Design, manufacture and optimization of a novel transferosome-based topical drug delivery system containing argirelin (acetylhexapeptide-3) (acetylhexapeptide-8) with the aim of improving skin permeability with anti-aging effect and eliminating wrinkles.

Abstract

Today, peptides have received much attention in the world with cosmetic and pharmaceutical approaches due to their high benefits and low disadvantages. Argireline peptide is one of the most widely used drugs in the treatment of skin wrinkles. Problems with the use of injectable products by individuals cause problems such as delays in using the appropriate dose due to the need to refer to a specialist and clinic or side effects related to the use of inappropriate doses of the drug. In case of topical application to the skin, the amount of changes, the desired areas of the face and also the desired corrections of the consumer can be applied. Transferosomes, as one of the new drug delivery formulations, are a suitable option for topical dermal drug delivery of large hydrophilic peptides due to their high stability and at the same time high flexibility in passing and penetrating through pores with a diameter 50 times smaller than their own size. In designing the transferosomal drug delivery system, the effort is to minimize the negative factors and obstacles to the drug reaching the desired skin layer without side effects and fluctuations in drug release. The advantages of this method include the lack of systemic absorption of the drug, greater stability and persistence of the transferosomal form in the site compared to the free form, reduced drug dosage, high efficiency and effectiveness with a 33% increase in skin penetration, low cost, and production and commercialization of the product within the country. The main objective of the present invention is to produce a topical dermal pharmaceutical formulation comprising transferosomal nanocarriers containing argirelin peptide.

Description

Description of the invention Title of the invention Design, manufacture and optimization of a novel transferosome-based topical drug delivery system containing argireline (acetylhexapeptide-3) (acetylhexapeptide-8) with the aim of improving skin permeability with anti-aging and wrinkle removal effects. Technical background of the relevant invention This invention relates to the field of pharmaceutical and cosmetic products, and in recent years, topical drug delivery has been the goal of many pharmaceutical and cosmetic companies. Topical drug delivery has several advantages over conventional dosage forms (oral and intravenous), such as avoiding first-pass metabolism (passing the hepatic route), ease of application, reduced enzymatic degradation associated with oral administration, improved patient acceptance, and controlled release of drugs (1). The skin is the largest and most important organ in the body for the local and systemic effects of drugs. The most important factor in the local cutaneous effect of a drug is the passage of the stratum corneum as the most important barrier to penetration into the body. The most common forms of topical drugs include ointments, creams, gels, and lotions. It is very important that the topical drug delivery system is selected based on the size and properties (wet / dry, mucosal / non-mucosal, healthy / diseased) of the skin to be treated in order to optimize the application and contact of the product with the skin. This can have profound effects on the potency, efficacy, and patient compliance (2).There are various systems for transdermal drug delivery that are selected based on molecular weight less than 500 Daltons, logarithm of octanol / water partition coefficient between 1 and 3, melting point less than 200 °C, aqueous solubility greater than 1 mg / mL, and absence or limited polar centers. So far, various products with appropriate efficacy and cost reduction have been used with this approach. These approaches include the use of chemical adsorption enhancers, methods of increasing physical penetration, encapsulation within hydrophobic carriers, and also chemical modification methods (3). Today, peptides have attracted much attention in the world with cosmetic and pharmaceutical approaches due to their high benefits and low disadvantages. Acetylhexapeptide-8 or (acetylhexapeptide-3) is known by the trade name Argireline, has the amino acid sequence Acetyl-Glu-Glu-Met-Gln-Arg-Arg-NH2 and, according to studies, prevents the release of neurotransmitters, which in turn has anti-wrinkle effects and seems to improve skin firmness and tone (4). HP-8. It is a copy of synaptosomal-associated protein 25 that competes for a position in the SNARE complex and destabilizes its formation without breaking any of its constituent components. In addition, it also inhibits