FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
"CULTURED MELANOCYTES ON BIOPOLYMER"
RELIANCE LIFE SCIENCES PVT.LTD
an Indian Company having its Registered Office at Chitrakoot, 2nd Floor, Shree Ram Mills Compound, Ganpath Rao Kadam Marg,Worli, Mumbai - 400 013, Maharashtra, India.
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-

FIELD OF THE INVENTION:
The present invention relates to a delivery system of skin melanocytes on a suitable carrier system, and its method of preparation. The present invention particularly relates to grafts of autologous melanocytes, its methods of preparation, transportation to hospitals and their therapeutic applications not limited to vitiligo.
BACKGROUND OF THE INVENTION Skin and Physiological role of Melanocytes
Skin is the largest organ of the body and is composed of an external epithelial component called the epidermis separated from an underlying connective tissue component, the dermis, by a basement membrane The portion of the dermis adjacent to the epidermis is called the reticular dermis and is composed primarily of collagen fibers produced by fibroblasts, microvessels and a few migrating leukocytes. The reticular dermis supplies all of the nutrition to the epidermis, which is devoid of blood vessels. The great majority of cells in the epidermis are keratinocytes, which are arranged in stratified layers. At the dermal-epidermal junction is a single layer of keratinocytes with a small number of interspersed melanocytes (approximately 1/30) called the stratum basale. Melanocytes are specialized cells that produce melanin, the brown pigment that gives our skin its rich color. These cells are present in the lowest sublayer of the epidermis, or the basal cell layer. The primary purpose of the melanocyte cell is to make melanin. However, each melanocyte via its dendrites supplies melanin to approximately 3' nearby keratinocytes. Melanocytes contain an enzyme called tyrosinase that is able to form a pigmented substance called melanin. The part of the melanocyte cell that forms melanin or pigment is called the melanosome. As pigment granules form, they are transferred from the melanocytes to the keratinocytes and then are carried to the surface of the epidermis. Major function of melanin is to absorb ultraviolet radiation and provide protection to newly dividing cells in the deeper layers. The melanocytes and their activity is the major determinant of the color of the hair and skin. Melanocytes originate in the neural crest and migrate to the basal layer of the epidermis and the hair matrices during

embryogenesis. These neural crest-derived cells also populate the inner ear, uveal tract and leptomeninges
Melanin production takes place in unique organelles (tiny structures within the cell) known as melanosomes. Darkly pigmented skin, hair and eyes have melanosomes that contain more melananin.
The melanosome: Melanosomes rearrange themselves within the cell in response to external cues such as ultraviolet rays or mutations in their transport system. Melanosomes usually cluster together near the center of the cell but can rapidly redistribute themselves to the ends of dendritic (tooth-like) processes projecting from the cell. Melanosome redistribution involves long-range transport from the center along microtubules and, along the periphery, short-range capture and transport. Each melanocyte supplies melanin to approximately 30 nearby keratinocytes via its dendrites. There are two major forms of melanin — eumelanin and pheomelanin. Eumelanin is brown to black while pheomelanin is yellow to red. The regulation of the production of eumelanin versus pheomelanin involves the interaction of the melanocortin 1 receptor (MC1R) on the surface of the melanocyte with melanocyte stimulating hormone (MSH) or with the agouti signaling protein. The binding of MSH to MC1R results in the formation of eumelanin while the binding of the agouti protein to MC1R leads to the production of pheomelanin. Tanning of the skin due to UV exposure represents an increase in the content of eumelanin within the epidermis and its major purpose is increased photoprotection. These can result from a change in the number of melanocytes or a decrease or increase in their activity. Leukoderma in association with inflammatory disorders of the skin such as atopic dermatitis and vitiligo are two of the more common disorders of hypopigmentation.
Recent studies have examined the nucleotide sequence of the MC1R receptor in various populations. There are five forms of the MCR but only MC1R is found on melanocytes. Variant sequences were found in over 80% of individuals with red hair and/or fair skin that tanned poorly, whereas these variant sequences were observed in less than 20% of persons with darkly pigmented hair and less than 4% of those with a good tanning response. Ultraviolet-induced tanning represents an increase in the content of eumelanin
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within the epidermis and its major purpose is increased photoprotection. The possibility exists that differences in the interactions between MSH or the agouti protein and the variant forms of MC1R could explain different pigmentation phenotypes in humans. Whether these variations in MC1R play a role in determining melanoma risk (beyond determining the degree of cutaneous pigment production) remains speculative.
Etiology and Pathogenesis
To date, three important hypotheses have been formulated on destruction of melanocytes, the pigment producing cells of the skin.
The autoimmune hypothesis originated from the observation that vitiligo is associated with some autoimmune diseases. Both cellular and humoral factors responsible for autoimmune damage to melanocytes have been demonstrated.
The autocytotoxic or self-destruct hypothesis suggests that some toxic molecules produced during the biosynthesis of melanin are responsible for melanocyte damage in susceptible individuals.
The neural hypothesis postulates that neurochemicals liberated from nerve endings are toxic to melanocytes.
The varied clinical and laboratory findings, however, indicate that multiple mechanisms may be responsible for the causation of vitiligo in an individual.
