A dermal filler for soft tissue augmentation

Abstract

The present invention relates to a dermal filler for soft tissue augmentation comprising 85-99% water and 1-12% microbial cellulose wherein the said cellulose is Acetobacter xylinum fermented in coconut milk culture media. The microbial cellulose is reduced to a nano particle having a diameter predominantly 100 nm and has a nano porous mesh structures and fibrils aggregate forming tunnels in between the microbial cellulose fibers for gaseous and fluid exchange; has a high cellulose crystallinity (60-80%) and has an enormous mechanical strength for the extended biodegradability, and biocompatibility. Said microbial cellulose has a preservative in the form of a natural coconut derived active ingredient where monolaurin is present in the amount of 1%. It is pure, natural, hydrophilic and characterized by the absence of lignin. It can be easily injected and pass through a gauge 30 needle with minimal reaction and discomfort at the site of the injection. It is safe and has a porosity benefit which makes it more permeable and penetrable for effective tissue augmentation and aesthetic correction of wrinkles or loss of muscle volume including nasolabial furrow, wrinkled cheeks, deep smile lines and lipoatrophy correction. It is non¬ phototoxic, no toxicity and safe on the area around the eyes (Optisafe). It can be administered intradermally or sub-epidermally. The cellulose suspension is packed in glass vials of 3 ml, 8%, 10% and 12% concentration.

Description

[0001] A DERMAL FILLER FOR SOFT TISSUE AUGMENTATION

[0002] SPECIFICATION

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] This invention relates in general to cosmetic enhancements, and more particularly to a dermal filler for soft tissue augmentation using a microbial cellulose nanoparticle which can be administered intradermally and sub-epidermally.

[0005] BACKGROUND OF THE INVENTION

[0006] As we grow old, our body starts to lose collagen which is an important substance that exists all over our body including our skin, muscles, bones and connective tissues. This decrease of collagen causes skin laxity and loose of volume resulting to thinner, less elastic and sagging skin.

[0007] The above condition can be medically remedied by the use of dermal fillers which add volume to sagging skin; make facial features more symmetrical; plump up lips and cheeks; and smooth wrinkles and creases in the face.

[0008] On the other hand, the key to growing old gracefully is to accept changes seen in the body. Unfortunately, the aging process brings about changes in the mature face, which many men and women cannot accept. The face acquires new attributes like wrinkles, which are very visible in the face.

[0009] Nonsurgical facial rejuvenation treatment methods are now the most common aesthetic treatments performed worldwide, and dermal fillers for soft tissue augmentation are second to botulinum toxin ( Botox) as the procedure chosen by patients to battle signs of aging. Dermal fillers are used to reduce wrinkle / facial lines.

[0010] Many types of dermal fillers are available in the market and these include hyaluronic acid ( HA), calcium hydroxylaptite ( CaHA), poly-L-lactice acid ( PLLA), and polymethylmedthacrylate (PMMA). HA is currently the most commonly used soft-tissue filler because of the vast number of products available, it is easy to use, and has overall low rates of complication. HA, which is a glycosaminoglycan found in the skin, attracts, and binds very well to water (hydrophilic). Thus, when HA is injected into the skin, it volumizes and hydrates. Moreover, HA has hydrophilic property and can create a gelatinous matrix which holds collagen and elastin fibers that impacts on skin turgor. This HA dermal filler usually last six (6) months to a year.

[0011] Because of high moisture retention, biocompatibility and viscoelasticity properties of this polymer, HA has become an important component of biomedical and cosmetic products from successful and lucrative pharmaceutical companies worldwide. Initially extracted from avian sources like rooster combs, large scale production of HA now involves the use of bacterial expression systems in Streptococci and the use of endotoxin-free microorganisms such as Bacilli and Escherichia coli. One of the limitations of HA is the rapid turnover within the tissues because of the presence of the enzyme hyaluronidase. To lessen the rapid turnover, HA has been modified by cross-linking the HA chains to certain chemicals like 1 ,4-butanediol diglycidyl ether (BDDE) which is the most commonly employed cross-linking agent. HA then becomes more stable after cross-linking and this increases its duration of action from four to greater than 6 months, depending on the type.

[0012] Despite the ease of use of HA-based dermal fillers, the occurrence of mild side effects usually disappear after a few days, with no downtime from the procedure. However HA filler treatment have occasional drawback, the migration or displacement of the filler from the appointed injection site which impairs the aesthetic outcome.

