Field of the Invention

The present invention relates to injectable viscoelastic formulations which are developed by means of synergistic effect of hyaluronic acid (HA), vitamins, minerals, antioxidants and osmoprotectants, and which can be used in intraarticular, intradermal and ophthalmic applications.

Background of the Invention Hyaluronic acid (HA)

Hyaluronic acid (HA) is a polyanionic, unsulfated glycosaminoglycan formed of N-acetyl-D-glucosamine and beta-glucoronic acid naturally. The said material, whose molecular weight varies in range of 0.1-10 million Daltons, has a special rheological character due to its high molecular weight, polyanionic character and unbranched chain structure. Its molecular volume grows approximately 10,000 times when hydrated compared to its dry state. HA is the most important and essential element of the extracellular matrix in connective tissue. It is present in the vitreous fluid of the eye, hyaline cartilage, synovial fluid, dermis and epidermis. It increases the volume in the vitreous fluid, serves as a lubricant in the tendons and muscles, thereby providing lubrication to the joints; it increases the stability of the spine and enables a relationship between the mother and the fetus inside the umbilical cord. It also plays regulatory roles in cell motility, cellular proliferation, morphogenesis, embryonic development, cancer metastasis and inflammation. HA's unique viscoelastic properties, its biocompatibility, and its non-immunogenicity have led to its use in a number of clinical applications, such as as a supplement for synovial fluid in joint arthritis, as a viscoelastic adjuvant in eye surgery, and such as facilitating the healing and regeneration of surgical wounds. Recently, HA has been studied as a drug delivery agent for various means of administration, including ophthalmic, nasal, pulmonary, parenteral, and topical administrations.

The use of HA in intraarticular applications

One of the uses of HA in intraarticular applications is for the treatment of Osteoarthritis. Osteoarthritis (OA) is the most common, chronic (long-term) joint disease affecting millions of people worldwide. The joint is where two bones are connected and the ends of these bones are covered with a protective tissue called cartilage. Cartilage is a durable and slippery tissue which allows joint movement to occur almost without friction. Synovial fluid, which provides lubricity between bone ends and cartilage, is comprised of Hyaluronic Acid (HA) that is naturally present in the body. The functions of synovial fluid are to reduce friction by lubricating the joint, to absorb shocks, to supply oxygen and nutrients to the chondrocytes of the articular cartilage, and to remove carbon dioxide and metabolic waste since the cartilage is not vascularized. The viscosity, lubrication and shock absorbing properties of the said synovial fluid decrease over time due to mechanical effects and the cartilages begin to rub against each other and dissolve. As the cartilage wears off and breaks down over time, the friction between the bones increases and OA progresses. OA affects the entire joint, as well as cartilage breakdown. It causes changes in the bone structure and the deterioration of the connective tissues that hold the joint together and connect the muscles to the bone; and it also causes inflammation of the joint capsule. Injections of HA, which is a component found naturally in the synovial fluid, exhibit a buffering effect on the knee and alleviate the severity of pain.

The use of HA in intradermal applications

Hyaluronic acid (HA) is a polysaccharide which is effective in many topical and subcutaneous anti-aging treatments such as dermal fdlers, and which is frequently used by utilizing its unique viscoelastic properties for soft tissue augmentation. When applied subcutaneously, HA gives the skin a fuller, younger appearance and fdls in wrinkles. Due to aging, gravity, sun exposure, muscle movements such as laughing and chewing, the underlying tissues which make our skin look younger begin to deteriorate. For this reason, wrinkles around the eyes, laugh lines, crow's feet and facial wrinkles occur. Soft tissue fillers help fill in these lines and wrinkles by means of temporarily restoring a smoother, younger-looking appearance. HA has properties which make it an appealing material for the use as dermal fillers due to the reasons such as its ability to bind large amounts of water, its natural presence in the skin, and its lack of adverse reactions.

The use of HA in ophthalmic applications

The uses of HA in ophthalmic applications are also known from the state of the art. Hyaluronic acid is an important component of the vitreous part of the eye and it is an important macromolecule used in ophthalmology. HA is used in a whole range of important ophthalmic surgeries due to its viscoelastic properties. Ophthalmic viscosurgical devices (OVDs) containing HA are transparent gels having viscous and elastic properties and used in cataract surgery. OVDs create and maintain anterior chamber depth and visibility. It also protects the comeal endothelium and other intraocular tissues during surgery. They minimize interaction between tissues and instruments, thereby providing high tissue integrity.

Dermis - cartilage - collagen synthesis - remodeling

The ultraviolet (UV) component in sunlight comprises short wavelength UVB (290-320 nm) and long wavelength UVA (320-400 nm). UVA, which is the main UV component, penetrates deeper into the dermis of the skin than the UVB, which essentially penetrates into the epidermis, triggering sunburn and cancer after a long period of time. Contrary to this, chronic exposure of normal human skin to UVA irradiation causes the characteristic features of aged skin, namely deep wrinkle formation and low elasticity of the dermis. This situation is called photoaging. The primary changes due to photoaging are the decrease in the amount of collagen matrix in the top and middle parts of the dermis and the replacement of collagen matrix with an elastotic material called actinic elastosis. In the photoaged skin, UVA-induced matrix metalloproteinase- 1 (MMP-1) expression, controlled by the transcription factor AP-1, reduces the amount of dermal collagen. Contrary to this, for collagenogenesis, ascorbic acid, which is an essential vitamin for collagen matrix synthesis in connective tissue, can produce prolyl and lysyl hydroxy lations necessary for the maturation of collagen peptide. It is well known that the expression of ascorbic acid transporters, namely the sodium ascorbate co-transporters, SVCT1 and SVCT2 expression-which play an important role in the ascorbic acid aggregation in the cytoplasm of fibroblasts- and the plasma ascorbic acid concentration decrease together with advancing age. For this reason, it is logical to predict that the concentration of ascorbic acid in the fibroblast cytoplasm is also reduced; this will be an important factor in the decreased synthesis of the dermal collagen matrix observed in the atrophic change of intrinsic skin aging.