The failure of melanocytes to migrate to these locations explains the association of congenital white spotting of the skin (piebaldism) with heterochromia of the irises as well as congenital deafness in Waardenburg syndrome.
Disorders of hypo- and hyperpigmentation can result from a change in the number of melanocytes or a decrease or increase in the activity of the melanocytes. Leukoderma in association with inflammatory disorders of the skin, e.g. atopic dermatitis, and vitiligo are two of the more common disorders of hypopigmentation. Presumably, given the
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autoimmune nature of vitiligo, the melanocytes have been destroyed by autoreactive T lymphocytes. One of the most common disorders of hyperpigmentation is melasma (also known as mask of pregnancy) that is seen primarily, but not exclusively in women and may or may not have its onset during pregnancy. Exposure to the sun plays a very important role in the induction and maintenance of these areas of hyperpigmentation on the face. Without protection, the UV radiation can cause the cell's genetic coding or DNA to mutate. This can significantly alter the manner in which the cell functions, resulting in changes associated with rosacea, aging skin or compromised immune function.
In addition, both inflammations resulting from rosacea, laser therapies as well as UV exposure can affect the functioning of the melanocytes, resulting in abnormal increase in the production of melanin or pigment. This may be visually manifested as freckles, uneven patchy coloring or solar lentigos (often referred to as age spots). Medical research has focused on methods of regulating melanin in order to control abnormal production and resolve the appearance of hyperpigmented lesions.
While there are a number of topical agents that inhibit the synthesis of melanin, researchers are focusing on breakthroughs that directly control the output of the tyrosinase enzyme resulting in greater control over pigmentary changes. Topical skin care preparations that assist in increasing the rate of cellular turnover and normalization of the epidermis also can lessen the appearance of abnormally pigmented areas because newer cells appear to contain fewer pigment granules. However, preliminary investigations have reported repigmentation of vitiliginous lesions with such topical skin care preparations containing isoprinosine, levamisole, suplatast tosilate, cyclosporine.
Vitiligo (vit-ill-eye-go) is the most common pigmentation disorder in which melanocytes in the skin, the tissues that line the inside of the mouth and nose and genital and rectal areas (mucous membranes), and the retina of the eyes are destroyed. As a result, white patches of skin appear on different parts of the body. The hair that grows in areas affected by vitligo may turn white.
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Vitiligo is fairly common affecting 1 out of every 100 people. About 1 to 2 percent of the world's population, or 40 to 50 million people, have vitligo. Vitiligo can be found in all parts of the world. It affects all ethnic groups, but is far more disabling in those who have dark skin. This disease affects males and females equally. The usual age of onset is between 10 and 30 years old, but the condition can start at any age. Ninety-five percent of people who have vitiligo develop it before 40 years of age.
The cause of vitiligo is not known, but doctors and researchers have several different theories. One theory is that people develop antibodies that destroy the melanocytes in their own bodies. Another theory is that melanocytes destroy themselves. Finally, some people have reported that sunburn or emotional distress can trigger vitiligo. However, these events have not been scientifically proven to cause vitiligo.
The change in appearance caused by vitiligo can affect a person's emotional and psychological well-being and may create difficulty getting or keeping a job. People with this disorder can experience emotional stress, particularly if vitiligo develops on visible areas of the body such as the face, hands, arms, feet, or on the genitals. Adolescents, who are often particularly concerned about their appearance, can be devastated by widespread vitiligo. Some people who have vitligo feel embarrassed, ashamed, depressed, or worried about how others will react.
The goal of treating vitligo is to restore the function of the skin and to improve the patient's appearance. Therapy for vitiligo takes a long time-it usually extending for 6 to 18 months. The choice of therapy depends on the number of white patches and how widespread they are, and on the patient's preference for treatment. Each patient responds differently to therapy and a particular treatment may not work in everyone.
Treatments available
Presently, there is no universally effective medical or surgical modality for vitiligo therapy; however, there are a number of active therapeutic approaches that are known to be effective. In addition to these medical and surgical therapies that are listed later in text,
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one has to always keep in mind adjunct therapies, such as broad-spectrum sunscreens to prevent photo damage of vitiliginous skin, and cosmetic camouflage of disfiguring skin with stains or make-up in exposed areas of vitiligo.
Medical Treatments
Topical steroid therapy are helpful in re-pigmenting (returning the color to white patches) the skin, particularly if started early in the disease. Corticosteroids are a group of drugs similar to the hormones produced by the adrenal glands (such as cortisone). Patients need to apply the steroid topical cream to the white patches on their skin for at least 3 months before seeing any results. Even though it is the simplest and safest treatment but not as effective as psoralen photochemotherapy and there is a risk of J effects such as skin shrinkage and skin striae (streaks or lines on the skin) for which the doctor has to closely monitor.
Psoralen photochemotherapy involves taking psoralen by mouth (orally) or applying it to the skin (topically). This is followed by carefully timed exposure f J ultraviolet A (UVA) light from a special lamp or to sunlight. Patients usually receive treatments in their doctors' offices so that they can be carefully watched for any side effects. Patients must minimize exposure to sunlight at other times. However, it is time-consuming-and care must be taken to avoid side effects, which can sometimes be severe. Psoralens are drugs that contain chemicals that react with ultraviolet light to cause darkening of the skin. There are two major potential side effects of topical PUVA therapy: (1) severe sunburn and blistering and (2) too much re-pigmentation or darkening of the treated patches or the normal skin surrounding the vitiligo (hyperpigmentation). Patients can minimize their chances of sunburn if they avoid exposure to direct sunlight after each treatment.
