WO2023052808A2 - Gel containing dual l-carnosine/aloe vera nanophytosomes for accelerating diabetic wound healing - Google Patents

Abstract

Present patent of invention's application is related to an extraordinary gel, containing dual Aloe barbadensis whole leaf extract and L-Carnosine dipeptide phytosomes for curative treatment of different types of wound, especially the ones associates with diabetes and describing preparation process of a pharmaceutical formulation of topically applied dual L-Carnosine/ A. vera phytosomal gel that utilizes therapeutical effects of the A. vera whole leaf extract and L-Carnosine in management of different types of wound, especially the ones associates with diabetes by providing anti-inflammatory, soothing, antimicrobial, analgesic, proangiogenic effects and cyto-protective activity on proliferative cells in the wound site.

Description

Gel containing Dual L-Carnosine/Aloe vera nanophytosomes for accelerating diabetic wound healing

Current invention is classified in the section of human needs; to the health field, more particularly, to medicinal preparation owing to its pharmaceutical application which is to achieve a treatment for diabetic ulcers with much greater rate of succession and a better wound healing outcome

In the present state of the art, few studies exist that mention the application of A. barbadensis in cosmetic pharmaceutical products. Nevertheless, none of them is related to a gel in which the main composition is composed of a combination of A. barbadensis and L-Carnosine in combination and in the form of dual phytosomes to be used for accelerating diabetic wounds healing. Other prior arts also exist that describe compositions capable of healing diabetic wounds, despite this, none of them is related to a gel in which the main composition is composed of a combination of A. barbadensis and L-Carnosine in combination and in the form of dual phytosomes to be used for accelerating diabetic wounds healing. Finally, other prior arts exist that introduce A. vera or L-Carnosine as wound healing agents, but none of them is related to a gel in which the main composition is composed of a combination of A. barbadensis and L-Carnosine in combination and in the form of dual phytosomes to be used for accelerating diabetic wounds healing.

The document WO2015007922A1, entitled “Dressings and plasters with aloe vera for wounds and ulcers” describes designing of a range of dressings and plasters containing a high amount of A. vera which combinne the efficiency of the mechanical properties of silicone or polyurethane with the biological properties of A. vera, for treating and preventing infections, reducing pain and attenuating complications observed during healing period of surgical and traumatic wounds as a consequence of burns, herpes and ulcers. Nevertheless, this invention is not related to a gel pharmaceutical formulation and is not composed of a combination of L-Carnosine/A. vera dual nanophytosomes and is not specially intended for application in treatment of diabetic wounds.

The document US4725438A, entitled “Aloe vera ointment” describes an ointment composed of a mixture of the raw gel of the aloe vera plant, beneficial in management of skin irritations and enhancement of the extent of wound healing. Nevertheless, this invention is not related to a gel pharmaceutical formulation and is not composed of a combination of L-Carnosine/A. vera dual nanophytosomes and is not specially intended for application in treatment of diabetic wounds.

The document KR102073009B1 explains a phytosomal formulation of extract of Mori cortex and Centella asiatica extracts for amelioration and treatment of skin inflammatory disorders including atopic dermatitis. Nevertheless, this invention is not related to a gel pharmaceutical formulation and is not composed of a combination of L-Carnosine/A. vera dual nanophytosomes and is not specially intended for application in treatment of diabetic wounds.

Present patent of invention’s application is related to an extraordinary gel, containing Aloe barbadensis whole leaf extract (A. vera) and L-Carnosine dipeptide for curative treatment of diabetic wounds, describing a pharmaceutical formulation of topically applied dual L-Carnosine/ A. vera gel that utilizes therapeutical effects of the A. vera whole leaf extract and L-Carnosine in management of diabetic wounds to provide anti-inflammatory, soothing, antimicrobial, analgesic, proangiogenic and cyto-protective effect on proliferative cells in the wound site.

