Patent ID: 12226549

DETAILED DESCRIPTION OF THE INVENTION

The term “synergistically cooperative”, used herein in respect of a composition or product means a composition or product wherein its components interact with one another in such a way that one component enhances the viscoelastic performance of a product made by other components; in the present invention, this property is obtained by formulating the composition or product in accordance with the present claims.

The term “aqueous”, referred to a solution, indicates a solution containing water for more than 50%, preferably more than 85%, more preferably more than 95% of its weight, in association with a water-miscible solvent, e.g ethanol, n-propanol, i-propanol, etc.; most preferably the term “aqueous” means water as the sole solvent; the aqueous solution can also be a buffered aqueous solution, for example a phosphate buffer solution (PBS), e.g. buffered at a physiologically compatible pH. From the physical point of view, the term “solution” is broadly used herein to indicate a homogeneous liquid system whose components are therein dissolved and/or finely dispersed. e.g. emulsified.

The term “composition” used herein, means the result of a physical mixing of its components (glycosaminoglycan, vitamin E and stabilizer) in which each of them maintains its individuality as a single molecule. Consequently, the term “composition” excludes products characterized by a formal chemical binding among the above components, such as realized by covalent binding; the term “composition” remains yet compatible with products in which said components are in free state or they are coordinated via electrostatic forces, e.g. hydrogen bonds, hydrophobic interactions, Van der Waals interactions, solvation forces, etc.; they may also be engaged via other forms of physical binding e.g. by incorporation, inclusion, emulsion, etc. The same conditions apply also to pharmaceutically or cosmetically active ingredients (optionally) present in the compositions.

The present compositions are also described herein as “complexes”, wherein this term includes coordination complexes and extends to functional complexes, i.e. any system in which the single components, retaining their individual molecular character, cooperate synergistically to improve the viscoelastic properties of the composition.

The term “glycosaminoglycan” means herein, in agreement with the technical literature, a polysaccharide containing a repeating disaccharide unit, said repeating unit containing an amino sugar (e.g. N-acetylglucosamine or N-acetylgalactosamine) along with an uronic sugar (e.g. glucuronic acid or iduronic acid) or galactose. Glycosaminoglycans are highly polar and attract water. They are useful to the body as a lubricant or as a shock absorber. Examples of glycosaminoglycans useful for the purpose of the present invention are hyaluronic acid and salts thereof, as well as chondroitin sulfate, chondroitin salts (e.g. sodium chondroitin), dermatan sulfate, heparan sulfate, etc. Mixtures of one or more of glycosaminoglycans can also be used in the present invention; particularly preferred are mixtures of hyaluronic acid with one or more glycosaminoglycans different from hyaluronic acid, in particular chondroitin sulfate. The glycosaminoglycan is used in the present solutions at a weight concentration of 0.01 to 25% by weight of the solution, preferably 0.01 to 10%, more preferably 0.1 to 10%; when mixtures of two or more glycosaminoglycans are used, the above intervals of concentration are meant to be referred to the total weight of glycosaminoglycans by weight of the solution.

Hyaluronic acid is a preferred glycosaminoglycan for use in the present invention. The term “hyaluronic acid” means herein, in agreement with common general knowledge, a glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) linked together by alternating beta-1,4 and beta-1,3 glycosidic bonds. Hyaluronic acid is also known as hyaluronan, hyaluronate, or HA. The terms hyaluronan, hyaluronic acid and HA are used interchangeably herein. Hyaluronic acid can be used as such or in a salt form (hyaluronate) and has average molecular weight preferably comprised between 40 kDa to 4000 kDa; a particularly preferred product for use in the invention is the Ultrapure sodium hyaluronate produced in accordance with the patent publication WO2014/005822 herein incorporated by reference. The HA is used in the invention within the concentration ranges referred above for glycosaminoglycans. Other preferred glycosaminoglycans are chondroitin sulfate and chondroitin salts (e.g. sodium chondroitin), dermatan sulfate, heparan sulfate and derivatives thereof.