Hyperpigmentation is usually a temporary problem and eventually disappears when treatment is stopped. Known side effects of oral psoralen include sunburn, nausea and vomiting, itching, abnormal hair growth, and hyperpigmentation. Oral psoralen photochemotherapy may increase the risk of skin cancer. To avoid sunburn and reduce the risk of skin cancer, patients undergoing oral PUVA therapy should apply sunscreen
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and avoid direct sunlight for 24 to 48 hours after each treatment. Patients should also wear protective UVA sunglasses for 18 to 24 hours after each treatment to avoid eye damage, particularly cataracts.
Depigmentation involves fading the rest of the skin on the body to match the already white areas. For people who have vitligo on more than 50 percent of their body, depigmentation may be the best treatment option. Patients apply the drug monobenzylether of hydroquinone (monobenzone or Benoquin) twice a day to pigmented areas until they match the already depigmented areas. Patients must avoid direct skin-to-skin contact with other people for at least 2 hours after applying the drug. The major side effect of de-pigmentation therapy is inflammation (redness and swelling) of the skin. Patients may experience itching, dry skin or abnormal darkening of the membrane that covers the white of the eye. De-pigmentation is permanent and cannot be reversed. In addition, a person who undergoes de-pigmentation will always be abnormally sensitive to sunlight.
Surgical treatments
Several surgical procedures for the treatment of de-pigmented skin have been reported to be effective in patients who have experienced difficulty in receiving good responses from medical treatments. The surgical therapeutic approaches include skin grafting. During skin grafting, the doctor removes sections of the normal, pigmented skin (donor sites) and places them on the de-pigmented areas (recipient sites). However, possible complications of autologous skin grafting include infection at the donor or recipient sites.
Techniques for surgical grafting include:
1. Suction Blister grafts: Blisters are created on patient's normal pigmented skin using heat, suction, or freezing. The tops of the blisters are then cut out and transplanted to a de-pigmented skin area. The risks of blister grafting include the development of a cobblestone appearance, scarring, and lack of re-pigmentation. However, there is less risk of scarring with this procedure than with other types of grafting.
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2. Punch grafts: The punch grafts have been used to replace de-pigmented skin. This
technique although effective, would lead to cobblestone appearance and scarring.
3. Tatto grafts: This procedure works best for the lip area, particularly in people with
dark skin; however, it is difficult for the doctor to match perfectly the color of the skin of the surrounding area. Tattooing tends to fade over time. In addition, tattooing of the lips may lead to episodes of blister outbreaks caused by the herpes simplex virus.
US patent number 4866038 has provided a method of stimulating the menalonycte production by external application of alpha -MSH analogs. However, in some cases of vitiligo, melanocytes are not capable of producing melanin even after stimulating melanin pathway.
US patent number 6673603 demonstrates the use of cell paste comprising keratinocytes and fibroblasts. This technique is more focussed on the wound healing and the induction of pigmentation is not achieved suitably.
US patent 20020106353 relates to a cell suspension and method for its preparation. The delivery of the cells are done using a device which is demonstrated to reduce the complexity associated with conventional grafting technology. The nozzle and device, are designed for delivery of cells with out subjecting to shear forces and it is not mixed with conventional propellants. However this type of delivery does not control / deliver the desired amount of cells at the site.
US patent 6039972 describes a wound dressing containing mammalian cells anchored on hydrophobic synthetic polymer film. The polymers presented herein are perforated hydrophobic polymers, which are suitable for culturing of cells and not for delivery to the skin.
US patent number 5700450 describes a method of increasing pigmentation comprising the implantation of skin grafts on the vitiliginous patches, the skin grafts comprising
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melanocytes activated by contact with an effective amount of a diacylglycerol. With this
methodology, the donor skin area to be taken for grafting depends upon the de-
pigmented area that needs to be covered by the skin graft, i.e. a larger donor skin would be required for the coverage of large de-pigmented areas.
US20010006813A1 demonstrates the culture of melanocytes and keratinocytes that are delivered using a carrier matrix. The application prefers the use of fibrin glue, which has a gel like consistency as a carrier matrix.
US6660305B1 claims a composition for stimulating the synthesis of a mammalian's melanic pigment, comprising: alcohol, an extract of human placenta, and calcium chloride.
WO2004096981A2 demonstrates treatment of occulocutenous pigment deficit. The idea is to deliver melanin pigment exogenously similar to insulin use in diabetics.
WO2005011676A1 proposes a composition that contains L-proline amino acid as active ingredient, which stimulates the synthesis of melanic pigment of the skin as well as the proliferation of melanocytes.
Other techniques include pure melanocyte cultures, and cocultures of melanocytes and keratinocytes. These techniques are indeed beneficial for localized lesions
Recent advances in culturing pigment cells from humans have made it possible to transplant autologous melanocytes obtained from areas of normal skin color into areas of skin that are de-pigmented. In direct melanocyte transplant procedure, a sample of the patient's normally pigmented skin is taken and subjected to enzymatic dissociation. The resulting cell suspension that includes keratinocytes as well as melanocytes besides other skin cells is then directly added to de-pigmented areas that have been debrided.
