Compositions comprising gelatin and hyaluronic acid and uses thereof

Abstract

Disclosed herein are compositions comprising hyaluronic acid co-crosslinked with gelatin, methods of making same, and their uses. The compositions are suitable for use as injectable dermal products.

Description

COMPOSITIONS COMPRISING GELATIN AND HYALURONIC ACID AND USES THEREOFTechnical field

[0001] The present invention relates to injectable dermal fillers, and more specifically to homogeneous compositions comprising gelatin, e.g., partially hydrolyzed gelatin, and hyaluronic acid. The compositions are at least partially crosslinked, and preferably are characterized by pH of 6-8, osmolarity of 250-400 mOsm / L, and extrusion force of 5-30 Newton. Provided herein also a process for the preparation of the injectable dermal fillers and uses thereof.Background art

[0002] Injectable dermal fillers are commonly used in aesthetic medicine to restore volume, smooth wrinkles, and enhance facial contours. These fillers are injected into or under the skin to provide a more youthful appearance by augmenting soft tissue and improving skin texture. Over the years, a variety of substances have been used as dermal fillers, including collagen, polylactic acid, and hyaluronic acid (HA), each with its unique properties and applications. Typical treatment with dermal fillers involves injecting small amounts of the filler substance into target areas of the skin, e.g., wrinkles or other imperfections, using fine needles or cannulas. The dosage and frequency of treatments vary depending on the patient’s needs and the specific filler used, but generally, 0.1-1 mL is injected per treated area, depending on the required volume, and treatments are performed every 6 to 18 months to maintain desired results.

[0003] Skin boosters belong to a category of injectable treatments aimed at improving overall skin quality by enhancing hydration, elasticity, and texture. Unlike traditional dermal fillers that primarily focus on adding volume or contouring specific areas, skin boosters, which are typically composed of HA with optional additional bio-stimulating ingredients, are injected more superficially, into the skin. This approach targets skin health on a cellular level, stimulating collagen production and improving the skin’s ability to retain moisture. The result is a more radiant, hydrated, and youthful complexion. Skin boosters can be used on the face, neck, hands, and decolletage, making them a versatile option for rejuvenating various areas of the body. Typical treatment with skin boosters involves multiple microinjections of the booster substance into the superficial layers of the skin. The dosage andfrequency of treatments vary, but they are generally administered in a series of sessions spaced a few weeks apart, with maintenance treatments recommended every 6 to 12 months to sustain improved skin quality.

[0004] HA is a naturally occurring polysaccharide found in connective tissues, skin, and eyes. It plays a crucial role in maintaining skin hydration and elasticity, in part due to its ability to retain large amounts of water. In injectable dermal fillers, crosslinked HA is often used to enhance its stability and longevity in the skin, providing longer-lasting results compared to non-crosslinked, native HA. The crosslinking process involves chemically bonding HA strands (molecules), which increases their resistance to enzymatic degradation and prolongs their presence in the dermal layer. Gelatin, on the other hand, is a protein derived from collagen, typically sourced from animal skin, bones, and connective tissues. It is widely used in pharmaceuticals, food products, and cosmetics due to its biocompatibility, biodegradability, and non-immunogenic nature. Due to the disassembly of the collagen’s triple helix structure and the fragmentation of its chains during the gelatin production, it is less immunogenic and more suitable for injectable and implantable applications, compared to collagen. Gelatin has also been used as an ingredient in various formulations, including those used in aesthetic medicine.

[0005] The incorporation of gelatin in injectable dermal fillers offers several potential benefits. Gelatin can enhance the mechanical properties of the filler, providing better structural support and improved elasticity. Additionally, gelatin may promote cellular interactions and tissue integration, due to the presence of several arginine-glycine-aspartic acid (RGD) tripeptides, a motif known to interact with fibroblast receptors and to induce cell adhesion and proliferation, leading to more natural and long-lasting results. Furthermore, its biodegradability ensures that the filler gradually breaks down over time, minimizing the risk of adverse reactions. On the other hand, crosslinking the gelatin molecules, both to themselves and to the HA molecules, may provide prolonged desired biological effects.

[0006] However, to date, no dermal filler or skin booster based on crosslinked HA and gelatin, either to itself (homo-crosslinked) and / or to each other (inter-crosslinked), is commercially available. As demonstrated in the appended examples, the physicochemical properties of such compositions are suboptimal for this intended use.

[0007] There is therefore a need in the art to provide compositions harnessing the full potential of gelatin inter-crosslinked with hyaluronic acid, preferably as injectable dermal compositions, with suitable uniform extrusion force, and flow properties. There is a need to provide processes for manufacturing these compositions.Summary of invention

[0008] It has now been unexpectedly found that the properties of both and the potential synergism of HA and gelatin and their inter-crosslinking, provided some critical properties are addressed, results in a series of dermal products, both filler and skin boosters, with enhanced performance and biocompatibility. As demonstrated in the appended examples, introducing partially hydrolyzed gelatin prior to cross-linking it into the HA system dramatically improves the flow properties and in fact enables the use of these systems in dermal applications. The results of application of these compositions demonstrated superior properties in reference to existing skin care products.

[0009] Therefore, in one aspect, disclosed herein is a process for the preparation of a composition comprising, as active components, gelatin in an amount of from about 0.5% to about 5% by weight, and HA in an amount of from about 0.5% to about 4% by weight, wherein a partial amount of each one of said components is crosslinked to itself and / or to the other component, said process comprising: providing (1) gelatin having an average molecular weight of from about 50 kDa to about 300 kDa, and characterized by a lysine content of from about 1% to about 5%, a viscosity of from about 2 to about 8 millipascal seconds (mPa-sec; also referred to as centipoise) when measured at 60°C and a concentration in water of 6.67%, and a Bloom value (gel strength) higher than 200 g; and (2) a slurry made of HA or a salt thereof, admixed with an aqueous solution of a crosslinker capable of crosslinking each one of said components both to itself and to the other component, wherein the molar ratio between said HA or salt thereof and said crosslinker is from about 88:1 to about 3:1, respectively; dissolving and mixing said gelatin in an aqueous solution of an alkali metal base having a molarity of from about 0.05 M to about 0.2 M, at a temperature of from about 70°C to about 95°C, wherein the ratio between said gelatin and said aqueous solution is from about 1:3.5 to about 1:7 by weight, respectively, to thereby hydrolyze said gelatin and obtain a homogeneous solution thereof having viscosity of from about 50 to about 150 mPa-sec when measured at 50°C with a rheometer; mixing said homogeneous solution with said slurry, at a ratio of from about 7:1 to about 1.5:1 by weight, respectively, until reaching a grainy homogeneous mass, followed by adding an aqueous solution of said alkali metal base having a molarity of from about 0.5 M to about 2 M, wherein the ratio between said aqueous solution and said grainy homogeneous mass is from about 1:3 to about 1:7, by weight, respectively, and mixing until reaching a homogeneous mass; and incubating said homogeneous mass to thereby obtain an intermediate product containing a partial amount of each one of said active components crosslinked to itself and / or to the other component, andhaving a pH of from about 11.5 to about 13; and diluting and neutralizing said intermediate product to thereby obtain the desired composition.

[0010] In a further aspect, provided herein is a process for the preparation of a composition comprising a cross-linked gel of gelatin and hyaluronic acid, e.g., crosslinked with a bifunctional epoxide crosslinker, said process comprising (i) combining in water hyaluronic acid or a salt thereof (HA) and crosslinker, to furnish HA slurry, (ii) combining said HA slurry with an alkaline gelatin solution and mixing to dissolution of HA, to furnish HA-gelatin homogenous mass, (iii) alkalizing said HA-gelatin homogenous mass, to yield alkalized HA-gelatin homogenous mass, (iv) incubating said alkalized HA-gelatin homogenous mass for a time interval sufficient to effect the crosslinking between said HA and said gelatin with said bifunctional epoxide crosslinker, to furnish cross-linked mass, and (v) neutralizing said homogenous mass, and optionally, to yield said injectable dermal composition, at least one of (vi) milling said neutralized homogenous mass, (vii) combining said neutralized homogenous mass with a local anesthetic, optionally with lidocaine hydrochloride, wherein said gelatin in said alkaline gelatin solution is a partially hydrolyzed medium-Bloom or high-Bloom gelatin, or is a low-Bloom gelatin, and optionally wherein said composition is an injectable dermal composition. Preferably, the process is wherein said alkaline gelatin solution has a viscosity of from about 50 to about 180 mPa-s when measured at 50°C with a rheometer at a shearing rate of 50 reciprocal seconds, and gelatin content of between from about 1:3.5 to about 1:7 by weight, optionally about 1:6.

[0011] In another aspect, disclosed herein is a composition comprising, as active components, gelatin in an amount of from about 0.5% to about 5% by weight, and HA in an amount of from about 0.5% to about 4% by weight, wherein a partial amount of each one of said active components is crosslinked to itself and / or to the other active component, wherein said composition is obtained by the process as defined above. In related aspect, provided herein a composition obtainable by any one of the processes as described herein and above.

[0012] In a further aspect, disclosed herein is a homogeneous composition comprising, as active components, gelatin in an amount of from about 0.5% to about 5% by weight, and HA in an amount of from about 0.5% to about 4% by weight, wherein a partial amount of each one of said active components is crosslinked to itself and / or to the other active component, and said composition is being characterized by pH in the range of 6-8, e.g., 6.8-7.2, osmolarity in the range of 250-400 mOsm / L, e.g., 320-350 mOsm / L, and extrusion force (EF) in the range of 5-30 N.

[0013] In a further aspect, provided herein is a composition comprising (a.) between about 0.5% to about 4% by weight of hyaluronic acid or a salt thereof having a molecular weight from about 1 MDa to about 3 MDa, optionally about 1.5 MDa or about 2 MDa, (b.) between about 0.5% to about 5% by weight of a partially hydrolyzed medium -Bloom or high-Bloom gelatin, or a low-Bloom gelatin, optionally wherein said partially hydrolyzed gelatin exhibiting a viscosity of from about 50 to about 180 mPa-s when measured at 50°C with a rheometer at a shearing rate of 50 reciprocal seconds in an aqueous solution having gelatin to water ratio of about 1:6, and (c.) an aqueous medium comprising a buffer, and optionally (d.) a local anesthetic, optionally lidocaine, and further optionally wherein said lidocaine is in a concentration from about 0.15% to about 0.45% by weight of said composition, wherein said gelatin and said hyaluronic acid are crosslinked with a crosslinker, optionally with BDDE (1,4-butanediol diglycidyl ether), such that a ratio between said hyaluronic acid and said crosslinker being from about 1 :0.150 to about 1 :0.005 by weight, and optionally wherein said gelatin is hydrolyzed from an acidic porcine gelatin having an average molecular weight of between 120 and 350 kDa, optionally between 160 and 330 kDa. Preferably, the composition is characterized in that, that a variability of an applied force recorded at 5 readings per second upon extrusion of said composition via 25G / 16 mm regular-wall hypodermic needle from 1-ml syringe having an internal diameter of 6.5 mm and the barrel length of 64.5 mm (Schott™ 1-mL long), at a speed of 0.33 mm / s (about 0.011 ml / s) over last 30 seconds of a 60-seconds time interval, displays a relative standard deviation of mean of less than 10 %, and optionally less than 5%.

[0014] The composition disclosed herein, upon steam sterilization, may be characterized by extrusion force in the range of 5-15 N, and a viscosity from about 100 to about 500 mPa’sec, when measured with a rheometer using a 50mm diameter plate-to-plate configuration at 37°C with a gap of 1mm through a shear rate range of 0.1 to 100 Hz. In particular embodiments, said composition is being characterized by EF in the range of 8-9N, and a viscosity from about 250 to about 400 mPa’sec when measured with a rheometer using a 50mm diameter plate-to-plate configuration at 37°C with a gap of 1mm through a shear rate of 1Hz. Such compositions are optionally in the form of a transparent particle-free mass. A particular such composition comprising 3% gelatin and 2% HA is exemplified herein and referred to as “Skin booster”. An further booster composition may be wherein said hyaluronic acid is present at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and said ratio between said hyaluronic acid and said BDDE is between 1:0.015 and 1:0.021, optionally about 1:0.018. Preferably, said boostercomposition may be characterized by average extrusion force of between 8 and 12 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a viscosity of between 250 and 1000 mPa*s measured using a 25 mm diameter plate-to-plate rheometer configuration at 37°C with a gap of 1 mm at a shear rate of 1 reciprocal second.

[0015] Alternatively, the composition disclosed herein, upon steam sterilization, may be characterized by extrusion force in the range of 12-3 ON, and a G’ (storage modulus) from about 100 to about 500 Pa, when measured with a rheometer using a 25 mm plate-to-plate configuration at 37°C through a shear rate of 1Hz. In particular embodiments, said composition is being characterized by EF in the range of 15-25 N; and a G’ from about 200 to about 350 Pa. Such compositions are optionally in the form of an opaque gel. A particular such composition comprising 3% gelatin and 2% HA is exemplified herein and referred to as “Dermal filler”. Further dermal fillers may comprise said hyaluronic acid at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and have the ratio between said hyaluronic acid and said BDDE of between 1:0.04 and 1:0.06, optionally about 1:0.05; or between 1:0.07 and 1:0.11, optionally about 1:0.09; or between 1:0.115 and 1:0.17, optionally about 1:0.14. Preferably these fillers may be characterized by a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and an average extrusion force of between 10 and 25 Newtons, optionally between 17 and 23 Newtons, and a storage modulus G’ of between 50 and 200 Pa, optionally between 75 and 150 Pa, as measured with a rheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %; or by average extrusion force of between 15 and 30 Newtons, optionally between 20 and 26 Newtons and a storage modulus G’ of between 200 and 400 Pa, optionally between 250 and 350 Pa, or by an by average extrusion force of between 20 and 35 Newtons, optionally between 25 and 33 Newtons, and a storage modulus G’ of between 300 and 600 Pa, optionally between 350 and 500 Pa, measured as described herein.

[0016] The compositions disclosed herein, regardless of whether defined per se or as a product-by process, are useful as injectable treatments in aesthetic medicine, to restore volume, smooth wrinkles, and enhance facial contours, and / or to improve overall skin quality by enhancing hydration, elasticity, and texture.

[0017] In yet another aspect, disclosed herein is a method for restoring volume, reducing smooth wrinkles, and / or enhancing facial contours in an individual, i.e., human being, in need thereof, said method comprising injecting into the skin of said individual an effective amount of a composition as defined above, e.g., a composition being characterized by pH inthe range of 6-8; osmolarity in the range of 250-400 mOsm / L; EF in the range of 12-30N; and G’ in the range of about 100-500 Pa.

[0018] In still another aspect, the disclosed herein is a method for improving skin quality of an individual, i.e., human being, in need thereof, by enhancing hydration, elasticity, and texture of said skin, said method comprising injecting into a superficial layer of a skin of said individual, e.g., a superficial layer of a skin in the face, neck, hands, or decolletage of said individual, an effective amount of a composition as defined above, e.g., a composition being characterized by pH in the range of 6-8; osmolarity in the range of 250-400 mOsm / L; EF in the range of 5-15N; and viscosity in the range of about 100-500 mPa’sec.Brief description of figures

[0019] Figure 1 demonstrates an output of extrusion force measurement of a composition according to an embodiment of the invention.

[0020] Figure 2 demonstrates a rheogram obtained for a composition according to an embodiment of the invention.

[0021] Figure 3 demonstrates an output of extrusion force measurement of a further composition according to an embodiment of the invention.

[0022] Figure 4 demonstrates measurements of storage modulus C’ obtained for a composition according to an embodiment of the invention.

[0023] Figure 5 demonstrates a comparison of measurements of storage moduli C’ obtained for a composition according to an embodiment of the invention and comparative compositions.

[0024] Figure 6 demonstrates an output of extrusion force measurement of a comparative composition.

[0025] Figure 7 demonstrates a comparison of measurements of storage moduli C’ obtained for compositions according to an embodiment of the invention with different starting materials.

[0026] Figure 8 demonstrates a comparison of measurements of storage moduli C’ obtained for compositions according to an embodiment of the invention and a comparative composition.

[0027] Figure 9 demonstrates an output of extrusion force measurement of a composition according to an embodiment of the invention and a comparative composition.

[0028] Figure 10 demonstrates a comparison of measurements of storage moduli C’ obtained for a composition according to an embodiment of the invention and a further comparative composition.

[0029] Figure 11 demonstrates an output of extrusion force measurement of a further comparative composition.

[0030] Figure 12 demonstrates an output of extrusion force measurement of a yet further comparative composition.

[0031] Figure 13 demonstrates a comparison of measurements of storage moduli C’ obtained for a composition according to an embodiment of the invention and a yet further comparative composition.

[0032] Figure 14 demonstrates results of clinical evaluation of a composition according to the invention.

[0033] Figure 15 demonstrates further results of clinical evaluation of a composition according to the invention.