“Vitamin E” refers, as well known in the art, to a group of compounds that include both tocopherols and tocotrienols (in particular, α-, β-, γ-, or δ-tocopherols and α-, β-, γ-, or δ-tocotrienols). The term “Vitamin E” means herein any of these compounds taken alone or any of its mixtures, irrespective of whether such compounds or mixtures occur in nature; the term “Vitamin E” also includes any possible salts and derivatives of the above compounds, e.g. Vitamin E esters, such as tocopherol acetate. Vitamin E is well-known as a peroxyl radical scavenger, disabling the production of damaging free radicals in tissues, by reacting with them to form a tocopheryl radical, which will then be reduced by a hydrogen donor and thus return to its reduced state. As it is fat-soluble, it is incorporated into cell membranes, which protects them from oxidative damage. In the present invention, the Vitamin E is used at a weight concentration from 0.0001 to 15%, preferably 0.1 to 15%, more preferably 0.1 to 10%, by weight of the solution. When mixtures of tocopherols and/or tocotrienols are used, the above intervals of concentration are meant to be referred to the total weight of said tocopherols and/or tocotrienols by weight of the solution.

The stabilizer is used in the present invention in a weight concentration range from 0.01 to 25% preferably 0.01 to 10%, more preferably 0.1 to 10% by weight of the solution. The term “stabilizer” refers herein in general to selected products capable to enhance/stabilize the viscoelastic performance of a physical mixture of glycosaminoglycan and vitamin E. Stabilizers according to the present invention are chosen from polyether clathrates, e.g. a cyclodextrin, or a mixture of fatty acid with an emulsifier.

“Polyether clathrates” are herein defined as structures comprising one or more macrocyclic rings (i.e. containing at least 12 atoms) said ring comprising, separated from each other, two or more oxygen heteroatoms (i.e. the ring comprises at least two ether bonds). From the functional point of view, as known in the art, clathrates form a molecular cage capable of hosting foreign molecules, compatibly with the mutual dimensions. Preferred examples of polyether clathrates for use in the present invention are cyclodextrins. Cyclodextrins are hydrophobic inside and hydrophilic outside, they can form complexes with hydrophobic compounds; they can enhance the solubility and bioavailability of such compounds. Cyclodextrins are able to form host-guest complexes with hydrophobic molecules given the nature imparted by their structure. This is of high interest for pharmaceutical as well as dietary supplement applications in which hydrophobic compounds shall be delivered. Examples of cyclodextrins are α-, β-, or γ-cyclodextrin (in which the macrocyclic ring as described above contains, respectively 30, 35 or 40 atoms). Particularly preferred for use in the present invention are derivatized cyclodextrin, such as propyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, sulfobutyl ether 4-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin; mixtures of cyclodextrins are also contemplated by the invention. Other examples of polyether clathrates, different from cyclodextrins, are crown ethers, for example, 12-crown-4, 15-crown-5, 18-crown-6, dibenzo-18-crown-6 ether.

Another stabilizer which can be used in the present invention is a combination of fatty acid with an emulsifier: these two ingredients are regarded herein as one component (“stabilizer”), even when they are separately added to the composition. In all these cases, the above given ranges of concentration for the stabilizer (0.01 to 25% preferably 0.01 to 15%, more preferably 0.1 to 10%) are meant to refer to the total weight of fatty acid and emulsifier, by weight of the solution; the whole of fatty acid and emulsifier, generally contains from 0.0995 to 9.5% by weight of fatty acid, the remainder being the emulsifier. The combination of fatty acid and emulsifiers can be prepared apart and added to the other components of the cooperative composition or, in alternative, the fatty acid and the emulsifier can be added separately thereto; in both cases the fatty acid and the emulsifier are able to interact and form an emulsion, whereby the fatty acid is homogeneously dispersed within the present solutions.

Fatty acids can be organic, monobasic acids derived from hydrocarbons by the equivalent of oxidation of a methyl group to an alcohol, aldehyde, and then acid. Fatty acids can be saturated and unsaturated. Preferred fatty acids are ω-3 fatty acids. The term “fatty acids” used herein includes, for example, lipoic acid, oleic acid, linoleic acid, linolenic acid, α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid or other omega-3-fatty acids; the term “fatty acids” also extends to molecules which are derivatized with fatty acids, such as triglycerides, phospholipids etc. Suitable non-ionic emulsifier include poly(oxyethylene)-poly(oxypropylene) block copolymers, commercially known as poloxamer and pluronic; polysorbates, such as Tween 20 or Tween 80. Amphoteric surfactants include quaternized imidazole derivatives.