At present, more than 425 million people are suffering from diabetes worldwide and the prevalence is predicted to be raised to 629 million by the year 2045 (1). Among this number, the chance of developing a lifetime risk of chronic nonhealing wound, including diabetic foot ulcer (DFU), is about 25% which highly imposes affected patients at risk of amputation. Moreover, 68% of diabetic patients with non-healing chronic wounds will have a survive rate of less than 5 years following development of injuries (2). The hyperglycemic state of diabetic patients in most cases is associated with microvascular endothelial damage, impaired vascular functioning and insufficient capability of oxygen delivery to the tissues which in turn, can promote chronic ischemia in affected area and delay the process of wound healing (3). Moreover, inadequate oxygen and nutrient supply to the wounds as a consequence of insufficient angiogenic response in diabetic patients, hampers fibroblasts and keratinocytes growth, proliferation and migration to the affected area, reduces the rate of extracellular matrix production and deposition, leads to development of non-healing diabetic associated chronic wounds (4).

While the exact pathogenesis of the diabetic ulcers has not been fully identified yet, relative hypoxia has been mentioned as a pivotal stimulator of the physiological wound healing process (5). In this context, achieving a sufficient angiogenic response during healing process has shown to be highly dependent of maintaining a constant hypoxic gradient in injured area (6). Moreover, hypoxia can initiate production of a range of proangiogenic cytokines, growth factors and enzymes including vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), basic fibroblast growth factor (bFGF), transforming growth factor-b (TGF-b), angiopoietin-2 (ANG-2) and MMPs (7) to facilitate migration of fibroblasts and keratinocytes to the site of injury (8).

Hypoxia-inducible factor (HIF) 1, is a heterodimeric master transcription factor, composed of α and β subunits which has shown to be the main element responsible for initiation of cellular adaptive responses to hypoxia. Generally, in normoxic condition, O2-dependent hydroxylation of one or both of the proline residues (9) of the α subunit of HIF-1 protein by the activity of a specific Fe2+- and oxoglutarate-dependent prolyl hydroxylase (PHDs) enzyme (10, 11), results in attachment of the hydroxylated HIF-1α to the von Hippel-Lindau (VHL) tumor suppressor protein and its degradation by proteasome system (12). Contrarily, during hypoxia, HIF-1α is stabilized and upon translocation to the nucleus, activates transcription of a range of pro-angiogenic genes, ending in enhancement of pro-angiogenic responses, cellular motility and endothelial progenitor cells recruitment to the wound site (13-16). Unfortunately, based on recent studies, hyperglycemia has shown to significantly reduce stability and impair function of HIF-1α. Moreover, obtained data from clinical trials demonstrates lower amounts of HIF-1α in diabetic patients compared to the normal subjects (8). Hence, restoring the diminished HIF-1α content in injured sites may efficiently accelerate diabetic wounds healing in patients.

L-Carnosine and Aloe vera (A. vera) are two over-the counter food supplements, widely used for enhancing exercise performance of athletes and accelerating healing of cutaneous injuries respectively (17, 18). Moreover, free radical scavenging and anti-inflammatory characteristics of these agents have also made them to be useful in management of DM (19, 20). In light of recent studies, L-Carnosine is capable of preventing proteins crosslinking and oxidation by a group of potentially toxic aldehydes such as aldose and ketose sugars, trioses, malondialdehyde, acetaldehyde and formaldehyde owing to its preferential reactivity with aldehydes. Similarly, L-Carnosine is also capable of preventing the destructive effects induced by Methyl glyoxal (MGO), the main byproduct of the triosephosphate’s non-enzymatic glycolysis which is excessively accumulated in body during diabetes. Presence of an amine group next to the imidazole moiety and a carboxylic acid group at the end of the molecule, confers L-Carnosine the ability of mimicking preferred glycation sites of proteins and turns L-Carnosine to a decoy for trapping free MGO and preventing from AGE formation. Thus, L-Carnosine can effectively suppress formation of AGEs by inhibiting first step of protein glycation (21). Notably, L-Carnosine is also capable of restricting interaction of MGO-modified proteins with other normal, unmodified proteins. In this context, spectrophotometric studies performed by Hipkiss et al. clearly demonstrated that L-Carnosine was capable of diminishing color changes induced by formation of protein (crosslinked from lysine moiety)-MGO-AGE complexes. Hence, L-Carnosine is also capable of inhibition the propagation step of protein’s glycation and formation of MGO oligo/polymeric protein adducts (21).