The disadvantages of this method include:
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Cell number identification:
As the resulting cell suspension contains all types of epidermal skin cells, the
identification of the number of melanocytes administered at the de-pigmented site
remains unclear.
Retaining cells at the de-pigmented site:
In this method, the cells are sprayed or poured on the de-pigmented area. This might
result is cells getting lost in the dressing. Hence, the number of cells retained on the
debrided area is unclear.
Area coverage:
If the initial biopsy taken is small compared to the de-pigmented area, then the number of
melanocytes administered to the de-pigmented site would not be sufficient to produce
satisfactory levels of pigmentation.
Some of the above disadvantages are overcome by developing a method to culture melanocytes in vitro. Following the multiplication of melanocytes in the culture dish, the cells are dissociated from the dish and the cells transplanted onto the patient's debrided de-pigmented skin patches. To date, all the studies on melanocytes transplantation has been conducted either in hospitals or in medical centers attached to research centers. These cells are delivered to the skin as a suspension. By this delivery method, the chances of loosing cells is high as there are chances of cells not sticking to the wound bed due to which these cell die or get trapped in the gauze.
Although strides have been made into identifying keys to the pathogenesis of vitiligo, treatment remains a challenge. Newer modalities and combination therapies have been studied, which give the clinician more treatment options and give patients hope for this psychologically devastating condition. As stated above, current treatment options focus on four main categories: sunscreens, camouflage, depigmentation, and repigmentation. Sunscreens are not a therapy per se but do serve a central purpose in vitiligo therapy. They can minimize Koebner phenomenon secondary to sunburn seen in vitiligo, and they provide photoprotection of depigmented areas that have lost their innate sun protection
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from loss of melanocytes and hence melanin production. Broad spectrum sunscreens or sunblock to protect from UVA and UVB are recommended.
Unfortunately, many of the commercially available products in today's market are only marginally effective. The ability to modify the expression of pigment content in the skin, to promote an even-looking skin tone, is highly desired in today's society.
Looking to the need of the hour, the inventors of the present invention have developed a delivery system to deliver functional melanocytes at the vitiliginous areas to induce repigmentation, obviating the disadvantages of the existing cell delivery systems.
The present invention is directed in part to a composition to deliver grafts of functional melanocytes and/or in combination with keratinocytes for transplantation from a centralized processing center to hospitals located in various other locations. The melanocytes as developed under the present invention is meant for delivery at the vitiliginous areas to induce repigmentation. Previous studies have all been carried out in hospitals attached to research centers. None of the literature available indicates the mode of transportation of these cells to the hospitals. Thus, there remains a need to have a system that would enable cells to be processed in a centralized facility and delivered to various hospitals. Retaining the viability of the cells during transport is critical for the success of grafting. The inventors of the present invention have developed a system that focuses on the biopsy transport; melanocytes culture, its transport and delivery on a biopolymer. The delivery system of the present invention has focused in providing the cell to the wound bed, which facilitates melanocyte migration and colonization of the wound bed, resulting in re-pigmentation.
The inventors of the present invention have thus been successful in generating such a system that would greatly reduce the cost of therapy thus benefiting patients.

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Using grafts of autologous cultured melanocytes in addition with keratinocytes, as developed by the present invention could achieve pigmentation with good healing thereby alleviating hypopigmented conditions such as vitiligo.
OBJECT OF THE INVENTION
The present invention aims to provide grafts comprising an epidermal component.
The present invention aims to provide a graft, which may be used as a tissue substitute.
The present invention aims to provide a graft, which would adhere well to the wound.
The present invention aims to provide a graft, which mimics the physiology and some of
the mechanics of the normal skin.
The present invention aims to produce a graft, which would be effective in accelerating
skin pigmentation.
The graft of the present invention aims to address the clinical conditions of
depigmentation such as vitiligo, leucoderma etc.
The graft of the present invention aims to deliver a layer of cultured melanocytes on a
biocompatible polymer.
The graft of the present invention aims in retaining the viability of the cells for a
minimum of 96 hours under transport conditions before grafting.
SUMMARY OF THE INVENTION
The present disclosure relates to a graft comprising proliferative melanocytes cultured on a transparent biocompatible film. The cells are directly delivered to the de-pigmented site by inverting the film so that the cells are in opposition to the debrided wound bed.
In preferred embodiment, the process of preparing the graft involves the optimization of scaffolds onto which cells are seeded. Scaffolds can be selected from the group comprising of natural materials such as collagen and fibrin or synthetic materials such as degradable polyesters used in surgical sutures. Scaffolds take forms ranging from spongelike sheets and fabrics to gels to highly complex structures with intricate pores and
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channels made with new materials processing technologies. The spatial and compositional properties of the scaffold, the porosity of the scaffold and interconnectivity of the pores are all required to enable cell penetration into the scaffold as well as the transport of nutrients and waste products.
In another embodiment, the melanocytes that form part of the differentiated keratinocytes cell culture process are delivered as 4-5 layer thick tissue, which can then be enzymatically detached from the culture dish and transplanted onto the de-pigmented area following debridement.
