:

[0162] 100 mg of Carbopol 980NF was dispersed by magnetic stirring in 90 ml of distilled water, then 252 mg of Phenytoin was added and thoroughly mixed, then 2% NaOH (5-10 ml) was added dropwise to obtain a pH 6.5-7.5, obtaining a clear gel, then further distilled water was added (if necessary) to 100 ml.

[0163] CARBOPOL 0.252% Phenytoin Gel PROCEDURE FOR PREPARING 0.504% PHENYTOIN GEL:

[0164] 100 mg of Carbopol 980NF was dispersed by magnetic stirring in 90 ml of distilled water, after 504 mg of Phenytoin was added and thoroughly mixed, then 2% NaOH (5-10 ml) was added dropwise to have pH 6.5-7.5 obtaining a clear gel, moreover distilled water was added (if necessary) to 100 ml.

[0165] PROCEDURE FOR PREPARING 0.756% PHENYTOIN GEL

[0166] 100 mg of Carbopol 980NF was dispersed by magnetic stirring in 90 ml of distilled water, then 756 mg of Phenytoin was added and thoroughly mixed, then 2% NaOH (5-10 ml) was added dropwise to have pH 6.5-7.5 obtaining a clear gel, moreover distilled water was added (if necessary) to 100 ml.

[0167] In order to expand the concentration of Phenytoin included in the Carbopol gel, the following formulation was prepared as described in the previous preparations, increasing the concentration of Carbopol 980 NF.

[0168] 5 12 ml glass vials were filled with 10 g of each gel, then the vials, suitably screwed, were sterilized, at 121°C for 15 minutes at 1 Atm: a structured transparent gel was obtained. It should be noted that the Gel preparation technique was developed using Carbopol NF 980, but can be usefully expanded with the use of other Carbopols, such as Carbopol Ultrez 10 NF or Carbopol ETD 2020 NF.

[0169] FOSPHENYTOIN

[0170] 0.5% FOSPHENYTOIN EYE DROPS, HYALURONIC ACID

[0171] 0.5% FOSPHENYTOIN EYE DROPS, POLOXAMER 407 75% FOSPHENYTOIN EYE DROPS, POLOXAMER 407

[0172] 0.75 FOSPHENYTOIN EYE DROPS, HYALURONIC ACID

[0173] References

[0174] 1) Rada JA, Shelton S, Norton TT. The sclera and myopia.

[0175] Exp Eye Res 2006;82:185-200.

[0176] 2) Thale A, Tillmann B. The collagen architecture of the - sclera SEM and immunohistochemical studies. Anat

[0177] Anzeiger 1993;175:215-20.

[0178] 3) Thale A, Tillmann B, Rochels R. Scanning electron microscopic studies of the collagen architecture of the human sclera - normal and pathological findings. Ophthalmologies 1996;210:137-41.

[0179] 4) Curtin BJ, Teng CC. Scleral changes in pathological myopia. Trans Am Acad Ophthalmol Otolaryngol 1957;62 :777-88.

[0180] 5) Curtin BJ, Iwamoto T, Renaldo DP. Normal and staphylomatous sclera of high myopia. Arch Ophthalmol 1979;97 (5):912-5.

[0181] 6) McBrien NA, Gentle A. Role of the sclera in the development and pathological complications of myopia. Prog Ret Eye Res 2003;22:307-38.

[0182] 7) Wessel H, Anderson S, Fite D, Halvas E, Hempel J, SundarRaj N. Type XII collagen contributes to diversities in human corneal and limbal extracellular matrices. Invest Ophthalmol Vis Sci 1997;38(11):2408-22.

[0183] 8) Sandberg-Lail M, Hagg PO, Wahlstrom I, Pihlajaniemi T. Type XIII collagen is widely expressed in the adult and developing human eye and accentuated in the ciliary muscle, the optic nerve and the neural retina. Exp Eye Res 2000;70(4):401-10.

[0184] 9) Siegwart Jr JT, Norton TT. The time course of changes in mRNA levels in tree shrew sclera during induced myopia and recovery. Invest Ophthalmol Vis Sci 2002;43:2067-75.

