WO2023220003A1 - Topical ophthalmic pirfenidone-loaded liposomes to increase the therapeutic activity of the drug in cornea and useful for prevention or therapy of corneal haze - Google Patents

Topical ophthalmic pirfenidone-loaded liposomes to increase the therapeutic activity of the drug in cornea and useful for prevention or therapy of corneal haze Download PDF

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WO2023220003A1
WO2023220003A1 PCT/US2023/021454 US2023021454W WO2023220003A1 WO 2023220003 A1 WO2023220003 A1 WO 2023220003A1 US 2023021454 W US2023021454 W US 2023021454W WO 2023220003 A1 WO2023220003 A1 WO 2023220003A1
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corneal
pfd
topical ophthalmic
peg
pirfenidone
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PCT/US2023/021454
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French (fr)
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Arturo SANTOS
Jose NAVARRO PARTIDA
Juan Armendariz Borunda
Phillip Frost
Jane H. HSIAO
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Opko Pharmaceuticals, Llc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Definitions

  • Corneal transparency depends mainly on stromal structure. Corneal stroma is composed of about 90% of extracellular matrix (ECM), principally collagen. This collagen is regularly packed into small diameter (• ⁇ 25 nm) fibrils packed as lamellae, this arrangement minimizes light scatering and permits transparency. Corneal collagen fibrils are composed mostly of collagen [ and lesser amounts of collagen V, as well as some proteoglycans. These features allow collagen fibrils to maintain their diameter and separation. [2]
  • ECM extracellular matrix
  • C iorneal injury induced by infection, trauma., chemicals or surgery such as PRK (photorefractive keratectomy), LASER (laser epithelial keratomileusis), and epi-LASIK (epithelial laser in-situ keratomileusis) triggers a complex series of processes, known as wound healing response, whose purpose is to restore the normal structure and function of the cornea.
  • PRK photorefractive keratectomy
  • LASER laser epithelial keratomileusis
  • epi-LASIK epi-LASIK
  • Myofibroblasts characterized by aS MA expression, guide the corneal haze phenomenon. These cells are highly contractile able to synthesize and deposit great amounts of ECM components, especially collagen I and II I, changing collagen diameter fibrils and disarranging stromal structure of the cornea. [4,5] The cornea does not have myofibroblasts present under normal conditions, they derive from resident keratocytes after injury in response to TGF-p1 released by epithelial cells. [6]
  • PFD 1 Perfenidone
  • CNV choroidal neovascularization
  • Liposomes small vesicles (approximate size of 30-1000 nm) which are prepared with phospholipids, offer an efficient option for PFD drug delivery. Liposomes have the advantage to be biodegradable with a relatively non-toxic behavior, which enhances drug permeation by binding to the corneal surface. [16] We anticipate that due to these nanoparticles, PFD penetration will increase the time of interaction with the ocular surface and would improve the therapeutic effect of PFD.
  • the topical ophthalmic compositions of the present invention comprise a combination of PFD as the active pharmaceutical ingredient and a thennodynamically stable liposome. Additional components such as surfactants (ionic or uon-ionic), penetration enhancers, buffers, tonicity agents and solvents suitable for use in ophthalmic treatment can be added as necessary- .
  • the invention comprises use of a topical ophthalmic formulation
  • SUBSTITUTE SHEET (RULE 26) which comprises a combination of PFD and a liposome in the treatment of an ophthalmic disease in which corneal haze is a related symptom or condition.
  • the liposome comprises a diacylglycerol-PEG compound.
  • the PEGylated compounds preferably comprise PEG-12-GDM or PEG- 12 GDO (glycerol dioleate) Preferred formulations comprise PFD and.
  • polyethyleneglycol (PEG-12) glyceryl dimyristate as the diacylglyverol-PEG compound
  • ethyl alcohol as organic solvent for liposomes generation
  • kolliphor IIS 15 as penetration enhancer
  • citric acid anhydrous and sodium citrate dehydrate as buffers
  • benzalkonium chloride as preservative
  • grade 2 purified water as solvent.
  • the ophthalmic formulations of the present invention have been found to increase the therapeutic activity of topically administered PED and are thus useful for prevention or treatment of corneal haze
  • FIG. 1 shows liposomal encapsulation of pirfenidone crystals.
  • FIG. 2 shows a comparison of corneal haze effects induced by alkali and recovery or lack thereof after 15 days of tr eatment with various reagents or drugs versus an eye which was not alkali treated (FIG 2 A).
  • the data dearly shows that the pirfenidone-loaded liposomes (PL) have a greater reduction in haze extension and density than those eyes treated with PBS (FIG. 2B), empty liposomes (FIG. 2C) and dexamethasone (FIG. 2D).
  • FIG. 3 shows the evaluation of corneal thickness, edema and inflammation in corneal tissue
  • A Repre-sentative microphotographs from the different experimental groups are presented and are as fol-lows A. non-burned eye (NB), B. PBS treated eye (PBS), C. empty liposome treated eye (EL), D. dexamethasone treated eye (DEX), E. 0.02% PFD treated eye, F. 0.1 % PFD treated eye, G. 0.02% PFD-loaded liposomes (PLs) treated eye and II. 0. 1 % PL treated eye.
  • B Quantitative analysis of corneal thickness, edema and corneal inflammation.
  • FIG. 4 shows a comparison of aSMA, TGF-P and IL- ip expression in corneal tissue among different treatments by immunoflaorescence.
  • Aj PFD suppresses uSMA in a dosedependent manner, and liposomes improve its effect. Representative immunofluorescence images obtained by confocal microscopy.
  • B Semi-quantification of aSM.A protein expression by fluorescence intensity analy-sis. Data expressed as mean standard deviation.
  • C PFD suppresses TGF-p in a dose-dependent manner and liposomes improve this effect.
  • PFD restores normal epithelial location of IL- 1(1 Non-bumed eyes (NB), PBS treated eyes (PBS), empty liposome treated eyes (EL), dexame-thasone treated eyes (DEX), 0.02% PFD treated eyes, 0.1% PFD treated eyes, 0.02% PFD-loaded liposome (PL) treated eyes and 0.1% PL treated eyes.
  • aSMA smooth muscle actin
  • PBS phosphate-buffered saline
  • EL empty liposome
  • DEX dexamethasone
  • PFD pirfenidone
  • PL pirfenidone-loaded liposome
  • compositions of the present invention contain a pharmaceutically effective amount of pirfenidone (PFD).
  • PFD pirfenidone
  • concentration of PFD in liposomes formulations ranges from 0.01 to 2.00% (w/v).
  • PFD (5-rneihyI-l-phenylpyridin-2( 1 ld)-one is a known.
  • pyridinone with antifribroic, anti-inflammatoiy and antioxidant activity with an empirical formula of CaHnNO and a molecular weight of 185.22 Da.
  • Polyethyleneglycol (PEG- 12) glyceryl dimyristate is used as structural constituent of liposomes in a concentration of 5-15% (w/v)
  • SUBSTITUTE SHEET (RULE 26) and ethyl alcohol is used as organic solvent for liposomes generation in a concentration of 0.7 to 2.1% (v%).
