WO2006116804A1

WO2006116804A1 - Processed hyoscyamus seed agent for the treatment of tissue disruption

Info

Publication number: WO2006116804A1
Application number: PCT/AU2006/000563
Authority: WO
Inventors: Chin Joo Goh; Kay Fei Chan; Jeffrey David Edwards
Original assignee: Asia Pacific Life Sciences Pte Ltd
Priority date: 2005-04-29
Filing date: 2006-04-28
Publication date: 2006-11-09

Abstract

The present invention relates to an agent for the treatment of a tissue disruption and provides a composition comprising a processed Hyoscyamus seed as well as methods of using a processed Hyoscyamus seed.

Description

PROCESSED HYOSCYAMUS SEED AGENT FOR THE TREATMENT OF TISSUE DISRUPTION

FIELD

The present invention relates to an agent for the treatment of a tissue disruption. In particular, the present invention relates to a processed Hyoscyamus seed which is effective in increasing the growth of fibroblasts and the treatment of wounds, lesions, and the like.

BACKGROUND

The treatment of many tissues using conventional therapeutics and/or dressings is less than satisfactory. Part of the problem is a lack of understanding of the healing process. For example, many of the current wound treatments have difficulty addressing the optimum requirements with respect to the acceleration of the rate of wound contraction, increasing the rate of epithelialisation and increasing the rate of maturation of granulation material, thereby ultimately reducing the time to full maturity of the healed wound.

Therefore, there is a continuing need to develop wound treatments and/or dressings that reduce the time to full maturity of a wound by accelerating the rate of wound contraction, increasing the rate of wound epithelialisation and increasing the rate of maturation of the granulation material .

Phyto-drugs derived from the genus Hyoscyamus (Solanaceae) have traditionally been used as sedatives. In particular, they have been used to relieve gastrointestinal spasms. These medicinal properties arose from the fact that plants of Hyoscyamus sp. are capable of biosynthesising anticholinergic tropane alkaloids. Henbane, also known as Hyoscyamus niger, is one such plant that had been used for the production of herbal alkaloids. In general, the leaves of this plant have been used for the extraction of the tropane alkaloids. In recent years, the biosynthetic pathways for these secondary metabolites, and the enzymes that are involved, have been studied extensively.

The use of oleum hyoscyami, oil extracted from the leaves of Hyoscyamus species, has been described for the manufacture of a topical cream or emulsion for the treatment or prevention of scar tissue. While the use of oleum hyoscyami is described there is no mention of the use of the seeds from the Hyoscyamus plants per se. In particular, the wound-healing properties that are unique to the seeds, especially their ability to form wound- healing phyto-membranes in situ, are not mentioned.

In the practice of traditional Chinese medicine (TCM) , the seeds of Hyoscyamus niger have been prescribed as an antispasmodic, analgesic, and sedative agent. Other beneficial properties of these seeds include their ability to reduce stomach cramps and heavy coughing. Occasionally, TCM practitioners have prescribed the seeds for treatment of neuralgia and manic psychosis. Due to the high potency of the anti-cholinergic alkaloids, a narrow therapeutic dosage range for the seeds and the potential toxicity, these herbal seeds were therefore classified (under TCM standards) as a "toxic drug" if they were consumed orally. However, when the herbal preparations were used externally as topical applications for neuraligia and manic psychosis, the concerns were no longer an issue.

The inventors have discovered that the seeds of Hyoscyamus species have tissue healing properties which overcome or at least alleviate some of the problems associated with current tissue healing therapeutics. SUMMARY

Accordingly, in a first aspect the invention provides a composition comprising a processed Hyoscyamus seed. The composition may be used in the treatment of a tissue disruption and/or in the growth of a fibroblast .

The seed of the composition may be obtained from a plant selected from the group consisting of Hyoscyamus niger, Hyoscyamus semen, Hyoscyamus muticus, and Hyoscyamus aureus. In some embodiments processing the seeds forms a hydroscopic powder.

The composition may further comprise an anti-microbial agent, anti-viral agent, growth factor, anti-dehydration agent, and/or antiseptic agent. In some embodiments the composition further comprises a pharmaceutically acceptable carrier.

In a second aspect the invention provides a method of treating a disease or disorder, comprising the step of administering to a subject a composition comprising a processed Hyoscyamus seed. The disease or disorder may be associated with a lesion, wound, microbial infection, burn, ulcer, and/or dermal condition and/or require increased fibroblast growth.