[0034] Figure 16 demonstrates yet further results of a further clinical evaluation of a composition according to the invention.Detailed description

[0035] In one aspect, the present invention relates to a process for the preparation of a composition comprising, as active components, gelatin in an amount of about 0.5-5% by weight, and HA in an amount of about 0.5-4% by weight, wherein a partial amount of each one of said components is crosslinked to itself and / or to the other component, said process comprising:(i) providing (1) gelatin having an average molecular weight of from about 50 kDa to about 300 kDa, and characterized by a lysine content of from about 1% to about 5%, a viscosity of from about 2 to about 8 mPa’sec when measured at 60°C and a concentration in water of 6.67%, and a Bloom value higher than 200 g; and (2) a slurry made of HA or a salt thereof, admixed with an aqueous solution of a crosslinker capable of crosslinking each one of said components both to itself and to the other component, wherein the molar ratio between said HA or salt thereof and said crosslinker is from about 88:1 to about 3:1, respectively;(ii) dissolving and mixing said gelatin in an aqueous solution of an alkali metal base having a molarity of from about 0.05 M to about 0.2 M, at a temperature of from about 70°C to about 95°C, wherein the ratio between said gelatin and said aqueous solution is from about 1 :3.5 to about 1 :7 by weight, respectively, to thereby hydrolyze said gelatin and obtain a homogeneous solution thereof having viscosity of from about 50 to about 150 mPa-sec when measured at 50°C with a rheometer;(iii) mixing said homogeneous solution with said slurry, at a ratio of from about 7:1 to about 1.5:1 by weight, respectively, until reaching a grainy homogeneous mass, followed by adding an aqueous solution of said alkali metal base having a molarity of from about 0.5 M to about 2 M, wherein the ratio between said aqueous solution and said grainy homogeneous mass is from about 1:3 to about 1:7, by weight, respectively, and mixing until reaching a homogeneous mass; and incubating said homogeneous mass to thereby obtain an intermediate product containing a partial amount of each one of said active components crosslinked to itself and / or to the other component, and having a pH of from about 11.5 to about 13; and(iv) diluting and neutralizing said intermediate product to thereby obtain the desired composition.

[0036] The process disclosed herein is aimed at preparing a composition comprising gelatin and HA as active components, each in an amount within a particular range as defined above, wherein a partial amount of each one of the components, e.g., up to 1%, up to 2%, up to 3%, up to 4%, up to 5%, up to 6%, up to 7%, up to 8% up to 9%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or more, of each one of the active components independently, is crosslinked to itself and / or to the other component.

[0037] The gelatin provided in step (i) of the process disclosed may be any gelatin regardless of the source thereof, provided that it has an average molecular weight of from about 50 kDa to about 300 kDa, and is characterized by a lysine content of at least from about 1% to about 5%, a viscosity of from about 2 to about 8 mPa’sec when measured as described above, and a Bloom value higher than 200 g. Such a gelatin is usually provided as a dry material, e.g., as powder, sheets or flakes, but preferably as powder. Alternatively, gelatin may have higher lysine content. The term “lysine content” as used herein with respectto gelatin denotes the number, more specifically the percentage, of lysine residues out of the overall number of amino acid residues in the gelatin molecule. The term “Bloom value” as used herein with respect to gelatin refers to the strength of the gelatin obtained with Bloom test as known in the art, as measured in grams, and may vary between 50 Bloom and 325 Bloom. Gelatins are classified as “Low Bloom gelatin” (Bloom value between 50-125), “Medium Bloom gelatin” (Bloom value between 175-225) and “High Bloom gelatin” (Bloom value between 225-325), wherein the higher a Bloom value, the higher the melting and gelling points of a gel, and the shorter its gelling times. The gelatin provided in step (i), which has Bloom value higher than 200 g, is thus High Bloom gelatin and in most cases is obtained from cow or pig collagen, by hydrolysis.

[0038] In certain embodiments, disclosed herein is a process as defined above, wherein the gelatin provided in step (i) has been obtained by acidic hydrolysis of type I collagen, porcine, with a molecular weight of about 330 kDa. Generally, for use in step (i), gelatin may have a molecular weight of between 50 kDa and 300 kDa, preferably between 100 kDa and 300 kDa, e.g., between 120 and 280 kDa.

[0039] In certain embodiments, disclosed herein is a process as defined above, wherein the gelatin provided in step (i) has an average molecular weight of about 160 kDa; and may further be characterized by at least one of a lysine content of about 2.7%, a viscosity of about 4.5 mPa’sec, and a Bloom value higher than 300 g, e.g. of about 320 g.

[0040] Hyaluronic acid (HA), also called hyaluronan, is a large, polyanionic and linear polymer (glycosaminoglycan) of the repeating disaccharide monomers (D-glucuronic acid and N-acetylglucosamine, linked together through alternating beta-1,4 and beta-1,3 glycosidic bonds), which is widely distributed throughout connective, epithelial and neural tissues. The slurry provided in step (i) of the process may be made of either HA or a salt thereof, e.g., an alkali metal (such as sodium and potassium) salt thereof. In certain embodiments, the HA composing said slurry is the HA sodium salt (sodium hyaluronate; NaHA). For the sake of clarity, all references made herein to HA should be understood as referring to both HA as well as a salt thereof.

[0041] The slurry provided in step (i) of the process disclosed comprises, or consists of, water-wetted HA or a salt thereof, more specifically HA or a salt thereof. The slurry preferably is admixed with an aqueous solution of a crosslinker capable of crosslinking each one of the components (gelatin and HA) both to itself and to the other component, wherein the ratio between said HA (or salt thereof) and said aqueous solution is determined such that the mixture obtained is in the form of a slurry, i.e., a mixture of denser solids suspended inan aqueous liquid, wherein said aqueous solution displaces the gas to maintain contact with the solid surface.

[0042] In certain embodiments, disclosed herein is a process as defined above, wherein said slurry is made of HA or a salt thereof, preferably NaHA, admixed with an aqueous solution of said crosslinker, at a ratio of from about 1:5 to about 1:1 (HA / NaHA: aqueous solution) by weight, respectively. In certain embodiments, disclosed herein is a process as defined above, wherein said slurry is made of NaHA admixed with an aqueous solution of said crosslinker, at a ratio of about 1:1.5 (NaHA:aqueous solution) by weight, respectively. In particular such embodiments, the molecular weight of said NaHA is from about 1 MDa to about 3 MDa, e.g., about 1.5 MDa or about 2 MDa. Examples of NaHA having molecular weight of about 1.5 MDa include, without being limited to, HA-EP1.8, manufactured by Bloomage Biotech, China (herein: “EPl.8”); and examples of NaHA having molecular weight of about 2 MDa include, without limiting, GS-200, manufactured by Kikkoman, Japan (herein: “GS-200”).

[0043] In certain embodiments, disclosed herein is a process as defined above, wherein the crosslinker being capable of crosslinking each one of the gelatin and HA both to itself and to the other one is a bis-epoxide compound such as a homobifunctional epoxide compound (i.e., a bisoxirane), or divinyl sulfone (DVS). In particular and preferred embodiments, said crosslinker is the homobifunctional epoxide compound BDDE (1,4-butanediol diglycidyl ether). Other homobifunctional epoxide compounds include PEG-diglycilyl ethers with PEG being polyethylene glycol chain of a varying length, diepoxyoctane, diglycidyl phthalate, and others.

[0044] According to the process disclosed, the molar ratio between the HA and the crosslinker dissolved in the aqueous solution wetting said HA ranges from about 88:1 to about 3:1, respectively. In certain embodiments, said molar ratio is from about 80: 1 to about 4:1, from about 75:1 to about 5:1, from about 70:1 to about 6:1, from about 65:1 to about 7:1, from about 60:1 to about 8:1, from about 55:1 to about 9:1, from about 50:1 to about 10:1, from about 45 : 1 to about 12:1, from about 40 : 1 to about 15:1, from about 35 : 1 to about 18:1, from about 30: 1 to about 20: 1, from about 25: 1 to about 22: 1, from about 18: 1 to about 3:1, from about 12 : 1 to about 4 : 1 , or from about 8 : 1 to about 5 : 1. For the sake of clarity, the molar weight of HA as referred to herein in reference to crosslinker reactivity, is the weight of its disaccharide monomer (or salt thereof) regardless of the overall weight of the polymer.

[0045] In certain embodiments, disclosed herein is a process as defined above, wherein said crosslinker is BDDE; the slurry provided in step (i) is made of NaHA admixed with anaqueous solution of said crosslinker, at a ratio of about 1:1.5 (NaHA:aqueous solution) by weight, respectively; and the ratio between said NaHA and said BDDE is from about 1 :0.150 to about 1:0.005 by weight, respectively. In particular such embodiments, the ratio between said NaHA and said BDDE is from about 1:0.100 to about 1:0.010 by weight, respectively. More particular such embodiments are those wherein the ratio between said NaHA and said BDDE is from about 1:0.1 to about 1:0.06, e.g., about 1:0.08; from about 1:0.080 to about 1:0.048, e.g., about 1:0.064; from about 1:0.060 to about 1:0.036, e.g., about 1:0.048; from about 1:0.040 to about 1:0.024, e.g., about 1:0.032, or from about 1:0.020 to about 1:0.012, e.g., about 1:0.016, by weight, respectively.

[0046] In step (ii) of the process disclosed, the gelatin provided is further hydrolyzed to thereby obtain a homogeneous solution thereof having viscosity of about 50-250 mPa’sec, e.g., 50-180 mPa*sec, or 50-150 mPa*s, which upon mixing at a particular ratio with said slurry (containing said HA and crosslinker) will provide a homogeneous mass as required. Said hydrolysis is carried out by dissolving and mixing the gelatin provided in an aqueous solution of an alkali metal base (such as NaOA and KOH) having a molarity of about 0.005-0.2 M, e.g., about 0.01 M, or about 0.1 M, at a temperature of about 70-95°C, wherein the ratio between said gelatin and said aqueous solution is from about 1:3.5 to about 1:7 by weight, respectively, and wherein said mixing is carried out in a closed system using an overhead stirrer.

[0047] In certain embodiments, disclosed herein is a process as defined above, wherein said alkali metal base is NaOH. In particular such embodiments, the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is a solution of NaOH 0.1M. The molarity of the alkali metal base solution used for the hydrolysis of the gelatin affects the hydrolysis efficiency and may consequently affect the mechanical properties of the final composition obtained. As found in accordance with the present invention, NaOH solution with a molarity substantially lower than 0.1 M results in insufficient hydrolysis, which nonetheless may be advantageously employed when needed, whereas NaOH with a molarity substantially higher than 0.1 M leads to a mechanically weak product.

[0048] In certain embodiments, disclosed herein is a process as defined above, wherein the ratio between the gelatin and the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is about 1:5 by weight, respectively, i.e., the concentration of the gelatin in the aqueous solution thus obtained is about 16.6% by weight. Without being bound by a particular theory, it is currently believed that an aqueous solution containing a substantially higher concentration of gelatin may cause less homogeneous hydrolysis that will result in amore polydisperse gelatin. Furthermore, such a solution might become hard and elastic, and the following mixing thereof with the HA-containing slurry might thus be challenging. On the other hand, an aqueous solution containing a substantially lower concentration of gelatin might be too diluted and consequently reduce the efficiency of the reaction with the crosslinker.

[0049] In certain embodiments, disclosed herein is a process as defined above, wherein the mixing in step (ii) is carried out at a temperature of about 85°C. In particular such embodiments, said mixing step is carried out for a period of from about 15 minutes to 4 hours, e.g., for about 1 hour. It is believed, without being bound by a particular theory, that a temperature substantially higher than 85°C during this step may lead to excessive hydrolysis which will result in lower mechanical strength of the final composition. On the other hand, a substantially lower temperature will lead to insufficient hydrolysis, which will result in a non-homogeneous final composition, manifested by, e.g., substantial variance of the measured extrusion force, e.g., above 15%, or even above 20% of relative standard deviation (seen as excursion from an essentially flat horizontal line of the graph representing the extrusion force, vide infra), and having a too high G’ (storage modulus) value.

[0050] The homogeneous solution obtained after hydrolysis of the gelatin in step (ii) has a viscosity of about 50-200, about 50-180, or about 50-150, e.g., about 55-145, 60-140, 65-130, 70-120, 75-110, or 80-100, mPa’sec, depending on the specific gelatin used and the hydrolysis parameters. In certain embodiments, disclosed herein is a process as defined above, wherein the viscosity of the homogeneous solution obtained in step (ii) is from about 80 to about 100 mPa’sec, or between 80 and 120 mPa*s. It is currently believed without being bound by a particular theory, that a substantially lower viscosity of the solution obtained in step (ii) may indicate excessive hydrolysis, which may result in lower mechanical strength of the final composition. On the other hand, a substantially higher viscosity of said solution may indicate insufficient hydrolysis, which may result in a non-homogeneous final composition having too high G’ .

[0051] In certain embodiments, disclosed herein is a process as defined above, wherein the hydrolysis step (ii) is carried out for about 1 hour. As found in accordance with the present invention, a substantially longer hydrolysis step may result in excessive hydrolysis and consequently lower mechanical strength of the final composition. On the other hand, a substantially shorter hydrolysis step may result in a non-homogeneous final composition having too high G’ .

[0052] Although step (ii) of the process is referred to as the hydrolysis step, it should be noted that the gelatin hydrolysis may in fact continue during the mixing and incubating step (iii) as well, due to the relatively high pH maintained and the temperature at which said incubation is carried out, and stops completely only upon neutralization of the intermediate product in step (iv).

[0053] In certain embodiments, disclosed herein is a process as defined above, wherein the mixing in step (ii) is carried out in a closed system using an overhead stirrer.

[0054] In step (iii) of the process disclosed, the gelatin solution obtained in step (ii) and the slurry provided in step (i) are thoroughly mixed at a ratio of from about 7:1 to about 1.5:1, e.g., from about 6.5:1 to about 1.5:1, from about 6:1 to about 1.5:1, from about 5.5:1 to about 1.5:1, from about 5:1 to about 1.5:1, from about 4.5:1 to about 1.5:1, from about 4.4:1 to about 1.5:1, from about 4.3:1 to about 1.5:1, from about 4.2:1 to about 1.5:1, from about 4.1:1 to about 1.5:1, from about 4:1 to about 1.5:1, from about 3.9:1 to about 1.5:1, from about 3.8:1 to about 1.5:1, from about 3.7:1 to about 1.5:1, from about 3.6:1 to about 1.5:1, from about 3.5:1 to about 1.5:1, from about 3.4:1 to about 1.5:1, from about 3.3:1 to about 1.5:1, from about 3.2:1 to about 1.5:1, from about 3.1:1 to about 1.5:1, from about 3:1 to about 1.5:1, from about 2.9:1 to about 1.5:1, from about 2.8:1 to about 1.5:1, from about 2.7:1 to about 1.5:1, from about 2.6:1 to about 1.5:1, from about 2.5:1 to about 1.5:1, or from about 2:1 to about 1.5:1, by weight, respectively, until reaching a homogeneous yet grainy mass. Then, a solution of an alkali metal base (such as NaOH and KOH) having a molarity of about 0.5-2 M is added to the mass and mixed therewith, until reaching a homogeneous mass that is then incubated to allow crosslinking of a partial amount of each one of the components (HA and gelatin) to itself and / or to the other component, and obtain an intermediate product having a pH of from about 11.5 to about 13.

[0055] The term “homogenous” as generally used herein does not necessarily imply complete homogeneity on molecular level, rather the uniformity of distribution of the structural components of the gel, and a general inseparability thereof one from another, e.g., of crosslinked gelatin from crosslinker hyaluronic acid, from co-crosslinked gelatin and hyaluronic acid. Generally, any reasonably small sample taken from the bulk, e.g., about SO-SOO microliters, will have the same qualitative and quantitative composition.

[0056] In certain embodiments, disclosed herein is a process as defined above, wherein said homogeneous solution and said slurry are mixed in step (iii) at a ratio of from about 4:1 to about 3 : 1 by weight, respectively. As found in accordance with the present invention, a ratio substantially lower than that (<3:1 by weight, respectively) will affect the ability of thegelatin, HA and crosslinker to mix well, and may result in non-homogeneous crosslinking that might lead to a particles-containing final composition. On the other hand, a ratio substantially higher than that (>4:1 by weight, respectively) will lead to excessive dilution and might result in an inefficient crosslinking process and consequently weak final composition. In case the crosslinker used is BDDE, this situation may further result in relatively high amount of 1,4-butanediol di-(propan-2,3-diolyl) ether (BDPE), which is the major impurity obtained from the HA-BDDE crosslinking process.

[0057] In certain embodiments, disclosed herein is a process as defined above, wherein the aqueous solution added in step (iii) is a solution of NaOH IM.

[0058] In certain embodiments, disclosed herein is a process as defined above, wherein said homogeneous mass is incubated in step (iii) first at a temperature of about 30-50°C, e.g., at a temperature of about 40°C for, e.g., about 4 hours, and then at room temperature, e.g., for about 15-18 hours.

[0059] In certain embodiments, disclosed herein is a process as defined above, wherein the intermediate product obtained in step (iii) has a pH of about 12.5.

[0060] As found in accordance with the present invention, complete mixing in step (iii) is crucial for obtaining a homogenous mass and consequently a homogeneous final composition. While inefficient or insufficient mixing might result in a particles-containing final composition. In certain embodiments, disclosed herein is a process as defined above, wherein the mixing in step (iii) thus comprises thorough and high-shear mixing using, e.g., a GAKO UNGUATOR® mixer. The high-shear mixing is aimed at achieving efficient mixing, dispersion, and particle size reduction, and involves the application of intense mechanical forces, such as high-speed rotation, to generate significant shear forces.