[0185] 10) Gentle A, Liu Y, Martin JE, Conti GL, McBrien NA. Collagen gene expression and the altered accumulation of scleral collagen during the development of high myopia. J BiolChem 2003;278(19):16587-94.

[0186] 11) Silverman AK, Fairley J, Wong RC. Cutaneous and immunologic reactions to phenytoin. J Am Acad Dermatol. 1988;18:721-41 .

[0187] 12) Kero M. Epidermolysis bullosa in Finland. Clinical features, morphology, and relation to collagen metabolism. Acta Derm Venereol Suppl (Stockh). 1984;110:1-51 .

[0188] 13) Eisenberg M, Stevens LH, Schofield PJ. Epidermolysis bullosa--new therapeutic approaches. Australas J Dermatol. 1978;19:1-8.

[0189] 14) Bauer EA, Gedde-Dahl T Jr, Eisen AZ The role of human skin collagenase in epidermolysis bullosa. J Invest Dermatol. 1977;68:119-24.

[0190] 15) Talas G, Adams TS, Eastwood M, Rubio G, Brown RA. Phenytoin reduces the contraction of recessive dystrophic epidermolysis bullosa fibroblast populated collagen gels. Int J Biochem Cell Biol. 1997;29:261-70.

[0191] 16) Moy LS, Tan EM, Holness R, Uitto J. Phenytoin modulates connective tissue metabolism and cell proliferation in human skin fibroblast cultures. Arch Dermatol. 1985;121:79-83.

[0192] 17) Genever PG, Cunliffe WJ, Wood EJ. Influence of the extracellular matrix on fibroblast responsiveness to phenytoin using in vitro wound healing models. Br J Dermatol. 1995 ;133:231-5.

[0193] 18) Muthukumarasamy MG, Sivakumar G, Manoharan G. Topical phenytoin in diabetic foot ulcers. Diabetes Care. 1991;14:909-11.

[0194] 19) Talas G, Brown RA, McGrouther DA. Role of phenytoin in wound healing--a wound pharmacology perspective. Biochem Pharmacol. 1999;57:1085-94.

[0195] 20) Dill RE, Miller EK, Weil T, Lesley S, Farmer GR, lacopino AM. Phenytoin increases gene expression for platelet-derived growth factor B chain in macrophages and monocytes. J Periodontal. 1993;64:169-73.

[0196] 21) Pendse AK, Sharma A, Sodani A, Hada S. Topical phenytoin in wound healing. Int J Dermatol. 1993;32:214-7

[0197] 22) Anstead GM, Hart LM, Sunahara JF, Liter ME. Phenytoin in wound healing. Ann Pharmacother. 1996;30:768-75.

[0198] 23). Bazin S. Delaunay A. Effect of phenytoin on the maturation of collagen in normal skin and granulomatous tissue. C R Acad Sci Paris, Series D 1972;275:509-11),

[0199] 24) Vernillo AT, Ramamurthy NS, Lee HM, Rifkin Br: The effect of phenytoin on collagenase and gelatinase activities in UMR 106-01 rat osteoblastic osteosarcoma cells. Matrix 10(l):27-32, 1990).

[0200] 25) Bekesi N, Kochevar IE, Marcos S. Corneal biomechanical response following collagen cross-linking with Rose Bengal-green light and riboflavin-UVA Invest Ophthalmol Vis Sci. 2016;57:992- 1001.

[0201] 26) Yuan Wu 1, Wenjing Song 1, Yun Tang 1, Ahmed Elsheikh 2, Yingfeng Shao 3 e Xiaoming Yan 1 Efficacy and safety of transglutaminase-induced corneal stiffening in rabbits, https: / / doi.org / 10.1167 / tvst.8.6. 27)

[0202] 27) Handbook methods for oxygen radical research 1985 "Nitroblue tetrazolio Reduction -Cristian Auclair and Emmanuelle Voisin" pp. 123-132).

[0203] 28) Cristoni S, Bernardi LR. Bioinformatics in mass spectrometry data analysis for proteomics studies. Expert Rev Proteomics. 2004 dec;1(4):469-83 ).