  • the liposomes formulation contains polyethylene glycol (15)- hydroxystearate or KolliphorHS 15 from 2.5 - 7.5% (w/v), as a potent non-ionic solubilizer and emulsifying agent, with low toxicity proposed to act as a permeability enhancer.
  • KolliphorHS 15 promotes drug transport across cell membranes (increasing the endocytosis rate) and stimulates drug translocation through the paracellular route (affects actin organization on the cell cytoskeleton with the subsequent tight junction opening).
  • the aqueous compositions of the present invention optionally comprise more excipients selected from the group consisting of buffering agents, pH-adjusting agents, and preservatives.
  • Citric acid anhydrous (0,04 - 0.16%) and sodium citrate dehydrate (0,23 - 0.69%) are used as buffers, whereas benzalkonium chloride (0.001 - 0.015%) as preservative Al of these compounds in units of % w/v
  • the pH ranges from about 5.0 to about 7,0, The ranges and values selected from 5.0, 5,1, 5,2, 5.3, 5.6, 5.7, 5.8, 5,9, 6,0, 6, 1, 6.2, 63, 6.4, 6.5, 6.6, 6,7, 6.8, 6,9 and 7.0 may be selected as preferred pHs for the buffered solution.
  • the viscosity of the suspension may range from about 25.0 to about 45.0 mPas.
  • the ranges and values selected from 25.1 , 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26.0 through 45.0 in the same progression may be selected as preferred viscosities.
  • Table 3 supra provides the most preferred for two dilutions of the liposoma I formulation.
  • compositions of the present invention may be prepared by conventional methods of preparing pharmaceutical suspension compositions.
  • the drug PED
  • PEG-12 polyethyleneglycol
  • ethyl alcohol a lipid mixture containing polyethyleneglycol (PEG-12) glyceryl dimyristate and ethyl alcohol.
  • An aqueous mixture having grade 2 purified water, polyethylene glycol (15)-hydroxystearate (KolliphorHS 15), citric acid anhydrous, sodium citrate dehydrate and benzalkonium chloride was commingled in a flask and set aside for compounding. The water mixture is gently added to the lipid mixture to obtain the finalformulation.
  • the invention further comprises a liposomal formulation of perfenidone which comprises perfenidone and a liposome.
  • the preferred liposome is a thermodynamically stable liposome which is disclosed in U.S. Pat. No. 6,958,160 which is hereby incorporated
  • SUBSTITUTE SHEET (RULE 26) by reference in its entirety.
  • liposomal formulations are suitable to treat any disease or condition that is treatable by perfenidorie. This includes pulmonary diseases such as idiopathic pulmonary edema.
  • pirfenidone-loaded liposomes formulation shown below is representative of the compositions of the present invention.
  • SUBSTITUTE SHEET (RULE 26)
  • the formulations shown in the EXAMPLES I and 2 (PL 0.1% and PL 0.02%, respectively) were prepared and subjected to characterization. Firstly, a microscopic characterization of PL formulation was carried out. Morphology of PFD in aqueous solution, in ethanolic solution and in liposomes was explored through scanning election microscopy (SEM) and transmission electron microscopy (TEM). Aqueous and ethanolic solutions of PED were prepared by adding ultrapure water or ethanol to the required quantity of PFD crystals to achieve the concentrations of 0.1%.
  • TESCAN MIRA3 EMU FE-SEM device was used (Tescan Orsay Holding, a.s., Bmo-Kohoutovice, Czech Republic), while for TEM studies a .
  • IEOL JEM- 1010 electron microscope was used (Jeol US A. Peabody, MA, USA).
  • PFD-loaded liposome (PL) formulations revealed interesting findings (FIG. 1 ).
  • PFD crystal structure is altered when in solution with ethanol, whereas it is completely dissolved by the liposomal formulation, as observed by SEM.
  • TEM images revealed that PFD crystal size was smaller in ethanol solution than in aqueous solution, and PFD crystals were seen inside liposomes.
  • PIG. 1. shows liposomal encapsulation of pirfenidone.
  • A SEM shows that PFD crystal shape is modified by ethanolic solution, whereas liposomal formulation completely dissolves it.
  • B TEM images reveal that PED crystal size is reduced by ethanol solution, compared with aqueous solu-tion while PFD crystals are observed inside liposomes.
  • C LEM images of liposomes containing PFD crystals are presented in different magnifications.
  • PFD pirfenidone
  • SEM scanning electron microscopy
  • TEM transmission electron microscopy.
  • the average particle size and Pdl of diluted PL formulations was 263 ⁇ 10 nm for 0,1% liposomes (1/200 v/v) and 0.37 ⁇ 0.04 in 1 mM PBS buffer, respectively.
  • the average particle size (256 ⁇ 2.6 nm) and Pdl (0.28 ⁇ 0.01 ) recorded were slightly lower, both with a uniform sample distribution.
  • the average particle size was 214 ⁇ 2.8 nm, with a Pdl value of 0.29 ⁇ 0.03 in PBS, while in ddH2O the particle size was 253 ⁇ 5.0 nm, with a Pdl value of 0.35 ⁇ 0.01.
  • the average zeta potential measured in PBS buffer was -20.4 ⁇ 0.1 and -20.9 ⁇ 0.7 mV for 0.1% and 0.02% liposomal formulations, respectively. Both PL formulations had a negative surface charge. It is important to emphasize that, although negative and neutral charged liposomes in ocular systems are easily drained from the precorneal area due to the negative charge of the cornea! epithelium [ 18], the viscosity of PL samples at 33 °C (discussed in the next section) was similar to a soft gel. Therefore, it is expected that this characteristic increases the residence time of the formulation at the ocular surface, and its bioavailability in consequence. [19]
  • SUBSTITUTE SHEET (RULE 26) proved that a maximum increase of penetration through the cornea by an eye drop solution takes place when viscosity falls into the range of 15 to 150 mPa s.
  • the viscosity at a shear rate of 100 s ⁇ l for 0.02% PL and 0.1% PL at 33 °C was 42.2 and 32.9 mPa's, respectively.
  • Osmolarity values were similar for both PL formulations (•'•100 mmol/kg). A substance is considered non-irritating to the eye when the osmolarity values fall within 205 and 684 mmol/L.
  • hypotonic formulations can be found in dry eye formulations and no adverse reactions to hypotonic solutions have been observed.
  • the claimed invention thus comprises a perfenidone liposomal formulation having at least one of the variables shown in Table 3 below including, without limitation, the pH range shown there and as further described herein as well as the viscosity range and specific pHs and viscosities for PL 0.1% and PL 0.02% compositions.
  • the formulations shown in Tables 1 and 2 may be modified accordingly with different pH adjusters (buffers), surfactants, absorption enhancers, tonicity agents and preservatives.
  • SUBSTITUTE SHEET (RULE 26) 0.5 M NaOH was placed on the cornea (right eye) for 30 s and the eye was then washed immediately with abundant sterile saline solution. Then, animals were assigned to 7 groups (5 mice per group): (1) PBS treated eyes (PBS), (2) Empty liposome treated eyes (EL), (3) Dexame-thasone treated eyes (DEX), (4) PFD treated eyes with PFD in aqueous solution at 0.02% (0.02% PFD), (5) PFD treated eyes with PFD in aqueous solution at 0.1 % (0.1% PFD), [6] PFD treated eyes with PFD in liposomes at 0.02% (0.02% PL) and (7) PFD treated eyes with PFD in liposomes at 0.1 % (0.1% PL). Additionally, 5 mice with non-burned eyes (NB) were used as healthy controls.
