US20180221404A1 - Ophthalmic formulations comprising cooperative complexes of low- and high-molecular-weight hyaluronic acid - Google Patents

Ophthalmic formulations comprising cooperative complexes of low- and high-molecular-weight hyaluronic acid Download PDF

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US20180221404A1
US20180221404A1 US15/747,157 US201615747157A US2018221404A1 US 20180221404 A1 US20180221404 A1 US 20180221404A1 US 201615747157 A US201615747157 A US 201615747157A US 2018221404 A1 US2018221404 A1 US 2018221404A1
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ophthalmic formulations
molecular weight
ophthalmic
formulations according
viscosity
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Mario De Rosa
Chiara Schiraldi
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Altergon SA
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • 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
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • 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
    • A61K9/08—Solutions
    • 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
    • A61K9/12—Aerosols; Foams
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/04—Artificial tears; Irrigation solutions

Definitions

  • the present invention relates to ophthalmic formulations comprising cooperative complexes of low- and high-molecular-weight glycosaminoglycans.
  • Disorders of the ocular apparatus are continually increasing due to growing environmental pollution, widespread use of contact lenses, increased resistance to antibiotics by infectious micro-organisms, and the increase in disorders such as diabetes, which causes severe eye damage.
  • eyedrops consist of a sterile aqueous or oily suspension or solution containing one or more active ingredients and various additives and rheological ingredients. Eyedrops are instilled into the lower conjunctival sac, and represent the preferred pharmaceutical form for the treatment of eye disorders of various aetiologies, such as dry eye, inflammation, infection, irritation, glaucoma and conjunctivitis, and for use before diagnostic procedures or after surgical operations.
  • Eyedrops contain the following types of excipient:
  • Tonicity adjusters must normally be isotonic with the lacrimal fluid. An ophthalmic solution is considered isotonic when its tonicity is equal to that of a saline solution (0.9% w/w sodium chloride). Sodium chloride is the most widely used tonicity adjuster, but the eye also tolerates other compounds, provided that their tonicity is equivalent to that of sodium chloride concentrations ranging between 0.5% and 1.8% w/w.
  • Viscosity-controlling agents Viscosity control in an ophthalmic formulation plays a strategic role in the efficacy of the product because preparations with low viscosity reduce the bioavailability of the active substances, due to the shorter residence time of the product on the eye surface, caused by blinking, during which it is estimated that there is a shear rate of 20000 s ⁇ 1 , and by its passage through the nasolacrimal duct.
  • Polymers such as hyaluronic acid, polyacrylates, chitosan, cellulose derivatives, pectins, alginate, polyvinyl alcohol, polyvinylpyrrolidone, etc. are usually employed to control viscosity and mucoadhesion.
  • the viscosity In the design of an ophthalmic formulation, the viscosity must not exceed 30 mPa ⁇ s, to prevent discomfort caused by excessive resistance of the viscous solution to the movement of the eyelids, and blurred vision.
  • Factors such as the hydration, molecular weight, shape and concentration of the polymer, and the presence of particular functional groups on the chain, strongly influence the mucoadhesion of the formulation, which is mainly generated by a process of molecular entanglement between the polymer chains and the macromolecular component of the mucous layer, especially mucin.
  • the minimum length of the polymer chain of the viscosity-controlling agent must therefore be at least 100 KDa, and macromolecular ingredients with strong crosslinking, which prevent effective entanglement, should not be used.
  • the greater the flexibility of the polymer chain of the viscosity-controlling agent the greater its spread in the mucous layer and entanglement with mucin, both of which factors combine to generate high mucoadhesion, ensuring the optimum residence time of the product applied to the eye surface.
  • pH stabilisers The purpose of these excipients is to keep the product isohydric with the lacrimal fluid. Ophthalmic preparations with a pH below 4 or above 10 cause irritation and intense lacrimation, especially when the pH is strongly alkaline. When choosing the type of buffer to be added to the ophthalmic formulation, the stability of the active ingredient at physiological pH values should be borne in mind, because drugs like pilocarpine, used in the treatment of glaucoma, require a pH of 4-5 to ensure adequate chemical stability of the molecule.
