METHOD FOR PREPARING A LIPOSOMAL COMPOSITION COMPRISING LATANOPROST, AND THE PHARMACOLOGICALLY ACTIVE LIPOSOMAL COMPOSITION FOR OPHTHALMOTHERAPY PREPARED BY THIS METHOD

Abstract

A group of inventions relates to pharmaceutics and refers to a method for preparing a liposomal composition and a pharmacologically active liposomal composition for ophthalmotherapy prepared by this method. The method is implemented by creating a mixture of solutions of latanoprost, egg phosphatidylcholine and cholesterol in organic solvents, vacuum drying thereof, emulsifying in an aqueous medium, and dispersing an emulsion under pressure. According to the invention, a solution of dipalmytoyl phosphatidylglycerol is introduced into the mixture of solutions, wherein latanoprost, egg phosphatidylcholine, and cholesterol are dissolved in ethyl alcohol, while dipalmytoyl phosphatidylglycerol is dissolved in a mixture of ethyl alcohol and chloroform. The emulsifying of the dried mixture is conducted by a lactose solution in a pH 7.1 buffer, while the dispersing of the emulsion is conducted at a stepwise pressure increase from 300 to 800 at followed by a sterilizing filtration and a freeze drying.

The composition prepared by this method is a frozen-dried powder and a formulation thereof comprises:

TABLE-US-00001 Latanoprost 1, Egg phosphatidylcholine 20.0-30.0, Dipalmytoyl  0.6-0.75, phosphatidylglycerol Cholesterol 0.5-0.9, Lactose 40.0-60.0, Water residue the remainder.

The pharmacologically active liposomal composition has a prolonged antihypertensive and neuroprotective action in case of ocular hypertension and glaucoma, and it is suitable for preparation a solution for an instillation use and/or injectable subconjunctival administration.

2 ind.cls., 2 d.cls.

Claims

1. A method for preparing a liposomal composition comprising: creating a mixture of solutions of latanoprost, egg phosphatidylcholine, and cholesterol in organic solvents, by combining: latanoprost, egg phosphatidylcholine, and cholesterol that are provided in ethyl alcohol; and dipalmytoyl phosphatidylglycerol provided in a mixture of ethyl alcohol and chloroform to form the mixture of solutions; vacuum drying the mixture of solutions; emulsifying the dried mixture of solutions in an aqueous lactose solution in a pH 7.1 buffer to form an emulsion; dispersing the emulsion at a stepwise pressure increase from 300 to 800 at; performing a sterilizing filtration; and freeze drying to form a freeze-dried liposomal composition under compliance of the following mass ratios: egg phosphatidylcholine:lactose being 1:2; egg phosphatidylcholine:dipalmytoyl phosphatidylglycerol:latanoprost being 1:0.02-0.04:0.03-0.05; and egg phosphatidylcholine, dipalmytoyl phosphatidylglycerol, and latanoprost:cholesterol being 1:0.02-0.04.

2. A pharmacologically active liposomal composition for ophthalmotherapy prepared by the method of claim 1, the composition comprising latanoprost, egg phosphatidylcholine, cholesterol, dipalmytoyl phosphatidylglycerol, and lactose, wherein the composition is a freeze-dried powder having the following mass ratio: TABLE-US-00013 Latanoprost 1, Egg phosphatidylcholine 20.0-30.0, Dipalmytoyl phosphatidylglycerol  0.6-0.75, Cholesterol 0.5-0.9, Lactose 40.0-60.0, Water residue the remainder.

3. The composition according to claim 2, wherein the composition has an antihypertensive and neuroprotective action in case of ocular hypertension and glaucoma.

4. The composition according to claim 2, wherein the composition is suitable for preparation of a solution for an instillation use and/or an injectable subconjunctival administration.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0108] Inspection results are illustrated by drawings, which depict the following:

[0109] FIG. 1 is a condition of the anterior chamber of the rabbit eye in an hour after the subconjunctival injection with the liposomal composition of LP prepared by the claimed method (Example No. 3).

[0110] FIG. 2 is a condition of the anterior chamber of the rabbit eye in an hour after the subconjunctival injection with the liposomal composition of LP prepared by the prototype method (Example No. 16).

[0111] FIG. 3 is a condition of the anterior chamber of the rabbit eye in a day after the subconjunctival injection with the liposomal composition of LP prepared by the claimed method (Example No. 1).

[0112] FIG. 4 is a condition of the anterior chamber of the rabbit eye in a day after the subconjunctival injection with the liposomal form of LP prepared by the prototype (Example No. 16).

[0113] FIG. 5 is a condition of the anterior chamber of the rabbit eye in a day after the subconjunctival injection with the liposomal form of LP prepared by the claimed method (Example No. 15).

[0114] FIG. 6 is an OCT scan image of the anterior chamber of the rabbit eye having the ocular hypertension immediately after the subconjunctival injection with the liposomal composition of LP prepared by the claimed method (Example No. 1). The arrow indicates a site for the product administration. The regimen is Radial 7×7 mm ANTERIOR Multi B-scan.

[0115] FIG. 7 is an OCT scan image of the anterior chamber of the rabbit eye having the ocular hypertension in 90 days after the subconjunctival injection with the liposomal composition of LP prepared by the claimed method (Example No. 1).

[0116] FIG. 8 is an OCT scan image of the anterior chamber of the rabbit eye having the ocular hypertension in 120 days after the subconjunctival injection with the liposomal composition of LP prepared by the claimed method (Example No. 1). The arrows indicate epithelium (1), connective-tissue stroma (2), sclera (3).