In a third aspect the invention provides a method of treating a tissue disruption, comprising the step of contacting the tissue disruption with a processed

Hyoscyamus seed. In some embodiments the tissue disruption is a wound.

In a fourth aspect the invention provides a method of increasing the growth of a fibroblast, comprising the step of contacting the fibroblast with a processed Hyoscyamus seed. In a method of the second, third, or fourth aspects the Hyoscyamus seed may be isolated from a plant selected from the group consisting of Hyoscyamus niger, Hyoscyamus semen, Hyoscyamus muticus, and Hyoscyamus aureus.

In some embodiments the seed is processed to form particles of less than 500 microns. In some embodiments the processed Hyoscyamus seed is provided in a composition.

The composition for use in a method of the second, third, or further aspects may further comprise one or more of an anti-microbial agent, anti-viral agent, growth factor, anti-dehydration agent, and/or antiseptic agent. The composition may comprise a pharmaceutically acceptable carrier.

The subject may be a mammal. In some embodiments the subject is a non-human primate, and may be a chimpanzee, ape, monkey. Alternatively the mammal may be a human. The mammal may be a farm animal or domestic animal.

In a fifth aspect the invention provides a dressing comprising a processed Hyoscyamus seed.

In a sixth aspect the invention provides a method for producing a dressing, comprising processing a Hyoscyamus seed.

In a seventh aspect the invention provides a medical device coated with a processed Hyoscyamus seed. In some embodiments the device is selected from the group consisting of a catheter, guide channel, probe, cardiac valve, soft tissue replacement, replacement of animal origin, artificial tendon, bone replacement, cardiac replacement, contact lens, blood oxygenator, artificial kidney, artificial heart, artificial pancreas, artificial liver, blood bag, syringe, surgical instrument, filtration system, laboratory instrument, container for cell or tissue culture and/or regeneration, support for a peptide, support for a protein, and support for an antibody.

In an eighth aspect the invention provides a method for producing a phyto-membrane, comprising: producing an extract of a Hyoscyamus seed; and combining said extract with an aqueous solution to form said phyto-membrane .

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a graph of fibroblast growth on days 5 and 7 of culture.

Figure 2 shows a graph of fibroblast growth on days 5 and 7 of culture .

Figure 3 shows micro-photographs of fibroblasts cultured in 0%, 10%, or 20% processed Hyoscyamus seed.

Figure 4 shows micro-photographs of fibroblasts cultured in 0%, 10%, or 20% processed Hyoscyamus seed.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present methods are described, it is to be understood that this invention is not limited to the particular materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a seed" is a reference to one or more seeds. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

All publications mentioned herein are cited for the purpose of describing and disclosing the methods, protocols and reagents which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

In its broadest aspect the present invention relates to the use of processed seed from a Hyoscyamus plant for the growth of fibroblasts and treatment of tissue disruptions.

Without wishing to be bound by any theory or hypothesis, the inventors have identified that processed Hyoscyamus seeds are able to increase the growth of fibroblasts and hence facilitate would healing. The inventors have further identified that the processed Hyoscyamus seeds are able to form a phyto-membrane which further facilitates wound healing and propose that the phyto-membrane is formed by polymerisation of one or more of the glycoproteins in the Hyoscyamus seeds .

As used herein the term "Hyoscyamus" refers to plants of the genus Hyoscyamus in the family Solanaceae. The genus Hyoscyamus comprises about 86 species including Hyoscyamus niger, Hyoscyamus semen, Hyoscyamus muticus, and Hyoscyamus aureus. Seeds from any plant of the genus Hyoscyamus may be used in the invention as seeds from all Hyoscyamus plants comprise alkaloids and glycoproteins suitable for use in the invention.

The main features of the seeds of Hyoscyamus plants are the ability to increase the growth and orientation of fibroblasts, to treat a tissue disruption, and to form a hydroscopic powder which can form a phyto-membrane . Therefore, as used herein the term "Hyoscyamus seed" refers to any seed from any plant of the genus Hyoscyamus that is capable of one or more of the features described herein.

The term "seed" as used herein refers to one or more parts of a fertilised ovule of a Hyoscyamus plant including the embryo and testa. In one preferred embodiment, the seeds are ripe when harvested, as ripe seeds generally contain greater amounts of alkaloids and glycoproteins compared to less ripe seeds .

Once the seed has been harvested it is processed prior to use. The seed may be processed directly or may be at least partially dried prior to processing. In one embodiment, the seed is heated to approximately 70⁰C for 8 to 10 minutes to reduce or eliminate moisture prior to processing.