  • PBS PBS treated eyes
  • EL Empty liposome treated eyes
  • DEX Dexame-th
  • Edema was measured by quantifying blank pixels between collagen fibers of the corneal stroma, which correspond to the areas with edema. Subsequently, all colored pixels that make up the corneal tissue were quantified. The percentage of empty pixels within the cornea corresponds to tissue edema. Inflammation was assessed by manually identifying and measuring areas with inflammatory infiltrate of mononuclear and polymorphonuclear cells, which are distinguishable from the spindle morphology of comeal fibroblasts. The percentage of area with inflammatory infiltrate within the entire cornea corresponds to tissue inflammation.
  • Corneal thickness was measured at the central cornea using Image) software (http://imagej.nih.gov/ij/index.html accessed on 11 March 2021 ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).
  • SUBSTITUTE SHEET (RULE 26) thickness of about 100 gm in the PBS group compared with non-burned (NB) corneas (p ⁇ 0.01), meanwhile treatment groups reduced this thickening by about 50 gin compared with the PBS group.
  • NB non-burned
  • the PBS group had an evident increase in corneal edema compared with the NB group (p ⁇ 0.01).
  • treatments including dexamethasone, PFD and PL showed a significant decrease in corneal edema escalation.
  • FIG. 2 provides a comparison of comeal haze induced by alkali burn after 15 days of treatment.
  • Repre-sehtaiive photographs from the different experimental groups are presented and are as follow;
  • (A) non-burned eye
  • (B) PBS treated eye
  • (C) empty liposome treated eye
  • (D) dexamethasone treated eye
  • (E) 0.02% PFD treated eye
  • G 0.02% PL treated eye
  • H 0.1% PL treated eye.
  • PFD (E and F) and PL (G and H) treated eyes have a greater reduction in haze ex-tension and density than those eyes treated with PBS (B), empty liposomes (C) and dexame-thasone (D).
  • PBS phosphate buffered saline
  • PFD pirfenidone
  • PL pirfenidone-loaded liposome.
  • [0034] 3 shows valuation of corneal thickness, edema and inflammation in corneal tissue.
  • A Repre-sentative microphotographs from the different experimental groups are presented and are as fol-lows A. non-burned eye (NB), B. PBS treated eye (PBS), C. empty liposome treated eye (EL), D. dexamethasone treated eye (DEX), E. 0.02% PFD treated eye, F. 0.1% PFD treated eye, G. 0.02% PFD-loaded liposomes (PLs) treated eye and H. 0.1% PL treated eye.
  • B Quantitative analysis of corneal thickness, edema and corneal inflammation.
  • corneal tissue slides were used for immunofluorescence analysis to identify fibrosis marks. Briefly, corneal tissue slides were deparaffinized in xylene, rehydrated in a series of graded alcohol, boiled in 10 mM sodium citrate buffer pH 6.0 for 10 min, cooled and washed with distilled water. Slides were blocked and pemieabilized using blocking buffer (5% albumin, 0.1% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA) for 1 h, and incubated with anti-aSMA, anti-TGF-p and anti-Interleiikin- Ip (an-ti-ILl p) primary antibodies (Cell Signaling) at 4 °C overnight.
  • blocking buffer 5% albumin, 0.1% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA
  • TGF-p is one of the most important pro-fibrogenic cytokines; an increased expression of this cytokine induces myofibroblast transformation and collagen I secretion.
  • ILl-p a pro-inflammatory cytokine secreted after corneal damage, regulates myofibroblast presence by inducing apoptosis of resident fibroblasts.
  • DEX dexamethasone
  • FIG. 4 shows a comparison of aSMA, TGF-fJ and IL-1 p expression in comeal tissue among different treatments by immunofluorescence.
  • A PFD suppresses uSMA in a dose-dependent manner, and liposomes improve its effect. Representative immunofluorescence images obtained by confocal microscopy.
  • B Semi-quantification of aSMA protein, expression by fluorescence intensity analy-sis. Data expressed as mean ⁇ standard deviation.
  • C PFD suppresses TGF-p in a dose-dependent manner and liposomes improve this effect.
  • PFD restores normal epithelial location of IL- 1 (1 Nonburned eyes (NB), PBS treated eyes (PBS), empty liposome treated eyes (EL), dexamethasone treated eyes (DEX), 0.02% PFD treated eyes, 0.1% PFD treated eyes, 0.02% PFD- loaded liposome (PL) treated eyes and 0.1% PL treated eyes.
  • aSMA smooth muscle actin
  • TGF-p transforming growth factor-p
  • IL- ip interleukin- 1(5; DA.PI, 4 f ,6-diamidino-2 ⁇ phenylindole
  • NB non-burned
  • PBS phosphate-buflered saline
  • EL empty liposome
  • DEX dexamethasone
  • PFD pirfenidone
  • PL pirfenidone-loaded liposome.
  • PFD shows a notorious anti-fibrotic and antiinflammatory effects in corneal alkali burns. Moreover, the use of liposomes remarkably improved these effects. Reduction of corneal haze (recovery of corneal transparency), edema, fibrosis, as well as the decline expression of TGF-p are improved by PFD-Ioaded liposomes.
  • the corneal haze or corneal fibrosis induced by infection, trauma, chemicals or surgery such as PRK (photbrefractive keratectomy), LASER (laser epithelial keratomileusis), and epi-LASIK (epithelial laser in-site keratomileusis) can be avoided or treated by pirfenidone-loaded liposomes.
  • PRK photbrefractive keratectomy
  • LASER laser epithelial keratomileusis
  • epi-LASIK epi-LASIK
  • SUBSTITUTE SHEET (RULE 26) haze (reduction of fibrosis and recovery of corneal transparency).
  • a method of treating patients involves administering 1 to 6 drops per day of the topical ophthalmic formulation comprising pirfenidone in a concentration range of about 0.1 mg/ml to 20 mg/ml [0.01 to 2.00% (w/v)], in a thermodynamically stable liposome formulation which optionally comprises at.
  • At least one excipient selected from the group consisting of a surfactant, absorption enhancer, buffer, preservative, solvent and tonicity agent (pirfenidone-foaded liposomes formulation or PL), in the eye wherein the patient exhibits corneal fibrosis or haze secondary to corneal infections, trauma, chemical injury, or surgery such as PRK (photorefractive keratectomy), LASER (laser epithelial keratomileusis), and epi-LASIK (epithelial laser in- situ keratomileusis), among others corneal pathologies.
  • PRK photorefractive keratectomy
  • LASER laser epithelial keratomileusis
  • epi-LASIK epi-LASIK
  • Each drop (50 pl) of the topical ophthalmic pirfeiiidone-loaded liposomes formulation (PL) contains from about 5 to about 1000 pg of pirfenidone.
  • the dosing amount depends upon the severity or relative severity of the fibrosis related condition in the eye.
  • the drops are administered on a daily basis until the condition approves.