  • Preservatives Ultraservatives—Used to guarantee that the sterility of the formulation is maintained, this being a crucial requirement for ophthalmic preparations. This type of excipient is only used in multi-dose formulations, because once the container is opened, sterility is not guaranteed over time. Examples of preservatives are phenethyl alcohol, chlorhexidine acetate, chlorhexidine gluconate, chlorobutanol, benzalkonium chloride, phenylmercuric nitrate, etc.
  • Solubilisers and suspending agents Ultrased for formulations in suspension when the active ingredient is poorly soluble. Examples of such products are polysorbates, sodium lauryl sulphate and sorbitan monoleate.
  • Hyaluronic acid and the salts thereof are widely used to prepare ophthalmic products due to its viscosity-controlling, mucoadhesive and hydrating action.
  • the pseudoplastic behaviour of HA is particularly important, as it leads to high viscosity values at rest and low viscosity values during rapid blinking, an ideal characteristic to reduce resistance to eyelid movements during blinking, at the same time ensuring that the product remains on the eye surface for a sufficient time.
  • HA hydroxyacetylcholine
  • active ingredients such as pilocarpine, timolol, aceclidine (glaucoma treatment), tropicamide (a mydriatic agent), arecoline (a mitotic agent), gentamicin and tobramycin (antimicrobials)
  • pilocarpine timolol
  • aceclidine glaucoma treatment
  • tropicamide a mydriatic agent
  • arecoline a mitotic agent
  • gentamicin and tobramycin antiimicrobials
  • HA solutions are successfully used as artificial tears in cases of dry eye, due to the ability of the polymer to bind water and epithelial cells, thus considerably increasing the stability of the lacrimal fluid, especially in cases where the mucin component is deficient.
  • ophthalmic products containing HA designed to supplement a tear secretion which is deficient due to mechanical, environmental or visual stress and to restore the physiological conditions of the tear film, are Blugel® and Bluyal®, two eyedrop brands consisting of HA and N-hydroxymethylglycinate combined with sodium edetate as preservative; Hyalistil® and Irlens®, used to improve the tolerability of contact lenses and in the symptomatic treatment of dry eye syndrome; Artelac Splash®, a soothing, hydrating, revitalising product used for dry, tired, irritated, red eyes; and Nebuvis®, a lubricant for tired, red eyes, used in case of poor lacrimation, long-term use of contact lenses, time spent in closed, smo
  • EP 2614090 discloses cooperative complexes of hyaluronic acid with high (H-HA) and low (L-HA) molecular weight, which are useful for intradermal skin biorevitalisation treatments, intraarticular viscosupplementation treatments, intravesical cystitis treatments, and treatments for inflammatory disorders of the vagina, alveolar disorders and disorders of the oral cavity.
  • HA molecules in solution are characterised by cooperative interaction phenomena based on the formation of hydrophobic bonds and interchain hydrogen bonds.
  • the cooperativity of said interactions depends on the length, and therefore the molecular weight of the chains.
  • the long chains of H-HA give stable interactions with one another, which affect all the molecules present in solution, giving rise to a three-dimensional network, whereas L-HA molecules give less stable interactions, which lead to aggregation systems that do not simultaneously involve all the molecules present, which interact with one another in clusters.
  • This different type of aggregation of H-HA and L-HA in solution is responsible for very different rheological behaviours, such as viscosity, a very important property for numerous applications, especially in the medical field.
  • the rapid decline in the viscosity of HA solutions according to molecular weight depends on this different intermolecular interaction capacity due to which, concentration being equal, the viscosity of H-HA solutions with a molecular weight greater than 1 ⁇ 10 6 Da is orders of magnitude greater than those of L-HA solutions with a molecular weight ranging between 5 ⁇ 10 3 and 5 ⁇ 10 5 Da.
  • Said L/H-HA cooperative complexes are formed by subjecting aqueous solutions containing both H-HA and L-HA to a suitably configured heat cycle.