The seed is processed by breaking it up such that particles of the seed are formed. The seed may be broken- up by any method known in the art. For example, the seed may be ground, crushed, minced, or pounded to form seed particles. In some embodiments the processed Hyoscyamus seeds are dissociated to form seed particles of less than 500 microns in size. In other embodiments the seed particles are between 20 and 250 microns in size.

The seed may be processed further and in some embodiments is subjected to an extractive process such as phenol/chloroform extraction, chromatography, ion exchange, centrifugation and the like.

In some embodiments, the processed Hyoscyamus seed may be used to increase the growth of a fibroblast. A "fibroblast" is a cell typically found in connective tissue that secretes an extracellular matrix rich in collagen and other macromolecules . A fibroblast has a branched cytoplasm surrounding an elliptical, speckled nucleus having 1 or 2 nucleoli. Active fibroblasts can be recognized by their abundant rough endoplasmic reticulum. Inactive fibroblasts, which are also called fibrocytes, are smaller and spindle shaped. They have a reduced rough endoplasmic reticulum. Fibroblasts migrate and proliferate readily in wound repair and in tissue culture.

The term "growth" as used herein means an increase in size and/or number. Hence an increase in the growth of a fibroblast means that the fibroblast has increased in size and/or number.

The inventors have identified that when a Hyoscyamus seed is processed according to the invention a hydroscopic powder is formed. The term "hydroscopic powder" as used herein refers to the ability of the processed seed to attract and absorb fluid. The fluid may be provided by an aqueous solution or by a tissue disruption, for example by tissue fluid or a wound exudate.

In some embodiments the processed seed is combined with other agents, such as other therapeutically active agents. Suitable agents include antimicrobial agents, including but not limited to antifungal agents, antibacterial agents, anti-viral agents and antiparasitic agents; growth factors; angiogenic factors; anaesthetics; mucopolysaccharides; metals; and other agents which have therapeutic activity.

Examples of antimicrobial agents that can be used in the present invention include, but are not limited to, isoniazid, ethambutol, pyrazinamnide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin, dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline, ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclazaril, acyclovir, trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir, iatroconazole, miconazole, Zn-pyrithione, heavy metals including, but not limited to, gold, platinum, silver, zinc and copper, and their combined forms including, salts, such as chloride, bromide, iodide and periodate, and complexes with carriers, and other forms.

Growth factor agents that may be incorporated into the wound dressing devices of the present invention include, but are not limited to, basic fibroblast growth factor (bFGF) , acidic fibroblast growth factor (aFGF) , nerve growth factor (NGF) , epidermal growth factor (EGF) , insulin-like growth factors 1 and 2 (IGF-I and IGF-2) , platelet derived growth factor (PDGF) , tumor angiogenesis factor (TAF) , vascular endothelial growth factor (VEGF) , corticotropin releasing factor (CRF) , transforming growth factors α and β (TGF-α and TGF-β), an interleukin e.g. interleukin-8 (IL-8) , granulocyte-macrophage colony stimulating factor (GM-CSF) , and an interferon.

Other agents that may be incorporated into the wound dressings of the present invention are acid mucopolysaccharides including, but not limited to, heparin, heparin sulfate, heparinoids, dermatan sulfate, pentosan polysulfate, cellulose, agarose, chitin, dextran, carrageenin, linoleic acid, and allantoin.

The therapeutically active agents may be bound either physically or chemically to the processed seed by methods well known in the art. Alternatively, they may be simply admixed.

Similarly, the processed seed may be bound to or admixed with a pharmaceutically acceptable carrier, ie to form a pharmaceutical composition, and may be formulated for administration as a pharmaceutical composition in accordance with known techniques. See, eg., Remington, The Science And Practice of Pharmacy (9^th Ed. 1995) .

In the manufacture of a pharmaceutical composition according to embodiments of the invention the processed Hyoscyamus seed is typically admixed with, inter alia, a pharmaceutically acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the pharmaceutical composition and should not be deleterious to the mammal being treated. The carrier may be a solid or a liquid, or both, and is preferably formulated with the processed Hyoscyamus seed as a unit-dose formulation. The pharmaceutical composition may be prepared by any of the well-known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients .

The pharmaceutical composition may be formulated for oral, buccal (sub-lingual) , parenteral, rectal, topical, and/or transdermal administration.

In some embodiments the pharmaceutical composition is formulated for topical administration. Pharmaceutical compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof .

In other embodiments the pharmaceutical composition is formulated for transdermal administration. Pharmaceutical compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Compositions suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6) :318 (1986)) and typically take the form of an optionally buffered aqueous solution of the processed

Hyoscyamus seed. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water .

In addition to any of the ingredients listed above, a composition of the invention may further comprise other agents. For example, agents such as binders, sweeteners, thickeners, flavouring agents, disintegrating agents, coating agents, preservatives, lubricants, and/or time delay agents .

In some embodiments a processed Hyoscyamus seed and/or composition of the invention may be applied as a "puff-on bandage" . As such, the processed seed and/or composition can be housed in a sterile gas-propelled canister. Alternatively, it can be delivered as a single use dosage (sterile sealed in an ampoule) for administration to a tissue disruption.

In some embodiments, the processed Hyoscyamus seed is stored at 16⁰C to 20°C then, when required, is mixed with an aqueous solution to form a phyto-membrane ex-vivo or ex situ. A "phyto-membrane" is a plant derived membrane. Once the phyto-membrane has been formed it is dried to form a "phyto-membranous wound dressing" . As used herein, the term "dried" refers to any method for the removal of water from the phyto-membrane . It is intended that the term encompasses methods including, but not limited to, air-drying, freeze-drying or heating. Some or all of the water may be removed from the phyto-membrane.

The phyto-membrane may serve as a packing material and, if required, may be covered with any suitable secondary wound dressing such as a wrap, tape, gauze, or pad. In some embodiments the phyto-membrane is temporary and is not intended for permanent incorporation into the healed tissues. When necessary, the phyto-membrane is removed by first removing any over-dressing material and then removing the phyto-membrane by washing the affected area with sterile saline, antiseptic solution or the like. The phyto-membrane of the present invention may then be replaced by a fresh dressing or other suitable wound covering.

A phyto-membrane of the invention may be used as a dressing. The term "dressing" refers to a therapeutic or protective material applied to a tissue disruption.

In some embodiments a processed seed and/or composition of the invention is used directly as a dressing. The dressing may be placed in its entirety into a wound. The dressing may be cut, shaped, and modified to accommodate numerous uses and applications.

A phyto-membranous dressing of the invention is preferably in the form of a continuous sheet, similar to dressings known in the art . However the invention may also take other particular conformations. For example, dressings of the invention may be produced by cutting a desired design pattern from stock sheets of dried composition material of the invention. For example, the sheet may be die-cut from stock sheets of dressing material. The material may then be sterilized using techniques known in the art such as gamma radiation, steam and heat sterilization, electron beam or chemical sterilization (such as by use of ethylene oxide) .

A processed seed, composition, phyto-membrane, or dressing of the invention may be used in the treatment of a disease or disorder including a tissue disruption.

Generally, the terms "treating," "treatment" and the like are used herein to mean affecting a mammal or its tissue or cells to obtain a desired pharmacological and/or physiological effect. The effect is especially therapeutic in terms of a partial or complete cure of a condition such as a tissue disruption. "Treating" as used herein covers any treatment of a tissue disruption in a mammal, particularly a human, and includes: (a) inhibiting the tissue disruption, i.e., arresting its development; or (b) relieving or ameliorating the symptoms of the tissue disruption, i.e., cause regression of the symptoms of the tissue disruption.

All mammals comprise tissue which is subject to disruption and is healed, inter alia, by fibroblasts. Accordingly, the term "mammal" as used herein refers to any member of the class mammalia, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea 'pigs. The terms do not denote a particular age. Thus, both adult and newborn mammals are intended to be covered.

Thus, provided is the treatment of mammals such as humans, as well as those mammals of economical importance and/or social importance to humans, for instance, carnivores other than humans (such as cats and dogs) , swine (such as pigs, hogs, and wild boars) , ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses .

The term "tissue disruption" refers to any abnormal tissue condition affecting a mammal which can be treated using a processed Hyoscyamus seed. Accordingly, the treatment of a tissue disruption such as a wound, a lesion, a microbial infection, a burn, an ulcer, and a skin condition is within the scope of the invention.

A wound is described as "any disruption to anatomic or physiologic function of tissues" . The moment an injury occurs, a complex series of events is initiated, eventually progressing to tissue repair.