[0204] 29) Phenytoin and Phenytoin Sodium CAS Nos. 57-41-0 and 630-93-3. National Toxicology Program, Department of Health and Human Services Report on Carcinogens,

[0205] Fourteenth Edition.

Claims

CLAIMS1) A method of preparing a phenytoin HPβCD inclusion complex, characterized in that it includes the following steps: a) dissolving 252 mg of Phenytoin (1mM) in 500 ml of ethanol by magnetic stirring overnight; b) adding and dissolving 1.54 g of hydroxypropyl-β-cyclodextrin (1mM) at 70°C by rotavapor for at least 8 hours; c) repeating the mixing and heating in a rotavapor twice, until a clear solution is obtained; d) the Phenytoin solution included in hydroxypropyl-β-cyclodextrin thus obtained was lyophilized, obtaining 1.792 g of soft dry powder.2) A method of preparing 10 mM phenytoin HPβCD complex eye drops, characterized in that it includes the following steps:1.792 g of soft dry powder obtained according to the method of claim 1 are solubilized, with 100 ml of distilled water and other ingredients are added, to obtain the following eye drops formula:10 mM Phenytoin HPβCD COMPLEX:3) A method of preparing 20 mMK phenytoin HPβCD complex eye drops, characterized in that 3.584 g of soft powder, obtained according to the method of claim 1, are solubilized with 100 ml of distilled water and other ingredients are added to obtain the following formulation:Phenytoin 20 mM HPβCD COMPLEX:4) An ophthalmic composition for use in in a method of topically treating diseases such as keratoconus and progressive myopia (axial myopia), characterized in that it comprises a phenytoin hydroxypropyl β cyclodextrin complex obtained according to the method of claim 2 or 3.5) An ophthalmic composition according to claim 4 for use in a method of treating diseases such as keratoconus and progressive myopia (axial myopia), which includes directly strengthening, through a continuous topical administration, the structure of corneal and scleral collagen by means of the cross-linking thereof.6) An ophthalmic composition according to claim 4, characterized in that such a composition is administered topically before an irradiation of ultraviolet rays, preferably at 366 nm (Wood's light), to promote the cross-linking of eye collagen and counteract the weakness thereof.7) Products containing Phenytoin in the form of a phenytoin HPHPβCD inclusion complex obtained according to the method of claim 1, to obtain and improve the solubilization thereof, for topical use in a method of treating eye diseases such as Keratoconus and Axial Myopia, which includes contacting them with collagen and ocular structures to obtain the cross-linking of the same collagen and counteract the weakness thereof.8) An ophthalmic composition in the form of a phenytoin HPβCD cyclodextrin inclusion complex obtained accordingto the method of claim 1, where phenytoin is present in concentrations between 0.0252 and 0.504% by weight, on the total weight of the composition, in borate buffer.9) An eye drops formulation where phenytoin is included in an HPBCD cyclodextrin inclusion complex obtained according to the method of claim 1.10) A method of preparing an HPβCD RIBOFLAVIN inclusion complex, characterized in that it includes the following steps a) dissolving 37.6 mg of RIBOFLAVIN (0.1mM) in 500 ml of ethanol by magnetic stirring overnight; b) 154.0 mg of hydroxypropyl-β-cyclodextrin (0.1mM) were added and dissolved at 70°C by rotavapor for at least 8 hours; c) repeating the mixing and heating in a rotavapor twice, until a clear solution (191.6 mg) is obtained; d) the RIBOFLAVIN solution included in hydroxypropyl-β-cyclodextrin thus obtained was lyophilized, obtaining a soft dry powder.11) A method of preparing two-component (bimodal) eye drops with a Phenytoin and Riboflavin cyclodextrin inclusion complex, characterized by solubilizing 1.792 g of dry soft powder of PHENYTOIN IN BETA-CYCLODEXTRINS obtained according to the method of claim 1 and 0.191 g of dry soft powder of RIBOFLAVIN IN BETA-CYCLODEXTRINS, obtained according to the method of claim 10, with 100 ml of distilled water and adding other ingredients, to obtain the following eye drops formula: 10mM Phenytoin in HPβCD COMPLEX + 1mM Riboflavin in HPβCDCOMPLEX

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