catecholamine secretion. Its effect is manifested by blocking acetylcholine receptors and ultimately the lack of muscle contraction, which is the apparent result of the removal of wrinkles (5). Argireline-containing products are marketed under different names and in different dosage forms such as serum or cream. Due to the technical characteristics of this peptide, including its high molecular weight and high polarity, it does not penetrate the skin properly, and due to the user's cost, the appropriate result occurs with a long time, high consumption and low efficacy. The injectable form of this peptide is used under the name Mosport and has a good effect similar to the effects of Botox type A in cosmetic uses (anti-wrinkle) and therapeutic effects (treatment of sweating problems and migraines). Considering that using the injectable form also involves considerations such as the requirement to visit a clinic or hospital, it makes use difficult for the patient.Due to the hydrophilic nature of argirelin, the high polarity of this peptide, and its size of 889 kDa, it is difficult and inefficient to cross the stratum corneum. The use of modern drug delivery systems such as transferosomes overcomes this problem. Transferosomes, due to their high stability and at the same time high flexibility in passing and penetrating through pores with a diameter 50 times smaller than their size, are a suitable option for topical dermal drug delivery of this large hydrophilic peptide (6, 7). The main objective of the present invention is to produce a topical dermal pharmaceutical composition unit comprising transferosomal nanocarriers containing argireline peptide. Resources: 1. Nasrollahi SA, Taghibiglou C, Azizi E, Farboud ESJCb, design d. Cell-penetrating peptides as a novel transdermal drug delivery system. 2012;80(5):639-46. 2 #x200f.#x200f Barnes TM, Mijaljica D, Townley JP, Spada F, Harrison IPJP. Vehicles for drug delivery and cosmetic moisturizers: Review and comparison. 2021;13(12):2012. 3 #x200f.#x200f Grumezescu V, Negut I, Grumezescu AM, Ficai A, Dorcioman G, Socol G, et al. MAPLE fabricated coatings based on magnetite nanoparticles embedded into biopolymeric spheres resistant to microbial colonization. 2018;448:230-6 . 4 #x200f.#x200f Schagen SKJC. Topical peptide treatments with effective anti-aging results. 2017;4(2):16 . 5. Ruiz M, Clares B, Morales M, Cazalla S, Gallardo VJIJoCS. Preparation and stability of cosmetic formulations with an anti ‐ aging peptide. 2008;30(1):74 -. 6. Lim SH, Sun Y, Thiruvallur Madanagopal T, Rosa V, Kang LJSr. Enhanced skin permeation of anti-wrinkle peptides via molecular modification. 2018;8(1):1596 . 7. Opatha SAT, Titapiwatanakun V, Chutoprapat RJP. Transfersomes: A promising nanoencapsulation technique for transdermal drug delivery. 2020;12(9):855 . مشكل فني و بيان اهداف اختراع Argireline peptide is one of the most widely used drugs in the treatment of skin wrinkles. Its injectable formulation seems superior to its topical formulation (serum or cream). However, the problems of using injectable products by the user cause problems such as delay in using the appropriate dose due to the requirement to visit a specialist and clinic or side effects related to using an inappropriate dose of the drug (changes are visible after a few days), while the effective topical form of the skin eliminates these problems and obstacles. Also, in the case of topical use of the skin, the amount of changes, the desired areas of the face, and also the desired corrections of the consumer can be applied. In designing new dermal drug delivery systems, the effort is to minimize the negative factors and obstacles to the drug reaching the desired skin layer without side effects and fluctuations in drug release, and to make the release effective and predictable under different conditions. Given the current conditions, the only obstacle to the topical effectiveness of argireline in the skin in terms of cosmetic effects (anti-wrinkle) or therapeutic effects (treatment of sweating problems, migraines or blepharospasm) is the passage through the stratum corneum of the skin. Goals: 1-Preparation of a topical dermal drug delivery system including a drug compound unit containing the peptide acetylhexapeptide-3 and controlled release on the skin surface including wrinkles 2- Preparation of flexible transferases carrying argireline to facilitate dermal drug delivery 2- No systemic absorption of the drug and entry into the lower layers of the dermis and reduced side effects compared to the injection method 3- Targeted drug delivery and appropriate drug release and greater stability of the transferosomal form compared to the free form 4- Producing a product with high efficiency and effectiveness and low cost 5- Production and commercialization