[0061] In step (iv) of the process disclosed, the intermediate product obtained in step (iii) is diluted and neutralized by adding suitable acid and buffer, so as to obtain the desired composition. The acid added may be either an organic acid or an inorganic acid. Examples of suitable organic acids include, without limiting, acetic acid and citric acid; and nonlimiting examples of inorganic acids include hydrochloric acid (HC1) and phosphoric acid. Suitable buffers include, without being limited to, buffer phosphate such as phosphate buffered saline (PBS), buffer acetate, buffer citrate, and buffer glutamate. In particular embodiments, the intermediate product obtained in step (iii) is diluted and neutralized in step (iv) by adding HC1, more particularly HC10.1M, and buffer phosphate. The amounts of acid and buffer to be added may be calculated based on the HA concentration in the slurry provided in step (i); the gelatin concentration in the homogeneous solution obtained in step(ii); the ratio at which said homogeneous solution and said slurry are mixed in step (iii); and the ratio between the grainy homogeneous mass obtained in step (iii) and the aqueous base solution added, taking into consideration the specific amount of each one of the active components in the desired composition.

[0062] In certain embodiments, disclosed herein is a process as defined above, wherein the gelatin provided in step (i) has an average molecular weight of about 160 kDa; and is characterized by a lysine content of about 2.7%, a viscosity of about 4.5 mPa’sec, and a Bloom value higher than 300 g, e.g., of about 320 g; said slurry is made of HA or a salt thereof, preferably NaHA, admixed with an aqueous solution of said crosslinker, at a ratio of from about 1:5 to about 1:1 (HA / NaHA: aqueous solution) by weight, respectively; said crosslinker is a bis-epoxide compound orDVS; said alkali metal base isNaOH, e.g., wherein the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is a solution of NaOH 0. IM; the ratio between the gelatin and the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is about 1:5 by weight, respectively; said mixing is carried out at a temperature of about 85°C (for a period of, e.g., from about 15 minutes to 4 hours, e.g., about 1 hour); and the viscosity of the homogeneous solution obtained in step (ii) is from about 80 to about 100 mPa’sec; said homogeneous solution and said slurry are mixed in step (iii) at a ratio of from about 4:1 to about 3:1 by weight, respectively, the aqueous solution added in step (iii) is a solution of NaOH IM, and the homogeneous mass obtained is then incubated first at a temperature of about 30-50°C (e.g., at a temperature of about 40°C for, e.g., about 4 hours), and then at room temperature (e.g., for about 15-18 hours); and said intermediate product is diluted and neutralized in step (iv) by adding HC10.1M and a buffer phosphate.

[0063] In particular such embodiments, said slurry is made of NaHA admixed with an aqueous solution of said crosslinker, at a ratio of about 1:1.5 (NaHA:aqueous solution) by weight, respectively; said bis-epoxide compound is a homobifunctional epoxide compound such as BDDE; and the ratio between said NaHA and said crosslinker is from about 1:0.150 to about 1:0.005 by weight, respectively. More particular such processes are those wherein said crosslinker is BDDE; and the ratio between said NaHA and said crosslinker is from about 1:0.100 to about 1:0.010 by weight, respectively, preferably from about 1:0.1 to about 1:0.06, e.g., about 1:0.08; from about 1:0.080 to about 1:0.048, e.g., about 1:0.064; from about 1:0.060 to about 1:0.036, e.g., about 1:0.048; from about 1:0.040 to about 1:0.024, e.g., about 1:0.032, or from about 1:0.020 to about 1:0.012, e.g., about 1:0.016, by weight, respectively. In certain such embodiments, said slurry is made of NaHA admixed with anaqueous solution of BDDE, at a ratio of about 1:1.5 (NaHA: aqueous solution) by weight, respectively; the ratio between said NaHA and said BDDE is from about 1:0.1 to about 1:0.06, e.g., about 1:0.08; from about 1:0.080 to about 1:0.048, e.g., about 1:0.064; from about 1:0.060 to about 1:0.036, e.g., about 1:0.048; from about 1:0.040 to about 1:0.024, e.g., about 1:0.032, or from about 1:0.020 to about 1:0.012, e.g., about 1:0.016, by weight, respectively; and the intermediate product obtained in step (iii) has a pH of about 12.5.

[0064] In a particular such aspect, thus disclosed herein is a process according to any one of the embodiments above, for the preparation of a composition comprising, as active components, gelatin in an amount of about 0.5-5% by weight, and HA in an amount of about 0.5-4% by weight, wherein a partial amount of each one of said components is crosslinked to itself and / or to the other component, said process comprising: providing (1) gelatin having an average molecular weight of about 160 kDa, and characterized by a lysine content of about 2.7%, a viscosity of about 4.5 mPa’sec when measured at 60°C and a concentration in water of 6.67%, and a Bloom value of about 320 g; and (2) a slurry made of NaHA admixed with an aqueous solution of BDDE, at a ratio of about 1:1.5 (NaHA: aqueous solution) by weight, respectively, wherein the ratio between said NaHA and said BDDE is from about 1:0.1 to about 1:0.06, e.g., about 1:0.08; from about 1:0.080 to about 1:0.048, e.g., about 1:0.064; from about 1:0.060 to about 1:0.036, e.g., about 1:0.048; from about 1:0.040 to about 1:0.024, e.g., about 1:0.032, or from about 1:0.020 to about 1:0.012, e.g., about 1:0.016, by weight, respectively; dissolving and mixing said gelatin in an aqueous solution of NaOH 0.1M, at a temperature of about 85°C (e.g., for a period of from about 15 minutes to 4 hours, e.g., for about 1 hour), wherein the ratio between said gelatin and said aqueous solution is about 1:5 by weight, respectively, to thereby hydrolyze said gelatin and obtain a homogeneous solution thereof having viscosity of from about 80 to about 100 mPa’sec when measured at 50°C with a rheometer; mixing said homogeneous solution with said slurry, at a ratio of from about 4:1 to about 3:1 by weight, respectively, until reaching a grainy homogeneous mass, followed by adding an aqueous solution of said alkali metal base having a molarity of about IM, wherein the ratio between said aqueous solution and said grainy homogeneous mass is from about 1 :3 to about 1 :7, by weight, respectively, and mixing until reaching a homogeneous mass; and incubating said homogeneous mass, first at about 40°C (e.g., for about 4 hours) and then at room temperature (e.g., for about 15-18 hours) to thereby obtain an intermediate product containing a partial amount of each one of said components crosslinked to itself and / or to the other active component, and having a pH of about 12.5;and diluting and neutralizing said intermediate product by adding HC1 0.1M and a buffer phosphate, to thereby obtain the desired composition.

[0065] In certain embodiments, the molecular weight of the NaHA utilized in the process of the present invention is from about 1 MDa to about 3 MDa, e.g., about 1.5 MDa or about 2 MDa. In particular such embodiments, said NaHA having molecular weight of about 1.5 MDa is HA-EP1.8 (Bloomage Biotech, China); or said NaHA having molecular weight of about 2 MDa is GS-200 (Kikkoman, Japan).

[0066] Thus, provided herein is a process for the preparation of a composition comprising a cross-linked gel of gelatin and hyaluronic acid, e.g., a co-crosslinked composition of gelatin and hyaluronic acid, such that gelatin is at least partially hydrolyzed medium- or high-Bloom gelatin, or is a native low- to low-medium Bloom gelatin. As demonstrated in the appended examples, utilizing a partially hydrolyzed gelatin allows to form compositions suitable for use as injectable dermal products, e.g., having a homogenous structure and uniform properties, particularly the extrusion force (vide infra), enabling precise and repeatable administration technique. The gelatin is preferably such that a solution thereof in water exhibits particular viscosity. Such, at a concentration of between about 12 and 30 % by weight, e.g., between 14.3% and 28.6% (water to gelatin ration of between 1:3.5 to 1:7), preferably at a concentration of about 17% (e.g., 16.7%, corresponding to water to gelatin ratio of 1:6), and at 50 °C, the solution should have a viscosity of between 50 to about 180 mPa’s, at a shearing rate of 50 reciprocal seconds. Preferably, at this shearing rate the solution is in Newtonian flow, i.e., the viscosity does not substantially change upon change of the shearing rate.

[0067] Gelatin may be provided into the process as an alkaline solution. This alkaline solution may be directly a product of prior hydrolysis step, or may be a solution obtained by dissolving a suitable gelatin as described above, in an alkali metal solution. Thus, the process generally comprises (i) combining in water hyaluronic acid or a salt thereof (HA) and crosslinker, to furnish HA slurry, (ii) combining said HA slurry with an alkaline gelatin solution and mixing to dissolution of HA, to furnish HA-gelatin homogenous mass, (iii) alkalizing said HA-gelatin homogenous mass, to yield alkalized HA-gelatin homogenous mass, (iv) incubating said alkalized HA-gelatin homogenous mass for a time interval sufficient to effect the crosslinking between said HA and said gelatin with said bifunctional epoxide crosslinker, to furnish cross-linked mass, and (v) neutralizing said homogenous mass, and optionally, to yield said injectable dermal composition, at least one of (vi) milling said neutralized homogenous mass, (vii) combining said neutralized homogenous mass witha local anesthetic, optionally with lidocaine hydrochloride. It is currently believed without being bound by any particular theory, that effecting the dissolution of HA into the alkaline gelatin solution in presence of a crosslinker and mild alkaline conditions may significantly improve the homogeneity of the final composition.

[0068] The steps above, e.g., combining HA slurry with a crosslinker may be prepared as generally described herein.

[0069] Gelatin may be present in an amount of from about 0.5% to about 5% by weight of the final composition. It may have an average molecular weight of from about 50 kDa to about 300 kDa, provided its solution conforms to the viscosity requirement above. The lysine content of gelatin may be between about 1% and about 5%, but other lysine content may also be suitable, e.g., up to 10% by weight. The alkaline gelatin solution may be obtained by partially hydrolyzing gelatin in alkaline solution, by dissolving and mixing said gelatin in an aqueous solution of an alkali metal base having a molarity of from about 0.005 M to about 0.2 M, at a temperature of from about 70°C to about 95°C, for a time interval of between 30 to 90 minutes necessary to achieve the required viscosity. Some specific alkaline solutions suitable for gelatin hydrolysis include 0.1 M sodium hydroxide solution, and 0.01 M sodium hydroxide solution. The source gelatin for the hydrolysis may be having a viscosity of from about 2 to about 8 mPa*s at a concentration in water of 6.67%, when measured at 60°C and at a shearing rate of 1 reciprocal second. The gelatin may have a Bloom value higher than 200 g, e.g., 240 g, 260 g. 280 g, or higher than 300 g, e.g., 330 g, or 350 g, and it may be sourced by acidic hydrolysis of type I porcine collagen, and may have a molecular weight of about 330 kDa. Preferably, gelatin may have an average molecular weight of about 160 kDa, the viscosity of 4.5 mPa*s determined as above, and a high Bloom value.

[0070] The slurry of HA wherein it is added to the alkaline gelatin solution usually comprises also the crosslinker dissolved in water wherein HA is dispersed. Generally, HA may be in its salt form, e.g., sodium hyaluronate (however, for the sake of simplicity it is also referred to herein as “HA”). The amount of HA in the slurry, as described herein, is usually high, preferably with HA being at a ratio of from about 1:2 to about 1:1 by weight to a solution of crosslinker in water, preferably about 1:1.5. A ratio between HA and a crosslinker, e.g., BDDE, may be from about 1:0.15 to about 1:0.005 by weight.

[0071] The (ii) combining step may be further characterized by having a ratio of from about 7:1 to about 1.5:1 by weight of the alkaline gelatin solution to HA slurry, e.g., from about 4: 1 to about 3 : 1 by weight.

[0072] The (iii) alkalizing step may be further characterized by comprising adding an aqueous solution of an alkali metal base having a molarity of from about 0.5 M to about 2 M, e.g., about 1 M. The amount of the alkali base metal solution should be sufficient to raise the pH value to that of between 12.2 and 12.8, e.g., about 12.5. AS described above, the preferred solution of alkali base metal comprises sodium hydroxide.

[0073] The (iv) incubating step may be further characterized by being performed for two time periods, the time interval of 2-6 hours, e.g., 2.5 to 5.5 hours, or 3-5 hours, e.g., about 4 hours, and the second time interval. The temperature during the first time-interval may be between 30 °C and 50 °C, e.g., between 35 °C and 45 °C, e.g., about 40 °C. The second time interval may have a duration of between 15 to 18 hours, and be performed at a temperature between 15 and 25 °C, e.g., at a room temperature.

[0074] The (v) neutralizing step may be further characterized by being performed by adding an acidified buffer solution comprising between 0.05 M and 0.5 M of hydrochloric acid, e.g., an acidified phosphate buffer. Other buffers may also be used, as described herein (vide supra).

[0075] Generally, the preferred crosslinker belongs to bifunctional epoxide group (i.e., a bisoxirane). Particularly preferred crosslinker is BDDE (1,4-butanediol diglycidyl ether).

[0076] In another aspect, the present invention relates to a composition comprising, as active components, gelatin in an amount of about 0.5-5% by weight, and HA in an amount of about 0.5-4% by weight, wherein a partial amount of each one of said active components is crosslinked to itself and / or to the other active component, wherein said composition is obtained or obtainable by the process of the present invention according to any one of the embodiments defined above. The compositions obtainable by a process, may also be obtained by a different process, provided that the final composition and its properties are identical or very similar to the one obtained by the processes as generally described herein.

[0077] In a particular aspect, the invention relates to a composition as defined above, obtained or obtainable by a more particular process, comprising:providing (1) gelatin having an average molecular weight of about 160 kDa, and characterized by a lysine content of about 2.7%, a viscosity of about 4.5 mPa’sec when measured at 60°C and a concentration in water of 6.67%, and a Bloom value of about 320 g; and (2) a slurry made of NaHA admixed with an aqueous solution of BDDE, at a ratio of about 1:1.5 (NaHA:aqueous solution) by weight, respectively, wherein the ratio between said NaHA and said BDDE is from about 1:0.1 to about 1:0.06, e.g., about 1:0.08; from about 1:0.080 to about 1:0.048, e.g., about 1:0.064; from about 1:0.060 to about 1:0.036,e.g., about 1:0.048; from about 1:0.040 to about 1:0.024, e.g., about 1:0.032, or from about 1:0.020 to about 1:0.012, e.g., about 1:0.016, by weight, respectively;dissolving and mixing said gelatin in an aqueous solution of NaOH 0.1M, at a temperature of about 85°C (e.g., for a period of from about 15 minutes to 4 hours, e.g., for about 1 hour), wherein the ratio between said gelatin and said aqueous solution is about 1:5 by weight, respectively, to thereby hydrolyze said gelatin and obtain a homogeneous solution thereof having viscosity of from about 80 to about 100 mPa-sec when measured at 50°C with a rheometer;mixing said homogeneous solution with said slurry, at a ratio of from about 4: 1 to about 3 : 1 by weight, respectively, until reaching a grainy homogeneous mass, followed by adding an aqueous solution of said alkali metal base having a molarity of about IM, wherein the ratio between said aqueous solution and said grainy homogeneous mass is from about 1 :3 to about 1 :7, by weight, respectively, and mixing until reaching a homogeneous mass; and incubating said homogeneous mass, first at about 40°C (e.g., for about 4 hours) and then at room temperature (e.g., for about 15-18 hours), to thereby obtain an intermediate product containing a partial amount of each one of said components crosslinked to itself and / or to the other active component, and having a pH of about 12.5; anddiluting and neutralizing said intermediate product by adding HC1 0.1M and a buffer phosphate, to thereby obtain the desired composition.

[0078] In certain embodiments, the molecular weight of the NaHA utilized in the process by which said composition is obtained is about 1-3 MDa, e.g., about 1.5 MDa (such as HA-EP1.8, Bloomage Biotech, China) or about 2 MDa (such as GS-200, Kikkoman, Japan).

[0079] In a further aspect, the present invention provides a homogeneous composition comprising, as active components, gelatin in an amount of about 0.5-5% by weight, and HA in an amount of about 0.5-4% by weight, wherein a partial amount of each one of said components is crosslinked to itself and / or to the other component, and said composition is being characterized by pH in the range of 6-8, osmolarity in the range of 250-400 mOsm / L, and extrusion force in the range of 5-30 N.

[0080] In certain embodiments, disclosed herein is a composition as defined above, wherein said composition is being characterized by pH in the range of 6.8-7.2, and osmolarity in the range of 320-350 mOsm / L.

[0081] In certain particular such embodiments, the composition disclosed, upon steam sterilization, is characterized by extrusion force in the range of 5-15 N, and a viscosity from about 100 to about 500 mPa-sec, when measured with a rheometer using a 25 mm diameterplate-to-plate configuration at 37°C with a gap of 1 mm through a shear rate range of 0.1 to 100 reciprocal seconds. In more particular such embodiments, the composition disclosed, upon steam sterilization, is being characterized by extrusion force in the range of 8-9 N, and a viscosity from about 250 to about 400 mPa-sec, when measured with a rheometer using a 25 mm diameter plate-to-plate configuration at 37°C with a gap of 1 mm through a shear rate of 1 reciprocal second. Such compositions are optionally in the form of a transparent particle-free mass. An example of such a composition is the “Skin booster” exemplified herein, which is in the form of a clear, homogeneous, particle-free, and transparent mass, being characterized by pH in the range of 6.8-7.2, osmolarity in the range of 320-350 mOsm / L, extrusion force in the range of 8-9 N, and a viscosity in the range of from about 250 to about 400 mPa-sec when measured as described hereinabove.