[0117] FIG. 9 is an OCT scan image of the anterior chamber of the rabbit eye having the ocular hypertension in 90 days after the subconjunctival injection with the liposomal composition of LP prepared by the prototype (Example No. 16).

[0118] FIG. 10 is a retina of the rabbit having the stable OG and glaucoma (pathology control, 12 weeks). The extremely low density and non-uniformity of ganglion cells with plural dropout areas. The bipolar cells with dropout meshes of cells with a layer having a thin width with a reduced number of neurons. Magnification is 600×.

[0119] FIG. 11 is an influence of the subconjunctival injections of the liposomal composition of LP according to the claimed method (Example No. 1) onto the retina of the rabbit with the stable OG and glaucoma (the scheme in the Table 7, 12 weeks). The high density of the ganglion cells with a single dropout area. Layers of the bipolar cells having a uniform width (more than 3 layers of cells) are seen well in the entire image. Magnification is 100×.

[0120] FIG. 12 is an influence of the subconjunctival injections of the liposomal composition of LP according to the claimed method (Example No. 1) onto the retina of the rabbit with the stable OG and glaucoma (the scheme in the Table 7, 12 weeks). The high density of the ganglion cells. Layers of the bipolar cells having a stable width are well seen in the entire image. Magnification is 600×.

[0121] FIG. 13 is an influence of the subconjunctival injections of the liposomal composition of LP according to the prototype method (Example No. 16) onto the retina of the rabbit with the stable OG and glaucoma (the scheme in the Table 7, 12 weeks). The non-uniformity of arrangement and the relatively low density of the ganglion cells are seen. The internal nuclear layer is shortened to 1 series of the bipolar neurons having a cell-like dropout. Magnification is 100×.

[0122] FIG. 14 is an influence of the subconjunctival injections of the liposomal composition of LP according to the prototype method (Example No. 16) onto the retina of the rabbit with the stable OG and glaucoma (the scheme in the Table 7, 12 weeks). The low density of the non-uniformly arranged ganglion cells is seen. Thinning of the layer of the bipolar cells with separate areas of their dropout. Magnification is 600×.

[0123] When studying an acute and chronic ophthalmic toxicity of the drops, the product emulsion was instilled into the right eye of the rabbits each 15 minutes during 6 hours and once a day in the evening during 30 days, respectively. The left eye (control) was instilled with the physiological solution being a 0.9% sodium chloride solution. The eye condition was evaluated by means of the Draize test (in balls) in terms of an appearance and a conjunctival test [30].

[0124] After instillation of all the products, no changes of the condition of the cornea, conjunctiva, iris, as well as edema and eye discharges have been detected (0 balls for all) that provides evidence of an absence of the acute and chronic ophthalmotoxicity of the liposomal composition of LP in case of the drop administration.

[0125] Upon the subconjunctival injection of 0.1 ml of the product emulsion prepared by the claimed method, in an hour, no changes of the eye condition in 4 of 8 animals of the group have been detected. The condition of the tested eye of the rabbits from other groups, according to the Draize test, was, in average: cornea—0.4; conjunctiva—0.4; iris—0.2; edema—0.4; discharges—0 balls (FIG. 1).

[0126] In an hour after the subconjunctival injection of 0.1 ml of the product emulsion according to the Example No. 15 and to the prototype No. 16, the Draize test has shown the following: cornea—1; conjunctiva—1; iris—1; edema—1; discharges—0 balls (FIG. 2).

[0127] In a day after the subconjunctival injection, the condition of the tested eye in most of the animals was without any specific changes (0 balls for all) (FIG. 3). Signs of an irritating effect onto the cornea and conjunctiva with a minor edema were seen in 2 rabbits after injection of the product according to the Example 15 and according to the prototype (FIGS. 4, 5), although such changes were statistically unreliable in terms of the Mann-Whitney test for both the control and tested eyes.

[0128] The absence of significant negative changes in the eye condition after the instillations and subconjunctival single injections of the emulsion provides the evidence regarding the ophthalmic safety of the liposomal composition of LP with optimal parameters of harmlessness for the product of the claimed method.

[0129] Taking into consideration a non-triviality of the use method being the subconjunctival injection and the novelty of the liposomal composition of LP as a potential drug, the ophthalmologic safety has been confirmed by a demonstrative method of the optical coherent tomography (OCT) (tomograph SOCT Copernicus REVO NX 700 SoftwareVersion 9.0 2018).

[0130] In order to provide a prognostic clinical perception of the information by the OCT, a condition of a conjunctival-limbal area of the anterior chamber of the eye was appreciated in the animals with the OG pathology directly after the subconjunctival injection and within distant terms (90 and 120 days, under the IOP dynamics according to the Table 6). Therewith, it should be noted that the OCT data after the subconjunctival injection of the liposomal LP product stated in the prototype was received exclusively for normotensive animals, thereby putting in a doubt their correct clinical correlation with the OG.

[0131] Directly after the subconjunctival injection of the liposomal composition of LP to the rabbit with the OG, a change of the conjuctiva's relief in the form of a gangliform invagination was noted (FIG. 6, the product according to the Example No. 1) that provides evidence of a targeted introduction of the product emulsion to the conjuctiva's tissue. On top of the invagination, the sclera is not visualized, the connective-tissue stroma of the conjuctiva has a mixed OCT-signal, but the structure of the conjuctiva's epithelium is not changed.