Chronic exposure to UV alters the composition of the extracellular matrix (ECM) rich in dense collagen. The ECM is comprised of connective tissue and basement membrane proteins such as elastin, glycosaminoglycans, and interstitial collagen. In human skin, UV radiation can elevate the levels of various matrix metalloproteinases (MMPs), including MMP-1, MMP-3 and MMP-9. Among these, MMP-1, fibroblast collagenase, is the key enzyme responsible for the breakdown of dermal components in the ECM. When elevated levels of MMP-1 start the degradation of fibrillar type I and III collagen, MMP-3 and MMP-9 follow the subsequent processes. Therefore, MMP-1 plays an important role in the initiation of UVB-induced wrinkle formation via ECM degradation. UV-induced MMP-1 overexpression is followed by upregulation of the mitogen-activated protein kinase (MAPK) signal pathway via various factors such as cytokines and growth factor receptors. Activator protein (AP)-l is a transcription factor and an important effector of the MAPK pathway in the regulation of MMP expression. AP-1 forms heterodimer complexes between Jun, Fos or activating transcription factor proteins. Since chronic exposure to UVB can trigger photoaging, inhibition of MMP-1 expression may result in preventive effects. Human skin is a serious barrier in terms of using topical drugs. The stratum comeum is the main layer responsible for barrier function and consists of comeocytes in a highly organized lipid matrix. Since water corresponds to only 15-30% of the stratum comeum, a hydrophilic material can hardly penetrate into this hydrophobic layer and, as a result it cannot pass into other layers of the skin. For this reason, the use of topical drugs is limited for many molecules. For a drug to be effective topically, it must have the following characteristics:

• The molecular weight of the drug should be lower than almost 800 daltons.

• The drug should have hydrophilic and hydrophobic affinity in its chemical structure.

• Over-splitting property is not suitable for successful transdermal drug delivery.

• The drug should preferably have low melting point.

• pH value of the drug solution should preferably be in range of 4.2-5.6.

Due to the reasons mentioned above, many molecules cannot reach the dermis in effective amounts when they are used topically.

Vitamins

Vitamins are a large group of organic compounds which cannot usually be synthesized by the human body, but are essential for maintaining normal functions properly. Under normal conditions, we can obtain different vitamins from food and proper nutrition, however most of the time minimum nutritional requirements are not met, which means they must be obtained through supplements. Vitamins are essential for the growth and proper functioning of the body, as well as metabolism. Only vitamin D is produced by the body; all other vitamins are obtained from foods or supplements. Vitamins can be divided into two groups depending on their solubility: water-soluble vitamins and fat-soluble vitamins. The first group is comprised of B group and C group vitamins, and the second group is comprised of vitamin A, vitamin D, vitamin E and vitamin K. The family of vitamin B is large and includes vitamin B 1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B8 (biotin), vitamin B9 (folacin), and vitamin B12 (cobalamins). Vitamin C, known as ascorbic acid, can be found in the form of derivatives such as L-ascorbic acid, tetrahexyldecyl ascorbate, ascorbyl glucoside, ethylated ascorbic acid, ascorbyl palmitate, magnesium ascorbyl palmitate, magnesium ascorbyl phosphate, calcium ascorbate, sodium ascorbate, and sodium ascorbyl phosphate.

In collagen synthesis, hydroxylation of Proline to hydroxyproline has critical importance in order to obtain a stable triple helix, and at 37 °C stable folding in a triple conformation cannot be achieved without hydroxylating at least 90 prolyl residues. These hydroxylases have different requirements to be active and they can act especially when prolyl or lysyl residues occupy the correct position in the amino acid sequence of a chain and when the peptides are not in the triple helix configuration. Moreover, iron ions, molecular oxygen, α-ketoglutarate and ascorbic acid are required for these enzymes to be active (Figure 2). This requirement for ascorbic acid may explain some of the consequences of scurvy on wound healing.

It is known that ascorbic acid increases both the transcription rate of procollagen genes and the stability of procollagen mRNA, and also modulates the growth properties of cells. Asc 2-P-supplemented cultures stimulate the aggregation of intact collagen molecules, which is expected to lead to the formation of mature ECMs. Basic researches on biochemical pathways after a musculoskeletal system injury suggests that vitamin C, also known as ascorbic acid, may increase collagen synthesis and soft tissue healing. In addition, it prevents degradation of existing collagen by increasing the production of metalloproteinase inhibitors. However, the cutaneous administration of ascorbic acid is limited due to its being hydrophilic and characteristics of the stratum comeum. It is accepted that chondral injuries generally do not heal spontaneously due to their avascular environment, and various approaches have been tested in order to improve cartilage healing.

It has been reported that cells derived from the synovium exhibit high chondrogenic potential compared to cells derived from other mesenchymal tissue that have been studied. It is known that synovium MSCs (mesenchymal cells) cultured in BMP2 exhibit chondrogenic potential. Release of such MSC monolayers cultured in ascorbic acid leads to a 3D structure by means of contraction; this is similar to collagen gel contraction and the resulting tissue has been called as scaffold-free tissue engineering construct (TEC).

Carnitine serves as a primary cofactor for long-chain zinc fatty acid oxidation in the mitochondria of cells. It is responsible for providing cells with a critical source of free coenzyme A by means of maintaining homeostasis in theacyl coenzyme A pools of the cell. Studies have shown that after dermal bum, carnitine provides vascularity increase, fibroblast proliferation, and inflammatory cell increase [1].

Studies show that L-carnitine (-hydroxyacid) exhibits excellent skin regenerating properties at a lower pH. These features are probably resulted from the acceleration in the rate of epidermal cycle in the skin. The removal of dead skin cells along with the increased rate of skin regeneration will result in younger, healthier looking skin. Even small amounts of L-carnitine make the skin smooth, moisturized and soft due to its hygroscopic nature. The promising effects of fibroblast proliferation and carnitine's ability to moisturize the skin may be valuable in the healing of the aging skin [2] .

Ascorbate is a cofactor (-N-trimethyllysine hydroxylase and -y-butyrobetaine hydroxylase) for dioxygenase reactions which require two a-ketoglutarates in the carnitine biosynthesis pathway. Furthermore, ascorbic acid increases the production of endogenous carnitine. In addition to its role in collagen synthesis, vitamin C acts as a strong antioxidant by neutralizing toxic reactive oxygen species (ROS) responsible for cell apoptosis during the inflammatory phase (Figure 3). Oxidative stress has been defined as an imbalance between ROS and antioxidants and, it provides an unfavorable healing environment which negatively affects the viability and proliferation of collagen- producing cells and eventually promotes apoptosis. As an antioxidant, vitamin C can neutralize ROS through redox reactions by relieving oxidative stress due to inflammation.