[0082] In other particular such embodiments, the composition disclosed, upon steam sterilization, is characterized by extrusion force in the range of 12-30 N, and a G’ (storage modulus) of about 100-500 Pa, when measured with a rheometer using a 25 mm plate-to-plate configuration at 37°C by oscillation at 1 Hz at constant shear strain of 0.1%. In more particular such embodiments, the composition disclosed, upon steam sterilization, is being characterized by extrusion force in the range of 15-25 N, and a G’ from about 200 to about 350 Pa. Such compositions are optionally in the form of an opaque gel. An example of such a composition is the “Dermal filler” exemplified herein, which is in the form of a white opaque homogeneous gel, being characterized by pH in the range of 6.8-7.2, osmolarity in the range of 320-350 mOsm / L, extrusion force in the range of 15-25 N, and a G’ of about 200-350 Pa when measured as described hereinabove.

[0083] The term “osmolarity” or “osmotic concentration” used herein interchangeably, refers to the measure of solute concentration, defined as the number of osmoles (Osm) of solute per liter of solution (Osm / L). The osmolarity of a solution is usually expressed as Osm / L, in the same way that the molarity of a solution is expressed as "M".

[0084] The term “extrusion force” as used herein refers to the continuous force required to extrude the gel out of a syringe via a needle, simulating the force the physician applies when injecting the gel to the patient’s skin. Extrusion force may be demonstrated as a graph of the force required (measured in N units) during the time it takes to extrude the gel via the needle. Herein, as measured at 5 -Hz for 60 sec (total of 300 measured values), the gel is pushed out from a SCHOTT 1-ml long syringe having an internal diameter of 6.5 mm and the barrel length of 64.5 mm, through a 16-mm long / 25G regular wall hypodermic needle at a speed of 0.33 mm / s (about 0.011 ml / s).

[0085] The term “viscosity” as used herein refers to a measure of a fluid’s ratedependent resistance to a change in shape or to movement of its neighboring portions relative to one another, and is scientifically defined as a force multiplied by a time divided by an area. Thus, its units are newton-seconds per square meter, or pascal-seconds, also known as centipoise (cP).

[0086] The term “storage modulus” (also known as Young's modulus) as used herein refers to a mechanical property of a solid material that measures the tensile or compressive stiffness when the force is applied lengthwise. It is the modulus of elasticity for tension or axial compression. Young's modulus is defined as the ratio of the stress (force per unit area) applied to the object and the resulting axial strain (displacement or deformation) in the linear elastic region of the material. For viscoelastic compositions, such as gels, it is opposed to viscous modulus, G”, measuring the propensity of the composition to flow.

[0087] According to the present invention, a process similar to that disclosed herein may be carried out using, alternatively, a slurry made of a polysaccharide other than HA or a salt thereof, admixed with an aqueous solution of a crosslinker capable of crosslinking each one of the active components, i.e., gelatin and said polysaccharide, both to itself and to the other component, wherein the molar ratio between said polysaccharide or salt thereof and said crosslinker is as defined in the process of the present invention, i.e., from about 88: 1 to about 3:1, respectively. Such an alternative process will result in a composition similar to that obtained by the process of the present invention, i.e., a composition comprising, as active components, gelatin in an amount of about 0.5-5% by weight, and said polysaccharide in an amount of about 0.5-4% by weight, wherein a partial amount of each one of said components is crosslinked to itself and / or to the other component, and said composition is being characterized by pH in the range of 6-8, osmolarity in the range of 250-400 mOsm / L, and extrusion force in the range of 5-30 N. Examples of polysaccharides other than HA that may be utilized in such an alternative process include, without limiting, a glycosaminoglycan other than HA, cellulose, chitosan, a carrageenan, and an alginate, all linear polysaccharides consisting of a disaccharide repeating unit.

[0088] Thus, in particular embodiments, provided herein is a composition comprising (a.) between about 0.5% to about 4% by weight of hyaluronic acid or a salt thereof having a molecular weight from about 1 MDa to about 3 MDa, e.g., about 1.5 MDa or about 2 MDa, (b.) between about 0.5% to about 5% by weight of a partially hydrolyzed medium-Bloom or high-Bloom gelatin, or a low-Bloom gelatin, e.g., when the partially hydrolyzed gelatin exhibiting a viscosity of from about 50 to about 180 mPa-s when measured at 50°C with arheometer at a shearing rate of 50 reciprocal seconds in an aqueous solution having gelatin to water ratio of about 1:6 (i.e., about 17 %wt), and (c.) an aqueous medium comprising a buffer, e.g., a phosphate buffer or a different buffer as generally described herein. The gel preferably also comprises (d.) a local anesthetic, e.g., lidocaine, such as lidocaine hydrochloride, such that when lidocaine is present it is in a concentration from about 0.15% to about 0.45% by weight of the composition. In the composition, gelatin and hyaluronic acid are crosslinked and / or co-crosslinked, with a crosslinker as defined herein, e.g., with BDDE (1,4-butanediol diglycidyl ether), such that a ratio between hyaluronic acid and the crosslinker being from about 1 :0.150 to about 1 :0.005 by weight. The gelatin may preferably be a hydrolyzed gelatin, e.g., from an acidic porcine gelatin having an average molecular weight of between 120 and 350 kDa, e.g., between 160 and 330 kDa.

[0089] The particular advantage of the present compositions is their homogeneity that enables their used as effective injectable dermal compositions. For example, the compositions are preferably characterized in low variability of the extrusion force. That is, a variability of an applied force recorded at 5 readings per second upon extrusion of said composition via 25G / 16 mm regular- wall hypodermic needle from 1-ml syringe having an internal diameter of 6.5 mm and the barrel length of 64.5 mm (Schott™ 1-mL long), at a speed of 0.33 mm / s (about 0.011 ml / s) over last 30 seconds of a 60-seconds time interval, displays a relative standard deviation of mean of less than 15%, preferably less than 10%, or further preferably less than 7.5%, or even less than 5%, or even less than 2.5%. AS demonstrated in the appended examples, the compositions according to the invention provided RSD values for the extrusion force of below 2.5%, whereas the comparative compositions provided values of above 10%, or even above 20%.

[0090] In some preferred compositions, hyaluronic acid is present at between 1.5 and 2.5 weight percent, gelatin is present between 1.8 and 3.5 weight percent, they are co-crosslinked with BDDE, and the ratio between hyaluronic acid and BDDE is between 1:0.015 and 1:0.021, e.g., about 1:0.018. Such compositions are particularly useful as injectable skin boosters. They may be characterized by an average extrusion force of between 8 and 12 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a viscosity of between 250 and 1000 mPa*s measured using a 25 mm diameter plate-to-plate rheometer configuration at 37°C with a gap of 1 mm at a shear rate of 1 reciprocal second.

[0091] In some other preferred compositions, hyaluronic acid is present at between 1.5 and 2.5 weight percent, gelatin is present between 1.8 and 3.5 weight percent, and the ratiobetween hyaluronic acid and BDDE is between 1:0.04 and 1:0.06, e.g., about 1:0.05. These compositions may be particularly useful as injectable dermal fillers. These compositions may be characterized by average extrusion force of between 10 and 25 Newtons, e.g., between 17 and 23 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / L, and a storage modulus G’ of between 50 and 200 Pa, e.g., between 75 and 150 Pa, as measured with a rheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %.

[0092] In some other preferred compositions, hyaluronic acid is present at between 1.5 and 2.5 weight percent, gelatin is present between 1.8 and 3.5 weight percent, and the ratio between said hyaluronic acid and said BDDE is between 1:0.07 and 1:0.11, e.g., about 1 :0.09. These compositions may be also particularly useful as injectable dermal fillers. They may be characterized by average extrusion force of between 15 and 30 Newtons, e.g., between 20 and 26 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a storage modulus G’ of between 200 and 400 Pa, e.g., between 250 and 350 Pa, as measured with a rheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %.

[0093] In some further preferred compositions, hyaluronic acid is present at between 1.5 and 2.5 weight percent, gelatin is present between 1.8 and 3.5 weight percent, and the ratio between hyaluronic acid and said BDDE is between 1:0.115 and 1:0.17, e.g., about 1:0.14. These compositions may be also particularly useful as injectable dermal fillers. They may be characterized by average extrusion force of between 20 and 35 Newtons, e.g., between 25 and 33 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / L, and a storage modulus G’ of between 300 and 600 Pa, e.g., between 350 and 500 Pa, as measured with a rheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %.

[0094] The compositions disclosed herein, regardless of whether defined per se or as a product-by process, are useful as injectable treatments in aesthetic medicine, to restore volume, smooth wrinkles, and enhance facial contours, and to improve overall skin quality by enhancing hydration, elasticity, and texture, wherein the specific use of the composition will depend on the physicochemical properties thereof, e.g., its extrusion force and either viscosity or storage modulus (G’).

[0095] Typical treatment with dermal fillers, i.e., compositions as disclosed herein characterized by, e.g., extrusion force in the range of 12-30 N and a G’ of about 100-500 Pa, involves injecting small amounts of the filler substance into targeted areas of the skin usingfine needles or cannulas. The dosage and frequency of the treatment vary depending on the patient’s condition and needs, and the specific filler used, but generally, 0.1-1 mL is injected per treated area, depending on the required volume, and treatments are performed every 6-18 months to maintain desired results. Typical treatment with skin boosters, i.e., compositions as disclosed herein characterized by, e.g., extrusion force in the range of 8-9N and a viscosity from about 250 to about 400 mPa-sec, involves multiple micro-injections of the booster substance into the superficial layers of the skin. The dosage and frequency of the treatment vary depending on the patient’s condition and needs, and the specific booster used, but they are generally administered in a series of sessions spaced a few weeks apart, with maintenance treatments recommended every 6-12 months to sustain improved skin quality.

[0096] In certain embodiments, the compositions disclosed herein, regardless of whether defined per se or as a product-by process, further comprises lidocaine, a local anesthetic of the amino amide type which prevents pain by blocking the signals at the nerve endings in the skin. In particular such embodiments, the amount of lidocaine comprised within the composition is from about 0.15% to about 0.45% by weight of said composition. More particular such embodiments are those wherein said lidocaine constitutes from about 0.17% to about 0.43%, from about 0.19% to about 0.41%, from about 0.21% to about 0.39%, from about 0.23% to about 0.37%, from about 0.25% to about 0.35%, from about 0.27% to about 0.33%, from about 0.29% to about 0.31%, by weight, of said composition.

[0097] The safety of the compositions disclosed herein may be evaluated pre-clinically by a panel of bio-compatibility studies, e.g., according to ISO 10993, including various in vitro and in vivo tests. The efficacy of the compositions may be evaluated pre-clinically by various in vitro (e.g., quantifying fibroblast migration and proliferation due to the presence of gelatin, compared with compositions with none or less gelatin) and in vivo (e.g., quantifying tissue volume, skin hydration, etc., weeks and months post dermal and / or subdermal implantation of these compositions compared with compositions with none or less gelatin) tests.

[0098] The safety and efficacy of the compositions may be evaluated in prospective and retrospective clinical studies in which patients are treated with the compositions. The safety profile may be evaluated by following the patients for several years post implantation, and the clinical results are evaluated subjectively (e.g., using patient and / or physician questionnaires) and / or objectively (e.g., using imaging systems capable of measuring tissue volume, skin color tone, skin hydration, etc.), compared with baseline conditions (i.e., before treatment) and / or with currently available treatments (e.g., in a split-face clinical study,where the patient is implanted with the study device (composition) in one side of the face and with a control device (control composition) in the other side).

[0099] As demonstrated in the appended examples, the clinical testing of selected compositions successfully improved the desired clinical outcomes with no side effects attributable to the compositions beyond what is expected in esthetic medicine.

[0100] Thus, in yet another aspect, the present invention thus relates to a method for restoring volume, reducing smooth wrinkles, and / or enhancing facial contours in an individual, i.e., human being, in need thereof, said method comprising injecting into the skin of said individual an effective amount of a composition according to any one of the embodiments above, regardless of whether defined per se or as a product-by process. In certain embodiments, the composition injected by the method disclosed herein is characterized by pH in the range of 6-8, e.g., 6.8-7.2; osmolarity in the range of 250-400 mOsm / L, e.g., 320-350 mOsm / L; extrusion force in the range of 12-30 N; and G’ in the range of about 100-500 Pa.

[0101] In still another aspect, the present invention thus relates to a method for improving skin quality of an individual, i.e., human being, in need thereof, by enhancing hydration, elasticity, and texture of said skin, said method comprising injecting into a superficial layer of a skin of said individual, e.g., a superficial layer of a skin in the face, neck, hands, or decolletage of said individual, an effective amount of a composition according to any one of the embodiments above, regardless of whether defined per se or as a product-by process. In certain embodiments, the composition injected by the method disclosed herein is characterized by pH in the range of 6-8, e.g., 6.8-7.2; osmolarity in the range of 250-400 mOsm / L, e.g., 320-350 mOsm / L; extrusion force in the range of 5-15 N; and viscosity in the range of about 100-500 mPa-sec.