Alongside, the high content of phenolic compounds, glycosides (aloins), 1,8-dihydroxyanthraquinone derivatives (aloe-emodin), beta-1,4 acetylated mannan, mannose-phosphate and alprogen found in whole leaf extract of Aloe vera L. (Liliaceae), an endemic plant to North Africa, has turned it to a powerful anti-inflammatory, antioxidant, cytoprotective, cardiac stimulatory and immunomodulatory agent (22). Similarly, specific components of A. vera whole extract including acemannan and β-sitosterol are capable of stimulating expression of vascular endothelial growth factor (VEGF) and potentiating nitric oxide (NO) synthesis by affecting NO synthase (NOS) activity (23). Moreover, the antidiabetic and hypoglycemic properties of A. vera extract have also been confirmed in several studies (24, 25). In this context, anthraquinone components of A. vera leaf’s extract, including aloin and aloe emodin which comprise 0.1 to 21.5% dry weight of the extract, are responsible for the observed anti-inflammatory activities of the aloe species (26).

Among the naturally existing inhibitors of the AGEs formation (i.e., inhibitors of Maillard reaction) currently discovered, most of them belong to the anthraquinone and flavonoid families possessing IC50 values ranging between 90–200 mM (27, 28). For instance, emodin derived from Cassia tora seeds has shown anti-glycation and AGE formation inhibitory activities which were significantly stronger than those observed with the aminoguanidine in vitro (27). Very recently, it has been shown that aloin derived from A. vera and peptides derived from camel casein demonstrated a synergistically enhanced inhibitory effect on AGEs formation which was attributed to the formation of the aloin–peptide complex, demonstrating an outstanding antioxidant activity (29). Considering these pre-existing data, co-administration of L-Carnosine and A. vera can affect multiple stages of angiogenesis at same time and therefore, may synergistically promote angiogenesis in diabetes associated wounds.

Phytosomes are biocompatible complexes of drugs and phospholipids which upon introduction to aqueous media, self-assemble into vesicular structures. Contrary to liposomes, hydrophilic drugs are aligned with polar heads of phospholipids (i.e., positive ammonium and negative phosphate groups) through formation of electrostatic interactions and hydrogen bonds. The main beneficial properties of phytosomes include high drug to carrier loading capacity, stability, ease of storage in a solid ready-to-reconstruction state, small size and biocompatibility, rendering them to suitable carriers for high wate soluble drugs (30). Alongside, achieving a nanosized phytosomal formulation (nanophytosome) can further improve penetration of the biomolecules to the most inner layer of the skin were proliferating fibroblasts, keratinocytes and endothelial cells as well as mesenchymal stem cells exist. by enhancing bioavailability and biodistribution while reducing elimination of it from body. Considering the advantages of phytosomes, as well as the abovementioned benefits of L-Carnosine and A. vera whole extract in treatment of DM together with their Low-cost and accessibility, here, we developed an L-Carnosine/A. vera dual loaded nanophytosome to achieve a potent and affordable formulation to prevent and treated two important DM associated complications, including endothelial dysfunction and impaired angiogenesis.

Consequently, present invention focused on providing a pharmaceutical formulation, effective for treatment of diabetic wounds mainly based on enhancement of angiogenesis and providing protective effects against MGO-induced cytotoxic effects in parallel to induction of anti-inflammatory, anti-microbial, soothing and analgesic effects.

Present patent of invention application explains a pharmaceutical formulation in a specific type of dermatological gel formulation, developed by combining produced dual L-Carnosine/A. vera nanophytosomes with carboxymethylcellulose or Carbopol inert hydrogel, to be applied in the management of diabetic wounds to enhance angiogenesis and to provide protective effects against MGO-induced cytotoxic effects in parallel to induction of anti-inflammatory, anti-microbial, soothing and analgesic effects.