[0013] In addition, the production of large volumes of HA is hampered by the methods used to manufacture HA, namely extraction from animal tissues and microbial fermentation using bacterial strains. Thus, to this day, the cost of HA dermal fillers is high. Popular HA dermal fillers like Juvederm and Restylane cost 682 USD per I ml syringe For HA products, re treatment is recommended after 3-4 months to maintain the beneficial effect and for elder women, they will need 2 - 4 ml. per treatment The cost of the whole treatment might be around 1800 to 2000 USD.

[0014] HA filler treatment have occasional drawback, the migration or displacement of the filler from the appointed injection site which impairs the aesthetic outcome. SUMMARY OF THE INVENTION

[0015] The subject invention relates to a dermal filler for cosmetic enhancement. It comprises 85-99% water and 1-12% microbial cellulose. The microbial cellulose is derived from all natural raw material. Said microbial cellulose is Acetobacter xylinum fermented in coconut milk culture media. It is reduced to a nano particle having a diameter predominantly 100 nm. Thus, for this invention the term MCNP will be used which stands for Microbial Cellulose Nano Particle. It has a nano porous mesh structures and fibrils aggregate forming tunnels in between the microbial cellulose fibers for gaseous and fluid exchange; has a high cellulose crystallinity (60-80%) and has an enormous mechanical strength for the extended biodegradability, and biocompatibility. Said microbial cellulose has a preservative in the form of a natural coconut derived active ingredient where monolaurin is present in the amount of 1%. It is pure, natural, hydrophilic and characterized by the absence of lignin. It can be easily injected and pass through a gauge 30 needle with minimal reaction and discomfort at the site of the injection. It is safe and has a porosity benefit which makes it more permeable and penetrable for effective tissue augmentation and aesthetic correction of wrinkles or loss of muscle volume including nasolabial furrow, wrinkled cheeks, deep smile lines and lipoatrophy correction. It is non-phototoxic, no toxicity and safe on the area around the eyes (Optisafe). It can be administered intradermally or sub-epidermally. The cellulose suspension is packed in glass vials of 3 ml, 8%, 10% and 12% concentration.

[0016] It can be reasonably cost because of the availability of the raw materials locally and its uncomplicated set up and workable technology.

[0017] The MCNP dermal filler is essentially identical to the body’s own collagen and consistent with the body’s own tissue. It enhances the synthesis of an extracellular matrix and eventually for dermal tissue stimulatory effect. MCNP is biodegradable with an extended period due to its nanofibrillar tensile strength and stay in the body for prolonged period without causing any toxic effects and inflammatory reaction.

[0018] There are several benefits and advantages for MCNP filler compared to HA as shown by the table below.

[0019] *Be able to save on foreign echamge and generate more local emplyment

[0020] Currently, the HA intradermal filler is the most popular and widely used by dermatologist with a global market share of 77.3 %. MCNP dermal Filler, considering its additional benefits will be positioned as the promising alternative to HA intradermal filler. MCNP dermal filler is highly valued, promising and safe for soft tissue augmentation biomedical / tissue engineered applications i.e. local implant and voluminizing which may last up to more than 12 months

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] To illustrate the invention, an exemplary embodiment is presented by way of example in the accompanying drawing. It is understood, however, that the invention is not limited to the precise instrumentalities shown. Figure 1 shows the process flowchart of producing A. xylinum microbial cellulose nanoparticle (MNCP) dermal filler. DETAILED DESCRIPTION

[0023] There are numerous way on how to produce microbial cellulose (MNCP) utilizing different types of culture media such as yeast, molasses, corn steep liquor, pineapple extract, coconut water, and coconut milk.

[0024] This invention is focused on utilizing coconut milk as culture media to cultivate an all-natural nata de coco cellulose. The active ingredient or device preservative incorporated in the product is 1 % to 3% of coconut derivative known as monolaurin which is also an all natural and hypoallergenic ingredient. In order to produce an all-natural nata de coco cellulose, the ingredients are the following: sugar, water, acetic acid, mother liquor (Acetobacter xylinum bacteria), and coconut milk. Ingredients will be mixed and cultured on a plastic tray in a room with a controlled temperature. The allowable temperature for this cellulose to grow is 28°C - 38°C.

[0025] The yield of product with high cellulose content is very vital for tissue augmentation applications. The cellulose will be harvested on the 7thday to yield a good quality. This will undergo a cleaning process by removing the white gelatinous base part of the pellicle utilizing a plastic scrapper or splitting machine. The cleaned pellicle will be checked for its cellulose consistency, damage, opacity, tear, cellulose contamination and discoloration using a quality control light with white bulb. The appearance should be opaque white, smooth, no holes, no tears, and no ripped edges. Hole is defined as any break or void in the cellulose structure that goes completely to the pellicle, and can be any shape. Gouge define hole as - any partial thickness break or void in the cellulose structure that does not go completely through the pellicle. Ripped or torn edges - any rip or tear going completely to the width of the pellicle that originated from the edge.