of the product in Iran A description of the state of the prior art and the history of developments related to the claimed invention. In studies, novel topical dermal drug delivery systems include the formulation of argirelin in dosage forms such as serums or creams. Consumers are increasingly seeking “anti-aging” products that address wrinkles and skin folds. The advent of costly and painful cosmetic injections to treat expression lines has increased interest in finding effective, noninvasive topical alternatives. Expression lines are a specific type of wrinkle that develops on the facial skin in early adulthood. They are anatomically associated with the facial expression muscles in the periorbital, glabella, forehead, and perioral regions. The activity of these muscles during smiling, pursing, and frowning places greater physical stress on the overlying skin than other areas of the face. For this reason, expression lines are less responsive to topical treatments that focus on noncontractile elements of the skin anatomy, such as the epidermis. In order to most effectively treat expression lines, it must involve the inhibition of facial expression muscles and the muscle fiber elements associated with the dermis. In patent number WO2007102957A3 and entitled “Skin care compositions including hexapeptide complexes and methods of their manufacture”, a “safe and effective amount” of the drug is considered to mean a sufficient amount of the compound to significantly improve the condition being treated, but at the same time, it is low enough to avoid unwanted side effects. The latter is precisely the point that creates limitations in injectable products, since there is no possibility of correction after injection. In patent US7566464B2 entitled “Cosmetic composition to accelerate repair of functional wrinkles”, considering the different effects of argirelin in the treatment of some diseases such as blepharospasm and also in another study entitled “Topical delivery of acetyl hexapeptide-8 from different emulsions: Influence of emulsion composition and internal structure”, with the anti-wrinkle effects of argirelin and the problem of skin penetration of this highly polar peptide, improving its skin permeability is of interest. In a clinical study registered in the US Government Clinical Trials Center https: / / classic.clinicaltrials.gov / ct2 / show / NCT01750346 for the treatment of blepharospasm and the topical use of argirelin (AH-8), appropriate effectiveness has been reported. It is important to note the thickness and thinness of the eyelid skin and the possibility of greater penetration into the SC layer compared to other areas of the facial skin. This means that if argirelin succeeds in penetrating the skin, it will have a good effect in blocking acetylcholine receptors. EP 1856139 A2, entitled “Compounds which inhibit neuronal exocytosis”, describes the use of peptides derived from SNAP-25 that have been chemically modified to increase their effectiveness. Diseases such as hyperhidrosis, for which treatment with botulinum toxin has been effective, are other possible applications. The cosmetic and health care industry has made significant efforts to develop compounds that mimic the action of botulinum toxin in the treatment and prevention of wrinkles. EP 1180524 A1, entitled “Neuronal exocytosis inhibiting peptides and cosmetic and pharmaceutical compositions containing said peptides”, and international application WO 97 / 34620 describe peptides derived from the amino sequence. This six-amino acid peptide is capable of blocking acetylcholine receptors. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention The use of hydrophobic carriers to transport argirelin into the skin could be one of the appropriate solutions to solve the problem of dermal drug delivery. Given that increasing dermal drug delivery using liposomal nanocarriers has been reported to increase skin hydration. Topical liposomes, due to their nature, reduce skin roughness and intercellular lipids that lead to skin softening and smoothing. Transfersome nanocarriers, a member of the liposomal family, are composed of hydrophilic and hydrophobic parts, which leads to becoming a unique drug nanocarrier system that can carry therapeutic agents with a wide range of solubility. Transfersomes, due to their high stability and flexibility, can pass through the stratum corneum (the most important skin barrier) by deforming through narrow paths, from 5 to 50 times smaller than the diameter of the vesicle, due to their extraordinary deformability and elasticity.The high vesicle conformation facilitates drug delivery across the skin without any measurable loss of drug in intact vesicles and can be used for topical as well as systemic therapies. The transfersome carrier is highly