In brief, the main advantages of present invention include:

- Providing a topical pharmaceutical formulation which contains L-Carnosine and A. vera as active ingredients in the form of nanophytosomes in combination with CMC-based or Carbopol inert hydrogel;

- Providing a topical pharmaceutical formulation for accelerating healing of diabetic wounds with a simple development process;

- Providing a topical pharmaceutical formulation with stable and accelerated absorption for accelerating healing of diabetic wounds;

- Providing a topical pharmaceutical formulation for accelerating healing of diabetic wounds, demonstrating a rapid dissolution rate;

- Providing a topical pharmaceutical formulation for accelerating healing of diabetic wounds, possessing a much greater effectiveness in healing;

- Providing a topical pharmaceutical formulation for accelerating healing of diabetic wounds, to enhance angiogenesis and to provide protective effects against MGO-induced cytotoxic effects in parallel to induction of anti-inflammatory, anti-microbial, soothing and analgesic effects.

For better understanding of the invention, it will be described in preferred embodiments and references will be provided to the figures and flowchart:

Fig.1

The Flowchart of the process of preparing the dual L-Carnosine/A. vera nanophytosome gel;

Fig.2

The SEM and TEM Images obtained from dual L-Carnosine/A. vera nanophytosomes.

Fig.3

The particle size and zeta potential of dual L-Carnosine/A. vera nanophytosomes.

Fig.4

Accelerated and long-term stability of dual L-Carnosine/A. vera nanophytosomes.

Fig.5

In vitro cytoprotective activity of L-Carnosine/A. vera nanophytosomes against MGO induced toxicity.

Fig.6

In vitro pro-angiogenic activity of L-Carnosine/A. vera nanophytosomes against MGO induced impaired angiogenesis.

Fig.7

In vivo wound healing activity of dual L-Carnosine/A. vera nanophytosomes gel in diabetic rats.

Fig.8

In vivo anti-inflammatory activity of dual L-Carnosine/A. vera nanophytosomes gel in diabetic rats.

Fig.9

In vivo improving activity of dual L-Carnosine/A. vera nanophytosomes on collagen I deposition in diabetic rats.

Fig.10

In vivo improving activity of dual L-Carnosine/A. vera nanophytosomes on α-SMA (an angiogenesis marker) expression in diabetic rats.

Fig.11

In vivo improving activity of dual L-Carnosine/A. vera nanophytosomes on Cytokeratin-10 (a keratinocyte marker) expression in diabetic rats.

To obtain Dual L-Carnosine/A. vera nanophytosomal gels, L-Carnosine at concentration of 1-4% (g/g) and A. vera at concentration of 0.01-0.04% (g/g), were homogenized (E1) in 1% of polysorbate surfactant (T) in a mixture of 10:1 (v/v) DMSO: dH20 mixture. In parallel, phospholipids at concentration ratio of 1:1 to 10:1 to total drug content, and 0.1 to 10% (g/g) of the composition was dissolved in minimum amount of chloroform (E2). The mixture (E1) was added to the solution (E2) and stirred for about 4-12 h to become completely unified yellowish solution (E3). In a separate beaker, with a volume at least three times of that of E3 solution, a 1:1 to 10:1 ratio of acetonitrile: ethanol will be prepared and the E3 solution will be added to the beaker dropwise with a flow rate of 0.1 ml/min to 10 ml/min. appeared precipitates will then become collected either by centrifugation at 5000 rpm for 10 min or filtration, using a filtration paper. Precipitates will then become completely dried for a week in a desiccator and then will be added (E4) to the inert hydrogel of 0.1-5% carboxymethylcellulose (G) at a concentration between 97.5 and 98.5% of the total product, at a temperature adjusted between 25 to 30° C. while undergoing homogenization (E5) for 15 minutes. desired amounts of dried precipitates were dissolved in dH2O by vigorous stirring or vortexing for 5 min. Nanophytosomes in liquid state were further subjected to pharmacological assays or microscopic imaging. After this point, the PG in a semi-solid pharmaceutical dosage form, will be stabilized, and the product (PG) can be bottled (E6), packed (E7) and labeled (E8). The L-Carnosine/A. vera gel will be stable until 01 year.