[0026] The pellicles that passed in the quality control inspection will undergo deacidification process. This process pertains to the washing of cellulose pellicle with water until desired ph 7.4 is attained with the use of a pH meter. The deacidified pellicle will be placed properly in the cutting board by using a pneumatic or mechanical padlock puncher and cutting blade. The size of the coco cellulose is approximately 2-3” x 5”. The pre cut cellulose will undergo boiling in water for 20 minutes. Then set aside to cool in a sterile environment. Once cooled, the cellulose will undergo dehydration by placing the cellulose in the manual pressing machine or conveyor type presser. Manually press the cellulose pellicles until the water in the pellicle is extracted.

[0027] The dehydrated cellulose will undergo the next procedure which is the depyrogenic process. This procedure pertains to the preparation of a 5% solution of a hypochlorite in 100 liter container, that is 5 liters of hypochlorite solution in a 100 liters of water. A twenty (20) kg of cellulose will be soaked and washed in the container with 5% solution for 90 minutes. The cellulose will undergo another dehydration process utilizing the conveyor type presser or manual pressing until the water in the cellulose is totally extracted.

[0028] The dehydrated cellulose will undergo boiling in 1% to 3% monolaurin solution.

[0029] This solution pertains to the mixture of monolaurin pellets in alkalinized water. The mixture of monolaurin and alkalinized water is set to boiling point before the addition of cellulose. Once boiling, timer is set to 20 minutes. After boiling, the cellulose will be drained and allow to cool. A sterile dehydration technique will follow once the cellulose is at room temperature. The room will be sterilized including the equipment and material that will be used in the procedure utilizing UV light for 8 hours prior to starting the procedure.

[0030] Final quality control inspection of the cellulose is done in a sterile area. All viewing must take place over the quality control light. Magnification is not to detect aesthetic flaws. A viewing technique is observed by the following: hold the part 30 degrees from the horizontal plane at the distance of 18 inches from viewing, part must be indirect line with the eyes, rotate the part 30 degrees to the left and right. Finally, the microbial cellulose will be cut, punctured and crushed to microsize particle, size of 20-100 microns. The microbial cellulose will then be held in a hydrogel suspension and will be packaged in a vial. The finish products will undergo sterilization by retort or by gamma radiation.

[0031] Acetobacter xylinum, a gram-negative, aerobic, rod-shaped organism belongs to the family Acetobacteraceae. During fermentation in aerobic environment, a secondary metabolite that extracellularly assembles into a thick cellulosic mat or pellicle covering the entire surface of the medium.

[0032] Henceforth, the natural mechanical strength of the acetobacter pellicle needs an inventive step to process it into its nanoparticle size. The state of the art in processing to the desired nanoparticle size of the pellicle was originated by the inventor with the following procedures. After the pellicles that passed in the quality control inspection and deacidification, washing, the pellicle is cut to its 3”x 6” size using a pneumatic or mechanical padlock puncher and cutting blade, boiled and dehydrated. This is followed by depyrogenic process by soaking with Hypochlorite solution, soaked for 90 minutes, washed and dried. The pellicle at this point is the final product used for biomedical applications, the most common of which is for wound dressing. At 90 minutes exposure to Hypochlorite solution, it is not possible to process the pellicle to its nanoparticle size. The inventive step to prepare the pellicle to its nanoparticle size is brought about by extending the exposure to the Hypochlorite solution for a longer time of 6-8 hours. The pellicle now is ready for mechanical crushing giving rise to its nano size of 20 - 100 nm. To confirm the nanoparticle size, a specimen sample was sent to EMPA Materials & Science Technology, Zurich, Switzerland. The scanning electron microscopy showed the typical network of fibril aggregate, with diameter predominantly 100nm, the thinnest nanofibril structure visible under the electron microscope. Additional findings showed the nano porous mesh structures and fibrils aggregate due to strong hydrogen bonding of the surface OH group forming tunnels in between the microbial cellulose fibers for gaseous and fluid exchange. This ultrafine network of MCNP are uniaxiliary oriented with high cellulose crystallinity (60-80%) and has an enormous mechanical strength for the extended biodegradability and biocompatibility.