In certain embodiment, the melanocytes can be cultured directly onto a delivery membrane in a culture vessel, which is then peeled off when required for use.
In another embodiment of the present invention, the graft of cultured melanocyte on biopolymer include genetic modification of transplanted cells to improve pigmentation Biocompatible polymers can be either natural or synthetic. In general, synthetic polymers offer greater advantages than natural materials in that they can be tailored to give a wider range of properties and more predictable lot-to-lot uniformity than can materials from natural sources. Synthetic polymers also represent a more reliable source of raw materials, one free from concerns of immunogenicity.
Poly lactic acid (PLA), poly glycolic acid (PGA), poly lactic-co-glycolic acid (PLGA) and polyurethane are some of the preferred biocompatible polymers in this invention.
The polymers have the advantage of not requiring surgical removal after they serve their intended purposes. These polymers have found a broad range of pharmaceutical and biomedical applications based on their unique properties, including versatile degradation kinetics, non-toxicity, and biocompatibility. PGA is a highly crystalline polymer and the most hydrophilic among them. It has a very high melting point (224°C to 226°C), and the degradation rate of PGA is much higher than that of PLA. Random PLGA copolymers with different ratios of lactide (LA) and glycolide (GA) exhibit different degradation
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rates, and thus can be tailor-made for specific applications requiring specific degradation kinetics ranging from weeks to months. They are generally more amorphous than their homo-polymers and become most susceptible to hydrolysis when the two-monomer contents are the same. Polylactic acid (PLA) and polyglycolic acid (PGA) are among the few synthetic degradable polymers that have been approved for clinical use and these have been widely studied in tissue development. Segmented polyurethane elastomers have a wide use as biomaterials due to their excellent mechanical properties and great chemical versatility. The vast majority of research devoted to the development of biomedical polyurethanes has focused on long-term applications such as vascular grafts and pacemaker lead insulators. The polyurethane polymer is used in a number of different forms and in a range of applications, both in the biomedical field and others. The material is fabricated by casting or other molding techniques to form a substrate, which can be used along or combined with other substrates to form homogenous multi-layered materials. Such multilayered homogeneous polyurethane materials may be formed with layers having different degrees of degradability.
The factors affecting the mechanical performance of biodegradable polymers are those that are well known to the polymer scientist, and include monomer selection, initiator selection, process conditions, and the presence of additives. These factors in turn influence the polymer's hydrophilicity, crystallinity, melt and glass-transition temperatures, molecular weight, molecular-weight distribution, end groups, sequence distribution (random versus blocky), and presence of residual monomer or additives. In addition, the polymer scientist working with biodegradable materials must evaluate each of these variables for its effect on biodegradation. Although the characteristics of these polymers are well understood, they merely serve to provide a 3-D biocompatible structure onto which cells can attach and do not interact with the cells. In the body, cells are situated within an extracellular matrix (ECM), which provides tissues with the appropriate architecture as well as signaling pathways that influence key cell function such as migration, proliferation, and differentiation. Research continues on the utilization of matrix molecules along with specific growth factors to optimize cell adherence to the scaffolds and direct cell activity.
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Culturing melanocytes on the present delivery system can avoid some of the disadvantages faced by existing delivery system for melanocytes.
The use of polymers under the present invention, on which the skin cells are cultured and transferred, improves the ease of graft handling during cell culture and its transplantation. The polymer used in the present invention being transparent permits microscopic observation of cells during cell culture as well as the visualization of the underlying skin after its application on the de-pigmented skin.
In the most preferred embodiment, the present disclosure provides the culture and transfer of human epidermal melanocytes for the treatment of de-pigmented skin. The present invention aims at using a biocompatible polymer in lieu of its reported properties. Biodegradable polymers contemplated for use in the present invention, for wound healing or wound care compositions include polyesters, poly (amino acids), polyester amides, polyurethanes, or copolymers thereof. Besides, the polymer sheets selected from PLA, PGLA, and polyurethane have the following advantages of ease of handling during culture and application on the wound bed, and its ability to stick to the contours of the wound. The sheets of PLA, PGA, PGLA and polyurethane are transparent, permitting visualization of the cells during culture as well as the pigmentation process after its application on the wound site. Further it possesses barrier properties, preventing microbial contamination of the wounds.
The present invention provides the use of sheets made of biocompatible, biodegradable polymer such as PLA as a delivery system for melanocytes as well as the ability of using this system as a tool to deliver proliferating melanocytes to the depigmented skin.
In one of the most preferred embodiments, the wound cover of the present invention primarily comprises of cultured melanocytes on Polylactic acid (PLA) sheets figure 2 in specially designed container figure 3. The crude PLA is prepared by catalytic reaction using dilactide and is purified by re-dissolving in a solvent like acetone, chloroform
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which is then re-precipitated with water. In this process the unconverted dilactide, other monomers, and impurities will be removed along with some portion of catalyst used. The polymer films are made by using purified PLA.
The present disclosure expects to make available biocompatible and biodegradable polymers to device designers and physicians that will help speed patient recovery. The present inventions provide a composition of keratinocytes, which is designed to induce pigmentation as well as enhance the rate of epidermal regeneration.