Table 1. Comparison of aqueous humor content with plasma [3]

Aqueous humor comprises a variety of enzymatic and non-enzymatic antioxidant defenses. Antioxidants with low molecular weight such as glutathione, cysteine tyrosine and ascorbic acid are found in aqueous humor. Ascorbic acid, which is a molecule that can scavenge superoxide anion, hydroxyl radical and singlet oxygen, is found in high concentrations in aqueous humor (1-2 mM) (20-50 times as much as it is in plasma, Table 1). It is thought that free radical scavengers such as glutathione, α-tocopherol (vitamin E), and ascorbic acid (vitamin C) serve as components of an endogenous defense system which helps limit retinal damage caused by ultraviolet light (Figure 4).

Vitamin C is also abundant in the retina. However, intraocular wash solutions currently used during eye surgery comprise only oxidized glutathione as a comeal protector. Consequentially, these surgical solutions do not comprise vitamin C, even though human aqueous fluid comprises high concentrations of vitamins.

The importance of ascorbic acid as a protective antioxidant against the harmful effects of sunlight is known. Nevertheless, in addition to its antioxidant function, attention has been focused on quite high damping coefficient for UV radiation.

It is known that eyes of vertebrate comprise a number of UV-absorbing compounds (proteins, amino acids and their derivatives, ascorbic and uric acid) from the cornea to the aqueous humor and lens. Among them, ascorbate is known to be by far the strongest absorbent, followed by urate and tryptophan/tyrosine and other proteins [4] . The high concentration of ascorbate in mammalian aqueous humor is provided together with a selective secretion process driven by a specific ascorbate transport protein (Sodium -Dependent Vitamin C Transporter, SVCT) in the ciliary body. Ascorbic acid is quite important in the prevention of oxidative damage due to especially ultraviolet radiation and inflammation in the eye, from comeal endothelial cells to the trabecular meshwork, from the lens to the retina (Figure 5). By this means, it has a critical importance in terms of several comeal diseases, some types of glaucoma, cataracts and macular degeneration. Mineral salts

Mineral salts (zinc, calcium, magnesium, selenium, potassium) are necessary for the organism and constitute about 4% of our body mass. They provide oxygen to the body cells, thereby ensuring proper functioning of the organism. Mineral salts are very important for the organism, since they control the aqueous balance (osmotic pressure) and regulate the acid-alkaline balance.

Zinc 1-pyrrolidone carboxylate (Zinc PCA) has been used as a cosmetic ingredient due to its anti-microbial properties for a long time. Zinc L-pyrrolidone carboxylate inhibits matrix metalloproteinase- 1 production induced with UVA by in vitro cultured skin fibroblasts (Figure 6 and Figure 7), while increasing collagen synthesis in fibroblasts and osteoblasts (Figure 8).

Zinc applies protective effects on UV damage in keratinocytes and fibroblasts in vitro and in vivo. Additionally, it is an in vivo essential element controlling enzymatic activities and transcriptional factors (Fig. 9).

In addition, it provides antioxidative and anti-inflammatory effects (Figure 10). Regarding the functions of zinc in the suppression of photo-aging, it has been found that zinc compounds reduce the formation of UVA-induced reactive oxygen species in the skin of hairless mice, thereby exhibiting a protective effect against UVA-induced skin damage. Zinc also exhibits a protective effect against UVB- induced tissue damage, i.e. a zinc(II) -glycine complex reduces oxidative stress by inducing metallothionein expression. Most importantly, zinc induces the synthesis of sodium-dependent L-ascorbic acid transporter (SVCT) and ALFA 1 (I) procollagen in osteoblasts.

Available and sufficient zinc concentration serves in maintaining the structural integrity of the lens proteins necessary for transparency. It is believed that the alteration of lens proteins due to oxidative damage contributes to cataract formation, and zinc may exhibit its effect through antioxidant metalloenzyme system dependent on the key zinc, which provides protection from oxidative damage. Typically, the retina comprises the highest concentration of zinc, followed by the lens and corneal tissue. Zinc is found in the lens approximately 15 times more than serum. It is found in the retina approximately 2 times more than the lens.

In humans, it has been shown that zinc deficiency causes poor dark adaptation and night blindness, which in most cases is reversible with zinc supplement. There is also several evidence that zinc deficiency plays a role in the pathogenesis of age- related macular degeneration, a condition characterized by aggregation of membranous debris, which is suggested to result from oxidative stress on both sides of the retinal pigment epithelial basement membrane (Figure 11).

The results of AREDS1, AREDS2 (age related eye disease study) have shown that daily zinc supplement prevents macular degeneration (Figure 11). As a result, zinc supplement has been recommended. In addition, zinc provides neuroprotective activity in the retina through NMDA receptors and calcium channels.

Antioxidants

Antioxidants are a defense mechanism produced by the body in order to neutralize the effects of reactive oxygen species (ROS). Antioxidants can be of enzymatic or non-enzymatic types. Non-enzymatic sources of antioxidants comprise vitamin C, vitamin E, N-Acetylcamosine (NAC), epigallocatechin gallate (EGCG), coenzyme Q10 (CoQ10), quercetin, alpha lipoic acid, resveratrol, beta carotene, carotene, taurine, hypotaurine and glutathione. Enzymatic antioxidants include SOD, catalase, glutaredoxin and glutathione reductase [5]. However, as the body ages, antioxidant levels decrease, and this causes the balance between antioxidants and pro-oxidant molecules to be disrupted. This results in oxidative stress and in turn nullifies the cleansing capacity of antioxidants, either due to reduced antioxidants or excessive production of ROS. Furthermore, antioxidants can block the harmful effects of free radicals as a prophylactic measure, which causes normal production of the skin's structural proteins. Polyphenolic compounds (for example, trolox, hydroxytyrosol, tyrosol, ferulic acid, caffeic acid, rutin, diosmin, and melatonin) are substances commonly found in nature related to plant survival and continuation of species. Various studies have associated the chemical structure of phenolic compounds with antioxidant activity. The presence of phenolic nucleus serves as an efficient detector of reactive species, which reduces and chelates ferric ions catalyzing lipid peroxidation [6].