[0033] The preservative used in this product is Monolaurin, IIIPAC name, dodecanoic acid 2-3 dihydroxyester. It has been extremely studied as a non-toxic food preservative which prevent the growth of bacteria.

[0034] The nanofibrillar architecture of acetobacter microbial cellulose has similarities with human extracellular matrix, particularly the collagen which is an important aspect in the innovation of the intradermal filler. Fillers are considered as medical devices, injected intradermally, with capacity of creating volume, restoring and improving skin tissue, by filling wrinkles and skin depressions.

[0035] The physical properties of acetobacter microbial cellulose include chemically pure, free from lignin, hydrophilic property, biocompatibility, biodegradability, porosity and tensile strength which is brough about by the hydrogen binding between the fibrillar units, permeability and pyrogen free. As a natural polysaccharide it has a distinct advantage of being non immunogenic (low probability for sensitization / allergic reaction) . Therefore, microbial cellulose can be tailored to fit many potential biomedical application, i.e. soft tissue engineered products. Intradermally, the acetobacter microbial cellulose nanoparticle will provide a space-occupying intercellular matrix of connective tissue and results in the correction of skin depressions due to aging or scaring and in shaping of the contour of the face.

[0036] Biocompatibility is the ability of a material to perform with an appropriate host response in a specific application. Biocompatibility does not only refer to the quality of not having toxic effects on the biological systems, but also to the need of having an appropriate host response to ensure satisfactory performance on a specific application.

[0037] The biocompatibility of A. xylinum microbial cellulose was proven in several animal studies. Mendes et al have assessed the tissue reaction in the presence of a microbial cellulose membrane after subcutaneous implantation in mice They found no evidence of foreign body reaction throughout the study period. Another study by Helius et al implanted pieces of A. xylinum microbial cellulose on rats. They found no histologic signs of inflammation and absence of giant cells, with formation of new blood vessels around and inside the implanted cellulose was observed. In vivo tests for tubular shaped A. xylinum microbial cellulose for the replacement of blood vessels have shown similar results. No microscopic signs of inflammation around the implant were found, instead it was found that the fibroblast had actually infiltrated the bacterial cellulose. These findings are unquestionably applicable on the safety and efficacy of microbial cellulose nanoparticle (MCNP) intradermal filler. A. xylinum MCNP has porosity forming like tunnels in between the microcellulose mesh of fibers for gaseous and fluid exchange preventing microbial cellulose displacement or migration after intradermal injection.

[0038] A. xylinum MCNP is biodegradable in extended period due to its nanofibrillar tensile strength, and was found out to stay in the body for a prolonged period of time without causing any toxic and inflammatory reaction. In addition, the hydrophilic or high-water holding capacity of bacterial cellulose results with constantly exchange of water with the host tissue and that prevents fibrosis I granuloma formation. The longevity and efficacy of A. xylinum cellulose nanoparticle intradermal filler is expected to reach up to more than 12 months because of its tensile strength and its nonenzymatic degradation and not affected by cellulose breakdown mechanisms.

[0039] As a new generation and promising molecule, the following safety and toxicity tests were done, namely (i) Safety Data Sheet, (ii) Scanning Electron Microscopy, (iii) Human Safety tests, (non-irritating and hypoallergenic), (iv) In vitro Toxicology Tests (non toxic, safe for the eyes and not phototoxic) at MB at Research Labs Spinnerstonwn, PA, USA. All these results were favorable. (Please refer to enclosed full reports), and (vi) MCNP Certification of Analysis by HyClone Laboratoties Inc, UTah USA results showed the pH 7.0-7.2, Osmolarity 279 mOsm / kg (Normal 279), Endotoxin > 1.0 (Normal <0.01 , Bacteria and fungi, no growth, Cytotoxicity non toxic,

[0040] The Human Phase 1 trial, entitled A Phase I Open-labeled, Single-arm, Single dose Clinical Trial and Histopathologic Evaluation on the Safety of A. xilinium cellulose nanoparticle for Soft Tissue Augmentation, was submitted by Dr. Gertrude P. Chan and approved by UERMMC Research Institute for Health Sciences, Ethics Committee. Methodology. Twelve subjects were injectoid with MCNP Intradermal filler on the deltoid area of the upper arm. On the injection site, serial biopsy was done. The processed skin biopsy specimens were evaluated by 2 independent derm pathologist. The conclusion evidenced that A. xylinum cellulose nanoparticle is biocompatible, exhibit tissue integration and no toxic effects on the biological system.