The present compositions may optionally include one or more pharmaceutically and/or cosmetically active ingredients, where the term “active” identifies compounds or entities that alter, inhibit, activate or otherwise affect biological or chemical events obtaining, respectively, a pharmaceutical- or cosmetic effect. Preferably, the active ingredient is a drug for human or animal use, with no limitations as to the pharmacologic class. Preferred pharmaceutically active agents used in the invention are anti-inflammatory drugs: examples thereof are e.g. salicylic acid, aspirin, mefenamic acid, tolfenamic acid, flufenamic acid, diclofenac, diclofenac, sulindac, fenbufen, indometacin, acemetacin, amfenac, etodolac, felbinac, ibuprofen, flurbiprofen, ketoprofen, naproxen, pranoprofen, fenoprofen, tiaprofenic acid, oxaprozin, loxoprofen, alminoprofen, zaltoprofen, piroxicam, tenoxicam, lornoxicam, meloxicam, tiaramide, tolmetin, diflunisal, acetaminophen, floctafenine, tinoridine, actarit, pharmaceutically acceptable salts thereof (for example diclofenac sodium), and mixtures thereof. When present, the pharmaceutically or cosmetically active agent is at a weight concentration preferably comprised from 0.0001 to 10% by weight of the solution; it may be present in free form or in electrostatic interaction with one or more of the main components of the composition (glycosaminoglycan, vitamin E and stabilizer). When more pharmaceutically and/or cosmetically active agents are present, the above concentration range is meant to be referred to the total sum of such agents.

Further excipients can be optionally present in the compositions, depending on the specific type of formulation considered and its final use. Among the excipients, there can be mentioned: preservatives, viscosity adjusting agents (thickening or fluidifying agents), emulsifiers (if not already present as fatty acid/emulsifier mixture), chelating agents, buffering agents, tonicity adjusting agents, co-solvents, etc. further optional agents present in the compositions are antioxidants such as ascorbic acid, melatonin, vitamin C, proteins (e.g., serum hyaluronidase inhibitors), etc.

A further object of the present invention is a process to prepare a composition as above described. In its general scope, the process comprises forming an aqueous solution of: (a) a glycosaminoglycan or mixtures thereof, (b) a stabilizer as herein defined, and (c) one or more tocopherols, tocotrienols and mixtures thereof, wherein the glycosaminoglycan is present at a concentration of 0.01 to 25%; the stabilizer is present at a concentration of 0.01 to 25% by weight; the tocopherols, tocotrienols and mixtures thereof are present at a concentration of 0.0001 to 10% by weight of the solution. In a more detailed embodiment, the above process is performed by adding into a suitable mixer: the stabilizer, the glycosaminoglycan and the tocopherols/tocotrienols; when the stabilizer is a lipid with emulsifier, they can be added separately or in premixed form; the aqueous component can be added at any time, at once or preferably stepwise, during the above described procedure; typically the aqueous component is added (at least in part) together with the first component being introduced in the container and the remainder (if any) is added in one or more steps during the rest of the process. All the above operations are suitably performed under agitation, which can be continued after the last addition for a time sufficient to obtain a single homogeneous phase, typically 2-10 hours; the whole process can be conveniently performed at ambient temperature (20-25° C.).

The compositions of the invention may be finally sterilized to obtain a product of pharmaceutical/cosmetic grade. All sterilization procedure can be used, e.g. ultrafiltration, dry heat, wet heat, γ-radiation, etc. Advantageously, the above referred components cooperate synergistically in protecting the resulting solution, in particular its viscoelastic profile, from thermal degradation: the solutions are thus treatable in autoclave procedures (as examples a typical autoclaving cycle involves treatment at 121° C., at a pressure of about 1 atm for 20 minutes or equivalent validated combination to obtain a sterile product) or by other thermal methods, with lesser influence on their final viscoelastic properties, compared to glycosaminoglycan solutions currently used for viscosupplementation; the increased thermal resistance can be verified by standard means, in particular in terms of preservation of elastic modulus G′ and the viscous modulus G″ in a frequency range from 0.01 to 10 Hz.