  • the dosage range can be titrated upward if treatment at a particular range is not working and provided there are no evident side effects of the drug.

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Abstract

The disclosed invention relates to topical liposomal formulations comprising perfenidone and thermodynamically stable liposomes. The topical ophthalmic formulations are useful to treat certain ophthalmic disorders including fibrosis related diseases, disorders and conditions. Patients having corneal haze or corneal fibrosis secondary to corneal infections, trauma or chemical injury can be treated with the topical ophthalmic liposomal formulation of perfenidone.

Description

TOPIC AL OPHTHALMIC PIRFENIDONE-LOADED LIPOSOMES TO INCREASE THE THERAPEUTIC ACTIVITY OF THE DRUG IN CORNEA AND USEFUL FOR PREVENTION OR THERAPY OF CORNEAL HAZE
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/340,043, filed May 10, 2022, and U.S. Provisional Application No. 63/445,453, filed February 14, 2023, each of which is herein incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] The cornea is the most important refractive structure in the eye Loss of corneal transparency is the second leading cause of blindness worldwide, only after cataracts. [1] Corneal transparency depends mainly on stromal structure. Corneal stroma is composed of about 90% of extracellular matrix (ECM), principally collagen. This collagen is regularly packed into small diameter (•~25 nm) fibrils packed as lamellae, this arrangement minimizes light scatering and permits transparency. Corneal collagen fibrils are composed mostly of collagen [ and lesser amounts of collagen V, as well as some proteoglycans. These features allow collagen fibrils to maintain their diameter and separation. [2]
[0003] C iorneal injury induced by infection, trauma., chemicals or surgery such as PRK (photorefractive keratectomy), LASER (laser epithelial keratomileusis), and epi-LASIK (epithelial laser in-situ keratomileusis) triggers a complex series of processes, known as wound healing response, whose purpose is to restore the normal structure and function of the cornea. However, an abnormal wound healing response leads to loss of corneal transparency or haze development due to myofibroblast transformation, the crucial factor that leads to corneal stromal fibrosis. [3] The corneal fibrosis or haze conditions a cloudy, opaque appearance of the cornea, that obstructs the vision and induces corneal blindness. [3]
[0004] Myofibroblasts, characterized by aS MA expression, guide the corneal haze phenomenon. These cells are highly contractile able to synthesize and deposit great amounts of ECM components, especially collagen I and II I, changing collagen diameter fibrils and disarranging stromal structure of the cornea. [4,5] The cornea does not have myofibroblasts present under normal conditions, they derive from resident keratocytes after injury in response to TGF-p1 released by epithelial cells. [6]
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SUBSTITUTE SHEET ( RULE 26) [0005] Perfenidone (“PFD1’) has shown anti-fibrotic effects reducing TGF-
Figure imgf000004_0001
expression and ECM deposit in different organs. [7] Different precllnical studies have exhibited PFD therapeutic potential in the treatment of fibrotic and proliferative eye disorders including corneal chemical burns [8], conjunctival scarring [9-11], and choroidal neovascularization (CNV) [I 2] .
[0006] Due to dynamic barriers in the precorneal area such as tear turnover and lacrimal drainage via the nasolacrimal drainage system, the water-based PFD eye drops exhibited a short half-life and poor bioavailability. [13] Consequently, several strategies have been developed to increase PFD bioavailability in the cornea and conjunctiva to achieve a needed therapeutic effect in conreal haze. One of them is the use of viscosity-enhancers such as hydroxypropyl methylcellulose to extend the ocular residence time of PFD. This approach has been recognized to prolong the residence time from 10 min to more than 20 min resulting in increased PFD levels in cornea, conjunctiva, sclera and aqueous humor until 90 min after topical administration. [14] Additionally, the use of nanotechnology is a promising strategy that, has proven to increase the bioavai lability and the therapeutic effect of PFD. For example, PFD-loaded poly iactic-co-glycolic acid (PLGA) nanoparticles significantly reduced collagen 1 level, corneal haze and the time for cornea! re-epithelialization following alkali burn in rats, as opposite to free pirfenidone. [15] Thus, a PFD formulation increasing comeal bioavailability will have the best chance to succeed.
[0007] Liposomes, small vesicles (approximate size of 30-1000 nm) which are prepared with phospholipids, offer an efficient option for PFD drug delivery. Liposomes have the advantage to be biodegradable with a relatively non-toxic behavior, which enhances drug permeation by binding to the corneal surface. [16] We anticipate that due to these nanoparticles, PFD penetration will increase the time of interaction with the ocular surface and would improve the therapeutic effect of PFD.
SUMMARY OF THE INVENTION
[0008] The topical ophthalmic compositions of the present invention comprise a combination of PFD as the active pharmaceutical ingredient and a thennodynamically stable liposome. Additional components such as surfactants (ionic or uon-ionic), penetration enhancers, buffers, tonicity agents and solvents suitable for use in ophthalmic treatment can be added as necessary- . The invention comprises use of a topical ophthalmic formulation
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SUBSTITUTE SHEET ( RULE 26) which comprises a combination of PFD and a liposome in the treatment of an ophthalmic disease in which corneal haze is a related symptom or condition. In a preferred embodiment, the liposome comprises a diacylglycerol-PEG compound. The PEGylated compounds preferably comprise PEG-12-GDM or PEG- 12 GDO (glycerol dioleate) Preferred formulations comprise PFD and. polyethyleneglycol (PEG-12) glyceryl dimyristate as the diacylglyverol-PEG compound, ethyl alcohol as organic solvent for liposomes generation, kolliphor IIS 15 as penetration enhancer, citric acid anhydrous and sodium citrate dehydrate as buffers, benzalkonium chloride as preservative, and grade 2 purified water as solvent.
[0009] The ophthalmic formulations of the present invention have been found to increase the therapeutic activity of topically administered PED and are thus useful for prevention or treatment of corneal haze
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows liposomal encapsulation of pirfenidone crystals.
[0011] FIG. 2 shows a comparison of corneal haze effects induced by alkali and recovery or lack thereof after 15 days of tr eatment with various reagents or drugs versus an eye which was not alkali treated (FIG 2 A). The data dearly shows that the pirfenidone-loaded liposomes (PL) have a greater reduction in haze extension and density than those eyes treated with PBS (FIG. 2B), empty liposomes (FIG. 2C) and dexamethasone (FIG. 2D).
[0012] FIG. 3 shows the evaluation of corneal thickness, edema and inflammation in corneal tissue (A) Repre-sentative microphotographs from the different experimental groups are presented and are as fol-lows A. non-burned eye (NB), B. PBS treated eye (PBS), C. empty liposome treated eye (EL), D. dexamethasone treated eye (DEX), E. 0.02% PFD treated eye, F. 0.1 % PFD treated eye, G. 0.02% PFD-loaded liposomes (PLs) treated eye and II. 0. 1 % PL treated eye. (B) Quantitative analysis of corneal thickness, edema and corneal inflammation. When comparing PBS group with NB group, a significant increase of corneal thickness, edema degree and inflammatory infiltrated cells quantity is evident. PFD decreased edema and reduced corneal thickness. Additionally, PL had a more significant reduction of corneal inflammation (p < 0.01) than PFD at matched concentrations (p < 0.05). Data expressed as mean ± standard deviation. NB, non-bumed; PBS, phos-phate-buffered saline: EL, empty liposome; DEX, dexamethasone; PFD, pirfenidone; PL, pirfenidone-loaded liposome. * p < 0.05 vs. PBS group; ** p < 0.01 vs. PBS group; *** p < 0.001 vs. PBS group.