[0132] In 90 days after the subconjunctival injection of the product of the claimed method, the local deformation of the conjuctiva's relief is not significant anymore, the OCT scan lacks thinnings of the connective-tissue stroma and cicatrical changes in the epithelium and in the sclera (FIG. 7). At the 120th day of the follow-up, the OCT picture of the conjunctival-limbal area of the eye is normal: the tomogram is structural, three contrast layers (conjuctiva's epithelium, connective-tissue stroma, sclera) are well visualized with a high and moderate level of the OCT-signal, their boundaries are straight and continuous (FIG. 8).

[0133] The OCT picture of the conjunctival-limbal area of the eye of the animals after the subconjunctival injection of the prototype composition within all terms is close to the one described above, however, at the 90th day after the injection, the intensity of the OCT-signal of the connective-tissue stroma is decreased (FIG. 9).

[0134] Results of the OCT studies not only has proven the absence of the negative changes of the conjuctiva's structure within distant terms after the subconjunctival injection of the liposomal composition of LP to animals with OG (the distant ophthalmic safety), but they also have visually demonstrated the delivery of the product to the predicted target zone of the pharmacological intervention.

[0135] The experimental results of the determination of the pharmacological activity of the liposomal composition of LP created according to the claimed method and to the prototype method are stated in the Tables 5-8. All the studied examples of the composition reveal the therapeutic effect as to the signs of the OG and glaucoma, however, the implementation of the claimed method particularly provides the creation of the product having more extended and high functional pharmacological activity:

[0136] 1. At daily drop administration (Table 5), the liposomal composition of LP according to the claimed method (Examples 1-3) in the OG model provides an average 3.5 and 6.6 mm Hg decrease of the increased IOP at 1st and 12th week of the follow-up respectively, thereby exceeding the antihypertensive effect of the prototype product by 1.2 and 1.4 times, and by 1.2 and 1.8 times for the analogue drug with the same follow-up terms. The valuable level of the pharmacological activity of the instillations of all of the products is seen during 12 weeks of the IOP monitoring, although only the liposomal composition of LP according to the Examples 1-3 almost provides the end normalization of the IOP up to the intact control.

[0137] It should be noted that the prototype does not discuss and does not propose the pharmacological activity of the product in case of the drop administration, thus, this effect was not expected, and its reveal in the LP composition according to the claimed method provides the advantage as to the creation of a “perfect” clinical compliance due to the possibility of combination of the drop and injection use.

[0138] 2. At single subconjunctival injection administration (Table 6) in the OG model, the liposomal LP composition according to the claimed method exhibits a prolonged antihypertensive effect during all follow-up 12 weeks with the 6.2-7.3 mm Hg decrease of the IOP up to the intact control. The product of the prototype method is inferior to the claimed one in terms of the IOP decrease level in 3.3-5.5 mm Hg, as well as in terms of the duration of the antihypertensive effect, since starting from the 9th week after injection thereof a gradual recovery of the OG level at 0.5-2.5 mm Hg is noted.

[0139] 3. In case of the experimental stable OG with glaucoma, the single subconjunctival injections of the liposomal LP composition according to the claimed method and to the prototype (Table 7) exhibit the prolonged antihypertensive effect during 6 weeks of the follow-up with the 33.0-39.3% and 14.4-20.0% IOP decrease, respectively, as compared to the pathology control (the initial TOP is 49% increased). The second injection of the LP composition according to the claimed method maintains the 34.4-39.3% IOP decrease with a tendency to normalize the ocular pressure up to the 12th week of the follow-up, while for the prototype, only 16.3% IOP decrease was registered at the 7th week with a slight gradual pressure increase up to the 12th week of the follow-up, i.e. the prototype is strongly inferior to the product of the claimed method in terms of the duration and the level of the effect.

[0140] 4. The prolonged antihypertensive activity of the subconjunctival injections of the liposomal composition of LP (according to the Table 7, 12 follow-up weeks) is accompanied by the neuroprotective action according to the specific histomorphologic parameters of the retina, which provide an evidence of the antiglaucomic effect of the product of the claimed method with the advantages over the prototype product (Table 8): [0141] preservation of the relative uniformity and high density of the neurons of the ganglionic layer that exceeds the one for the prototype by more than 2 and 3 times under small and large magnification, respectively, with almost no dropout areas of the ganglion cells, which exist under the prototype's influence (FIGS. 10-14); [0142] preservation of the layer width and only insignificant reduction of the number of neurons within the layer of the bipolar cells as compared to the notable thinning of the width of this layer and the presence of areas with the reduced number of neurons under the action of the prototype (FIGS. 10-14).

[0143] In should be noted that the neuroprotective effect of the product of the claimed method is obvious when comparing to the histomorphologic picture of the animal retina with the stable OG (pathology control—FIG. 10) that, according to main criteria, corresponds to the human retina condition with glaucoma [30] with a non-uniform distribution and a sharp decrease of the density of neurons of the ganglionic layer with the dropout areas, reduction of the number of neurons, and thinning of the layer of the bipolar cells.

[0144] 5. The liposomal composition of LP, on top of the prolonged antihypertensive action, develops the neuroprotective effect affecting the specific biochemical neurotoxicity markers being glutamate and MDA, while changes in their content in the retina provide evidence of the advantage of the product of the claimed method over the prototype product (Table 8): [0145] 40-43% reduction of the glutamate content in the retina relative to the pathology control with a tendency to normalize the parameter (only 2% increase relative to the intact control), while the same parameters of the prototype are 24% and 39% respectively; [0146] 37-42% reduction of the MDA content in the retina relative to the pathology control with a tendency to normalize the parameter (only 5% increase relative to the intact control), while the same parameters of the prototype are 25% and 33% respectively.