A further set of embodiments of the present invention is described by the following clauses 1-17.1. A synergistically cooperative composition, in aqueous solution form, comprising: (a) glycosaminoglycan or mixtures thereof at a weight concentration of 0.01 to 25%, (b) cyclodextrin at a weight concentration of 0.01 to 25%, (c) a lipid with emulsifier a weight concentration of 0.0001 to 15%, by weight of the solution, wherein said components (a), (b) and (c) are not engaged in formal chemical binding with each other.2. Composition according to clause 1, wherein the glycosaminoglycan is selected from hyaluronic acid or salt thereof, chondroitin sulfate, sodium chondroitin, dermatan sulfate or heparan sulfate.3. Composition according to clauses 1-2, wherein the hyaluronic acid in form of two or more fractions thereof with different average molecular weights, each comprised between 40 to 4000 kDa.4. Composition according to clauses 1-3 wherein the hyaluronic acid salt is selected from sodium, potassium, ammonium, calcium, magnesium, zinc and cobalt salts and mixtures thereof.5. Composition according to clauses 1-4, wherein the lipid comprises one or more of lipoic acid, oleic acid, linoleic acid, linolenic acid, α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid or other omega-3-fatty acids, triglycerides and phospholipids.6. Composition according to clauses 1-5, wherein the emulsifier is selected from phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol (DPPG); hexanedecanol; polyoxyethylene-9-lauryl ether; sorbitan trioleate (Span 85) glycocholate; sorbitan monolaurate (Span 20); polysorbate 20 (Tween-20); polysorbate 60 (Tween-60); polysorbate 65 (Tween-65); polysorbate 80 (Tween-80); polysorbate 85 (Tween-85); poloxomers or pluronics; sorbitan fatty acid ester such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol; poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethylene glycol)400-monostearate; phospholipids; synthetic and/or natural detergents having high surfactant properties; deoxycholates; cyclodextrins; chaotropic salts; ion pairing agents; and mixtures thereof.7. Composition according to clauses 1-6, wherein the tocopherol is chosen from an α-, β-, γ- or δ-tocopherol and mixtures thereof.8. Composition according to clauses 1-7, wherein the tocotrienol is chosen from an α-, β-, γ- and δ-tocotrienol and mixtures thereof.9. Composition according to clauses 1-8, further comprising a pharmaceutically- or cosmetically active agent.10. Compositions according to clause 9, wherein the pharmaceutically active agent includes one or more non-steroidal anti-inflammatory drugs.11. Composition according to clause 10, wherein the anti-inflammatory drug is selected from salicylic acid, aspirin, mefenamic acid, tolfenamic acid, flufenamic acid, diclofenac, sulindac, fenbufen, indometacin, acemetacin, amfenac, etodolac, felbinac, ibuprofen, flurbiprofen, ketoprofen, naproxen, pranoprofen, fenoprofen, tiaprofenic acid, oxaprozin, loxoprofen, alminoprofen, zaltoprofen, piroxicam, tenoxicam, lornoxicam, meloxicam, tiaramide, tolmetin, diflunisal, acetaminophen, floctafenine, tinoridine, actarit, pharmaceutically acceptable salts thereof and mixtures thereof, in concentration of 0.0001 to 10% by weight of the solution.12. Composition according to clause 11, wherein the antiinflammatory drug is diclofenac sodium.13. A stabilized cooperative complex in accordance with clauses 1-12.14. A process of preparing a composition or complex according to clauses 1-13, comprising forming an aqueous solution of a glycosaminoglycan or mixtures thereof at a weight concentration of 0.01 to 25%, a fatty acid and emulsifier at overall weight concentration of 0.01 to 25% by weight and one or more tocopherols or tocotrienols and mixtures thereof at a weight concentration of 0.0001 to 15%, by weight of the solution.15. The composition or complex according to clauses 1-13, for use in therapy.16. The composition or complex according to clauses 1-13, for use in a medical method of treatment selected from: dermo-cosmetic, esthetic, soft tissue augmentation, viscosupplementation, dermal-filling, regenerative treatments and drug delivery applications.17. Use of the composition or complex according to clauses 1-13, in a cosmetic method of treatment selected from: dermo-cosmetic, esthetic, soft tissue augmentation, viscosupplementation, dermal-filling, regenerative treatments.