The term "wound" as used herein refers to a tissue disruption that exposes underlying epidermal, dermal, muscular or adipoidal tissue to the air, such as in the skin or oral mucosal layer or the surface layers of the eye including the conjunctiva and cornea. Wounds include, but are not limited to, a puncture wound, an incision, a laceration, a penetrating wound, a perforating wound, a tunnel wound and the like. Wounds also include open wounds that have been sutured or otherwise mechanically closed but have not healed or repaired.

The term "wound management" refers to therapeutic methods that induce and/or promote repair of a wound including, but not limited to, arresting tissue damage such as necrotization, promoting tissue growth and repair, reduction or elimination of an established microbial infection of the wound and prevention of new or additional microbial infection or colonization. The term may further include reducing or eliminating the sensation of pain attributable to a wound.

The terras "wound healing" and "wound repair" refer to a process involving tissue growth that partially or totally closes a wound, repairs a breach in the dermis or epidermis and partially or totally restores the barrier properties of the skin or the repair of the surface layers of the eye including the conjunctiva and cornea.

The terms "lesion" and "surface lesion" as used herein refer to a circumscribed area of pathologically altered tissue, an injury or wound. Primary lesions are the immediate result of the pathological condition and include, but are not limited to, cuts, abrasions, vesicles, blebs, bullae chancres, pustules, tubercles or any other such condition of the skin or a surface of the mouth, nose, anus or any other orifice of the body of a human or animal, or to the surface layers of the eye including the conjunctiva and cornea, or secondary lesions that later develop from a primary lesion and includes, but is not limited to, fissures and ulcers and other wounds.

The term "microbial infection" as used herein refers to any pathological presence of at least one bacterial species on or in a tissue disruption of a mammal. It is further understood that a "microbial infection" may include any systemic infection that is amenable to inhibition by application of the antimicrobial compositions of the present invention.

The term "burn" as used herein refers to tissue injury of the skin caused by thermal, chemical, or radiation exposure or abrasive friction. A burn may be a "first- degree burn" with superficial damage to the outer cornified layer, a "second-degree burn" with damage extends down into the epidermal layer of cells but is not of sufficient extent that regeneration of the skin is prevented, or a "third-degree burn" where the injury extends below the dermis to the underlying tissue and wherein repair of the skin is not possible without grafting.

The term "ulcer" as used herein refers to an open sore or lesion of the skin or a mucous membrane that involves the sloughing off of inflamed and necrotised tissue and includes, but is not limited to, callous ulcers, chronic leg ulcers, decubitus, denture ulcers of the oral mucosa, traumatic ulcers of the mouth, infections stomatitis of the mouth and any type of secondary lesion that is a breach of the cornified and the epidermal layer of the skin or the mucosal surface of the mouth.

The term "skin condition" refers to a condition affecting one or more layers of the skin, including conditions affecting the texture and/or appearance of the skin. Examples of skin conditions include acantholysis, acanthosis, acne, dermatosis, eczema, erthrodema, furunculosis, hair loss, impetigo, jungle rot, keratoderma, keratonosis, keratosis, leukoderma, lichen, livedo, lupus, melanism, molluscum, necrobiosis lipoidica, pemphigus, prurigo, psoriasis, rhagades, Saint Anthony's fire, seborrhoea, vitiligo, xanthoma, and xanthosis.

A processed seed, composition, phyto-membrane or dressing of the invention may also be applied to any of a wide variety of contacting surfaces of medical devices.

Contacting surfaces include, but are not limited to, surfaces that are intended to contact blood, cells or other bodily fluids or tissues of a mammal, including specifically a human. Suitable contacting surfaces include one or more surfaces of medical devices that are intended to contact blood or other tissues. The medical devices include aneurysm coils, artificial blood vessels, artificial hearts, artificial valves, artificial kidneys, artificial tendons, blood bags, blood oxygenators, bone and cardiovascular replacements, bone prostheses, bone waxes, cardiovascular grafts, cartilage replacement devices, catheters, contact lenses, containers for cell and tissue culture and regeneration, embolization particles, filtration systems, grafts, guide channels, indwelling catheters, laboratory instruments, microbeads, nerve-growth guides, ophthalmic implants, orthopedic implants, pacemaker leads, probes, prosthetics, shunts, stents, supports for peptides, surgical instruments, sutures, syringes, urinary tract replacements, wound coverings, wound dressings, wound healing devices and other medical devices known in the art .