[0147] Generally, advantages of the pharmacological action of the liposomal composition of LP prepared by the claimed method have been demonstrated in comparison to the composition prepared by the prototype method in terms of the level and duration of the TOP reduction and recovery of histomorphologic and biochemical parameters of the retina condition, which provide evidence of the antihypertensive and antiglaucomic activity with a focus on the subconjunctival use that provides the prolonged effect. Said properties are inherent to all studied products of implementation of the claimed method, although the generalized high level of the pharmacological activity is provided by the liposomal composition of LP according to the Examples No. 1-3, for which the advantages of the pharmaceutical quality, which have been already described, are established.

[0148] This comparative conclusion has been made on the basis of the analysis of the parameters of the pharmaceutical and pharmacological quality, which have been established ex tempora after preparation of the liposomal composition of LP both by the claimed method and by the prototype method. Therewith, the reliable solution of the task of the invention is possible only after the long-term stability and the quality of the composition are confirmed, considering the provided target pharmaceutical purpose of the product and its pharmacotherapeutic use.

[0149] To this end, the stability of the quality of the liposomal LP composition prepared by the claimed method and by the prototype method has been determined when storing the target product during 6-12 months with a standard climate control.

[0150] Considering the long period of the works, the evaluation of the stability has been conducted for the illustrative examples of the liposomal composition of LP prepared by the claimed method (Examples 1 and 3 with the highest pharmaceutical quality established ex tempora, Examples 13 and 15—for comparison), as well as for the composition according to the prototype method (Example 16). The temperature regimen for storing the liquid-phase products according to the Examples Nos. 15 and 16-prototype (4° C.) is proposed in the description of the prototype, while for the frozen-dried products according to the Examples No. 1, 3, and 13 the Applicant has selected the temperature of −20° C.

[0151] Dynamics of the demonstrative parameters of the pharmaceutical and pharmacological quality when storing the liposomal LP composition (Table 10) provides evidence of a rather high stability of the product of the claimed method according to the Examples Nos. 1, 3, and 13 during 12 months, while essential changes of the parameters have been noted for the Examples Nos. 15 and 16-prototype at the 6th follow-up month already: [0152] the content of the composition components relative to the initial one changes within the range of 1.1-4% only as compared to 5-26% for the Examples Nos. 15 and 16-prototype, while the largest deviations are seen for the CHOL. Thus, the mass ratio of the components (the formulation) remains stable that does not differ from the one for the product ex tempora (Table 4); [0153] the sum of non-identified impurities in the product raises by 1.6 times, while it raises by 4 and 5 times for the Examples Nos. 15 and 16-prototype respectively; [0154] the oxidation index remains almost unchanged, while it has 20% and 34% increase for the Examples Nos. 15 and 16-prototype respectively; [0155] the dispersity profile has only 5-7% change, and almost retaining the initial size of the liposomes, while the proportion of the liposomes of the initial size for the Examples Nos. 15 and 16-prototype is not more than 50-78% with appearance of up to 30% of small liposomes. It should be noted that the prototype being the only one confirmation of the product's stability proposes only retention of the liposomes of a certain size (namely, 88 nm) leaving alone the appearance of the significant proportion of the liposomes of other sizes (up to 50%) during storage; [0156] the prolonged antihypertensive activity of the single subconjunctival injection of the composition is 2.7% reduced, while the decrease of the effect regarding the IOP reaches 9-27% and 23-33% for the Examples Nos. 15 and 16-prototype respectively; [0157] the normalizing influence of the composition onto the content of the neurotoxicity markers being glutamate and MDA in the retina is 2.1% and 0.5-1.2% reduced respectively, while these parameters of the antiglaucomic activity are 12.3%-10.1% and 4.2%-87.1% decreased for the Examples Nos. 15 and 16-prototype respectively.

[0158] The data of the conducted comparative analysis confirms the liposomal identity, stability of the formulation and of the pharmaceutical and pharmacological quality of the liposomal composition of LP prepared by the claimed method when storing during 12 months (Table 10). The composition according to the prototype, as well as the product of the claimed method No. 15, are not stable, have significant changes of the formulation and quality parameters already on the 6th month of the follow-up.

[0159] Generally, the highest quality is peculiar to the liposomal composition of LP that is created by the claimed method under compliance with the parameters, which are defined by the Examples 1-3 and differ from the ones provided by the prototype method, namely: when creating the mixture of the solutions, LP, ePC, and CHOL are dissolved in ethyl alcohol, the DPPG solution is introduced into the mixture of solutions in the mixture of ethyl alcohol and chloroform, the mass ratio of ePC:DPPG:LP being 1:0.02-0.04:0.03-0.06 and the mass ratio of ePC+DPPG+LP:CHOL being 1:0.02-0.04 are used, a emulsifying medium is the lactose solution in the pH 7.1 buffer at the mass ratio of ePC:lactose being 1:2, and the emulsion is dispersed at the stepwise pressure increase from 300 to 800 at followed by the sterilizing filtration and the freeze drying.

[0160] In case of the deviation from said parameters of the method (Examples 4-15 and the prototype being the Example No. 16), the desirable pharmaceutical quality of the target product being the stable liposomal composition of LP is not achieved (Tables 3 and 10).

[0161] Thus, the high pharmaceutical quality of the target product defines the functional pharmacological activity being the long-term and high antihypertensive effect in case of OG and the antiglaucomic action of the liposomal composition comprising LP, ePC, and CHOL, when providing parameters of its formulation, which are defined by the Examples Nos. 1-3 and differ from the ones for the composition created according to the prototype method, namely: the frozen-dried composition comprises DPPG and lactose, and the mass ratio of LP:ePC:DPPG:CHOL:lactose is 1:29.0-39.0:0.60-0.75:0.50-0.90:40.0-60.0.