The invention is further described in non-limitative manner by the following examples.

EXAMPLES

1. Formulations and Preparation

The following Table shows the composition of the Formulation Examples 1 to 5.

TABLE 1Composition of Formulation Examples 1 to 5.ExampleExampleExampleExampleExample12345Compositionw/w %HA22222FA (omega 311———mixtures)Pluronic0.020.02———CD——22—VE—2—22HA = hyaluronic acid;FA = fatty acid;CD = cyclodextrin;VE = Vitamin E

1.1. Examples 1 (Reference) and 2 (Invention)

Solutions according to Examples 1 and 2 were obtained by the following procedure:Suitable emulsifier was weighed into an appropriate container and then diluted in PBS.FA was weighed and added to the previous solution.HA according to the formulation Examples, and Vitamin E if present, were added to the previous mixture at room temperature.Finally, the previous mixture was diluted by PBS, maintaining stirring for at least 3 to 8 hours.The product resulting from described procedure is homogeneous with one phase.

1.2. Examples 3 (Reference) and 4 (Invention)

Solutions according to Examples 3 and 4 were obtained by the following procedure:CD was weighed into an appropriate container and then diluted in Phosphate Buffer Solution (PBS).HA according to the formulation Examples, and Vitamin E if present, were added to the previous solution at room temperature.Finally, the previous mixture was diluted by PBS, maintaining stirring for at least 3 to 8 hours.The product resulting from described procedure is homogeneous with one phase.

1.3. Example 5 (Reference)

The solution according to Example 5 was obtained by the following procedure:HA according to the formulation Example, and Vit E, were added to PBS, maintaining stirring for at least 3 to 8 hours.The product resulting from described procedure is homogeneous with one phase.

2. Assessment of Viscoelastic Properties

Viscoelastic properties measurements were carried out through a strain controlled rotational rheometer (Mars III, HAAKE Rheometer, Waltham, MA USA), using a parallel plate geometry at 20 and 37° C. The frequency was in the range from 0.01 to 10 Hz. In order to identify the linear viscoelastic response range of the materials, preliminary strain sweep tests were performed on the samples, at the oscillation frequency of 1 Hz. The tests were repeated at least three times on each sample.

The dependence of the elastic modulus G′ and the viscous modulus G″ as function of frequency, the so called “mechanical spectra” are reported in theFIGS.1-4. In particular:

FIG.1compares the mechanical spectra of Example 2 (invention) and 5 (reference), prior/after performing the autoclaving (AC) cycle: the spectra are recorded at 20° C. (FIG.1A,1B) or 37° C. (FIG.1C, D).

FIG.2compares the mechanical spectra of Example 4 (invention) and 5 (reference), prior/after performing the autoclaving (AC) cycle: the spectra are recorded at 20° C. (FIG.2A,2B) or 37° C. (FIG.2C,2D).

FromFIG.1A(or1C) it is evident that the addition of the stabilizer (FA/emulsifier) to the reference solution containing HA+Vitamin E enhances the viscoelastic properties (both G′, G″) of the resulting solution.

After the AC treatment, the above difference becomes even larger: cf.FIG.1Avs.1B (orFIG.1Cvs.1D) showing that, for the solution of the invention, the values G′ and G″ are highly preserved after the AC treatment, whereas in the reference solution they undergo a clear decline.

FromFIG.2A(or2C) it is evident that the addition of the stabilizer (CD) to the reference solution containing HA+Vitamin E enhances the viscoelastic properties (both G′, G″) of the resulting solution.

After the AC treatment, the above difference becomes even larger: cf.FIG.2Avs.2B (orFIG.2Cvs.2D) showing that, for the solution of the invention, the values G′ and G″ are highly preserved after the AC treatment, whereas in the reference solution they undergo a clear decline.

In the following table 2 the ratio of elastic modulus of Ex4 and Ex 2 respect to Ex5 at 1 Hz are reported. The data show that the addition of CD (or FA+emulsifier) to a reference solution of HA and Vitamin E leads to an increase of, at the least twice, the elastic modulus ratio. This result indicates that both CD or FA interact with vitamin E and HA through secondary bonds cooperating in the formation of complexes among the molecules that stabilize the network. These cooperating complexes are further stabilised (i.e. better protected) when the formulations are thermically processed by heating at a temperature between 80° C. and 130° C. for a processing time between 10 and 30′ and then quickly cooled at 20/37° C. After the thermal treatment and the quenching there is an increase of the elastic modulus ratio that is from 4 to 12 times due to the presence of CD or FA+emulsifier.