[0102] Various features according to the invention as described herein for the aspect of method of manufacturing a composition are applicable mutatis mutandis to the compositions according to the teachings herein. Generally, various features according to the invention as described herein for one aspect are applicable mutatis mutandis to other disclosed aspects according to the teachings herein. The herein described preferred embodiments provided herein demonstrating some of the embodiments of the present disclosure are provided to better understand the present disclosure, which however does not limit the invention in any respect. Some variants and equivalents may be readily envisaged by the skilled artisan; the invention therefore encompasses all these variations and equivalents. It must also be noted that, as used in this specification and the appended claims: all scientific and technical termshave meanings commonly used in the art unless otherwise specified; the definitions as provided herein are given with the purpose to facilitate understanding of certain terms used frequently herein and are not necessarily meant to limit the scope of the present disclosure; as used herein the term "about", “c.a ”, and like, as used interchangeably herein, refers to the value and the range of ± 10 %; the terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to", with this terms also encompassing the terms "consisting of and "consisting essentially of', which have their narrower meaning as known in the art, thus an embodiment described as comprising something also discloses embodiments consisting essentially of same and consisting exclusively of same; the singular forms “a”, “an”, and “the”, include plural referents unless the content clearly dictates otherwise; as used herein, a phrase in the form “A and / or B” means a selection from the group consisting of (A), (B) or (A and B); as used herein, a phrase in the form “at least one of A, B, and C” means a selection from the group consisting of (A), (B), (C), (A and B), (A and C), (B and C) or (A, and B, and C), and further combinations are envisaged for the lists comprising larger number of terms. It is appreciated that, certain features of the invention, which are, for brevity, described in the context of separate embodiments, may also be provided in combination with other features in a single embodiment, unless technically infeasible. Conversely, features described in specific combinations of various features, which are, for clarity and demonstration, are described in the context of a single embodiment, may also be provided as separate embodiments individually or in any suitable sub-combination with other features and / or embodiments, as reasonable to the skilled artisan, suitable and operative. Certain features described in the context of various embodiments, including preferred features, are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0103] The invention may be described by the list of following numbered embodiments: 1. A process for the preparation of a composition comprising, as active components, gelatin in an amount of from about 0.5% to about 5% by weight, and hyaluronic acid (HA) in an amount of from about 0.5% to about 4% by weight, wherein a portion of each one of said components is crosslinked to itself and / or to the other component, said process comprising:(i) providing (1) gelatin having an average molecular weight of from about 50 kDa to about 300 kDa, and characterized by a lysine content of from about 1% to about 5%, a viscosity of from about 2 to about 8 millipascal seconds (mPa-sec)when measured at 60°C and a concentration in water of 6.67%, and a Bloom value (gel strength) higher than 200 g; and (2) a slurry made of HA or a salt thereof, admixed with an aqueous solution of a crosslinker capable of crosslinking each one of said components both to itself and to the other component, wherein the molar ratio between said HA or salt thereof and said crosslinker is from about 88:1 to about 3:1, respectively;(ii) dissolving and mixing said gelatin in an aqueous solution of an alkali metal base having a molarity of from about 0.05 M to about 0.2 M, at a temperature of from about 70°C to about 95°C, wherein the ratio between said gelatin and said aqueous solution is from about 1:3.5 to about 1:7 by weight, respectively, to thereby hydrolyze said gelatin and obtain a homogeneous solution thereof having viscosity of from about 50 to about 150 mPa-sec when measured at 50°C with a rheometer;(iii) mixing said homogeneous solution with said slurry, at a ratio of from about 7 : 1 to about 1.5:1 by weight, respectively, until reaching a grainy homogeneous mass, followed by adding an aqueous solution of said alkali metal base having a molarity of from about 0.5 M to about 2 M, wherein the ratio between said aqueous solution and said grainy homogeneous mass is from about 1 :3 to about 1:7, by weight, respectively, and mixing until reaching a homogeneous mass; and incubating said homogeneous mass to thereby obtain an intermediate product containing a partial amount of each one of said active components crosslinked to itself and / or to the other component, and having a pH of from about 11.5 to about 13; and(iv) diluting and neutralizing said intermediate product to thereby obtain the desired composition.2. The process of embodiment 1, wherein the gelatin provided in step (i) has been obtained by acidic hydrolysis of type I collagen, porcine, with a molecular weight of about 330 kDa.3. The process of embodiment 1, wherein the gelatin provided in step (i) has an average molecular weight of about 160 kDa; and is characterized by a lysine content of about 2.7%, a viscosity of about 4.5 mPa'sec, and a Bloom value higher than 300 g, optionally of about 320 g.4. The process of embodiment 1, wherein said slurry is made of HA or a salt thereof, preferably HA sodium salt (NaHA), admixed with an aqueous solution of said crosslinker,at a ratio of from about 1:5 to about 1:1 (HA / NaHA: aqueous solution) by weight, respectively.5. The process of embodiment 1, wherein said slurry is made of NaHA admixed with an aqueous solution of said crosslinker, at a ratio of about 1:1.5 (NaHA:aqueous solution) by weight, respectively.6. The process of embodiment 5, wherein the molecular weight of said NaHA is from about 1 MDa to about 3 MDa, optionally about 1.5 MDa or about 2 MDa.7. The process of embodiment 1, wherein said crosslinker is a bis-epoxide compound or divinyl sulfone (DVS).8. The process of embodiment 7, wherein said bis-epoxide compound is a homobifunctional epoxide compound, optionally BDDE (1,4-butanediol diglycidyl ether).9. The process of embodiment 1, wherein said crosslinker is BDDE, said slurry is made of NaHA admixed with an aqueous solution of said crosslinker, at a ratio of about 1:1.5 (NaHA:aqueous solution) by weight, respectively, and the ratio between said NaHA and said BDDE is from about 1:0.150 to about 1:0.005 by weight, respectively.10. The process of embodiment 9, wherein the ratio between said NaHA and said BDDE is from about 1:0.100 to about 1:0.010 by weight, respectively, optionally selected from the group consisting of from about 1:0.1 to about 1:0.06, optionally, about 1:0.08; from about 1:0.080 to about 1:0.048, optionally, about 1:0.064; from about 1:0.060 to about 1:0.036, optionally, about 1:0.048; from about 1:0.040 to about 1:0.024, optionally, about 1:0.032, or from about 1:0.020 to about 1:0.012, optionally, about 1:0.016, by weight.11. The process of embodiment 1, wherein said alkali metal base is NaOH.12. The process of embodiment 11, wherein the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is a solution of NaOH 0.1M.13. The process of embodiment 1, wherein the ratio between the gelatin and the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is about 1:5 by weight, respectively.14. The process of embodiment 1, wherein the mixing in step (ii) is carried out at a temperature of about 85°C.15. The process of embodiment 1, wherein the viscosity of the homogeneous solution obtained in step (ii) is from about 80 to about 100 mPa’sec.16. The process of embodiment 1, wherein the mixing in step (ii) is carried out in a closed system using an overhead stirrer.17. The process of embodiment 1, wherein said homogeneous solution and said slurry are mixed in step (iii) at a ratio of from about 4: 1 to about 3 : 1 by weight, respectively. 18. The process of embodiment 1, wherein the aqueous solution added in step (iii) is a solution of NaOH IM.19. The process of embodiment 1, wherein said homogeneous mass is incubated in step (iii) first at a temperature of about 30-50°C, and then at room temperature.20. The process of embodiment 1, wherein the intermediate product obtained in step (iii) has a pH of about 12.5.21. The process of embodiment 1, wherein the mixing in step (iii) comprises thorough and high-shear mixing using, e.g., a GAKO UNGUATOR® mixer.22. The process of embodiment 1, wherein said intermediate product is diluted and neutralized in step (iv) by adding hydrochloric acid (HC1) 0.1M and buffer phosphate. 23. The process of embodiment 1, wherein:the gelatin provided in step (i) has an average molecular weight of about 160 kDa; and is characterized by a lysine content of about 2.7%, a viscosity of about 4.5 mPa* sec, and a Bloom value higher than 300 g, optionally of about 320 g;said slurry is made of HA or a salt thereof, optionally NaHA, admixed with an aqueous solution of said crosslinker, at a ratio of from about 1:5 to about 1:1 (HA / NaHA: aqueous solution) by weight, respectively;said crosslinker is a bis-epoxide compound or DVS;said alkali metal base is NaOH, optionally, wherein the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is a solution of NaOH 0. IM;the ratio between the gelatin and the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is about 1:5 by weight, respectively; said mixing is carried out at a temperature of about 85°C; and the viscosity of the homogeneous solution obtained in step (ii) is from about 80 to about 100 mPa-sec;said homogeneous solution and said slurry are mixed in step (iii) at a ratio of from about 4:1 to about 3:1 by weight, respectively, the aqueous solution added in step (iii) is a solution of NaOH IM, and the homogeneous mass obtained is then incubated first at a temperature of about 30-50°C, and then at room temperature; andsaid intermediate product is diluted and neutralized in step (iv) by adding HC1 0.1M and a buffer phosphate.24. The process of embodiment 23, wherein:said slurry is made of NaHA admixed with an aqueous solution of said crosslinker, at a ratio of about 1:1.5 (NaHA: aqueous solution) by weight, respectively;said bis-epoxide compound is a homobifunctional epoxide compound, optionally BDDE; andthe ratio between said NaHA and said crosslinker is from about 1:0.150 to about 1:0.005 by weight, respectively.25. The process of embodiment 23, wherein:said crosslinker is BDDE; andthe ratio between said NaHA and said crosslinker is from about 1:0.100 to about 1:0.010 by weight, respectively, optionally selected from the group consisting of is from about 1:0.1 to about 1:0.06, optionally, about 1:0.08; from about 1:0.080 to about 1:0.048, optionally, about 1:0.064; from about 1:0.060 to about 1:0.036, optionally, about 1:0.048; from about 1:0.040 to about 1:0.024, optionally, about 1:0.032, or from about 1:0.020 to about 1:0.012, optionally, about 1:0.016, by weight, respectively.26. The process of embodiment 25, wherein the intermediate product obtained in step (iii) has a pH of about 12.5.27. The process of any one of embodiments 23-25, comprising:(i) providing (1) gelatin having an average molecular weight of about 160 kDa, and characterized by a lysine content of about 2.7%, a viscosity of about 4.5 mPa’sec, and a Bloom value of about 320 g; and (2) a slurry made of NaHA admixed with an aqueous solution of BDDE, at a ratio of about 1:1.5 (NaHA: aqueous solution) by weight, respectively, wherein the ratio between said NaHA and said BDDE is from about 1:0.1 to about 1:0.06, optionally, about 1:0.08; from about 1:0.080 to about 1:0.048, optionally, about 1:0.064; from about 1:0.060 to about 1:0.036, optionally, about 1:0.048; from about 1:0.040 to about 1:0.024, optionally, about 1:0.032, or from about 1:0.020 to about 1:0.012, optionally, about 1:0.016, by weight, respectively;(ii) dissolving and mixing said gelatin in an aqueous solution of NaOH 0.1M, at a temperature of about 85°C, wherein the ratio between said gelatin and said aqueous solution is about 1 :5 by weight, respectively, to thereby hydrolyze said gelatin and obtain a homogeneous solution thereof having viscosity of from about 80 to about 100 mPa’sec;(iii) mixing said homogeneous solution with said slurry, at a ratio of from about 4: 1 to about 3:1 by weight, respectively, until reaching a grainy homogeneousmass, followed by adding an aqueous solution of said alkali metal base having a molarity of about IM, wherein the ratio between said aqueous solution and said grainy homogeneous mass is from about 1:3 to about 1:7, by weight, respectively, and mixing until reaching a homogeneous mass; and incubating said homogeneous mass, first at about 40°C and then at room temperature, to thereby obtain an intermediate product containing a partial amount of each one of said components crosslinked to itself and / or to the other active component, and having a pH of about 12.5; and(iv) diluting and neutralizing said intermediate product by adding HC1 0.1M and a buffer phosphate, to thereby obtain the desired composition.28. The process of embodiment 27, wherein the molecular weight of said NaHA is from about 1 MDa to about 3 MDa, optionally, about 1.5 MDa or about 2 MDa.29. A process for the preparation of a composition comprising a cross-linked gel of gelatin and hyaluronic acid, optionally crosslinked with a bifunctional epoxide crosslinker, said process comprising(i) combining in water hyaluronic acid or a salt thereof (HA) and crosslinker, to furnish HA slurry,(ii) combining said HA slurry with an alkaline gelatin solution and mixing to dissolution of HA, to furnish HA-gelatin homogenous mass,(iii) alkalizing said HA-gelatin homogenous mass, to yield alkalized HA-gelatin homogenous mass,(iv) incubating said alkalized HA-gelatin homogenous mass for a time interval sufficient to effect the crosslinking between said HA and said gelatin with said bifunctional epoxide crosslinker, to furnish cross-linked mass, and(v) neutralizing said homogenous mass, and optionally, to yield said injectable dermal composition, at least one of(vi) milling said neutralized homogenous mass,(vii) combining said neutralized homogenous mass with a local anesthetic, optionally with lidocaine hydrochloride,whereinsaid gelatin in said alkaline gelatin solution is a partially hydrolyzed medium-Bloom or high-Bloom gelatin, or is a low-Bloom gelatin, and optionally wherein said composition is an injectable dermal composition.30. The process according to embodiment 29, wherein said alkaline gelatin solution has a viscosity of from about 50 to about 180 mPa-s when measured at 50°C with a rheometer at a shearing rate of 50 reciprocal seconds, and gelatin content of between from about 1:3.5 to about 1:7 by weight, optionally about 1:6.31. The process according to any one of embodiments 29-30, wherein said gelatin being characterized by at least one of the below:- being in an amount of from about 0.5% to about 5% by weight of the final composition; - having an average molecular weight of from about 50 kDa to about 300 kDa;- having a lysine content of from about 1% to about 5%.32. The process according to any one of embodiments 29-30, wherein said alkaline gelatin solution is obtained by partially hydrolyzing said gelatin in alkaline solution, by dissolving and mixing said gelatin in an aqueous solution of an alkali metal base having a molarity of from about 0.005 M to about 0.2 M, at a temperature of from about 70°C to about 95°C, for a time interval of between 30 to 90 minutes necessary to achieve said viscosity, optionally wherein said metal base is sodium hydroxide.33. The process according to embodiment 32, wherein said gelatin being characterized by at least one of the below:- at a concentration in water of 6.67% having a viscosity of from about 2 to about 8 mPa*s when measured at 60°C and at a shearing rate of 1 reciprocal second;- having a Bloom value higher than 200 g;- sourced by acidic hydrolysis of type I porcine collagen, with a molecular weight of about 330 kDa.34. The process according to embodiment 33, wherein said gelatin being further characterized by at least one of the below:- an average molecular weight of about 160 kDa;- a lysine content of about 2.7%;- a viscosity of about 4.5 mPa-sec;- a Bloom value higher than 300 g, optionally of about 320 g.35. The process according to any one embodiments 29-34, wherein said (i) combining being characterized by at least one of the below:- said HA or salt thereof being in an amount of from about 0.5% to about 4% by weight of the final composition;- the molar ratio between said HA or salt thereof and said crosslinker being from about 88:1 to about 3:1;- said HA or salt thereof being at a ratio of from about 1 :5 to about 1 : 1 by weight to a solution of said crosslinker in said water;- said HA being in a form of sodium hyaluronate having a molecular weight from about 1 MDa to about 3 MDa, optionally about 1.5 MDa or about 2 MDa.36. The process according to embodiment 35, wherein said HA or salt thereof being at a ratio of from about 1:2 to about 1:1 by weight to a solution of said crosslinker in said water, optionally about 1:1.5.37. The process according to any one embodiments 29-36, further characterized by at least one of the below:- said (ii) combining step being at a ratio of from about 7:1 to about 1.5:1 by weight of said alkaline gelatin solution to said HA slurry, optionally from about 4: 1 to about 3 : 1 by weight; - said (iii) alkalizing step comprising adding an aqueous solution of an alkali metal base having a molarity of from about 0.5 M to about 2 M, optionally about 1 M, further optionally to a pH value of between 12.2 and 12.8, optionally about 12.5;- said (iv) incubating being performed for a time interval of 2-6 hours, optionally about 4 hours, at a temperature of between 30 °C and 50 °C, followed by a time interval of between 15 to 18 hours at a temperature between 15 and 25 °C;- said (v) neutralizing being performed by adding an acidified buffer solution comprising between 0.05 M and 0.5 M of hydrochloric acid, optionally an acidified phosphate buffer.38. The process according to any one embodiments 29-37, wherein said crosslinker is a bifunctional epoxide crosslinker, optionally BDDE (1,4-butanediol diglycidyl ether).39. The process according to embodiment 38, wherein said (i) combining comprises mixing sodium hyaluronate with water and BDDE, at a ratio of about 1:1.5 of said sodium hyaluronate to water by weight, and at a ratio between said sodium hyaluronate and said BDDE from about 1:0.15 to about 1:0.005 by weight.40. The process according to any one embodiments 29-39, further comprising at least one of (viii) degassing said injectable dermal composition, (ix) filling said injectable dermal composition into syringes, (x) degassing filled syringes, and (xi) sterilizing said filled syringes to furnish an injectable dermal product.41. A composition comprising, as active components, gelatin in an amount of from about 0.5% to about 5% by weight, and hyaluronic acid in an amount of from about 0.5% to about 4% by weight, wherein a partial amount of each one of said components is crosslinked to itself and / or to the other component, obtainable by the process of any one of embodiments 1-28.42. A composition obtainable by a process according to any one of embodiments 29-40. 43. A homogeneous composition comprising, as active components, gelatin in an amount of from about 0.5% to about 5% by weight, and hyaluronic acid (HA) in an amount of from about 0.5% to about 4% by weight, wherein a partial amount of each one of said components is crosslinked to itself and / or to the other component, and said composition is being characterized by pH in the range of 6-8, osmolarity in the range of 250-400 mOsm / L, and extrusion force in the range of 5-30 N.44. The composition of embodiment 43, wherein said composition is being characterized by pH in the range of 6.8-7.2, and osmolarity in the range of 320-350 mOsm / L.45. The composition of embodiment 44, wherein upon steam sterilization, said composition is being characterized by extrusion force in the range of 5-15 N, and a viscosity from about 100 to about 500 mPa-sec, when measured with a rheometer using a 50-mm diameter plate-to-plate configuration at 37°C with a gap of 1mm through a shear rate range of 0.1 to 100 Hz.46. The composition of embodiment 45, wherein said composition is being characterized by extrusion force in the range of 8-9 N, and has a viscosity from about 250 to about 400 mPa-sec, when measured with a rheometer using a 50mm diameter plate-to-plate configuration at 37°C with a gap of 1mm through a shear rate of 1 Hz.47. The composition of any one of embodiments 43-46, in the form of a transparent particle-free mass.48. The composition of embodiment 43, wherein upon steam sterilization, said composition is being characterized by extrusion force in the range of 12-30 N, and a G’ (storage modulus) from about 100 to about 500 Pa, when measured with a rheometer using a 25 mm plate-to-plate configuration at 37°C through a shear rate of 1 Hz.49. The composition of embodiment 48, wherein said composition is being characterized by extrusion force in the range of 15-25 N, and a G’ from about 200 to about 350 Pa.50. The composition of any one of embodiments 44 or 48-49, in the form of an opaque gel.51. The composition of any one of embodiments 43-50, further comprising lidocaine. 52. The composition of embodiment 51, wherein said lidocaine constitutes from about 0.15% to about 0.45% by weight of said composition.53. A composition comprisinga. between about 0.5% to about 4% by weight of hyaluronic acid or a salt thereof having a molecular weight from about 1 MDa to about 3 MDa, optionally about 1.5 MDa or about 2 MDa,b. between about 0.5% to about 5% by weight of a partially hydrolyzed medium-Bloom or high-Bloom gelatin, or a low-Bloom gelatin, optionally wherein said partially hydrolyzed gelatin exhibiting a viscosity of from about 50 to about 180 mPa-s when measured at 50°C with a rheometer at a shearing rate of 50 reciprocal seconds in an aqueous solution having gelatin to water ratio of about 1 :6, andc. an aqueous medium comprising a buffer, and optionallyd. a local anesthetic, optionally lidocaine, and further optionally wherein said lidocaine is in a concentration from about 0.15% to about 0.45% by weight of said composition,wherein said gelatin and said hyaluronic acid are crosslinked with a crosslinker, optionally with BDDE (1,4-butanediol diglycidyl ether), such that a ratio between said hyaluronic acid and said crosslinker being from about 1:0.150 to about 1:0.005 by weight, and optionallywherein said gelatin is hydrolyzed from an acidic porcine gelatin having an average molecular weight of between 120 and 350 kDa, optionally between 160 and 330 kDa. 54. The composition according to embodiment 53, characterized in that, that a variability of an applied force recorded at 5 readings per second upon extrusion of said composition via 25G / 16 mm regular-wall hypodermic needle from 1-ml syringe having an internal diameter of 6.5 mm and the barrel length of 64.5 mm (Schott™ 1-mL long), at a speed of 0.33 mm / s (about 0.011 ml / s) over last 30 seconds of a 60-seconds time interval, displays a relative standard deviation of mean of less than 10 %, and optionally less than 5%.55. The composition according to any one of embodiments 53 to 54, wherein said hyaluronic acid is present at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and said ratio between said hyaluronic acid and said BDDE is between 1:0.015 and 1:0.021, optionally about 1:0.018.56. The composition according to embodiment 55, characterized by average extrusion force of between 8 and 12 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a viscosity of between 250 and 1000 mPa*s measured using a 25 mm diameter plate-to-plate rheometer configuration at 37°C with a gap of 1 mm at a shear rate of 1 reciprocal second.57. The composition according to any one of embodiments 53 to 54, wherein said hyaluronic acid is present at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and said ratio between said hyaluronic acid and said BDDE is between 1:0.04 and 1:0.06, optionally about 1:0.05.58. The composition according to embodiment 57, characterized by average extrusion force of between 10 and 25 Newtons, optionally between 17 and 23 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a storage modulus G’ of between 50 and 200 Pa, optionally between 75 and 150 Pa, as measured with a rheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %.59. The composition according to any one of embodiments 53 to 54, wherein said hyaluronic acid is present at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and said ratio between said hyaluronic acid and said BDDE is between 1:0.07 and 1:0.11, optionally about 1:0.09.60. The composition according to embodiment 59, characterized by average extrusion force of between 15 and 30 Newtons, optionally between 20 and 26 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a storage modulus G’ of between 200 and 400 Pa, optionally between 250 and 350 Pa, as measured with a rheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %.61. The injectable composition according to any one of embodiments 53 to 54, wherein said hyaluronic acid is present at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and said ratio between said hyaluronic acid and said BDDE is between 1:0.115 and 1:0.17, optionally about 1:0.14.62. The injectable composition according to embodiment 61, characterized by average extrusion force of between 20 and 35 Newtons, optionally between 25 and 33 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a storage modulus G’ of between 300 and 600 Pa, optionally between 350 and 500 Pa, as measured with a rheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %.63. A method for restoring volume, reducing smooth wrinkles, and / or enhancing facial contours in an individual in need thereof, said method comprising injecting into the skin of said individual an effective amount of a composition according to any one of embodiments 38-59.64. The method of embodiment 63, wherein said composition is characterized by pH in the range of 6-8, optionally, 6.8-7.2; osmolarity in the range of 250-400 mOsm / L, optionally, 320-350 mOsm / L; extrusion force in the range of 12-30N; and G’ in the range of about 100-500 Pa.65. A method for improving skin quality of an individual in need thereof, by enhancing hydration, elasticity, and texture of said skin, said method comprising injecting into a superficial layer of a skin of said individual an effective amount of a composition according to any one of embodiments 41-62.66. The method of embodiment 65, wherein said composition is injected into a superficial layer of a skin in the face, neck, hands, or decolletage of said individual.67. The method of embodiment 65 or 66, wherein said composition is characterized by pH in the range of 6-8, optionally, 6.8-7.2; osmolarity in the range of 250-400 mOsm / L, optionally, 320-350 mOsm / L; extrusion force in the range of 5-15N; and viscosity in the range of about 100-500 mPa-sec.