[0162] The deviation from said parameters of the composition formulation (Examples 4-9 and the prototype being the Example No. 16, including the absence of the DPPG in the formulation being the Example No. 13) or its provision in the liquid-phase form (Example No. 15) reduces the pharmacological quality, namely the level and duration of the pharmacological effects.

[0163] The achieved optimal combination of the beneficial performance and elements of novelty of the method implementation with the reliable pharmaceutical identity and positive pharmacological properties of the target product proves the advantages of the claimed method in resolving the task that lies in the preparation of the liposomal drug comprising LP.

[0164] The stable liposomal composition of LP created according to the proposed method having the identified formulation, for which the ophthalmic harmlessness and pharmacological activity in case of OG and glaucoma have been established, beneficially differs from the liposomal product comprising LP prepared according to the prototype method. The high and prolonged pharmacological effect has been proven when using the created liposomal composition of LP in the form of drops and subconjunctival injection.

[0165] The stated information provides evidence of the expediency of use of the claimed method to prepare the stable and proper liposomal product comprising LP and incorporation of the liposomal composition of LP prepared according to this method as a drug having a rational compliance for the pharmacotherapy of OG and glaucoma.

TABLE-US-00003 TABLE 1 Parameters of the implementation of the claimed method for preparing the liposomal composition comprising LP and the prototype method for the ratios of the components of the composition being created, which were used when carrying out the process Ratio of the components* Example ePC:DPPG: ePC + DPPG + ePC:lactose No. LP (wt.) LP:CHOL (wt.) (wt.) The claimed method 1 1:0.02:0.03 1:0.03 1:2.0 2 1:0.04:0.05 1:0.04 1:2.0 3 1:0.03:0.04 1:0.02 1:2.0 4 1:0.01:0.03 1:0.02 1:2.0 5 1:0.016:0.03 1:0.01 1:2.0 6 1:0.02:0.03 1:0.05 1:2.0 7 1:0.02:0.025 1:0.04 1:2.0 8 1:0.05:0.03 1:0.03 1:2.0 9 1:0.02:0.03 1:0.01 1:2.0 10 1:0.02:0.03 1:0.03 1:2.0 11 1:0.02:0.03 1:0.03 1:2.0 12 1:0.02:0.03 1:0.03 1:2.0 13 1:n/u 1:0.03 1:2.0 (DPPG):0.03 14 1:0.02:0.03 1:0.03 1:2.0 15 1:0.02:0.03 1:0.03 1:2.0 The prototype method 16 The 1(ePC): 1(ePC + LP): n/u lactose proto- n/u(DPPG): 0.11(CHOL) type 0.086(LP) *n/u-the component is not used when creating the composition

TABLE-US-00004 TABLE 2 Parameters of the preparation of the liposomal composition of LP according to the claimed method and to the prototype method, which were used during carrying out the process (* n/u-not used in the process) Example No. The claimed method 16-The 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 prototype Components involved into the method implementation: LP + + + + + + + + + + + + + + + + ePC + + + + + + + + + + + + + + + + DPPG + + + + + + + + + + + + n/u* + + n/u* CHOL + + + + + + + + + + + + + + + + Lactose + + + + + + + + + + + + + + + n/u* Dissolution medium For LP: ethylalcohol + + + + + + + + + − + + + + + − acetonitrile − − − − − − − − − + − − − − − + For ePC: ethylalcohol + + + + + + + + + + − + + + + − chloroform + methyl − − − − − − − − − − + − − − − + alcohol 2:1 (vol.) For DPPG: ethyl alcohol + + + + + + + + + + + + + n/u* + + n/u* chloroform 1:9 (vol.) For CHOL: ethyl alcohol + + + + + + + + − + + + + + + − chloroform + methyl − − − − − − − − + − − − − − − + alcohol 2:1 (vol.) Emulsifying medium: lactose solution (0.06 g/ml) in the phosphate buffer pH 7.1 + + + + + + + + + + − + + + + − pH 5.5 − − − − − − − − − − + − − − − − the pH 5.5 − − − − − − − − − − − − − − − + phosphate buffer solution Emulsion dispersing parameters: A) Pressure, at: 1-3rd cycle 300 300 300 300 300 300 200 300 300 300 300 800 300 200 300 800 4-7th cycle 800 800 800 800 600 800 800 800 800 800 800 the 800 700 800 the entire entire process process b) Particle size after ≤220 ≤220 ≤200 ≤200 ≤230 ≤200 ≤200 ≤200 ≤200 ≤180 ≤180 <180 ≤200 <300 ≤200 <125 dispersing, nm Freeze drying of the + + + + + + + + + + + + + + n/u* n/u* emulsion after dispersing