versatile and efficient in accommodating a variety of agents almost independent of their size, structure, molecular weight or polarity. The transfersome is composed of natural phospholipids and the EA lipoactivator, and is therefore highly biocompatible and biodegradable. In one or more embodiments of the present invention, the formulation and physicochemical properties of argirelin-containing transferosomes are prepared and the results obtained are as follows: 1-Preparation and investigation of physicochemical properties of transferosomes containing argirelin Transferosomes were prepared using the rotary film evaporation method, originally developed by Bangham. In this process, the required amount of phospholipids and surfactants (as EA) to form a thin film are dissolved in an organic solvent, which is a mixture of chloroform and methanol. The prepared solution is transferred to a round-bottomed flask, which is rotated at a constant temperature (above the lipid transfer temperature) and with reduced pressure. A layer of lipids and EA forms on the wall of the flask in the form of a thin film. The twisted film is then hydrated using aqueous media containing the drug. Addition of an aqueous solvent carrying the drug results in the formation of lipid bilayer vesicles called transferosomes, which are reduced to the desired size using extrusion or sonication. Phospholipids used in the preparation of transferosomal formulations include DPPC, DOPC, Egg PC, and a mixture of the aforementioned phospholipids. Surfactants such as Tween80, Span60, and Sodium deoxycholate were also used in different percentages according to the experimental design. The effective factors are listed in Table 2. 2-Determining the percentage of argirelin retention in the transferosome In order to investigate the retention rate of argirelin, (EE%) a standard curve was first drawn by preparing different concentrations of standard argirelin in methanol and determining the absorption in a UV-visible spectrophotometer at a wavelength of 215 nm. To investigate the retention rate of the drug in transferosomes, a standard curve of argirelin in methanol was first drawn as follows: From the 1 mg / ml stock solution, different concentrations of 5, 10, 12.5, 25, 50, 75, 100, 125 µg / ml were prepared and their UV absorbance was determined. To draw the calibration graph, the preparation and UV absorbance measurement step was repeated at least 3 times and the intra- and inter-day reproducibility of the analysis method was examined. The prepared transferosomes were thoroughly mixed and disrupted with methanol (v / v) in a ratio of 1:10 and their UV absorbance at 215 nm was determined using a visible-ultraviolet spectrometer and finally the argirelin concentration was obtained according to the standard curve equation. Then, the percentage of drug accumulated in the transfersome was measured using the equation below and is shown in Figure 6. It should be noted that in order to ensure that there was no possible interference of the components of the transfersome in the UV absorption with argirelin, an empty transfersome was prepared and after performing the above-mentioned steps, the absorption of the resulting sample was examined at a wavelength of 215 nm, which did not show any significant absorption. Argirelin was also added to the above-mentioned empty transfersome sample at a certain concentration and after complete mixing, the absorption and UV spectrum of the sample were prepared and examined, and no change was observed in the maximum of argirelin and its absorption rate. 3-Investigating the particle size distribution and zeta potential of formulations The particle size distribution of the transferosome nanoparticles was measured and reported using a Malvern Zetasizer. It is shown in Figure 12. 4-Investigation of the release of argirelin from transfersomes in vitro To investigate drug release from transferosomes, 500 μL of each of the formulations containing argirelin was placed inside a dialysis bag and placed in 50 ml of PBS medium with pH=7.4, pH=5.8, and deionized water at 37°C and 50 rpm rotation. At specific times (0, 2, 4, 6, 12, and 24 hours), samples were taken from the release medium and the medium was replaced according to the amount of sample taken, and then the amount of drug released was determined using HPLC and a calibration chart. 