[0104] The invention will now be illustrated by the following non-limiting Examples.EXAMPLESExample 1. Preparation of 500 g crosslinked HA-gelatin skin boosterStep 1: Hydrolysis and crosslinking

[0105] A glass bottle equipped with an overhead stirrer cap was loaded with 0.1 M NaOH solution (75 g) and gelatin (15 g, dry weight), in this order. The bottle was closed, placed in a water-bath set to 85°C, and connected to an overhead mixer. The mixture was left stirring for 45 minutes at 100 revolutions per minute (rpm), to effect a partial hydrolysis of gelatin. Five minutes before the end of the hydrolysis, sodium hyaluronate (10 g, dry weight) and a solution of BDDE (0.166 mL) in distilled water (15 mL) were added into a 200-g jar (i.e., the size suitable according to the batch size) for Gako Unguator equipped with a standard mixing blade. At the end of the 45 minutes, 10 grams of the partially hydrolyzed gelatin solution were transferred to the jar. The mass was mixed at 600 rpm for 8 minutes (i.e., the mixing time suitable according to the jar size and the height used; a smaller batch in a shorter jar would be mixed for shorter time intervals). The glass bottle containing the remaining gelatin solution was connected again to the overhead stirrer in the hot bath and mixed for another 15 minutes at 100 rpm. The remaining gelatin solution was then poured into the 200-g jar. The combined mass was mixed with a Gako Unguator for another 24 minutes at 600 rpm. Thereafter, NaOH 1-M solution (22 g) was added to the 200-g jar, and the mass was mixed with the Gako Unguator for 3 cycles of 10 minutes at 600 rpm followed by 1 minute at 1400 rpm. The 200-g jar was then placed in a 40°C hot bath for 4hours. After 4 hours the heating was turned off and the jar was left cooling and incubating in the bath for 15-18 hours at room temperature.Step 2: neutralization and ending of preparation.

[0106] After the RT incubation, the 200-g jar was mixed in the Gako Unguator for 1.5 min at 300 rpm and 1.5 min at 600 rpm, and the mass was transferred to a 500-g Unguator jar. To this mass, a first portion of HC1 0.1M solution (111.25 g) was added, and mixed for 2 cycles of 10 minutes at 600 rpm followed by 1 minute at 1400 rpm. The second portion of HC1 0.1M solution (111.25 g) was added to the 500-g jar, and the mass was then mixed for 20 minutes at 600 rpm. Lidocaine HC1 1.5 g (dry weight) was dissolved in phosphate-buffered saline (PBS) and added into the 500-g jar and mixed for 16 minutes at 600 rpm. The amount of buffer added at the end of the procedure was calculated based on the water content (loss on drying) of the raw materials, such that the final amount of the mass was 500 grams.Table 1Step 3: Refining of final product

[0108] The mass is passed through a net with 100-micron size orifices and then a net with 50-micron size orifices to filter / break down possible particles or clusters. The mass was then degassed under vacuum for 20 minutes, and distributed into 1 ,25-mL syringes. The syringes were then centrifuged for further degassing (4 minutes at 1500 rpm), and steam-sterilized in an autoclave at 121°C for 15 minutes. The composition of the skin booster is presented in Table 1 above.Example 2. Physicochemical characteristics of crosslinked HA-gelatin skin booster

[0109] The product obtained in Example 1 was evaluated to conform to the following. Appearance of product: clear, homogeneous and transparent mass, without any particles. pH: 6.8-7.2. Osmolarity: 320-350 mOsm / L. Extrusion force: 8-9 N. The measured force should be consistent, without any significant higher / lower values, to indicate that the gel is homogenous.

[0110] Extrusion was measured at 5 Hz for 60 sec (total of 300 measured values) by forcing the gel out from a SCHOTT 1-ml long syringe through a 16-mm long / 25G regular wall hypodermic needle, at a speed of 0.33 mm / s (about 0.011 ml / s). The extrusion force graph is presented in Figure 1. In the figure, the extrusion force as measured in Newtons (designated “N (Newton)”) is plotted along the ordinate axis, whereas the time elapsed from the start of the measurement is plotted along abscissa axis, designated “time (s)”. The measured value for the whole duration of the test was between 8 and 9 Newton.

[0111] The viscosity of the product after autoclave was set to 250-400 mPa*s at 1 1 / s. Viscosity was measured with a plate-to-plate rheometer configuration at 37°C with a gap of 1 mm, through a shear rate sweep within 0.1 to 100 1 / s range. The obtained rheogram is presented in Figure 2. In the figure, the viscosity as measured in millipascal-seconds (designated “Viscosity r| in mPa*S”) is plotted along the ordinate axis, whereas the shearing rate is plotted along abscissa axis, designated “Shear Rate y in 1 / s”. The observed viscosity at 1 reciprocal second was about 300 mPa*s.Example 3. Preparation of 100 g crosslinked HA-gelatin dermal fillerStep 1: Hydrolysis and crosslinking

[0112] The process was carried out as generally described in Example 1, with the following alternations. In particular, a capped 100 mL plastic vial equipped with a magnetic stir bar were added NaOH 0.1 M solution (15 g) and gelatin (3 g, dry weight) in this order. The vial was closed, placed in a 85°C water-bath on a magnetic stirring plate and stirred for 45 minutes at 300 rpm. Five minutes before the end of hydrolysis, in a 100-g jar for Gako Unguator equipped with a standard mixing blade were added sodium hyaluronate (2 g, dry weight, EPl.8 by Bloomage™, intrinsic viscosity 1.6-2.2 m3 / kg) and a solution of BDDE (0.166 mL) in distilled water (3 mL). At the end of the 45 minutes, 2.5 g of gelatin solution were transferred to the jar. The mass was mixed for 6 minutes at 600 rpm. The vial containing the remaining gelatin solution was placed again in the hot bath and stirred for another 15 minutes at 300 rpm. The remaining gelatin solution was then poured into the 100-g jar. Thecombined mass was mixed with a Gako Unguator for another 12 minutes at 600 rpm. NaOH 1 M solution (4.4g) was added to the 100-g jar. The mass was mixed with the Gako Unguator for 24 min at 600 rpm. The jar was placed in a 40°C hot bath for 4 hours. After 4 hours the heating was turned off and the j ar was left cooling and resting in the bath for 15 - 18 h at room temperature.Step 2: neutralization and ending of preparation

[0113] After the incubation, the 100-g jar was mixed in the Gako Unguator for 1 min at 300 rpm and 1 min at 600 rpm. HC1 0.1M solution (44.5 g) was added to the jar. The mass was mixed with Gako Unguator for 3 times x (8 minutes at 600 rpm + 1 minute at 1400 rpm). The amount of buffer added at the end of the procedure depends on the water content (loss on drying) of the raw materials, as in Example 1, to the final amount of the mass of 100 g. Lidocaine HC1 (0.3 g, dry weight) is dissolved in PBS, added into the jar and mixed for 12 minutes at 600 rpm. The composition of the dermal filler is presented in Table 2.Table 2Step 3: Refining of final product

[0115] The mass was passed twice through a net with 200-micron size holes to filter possible particles or clusters. The mass was then degassed under vacuum for 20 minutes before distribution into 1.25 mL syringes. The syringes were then centrifuged for further degassing (8 minutes at 1500 rpm). The syringes were steam-sterilized in an autoclave at 121 °C for 15 minutes.Example 4. Physicochemical characteristics of crosslinked HA-gelatin dermal filler

[0116] The product obtained in Example 3 was evaluated to conform to the following. Appearance: a white opaque homogeneous gel. pH: 6.8-7.2. Osmolarity: 320-350 mOsm / L. Extrusion force: 15-25 N. The measured force should be consistent, without any significant higher / lower values, to indicate that the gel is homogenous.

[0117] Extrusion was measured as per example 2. The extrusion force graph is presented in Figure 3. In the figure, the extrusion force as measured in Newtons (designated “N (Newton)”) is plotted along the ordinate axis, whereas the time elapsed from the start of the measurement is plotted along abscissa axis, designated “time (s)”. The measured value for the whole duration of the test was between 20 and 22 Newton.

[0118] Elastic storage G’ modulus at 1 Hz and 37°C, as described herein above: 200-350 Pa. G’ represents the ability of a gel to recover its shape after a shear deformation, i.e., its elasticity. When a gel is deformed by a shear force, a fraction of the energy invested is lost in permanent deformation (loss modulus, G”), while the other fraction is regained through the gel restoring its previous shape (storage modulus G’). A gel with higher G’, or elasticity, will have better resistance upon deformation and, thus, better volumizing and filling capacity. The storage modulus G’ graph is presented in Figure 4. In the figure, the storage modulus as measured in Pascals (designated “Pa”) is plotted along the ordinate axis, whereas the oscillation frequency for each measurement is plotted along abscissa axis, designated “Hz”. The measured value varied between 161 Pa for 0.1 Hz, via 256 Pa for 1 Hz, and up to 470 Pa for 10 Hz, for 25-mm plate-to-plate configuration with 1 mm gap and shearing under constant strain of 0.1%.Example 5. Examples of products prepared with different methods

[0119] Example 5.1, comparative', when HA-gelatin skin booster 500 g was prepared as in Example 1, but by dissolving BDDE in 10 mL of water instead of 15 mL, and it was mixed 8 minutes instead of 24 minutes after addition of NaOH IM, the final product was not clear, homogeneous and transparent but rather presented white particles, indicating it is not usable.

[0120] Example 5.2, comparative', when HC1 was added in a single step rather than in two steps as described in Example 1, the final product was not clear and homogeneous, and it contained white particles.

[0121] Example 5.3, comparative'. For HA-gelatin dermal filler prepared according to Example 3, a hydrolysis time longer or shorter than 60 minutes failed to yield a product with G’ in the desired range (200-350 Pa at 1 Hz 37°C). Likewise, autoclaving time affected thestorage modulus of the final product. Four gels were prepared as generally outlined in Example 3, with the following differences: HA raw material: FCH200 (Kikkoman, intrinsic viscosity 2.5-3.3 m3 / kg) was used instead of EPl.8 (Bloomage, intrinsic viscosity 1.6-2.2 m3 / kg), and the amount of NaOH IM was 3 g instead of 4.4 g. Gel 1 contained gelatin that was not hydrolyzed, gel 2 was hydrolyzed for 60 minutes as described in Example 3, gel 3 was hydrolyzed for 90 minutes, and gel 4 was hydrolyzed for 180 minutes.

[0122] The storage moduli G’ graph of the gels 1-4 tested either before or after autoclaving is presented in Figure 5. In the figure, the storage modulus as measured in Pascals (designated “Pa”) is plotted along the ordinate axis, whereas the oscillation frequency for each measurement is plotted along abscissa axis, designated “Hz”. The lines represent the following gels, from top to bottom: gel 1 before autoclave results are shown in markers “x”, gel 2 prior to autoclaving is marked with closed triangles (A), gel 2 after autoclaving is shown in solid squares (■), gel 3 after autoclaving is shown with asterisks (*), and the lowest curve is gel 4 after autoclaving, shown in closed circles (•). The numerical values of G’ at 1 Hz are demonstrated in Table 3 below.Table 3

[0124] For HA-gelatin dermal filler and skin booster, if gelatin is not hydrolyzed (gel 1 of example 5.1) but just dissolved in NaOH 0.1M, the resulting gel (before and after autoclave) is coarse, non-injectable, clustered and rigid, as shown in Figure 6. In the figure, the extrusion force as measured in Newtons (designated “N (Newton)”) is plotted along the ordinate axis, whereas the time elapsed from the start of the measurement is plotted along abscissa axis, designated “time (s)”. It can be readily seen that in both cases the ejection forces are erratic, with excursions between ca. 20 to ca. 60 Newton. The relative standard deviation of the last 30 seconds of the measurements was about 10%.

[0125] Example 5.4, not comparative'. For HA-Gelatin filler, prepared according to Example 3 using a lower intrinsic viscosity HA (substitution of FCH 200 with EPl.8 as described above) results in a lower G’ final product. Combinations of these two polymersshould probably result in intermediate G’ values. Using FCH200-100%: a value for G’ of 278 Pa was obtained after autoclave. Using EPl.8-100%: a value for G’ of 86 Pa was obtained after autoclave. The storage moduli G’ graph of the gels is presented in Figure 7. In the figure, the storage modulus as measured in Pascals (designated “Pa”) is plotted along the ordinate axis, whereas the oscillation frequency for each measurement is plotted along abscissa axis, designated “Hz”.

[0126] Example 5.5, comparative: the gel was prepared according to Example 3, but with hydrolysis of performed in an equivalent amount of water at 70°C for one hour (instead of NaOH 0.1 M at 85°C). Additionally, gelatin was Qualipure grade, Rousselot, [6.67%, 60 °C] 4.8 mPa*sec, Bloom [6.67%, 10°C, 17h] 325 g). A reference composition according to Example 3 was also prepared. The final G’ at 1 Hz after autoclave was much higher (220 Pa versus 80 Pa for the reference according to Example 3), as shown in in Figure 8, where the storage modulus as measured in Pascals (designated “Pa”) is plotted along the ordinate axis, whereas the oscillation frequency for each measurement is plotted along abscissa axis, designated “Hz”, and the lines represent the gels, from top to bottom, gel in water results are shown with solid diamonds (♦), designated as “DDW-70C”, and reference gel is shown below the water hydrolysis result, in solid squares (■), designated as “NaOH 0.1M-85C”. The product texture was coarser and non-homogeneous, as evidenced by spikes in Figure 9. In the figure, the extrusion force as measured in Newtons (designated “N (Newton)”) is plotted along the ordinate axis, whereas the time elapsed from the start of the measurement is plotted along abscissa axis, designated “time (s)”. The measured value for the whole duration of the test was between 16.52 and 14.58 Newton, with the relative standard deviation of the plateau region of about 17%. The excursions of the water-treated gelatin are significantly higher, between 16.12 and 28.8 N, with the relative standard deviation of the plateau region of about 25%.

[0127] Example 5.6, comparative: the gel was prepared according to Example 3 with pH decreased from 12.5 to 12 during crosslinking of the HA-gelatin filler, by adding 3 g NaOH 1 M instead of 4.4 g. FCH200™ (vide supra) was used for hyaluronic acid, and Qualipure gelatin was used instead of HBLV. Apparently, the crosslinking is less effective, as evident by a lower G’ values after autoclave: after addition of 4.4 g NaOH IM (pH 12.6): 530 Pa (1Hz), and after addition of 3 g NaOH IM (pH12): 278 Pa (1Hz). The frequency sweep of the gels is shown in Figure 10. In the figure, the storage modulus as measured in Pascals (designated “Pa”) is plotted along the ordinate axis, whereas the oscillation frequency foreach measurement is plotted along abscissa axis, designated “Hz”. Gel 6 is designated with solid triangles (A).

[0128] Example 5.7, comparative: the gel was prepared under more basic gelatin hydrolysis conditions (with double the amount of NaOH 0.1M, 8.8 g instead of 4.4 g), according to Example 3. The procedure resulted in a complete excessive and hydrolysis such that after the overnight incubation the mass was an unusable thin liquid.