Aloe vera whole leaf extract powder, refers to the powder obtained following freeze-drying of the A. vera’s leaf pulp gel according to the previously established procedures, comprising steps of washing, decontaminating, separating the skin, crushing the gel, juice squeezing, enzyme deactivating and decolorating, filtrating, concentrating and finally, vacuum freeze drying. Moreover, the Aloe vera extract powder of this invention is standardized according to the polysaccharide content of the aloe product and must not contain carbohydrate amounts less than 9%. Standardization is performed by GC-MS and HPLC (31).

Alongside, quality of the carboxymethylcellulose based hydrogel was evaluated using samples of calcium alginate hydrogels already existing in clinic. To evaluate physiochemical properties of dual phytosomal gels (PG), the organoleptic characteristics, density, pH, viscosity, unit, dry residue and granulometry were analyzed according to the instructions provided by Cosmetic Products Quality Control Guides (32).

The stability of dual L-Carnosine/A. vera nanophytosomes was evaluated by both short-term and long-term methods. In short term method, 2 mL of a freshly prepared 10 mg/mL optimized nanophytosome was placed in glass bottles and stored at 25 or 4 °C for a duration of 14 days. Residual drug loading content, as one of the main evaluators of stability of a formulation, was then determined at specific time points and used for plotting short-term stability curves and understanding drug loosing pattern of formulation. For long-term stability analysis, dual nanophytosomes were lyophilized and stored for 6 months at constant temperature of 25 °C or 4 °C. At the end of each month, long-term stability was evaluated by measuring mean particle size and residual drug-loading content of formulations.

Moreover, the stability of phytosomal gels was evaluated based on the Cosmetic Product Stability Guide (33). In this context, multiple samples were produced and incubated either at ambient temperature, (25 ± 3° C) or an oven at 40± 4° C. At the end of 0, 15, 30, 60 and 90 days post incubation, changes in color, odor, pH (measured in 10% solution of samples) and humidity (Karl Fischer method) were evaluated in each sample.

Moisture test was also performed by immersing the phytosomal hydrogel in water for 2 hours at room temperature. Following this period, hydrogels were removed and dried in room temperature for 24 h after induction of slight scraps on the surface of the gels for removing dissolved moiety and the part not diluted in water. In addition, phytosomal gels (PG) were also evaluated for the presence of moisture, viscosity, homogeneity and color, odor, and weighed to evaluate the loss of mass.

Dual L-Carnosine/A. vera nanophytosomal gels are stable pharmaceutical formulations intended for use as topical application in treatment of diabetic wounds, providing the advantages of its main active components, L-Carnosine and A. vera. Administration of this composition brings therapeutic benefits to the management of diabetic ulcers, and hence, it provides greater effectiveness in management of diabetic wound owing to the anti-inflammatory, analgesic, healing, antimicrobial, and most importantly, proangiogenic activities.

Preclinical studies were performed with the purpose of evaluating accelerating activity of the phytosomal hydrogel on diabetic wound activity following approval of the study by council on animal experimentation of IUMS. The acute toxicity of the phytosomal hydrogel was assessed PO in mice of Mus musculus species for determination of LD50 values based on the protocol described by Litchfield and Wilcoxon (34). In this context, animals were fast for 18 hours and 1 hour following administration of the dual L-Carnosine/A. vera phytosomes, animals were subjected to muscle tone test (stick test) and exploratory activity test (open field test) and observations continued for 14 days, on morning time (1 hour).

Primary and cumulative dermal toxicity was evaluated according to the established Cosmetic Product Safety Assessment Guides. Initial skin irritation tests were carried out in mice (20 to 30 g) and rats (180 to 300 g) which had been previously shaved. 4 regions of the laboratory animals, including the dorsal skin and ear regions were demacerated, two of which were superficially scratched by a sterile needle while thw other remaining two marked regions remained intact and used as control. and edema of the paw and ear of the laboratory animals had four regions demarcated, two of them superficially scratched with the aid of a sterile needle and the other two remained intact, being used as control. Phytosomal hydrogel samples were then applied in these regions utilizing an occlusive patch, composed of fixed sterile gauze; 24- to 72 hours later, erythema and/or edema occurrence in the skin was evaluated. For chronic exposure, the same procedure was repeated except for applying samples every 24 and 72 hours for 12 to 14 cycles.