  • Solutions of L/H-HA cooperative hybrids are characterised by viscosities that do not change over time and are considerably lower than those before the heat cycle, wherein energy conditions are created which are able to simultaneously break all the interactions between the H-HA chains and those between the L-HA chains. Under said conditions the pre-requisites no longer exist for the weak interactions that develop between the molecules in solution to be cooperative, and the polymer chains act as independent entities.
  • L/H-HA Complexes of the same type of L/H-HA can be obtained by replacing L-HA with other low-molecular-weight glycosaminoglycans (15-150 KDa) such as chondroitin sulphate (CS), keratan sulphate (KS) and chondroitin (C).
  • CS chondroitin sulphate
  • KS keratan sulphate
  • C chondroitin
  • the invention therefore relates to ophthalmic formulations comprising, as active ingredients, hybrid cooperative complexes (L/H-HA) obtainable by heating, at 100-130° C. for 10-30 min, a mixture of aqueous solutions of at least one fraction (L-HA) of hyaluronic acid or of chondroitin sulphate, keratan sulphate or chondroitin (CS, KS, C), said fraction having an average molecular weight ranging between 1 ⁇ 10 4 and 5 ⁇ 10 5 Da, and an aqueous solution of at least one fraction (H-HA) of hyaluronic acid having an average molecular weight at least 5 times higher than that of L-HA and in any event ranging between 5 ⁇ 10 4 Da and 5 ⁇ 10 6 Da, the weight ratio between L-HA and H-HA in the L/H-HA complex ranging between 0.5 and 2.
  • hybrid cooperative complexes L/H-HA
  • the average molecular weight of the H-HA fraction preferably ranges between 5 ⁇ 10 5 Da and 3 ⁇ 10 6 Da, while the average molecular weight of the L-HA fraction preferably ranges between 3 ⁇ 10 4 Da and 1 ⁇ 10 5 Da.
  • the low-molecular-weight fraction does not consist of hyaluronic acid but of other glycosaminoglycans, its average molecular weight preferably ranges between 1 ⁇ 10 4 and 1 ⁇ 10 5 Da.
  • formulations according to the invention typically in the form of eyedrops, ointments or sprays, preferably contain water as solvent, and are characterised by a viscosity not exceeding 100 mPa s, preferably not exceeding 30 mPa ⁇ S.
  • concentration of the complexes as defined above in the formulations according to the invention can range from 0.1 to 1% by weight.
  • the formulations according to the invention can include other active ingredients in ophthalmic use (non-steroidal anti-inflammatory drugs, antibiotics, beta-blockers, antihistamines, etc.), buffer systems, salts, osmoregulators, preservatives, soothing agents and rheological reagents.
  • active ingredients in ophthalmic use non-steroidal anti-inflammatory drugs, antibiotics, beta-blockers, antihistamines, etc.
  • buffer systems salts, osmoregulators, preservatives, soothing agents and rheological reagents.
  • the formulations according to the invention are particularly useful as tear substitutes for the treatment of dry eye syndrome.
  • H-HA MW 1.36 ⁇ 10 6 Da; Mw/Mn 1.43
  • L-HA MW 8.41 ⁇ 10 4 Da; Mw/Mn 1.75
  • the resulting solutions undergo the following heat cycle in a pressurised system: from 20° C. to 120° C. in 12 min, for 1 min at 120° C., from 120° C. to 20° C. in 15 min.
  • the dynamic viscosity of the samples, the MW and polydispersity index Mw/Mn of L-HA, H-HA and L/H-HA are determined with the Viscotek system described in detail below.
  • the data in Table 1 demonstrates that the viscosity of the L/H-HA cooperative complexes depends on the L-HA/H-HA ratio; the higher the ratio, the lower the viscosity. In any event the most important variation in ⁇ takes place with the formation of the L/H-HA complex, which is already significant as from the lowest value of the ratio (L-HA/H-HA w/w).
  • Ophthalmic formulations comprising the L/H-HA complexes described can be used to prepare novel eyedrops wherein the total quantity of HA can be varied simply, without causing discomfort for the patient.