[0129] Example 5.8, comparative', for HA-gelatin filler (and also expected without being bound by a particular theory, for the skin booster) with the composition according to Example 3, if the gelatin is not hydrolyzed but rather just dissolved in the same amount of NaOH 0.1M, and then the mixture is cooled down to a gel, grinded and mixed with HA and BDDE for crosslinking, the outcome is a coarse, non-homogenous and non-swelling mass. The extrusion force graph is demonstrated in Figure 11. In the figure, the extrusion force as measured in Newtons (designated “N (Newton)”) is plotted along the ordinate axis, whereas the time elapsed from the start of the measurement is plotted along abscissa axis, designated “time (s)”

[0130] Example 5.9, comparative: if dry gelatin and HA powders are mixed according the composition of example 3, and DDW, BDDE and NaOH are then added according to the procedure above, a non-usable, non-homogenous, coarse mixture is obtained. The extrusion force graph is demonstrated in Figure 12. In the figure, the extrusion force as measured in Newtons (designated “N (Newton)”) is plotted along the ordinate axis, whereas the time elapsed from the start of the measurement is plotted along abscissa axis, designated “(s)”

[0131] Example 5.10, comparative'. In the HA-gelatin filler the order of addition of reactants has a major influence on the outcome. If instead of the following order: (1) mixing BDDE solution with HA; (2) adding hydrolyzed gelatin solution into HA mixture; and (3) adding NaOH IM, the order is switched to: (1) adding HA to gelatin solution; (2) adding NaOH IM to mixture; and (3) adding BDDE solution, the crosslinking is not successful and the G’ at 1 Hz drops from 85 Pa to 17 Pa at 1Hz. The result is a viscous solution rather than a gel. The storage moduli G’ graph of the gels is presented in Figure 13, with values for order (1) being filled diamonds (♦), and for the comparative order filled squares (■). In the figure, the storage modulus as measured in Pascals (designated “Pa”) is plotted along the ordinate axis, whereas the oscillation frequency for each measurement is plotted along abscissa axis, designated “Hz”.

[0132] Example 5.11, comparative', in both booster (according to Example 1) and filler (according to Example 3), if gelatin is dissolved in half the amount of NaOH 0. IM, the mass doesn’t dissolve, and mixing is difficult and ineffective.Example 6. The efficacy of the Skin booster

[0133] 29 patients were dermally injected with the skin booster according to Example 1, for either 1 treatment (N=l), 2 consecutive treatments (N=10), or 3 consecutive treatments (N=18), 2-4 weeks between treatments. Treatment areas were low cheek lines (N=28), perioral lines (N=25), lips (N=4), nasolabial folds (N=3), chin (N=l), and glabella (N=l). In each follow-up both physician and patient subjectively evaluated the treated areas appearance relative to the baseline (before any treatment) using a Global Aesthetic Improvement Scale (GAIS) score (l=very much improved, 2=much improved, 3=improved, 4=no change, 5=worse), and the patients further rated their overall satisfaction from the treatment (0=very dissatisfied, l=dis satisfied, 2=satisfied, 3=very satisfied).

[0134] Overall results were favorable and significant: Average physician GAIS scores were 2.12 in the first follow-up (2-3 weeks post first treatment, N=25), 2.05 in the second follow-up (4-7 weeks post first treatment, N=19) and 2.50 in the third follow-up (15-22 weeks post first treatment, N=6); Average patient GAIS scores were 3.08 in the first followup (N=25), 2.45 in the second follow-up (N=20) and 2.00 in the third follow-up (N=7); Average patient overall satisfaction scores were 2.42 in the first follow-up (N=26), 2.40 in the second follow-up (N=20) and 2.00 in the third follow-up (N=7).Example 7 - further compositions

[0135] Following the general principles as described herein, the following preparations having very similar molecular composition but tunable different properties, suitable both for skin booster and dermal filler, are presented. Generally, utilizing partially hydrolyzed gelatin it was possible to achieve the preparations suitable for use as dermal products, having very low extrusion force and weak gelling properties (e.g., for use as skin booster), as well as gels with three distinct storage moduli, while retaining the flow properties and the extrusion force within the limits required for easy and robust administration.

[0136] The compositions have been prepared according to the Table 4 below. Comments regarding the components of the compositions are presented in the ensuing text.Table 4

[0137] (*) For booster, HA used was GS200 Kikkoman HA, and for the fillers EPl.8 Bloomage. (**) The 0.1 M hydrochloric acid solution was prepared by tenfold diluting 1-M solution with respective phosphate buffer, yielding pH 1.6-1.9 for booster preparation and 2.0-2.3 for fillers (***) For booster, 0.08 M phosphate buffer with pH 6.8-7.2 was used, for the fillers 0.084 M buffer with pH 7.0-7.4 was used. The gelatin used was HBLV (High bloom low viscosity) gelatin (Rousselot, mean Mw=160 kDa, viscosity [6.67%, 60 °C] 4.7 mPa*sec, Bloom [6.67%, 10°C, 17h] 354 g).

[0138] The process followed that of Examples 1 and 3. All the liquids have been filtered through 0.22 pm filter. A water bath was used, preheated to 85 °C, and an incubation oven was preheated to 40 °C. Requisite amounts of gelatin and 0.1 M or 0.01 M solution of sodium hydroxide were loaded into either glass spinner reactor bottle (booster), or Duran glass bottle with pressure-resistant cap (fillers), and the container was placed under mixing with magnetic stirrer into the water bath, for a total time of Ih and stirring (booster, filler 1, and filler 2) or 30 minutes (filler 3). Stirring speed of booster hydrolysis was 300 rpm and 150 rpm for the three fillers. Shortly after the end of the hydrolysis, sodium hyaluronate was placed into Gako Unguator jar, followed by BDDE dissolved in the water for irrigation, and an aliquot of 10 %wt of the gelatin hydrolysis solution, and mixed at 600 rpm. The remainder of gelatin hydrolysate solution was returned to the water bath to the total hydrolysis time of 60 minutes, whereupon the solution was cooled for 5 minutes at ambience, and transferred into the Unguator jar for a further 18-24 minutes of mixing at 600 rpm. Thereafter, sodium hydroxide solution 1 M was added to the jar and the composition was mixed for additional 24 minutes at 600 rpm. The jar was then placed in the preheated oven (40°C) and incubatedfor 4 hours, and then further incubated at room temperature for additional 16 hours (at ca.25°).

[0139] After the incubation, the booster mass was mixed in Unguator for 2 minutes at 300 rpm, and for additional 1.5-2 minutes at 600 rpm, while the fillers' mass was mixed at 600 rpm for 12 minutes. The mass was transferred into a larger jar (one size scale-up) and mixed with 0.1 M of hydrochloric acid in phosphate buffer for 24 minutes at 300 rpm of 24 minutes (booster), while fillers mixed 30 minutes at 600 rpm +1400 rpm intermittent speeds ("GEL" mixing program). Thereafter, lidocaine hydrochloride monohydrate was dissolved in the phosphate buffer, filtered through 0.22 pm filters, followed by the remainder of phosphate buffer, and mixing for additional 24 minutes at 600 rpm (booster) or "GEL" program (fillers).

[0140] The ready booster was passed through a 100-pm steel mesh, followed by 50 pm mesh; the fillers were passed through two 200 pm steel meshes. The products were degassed under vacuum for 20 minutes, filled into 1-mL syringes, and sterilized for 15 minutes at 121 °C in autoclave.

[0141] The booster composition was clear and transparent, with an average extrusion force of between 8 and 12 Newtons, pH of between 6.5 and 7.5, and osmolarity on range of 270-370 mOsm / kg. The viscosity measured using a 25 mm diameter plate-to-plate configuration at 37°C with a gap of 1 mm at a shear rate of 1 reciprocal second, was between 250 and 1000 mPa*s. The filler compositions had the same pH and osmolarity ranges, and were characterized by an increasing extrusion force and storage modulus G’ : filler 1 had 17-23 N and 75-150 Pa, filler 2 had 20-26 N and 250-350 Pa, and filler 3 had 25-33 N and 350-500 Pa, using the methods as defined herein above. The relative standard deviation of the extrusion force values for the booster was 1.2%, and for the boosters - 1.96%.Example 8 - the efficacy of booster of Example 7

[0142] The objective of the study was to assess safety and efficacy in improving skin texture and fine lines using booster as prepared according to Example 7. The patients (N=24) were treated in two to three sessions, spaced four to six weeks apart, with a further maintenance session at six months. The treated areas included cheeks, perioral lines, lips, chin, and glabella. An average of 0.68 mL of booster was applied per side using microinjections and canula sheeting methods. The patients were followed-up after 2 weeks, 1 month, 6 months, and 12 months.

[0143] The results are shown in Figure 14. Average GAIS scores plotted along the ordinate axis and designated as “Average Score”, are presented as columns, with the error bars represent the two-sided 95% confidence interval. The abscissa axis houses the labels for the assessment, with the follow-up times (designated as “2W”, “IM”, “6M”, and “12M”), with patient scoring grouped on the left side (designated “iGAIS”), and physician’s assessment grouped on the right side (designated “sGAIS”). Physician GAIS assessments demonstrated “much improved” outcomes, and patients rated as “improved” after first session and “much improved” afterwards, with the degree of improvement increasing over time. The results demonstrated rapid onset and increasing clinical benefit. Such, 59.1% of physicians rated “very much improved” at 12 months, and 100% (!) of physicians rated improvement at 12 months, whereas 84% of physician and 72% of patients reported improvement after only 1 treatment. The percentiles of the reported improvement are presented in Figure 15. Percentile of subjects experiencing improvement is plotted along the ordinate axis and designated as “Improved Subjects”, are presented as stacked columns, with the segments from top to bottom indicating “improved” score (top), “much improved” score (middle), and “very much improved” score (bottom). The abscissa axis houses the labels for the assessment, with the follow-up times (designated as “2W”, “IM”, “6M”, and “12M”), with patient scoring grouped on the left side (designated “iGAIS”), and physician’s assessment grouped on the right side (designated “sGAIS”).Example 9 - the efficacy of “filler 2” of Example 7

[0144] The objective of the study was to assess safety and efficacy in improving the correction of moderate to deep wrinkles and folds using filler 2 as prepared according to Example 7. The patients (N=12) were treated in a single session, with an optional touch-up. The treated areas included cheeks, marionettes, perioral lines, nasolabial folds, temples, chin and jawline. An average of 1.36 mL of booster was applied cumulatively across all face, using micro-injections and canula sheeting methods. The patients were followed-up after 3 months, 6 months, and 12 months. Wrinkle Severity Rating Scale (WSRS) was employed to evaluate the efficacy of the treatment.

[0145] The results are shown in Figure 16. In the Figure, average WSRS scores plotted along the ordinate axis and designated as “Average WSRS Score”, are presented as columns, with the error bars represent the upper 95% confidence interval limit. The abscissa axis houses the labels for the assessment, with the follow-up times (designated as “Baseline”, “3Months”, “6 Months”, and “12 Months”). Clinically significant wrinkle reduction was observed and sustained for 12 months. Wrinkle severity significantly reduced after treatment, and clinically meaningful improvement was achieved despite very low injection volume. About 80% of patients rated the Global Aesthetic Improvement Scale (GAIS) as improved at 12 months, with physicians seconding them at 75. The effect was sustained through 12-month follow-up, with very encouraging preliminary safety results, with 0% adverse effects that are not considered a common treatment reaction.Example 10 - characterizing the hydrolyzed gelatin

[0146] Gelatin (HBLV, Rousselot) was dissolved at 50 °C to the final concentration of 17 % by weight, and was subjected to hydrolysis at 85 °C for 1 hour, in either (i) pure water, (ii) 0.1 M of NaOH, or (iii) for 30 minutes in 0.01 M NaOH. Viscosity was measured using Anton Paar rheometer MCR 102e, configuration at 50°C, by scanning the shearing rate from 0.1 1 / s to 100 1 / s, using a 25-mm diameter plate-to-plate rheometer with a gap of 1 mm.

[0147] Native gelatin solution demonstrated a pseudoplastic behavior between 1 and 100 reciprocal seconds, with viscosity declining from about 1000 mPa*s to about 200, with the value of 468 mPa*s at 50 reciprocal seconds. Hydrolyzed gelatins demonstrated an essentially Newtonian behavior between ca. 0.5 and 100 reciprocal seconds, with (i) yielding values of about 155 mPa*s, (ii) 86 mPa*s, and (iii) 118 mPa*s at 50 1 / s.

Claims

CLAIMS1. A process for the preparation of a composition comprising, as active components, gelatin in an amount of from about 0.5% to about 5% by weight, and hyaluronic acid (HA) in an amount of from about 0.5% to about 4% by weight, wherein a portion of each one of said components is crosslinked to itself and / or to the other component, said process comprising:(v) providing (1) gelatin having an average molecular weight of from about 50 kDa to about 300 kDa, and characterized by a lysine content of from about 1% to about 5%, a viscosity of from about 2 to about 8 millipascal seconds (mPa-sec) when measured at 60°C and a concentration in water of 6.67%, and a Bloom value (gel strength) higher than 200 g; and (2) a slurry made of HA or a salt thereof, admixed with an aqueous solution of a crosslinker capable of crosslinking each one of said components both to itself and to the other component, wherein the molar ratio between said HA or salt thereof and said crosslinker is from about 88:1 to about 3:1, respectively;(vi) dissolving and mixing said gelatin in an aqueous solution of an alkali metal base having a molarity of from about 0.05 M to about 0.2 M, at a temperature of from about 70°C to about 95°C, wherein the ratio between said gelatin and said aqueous solution is from about 1:3.5 to about 1:7 by weight, respectively, to thereby hydrolyze said gelatin and obtain a homogeneous solution thereof having viscosity of from about 50 to about 150 mPa-sec when measured at 50°C with a rheometer;(vii) mixing said homogeneous solution with said slurry, at a ratio of from about 7: 1 to about 1.5:1 by weight, respectively, until reaching a grainy homogeneous mass, followed by adding an aqueous solution of said alkali metal base having a molarity of from about 0.5 M to about 2 M, wherein the ratio between said aqueous solution and said grainy homogeneous mass is from about 1 :3 to about 1:7, by weight, respectively, and mixing until reaching a homogeneous mass; and incubating said homogeneous mass to thereby obtain an intermediate product containing a partial amount of each one of said active components crosslinked to itself and / or to the other component, and having a pH of from about 11.5 to about 13; and(viii) diluting and neutralizing said intermediate product to thereby obtain the desired composition.

2. The process of claim 1, wherein the gelatin provided in step (i) has been obtained by acidic hydrolysis of type I collagen, porcine, with a molecular weight of about 330 kDa.

3. The process of claim 1, wherein the gelatin provided in step (i) has an average molecular weight of about 160 kDa; and is characterized by a lysine content of about 2.7%, a viscosity of about 4.5 mPa'sec, and a Bloom value higher than 300 g, optionally of about 320 g.

4. The process of claim 1 , wherein said slurry is made of HA or a salt thereof, preferably HA sodium salt (NaHA), admixed with an aqueous solution of said crosslinker, at a ratio of from about 1:5 to about 1:1 (HA / NaHA: aqueous solution) by weight, respectively.

5. The process of claim 1, wherein said slurry is made of NaHA admixed with an aqueous solution of said crosslinker, at a ratio of about 1:1.5 (NaHA:aqueous solution) by weight, respectively.

6. The process of claim 5, wherein the molecular weight of said NaHA is from about 1 MDa to about 3 MDa, optionally about 1.5 MDa or about 2 MDa.

7. The process of claim 1, wherein said crosslinker is a bis-epoxide compound or di vinyl sulfone (DVS).

8. The process of claim 7, wherein said bis-epoxide compound is a homobifunctional epoxide compound, optionally BDDE (1,4-butanediol diglycidyl ether).

9. The process of claim 1, wherein said crosslinker is BDDE, said slurry is made of NaHA admixed with an aqueous solution of said crosslinker, at a ratio of about 1:1.5 (NaHA:aqueous solution) by weight, respectively, and the ratio between said NaHA and said BDDE is from about 1:0.150 to about 1:0.005 by weight, respectively.

10. The process of claim 9, wherein the ratio between said NaHA and said BDDE is from about 1:0.100 to about 1:0.010 by weight, respectively, optionally selected from the group consisting of from about 1:0.1 to about 1:0.06, optionally, about 1:0.08; from about 1:0.080 to about 1:0.048, optionally, about 1:0.064; from about 1:0.060 to about 1:0.036, optionally, about 1:0.048; from about 1:0.040 to about 1:0.024, optionally, about 1:0.032, or from about 1:0.020 to about 1:0.012, optionally, about 1:0.016, by weight.

11. The process of claim 1, wherein said alkali metal base is NaOH.

12. The process of claim 11, wherein the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is a solution of NaOH 0.1M.

13. The process of claim 1, wherein the ratio between the gelatin and the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is about 1:5 by weight, respectively.

14. The process of claim 1, wherein the mixing in step (ii) is carried out at a temperature of about 85°C.

15. The process of claim 1, wherein the viscosity of the homogeneous solution obtained in step (ii) is from about 80 to about 100 mPa-sec.

16. The process of claim 1, wherein the mixing in step (ii) is carried out in a closed system using an overhead stirrer.

17. The process of claim 1, wherein said homogeneous solution and said slurry are mixed in step (iii) at a ratio of from about 4: 1 to about 3 : 1 by weight, respectively.

18. The process of claim 1, wherein the aqueous solution added in step (iii) is a solution of NaOH IM.

19. The process of claim 1, wherein said homogeneous mass is incubated in step (iii) first at a temperature of about 30-50°C, and then at room temperature.