Examined in related pharmacological toxicity assays and preclinical studies related to acute and subchronic toxicity in mice and rats, A. vera whole extract and L-Carnosine did not induce mortality and no toxic effects were observable at doses higher than 5000 mg/kg. In tests related to the assessment of analgesia, oral and intraperitoneal administration of phytosomal gel demonstrated a dose-dependent analgesic effect in acetic acid, formalin and capsaicin pain models. Moreover, administration of phytosomal gel could meaningfully increase animal’s latency in hot plate test model of pain (49-54° C.). a statistically significant anti-inflammatory effect from the L-Carnosine/A. vera dual phytosome was demonstrated in the model of the edemological inflammatory process induced upon administration of carrageenan, croton oil and arachidonic acid. Consequently, based on results, administration of dual phytosomes resulted in induction of a significant anti-inflammatory effects and did not present toxicity in laboratory animals orally. Physiochemical characteristics of dual L-Carnosine/A. vera gel (PG) includes: a bitter spicy tase; white to perish yellow color; odorless, in a liquid to solid state. In the skin sensitivity test in mice, no irritation was observable in wound site. Moreover, applying dual phytosomal gels at concentration between 1 to 4% in human patients did not cause any dermal sensitivity or skin irritation capable of inducing cytotoxic effects compared to the non-treated group as well as the standard calcium alginate hydrogel group.

Preclinical studies performed with dual L-Carnosine/A. vera on full-tichness wounds of diabetic mice demonstrated a statistically significant improvement in healing rate of the groups treated with 1-4% phytosomal gels. The group treated with 4% dual phytosomal gels reduced healing time observed with calcium chloride Alginate hydrogels from 21 days to about 14 days, while 2% phytosamal gel could reduce healing time to 17 days. In these groups, deposition of collagen type I and α-SMA was significantly increased and inflammation was effectively subsided. Finally, 0.25% phytosomal gel could reduce the treatment period to about 19 days. No meaningful alterations in toxicological parameters including those related to liver, kiney and cardiac function was observed in all studied groups.

In a presently preferred exemplary embodiment, L-Carnosine is provided in amounts ranging from 0.01 to 10% of the dual L-Carnosine/A. vera phytosomal gel. More preferably, L-Carnosine is provided in amounts ranging from 0.05% to 5% of the dual L-Carnosine/A. vera phytosomal gel. Even more preferable, L-Carnosinne is provided in amounts ranging from 0.25 to 1% of the dual L-Carnosine/A. vera phytosomal gel. L-Carnosine is a dipeptide molecule, composed of the amino acid’s beta-alanine and histidine which is naturally synthesized in human liver from its precursor. It is highly accumulated in brain and muscles and demonstrates effective anti-oxidative and anti-inflammatory activity in vitro.

In a presently preferred exemplary embodiment, A. vera is provided in amounts ranging from 0.0001 to 0.1% of the dual L-Carnosine/A. vera phytosomal gel. More preferably, A. vera is provided in amounts ranging from 0.0005% to 0.05% of the dual L-Carnosine/A. vera phytosomal gel. Even more preferable, A. vera is provided in amounts ranging from 0.0025 to 0.01% of the dual L-Carnosine/A. vera phytosomal gel.

In a presently preferred exemplary embodiment, phospholipids are provided in amounts ranging from 0.03 to 30% of the dual L-Carnosine/A. vera phytosomal gel. More preferably, L-Carnosine is provided in amounts ranging from 0.15% to 15% of the dual L-Carnosine/A. vera phytosomal gel. Even more preferable, L-Carnosinne is provided in amounts ranging from 0.75 to 3% of the dual L-Carnosine/A. vera phytosomal gel. Phospholipids, also referred as phosphatides, are a specific group of lipids whose members have a hydrophilic phosphate bearing "head", and two hydrophobic fatty acid "tails" linked through a glycerol moiety. In present invention, phospholipids consist of, but not limited to phosphatidylcholines, phosphatidyl serins, phosphatidylethanolamines, etc. the phospholipid in present invention is more preferably chosen from phosphatidylcholine molecules. The phospholipid in present study is even more preferably lecithin from soybean and egg yolk.