Other examples of medical devices that would benefit from the application of the present invention will be readily apparent to those skilled in the art of surgical and medical procedures and are therefore contemplated by the invention. The contacting surface may include a mesh, coil, wire, inflatable balloon, or any other structure which is capable of being implanted at a target location, including intravascular locations, intra-lumenal locations, locations within solid tissue, and the like. The implantable device can be intended for permanent or temporary implantation. Such devices may be delivered by or incorporated into intravascular and other medical catheters .

The process of coating the surfaces of such devices can be performed by the plasma coating technique, as described in the International patent application No. WO96/24392.

Throughout the specification, the word "comprise" and variations of the word, such as "comprising" and

"comprises" , means "including but not limited to" and is not intended to exclude other additives, components, integers or steps. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

The invention will now be further described by way of reference only to the following non-limiting examples. It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above. For example, while the majority of the examples relate to seeds from Hyoscyamus niger, it is to be understood that the invention can also seeds from other Hyoscyamus species as disclosed herein.

EXAMPLE 1 PREPARATION OF PROCESSED HYOSCYAMUS SEEDS

The working environment for the seed processing was kept cool at between about 20⁰C to 25°C and dry. The desired relative humidity was between 50% to 65%.

The initial raw preparation of Hyoscyamus niger seeds contained excessive debris from other parts of the plant and sieving of this bulk raw material was required. The bulk raw materials were passed through size sieving apparatus fitted with 14G to 18G particle sieves. Unwanted debris were removed and discarded accordingly and the seeds were collected in stainless steel trays under regulated environment conditions .

The seeds were then pre-heated to 70⁰C for 8 to 10 minutes, to eliminate any moisture prior to the grinding procedures .

The pre-treated seeds were then introduced into a commercial grinder under the regulated environmental conditions . Strict anhydrous conditions were maintained at this stage. A single passage through the grinder was usually sufficient. However, if the resulted powder was too coarse, a second passage through the grinder was undertaken.

The herbal powder was then collected into plastic containers layered with desiccants (anhydrous silica gels) packed in pouches. These powders were then stored at 16°C to 20°C for further processing.

EXAMPLE 2 CYTOTXOCITY EVALUATION OF PHYTO MEMBRANE ON PRIMARY HUMAN SKIN FIBROBLAST CULTURE

The effects of a phyto-membrane containing processed Hyoscyamus seed on the proliferation of fibroblasts was determined using direct and indirect cell culture assays and two concentrations of processed Hyoscyamus seed, 10% w/v and 20% w/v (respectively equivalent to 200mg and 400mg phyto-membrane dry weight) .

Phyto-membrane materials were provided by Dr Goh, Asia Pacific Life Sciences (S) Pte Ltd. DMEM, FCS and EpiLife™ cell culture media were purchased from Invitrogen or Cascade Biologies, respectively. Primary skin fibroblasts were provided by CellResearch Corporation Pte Ltd,

Singapore. All other reagents were purchased from Sigma. Dried powdered Hyoscyamus seed prepared as described in Example 1 was weighed and mixed with basal media of DMEM or EpiLife™ to form a gel containing either 10% or 20% powdered Hyoscyamus seed. Each gel was spread over 10cm- tissue culture dishes (Nunc) , allowed to dry ie form a membrane, then kept in a flow lamina hood until used.

Normal human fibroblasts at early passage [NF-117, Passage-1; NF-118, Passage-4 and NF-118, Passage-6] were I seeded onto the phyto-membrane at 1 x 10⁶ cells per dish}-. One group of cells was not stabilized in growth media (DMEM/10% FCS or Supplemented EpiLife™) in advance, and therefore these cells were potentially more sensitive to phyto-membrane cytotoxicity. Another group of cells were stabilized in 10ml of DMEM/10%FCS or EpiLife™ medium prior to seeding. Another group of cells was cultured in dishes that had not been coated with the phyto-membrane.

The cells were cultured at 37°C, 10% CO₂ for up to 5 days. At days 1 or 5, cells were fixed with methanol and stained with rhodamine . Cell viability and proliferation were monitored using a light inverted microscope. Micro-photos were taken.

Fibroblasts were seeded in tissue culture inserts

(Milipore) at a density of 1 x 10⁴ cells per insert in 1 ml of DMEM basal medium containing 10% FCS or EpiLife™ medium and maintained for 2 days in DMEM/10% FCS or EpiLife™ supplemented with 10% FCS. The cells were then seeded onto plates coated with the phyto-membrane as outlined above then cultured in DMEM/l0%FCS or Supplemented EpiLife™. In this method, the cells indirectly interacted with the phyto-membrane via the media liquid phase. One set of cell-insert constructs were cultured as a control in a dish which had not been coated with the phyto- membrane . The cells were monitored daily by microscope. The MTT [3- (4.5-dimethyl thiazol-2-yl) -2 , 5-diphenyl tetrazolium bromide] assay was used to assess the fibroblast cell viability at day 5 and day 7.