20. The process of claim 1, wherein the intermediate product obtained in step (iii) has a pH of about 12.5.

21. The process of claim 1 , wherein the mixing in step (iii) comprises thorough and high-shear mixing using, e.g., a GAKO UNGUATOR® mixer.

22. The process of claim 1, wherein said intermediate product is diluted and neutralized in step (iv) by adding hydrochloric acid (HC1) 0. IM and buffer phosphate.

23. The process of claim 1, wherein:the gelatin provided in step (i) has an average molecular weight of about 160 kDa; and is characterized by a lysine content of about 2.7%, a viscosity of about 4.5 mPa* sec, and a Bloom value higher than 300 g, optionally of about 320 g;said slurry is made of HA or a salt thereof, optionally NaHA, admixed with an aqueous solution of said crosslinker, at a ratio of from about 1:5 to about 1:1 (HA / NaHA: aqueous solution) by weight, respectively;said crosslinker is a bis-epoxide compound or DVS;said alkali metal base is NaOH, optionally, wherein the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is a solution of NaOH 0. IM;the ratio between the gelatin and the aqueous solution in which said gelatin is dissolved and mixed in step (ii) is about 1:5 by weight, respectively; said mixing is carried out at a temperature of about 85°C; and the viscosity of the homogeneous solution obtained in step (ii) is from about 80 to about 100 mPa-sec;said homogeneous solution and said slurry are mixed in step (iii) at a ratio of from about 4:1 to about 3:1 by weight, respectively, the aqueous solution added in step (iii) is a solution of NaOH IM, and the homogeneous mass obtained is then incubated first at a temperature of about 30-50°C, and then at room temperature; andsaid intermediate product is diluted and neutralized in step (iv) by adding HC1 0.1M and a buffer phosphate.

24. The process of claim 23, wherein:said slurry is made of NaHA admixed with an aqueous solution of said crosslinker, at a ratio of about 1:1.5 (NaHA: aqueous solution) by weight, respectively;said bis-epoxide compound is a homobifunctional epoxide compound, optionally BDDE; andthe ratio between said NaHA and said crosslinker is from about 1:0.150 to about 1:0.005 by weight, respectively.

25. The process of claim 23, wherein:said crosslinker is BDDE; andthe ratio between said NaHA and said crosslinker is from about 1:0.100 to about 1:0.010 by weight, respectively, optionally selected from the group consisting of is from about 1:0.1 to about 1:0.06, optionally, about 1:0.08; from about 1:0.080 to about 1:0.048, optionally, about 1:0.064; from about 1:0.060 to about 1:0.036, optionally, about 1:0.048;from about 1:0.040 to about 1:0.024, optionally, about 1:0.032, or from about 1:0.020 to about 1:0.012, optionally, about 1:0.016, by weight, respectively.

26. The process of claim 25, wherein the intermediate product obtained in step (iii) has a pH of about 12.5.

27. The process of any one of claims 23-25, comprising:(v) providing (1) gelatin having an average molecular weight of about 160 kDa, and characterized by a lysine content of about 2.7%, a viscosity of about 4.5 mPa’sec, and a Bloom value of about 320 g; and (2) a slurry made of NaHA admixed with an aqueous solution of BDDE, at a ratio of about 1:1.5 (NaHA: aqueous solution) by weight, respectively, wherein the ratio between said NaHA and said BDDE is from about 1:0.1 to about 1:0.06, optionally, about 1:0.08; from about 1:0.080 to about 1:0.048, optionally, about 1:0.064; from about 1:0.060 to about 1:0.036, optionally, about 1:0.048; from about 1:0.040 to about 1:0.024, optionally, about 1:0.032, or from about 1:0.020 to about 1:0.012, optionally, about 1:0.016, by weight, respectively;(vi) dissolving and mixing said gelatin in an aqueous solution of NaOH 0.1M, at a temperature of about 85°C, wherein the ratio between said gelatin and said aqueous solution is about 1 :5 by weight, respectively, to thereby hydrolyze said gelatin and obtain a homogeneous solution thereof having viscosity of from about 80 to about 100 mPa’sec;(vii) mixing said homogeneous solution with said slurry, at a ratio of from about 4: 1 to about 3:1 by weight, respectively, until reaching a grainy homogeneous mass, followed by adding an aqueous solution of said alkali metal base having a molarity of about IM, wherein the ratio between said aqueous solution and said grainy homogeneous mass is from about 1:3 to about 1:7, by weight, respectively, and mixing until reaching a homogeneous mass; and incubating said homogeneous mass, first at about 40°C and then at room temperature, to thereby obtain an intermediate product containing a partial amount of each one of said components crosslinked to itself and / or to the other active component, and having a pH of about 12.5; and(viii) diluting and neutralizing said intermediate product by adding HC1 0.1M and a buffer phosphate, to thereby obtain the desired composition.

28. The process of claim 27, wherein the molecular weight of said NaHA is from about 1 MDa to about 3 MDa, optionally, about 1.5 MDa or about 2 MDa.

29. A process for the preparation of a composition comprising a cross-linked gel of gelatin and hyaluronic acid, optionally crosslinked with a bifunctional epoxide crosslinker, said process comprising(i) combining in water hyaluronic acid or a salt thereof (HA) and crosslinker, to furnish HA slurry,(ii) combining said HA slurry with an alkaline gelatin solution and mixing to dissolution of HA, to furnish HA-gelatin homogenous mass,(iii) alkalizing said HA-gelatin homogenous mass, to yield alkalized HA-gelatin homogenous mass,(iv) incubating said alkalized HA-gelatin homogenous mass for a time interval sufficient to effect the crosslinking between said HA and said gelatin with said bifunctional epoxide crosslinker, to furnish cross-linked mass, and(v) neutralizing said homogenous mass, and optionally, to yield said injectable dermal composition, at least one of(vi) milling said neutralized homogenous mass,(vii) combining said neutralized homogenous mass with a local anesthetic, optionally with lidocaine hydrochloride,whereinsaid gelatin in said alkaline gelatin solution is a partially hydrolyzed medium-Bloom or high-Bloom gelatin, or is a low-Bloom gelatin, and optionally wherein said composition is an injectable dermal composition.

30. The process according to claim 29, wherein said alkaline gelatin solution has a viscosity of from about 50 to about 180 mPa-s when measured at 50°C with a rheometer at a shearing rate of 50 reciprocal seconds, and gelatin content of between from about 1:3.5 to about 1:7 by weight, optionally about 1:6.

31. The process according to any one of claims 29-30, wherein said gelatin being characterized by at least one of the below:- being in an amount of from about 0.5% to about 5% by weight of the final composition; - having an average molecular weight of from about 50 kDa to about 300 kDa;- having a lysine content of from about 1% to about 5%.

32. The process according to any one of claims 29-30, wherein said alkaline gelatin solution is obtained by partially hydrolyzing said gelatin in alkaline solution, by dissolving and mixing said gelatin in an aqueous solution of an alkali metal base having a molarity of from about 0.005 M to about 0.2 M, at a temperature of from about 70°C to about 95°C, for a time interval of between 30 to 90 minutes necessary to achieve said viscosity, optionally wherein said metal base is sodium hydroxide.

33. The process according to claim 32, wherein said gelatin being characterized by at least one of the below:- at a concentration in water of 6.67% having a viscosity of from about 2 to about 8 mPa*s when measured at 60°C and at a shearing rate of 1 reciprocal second;- having a Bloom value higher than 200 g;- sourced by acidic hydrolysis of type I porcine collagen, with a molecular weight of about 330 kDa.

34. The process according to claim 33, wherein said gelatin being further characterized by at least one of the below:- an average molecular weight of about 160 kDa;- a lysine content of about 2.7%;- a viscosity of about 4.5 mPa-sec;- a Bloom value higher than 300 g, optionally of about 320 g.

35. The process according to any one claims 29-34, wherein said (i) combining being characterized by at least one of the below:- said HA or salt thereof being in an amount of from about 0.5% to about 4% by weight of the final composition;- the molar ratio between said HA or salt thereof and said crosslinker being from about 88:1 to about 3:1;- said HA or salt thereof being at a ratio of from about 1 :5 to about 1 : 1 by weight to a solution of said crosslinker in said water;- said HA being in a form of sodium hyaluronate having a molecular weight from about 1 MDa to about 3 MDa, optionally about 1.5 MDa or about 2 MDa.

36. The process according to claim 35, wherein said HA or salt thereof being at a ratio of from about 1:2 to about 1:1 by weight to a solution of said crosslinker in said water, optionally about 1:1.5.

37. The process according to any one claims 29-36, further characterized by at least one of the below:- said (ii) combining step being at a ratio of from about 7:1 to about 1.5:1 by weight of said alkaline gelatin solution to said HA slurry, optionally from about 4: 1 to about 3 : 1 by weight; - said (iii) alkalizing step comprising adding an aqueous solution of an alkali metal base having a molarity of from about 0.5 M to about 2 M, optionally about 1 M, further optionally to a pH value of between 12.2 and 12.8, optionally about 12.5;- said (iv) incubating being performed for a time interval of 2-6 hours, optionally about 4 hours, at a temperature of between 30 °C and 50 °C, followed by a time interval of between 15 to 18 hours at a temperature between 15 and 25 °C;- said (v) neutralizing being performed by adding an acidified buffer solution comprising between 0.05 M and 0.5 M of hydrochloric acid, optionally an acidified phosphate buffer.

38. The process according to any one claims 29-37, wherein said crosslinker is a bifunctional epoxide crosslinker, optionally BDDE (1,4-butanediol diglycidyl ether).

39. The process according to claim 38, wherein said (i) combining comprises mixing sodium hyaluronate with water and BDDE, at a ratio of about 1:1.5 of said sodium hyaluronate to water by weight, and at a ratio between said sodium hyaluronate and said BDDE from about 1:0.15 to about 1:0.005 by weight.

40. The process according to any one claims 29-39, further comprising at least one of (viii) degassing said injectable dermal composition, (ix) filling said injectable dermal composition into syringes, (x) degassing filled syringes, and (xi) sterilizing said filled syringes to furnish an injectable dermal product.

41. A composition comprising, as active components, gelatin in an amount of from about 0.5% to about 5% by weight, and hyaluronic acid in an amount of from about 0.5% to about 4% by weight, wherein a partial amount of each one of said components is crosslinked to itself and / or to the other component, obtainable by the process of any one of claims 1-28.

42. A composition obtainable by a process according to any one of claims 29-40.

43. A homogeneous composition comprising, as active components, gelatin in an amount of from about 0.5% to about 5% by weight, and hyaluronic acid (HA) in an amount of from about 0.5% to about 4% by weight, wherein a partial amount of each one of said components is crosslinked to itself and / or to the other component, and said composition is being characterized by pH in the range of 6-8, osmolarity in the range of 250-400 mOsm / L, and extrusion force in the range of 5-30 N.

44. The composition of claim 43, wherein said composition is being characterized by pH in the range of 6.8-7.2, and osmolarity in the range of 320-350 mOsm / L.

45. The composition of claim 44, wherein upon steam sterilization, said composition is being characterized by extrusion force in the range of 5-15 N, and a viscosity from about 100 to about 500 mPa-sec, when measured with a rheometer using a 50-mm diameter plate-to-plate configuration at 37°C with a gap of 1mm through a shear rate range of 0.1 to 100 Hz.

46. The composition of claim 45, wherein said composition is being characterized by extrusion force in the range of 8-9 N, and has a viscosity from about 250 to about 400 mPa-sec, when measured with a rheometer using a 50mm diameter plate-to-plate configuration at 37°C with a gap of 1mm through a shear rate of 1 Hz.

47. The composition of any one of claims 43-46, in the form of a transparent particle-free mass.

48. The composition of claim 43, wherein upon steam sterilization, said composition is being characterized by extrusion force in the range of 12-30 N, and a G’ (storage modulus) from about 100 to about 500 Pa, when measured with a rheometer using a 25 mm plate-to-plate configuration at 37°C through a shear rate of 1 Hz.

49. The composition of claim 48, wherein said composition is being characterized by extrusion force in the range of 15-25 N, and a G’ from about 200 to about 350 Pa.

50. The composition of any one of claims 44 or 48-49, in the form of an opaque gel.

51. The composition of any one of claims 43-50, further comprising lidocaine.

52. The composition of claim 51, wherein said lidocaine constitutes from about 0.15% to about 0.45% by weight of said composition.

53. A composition comprisinga. between about 0.5% to about 4% by weight of hyaluronic acid or a salt thereof having a molecular weight from about 1 MDa to about 3 MDa, optionally about 1.5 MDa or about 2 MDa,b. between about 0.5% to about 5% by weight of a partially hydrolyzed medium-Bloom or high-Bloom gelatin, or a low-Bloom gelatin, optionally wherein said partially hydrolyzed gelatin exhibiting a viscosity of from about 50 to about 180 mPa-s when measured at 50°C with a rheometer at a shearing rate of 50 reciprocal seconds in an aqueous solution having gelatin to water ratio of about 1 :6, andc. an aqueous medium comprising a buffer, and optionallyd. a local anesthetic, optionally lidocaine, and further optionally wherein said lidocaine is in a concentration from about 0.15% to about 0.45% by weight of said composition,wherein said gelatin and said hyaluronic acid are crosslinked with a crosslinker, optionally with BDDE (1,4-butanediol diglycidyl ether), such that a ratio between said hyaluronic acid and said crosslinker being from about 1:0.150 to about 1:0.005 by weight, and optionallywherein said gelatin is hydrolyzed from an acidic porcine gelatin having an average molecular weight of between 120 and 350 kDa, optionally between 160 and 330 kDa.

54. The composition according to claim 53, characterized in that, that a variability of an applied force recorded at 5 readings per second upon extrusion of said composition via 25G / 16 mm regular-wall hypodermic needle from 1-ml syringe having an internal diameter of 6.5 mm and the barrel length of 64.5 mm (Schott™ 1-mL long), at a speed of 0.33 mm / s (about 0.011 ml / s) over last 30 seconds of a 60-seconds time interval, displays a relative standard deviation of mean of less than 10 %, and optionally less than 5%.

55. The composition according to any one of claims 53 to 54, wherein said hyaluronic acid is present at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and said ratio between said hyaluronic acid and said BDDE is between 1:0.015 and 1:0.021, optionally about 1:0.018.

56. The composition according to claim 55, characterized by average extrusion force of between 8 and 12 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a viscosity of between 250 and 1000 mPa*s measured using a 25mm diameter plate-to-plate rheometer configuration at 37°C with a gap of 1 mm at a shear rate of 1 reciprocal second.

57. The composition according to any one of claims 53 to 54, wherein said hyaluronic acid is present at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and said ratio between said hyaluronic acid and said BDDE is between 1:0.04 and 1:0.06, optionally about 1:0.05.

58. The composition according to claim 57, characterized by average extrusion force of between 10 and 25 Newtons, optionally between 17 and 23 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a storage modulus G’ of between 50 and 200 Pa, optionally between 75 and 150 Pa, as measured with a rheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %.

59. The composition according to any one of claims 53 to 54, wherein said hyaluronic acid is present at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and said ratio between said hyaluronic acid and said BDDE is between 1:0.07 and 1:0.11, optionally about 1:0.09.

60. The composition according to claim 59, characterized by average extrusion force of between 15 and 30 Newtons, optionally between 20 and 26 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a storage modulus G’ of between 200 and 400 Pa, optionally between 250 and 350 Pa, as measured with a rheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %.

61. The injectable composition according to any one of claims 53 to 54, wherein said hyaluronic acid is present at between 1.5 and 2.5 weight percent, said gelatin is present between 1.8 and 3.5 weight percent, and said ratio between said hyaluronic acid and said BDDE is between 1:0.115 and 1:0.17, optionally about 1:0.14.

62. The injectable composition according to claim 61, characterized by average extrusion force of between 20 and 35 Newtons, optionally between 25 and 33 Newtons, a pH value in the range of 6.5 to 7.5, osmolarity in the range of 270-370 mOsm / kg, and a storage modulus G’ of between 300 and 600 Pa, optionally between 350 and 500 Pa, as measured with arheometer in a 25-mm plate-to-plate configuration with 1 mm gap at 1 Hz and 37°C, and shearing under constant strain of 0.1 %.

63. A method for restoring volume, reducing smooth wrinkles, and / or enhancing facial contours in an individual in need thereof, said method comprising injecting into the skin of said individual an effective amount of a composition according to any one of claims 38-59.

64. The method of claim 63, wherein said composition is characterized by pH in the range of 6-8, optionally, 6.8-7.2; osmolarity in the range of 250-400 mOsm / L, optionally, 320-350 mOsm / L; extrusion force in the range of 12-30N; and G’ in the range of about 100-500 Pa.

65. A method for improving skin quality of an individual in need thereof, by enhancing hydration, elasticity, and texture of said skin, said method comprising injecting into a superficial layer of a skin of said individual an effective amount of a composition according to any one of claims 41-62.

66. The method of claim 65, wherein said composition is injected into a superficial layer of a skin in the face, neck, hands, or decolletage of said individual.

67. The method of claim 65 or 66, wherein said composition is characterized by pH in the range of 6-8, optionally, 6.8-7.2; osmolarity in the range of 250-400 mOsm / L, optionally, 320-350 mOsm / L; extrusion force in the range of 5-15N; and viscosity in the range of about 100-500 mPa’sec.

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