5-Determination of the amount of argirelin in the release medium by HPLC method Due to the slow release of the drug from the transfersome and the presence of low concentrations of argirelin in the release medium, HPLC analysis was used to determine the amount of argirelin in the samples taken, the conditions of which are as follows: Resident phase: C18 column Mobile phase: water and acetonitrile mixture (v:v): 50:50, 90:10, 95:5 with TFA1 / 0 (v / v)) Mobile phase speed: 0.7 ml / min Column temperature: 25℃ Wavelength: 215 nm Injection volume: 20 microliters 6- Drawing a standard curve for drug analysis in the release environment using HPLC. In order to investigate the release rate of argirelin from the transferosome, first, the calibration curve of argirelin in water and PBS at the mentioned pHs was drawn with an HPLC device. For this purpose, different concentrations of 10, 25, 100, 250, 500, 1000 and 2000 ng / ml were prepared. Then, to obtain a relationship between the concentration of argirelin and the area under the peaks, the calibration curve of argirelin was drawn. 7- Checking the accuracy and precision of the drug analysis method with HPLC equipment To investigate the two indicators of accuracy and precision, intraday and interday studies were conducted. This means that samples of all calibration concentrations were prepared and analyzed three times in one day and also on three consecutive days, respectively, and the results were reported as relative error percentage (RE%) and coefficient of variation percentage (RSD%), respectively. 8-Investigating the stability of argirelin under argirelin release test conditions There is no guideline in the Pharmacopoeia or USP regarding the stability of peptides. In order to ensure the stability of argirelin during the analysis period, especially the release time of argirelin, different concentrations of argirelin were investigated under release conditions (temperatures of 4, 25 and 37 ℃ in water and PBS medium) and the argirelin peptide was stable. 9- Morphological evaluation using field emission electron microscopy Field emission electron microscopy images of the optimized formulation of transferosomes containing argirelin peptide are shown in Figure 13. The round and circular structure like transferosomes can be seen. 10- Morphological evaluation using transmission electron microscopy Transmission electron microscopy images of the optimized formulation of transferosomes containing argirelin peptide are shown in Figure 14. The particle size and circular cross-section of the transferosomes can be seen. 11-Investigating possible interference between carrier ingredients and drug using FTIR method To identify the materials used in the formulation and to investigate the interference between the peptide and the carrier, FTIR spectra were used with an AgilentCary 360 FTIR instrument (USA). Each reported spectrum has an average of 64 scans with a resolution of 14 cm-1 in the spectral range from 1400 to 4000 cm-1. The results are shown in Figure 12. 12-Investigation of skin permeability of transferosomes containing argerelin (In-Vivo) To study skin permeability, human abdominal skin obtained from abdominoplasty surgery was used. The epidermis layer was separated from the skin and studied. Studies using Cell France and human skin samples were studied and examined over a 24-hour period (times 0, 24, 18, 12, 6) according to the following analysis method and using an LC-Mass device, the results of which are shown in Table 3. Analysis method: The isocratic method was performed with a 10 cm C18 column at a flow rate of 0.4 ml / min with a solvent ratio of 97% mobile phase A (deionized water with 0.1% TFA) and 3% mobile phase B (acetonitrile with 0.1% TFA) for 5 minutes with an injection volume of 20 μl. In order to investigate the MRM for the peptide, ion monitoring and determination of the masses of the appropriate daughter ions with appropriate analysis parameters were performed via product ion. After obtaining an optimal method, samples of different formulations were analyzed and measured at different times. It should be noted that in order to examine the accuracy and precision of the analysis method, the samples were examined intraday and interday. Explanation of shapes, maps and diagrams The drawings, manufacturing steps of the present invention, figures and tables presented herein illustrate one or more embodiments of the present invention by way of example only. Accordingly, the figures presented herein do not limit the scope of the present invention. Shapes: Figure 1- General schematic of the synthesis of argirelin-containing transferosomes and skin penetration study Figure 2 - Chemical structure of (Acetylhexapeptide-3)-(Acetylhexapeptide-8)-(Argireline) Figure 3 - Synthesis steps of argirelin-containing transferosomes Figure 4 - Visible-ultraviolet spectrum of standard argireline Figure 5- FTIR spectrum of argyreline Figure 6- Calibration curve of argirelin synthesized in methanol by ultraviolet spectrophotometer in the concentration range of 5-100 μg / ml (n=3) Figure 7 - HPLC spectrum, standard argirelin Figure 8- Argireline standard curve in release medium (phosphate buffer) (pH=5.8) Figure 9- Argireline standard curve in