Inflammation and inflammatory cytokines play an important role in osteoarthritis. Among these inflammatory cytokines, interleukin (IL)- 1β and tumor necrosis factor alpha (TNF-α), which control the degeneration of the articular cartilage matrix, have been identified as the main initiators of OA. Activated by factors such as inflammatory cytokines, chondrocytes produce several inflammatory response proteins such as IL-6, TNF-α, matrixdegrading enzymes including metalloproteinase (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS),3 inflammatory mediators such as nitric oxide (NO), and lipid-derived inflammatory mediators such as prostaglandin E2 (PGE2). Inhibition of IL-1β production or activity by IL- 1β receptor antagonists or soluble receptors has been tested as a therapeutic strategy in OA. In addition, weakening of IL- IB-induced inflammatory mediator may be a potential treatment strategy for osteoarthritis (OA). The nuclear factor kappa B (NF-κB) signaling pathway has a determinative role in the start and progression of OA. NF-κB signal transmission inhibits the anabolic activities of chondrocytes, decreases collagen and proteoglycan synthesis, triggers the secretion of various matrix degrading proteinases, and consequently increases joint damage through the induction of NO and PGE2.

Again, phosphatidylinsitol-3 -kinase (PI3K) and protein kinase B (Akt) play a role in regulation of cellular target in several pathways, such as activation of the NF- κB pathway through phosphorylation of IκBα. Inhibition of the PI3K/Akt pathway and NF-κB has been accepted as an option for the treatment of OA.

Allicin, which is the active component of garlic, has a number of biological activities including antioxidant, anti-inflammatory, neuroprotective, antimicrobial and antineoplastic effects. Allicin inhibits the PI3K/Akt/NF-κB pathway in chondrocytes, thereby inhibiting the expression of IL- 1β-induccd inflammatory mediators in clinical studies.

Its antibacterial activity was identified by Louis Pasteur in 1858. It is well known that allicin has different health-improving effects such as antibacterial, antifungal, antiviral and antiprotozoal effects. The antibacterial properties of allicin have been known for a long time; in fact, as early as 1944 Cavallito et al. noticed that dilute allicin solutions inhibited the growth of both Gram-negative and Gram-positive bacteria (Figure 12) [7].

Various scientific evidence shows that allicin has great potential as a therapeutic antimicrobial agent in pharmaceutical products due to the high prevalence rates of multi drug -resistant microorganisms. In the last two decades, attention has been significantly drawn to the antibacterial and antifungal potential of allicin against multidrug-resistant microorganisms (MDR). For antimicrobial activities, it has been found that allicin reduces toxin production in both methicillin-susceptible and resistant Staphylococcus and neutralizes the toxin stimulated by Streptococcus pneumoniae under in vitro experimental conditions [8] .

In chemical tests, it was discovered that allicin reduces the production of conjugated diene hydroperoxides, molecules derived from ROS, which implies the ability of allicin to suppress lipid peroxidation [9]. Allicin also increases the glutathione content with a direct effect. Allicin can be metabolized by the cell to yield allicin derivatives which induce glutathione production. Glutathione and its derivatives play an important role in the antioxidant effect of aqueous humor. In addition to its direct antioxidant effect, allicin also contributes by increasing the amount of glutathione.

Astaxanthin (ASX), which is a carotenoid without vitamin A activity, has potential clinical applications due to its antioxidant activity higher than (3,30- dihydroxy-b, b-carotene-4,40-dione) β-carotene and α-tocopherol (Figure 13). ASX is found abundantly in the red-orange pigment of marine animals such as salmon (and salmon eggs), and in the shells of crabs and shrimps. ASX and ASX- like products are generally identified as antioxidants and immune modulators.

ASX has many highly strong pharmacological effects. These include protection against UV-induced cell damage and chronic inflammatory diseases, promoting immunomodulatory activities, alleviating metabolic syndromes, cardioprotective effects, antidiabetic activity, inhibition of neuronal damage, antiaging effects on the skin, suppression of cell membrane peroxidation, and anticancer activity (Figure 14).

It is known that ASX is more effective than β-carotene, a representative carotenoid, for inhibiting ROS production, especially singlet oxygen, and lipid peroxidation in solutions and various biomembrane systems. Therefore, ASX is expected to be an effective antioxidant agent to prevent UV-induced skin damage.

There are several reports about the in vitro inhibitory effects of ASX on UV- induced skin damage [10, 11]. Strong antioxidative effect of liposomes comprising ASC is responsible for efficient protective effect against UV-induced skin damage, collagen degradation and melanin production. For this reason, ASX liposomes are a potentially useful formulation for protection against UV-induced skin disorders. ASX purified from haematococcus pluvialis promotes tissue regeneration by means of reducing oxidative stress and increasing collagen secretion in vitro and in vivo. It has been shown that a multi -component dietary supplement formulation comprised of krill oil, natural astaxanthin, and sodium hyaluronate significantly reduces pain in individuals suffering from chronic mild to moderate knee joint pain. In addition, it effectively suppresses the expression levels of matrix metalloproteinases (MMPs) at the transcriptional level in inflamed knee joint tissues. Hyun-Sun Yoon et al. reported that 12 weeks of dietary ASX supplementation increased collagen levels in human skin [12].

It has been reported that ASX increases collagen expression by inhibiting the expression of MMP-1 and MMP-9 protein in an animal model. In addition, it has been reported that ASX increases collagen content by inhibiting MMP-1 and MMP-3 protein expression in human dermal fibroblasts. When the morphological and histological results were evaluated, it was determined that cartilage degradation was reduced with astaxanthin. Astaxanthin inhibited the gene expression of matrix metalloproteinases- 1 (MMP-1), MMP-3 and MMP-13 in cartilage compared to the means group. The results suggest that astaxanthin can be considered as a pharmaceutical agent in the treatment of OA [13].