TABLE 2The elastic modulus ratio at 1 Hz of the examples formulations.20° C.37° C.Ratio20° C.after AC37° C.after ACG′Ex.2/G′Ex.5242.56G′Ex.4/G′Ex.52.162.312

3. Rheological Synergism of the Cooperating Complexes

The interactions between HA, VE, and CD or FA+emulsifier lead to cooperating complexes that stabilize the network and result in improved rheological properties. This improvement in the viscoelastic properties indicates that exists a rheological synergism between HA and Vit E and CD or HA and Vit E and FA. The rheological synergy can be quantified by the interaction parameter, that is the difference between the dynamic modulus values of the mixture evaluated by rheological test and the theoretical one given by adding the dynamic modulus values of the primary components. For the composition of Ex. 2 the synergistic parameter (ΔG′Synergistic) is described by the following formula (1), wherein a positive value of ΔG′Synergisticindicates the presence of synergism.
ΔG′Synergistic=G′Ex.2−(G′HA+VE+G′FA)  (1)

The results of the calculation of synergistic parameters for Ex. 2 at 20 and 37° C. before and after AC are reported in table 3.

TABLE 3the interaction parameters of Ex. 2 at 1 HzG′Ex.2G′HA+VEG′FAΔG′SynergisticCondition[Pa][Pa][Pa][Pa]Synergism20° C.150780.015+72YES20° C.55130.011+42YESAfter AC37° C.133530.015+80YES37° C.4980.011+41YESAfter AC

For the final composition of Ex. 4 the synergistic parameter ΔG′Synergisticis described by formula (2) at 20 and 37° C. before and after AC:
ΔG′Synergistic=G′Ex.4−(G′HA+VE+G′CD)  (2)

The results of the calculation of synergistic parameters for Ex. 4 at 20 and 37° C. before and after AC are reported in table 4.

TABLE 4the interaction parameters of Ex. 4 at 1 Hz.G′Ex.4G′HA+VEG′CDΔG′SynergisticCondition[Pa][Pa][Pa][Pa]Synergism20° C.166780.013+88YES20° C.78130.011+65YESAfter AC37° C.130530.013+77YES37° C.4880.013+40YESAfter AC

The data in table 3 (Ex. 2) and table 4 (Ex. 4) show a strongly positive (ΔG′Synergistic) parameter, thus indicating a strong synergism. This remains well evident for the solutions measured at different temperature (20 or 37° C.), either prior or after the autoclaving cycle.

4. Mechanical Stability of the Formulations

The comparison between the mechanical spectra of Ex. 1 and Ex. 2 as well as Ex. 3 and Ex. 4 are shown inFIGS.3and4, respectively. In particular:

FIG.3compares the mechanical spectra of Example 2 (invention) and 1 (reference), prior/after performing the autoclaving (AC) cycle: the spectra are recorded at 20° C. (FIG.3A,3B) or 37° C. (FIG.3C,3D).

FIG.4compares the mechanical spectra of Example 4 (invention) and 3 (reference), prior/after performing the autocalving (AC) cycle: the spectra are recorded at 20° C. (FIG.4A,4B) or 37° C. (FIG.4C,4D).

As it can be seen fromFIG.3A(or3C), the addition of Vit E increased the stability of the Ex. 2 formulation in comparison with the reference Ex. 1 which does not possess Vit E. Moreover, the same trend is observable when the mechanical spectra of Ex. 4 is compared to Ex. 3 that does not have Vit E (FIG.4). The presence of Vit E is thus essential for the formation of the cooperating complexes and for the stabilization of the network and the consequence improvement of the viscoelastic properties.