In another preferred embodiment, the present invention provides for the culturing of
melanocytes along with other cells like keratinocytes and its delivery on a biocompatible
polymer.
The present invention has provided a process for co-culturing and its use in
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transplantation in skin conditions such as -vitiligo. The advantage of co- delivering keratinocytes with melanocytes would be to enhance the rate of epithelialization of the wound bed. Besides, the growth factors secreted by keratinocytes would aid in earlier pigmentation of the skin
The potential advantages or the main features of the present invention are as follows:
1. The melanocyte graft aids in pigmentation.
2. The polymer film aids in healing by protecting the wound bed against microbial invasion.
3. Polymer film adheres to the wound bed and relieves pain by sealing the nerve endings.
4. Polymer film ensures a moist wound environment by preventing wound desiccation.
5. Transmission of infectious disease is minimised by using rigorous in-process controls.
6. The melanocytes cultured on the delivery system can then be transferred to the patient thus minimizing the handling of cells by the clinician.
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7. Polymer film used for cell delivery enables cell migration onto the wound bed, thereby preventing cell loss..
8. The polymer film provides sufficient strength for the graft during culture process as well as its application.
9. The transparent biopolymer ensures microscopic evaluation of the cells during cell culture process as well as visualization of the skin after its application.
10. Large areas of de-pigmented skin can be covered with a small biopsy obtained from the patient.
11. The viability of the graft can be retained for about 96 hours under transport conditions, thereby facilitating its delivery to various hospitals located far away from the central processing facility.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Fig.l: Illustrates the interactive biopolymer graft of the present invention comprising
cultured melanocytes on PL A sheets.
Figure 2: Illustrates the schematic representation of the transport container for the wound
cover of the present invention. The design of the transport container is registered under
the design patent act having number 195065 which is incorporated herein by reference.
Figure 3: Illustrates the comparative result of the mycoplasma testing of the cells as
shown in 3A: Mycoplasma positive control; 3 B: Cultured melanocytes showing absence
of mycoplasma
Figure 4: Illustrates the cell identity testing of the melanocytes by MEL-5 antibody
staining wherein, the melanocytes are identified as green cells. The cell nuclei are stained
withDAPI.
Figure 5: Illustrates the MART-1 gene expression in melanocytes by PCR analyses
Figure 6: Illustrates the functionality of cultured melanocytes by DOPA staining
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Figure 7: Illustrates the MHC-II (HLA-DR)gene expression in melanocytes by PCR
analyses
Figure 8: Illustrates the migration of melanocytes from PLA sheets to the wound cover
indicated by the presence of red fluorescent-labeled cells in the wound bed.
Figure 9: Table: 1. Illustrates the results of the in vitro tumorigenesis assay indicating the
safety of the cultured melanocytes for transplantation
Figure 10: Illustrates the viability of melanocytes under transport conditions.
Figure 11: Co-culture of melanocytes plus keratinocytes under phase contrast microscope
Figure 12: Identification of keratinocytes by keratin staining
Figure 13: Identification of MHC-II(HLA-DR) expression in keratinocytes by-PCR
Figure 14: Table: 2. Illustrates the results of the in vitro tumorigenesis assay indicating
the safety of the cultured keratinocytes for transplantation
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a composition and a delivery system comprising a cultured graft of actively growing epidermal cells such as melanocytes or melanocyte-keratinocytes in the ration of 1:1 on a biocompatible support material such as PLA, for transplantation from a centralized processing center to hospitals located in various other locations
Most of the raw materials used in the preparation of the grafts of cultured melanocytes on biopolymer of the present invention are sterile products purchased from well reputed US based biotech companies like Cascade Biologies, Invitrogen, Sigma etc. Source of fetal bovine serum used for patient and/or donor skin transport, is certified from BSE-free countries. All plasticware used in the manufacturing process are disposable and obtained from NUNC™ (USA) and Falcon™ (USA). In the grafts of cultured melanocyte on biopolymer of the present invention, PLA membrane serves as a carrier of melanocytes. PLA is cast into films and ETO sterilized. The grafts of cultured melanocytes on biopolymer of the present invention are transported in specially designed polycarbonate dishes. As part of the transport container for the wound cover of the present invention,
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silicone O-ring is used. All raw materials are tested to further ensure sterility of the materials.
The process of manufacturing the wound cover of the present invention is carried out in clean rooms under cGMP norms.
The following are involved in the preparation of the composition of graft of cultured melanocyte, under the present invention:
1. Isolation of melanocytes
2. Culture of melanocytes
3. Preparation of biopolymer films
4. Safety and efficacy studies.
The graft of cultured melanocyte on biopolymer of the present invention is for use in conditions where patients have de-pigmented or hypopigmented skin such as vitiligo, leucoderma etc.
The graft of cultured melanocyte on biopolymer of the present invention is available as approximately 16 sq. cm. circular film. However the size does not limit the scope of the invention, in that the graft can be of any size or shape. The film with the cells facing upwards is supported inside a specially designed transport dish. Each graft is processed aseptically and packed individually under sterile conditions in transport media. The dish is sealed in specially designed trays and shipped in insulated box that maintains a temperature of 8°C-25°C for a minimum of 96 hours.