Evidence that support an important role of Nrf2 in OA progression has recently begun to accumulate. Nrf2 is a stress response regulator applying antioxidative and anti-inflammatory effects in OA chondrocytes. Some of these studies have shown that ASX suppresses inflammation and oxidative stress in macrophages via Nrf2 (Figure 15). ASX also provides inhibitory effects on oxidative stress and apoptosis of hematopoietic progenitor cells through activation of Nrf2/HO-1. Regarding OA, previous studies had reported that ASX reduced IL-1β-induced MMP expression in chondrocytes and healed cartilage loss in experimental osteoarthritis [14],

ASX can protect cartilage homeostasis through modulation of Nrf2 signaling. ASX also provides anti -arthritic effects by inhibiting inflammation, oxidative stress and apoptosis in mouse OA chondrocytes (Figure 16). ASX treatment can alleviate in vivo cartilage degradation by activating Nrf2. These findings provide comprehensive information about the bioactivity of ASX in OA progression and suggest the use of ASX for the early clinical treatment of OA.

ASX can effectively suppress in vitro cell damage caused by free radicals and induction of (matrix metalloproteinase- 1) MMP-1 protein in human dermal fibroblasts after UV irradiation. In addition, it has been reported that ASX reduces the level of oxidative stress as demonstrated by decreased plasma malondialdehyde levels and reverses age-related changes in skin surface components of middle-aged subjects. Data obtained from the present study also show that the number of capillaries is significantly reduced after being exposed to UV, whereas oral administration with ASX prevents the reduction of capillaries in the skin of mice. Moreover, in UV-induced skin, dermal thickness increases while capillary density decreases. Additionally, they have found an inverse correlation between wrinkle and ROS levels and the number of capillaries in the skin of the mice. Therefore, photoaging may be associated with capillary regression in the skin [15].

Effects of ASX supplementation in ophthalmology; the suppression of inflammation after cataract surgery and the change in oxidation-related parameters, superoxide (02 · -) scavenging activity, hydrogen peroxide (H2O2) and total hydroperoxides (TH, levels in aqueous humor) and the relationship between these parameters were analyzed. Astaxanthin intake clearly increased superoxide scavenging activity and suppressed total hydroperoxide production in human aqueous humor, which suggests the possibility that astaxanthin may have suppressive effects on various oxidative stress-related diseases [16].

Unlike most antioxidants working on the interior (e.g. vitamin E and β-carotene) or exterior (e.g. vitamin C) of the cell membrane, astaxanthin extends across the double-layer membrane, scavenges reactive oxygen species (ROS) in both the inner and outer layers of the cellular membrane, thereby providing protection against oxidative stress. Several clinical studies that were carried out recently emphasizes the potential role of astaxanthin in improving eye health, as suggested by significant healing in the outcome of various ocular diseases, including diabetic retinopathy, age-related macular degeneration, glaucoma, and cataracts

[17].

The results suggest the possibility that ASX may have protective effects against glutamate neurotoxicity and oxidative stress in vivo. Since reducing intraocular pressure as much as possible is the only treatment currently available for NTG (normal tension glaucoma) in the clinical environment, results suggest that ASX supplementation may be effective to provide protection against certain factors involved in NTG, such as glutamate neurotoxicity and oxidative stress [18].

Osmoprotectants

Osmoprotectants are electrically neutral, highly soluble and non-toxic small molecules with low molecular weight at molar concentrations. Osmoprotectants regulate cellular osmotic regulation, reduce the risk of damage caused by ROS, prevent membrane damage, and stabilize proteins and enzymes. At the same time, they settle inside the cell and maintain the balance of the osmotic difference between the cell's environment and the cytosol. Osmoprotectants provide adaptation by increasing the osmotic pressure in the cytoplasm under various negative environmental conditions such as extreme salinity and temperature [19]. Generally, osmoprotectants are grouped as osmoprotectants comprising ammonium compounds (L-carnitine, putrescine, spermidine, spermine, glycinebetaine, b-alanine betaine, dimethyl-sulfonio propionate and choline-O- sulphate), sugar and sugar alcohols (trehalose, erythrol, fructan, mannitol, dextran and sorbitol) and osmoprotectants comprising amino acid (proline and ectoine).

Among these osmoprotectants, trehalose (TRE) is one of the primary osmoprotectants found in nature. Trehalose, which is a natural non-reducing disaccharide, is comprised of two glucoses with 1,1-glycosidic bond and it was first found in ergot. Besides mammals, TRE is commonly found in bacteria, yeasts, insects, invertebrates, fungi, algae and everyday foods such as bread, beer, mushrooms and seaweed. Trehalose has been used as a protectant for tissues in organ transplants, as it stabilizes and protects the cell membrane.

TRE has been recognized as a new type of food additive and it is widely applied in the food industry, due to its low sweetness, its anti-rot property, being easily digestible, its preventing starch aging, its preventing protein denaturation, frozen preservation and its many other excellent properties.

Recent studies have shown that trehalose has many biological and pharmacological activities such as neuroprotection, anti-oxidation, anti- inflammatory, prevention of heavy metal poisoning and induction of autophagy. Results of the clinical studies have shown that TRE (150 and 300 mM) can effectively prevent HaCaT (human immortal keratinocytes) cells from being damaged by UVB radiation. Furthermore, TRE reduces MMP production levels caused by UVB and improves procollagen I and TIMP-1 contents in HaCaT cells, reduces ROS-induced DNA damage, thereby inhibiting the NF-κB pathway. More importantly, it increases procollagen synthesis by activating TGF-β1 and smad2 / 3 (Figure 17), and Smad7 negative feedback regulates procollagen synthesis by inhibiting Smad2/3 phosphorylation [20].

Trehalose enables wound healing by protecting cells, especially cell membranes, from oxidative damage and desiccation. When the injured cornea is treated with trehalose, corneal inflammation, scar formation and comeal neovascularization are suppressed. In dry eye disease, trehalose reduced cell apoptosis and reduced oxidative, inflammatory and proteolytic activity on the ocular surface (Figure 18). In the UVB-irradiated cornea, trehalose suppressed photodamage caused by UVB rays. It reduces intracorneal inflammation and comeal neovascularization. Trehalose prevents postoperative fibrous scar formation after ocular surgery such as glaucoma filtration surgery [21]. European patent application document EP2670447, an application known in the state of the art, discloses a viscoelastic and injectable hyaluronic acid composition comprising a hyaluronic acid, and used for medical and non-medical purposes.

International patent document no WO2014064632, an application known in the state of the art, discloses sterile, injectable formulations which are designed in order to treat skin defects and wherein hyaluronic acid or its salt is used. The formulation of the invention comprises only crosslinked hyaluronic acid gels. The limited effectiveness of hyaluronic acid here is that it acts as a physical filler.