The culture conditions are summarized in Table 1.

TABLE 1

When MTT is applied to living cells, succinate dehydrogenase in the mitochondria of the cells will change the MTT to a blue formazan product that can be measured at 570nm after being eluted from the cells by a 20% SDS solution.

500μl of MTT (5mg/ml in PBS) solution was added to each cultured insert and the cells were incubated at 37⁰C for 2 hours. The medium was replaced with 100μl of 20% SDS solution and the cells were incubated at 37°C for half an hour. The supernatant was transferred from the cells to a 96 well plate and the optical density was measured by at 570nm. 20% SDS solution was used as a control. Each experiment was performed in duplicate and four inserts were tested for each concentration.

The results were determined by mean standard error of the mean.

Figure 1 shows that growth of normal human fibroblast indirectly cultured in 10% (less) phyto-membrane significantly increased by 128.11% and 122.76% (normalised to the control cell count, which was set to 100%) on days 5 and 7 respectively compared with the control. A decrease in growth of 9.966% and -0.7929% (normalised to the control cell count, which was set to 100%) on days 5 and 7 respectively was noted in the metabolism of fibroblasts treated with 20% (more) phyto-membrane compared with the control.

As shown in Figure 2, neither 10% (less) and 20% (more) phyto-membrane are toxic to cells, as determined by direct cell culture.

As shown in Figure 3, normal fibroblasts (NF-117) can coexist with a phyto-membrane of the invention. No evidence of cytotoxicity of the phyto-membrane to fibroblasts was shown at 10% (200mg) phyto-membrane, as determined by direct cell culture.

As shown in Figure 4, normal fibroblasts (NF-118) can coexist with the phyto-membrane of the invention. No evidence of cytotoxicity of the phyto-membrane was shown at either 10% or 20% phyto-membrane as determined by direct cell culture.

It could be concluded that there is no in vitro cytotoxicity to normal skin fibroblasts caused by a phyto- membrane of the invention when the cells are cultured by either a direct or indirect culture method. In fact, culture of the fibroblasts at 10% (200mg) showed a slight increase in cell proliferation. Therefore the phyto- membrane could be used in the early phase of wound healing, inflammation, and/or granulation tissue formation. It could also be used for cavity or other chronic wounds prior to re-epithelialization.

EXAMPLE 3 PRODUCTION OF TOPICAL AGENTS

The flow properties of the powder produced in Example 1 was not optimum for packaging and for use in some topical applications. Therefore, in order to achieve a desirable dosage form for large-scale commercial production, a suitable blend of the powder was developed. The best combination of Hyoscyamus powder and excipient was found to be at 75% of the crude powder and 25% of a talc excipient . This combination provided the dosage form for industrial production and topical applications. Thus, in the production protocol, 75% (by weight) of the Hyoscyamus powder was added to 25% (by weight) of talc powder in a commercial blender. If additional therapeutics were required these were added to the Hyoscyamus powder within the 25% portion of the total blend. The materials were mixed to close homogeneity for about 20 minutes. The whole process was carried out in an anhydrous environment. No clumping of the mixture was allowed at this stage of production. As an added precaution, a bag of anhydrous silica gel may be included in the blending chamber to reduce or eliminate moisture.

The blended powder was then packaged into autoclavable tubes, each containing approximately 5 grams of the powder, and placed in a stainless steel container with a lid. Two bags of anhydrous silica gels (5Og per bag) were then placed in the container together with the tubes . The container lid was replaced and the whole assembly was then put into the autoclave for sterilisation. A single cycle of 20 minutes at 120⁰C was sufficient to achieve sterility.

Alternatively, the packed powder was treated with gamma or X-ray irradiation by standard procedures.

Samples of the final powder was applied to agar plates using a sterile applicator under sterile conditions. The plates were then incubated at 37⁰C for 36 hours. A colony count was then performed.

The final powder product was then stored in a cool and dry environment. The temperature range being between 15°C to 25°C.

EXAMPLE 4 TREATMENT OF TOPICAL WOUNDS

The wound to be clinically managed is initially debrided as much as possible to remove necrotic tissue. If required biological specimens are sent off for microbiological and pathological studies. Radiological studies may also be done to ascertain underlying deep tissue involvement e.g. as in underlying osteomyelitis (or bone infections) .