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SUBSTITUTE SHEET ( RULE 26) [0013] FIG. 4 shows a comparison of aSMA, TGF-P and IL- ip expression in corneal tissue among different treatments by immunoflaorescence. (Aj PFD suppresses uSMA in a dosedependent manner, and liposomes improve its effect. Representative immunofluorescence images obtained by confocal microscopy. (B) Semi-quantification of aSM.A protein expression by fluorescence intensity analy-sis. Data expressed as mean standard deviation. (C) PFD suppresses TGF-p in a dose-dependent manner and liposomes improve this effect. Meanwhile, PFD restores normal epithelial location of IL- 1(1 Non-bumed eyes (NB), PBS treated eyes (PBS), empty liposome treated eyes (EL), dexame-thasone treated eyes (DEX), 0.02% PFD treated eyes, 0.1% PFD treated eyes, 0.02% PFD-loaded liposome (PL) treated eyes and 0.1% PL treated eyes. aSMA, a smooth muscle actin; TGF-(f transforming growth factor- IL-1 [3, interleukin-
Figure imgf000006_0001
DAPI, 4',6-diamidino-2-pheny1indole; NB, non-bumed; PBS, phosphate-buffered saline; EL, empty liposome; DEX, dexamethasone; PFD, pirfenidone; PL, pirfenidone-loaded liposome, * p < 0.05 vs. PBS group; ** p < 0.01 vs. PBS group; *** p <0.001 vs. P BS group. Comparison of corneal haze inducedby alkali bum after 15 days of treatment. Representative photographs from the different experimental groups are presented and are as follow; (A) Non-bumed eye, (B) PBS treated eye, (C) empty liposome treated eye, (D) dexa-metliasone treated eye, (E) 0.02% PFD treated eye, (F) 0.1 % PFD treated eye, (G) 0.02% PL treated eye and (Id) 0.1 % PL treated eye. PFD (E and F) and PL (G and H) treated eyes have a greater re-duction in haze extension and density than those eyes treated with PBS (B), empty' liposomes (C) and dexamethasone (D). PBS, phosphate buffered saline; PFD, pirfenidone; PL, pirfenidone-loaded liposome. Scale bar 10 (um for all panels.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Ingredient concentrations are presented in units of % weight/volume (% w/v) or % volume/volume (% v/v).
[0015] The compositions of the present invention contain a pharmaceutically effective amount of pirfenidone (PFD). The concentration of PFD in liposomes formulations ranges from 0.01 to 2.00% (w/v). PFD (5-rneihyI-l-phenylpyridin-2( 1 ld)-one is a known. pyridinone with antifribroic, anti-inflammatoiy and antioxidant activity, with an empirical formula of CaHnNO and a molecular weight of 185.22 Da. Polyethyleneglycol (PEG- 12) glyceryl dimyristate is used as structural constituent of liposomes in a concentration of 5-15% (w/v)
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SUBSTITUTE SHEET ( RULE 26) and ethyl alcohol is used as organic solvent for liposomes generation in a concentration of 0.7 to 2.1% (v%).
[0016] Besides, the liposomes formulation contains polyethylene glycol (15)- hydroxystearate or KolliphorHS 15 from 2.5 - 7.5% (w/v), as a potent non-ionic solubilizer and emulsifying agent, with low toxicity proposed to act as a permeability enhancer. KolliphorHS 15 promotes drug transport across cell membranes (increasing the endocytosis rate) and stimulates drug translocation through the paracellular route (affects actin organization on the cell cytoskeleton with the subsequent tight junction opening).
[0017] Additionally, the aqueous compositions of the present invention optionally comprise more excipients selected from the group consisting of buffering agents, pH-adjusting agents, and preservatives. Citric acid anhydrous (0,04 - 0.16%) and sodium citrate dehydrate (0,23 - 0.69%) are used as buffers, whereas benzalkonium chloride (0.001 - 0.015%) as preservative Al of these compounds in units of % w/v, In preferred embodiments, the pH ranges from about 5.0 to about 7,0, The ranges and values selected from 5.0, 5,1, 5,2, 5.3, 5.6, 5.7, 5.8, 5,9, 6,0, 6, 1, 6.2, 63, 6.4, 6.5, 6.6, 6,7, 6.8, 6,9 and 7.0 may be selected as preferred pHs for the buffered solution. The viscosity of the suspension may range from about 25.0 to about 45.0 mPas. The ranges and values selected from 25.1 , 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26.0 through 45.0 in the same progression may be selected as preferred viscosities. Table 3 supra provides the most preferred for two dilutions of the liposoma I formulation.
[0018] The compositions of the present invention may be prepared by conventional methods of preparing pharmaceutical suspension compositions. According to the preferred method, the drug (PED) is first added to a lipid mixture containing polyethyleneglycol (PEG-12) glyceryl dimyristate and ethyl alcohol. An aqueous mixture having grade 2 purified water, polyethylene glycol (15)-hydroxystearate (KolliphorHS 15), citric acid anhydrous, sodium citrate dehydrate and benzalkonium chloride was commingled in a flask and set aside for compounding. The water mixture is gently added to the lipid mixture to obtain the finalformulation.
|(W19| The invention further comprises a liposomal formulation of perfenidone which comprises perfenidone and a liposome. The preferred liposome is a thermodynamically stable liposome which is disclosed in U.S. Pat. No. 6,958,160 which is hereby incorporated
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SUBSTITUTE SHEET ( RULE 26) by reference in its entirety. Such liposomal formulations are suitable to treat any disease or condition that is treatable by perfenidorie. This includes pulmonary diseases such as idiopathic pulmonary edema.
[0020] The following examples are intended to illustrate. but not limit, the present invention.
EXAMPLE 1.
[0021] The pirfenidone-loaded liposomes formulation (PFD-LF) shown below is representative of the compositions of the present invention.
Figure imgf000008_0001
EXAMPLE 2
[0022] 'The pirfenidone-laaded liposomes forniulation (PFD-LF) shown below is representative of the compositions of the present invention.