United States patent document no US20110171286, an application known in the state of the art, discloses hyaluronic acid compositions which is used dermatologically and comprises at least one additional constituent selected from the group consisting of vitamin B, C and vitamin E. The vitamin E in this invention is an antioxidant with moderate activity. B group vitamins play an active role in the energy pathways of cell metabolism. They are also effective in division and proliferation.

South Korean patent document no KR20140071676, an application known in the state of the art, discloses a composition preventing inflammation for use in intraarticular applications. The formulation of the present invention comprises hyaluronic acid and Vitamins A, B, C, D and E or a combination thereof. The vitamin A in the invention is a derivative of retinol and essentially exhibits epithelial and antioxidant effects.

Summary of the Invention

The objective of the present invention is to create a viscoelastic gel formulation, which is developed with the synergistic effect of vitamins, minerals, antioxidants and osmoprotectants, in addition to hyaluronic acid, which is known to be used in intra-articular, intradermal and ophthalmic applications, and which is to be prepared in buffer solution with sterile injectors.

Another objective of the invention is to create a viscoelastic gel formulation to be used for synovial fluid support, decrease in inflammation due to osteoarthritis, decrease in cartilage surface destruction and regeneration of the cartilage surface in the field of orthopedics.

Yet another objective of the invention is to create a viscoelastic gel formulation to be used in the field of ophthalmology in order to prevent damage due to cataract surgery (ultrasonic power, surgical manipulation, etc.), to maintain the natural levels of antioxidant balance, to naturally block the permeability of the UV light spectrum and to prevent damage to the ocular structures due to possible inflammation after surgery.

A further objective of the invention is to create a viscoelastic gel formulation to be used in the field of dermatology in order to preserve the natural balance of the dermis in the process due to photoaging, to maintain the natural remodeling by increasing collagen synthesis and reducing its degradation, to minimize oxidative stress due to photoaging and inflammation, and to correct cosmetic defects.

Detailed Description of the Invention

INJECTABLE HYALURONIC ACID FORMULATIONS FOR USE IN INTRAARTICULAR, INTRADERMAL AND OPHTHALMIC APPLICATIONS” which is developed in order to achieve the objectives of the present invention is illustrated in the accompanying figures, in which:

Figure 1 Schematic view of the collagen synthesis.

Figure 2 Schematic view of the enzymatic reaction of Proline and Lysine hydroxylase. Figure 3 Schematic view of the reactive oxygen species (ROS) neutralization mechanism (Antioxidant Defense).

Figure 4 Schematic view of the antioxidant defense mechanism against UVA and UVB-induced ROS in aqueous humor.

Figure 5 Schematic view of the mechanisms of ROS formation in the eye. Figure 6 Schematic view of the relationship between different MMPs and zinc.

Figure 7 Schematic view of the regulation of MMP activity by zinc.

Figure 8 Schematic view of osteoblast collagen synthesis regulation.

Figure 9 Schematic view of the regulatory effect of zinc on transcriptional mechanisms.

Figure 10 Schematic view of the antioxidative and anti-inflammatory activity of zinc.

Figure 11 Schematic view of the effects of zinc on the prognosis of age- related macular degeneration (ARED Study-2001).

Figure 12 Schematic view of the antimicrobial activity of allicin.

Figure 13 Schematic view of the antioxidant activity of ASX.

Figure 14 Schematic view of multisystem activity of ASX through different receptors.

Figure 15 Schematic view of the effect of ASX on OA progression through Nrf2.

Figure 16 Schematic view of the relationship between ASX and apoptosis in chondrocytes.

Figure 17 Schematic view of the relationship between trehalose and collagen synthesis.