TABLE-US-00005 TABLE 3 Parameters of the identification and pharmaceutical quality of the liposomal composition prepared according to the claimed method and to the prototype method Example No. The claimed method 16-The 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 prototype Identification of the components: LP + + + + + + + + + + + + + + + + ePC + + + + + + + + + + + + + + + + DPPG + + + + + + + + + + + + n/c + + n/c CHOL + + + + + + + + + + + + + + + + Quantitative content of the components 1) The product- frozen-dried composition, mg/bottle: LP (±0.02) 0.50 0.50 0.52 0.50 0.51 0.50 0.48 0.50 0.49 0.50 0.47 0.50 0.51 0.50 − − ePC (±0.15) 15.0 10.0 13.0 14.9 17.5 15.1 19.2 15.1 14.9 15.0 13.82 15.0 15.2 14.87 − − DPPG (±0.02) 0.30 0.31 0.39 0.16 0.28 0.30 0.39 0.73 0.30 0.30 0.28 0.30 n/c 0.30 − − CHOL (±0.02) 0.45 0.42 0.30 0.32 0.19 0.61 0.77 0.49 0.16 0.45 0.42 0.44 0.47 0.40 − − 2) The product- emulsion (without freeze drying), mg/ml LP (±0.02) − − − − − − − − − − − − − − 1.01 1.01 ePC (±0.15) − − − − − − − − − − − − − − 29.93 10.70 DPPG (±0.02) − − − − − − − − − − − − − − 0.62 − CHOL (±0.02) − − − − − − − − − − − − − − 0.87 1.41 LP encapsulation 100 99 98 96 94 98 99 96 96 93 93 94 94 92 97 94 (% of the administered matter, (±3) Size of the 225/ 220/ 215/ 200/ 190/ 170/ 220/ 225/ 220/ 200/ 190/ 180/ 200/ 240/ 220/ 88/ liposomes 100 95 96 80 90 100 95 85 85 80 95 100 90 80 95 85 after preparation 147/ 150/ 120/ 100/ 100/ 90/ 85/ 80/ 60/ 55/ 160/ 50/ 70/ of the 5 4 20 10 5 15 5 20 5 10 20 5 15 composition, nm (±3)/% of the liposomes having the corresponding size Oxidation index, 0.22 0.21 0.20 0.24 0.25 0.26 0.27 0.26 0.25 0.26 0.26 0.27 0.27 0.27 0.28 0.28 c.u. * PH of the 7.1 7.1 7.0 7.1 7.1 7.0 7.0 7.0 7.0 7.0 5.5 7.1 7.0 7.0 7.1 5.5 emulsion* Residual solvents, %: * ethyl alcohol 0.02 0.02 <0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.01 <0.02 0.02 0.02 0.04 0.02 <0.06 methyl alcohol − − − − − − − − − − 0.01 − − − − 0.01 chloroform 0.01 0.01 <0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 <0.02 0.01 <0.04 acetonitrile − − − − − − − − − <0.01 − − − − − <10.02 Osmolality, * 320 300 310 300 310 310 320 300 300 280 280 310 320 330 300 280 mOsmol/kg Sterility + + + + + + + + + + + + + + + + * is not regulated by the prototype

TABLE-US-00006 TABLE 4 The formulation of the liposomal composition of LP prepared by the claimed method and the prototype method in terms of the mass ratio of the components determined according to the established parameters of the pharmaceutical quality.sup.1) Example Mass ratio No. LP:ePC:DPPG:CHOL:lactose.sup.2) The composition prepared according to the claimed method 1 1:30.0:0.60:0.90:60.0 2 1:20.0:0.62:0.85:40.0 3 1:25.0:0.75:0.50:50.0 4 1:30.0:0.30:0.63:60 5 1:34.3:0.54:0.37:68.5 6 1:30.0:0.60:1.60:60.0 7 1:40.0:0.80:1.65:80.0 8 1:30.0:1.50:0.98:60.0 9 1:30.0:0.60:0.33:60.0 10 1:30.0:0.60:0.90:60.0 11 1:28.1:0.60:0.90:60.0 12 1:30.0:0.60:0.90:60.0 13 1:30.0:n/c (DPPG):0.90:60.0 14 1:29.4:0.60:0.80:60.0 15 1:29.1:0.60:0.87:60.0 The composition prepared according to the prototype method 16 The 1(LP):10.60(ePC):n/c (DPPG): prototype 1.40 (CHOL):n/c (lactose) .sup.1)The formulation of the compositions is stated after preparation of the frozen-dried product according to the Examples Nos. 1-14 and the liquid product according to the Example No. 15 according to the claimed method, as well as the liquid product according to the Example No.16-prototype. Water content in the products is not stated .sup.2)Lactose content is taken according to the administered quantity .sup.3)n/c means that the composition does not comprise the component

TABLE-US-00007 TABLE 5 The efficiency of the claimed method and of the prototype method in terms of the pharmacological activity of the prepared liposomal composition of LP for the daily drop administration in terms of influence onto the IOP level in case of ophthalmic hypertension IOP level, mm Hg, (±0.60): Liposomal composition of LP.sup.1) According Follow-up to the Analogue term, According to the claimed method (Example No.): prototype drug week 1 2 3 13 15 No. 16 Lanotan.sup.2) The initial IOP prior to the experiment = 11.90 ± 0.33 (intact control) The initial IOP in case of ophthalmic hypertension = 18.88 ± 0.73 (pathology control) 1 15.30 15.40 15.06 15.44 15.67 16.23 16.80 2 14.45 14.73 14.70 14.88 14.89 15.44 16.00 3 13.65 14.00 14.23 14.52 14.38 15.05 15.68 4 13.00 13.58 13.15 13.80 13.89 14.60 15.00 5 12.55 12.80 12.76 13.26 13.10 13.85 14.47 6 12.30 12.41 12.38 13.00 12.70 13.27 14.05 7 12.10 12.10 12.10 13.00 12.39 13.10 14.01 8 12.10 12.05 12.15 12.81 12.30 13.10 14.00 9 12.00 12.17 12.15 12.45 12.20 13.00 13.85 10 12.05 12.29 12.00 12.56 12.27 12.91 13.61 11 12.15 12.30 12.10 12.70 12.20 12.80 13.50 12 12.10 12.45 12.10 12.75 12.15 12.86 13.44 .sup.1)data regarding the activity of the liposomal composition of LP according to the claimed method and to the prototype method are stated after their preparation and identification .sup.2)data regarding the activity of the analogue drug with actual expiration term is stated