release medium (phosphate buffer) (pH=7.4) Figure 10- Argireline standard curve in release medium (deionized water) Figure 11 - Investigation of argireline and transferosome interactions Figure 12 - Sample particle size distribution and zeta potential of several formulations. Figure 13 - FESEM microscope image of the optimal formulation Figure 14 - TEM microscope image of the optimal formulation Figure 15 - LC-Mass spectrum with MRM method of skin samples Tables: Table 1- Intra-day and inter-day accuracy and precision in the argirelin analysis method using the ultraviolet spectrometer in methanol (n=3) Table 2 - Factors affecting the formulation of Transfersome-Argireline Table 3- Investigation of skin permeability of argirelin peptide compared to argirelin-containing transferosomes using LC_Mass device A clear and precise statement of the advantages of the claimed invention over prior inventions. An invention based on the production of a pharmaceutical composition consisting of transferosomes containing the peptide argirelin (acetylhexapeptide-8 or acetylhexapeptide-3) for targeted drug delivery to the skin with the aim of reducing wrinkles and in treatment cases such as migraine or blepharospasm has not been registered. By pointing out the advantages of using transferosomes as a carrier for this peptide in improving wrinkles, the problems of common formulations in the incomplete delivery of this drug can be overcome; 1- Easy and green synthesis method using phospholipids and lecithin as drug carriers and stabilizers, with hydrophobic nature and high flexibility in passing through skin layers. 2- Slow release of the drug from nanoparticles and physicochemical stability in passage and release in the dermis layer of the skin 3- The method is cost-effective and non-toxic due to the use of biocompatible materials. 4- The effect of both nanocarrier and peptide components on the skin by creating anti-aging properties 5-Reducing the drug dosage due to drug delivery at the appropriate concentration for treatment Combining the nanocarrier with the drug (peptide) will not only prevent the release of the drug before reaching the target site, but also prevent the sudden release of the drug and cause slow and intermittent release in this area. Finally, ex-vivo studies show the proper performance of this drug delivery system (the present invention) in the treatment of skin wrinkles and some related diseases that can be treated by blocking acetylcholine receptors (paralyzing the target muscle). Description of at least one implementation method for implementing the invention Industrial production of transferosomal formulations containing argirelin with appropriate efficacy and favorable cost, with a cosmetic or therapeutic approach, is easily possible in clean conditions of pharmaceutical factories. Explicit mention of the industrial application of the invention The drug delivery system based on transferosomes containing argirelin can be used as a topical pharmaceutical and cosmetic composition with good skin penetration, high durability and stability. The transfersome formulation containing argirelin is used topically on the skin, and the good physicochemical stability, loading of appropriate amounts of drug, absence of investigated interactions and biodegradability have made this formulation a very suitable composition that can be used in the pharmaceutical industry and as a new cosmetic and pharmaceutical product.

Claims

Claims What is claimed: Claim 1) ] Design and manufacture of a novel topical drug delivery system based on transferosome containing argireline with the aim of improving skin permeability. Claim 2) Creating a pharmacopoeial feature according to claim 1, including a nanotransferosomal argirelin formulation, using various phospholipids including Egg PC, DOPC, DPPC, SPC cholesterol and various surfactants including Tween80, SDC and Span 60 by the thin film method and using a mixture of chloroform and methanol (70:30) and then prepared by the extrusion method. Claim 3) The formulation made according to claim 2 is in the form of a flexible yet stable reservoir particle system (transferosome), having a hydrodynamic size of 153 nm and PDI=0.

3. Claim 4) The pharmaceutical composition contains a certain percentage of the drug (argirelin) and transferosome (as a nanocarrier) that has the property of significantly improving skin penetration into skin cells and paralyzing tissue muscles for aesthetic (anti-wrinkle) or therapeutic (migraine or sweating) purposes. Claim 5) The transferosome made according to claim 3 is 33% more permeable to the skin than argirelin without a carrier, and the release of the drug from the carrier to the site takes about 24 hours in a slow release manner. [

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