  • Viscotek measurements the MW and polydispersity index Mw/Mn are determined with a size-exclusion chromatography system equipped with a multi-detector, consisting of a four-bridge viscometer, a refractometer, a right-angle light-scattering detector (RALS) and a low-angle light-scattering detector (LALS), made by Viscotek (www.viscotek.com).
  • the signal measured with LALS is proportional to the molecular weight and concentration
  • the signal measured with the viscometric detector is proportional to the concentration of the sample and the intrinsic viscosity, while the refractometer measures the concentration.
  • the dynamic viscosity measurements as a function of the shear rate are measured in a range from 0.1 s ⁇ 1 to 1000 s ⁇ 1 , acquiring 50 points in “no time setting” mode for each measurement.
  • ophthalmic formulations with an aqueous base which have, as active ingredient and rheological component, L/H-HA complexes with the same 1:1 w/w ratio between H-HA and L-HA but use L/HA of a different molecular weight.
  • Aqueous solutions of H-HA (MW 1.36 ⁇ 10 6 Da; Mw/Mn 1.43) and L-HA (MW 8.41 ⁇ 10 4 Da; Mw/Mn 1.75); L-HA (MW 2.12 ⁇ 10 5 Da; Mw/Mn 1.61) are prepared at the concentration of 2% w/v in distilled water, and used to prepare the various solutions reported in Table 2.
  • the resulting solutions undergo the following heat cycle in a pressurised system: from 20° C. to 120° C.
  • H-HA MW 1.36 ⁇ 10 6 Da; Mw/Mn 1.43
  • L-HA MW 8.41 ⁇ 10 4 Da; Mw/Mn 1.75
  • CS MW 3.81 ⁇ 10 4 Da; Mw/Mn 1.65
  • KS MW 3.45 ⁇ 10 4 Da, Mw/Mn 1.52
  • C Mw 2.9 ⁇ 10 4 Da Mw/Mn 1.66
  • glycosaminoglycans other than L-HA such as CS, KS and C
  • form cooperative complexes with H-HA albeit with a phenomenology involving a reduction in viscosity following the formation of the complex which is less marked than when the low-molecular-weight component is L-HA.
  • Ophthalmic formulations comprising complexes between H-HA and CS or KS or C can be used to prepare eyedrops wherein the total quantity of HA can varied simply, without causing discomfort for the patient.
  • Mucoadhesion is determined as reported by Hassan et al. (1990, Pharm Res. May; 7(5):491-5) and Oechsner et al. (1999, Eur J Pharm Biopharm. Mar; 47(2): 113-8).
  • a polymer can be described as mucoadhesive if the viscosity value of the solution containing the polymer and the mucin (solution 3) is greater than the sum of the viscosities of the polymer solution (solution 2) and the mucin solution (solution 1). This increase in viscosity is attributable to the polymer-mucin interaction; the extent of that increase indicates the mucoadhesive strength of the polymer (2015, Biomacromolecules. Mar 9; 16(3):924-35. doi:0.1021/bm501832y. Epub 2015 Feb 18).
  • Mucoadhesion is calculated in terms of ⁇ % using the following formula:
  • ⁇ (%) [ ⁇ muc+HA ⁇ ( ⁇ muc + ⁇ HA )]/( ⁇ muc + ⁇ HA )*100
  • ⁇ muc+HAS is the viscosity of the solution containing both mucin and HA (solution 3);
  • ⁇ muc is the viscosity of the solution of mucin only (solution 1);
  • ⁇ HA is the viscosity of the solution of HA only (solution 2).
  • the viscosity measurements are taken on 8 mucin solutions prepared independently.
  • H-HA MW 1.36 ⁇ 10 6 Da; Mw/Mn 1.43
  • L-HA MW 8.41 ⁇ 10 4 Da; Mw/Mn 1.75
  • L/H-HA 1:1 w/w obtained by using the above-mentioned H-HA and L-HA
  • the phosphate buffer used is prepared by adding 0.5M HCl to an 0.35 M solution of Na 3 PO 4 until a pH of 7.4 is reached, to give a salt concentration similar to that of the mucin solution (solution 1).