Clinically when used the graft is inverted on the wound bed so that the cells are in closed opposition on the dermabraded wound bed. The presence of functional melanocytes in the regenerated epithelia would result in pigmentation of the skin following exposure to sunlight or PUVA. The keratinocytes when presented as a cocultured graft would also migrate onto the wound bed, colonize and reconstitute the epidermis. The growth factors
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secreted by these cells aid in inducing the recipient cells to migrate, proliferate and reform the epidermis resulting in wound regeneration.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute .preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1: PREPARATION OF CULTURED MELANOCYTE GRAFT ON
BIOPOLYMER
Collection of the skin
A punch biopsy is collected from the person who needs to be grafted hereto referred to as patient. Patient's blood (5ml) is collected for infectious disease testing to determine the ID status of the patient to ensure safety to the operator as well as the patient at the time of transplantation.
Preparation of the Polymer films
The polymer solution is clarified to remove any suspended particles by centrifugation. The polymer film is then prepared by solvent casting method. The film is allowed to dry by evaporation of solvent at ambient temperature and sterilized by ETO. A typical preparation of the biopolymer sheet for example is described in detail in our co pending application number IN 205/MUM/2006 filed on Feb 14,2006.
Cell culture procedure
Melanocytes isolation and expansion involves the following steps:
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1) The patient skin is trimmed, decontaminated, and epidermis is separated from the underlying dermis by enzymatic digestion.
2) A single cell suspension of the epidermis is made.
3) The viable cells are assessed and seeded in tissue culture dishes in media that supports melanocyte growth.
4) When the cells reach confluency, the cells are passaged. Depending upon the area of the skin to be covered, the cells are passaged 2-3 times to obtain sufficient cell numbers. Before dispatching the graft, the cells are trypsinized and seeded on biopolymer films (Ixl04-lxl05 cells/cm2 graft) in specially designed containers (Design Number 198591)
5) The cells are allowed to attach and proliferate.
The figure 1 represents the biopolymer graft of the present invention wherein the cultured melanocytes are seen distributed on a PL A film.
In- process controls:
The safety of the cells to the recipient is ensured by continuously monitoring the sterility and endotoxin levels of the cells during the culture period.
Besides, to overrule that culture conditions produce abnormality in the cells, several batches of melanocytes were tested for in vitro tumorigenicity as well as karyotyping. All experiments showed no abnormality in cultured cells
EXAMPLE 2: CO-CULTURE TECHNIQUES
Application of keratinocytes to the wound site results in enhanced rate of epithelialization. To improve the rate of healing of the wound that is produced following debridement of the de-pigmented skin, co-application of melanocytes along with keratinocytes would result in pigmentation as well as early wound closure.
Keratinocytes would be obtained either from the patient undergoing melanocyte treatment or from another donor. Incase of using cultured keratinocytes obtained from the patient himself, the keratinocytes would continue to survive for a longer time and form part of the reconstituted epithelia. However, the application of an allogenic source of
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cultured keratinocytes though would enhance the rate of healing by stimulating the body to regenerate. These cells would however survive temporarily. Figure 11 represents the co-cultured cells on a biopolymer.
EXAMPLE 3: TRANSPORT OF THE GRAFT OF CULT RED MELANOCYTE BIOPOLYMER
The design of the transport container, its assembly and use in transport of cultured cells is elaborated in our co-pending patent application 60/MUM/2006, which is incorporated herein by reference. The design of the container is shown in figure 2
Before dispatching to hospitals, the media in the dishes are replaced with CO2 endued culture media. The dishes are secured air tight to ensure viability and sterility of the grafts during transport. The dishes are then placed in insulated boxes that maintain a temperature between 8-25°C for 96h and shipped to various hospitals for transplantation.
PRODUCT CHARACTERISATION AND TESTING EXAMPLE 4: SAFETY TESTING:
To ensure recipient safety, part of the donor cells is subjected to the following tests:
1. Karyotyping: Karyological analysis was conducted on cells to determine the number
of chromosomes and check for the presence of abnormalities. There was no evidence of
clinically significant numerical or structural chromosomal abnormalities.
2. Mycoplasma testing: This test was done by Hoechst staining. Using mycoplasma
stain kit, the cells were examined under fluorescence staining wherein the positive
cultures are identified by particulate or filamentous fluorescence around the cell nuclei (
fig 3A) and the negative cultures are identified by only nuclear staining as indicated
herein (fig 3B)
3. Testing for infectious diseases: To ensure patient's safety, the blood of the patient
(collected at the time of biopsy collection) is subjected to infectious disease testing by
ELISA method to check for HIV, HBV, CMV, HBSAg and Syphilis. The cells before
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dispatch to the hospitals/ recipients are also tested for infectious disease by PCR (polymerase chain reaction) method.
4. Sterility: The sterility of the product is ensured by stringent in-process testing like mycoplasma testing, sterility and endotoxin testing by LAL method
EXAMPLE 5: CELL CHARACTERIZATION
1. Identification: The cells were analyzed and identified by immunohistochemical
analysis.