Figure imgf000008_0002
SUBSTITUTE SHEET ( RULE 26) [0023] The formulations shown in the EXAMPLES I and 2 (PL 0.1% and PL 0.02%, respectively) were prepared and subjected to characterization. Firstly, a microscopic characterization of PL formulation was carried out.. Morphology of PFD in aqueous solution, in ethanolic solution and in liposomes was explored through scanning election microscopy (SEM) and transmission electron microscopy (TEM). Aqueous and ethanolic solutions of PED were prepared by adding ultrapure water or ethanol to the required quantity of PFD crystals to achieve the concentrations of 0.1%. For SEM analysis, a TESCAN MIRA3 EMU FE-SEM device was used (Tescan Orsay Holding, a.s., Bmo-Kohoutovice, Czech Republic), while for TEM studies a . IEOL JEM- 1010 electron microscope was used (Jeol US A. Peabody, MA, USA). SEM samples were kept at “4 °C before being mounted onto stubs and gold- coated using a Denton Vacuum Desk 11 sputter coater (SPI supplies, West Chester, PA, USA), TEM samples were previously treated using phosphotungstic acid as a negative staining agent in a 1 :1 dilution (v/v) and were de-posited onto FF 300 square mesh copper grids (Electron Microscopy Sciences, Fort Washington, PA, USA) for observation. Manual counting and measurement, of particles were performed using SEM micrographs at a view field of 63.6 mm to calculate the size and distribution of PFD-loaded liposome (PL) formulations (PLs). SEM and TEM examination of PFD-loaded liposome (PL) formulations revealed interesting findings (FIG. 1 ). PFD crystal structure is altered when in solution with ethanol, whereas it is completely dissolved by the liposomal formulation, as observed by SEM. TEM images revealed that PFD crystal size was smaller in ethanol solution than in aqueous solution, and PFD crystals were seen inside liposomes.
[0024] PIG. 1. shows liposomal encapsulation of pirfenidone. SEM and TEM images of PFD crystals in lip-osomes formulation, ethanolic and aqueous solutions. (A) SEM shows that PFD crystal shape is modified by ethanolic solution, whereas liposomal formulation completely dissolves it. (B) TEM images reveal that PED crystal size is reduced by ethanol solution, compared with aqueous solu-tion while PFD crystals are observed inside liposomes. (C) LEM images of liposomes containing PFD crystals are presented in different magnifications. PFD, pirfenidone; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
SUBSTITUTE SHEET ( RULE 26) [0025] The physicochemical properties, size distribution., and zeta potential of different PL formulations and diluted samples were detemiined at 33 °C, which is the ocular surface temperature. [17] The osmolality of 10 pL of sample was measured at room temperature using a Vapro 5600 vapor pressure osmometer (ELITechGroup, Paris, France). The viscosity of PL formulations was measured at a shear rate of 100 s- 1 using a stress-controlled AR-G2 rheometer (TA Instraments, New Castle, DE, USA) with a 60 mm cone-and-plate geometry of 2° angle. pH was monitored using an Orion Star A210 (Thermo Fisher Scientific, Waltham, MA, USA). Intensity-sized distributions, polydispersity index (Pdl), and zeta potential values of diluted liposomal formulations in double-distilled water (ddH2O) or 1 mM PBS buffer (pH 7.4) were determined via dynamic light scattering (DLS) in a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK), DLS measurements were performed using a dispersant refractive index of 1.33 and an ab-sorption index of 0.01, Zeta potential values were obtained using the same diluted samples in a disposable capillary cell (DTS1070). All experiments were performed in triplicate.
[0026] The average particle size and Pdl of diluted PL formulations was 263 ± 10 nm for 0,1% liposomes (1/200 v/v) and 0.37 ± 0.04 in 1 mM PBS buffer, respectively. In ddH2O, the average particle size (256 ± 2.6 nm) and Pdl (0.28 ± 0.01 ) recorded were slightly lower, both with a uniform sample distribution. For 0.02% liposomes, the average particle size was 214 ± 2.8 nm, with a Pdl value of 0.29 ± 0.03 in PBS, while in ddH2O the particle size was 253 ± 5.0 nm, with a Pdl value of 0.35 ± 0.01. The average zeta potential measured in PBS buffer was -20.4 ± 0.1 and -20.9 ± 0.7 mV for 0.1% and 0.02% liposomal formulations, respectively. Both PL formulations had a negative surface charge. It is important to emphasize that, although negative and neutral charged liposomes in ocular systems are easily drained from the precorneal area due to the negative charge of the cornea! epithelium [ 18], the viscosity of PL samples at 33 °C (discussed in the next section) was similar to a soft gel. Therefore, it is expected that this characteristic increases the residence time of the formulation at the ocular surface, and its bioavailability in consequence. [19]
[0027] In ddH2O, the average zeta potential for liposomes 0. 1% and 0.02% was -26.6 ± 0.7 and -19,4 ± 0.4, respectively.
[0028] The physiologic human tear pH range is located between 6.5 to 7.6, but pH values in the range of 4-8 are well tolerated by the eye. Regarding viscosity, the results obtained herein
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SUBSTITUTE SHEET ( RULE 26) proved that a maximum increase of penetration through the cornea by an eye drop solution takes place when viscosity falls into the range of 15 to 150 mPa s. [20] The viscosity at a shear rate of 100 s~l for 0.02% PL and 0.1% PL at 33 °C was 42.2 and 32.9 mPa's, respectively. Osmolarity values were similar for both PL formulations (•'•100 mmol/kg). A substance is considered non-irritating to the eye when the osmolarity values fall within 205 and 684 mmol/L. However, hypotonic formulations can be found in dry eye formulations and no adverse reactions to hypotonic solutions have been observed. Complete size distribution, zeta potential, and physicochemical characterization of PL are shown in Table 3. The claimed invention thus comprises a perfenidone liposomal formulation having at least one of the variables shown in Table 3 below including, without limitation, the pH range shown there and as further described herein as well as the viscosity range and specific pHs and viscosities for PL 0.1% and PL 0.02% compositions. The formulations shown in Tables 1 and 2 may be modified accordingly with different pH adjusters (buffers), surfactants, absorption enhancers, tonicity agents and preservatives.
Figure imgf000011_0001
[0029] Posteriorly, an evaluation of the therapeutic activity of PLs in a. mice model of corneal alkali burn was perfouned. To induce the corneal alkali bum mice model, the right cornea of thirty-five male C57BL/6 mice was exposed to NaOH (Sigma-Aldrich, St. Louis, MO, USA), First, animals were anesthetized using intraperitoneal Ketamine/Xylazine (40 mg/kg/5 mg/kg, PiSA Farmaceutica, Guadalajara, Mexico) and 2 tetracaine drops to the experimental eye. Subsequently, filter paper of approximately 2 mm of diameter soaked with
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SUBSTITUTE SHEET ( RULE 26) 0.5 M NaOH was placed on the cornea (right eye) for 30 s and the eye was then washed immediately with abundant sterile saline solution. Then, animals were assigned to 7 groups (5 mice per group): (1) PBS treated eyes (PBS), (2) Empty liposome treated eyes (EL), (3) Dexame-thasone treated eyes (DEX), (4) PFD treated eyes with PFD in aqueous solution at 0.02% (0.02% PFD), (5) PFD treated eyes with PFD in aqueous solution at 0.1 % (0.1% PFD), [6] PFD treated eyes with PFD in liposomes at 0.02% (0.02% PL) and (7) PFD treated eyes with PFD in liposomes at 0.1 % (0.1% PL). Additionally, 5 mice with non-burned eyes (NB) were used as healthy controls.
[0030] Treatments with PFD in aqueous solution, PFD contained in liposomes (0.02% and 0.1%), as well as empty liposomes and dexamethasone ( 1%), were instilled 4 times per day for 15 days in the right cornea. At the end of the treatment, all mice were sacrificed by an overdose of pentobarbital (100 mg/kg, PiSA Fannaceutica, Guadalajara, Mexico). Whole eyes were fixed in 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA) overnight and later, parallel sections were made, paraffin blocks were prepared and multiple slides were obtained from each block. Slides were stained by routine hematoxylin and eosin (H&E) staining. These slides were used to evaluate inflammation, edema and corneal thickness.