  • the H-HA solutions are prepared at 0.15, 0.23, 0.28 and 0.30% w/w, and the L-HA and L/H-HA solutions are prepared at 0.15, 0.30, 0.45, 0.80 and 1.03% w/w; each solution is prepared in duplicate.
  • a concentrated solution of the HA sample in water is added to the buffered mucin solution to obtain, after mixing, HA at the concentration of solution 2.
  • the solution is made up to the graduation mark with water.
  • Each solution is prepared in duplicate.
  • Table 4 shows the ⁇ % values for solutions of H-HA, L-HA and the L/H-HA complex at the same concentration value (0.30%) and at two different shear rate values (33.9 and 222.2 s ⁇ 1 ).
  • Table 5 shows the ⁇ % values for solutions of H-HA, L-HA and the L/H-HA complex at concentrations with the same dynamic viscosity value ( ⁇ ).
  • Mucoadhesion index ( ⁇ %) at two H-HA shear rate values (MW 1.36 ⁇ 10 6 Da; Mw/Mn 1.43), L-HA (MW 8.41 ⁇ 10 4 Da; Mw/Mn 1.75) and L/H-HA, stoichiometry L-HA/H-HA 1:1 w/w, all at the same concentration (0.30% w/w).
  • H-HA is the most mucoadhesive form of the biopolymer in a wide shear rate range (3-200 s ⁇ 1 ), while at higher values the mucoadhesion values of the various forms become comparable;
  • dynamic viscosity being equal, L/H-HA and L-HA are more mucoadhesive than H-HA throughout the shear rate range.
  • Preparation of primary corneal epithelial cell cultures from porcine eye The eyes of mini-pigs used for surgical training are removed at the time of euthanasia, and the corneas are removed. The corneas are then subjected to enzymatic digestion with a solution of 3 mg/mL collagenase and 4 mg/mL dispase diluted 1:5 in DMEM/F12 culture medium (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, GIBCO Invitrogen USA) 15% FBS (GIBCO Invitrogen, USA) under stirring (600 rpm) at 37° C.
  • DMEM/F12 culture medium Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, GIBCO Invitrogen USA
  • FBS GIBCO Invitrogen, USA
  • the medium used for the growth of the porcine corneal epithelial cells is DMEM/F12 with the addition of 15% foetal bovine serum (FBS), 10 ng/mL EGF (epidermal growth factor, GIBCO Invitrogen, USA) and 40 ⁇ (g/mL gentamicin sulphate (Fisiopharma, Italy). After 20 h the cells are filtered through 0.70 ⁇ m filters and centrifuged at 1500 rpm for 10 min. The pellet is resuspended in culture medium and the cells are seeded to amplify the culture, which mainly consists, 4 days after seeding, of endothelial cells with insignificant fibroblastoid contamination.
  • FBS foetal bovine serum
  • EGF epidermal growth factor
  • 40 ⁇ g/mL gentamicin sulphate
  • Wound-healing test The biological activity and effect of the L/H-HA complex, prepared as described in Example 1, compared with H-HA alone, is evaluated with a wound-healing test, monitoring the wound-healing process with time-lapse video microscopy (TLVM) wherein the incubator stage is maintained at 37° C. in a 5% CO2 atmosphere.
  • the injured cells are treated with: a) 0.3% w/v H-HA (1300-1400 KDa); b) the 0.3% L/H-HA complex, stoichiometry 1:1 w/w H-HA/L-HA; c) the 0.6% L/H-HA complex, stoichiometry 1:1 w/w H-HA/L-HA) in DMEM 1.5% FBS growth medium; the culture medium alone is therefore used as control (CTR).
  • CTR control
  • Comparing a and b provides indications, HA content being equal, regardless of its MW, whereas comparison of a and c analyses two formulations which have the same quantity of H-HA alone.
  • the solutions are sterilised by filtration, using 0.22 ⁇ m filters.
  • the plate thus prepared is housed in the incubator stage of the TLVM station, and 5 fields of view are selected for each well, a delay time of 60 min being set. Each test is conducted in triplicate, and the total duration of the experiment is set at 24 h, having observed that complete repair of the wound takes place after about 12 h for all treatments.