TABLE-US-00008 TABLE 6 The efficiency of the claimed method and of the prototype method in terms of the pharmacological activity of the prepared liposomal composition of LP upon the single subconjunctival administration in terms of influence onto the IOP level in case of ophthalmic hypertension.sup.1) The IOP level under action of the liposomal composition of LP, mm Hg (±0.66): Follow-up According to the terms, According to the claimed method (Example No.): prototype weeks 1 2 3 10 15 No. 16 The initial IOP prior to the experiment = 12.40 ± 0.33 (intact control) The initial IOP in case of ophthalmic hypertension = 19.39 ± 0.73 (pathology control) 1 12.04 12.38 12.18 13.15 12.60 13.86 2 11.97 12.20 12.00 12.49 12.57 13.73 3 11.68 12.24 12.00 12.45 12.52 13.44 4 12.06 12.35 11.97 12.30 12.49 13.45 5 12.14 12.20 12.06 12.35 12.53 13.46 6 12.08 12.25 12.00 12.40 12.58 13.90 7 11.82 12.17 11.88 12.45 12.44 13.90 8 12.16 12.10 11.80 12.53 12.59 13.79 9 12.36 12.27 11.96 12.51 12.60 13. 81 10 12.44 12.36 12.10 12.60 12.65 13.92 11 12.48 12.48 12.14 12.64 12.60 14.54 12 12.58 12.50 12.27 12.61 12.70 16.06 .sup.1)data regarding the activity of the liposomal composition of LP according to the claimed method and to the prototype method are stated after preparation and identification

TABLE-US-00009 TABLE 7 The efficiency of the claimed method and of the prototype method in terms of the pharmacological activity of the prepared liposomal composition of LP upon the injection subconjunctival administration in terms of influence onto the IOP level in case of stable ophthalmic hypertension with glaucoma.sup.1) IOP level, mm Hg (±0.73): Liposomal composition of LP OG According to Follow-up (pathology- According to the claimed method (Example No.): the prototype terms control) 1 2 3 10 15 No. 16 The initial IOP prior to the experiment = 16.44 ± 0.33 (intact control)  0 29.86 1st injection of the composition  3rd day 30.06 18.69 18.53 18.27 19.20 19.03 24.44  1 week 29.94 18.09 18.37 18.06 18.52 18.72 24.00  2 weeks 29.25 18.19 18.40 17.93 18.44 18.40 24.10  4 weeks 27.81 18.39 18.72 18.29 18.97 18.83 23.55  6 weeks 28.28 18.87 19.10 18.90 19.43 19.24 24.21 2nd injection of the composition  7 weeks 28.91 18.40 17.00 17.25 18.58 18.27 24.0  8 weeks 27.90 17.74 17.46 17.63 18.70 17.97 24.20 10 weeks 27.59 17.65 18.00 18.00 19.00 18.32 24.42 12 weeks 27.44 17.73 18.16 18.10 19.43 18.48 24.00 .sup.1)data regarding the activity of the liposomal composition of LP according to the claimed method and to the prototype method are stated after preparation and identification

TABLE-US-00010 TABLE 8 The efficiency of the claimed method and of the prototype method in terms of the pharmacological activity of the subconjunctival injections of the prepared liposomal composition of LP as to the influence onto the specific histomorphologic and biochemical parameters of theretina's condition in case of glaucoma with the stable OG* Parameter value Specific Prior to the Glaucoma Influence of the liposomal composition of LP parameters of the experiment (pathology- The claimed method (Example No.) The prototype retina's condition (intact control) control) 1 2 3 10 15 No. 16 Histomorphologic parameters Neurons of the ganglionic layer: Density, c.u.: magnification ≥20 <10 ≥18 ≥19 ≥20 ≥16 ≥18 ≥9 100x ≥12 ≤5-6 ≥10 ≥11 ≥10 ≥6 ≥9 ≥4 magnification 600x Uniformity of + − + + + +/− + +/− the cell distribution Presence of areas − + − − − − −/+ +/− dropout Bipolar cells: thinning of the − + − − − −/+ − +/− layer width areas with the − + − −/+ − −/+ −/+ + reduced number of neurons Biochemical parameters Glutamate 13.3 ± 24.4 ± 13.7 ± 13.8 ± 14.2 ± 15.6 ± 14.0 ± 15.6 ± content, μmol/g 1.1 1.9 1.2 1.3 1.1 1.2 1.2 1.7 MDA content, 832.6 ± 1465.8 ± 877.4 ± 847.5 ± 896.5 ± 911.5 ± 900.3 ± 1074.3 ± nmol/g 56.8 84.6 42.9 46.2 50.0 40.7 31.1 69.99 *the parameters were determined after the experiment ended according to the scheme stated in the Table 7.