[0031] Single-blind analysis of edema, inflammation and corneal thickness was performed using Image Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA). Edema was measured by quantifying blank pixels between collagen fibers of the corneal stroma, which correspond to the areas with edema. Subsequently, all colored pixels that make up the corneal tissue were quantified. The percentage of empty pixels within the cornea corresponds to tissue edema. Inflammation was assessed by manually identifying and measuring areas with inflammatory infiltrate of mononuclear and polymorphonuclear cells, which are distinguishable from the spindle morphology of comeal fibroblasts. The percentage of area with inflammatory infiltrate within the entire cornea corresponds to tissue inflammation. Corneal thickness was measured at the central cornea using Image) software (http://imagej.nih.gov/ij/index.html accessed on 11 March 2021 ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).
[0032] After 15 days of trea tment of the mice model of corneal chemical injury, we observed a reduction in haze size and density in the treated eyes which was more evident in those treated with PFD (FIG. 2). Histology analysis (FIG. 3) showed an increase in corneal
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SUBSTITUTE SHEET ( RULE 26) thickness of about 100 gm in the PBS group compared with non-burned (NB) corneas (p < 0.01), meanwhile treatment groups reduced this thickening by about 50 gin compared with the PBS group. Additionally, we evaluated the degree of edema and infiltration of inflammatory cells in the corneal stroma. We observed that the PBS group had an evident increase in corneal edema compared with the NB group (p < 0.01). However, treatments including dexamethasone, PFD and PL showed a significant decrease in corneal edema escalation. Finally, with the purpose of evaluating inflammation, we determined the inflammatory cells infiltrated in the corneal tissue. An evident increase of inflammatory cells in corneal tissue of the PBS group was observed when compared with the NB group (p < 0.001), nevertheless, PFD (p < 0.05) and PL (p < 0.01 ) showed a significant reduction of inflammatory infiltrated cells in corneal stroma. These results show PDF anti-inflammatory effect and its biologic action enhancement by liposomes.
[0033] Thus, FIG. 2 provides a comparison of comeal haze induced by alkali burn after 15 days of treatment. Repre-sehtaiive photographs from the different experimental groups are presented and are as follow; (A) non-burned eye, (B) PBS treated eye, (C) empty liposome treated eye, (D) dexamethasone treated eye, (E) 0.02% PFD treated eye, (F) 0.1% PFD treated eye, (G) 0.02% PL treated eye and (H) 0.1% PL treated eye. PFD (E and F) and PL (G and H) treated eyes have a greater reduction in haze ex-tension and density than those eyes treated with PBS (B), empty liposomes (C) and dexame-thasone (D). PBS, phosphate buffered saline; PFD, pirfenidone; PL, pirfenidone-loaded liposome.
[0034] 3 shows valuation of corneal thickness, edema and inflammation in corneal tissue. (A) Repre-sentative microphotographs from the different experimental groups are presented and are as fol-lows A. non-burned eye (NB), B. PBS treated eye (PBS), C. empty liposome treated eye (EL), D. dexamethasone treated eye (DEX), E. 0.02% PFD treated eye, F. 0.1% PFD treated eye, G. 0.02% PFD-loaded liposomes (PLs) treated eye and H. 0.1% PL treated eye. (B) Quantitative analysis of corneal thickness, edema and corneal inflammation. When comparing PBS group with NB group, a significant increase of comeal thickness, edema degree and inflammatory infiltrated cells quantity is evident. PFD decreased edema and reduced corneal thickness. Additionally, PL had a more significant reduction of corneal inflammation (p < 0.01) than PED at matched concentrations (p < 0.05). Data expressed as mean ± standard deviation. NB, non-burned; PBS, phosphate-buffered saline; EL, empty
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SUBSTITUTE SHEET ( RULE 26) liposome; DEX, dexametliasone; PFDS pirfenidone; PL, pirfenidone-loaded liposome. * p < 0.05 vs. PBS group; ** p < 0.01 vs. PBS group; *** p < 0.001 vs. PBS group.
[0035] Additional corneal tissue slides were used for immunofluorescence analysis to identify fibrosis marks. Briefly, corneal tissue slides were deparaffinized in xylene, rehydrated in a series of graded alcohol, boiled in 10 mM sodium citrate buffer pH 6.0 for 10 min, cooled and washed with distilled water. Slides were blocked and pemieabilized using blocking buffer (5% albumin, 0.1% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA) for 1 h, and incubated with anti-aSMA, anti-TGF-p and anti-Interleiikin- Ip (an-ti-ILl p) primary antibodies (Cell Signaling) at 4 °C overnight. After 3 washes with PBS, the slides were incubated with a fluorescent secondary antibody (Alexa Fluor Goat anti-rabbit IgG or antimouse IgG. Cell Signaling) for 2 h. Nuclei were stained with DAPI. Samples were examined under a confocal microscope ( Leica Microsystems),
[0036] We observed that PFD and PLs Suppress aSMA expression in comeal tissue. Increased expression of aSMA is a dear sign of a fibrogenic process, consequently, aSMA is one of the main profibrotic biomarkers, Immunofiuorescence image analysis shows that NB corneas do not express aSMA. Meanwhile, the PBS group shows an evident rise in aSMA expression. However, PFD showed a significant suppression in aSMA expression in a dose-dependent manner. Additionally, PL improved the effect of PFD, inducing a further decrease of aSMA expression (FIG. 4A, B).
[0037] On the other hand, PFD and PLs suppress TGF-P expression in corneal tissue and restores normal IL-ip expression. TGF-p is one of the most important pro-fibrogenic cytokines; an increased expression of this cytokine induces myofibroblast transformation and collagen I secretion. Besides, ILl-p, a pro-inflammatory cytokine secreted after corneal damage, regulates myofibroblast presence by inducing apoptosis of resident fibroblasts. In immunofluorescence images, we observed an increment of TGF-p expression in PBS, EL and dexamethasone (DEX) groups compared with NB group. Nevertheless, PFD suppressed TGF-p expression in a dose-dependent manner, while PL notoriously enhanced this effect at matched doses. On the other hand, IL-lp appeared to be confined to die interior of corneal epithelial cells of N B corneas. PBS, EL and DEX groups show loss of epithelial localization and expression of IL-lp, as well as presence in the corneal stroma. Noteworthy, PFD and PL restored expression and epithelial localization of IL- Ip (FIG. ■•!(' ).