Tables 5 and 6 report the values of G′ and G″ for Ex. 1, Ex. 2 and Ex 3, Ex 4 respectively, at 20 and 37° C. before and after AC. As it can be from the tables, before autoclaving, G′ of Ex 2 [150 Pa] and G′ of Ex 4 [166 Pa] are more than two folds higher than G′ Ex 1 and Ex 3. Furthermore, after the thermal processing the viscoelastic properties of the ternary systems are better preserved, indicating that the cooperating complexes that are created by the interactions among the HA and CD and Vit E or HA, Vit E and FA stabiles the network systems of these formulations. Indeed G′ of Ex 2 after autoclaving tested at 37° C. is 49 Pa while G′ of Ex 1 is 2 Pa and G′ of Ex 4 is 43 Pa while G′ of Ex 3 is 15 Pa.

TABLE 5The values of G′ and G″ at 1 Hz for formulations ofEx. 1 and Ex. 2.Before ACAfter ACBefore ACAfter ACat 20° C.at 20° C.at 37° C.at 37° C.EntryG′ [Pa]G″ [Pa]G′ [Pa]G″ [Pa]G′ [Pa]G″ [Pa]G′ [Pa]G″ [Pa]Example 1726315204951211(reference)Example 215010755801331004972(invention)

TABLE 6The values of G′ and G″ at 1 Hz for formulations ofEx. 3 and Ex. 4.Before ACAfter ACBefore ACAfter ACat 20at 20at 37at 37EntryG′ [Pa]G″ [Pa]G′ [Pa]G″ [Pa]G′ [Pa]G″ [Pa]G′ [Pa]G″ [Pa]Example 36657172453491520(reference)Example 41661197898126984367(invention)

The table 5 further shows a better viscoelastic performance for the example 2 of the invention, compared to example 1; the higher viscoelastic performance of Example 2 remains also after the AC, showing a better protection of viscoelastic properties compared to the Example 1.

The table 6 further shows a better viscoelastic performance for the example 4 of the invention, compared to example 3; the higher viscoelastic performance of Example 4 remains also after the AC, showing a better protection of viscoelastic properties compared to the Example 3.

5. Drug Solubility of the Formulations

Different amounts of DF-Na including 2.5, 5.7, and 20 mg/ml were added to the formulation of Example 4, the formulations were stirred until being completely homogenised. Once prepared they were kept for 24 hrs and after centrifuged (6000 rpm for 15 min). Finally, the supernatants were analyzed by means of UV spectrophotometer. The tests were performed in triplicate and the wavelengths used for the detection of diclofenac sodium was 276 nm. In order to associate the recorded absorbance to the amount of DF-Na in the supernatant, a calibration curve was constructed by plotting absorbance against predetermined concentration of DF-Na. Then, linear regression was used to determine the regression equation representing the calibration curve. The results of the dissolution tests are reported in table as solubilized fraction (SF) as the ratio between DF-Na which was found into the supernatant of the solution, and total DF-Na as expressed by:

SF⁢⁢%=solubilized⁢⁢DFTotal⁢⁢DF×100

The results of dissolution test are shown in table 7.

TABLE 7Solubilized fraction (SF) of Ex. 4 with differentconcentration of DF-NaTotal DF-Na,Solubilizedmg/mlfraction (SF), %2.5935.7892078

6. Drug Release Ability of the Formulations

The drug release profiles from the formulation of Ex 2 and Ex 4 loaded with diclofenac sodium (DF-Na) are shown inFIG.5. To perform the release test, 1 gr of the formulation containing DF-Na at 1% was inserted in a dialysis membrane, with cut off 500 to 1000 Da, that was immersed in PBS medium (18 ml) at the temperature of 37° C. At predetermined time intervals, 50 μL aliquots of the medium was withdrawn and the same volume of fresh medium was added. The drug concentration released into the PBS buffer was detected by UV spectrophotometer as a function of time. The calibration curve was constructed as explained in the previous paragraph. The tests were performed in triplicate and the wavelengths used for the detection of diclofenac sodium was 276 nm.

For the formulation of Ex. 2 with DF-Na (FIG.5A), it can be seen that after 8 hours 69% of the drug was released in the medium and after 24 hrs there is the complete release of the drug. Moreover, for the Ex. 4 with DF-Na (FIG.5B), it can be seen that after 10 hours 48% of the drug was released in the medium and after 24 hrs there is the complete release of the drug. These formulations are able to control the delivery of the drug molecules over the time. There was very good reproducibility between the triplicates. Prominently, no significant differences between samples was observed which indicate a homogenous diffusion in the prepared composition.