TABLE-US-00011 TABLE 9 The stability of the liposomal composition of LP prepared by the claimed method and by the prototype method in terms of the parameters of the pharmaceutical and pharmacological quality Value of the quality parameter of the liposomal composition of LP Example No. according to the claimed method: No. 16- 1 3 13 15 Prototype Storage term (month) at a temperature of (±0.5° C.): −20° C. 4° C. 4° C. Parameters 0 6 12 0 6 12 0 6 12 0 6 0 6 Pharmaceutical quality Content of the identified components * LP (±0.02) 0.50 0.50 0.50 0.52 0.49 0.49 0.51 0.50 0.49 1.03 0.97 1.01 0.90 ePC (±0.15) 15.0 15.0 14.8 13.0 13.1 12.9 15.2 15.0 14.8 29.93 27.25 10.70 8.40 DPPG (±0.02) 0.30 0.29 0.29 0.39 0.36 0.37 n/c n/c n/c 0.62 0.48 n/c n/c CHOL (±0.02) 0.45 0.45 0.43 0.30 0.30 0.29 0.46 0.47 0.45 0.87 0.64 1.41 1.01 Non-identified <0.02 <0.02 <0.03 <0.02 <0.02 <0.04 <0.02 <0.04 <0.04 <0.02 0.08 <0.02 0.10 impurities (±0.03) * LP encapsulation (% 100 99 97 99 98 96 94 92 92 98 86 94 72 of the overall content, (± 5) Size of the 225/ 223/ 220/ 220/ 220/ 218/ 210/ 210/ 210/ 220/ 200/ 88/ 94/ liposomes, 100 100 95 95 94 95 93 88 88 95 78 85 20 (nm (±3)/% of the 110/ 147/ 127/ 124/ 65/ 65/ 55/ 50/ 55/ 70/ 88/ liposomes having the 5 5 6 5 7 7 12 5 22 15 50 corresponding size) 56/ 50/ 5 30 Oxidation index, c.u. 0.23 0.24 0.25 0.22 0.26 0.26 0.30 0.31 0.32 0.28 0.33 0.29 0.40 pH of the emulsion 7.1 7.1 7.1 7.0 7.0 6.9 7.1 7.0 7.0 7.0 6.7 5.5 5.5 Osmolality, 320 315 310 310 300 300 320 310 300 300 250 280 250 mOsmol/kg Sterility + + + + + + + + + + +/− + +/− Pharmacological quality Effect of the single s/c injection as to the IOP level in case of OG, % up to the initial IOP (intact)/up to the IOP (pathology- control) (±5.3%): in 1 week 97.1/ 99.7/ 100.4/ 98.2/ 100.1/ 100.5/ 106.9/ 106.5/ 107.3/ 101.6 116.1/ 111.8/ 135.2/ 67.8/ 63.8 64.2 62.8 64.0 64.3 68.3 68.1 68.6 65.0 74.2 71.5 86.5 in 2 weeks 96.5/ 96.8/ 98.4/ 96.8/ 100.0/ 100.9/ 104.8 106.9/ 107.7/ 101.4/ 121.0/ 111.7/ 140.6/ 61.7 61.9 62.9 61.9 64.0 64.5 67.0 68.3 68.8 64.8 77.3 71.3 89.9 in 6 weeks 97.4/ 98.9/ 99.2/ 96.8/ 100.7/ 101.6/ 104.0/6 108.1/ 108.7/ 101.0/ 128.7/ 112.1/ 145.2/ 62.3 63.2 63.4 61.9 64.4 65.0 6.5 69.1 69.5 64.6 82.0 71.7 92.8 Effect as to the 103.0/ 106.0/ 107.2/ 102.3/ 103.8/ 104.5/ 106.7/ 108.3/ 107.5/ 105.3/ 127.6/ 117.3/ 136.8/ glutamate level in 56.1 57.8 58.2 55.7 56.6 57.0 58.2 59.0 58.6 57.4 69.7 63.9 74.6 the retina in 6 weeks after the single s/c injection, % up to the initial values (intact)/up to the values in the pathology-control (±4.5%) Effect as to the MDA 105.4/ 106.9/ 107.5/ 104.3/ 104.7/ 105.3/ 107.1/ 107.6/ 109.1/ 108.1/ 132.0/ 141.7/ 160.9/ level in the retina in 59.9 60.8 61.1 59.3 59.5 59.8 60.9 61.1 62.0 61.4 76.3 80.5 91.4 6 weeks after the single s/c injection, % up to the initial values (intact)/ up to the values in the pathology-control (±3.7%) * The quantitative content is stated in mg/bottle for the composition according to the Examples Nos. 1, 3, 13 and mg/ml for the Examples Nos. 15 and 16-prototype.

TABLE-US-00012 TABLE 10 The stability of the formulation of the liposomal composition of LP prepared by the claimed method and by the prototype method in terms of the mass ratio of the components Liposomal composition Mass ratio * of LP (Example No.) in LP:ePC:DPPG: the selected conditions CHOL:lactose The composition prepared according to the claimed method No. 1-Storage at −20° C. during (month): 0 months (ex tempora) 1:30.0:0.60:0.90:60.0 6 months 1:30.0:0.58:0.90:60.0 12 months 1:29.6:0.58:0.87:60.0 No. 3-Storage at −20° C. during (month): 0 months (ex tempora) 1:25.0:0.75:0.50:50.0 6 months 1:25.0:0.73:0.58:50.0 12 months 1:25.0:0.75:0.50:50.0 No. 13-Storage at −20° C. during (month): 0 months (ex tempora) 1:30.0:n/c (DPPG):0.90:60.0 6 months 1:30.0:n/c (DPPG):0.94:60.0 12 months 1:30.2:n/c (DPPG):0.91:60.0 No. 15-Storage at 4° C. during (month) 0 months (ex tempora) 1:29.1:0.60:0.87:60.0 6 months 1:28.1:0.49:0.77:60.0 The composition prepared according to the prototype method No. 16-Storage at 4° C. during (month): 0 months (ex tempora) 1:10.60:n/c (DPPG): 1.40:n/c (lactose) 6 months 1:9.30:n/c (DPPG): 1.04:n/c (lactose) * Mass ratio of the components is calculated according to the determined content of LP, ePC, DPPG, and CHOL (parameters of the pharmaceutical quality) at a certain storage term of the product. Lactose content is taken according to the administered quantity.

LITERATURE

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