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SUBSTITUTE SHEET ( RULE 26) [0038] Thus, FIG. 4 shows a comparison of aSMA, TGF-fJ and IL-1 p expression in comeal tissue among different treatments by immunofluorescence. (A) PFD suppresses uSMA in a dose-dependent manner, and liposomes improve its effect. Representative immunofluorescence images obtained by confocal microscopy. (B) Semi-quantification of aSMA protein, expression by fluorescence intensity analy-sis. Data expressed as mean ± standard deviation. (C) PFD suppresses TGF-p in a dose-dependent manner and liposomes improve this effect. Meanwhile, PFD restores normal epithelial location of IL- 1 (1 Nonburned eyes (NB), PBS treated eyes (PBS), empty liposome treated eyes (EL), dexamethasone treated eyes (DEX), 0.02% PFD treated eyes, 0.1% PFD treated eyes, 0.02% PFD- loaded liposome (PL) treated eyes and 0.1% PL treated eyes. aSMA, a smooth muscle actin; TGF-p, transforming growth factor-p; IL- ip, interleukin- 1(5; DA.PI, 4f,6-diamidino-2~ phenylindole; NB, non-burned; PBS, phosphate-buflered saline; EL, empty liposome; DEX, dexamethasone; PFD, pirfenidone; PL, pirfenidone-loaded liposome. * p < 0.05 vs. PBS group; ** p < 0.01 vs. PBS group; *** p < 0.001 vs. PBS group. Comparison of corneal haze induced by alkali bum after 15 days of treatment. Representative photographs from the different experimental groups are presented and are as follow; (A) Non-burned eye, (B) PBS treated eye, (C) empty liposome treated eye, ( D) dexa-methasone treated eye, (E) 0.02% PFD treated eye, (F) 0.1% PFD treated eye, (G) 0.02% PL treated eye and (H) 0.1% PL treated eye. PFD (E and F) and PL (G and H) treated eyes have a greater re-duction in haze extension and density than those eyes treated with PBS (B), empty liposomes (C) and dexamethasone (D). PBS, phosphate buffered saline; PFD, pirfenidone; PL, pirfeni done- loaded liposome. Scale bar 10 pm for all panels.
[0039] According to our findings, PFD shows a notorious anti-fibrotic and antiinflammatory effects in corneal alkali burns. Moreover, the use of liposomes remarkably improved these effects. Reduction of corneal haze (recovery of corneal transparency), edema, fibrosis, as well as the decline expression of TGF-p are improved by PFD-Ioaded liposomes. Therefore, the corneal haze or corneal fibrosis induced by infection, trauma, chemicals or surgery such as PRK (photbrefractive keratectomy), LASER (laser epithelial keratomileusis), and epi-LASIK (epithelial laser in-site keratomileusis) can be avoided or treated by pirfenidone-loaded liposomes. Besides, the pirfenidone-loaded liposomes is able to improve the visual acuity and visual contrast based in its therapeutic activity for co meal
SUBSTITUTE SHEET ( RULE 26) haze (reduction of fibrosis and recovery of corneal transparency). A method of treating patients involves administering 1 to 6 drops per day of the topical ophthalmic formulation comprising pirfenidone in a concentration range of about 0.1 mg/ml to 20 mg/ml [0.01 to 2.00% (w/v)], in a thermodynamically stable liposome formulation which optionally comprises at. least one excipient selected from the group consisting of a surfactant, absorption enhancer, buffer, preservative, solvent and tonicity agent (pirfenidone-foaded liposomes formulation or PL), in the eye wherein the patient exhibits corneal fibrosis or haze secondary to corneal infections, trauma, chemical injury, or surgery such as PRK (photorefractive keratectomy), LASER (laser epithelial keratomileusis), and epi-LASIK (epithelial laser in- situ keratomileusis), among others corneal pathologies. Each drop (50 pl) of the topical ophthalmic pirfeiiidone-loaded liposomes formulation (PL) contains from about 5 to about 1000 pg of pirfenidone. The dosing amount depends upon the severity or relative severity of the fibrosis related condition in the eye. The drops are administered on a daily basis until the condition approves. The dosage range can be titrated upward if treatment at a particular range is not working and provided there are no evident side effects of the drug.
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SUBSTITUTE SHEET ( RULE 26)

Claims

WHAT IS CLAIMED IS:
1. A topical ophthalmic formulation comprising pirfenidone, a thermodynamically stable liposome and. optionally, at least one excipient selected from the group consisting of a surfactant, absorption enhancer, buffer, preservative, solvent and tonicity agent.
2. The topical ophthalmic formulation according to claim 1 wherein the thermodynamically stable liposome comprises a diacylglycerol-PEG compound having a melting point below about 40 degrees C and the acyl chains are greater than or equal to about 14 carbons in length and the PEG chain has a molecular weight of between about 300 Daltons to 5000 Daltons.
3. The topical ophthalmic formulation according to claim 2 wherein the diacylglycerol- PEG compound is selected from the group consisting of PEG-12-GDM (polyethylene glycol 12-Glycerol Dimyristate) and PEG-12 GDO ( polyethylene glycol 12-glycerol dioleate).
4. The topical ophthalmic formulation according to claim 3 wherein the diacylglycerol- PEG compound is selected from PEG-12-GDM.
5. A topical ophthalmic formulation comprising; a) pirfenidone (PFD) from 0.01 to 2.00% (w/v). b) Polyethyleneglycol (PEG- 12) glyceryl dimyristate from 5-15% (w/v) c) Ethyl alcohol from 0.7 to 2.1% (v/v) d) Polyethylene glycol (15)-hydroxystearate (Kolliphor HS 15) 2.5 - 7.5% (w/v) e) Citric acid anhydrous from 0.04 ~ 0. 16% (w/v) f) Sodium citrate dehydrate from 0.23 - 0.69% (w/v) g) Benzalkonium chloride from 0.001 0,015% (w/v). h) Grade 2 purified water.
6. The topical ophthalmic formulation according to claims 1 -5 having a pH range of about. 5 to 7.
7. A method of treating a patient in need of treatment thereof with a topical ophthalmic formulation according to claims 1 -6, wherein the patient exhibits corneal fibrosis of haze secondary to corneal infections, trauma, chemical injury, or surgery such as PRK (photorefractive keratectomy), LASEK (laser epithelial keratomileusis), and epi-LASIK (epithelial laser in-situ keratomileusis), among others.
SUBSTITUTE SHEET ( RULE 26)
8. A method of improving visual outcome in a patient -suffering from impaired vision due to corneal fibrosis or injury comprising administering a topical ophthalmic formulation according to claims 1-5, wherein said treatment results in an improvement in the visual outcome selected from visual acuity and contrast sensitivity after PRK “photorefractive keratectomy”, LASEK. “laser epithelial keratomileusis”. and epi-L ASIK “epithelial laser in- situ keratomileusis” surgeries.
9. A method of treating a patient having corneal haze or corneal fibrosis secondary to corneal infections, trauma, or chemical injury comprising administering a pharmaceutically effective amount of a topical ophthalmic formulation according to claims 1.-5.
10. Use of a topical ophthalmic liposomal formulation comprising perfenidone in the treatment of an ophthalmic disease or condition selected from fibrosis-related ocular disease.
1.1. Use of a dtennodynamicaliy stable liposome formulation comprising perfenidone in the treatment of a fibrosis related disease or disorder.
SUBSTITUTE SHEET ( RULE 26)
PCT/US2023/021454 2022-05-10 2023-05-09 Topical ophthalmic pirfenidone-loaded liposomes to increase the therapeutic activity of the drug in cornea and useful for prevention or therapy of corneal haze WO2023220003A1 (en)

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