Melanocyte graft
To determine the purity of melanocyte cultures, the cells were identified by using antibodies to MEL-5. All cells test positive to MEL-5 antibody. MEL-5 (Ta99) monoclonal antibody detects a differentiation-related, pigmentation-associated glycoprotein (gp75), (75kD) expressed by melanoma cells, nomal melanocytes and nevi. The gp75 antigen is now considered as the tyrosinase-related protein (TRP-1). (Figure 4)
For the co-cultured graft
To determine the identity and the number of keratinocytes in the graft, the cells were determined by immuno-staining the cells with PAN-keratin antibody that specifically binds to keratinocytes. The positive cells are detected using FITC - conjugated anti-mouse secondary antibody and DAPI is used to identify the nucleus of the cells as seen in Figure 12.
2. MART-1 PCR: As shown in Fig 5, the expression of MART-1 in all passages of
cultured melanocytes indicates the ability of the cultured cells to induce pigmentation.
GAPDH (Glyceraldehyde phosphate 3-dehydrogenase) serves as internal control.
MART-1 forms a complex with Pmell7 (a melanosomal protein) and affects its
expression, stability, trafficking, and the processing which is critical to the formation of
Stage II melanosomes. MART-1 is thus indispensable for Pmell7 function and plays an
important role in regulating mammalian pigmentation.
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4. Functional assay: The ability of cultured melanocytes to convert DOPA to melanin indicates the functional characteristics of the cells. Functional melanocytes produce melanin in a chemical reaction in which tyrosinase catalyses the conversion of tyrosine to DOPA which is further converted to melanin, (figure 6)
5. MHC-II (HLA-DR) expression: MHC-II plays an important role in immunogenic response. Melanocytes loose expression of HLA-DR during culturing (figure 7). The identification of the MHC- class II (HLA-DR) expression in keratinocytes by PCR in cocultured product is illustrated in figurer 13.
6. Viability of the cells under transport conditions: The viability of the melanocytes under transport conditions was checked. The graft maintained the viability up to 4 days at a temperature of 8-35°C. (Figure 10)
EXAMPLE 6: EFFICACY TESTING BY PRE-CLINICAL STUDIES
1. Migration of the cells from the biopolymer
The ability of the keratinocytes and melanocytes to migrate into the wound cover bed was tested in a full thickness punch wound biopsy model in guinea pigs and SCID mice respectively. Visually, wounds which were treated with cells contracted on day 5 as compared to control wounds which contracted on the day 9—10. Histochemical analyses demonstrated that the cells had migrated into the wound bed and formed part of the regenerated epithelium. (Figure 8)
2. Toxicity testing: PLA also did not show any toxicity when tested on mice and guinea
pigs. The entire graft also did not show any toxic effect in animals.
1. Tumorigenicity: To ensure that the cells do not have any abnormalities / transformations that might lead to tumor formation in the recipient, tumorigencity assays were conducted in vitro. The ability of the cultured cells to form colonies in soft agar was assessed as an indicator of possible transformation and the potential ability for the cells to form tumors in humans. Cultures were monitored for 28 days for the formation of
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colonies of greater than 10 cells. No colonies were formed with the lots of cultured cells tested. Figure 9 illustrates the melanocytes culture and figure 14 illustrates the results of keratinocytes co cultured along with melanocytes.
The present invention also aims to address the following complications observed in conventional techniques in treating hypopigmentation by studying the autologous patients after transplantation.
1. Leaching
2. Color mismatch
3. Graft rejection
4. Scarring of the donor site
5. Post transplantation inflammation
6. Cobblestone appearance
REFERENCES
1. Boissy RE and Nordlund JJ. Biology of melanocytes. In: Cutaneous Medicine and Surgery. Arndt KA, LeBoit PE, Robinson JK, and Wintroub BU, eds. W.B. Saunders Co: Philadelphia, 1996, pp.1203-1218.
2. Bolognia JL and Shapiro PE. Albinism and other disorders of hypopigmentation. In: Cutaneous Medicine and Surgery. Arndt KA, LeBoit PE, Robinson JK, and Wintroub BU, eds. W.B. Saunders Co: Philadelphia, 1996, pp. 1219-1232.
3. Torres JE and Sanchez JL. Melasma and other disorders of hyperpigmentation. In: Cutaneous Medicine and Surgery. Arndt KA, LeBoit PE, Robinson JK, and Wintroub BU, eds. W.B. Saunders Co: Philadelphia, 1996, pp. 1233-1241.
4. Valverde P, Healy E, Jackson I, Rees JL, and Thody AJ. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nature Genet 11:328-330, 1997.
5. Toshihiko Hoashit, Hidenori Watabet, Jacqueline Mullers, Yuji Yamaguchil, Wilfred D. Vieirat, and Vincent J. Hearing MART-1 Is Required for the Function of the Melanosomal Matrix Protein PMEL17/GP100 and the Maturation of Melanosomes. J.Biol. Chem., Vol. 280, Issue 14, 14006-14016, April 8, 2005
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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Dated this 13th day of October 2006
For Reliance Life Sciences Pvt. Ltd
K. V. Subramaniam President
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ABSTRACT
The present invention relates to a graft wherein cultured autologous melanocytes are delivered using a biopolymer. The present invention describes the composition, method of preparation and its properties relating to safety, and efficacy. The graft of the present invention has a potential use in repigmenting skin