Examples

Example 1 demonstrates topical composition based on the present invention. The composition can be developed in conventional manner and it is appropriate for management of different types of wounds, more preferably, diabetic associated wounds use by induction of anti-inflammatory, anti-microbial, soothing and analgesic effects.

Component	% w/w
L-Carnosine	0.25%
A. vera whole leaf extract	0.0025%
Lipid Content	0.75%
Polysorbate
20	1%
Methyl Paraben	0.3%
CarboxymethylCellulose
	1%
Water	Up to 100

N/A

N/A

N/A

N/A

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WO2015007922A1

US4725438A

KR102073009B1

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Claims (9)

1. Gel containing dual L-Carnosine/Aloe barbadensis (A. vera) nanophytosomes for acceleration of diabetic wound healing, A gel encompassing Aloe vera’s (Aloe barbadensis) whole leaf extract powder and L-Carnosine pure powder (D) in a 1:1 to 1:1000 (w/w) ratio at a rate of 0.1 to 2% of the composition and phospholipids (L): total drug content in a 1:1 to 1:10 (w/w) ratio, comprising 0.1 to 10% of the composition; Polysorbate surfactant (T) at a rate of 1% of the composition; and 0.1-5% carboxymethylcellulose inert hydrogel (G) at a rate between 97.5 and 98.5% of the composition.
2. The gel encompassing dual L-Carnosine/A. vera nanophytosomes for accelerating diabetic wounds according to claim 1, wherein L-Carnosine can be replaced with its alternatives including but not limited to N-acetyl Carnosine, homocarnosine, Zinc-Carnosine, Anserine, beta-Alanine, L-histidine.
3. The gel encompassing dual L-Carnosine/A. vera nanophytosomes for accelerating diabetic wounds according to claim 1, wherein A. vera whole leaf extract can be replaced with its alternatives including but not limited to N-acetyl Carnosine, homocarnosine, Zinc-Carnosine, Anserine, beta-Alanine, L-histidine.
4. The gel encompassing dual L-Carnosine/A. vera nanophytosomes for accelerating diabetic wounds according to claim 1, wherein A. vera whole leaf extract can be replaced with other Aloe species and either parts of the leaf alone, including inner mucilage and/or outer rind of the leaf.
5. The gel encompassing dual L-Carnosine/A. vera nanophytosomes for accelerating diabetic wounds according to claim 1, wherein the phytosomal gel (PG) is presented as a gel of topical use, at a concentration level of 0.5 g of phytosomes (P) per 100 g of carboxymethylcellulose hydrogel (G).
6. The gel encompassing dual L-Carnosine/A. vera nanophytosomes for accelerating diabetic wounds according to claim 1, wherein the phytosomal gel (PG) is presented as a gel of topical use, at a concentration level of 1 g of phytosomes (P) per 100 g of carboxymethylcellulose hydrogel (G).
7. The gel encompassing dual L-Carnosine/A. vera nanophytosomes for accelerating diabetic wounds according to claim 1, wherein the phytosomal gel (PG) is presented as a gel of topical use, at a concentration level of 0.25 g of phytosomes (P) per 100 g of carboxymethylcellulose hydrogel (G).
8. The gel encompassing dual L-Carnosine/A. vera nanophytosomes for accelerating diabetic wounds according to claim 1, wherein the dual L-Carnosine/ A. vera nanophytosomal gel (PG) is applied for induction and acceleration of wound healing process.
9. The gel encompassing dual L-Carnosine/A. vera nanophytosomes for accelerating diabetic wounds according to claim 1, wherein the dual L-Carnosine/ A. vera nanophytosomal gel (PG) is applied for treatment of diabetic wounds.
   

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