Figure 18 Schematic view of inhibition of oxidative stress by trehalose at the ocular surface. The subject matter of the present invention is an injectable viscoelastic gel formulation suitable for use in intra-articular, intradermal and ophthalmic applications. The related formulation comprises hyaluronic acid (HA), vitamin, mineral, antioxidant and osmoprotectant components prepared in buffer solution, and the functions of these components in the formulation are as follows: - Hyaluronic acid (HA) a) Intradermal applications: It inhibits collagen degradation, and stimulates intrinsic HA production. It is responsible for the moisture balance (water content) of the dermis and the epidermis. It takes part in immune regulation and dermal vessel formation. It plays an active role in tissue repair and wound healing. b) Intra-articular applications: It provides the required lubrication. It prevents collagen degradation and enables to increase the synthesis of intrinsic HA. It plays a role in tissue repair and wound healing. It adapts to changing shear forces with dynamic viscosity. It acts as a shock absorber, exhibits elastic and suspensive behavior. c) Ophthalmic applications: It enables anterior chamber formation, to create a suitable area for surgery and to maintain intraocular pressure throughout the surgery. It ensures the safety of surgery (prevention of complications such as posterior capsule rupture, descement membrane detachment). It enables the corneal endothelial cells to be protected from high temperature and surgical manipulation with dispersive activity and also enables intraocular lens implantation. - Vitamins a) Intradermal applications: It is a cofactor for the hydroxylation of proline and lysine, which is critical in collagen synthesis. It increases the transcription of procollagen genes and the stability of procollagen mRNA. It prevents degradation of existing collagen by increasing the production of metalloproteinase inhibitors. Ascorbate is a cofactor for dioxygenase reactions which require two α-ketoglutarates in the carnitine biosynthesis pathway. It increases the production of endogenous carnitine. It prevents apoptosis with ROS neutralization and increases the viability of collagen-producing cells. b) Intra-articular applications: It increases collagen synthesis and soft tissue healing. It neutralizes ROS caused by inflammation. c) Ophthalmic applications: It scavenges superoxide anion, hydroxyl radical and singlet oxygen. It is present in the aqueous at a concentration of 20 times that of the plasma. It serves as components of an endogenous defense system which helps limit retinal damage caused by ultraviolet light. It is the strongest of a number of UV- absorbing compounds, from the cornea of vertebrate eyes to the aqueous humor and lens. It has a critical importance in terms of several comeal diseases, types of glaucoma, cataracts and macular degeneration. - Osmoprotectants a) Intradermal applications: It lowers MMP production levels caused by UVB. It improves procollagen I and TIMP-1 contents and reduces ROS-induced DNA damage. It inhibits NF-κB pathway. It increases procollagen synthesis by activating TGF-β1 and SMAD2/3, and Smad7 negative feedback inhibits SMAD2/3 phosphorylation, thereby regulating procollagen synthesis. b) Intra-articular applications: It exhibits antioxidative and anti- inflammatory activity. It reduces ROS induced DNA damage, and inhibits NF-κB pathway. It increases procollagen synthesis. c) Ophthalmic applications: It suppresses comeal inflammation, scar formation and comeal neovascularization. In dry eye disease, trehalose reduces cell apoptosis and reduces oxidative, inflammatory and proteolytic activity on the ocular surface. It prevents the formation of postoperative fibrous scarring after ocular surgery. Antioxidants a) Intradermal applications: It plays an active role in prevention of UV induced skin damage. It increases collagen content and supports tissue regeneration by reducing oxidative stress and inhibiting MMP 1-3-9. It prevents the decrease in capillaries caused by photoaging. It prevents apoptosis with ROS neutralization and increases the viability of collagen-producing cells. It reduces lipid peroxidation. b) Intra-articular applications: It significantly reduces mild to moderate chronic knee joint pain. It effectively suppresses the expression levels of matrix metalloproteinases (MMPs) at the transcriptional level in inflamed knee joint tissues. It reduces cartilage degradation by maintaining cartilage homeostasis through Nrf2 in osteoarthritis. It inhibits the PI3K / Akt / NF-κB pathway in chondrocytes and exhibits a therapeutic effect in osteoarthritis and reduces cartilage destruction by inhibiting the expression of IL-1β- induced inflammatory mediators. It neutralizes the toxic effects of UV- induced ROS on the anterior chamber lens and retina. c) Ophthalmic applications: It suppresses inflammation after cataract surgery. It exhibits active and strong activity in ROS neutralization. It has a curative effect on significant eye diseases such as diabetic retinopathy, age-related macular degeneration, glaucoma and cataracts. It prevents apoptosis by ROS neutralization. It exhibits strong antimicrobial and antioxidant activity. Minerals a) Intraderm al applications: It increases collagen synthesis while inhibiting UVA-induced matrix metalloproteinase- 1 production. It exhibits a protective effect against UVA-induced skin damage by reducing the formation of reactive oxygen species. It reduces oxidative stress by inducing metallothionein expression. It induces the synthesis of sodium-dependent L-ascorbic acid transporter (SVCT) and ALFA 1 (I) procollagen. b) Intra-articular applications: It increases collagen synthesis by regulating MMP activity and exhibits antioxidant and anti- inflammatory activity. c) Ophthalmic applications: It plays a role in maintaining the structural integrity of lens proteins necessary for transparency. It enhances adaptation to darkness and shows protective effect against night blindness. It prevents macular degeneration. It provides neuroprotective activity in the retina through NMDA receptors and calcium channels.

In one embodiment of the invention, the formulation comprises at least one osmoprotectant selected from the group consisting of L-carnitine, betaine, putrescine, spermidine, spermine, glycinebetaine, 6-alanine betaine, choline-O- sulfate, dimethyl -sulfonio propionate, trehalose, erythrole, fructan, mannitol, dextran, sorbitol, proline, ectoin and combinations thereof.

In one embodiment of the invention, the formulation comprises at least one antioxidant selected from a group consisting of glutathione, allicin, astaxanthin, N-Acetylcamosine (NAC), epigallocatechin gallate (EGCG), coenzyme Q10 (CoQ10), quercetin, alpha lipoic acid, resveratrol, alpha tocopherol, carotene, beta carotene, trolox, hydroxytyrosol tyrosol, ferulic acid, caffeic acid, rutin, diosmin, melatonin, taurine, hypotaurine, and combinations thereof.

In an embodiment of the invention, the formulation comprises at least one vitamin selected from the group consisting of vitamin A derivatives such as retinal, retinol, pro-vitamin A, retinoic acid, vitamin B derivatives such as vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B8 (biotin), vitamin B9 (folacin), vitamin B12 (cobalamins), vitamin C derivatives such as L-ascorbic acid, tetrahexyldecyl ascorbate, ascorbyl glucoside, ethylated ascorbic acid, ascorbyl palmitate, magnesium ascorbyl palmitate magnesium ascorbyl phosphate, calcium ascorbate, sodium ascorbate, and sodium ascorbyl phosphate, vitamin D and vitamin K derivatives, and combinations thereof. In one embodiment of the invention, the formulation comprises at least one mineral salt selected from the group consisting of zinc sulfate, zinc acetate, zinc glutamate, zinc PCA, calcium chloride, calcium carbonate and calcium phosphate, tricalcium citrate, calcium lactate, calcium lactate gluconate, calcium gluconate, magnesium oxide, magnesium citrate, magnesium gluconate, magnesium chloride, magnesium sulfate, magnesium lactate, magnesium aspartate hydrochloride, potassium chloride, potassium carbonate, selenium salts and combinations thereof.

In one embodiment of the invention, the formulation to be used in applications comprises lidocaine hydrochloride in order to provide anesthetic effect.

An injectable viscoelastic gel formulation of the invention is suitable for use in intra-articular, intradermal and ophthalmic applications and comprises sodium hyaluronate, antioxidant and vitamins. In addition these components, it comprises at least one auxiliary component selected from the group consisting of osmoprotectant, mineral salt and buffer solution and different combinations thereof. Within the scope of the invention, viscoelastic formulas to be prepared in buffer solution (preferably in phosphate buffer solution) with disposable sterile syringes comprise linear and/or cross-linked forms of sodium hyaluronate with a molecular weight of 10 kDa - 7 MDa to be in the range of 0.5 - 5% by weight in the formulation, and osmoprotectants, vitamins, antioxidants, mineral salts and / or combinations thereof in the range of 0.001% - 5% by weight in the formulation. In addition to these combinations, the formulations to be used in the applications comprise lidocaine hydrochloride in the range of 0.01% - 2% by weight in order to provide anesthetic effect. The components and derivatives which will have a synergistic effect with hyaluronic acid in the formulation are as follows:

• Osmoprotectants (L-carnitine, betain, putrescine, spermidine, spermine, glycinebetaine, b-alanine betaine, choline-O-sulfate, dimethyl-sulfonio propionate, trehalose, erythrole, fructan, mannitol, dextran, sorbitol, proline, ectoin)

• Antioxidants (glutathione, allicin, astaxanthin, N-Acetylcamosine (NAC), epigallocatechin gallate (EGCG), coenzyme Q10 (CoQ10), quercetin, alpha lipoic acid, resveratrol, alpha tocopherol, carotene, beta carotene, trolox, hydroxytyrosol tyrosol, ferulic acid, caffeic acid, rutin, diosmin, melatonin, taurine, hypotaurine)

• Vitamins (vitamin A derivatives such as retinal, retinol, pro-vitamin A, retinoic acid, vitamin B derivatives such as vitamin B 1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B8 (biotin), vitamin B9 (folacin), vitamin B12 (cobalamins), vitamin C derivatives such as L-ascorbic acid, tetrahexyldecyl ascorbate, ascorbyl glucoside, ethylated ascorbic acid, ascorbyl palmitate, magnesium ascorbyl palmitate magnesium ascorbyl phosphate, calcium ascorbate, sodium ascorbate, and sodium ascorbyl phosphate, vitamin D and vitamin K derivatives)

• Mineral salts (zinc sulfate, zinc acetate, zinc glutamate, zinc PCA, calcium chloride, calcium carbonate and calcium phosphate, tricalcium citrate, calcium lactate, calcium lactate gluconate, calcium gluconate, magnesium oxide, magnesium citrate, magnesium gluconate, magnesium chloride, magnesium sulfate, magnesium lactate, magnesium aspartate hydrochloride, potassium chloride, potassium carbonate, selenium salts)

Vitamin C acts as a cofactor in the proline and lysine hydroxy lation step in collagen synthesis and increases collagen synthesis. However, in order to recover from the degenerative findings due to photoaging in the dermis, it is required to establish the balance of collagen degradation and production (collagen remodeling), and also to increase the production rate of collagen with transcriptional effects and decrease the rate of degradation. Zinc in our present invention improves collagen remodeling especially through MMPs, decreases the rate of collagen degradation by modulating MMPs, and increases the rate of collagen production (through DNA) through transcriptional factors. In addition, trehalose in our present invention reduces UVB-induced MMP production levels and inhibits the NF-κB pathway by means of reducing ROS-induced DNA damage. Most importantly, it increases procollagen synthesis by activating TGF-β1 and smad2/3.

With its large amount of water-binding capacity, hyaluronic acid enables that wrinkles due to aging in the dermis are physically corrected and regain their natural appearance. However, there are increased collagen degradation, decreased collagen production, and generally slowed or stopped collagen remodeling in the basis of photoaging. This deterioration in the structure and function of collagen causes the formation of superficial and deep wrinkles in the dermis. Vitamin C, zinc and trehalose in our present invention increase collagen synthesis and reduce its degradation, while maintaining the remodeling as it should be, thereby preventing deterioration of collagen structure and function, which is the root cause of these wrinkles. Furthermore, allicin, astaxanthin and glutathione in our present invention neutralize free oxygen radicals caused by UV and inflammation, thereby minimizing cell and connective tissue damage.

Astaxanthin and glutathione in the present invention are much more powerful and effective antioxidants compared to vitamin E, and furthermore astaxanthin also shows activity in intracellular membranes. In addition, zinc and trehalose, which contribute to collagen remodeling, prevent deterioration of collagen structure and function, which is the basis of photoaging.

Astaxanthin in our present invention is a carotenoid derivative such as vitamin A and it is the only antioxidant which has activity both outside and inside the cell membrane. Vitamin D has modulatory effect on osteoclasts and osteoblasts. There is no proven efficacy of additional vitamin D supplementation in patients with normal vitamin D levels. Furthermore, vitamin D needs zinc in order to show its effectiveness properly through osteoclasts and osteoblasts. For the purpose of using an injectable viscoelastic gel formulation of the invention in the field of dermatology, it is a subcutaneous injectable viscoelastic gel which can be used with a cannula size in range of 18-30G in order to make the formulation suitable for use, and which is used in order to protect the natural balance of the dermis in the process due to photoaging, to increase collagen synthesis and reduce its degradation, and therefore to maintain natural remodeling, to minimize oxidative stress due to photoaging and inflammation, and to correct cosmetic defects.

For the purpose of using an injectable viscoelastic gel formulation of the invention in the field of orthopedics, it is a viscoelastic gel for intra-articular injection to patients which can be used with a cannula size in range of 18-30G in order to make the formulation suitable for use, and which is used for synovial fluid support, decrease in inflammation due to osteoarthritis, decrease in cartilage surface destruction and regeneration of the cartilage surface.

For the purpose of using an injectable viscoelastic gel formulation of the invention in the field of ophthalmology, it is an injectable viscoelastic gel which can be used with a cannula size in range of 18-30G in order to make the formulation suitable for use, and which is used for preventing damage due to cataract surgery (ultrasonic power, surgical manipulation, etc.), preserving the natural levels of antioxidant balance, naturally blocking the permeability of the UV light spectrum, and preventing damage to ocular structures due to possible inflammation after surgery.

The production method of an injectable viscoelastic gel formulation of the present invention comprises the steps of

- Mixing the components in the buffer solution with a rotational mixer at 10- 100 rpm for 2-50 hours,

- Degassing the mixture with vacuum, - Filling the mixture into syringes in the volume range of 0.5-2.0 ml for ophthalmic products, 1.0-10.0 ml for intra-articular products and 1.0-30.0 ml for intradermal products,

- Sterilizing ophthalmic products among filled products by ethylene oxide and steam sterilization, and intra-articular and intradermal products only by steam sterilization,

- Packing the final products. These formulations described above have been developed for use in the fields of Orthopedics, Dermatology and Ophthalmology. In the field of orthopedics; synovial fluid support, decrease in inflammation due to osteoarthritis, regeneration of the cartilage surface or decrease in cartilage destruction are provided. In the field of dermatology; cellular regeneration (anti-aging) in the dermis, collagen remodeling or correction of cosmetic defects are provided. In the field of ophthalmology; it is achieved that the oxidative damage caused by cataract surgery is prevented or the natural physiological levels of the anterior chamber antioxidant balance are preserved.

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