  • the cells are treated for 2 h with 0.3% w/w H-HA and the 0.6% w/w H-HA/L-HA complex used in the ratio of 1:1 w/w H-HA and L-HA as shown in Table 1.
  • the solutions are used “as is” and diluted 1:3, 1:10 and 1:30. All solutions are prepared in the corneal growth medium.
  • the cells After treatment, the cells are subjected to dehydration stress, being incubated dry and without a lid at 37° C. for 20 min.
  • the positive control (CTR) is represented by the cells not subjected to dehydration, while the negative control (NC) cells undergo dehydration, but not protective pre-treatment with H-HA or L/H-HA.
  • Presto Blue viability assay (Invitrogen, GIBCO), conducted by adding 1 mL of a solution of Presto Blue diluted 1:10 in growth medium to each well.
  • the presence of metabolically active cells is demonstrated by the conversion of the Presto Blue reagent (blue resazurin) to a fuchsia-coloured compound (resorufin).
  • the spectrophotometric readings at 570 nm (maximum absorption peak for resazurin) and 600 nm (maximum absorption peak for resorufin) allow cell viability to be quantified on the basis of the number of cells able to activate the reaction.
  • Eyedrops 2 0.6% w/w L/H-HA complex (stoichiometry L-HA/H-HA 1:1 w/w, prepared as reported in Example 1, in aqueous solution, pH 7.2, for phosphate buffer, final osmolarity 300 mOsmL ⁇ 1 , corrected with NaCl or another biocompatible osmolite containing a suitable concentration of an active ingredient with antimicrobial activity commonly used in the ophthalmic field.
  • Eyedrops 3 0.6% w/w L/H-HA complex (stoichiometry L-HA/H-HA 0.5:1 w/w, prepared as reported in Example 1, in aqueous solution, pH 7.2, for phosphate buffer, final osmolarity 300 mOsmL ⁇ 1 , corrected with NaCl or another biocompatible osmolite containing a suitable concentration of an active ingredient with anti-inflammatory activity commonly used in the ophthalmic field.
  • Eyedrops 4 0.4% w/w C/H-HA complex (stoichiometry C/H-HA 1:1 w/w, prepared as reported in Example 3, in aqueous solution, pH 7.2, for phosphate buffer, final osmolarity 300 mOsmL ⁇ 1 , corrected with NaCl or another biocompatible osmolite containing 0.1% w/w cortisone.
  • Eyedrops 6 0.3% w/w CS/H-HA complex (stoichiometry CS/H-HA 0.5:1 w/w, prepared as reported in Example 3, in aqueous solution, pH 7.2, for phosphate buffer, final osmolarity 300 mOsmL ⁇ 1 , corrected with NaCl or another biocompatible osmolite containing 0.1% w/w tetrazoline hydrochloride.
US15/747,157 2015-07-28 2016-07-13 Ophthalmic formulations comprising cooperative complexes of low- and high-molecular-weight hyaluronic acid Abandoned US20180221404A1 (en)

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IT102015000038988 2015-07-28
ITUB2015A002542A ITUB20152542A1 (it) 2015-07-28 2015-07-28 Formulati oftalmici a base di complessi cooperativi di acido ialuronico a basso e alto peso molecolare
PCT/EP2016/066639 WO2017016873A1 (fr) 2015-07-28 2016-07-13 Formulations ophtalmiques comprenant des complexes coopérants d'acide hyaluronique de poids moléculaire faible et élevé

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RU2733733C2 (ru) 2020-10-06
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EP3328396B1 (fr) 2021-10-20
WO2017016873A1 (fr) 2017-02-02
US11878030B2 (en) 2024-01-23
HK1253944A1 (zh) 2019-07-05
US20210386776A1 (en) 2021-12-16
CN107921056A (zh) 2018-04-17
RU2018102693A (ru) 2019-08-30
PL3328396T3 (pl) 2022-02-14
CN113616669A (zh) 2021-11-09
CA2993470A1 (fr) 2017-02-02
EP3328396A1 (fr) 2018-06-06

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