[0041] A Phase Il-Ill Randomized, Single Injection, Evaluator-Blind, to Determine the Efficacy and Tolerability of Microbial Cellulose Nanoparticle Filler compared to Hyaluronic Acid Filler was submitted to the Ethics Committee. Subjects were injected with MCNP on the right nasolabial fold and on the opposite nasolabial fold HA was injected. Data analysis utilizing the showed that MCNP Filler and HA Filler are parity for safety and efficacy.

[0042] The A. xylinum microbial cellulose intradermal filler is prepared in 3 concentrations, 8%, 10% and 12%. The preferred method will depend on the site of treatment. For soft tissue augmentation on the facial tissue, cheeks, superficial wrinkles or other areas covered by the skin, the 3% concentration is selected. The other site of treatment is in the mid dermis, including deep wrinkles, nasolabial folds and scars the appropriate concentration is 10% and the 12% in the deeper layer of the dermis and / or subcutis or supra-periosteal treatment site.

[0043] As shown in Figure 1 , the subject dermal filler is processed using the following steps: a. mixing and culturing the ingredient on plastic tray in a room with controlled temperature; b. cleaning the cellulose by removing the white gelatinous base part of the pellicle; c. washing the cellulose with water until the desired pH is attained; d. cutting and boiling the pre-cut cellulose in water and cooling said cellulose in a sterile environment; e. dehydrating the cellulose in the manual pressing machine or conveyor type presser; f. soaking and washing the cellulose in 5% solution of hypochlorite for 6-8 hours; g. draining, cooling and sterilizing the cellulose at room temperature; h. crushing the cellulose to micronized particles under high pressure sheering action; i. holding the microbial cellulose in a hydrogel suspension and packaging in glass syringes or vials; j. sterilizing the final product by gamma radiation or autoclave While specific embodiments have been disclosed, these should not be interpreted as limitations of scope of the subject invention, the true spirit and scope of which being defined by the appended claims.

Claims

CLAIMS1. A dermal filler for soft tissue augmentation comprising 85-99% water and 1-12% microbial cellulose wherein the said cellulose is Acetobacter xylinum fermented in coconut milk culture media.

2. The dermal filler as in claim 1 wherein the microbial cellulose is reduced to a nano particle having a diameter predominantly 100 nm.

3. The dermal filler as in claim 2 wherein the microbial cellulose has a nano porous mesh structures and fibrils aggregate forming tunnels in between the microbial cellulose fibers for gaseous and fluid exchange.

4. The dermal filler as in claim 3 wherein the micorbial cellulose has a high cellulose crystallinity (60-80%) and has an enormous mechanical strength for the extended biodegradability, and biocompatibility.

5. The dermal filler as in claim 4 wherein the microbial cellulose has a preservative in the form of a natural coconut derived active ingredient where monolaurin is present in the amount of 1 %.

6. The dermal filler as in claim 5 wherein the microrbial cellulose is pure, natural, hydrophilic and absence of lignin.

7. The derma filler as in claim 6 which can be easily injected and pass through a gauge 30 needle with minimal reaction and discomfort at the site of the injection.

8. The derma filler as in claim 7 which is safe and has a porosity benefit thereby making it more permeable, penetrable making it effective for tissue augmentation and aesthetic correction of wrinkles or loss of muscle volume including nasolabial furrow, wrinkled cheeks, deep smile lines and lipoatrophy correction.

9. The dermal filler as in claim 8 which is non-phototoxic, no toxicity and safe on the area around the eyes.

10. The dermal filler as in claim 9 which is administered intradermally or sub- epidermally.

11. The dermal filler as in claim 10 wherein the cellulose suspension is packed in glass vials of 3 ml, 8%, 10% and 12% concentration.

12. A process of producing A. xylinum microbial cellulose nanoparticle ( MNCP) dermal filler comprising the following steps: a. mixing and culturing the ingredient on plastic tray in a room with controlled temperature; b. cleaning the cellulose by removing the white gelatinous base part of the pellicle; c. washing the cellulose with water until the desired pH is attained; d. cutting and boiling the pre-cut cellulose in water and cooling said cellulose in a sterile environment; e. dehydrating the cellulose in the manual pressing machine or conveyor type presser;f. soaking and washing the cellulose in 5% solution of hypochlorite for 6-8 hours; g. draining, cooling and sterilizing the cellulose at room temperature; h. crushing the cellulose to micronized particles under high pressure sheering action; i. holding the microbial cellulose in a hydrogel suspension and packaging in glass syringes or vials; j. sterilizing the final product by gamma radiation or autoclave.

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