Assessment of the wound through a description of its shape and dimensions including depth (grading of wound) is obtained prior to treatment with the sterile Hyoscyamus powder and excipient. A diagram or photograph of the wound is obtained prior to treatment .

The sterile preparation of Hyoscyamus powder is then applied over the wound. Depending on granularity and the delivery platform, the granules are applied as uniformly as possible upon the exposed surface of the wound under treatment .

A light dressing is applied to protect the surface of the wound.

The wound is inspected regularly and the dressing changed according to the clinical need and amount of wound discharge.

The process of wound dressing change is as follows:

The wound is irrigated with normal saline solution before dressing is removed especially if the dressing is adherent to the underlying tissue. The phyto-membrane complex formed by the Hyoscyamus powder and the wound discharge would be removed either through irrigation with normal saline or with use of sterile forceps.

The wound is re-assessed periodically as to size, shape, depth and vitality of the healing scar tissue upon the wound bed. Repeat photographs and wound diagrams are documented to chart progress.

Use of the Hyoscyamus powder does not preclude the need for further surgical interventions, antibiotics, pressure relief measures, nutritional supplementation or current standard of care for complicated wounds .

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A composition comprising a processed Hyoscyamus seed.

2. A composition of claim 1, for use in the treatment of a tissue disruption.

3. A composition of claim 1 or claim 2, for use in the growth of a fibroblast.

4. A composition of any one of claims 1 to 3, wherein the seeds are obtained from a plant selected from the group consisting of Hyoscyamus niger, Hyoscyamus semen, Hyoscyamus muticus, and Hyoscyamus aureus.

5. A composition of any one of claims 1 to 4, wherein the seeds are processed to form a hydroscopic powder.

6. A composition of any one of claims 1 to 5, further comprising an anti-microbial agent, anti-viral agent, growth factor, anti-dehydration agent, and/or antiseptic agent .

7. A composition of any one of claims 1 to 6, further comprising a pharmaceutically acceptable carrier.

8. A method of treating a disease or disorder, comprising the step of administering to a subject a composition comprising a processed Hyoscyamus seed.

9. A method of claim 8, wherein the disease or disorder is associated with a lesion, wound, microbial infection, burn, ulcer, and/or dermal condition.

10. A method of treating a tissue disruption, comprising the step of contacting the tissue disruption with a processed Hyoscyamus seed.

11. A method of increasing the growth of a fibroblast, comprising the step of contacting the fibroblast with a processed Hyoscyamus seed.

12. A method of any one of claims 8 to 11, wherein the Hyoscyamus seed is isolated from a plant selected from the group consisting of Hyoscyamus niger, Hyoscyamus semen, Hyoscyamus muticus, and Hyoscyamus aureus.

13. A method of claim 12, wherein the seeds are processed to form particles of less than 500 microns.

14. A method of any one of claims 8 to 13, wherein the processed Hyoscyamus seed is provided in a composition.

15. A method of claim 14, wherein the composition further comprising one or more of an anti-microbial agent, antiviral agent, growth factor, anti-dehydration agent, and/or antiseptic agent .

16. A method of claim 14 or claim 15, wherein the composition comprises a pharmaceutically acceptable carrier.

17. A method of any one of claims 8 to 16, wherein the subject is a mammal.

18. A method of claim 17, wherein the mammal is a non- human primate.

19. A method of claim 18, wherein the non-human primate is a chimpanzee, ape, or monkey.

20. A method of claim 17, wherein the mammal is a human.

21. A method of claim 17, wherein the mammal is a farm animal or domestic animal.

22. A dressing comprising a processed Hyoscyamus seed.

23. A method for producing a dressing, comprising processing a Hyoscyamus seed.

24. A medical device coated with a processed Hyoscyamus seed.

25. A medical device of claim 24 selected from the group consisting of a catheter, guide channel, probe, cardiac valve, soft tissue replacement, replacement of animal origin, artificial tendon, bone replacement, cardiac replacement, contact lens, blood oxygenator, artificial kidney, artificial heart, artificial pancreas, artificial liver, blood bag, syringe, surgical instrument, filtration system, laboratory instrument, container for cell or tissue culture and/or regeneration, support for a peptide, support for a protein, and support for an antibody.

26. A method for producing a phyto-membrane, comprising: producing an extract of a Hyoscyamus seed; and combining said extract with an aqueous solution to form said phyto-membrane .