HK1066482B - Use of fucans in the treatment of adhesions, arthritis and psoriasis - Google Patents

Description

Use of fucoidan for treating adhesions, arthritis and psoriasis

Cross reference to related applications

This application claims priority from U.S. provisional patent application No. 60/315,362, filed on 8/29/2001.

Background

Surgical adhesions are scars, which often form between two parts of the body after surgery. Blocking can cause serious problems. For example, adhesions involving female reproductive organs (ovaries, fallopian tubes) can cause infertility, dyspareunia (dyspareunia), and severe pelvic pain. Adhesions occurring in the intestine can cause ileus or blockage, and can also form elsewhere such as in the heart, around the spine, and in the hands. In addition to surgery, adhesions can also be caused by other causes such as endometriosis, infection, chemotherapy, radiation and cancer.

Adhesions and other angiogenesis-related diseases such as arthritis and psoriasis can last for weeks, months or years, requiring long-term and costly care. See cobran, r.s., Kumar, v., Robbins, s.l. written Robbins clinical Basis of Disease, page 75 (w.b. saunders co., 1989). These diseases and conditions can progress to chronic inflammation with dire consequences for the psychological and physiological health of the patient. Unfortunately, there are few treatment options for patients with surgical adhesions, arthritis, and psoriasis. Patients are often treated with drugs such as steroidal or non-steroidal anti-inflammatory drugs to alleviate symptoms of the disease. However, these treatments may not provide adequate long-term benefits and may also lead to serious side effects if used too frequently (e.g. gastric ulceration with non-steroidal anti-inflammatory drugs or more severe toxicity with overuse of steroids). Other more potent antiproliferative and/or antiangiogenic drugs such as the anticancer drugs paclitaxel, methotrexate, doxorubicin, camptothecin, and etoposide may provide a positive form of treatment, but use of these drugs for non-life threatening diseases is limited by undesirable toxicity and side effects.

Thus, there is an unmet need for compounds, compositions, methods, etc. (including methods of delivery) that are preferably more effective and have fewer side effects for the treatment of one or more of these diseases. The compounds, compositions, methods, etc. of the present invention provide one or more of these advantages.

Summary of The Invention

Compositions and methods comprising fucoidan, particularly fucoidan (fucoidan), are useful for treating surgical adhesions, arthritis, and psoriasis. Fucoidan provides significant therapeutic effects and low side effects for each of these diseases.

In one aspect, the invention provides a method of treating adhesions in an animal (which may be a human or other subject in need thereof) comprising administering a therapeutically effective amount of a fucan (which may be a fucoidan) to a site of a disease at which the adhesion is likely to occur. The disease site can be a surgical site and the fucan can be administered as a composition directly to the disease site. The fucan can be administered substantially continuously to the disease site by controlled release from a polymeric dosage form which can be a film, patch, paste, microsphere, implant, gel, spray, or liquid formulation. The fucan can be administered as a pharmaceutical composition in a form comprising at least one of a cream, a paste, an injectable excipient, and a polymer. (unless otherwise indicated or clear from the context, all embodiments, aspects, features, etc. can be mixed and matched, combined, and arranged in any desired manner.)

The fucan can be administered as a pharmaceutical composition comprising the fucan and a therapeutically effective amount of at least one other drug. The drug may be at least one of paclitaxel, doxorubicin, camptothecin, etoposide, mitoxantrone, methotrexate, menadione, plumbagin, juglone, beta-lapachone, cyclosporine, sulfasalazine, steroids, rapamycin, retinoids, docetaxel, colchicine, antisense oligonucleotides, ribozymes. A therapeutically effective amount of fucan can be delivered as part of the composition, and the fucan can comprise about 0.1% to 35%, 5% to 50%, 20-80%, 80% -100% (w/v) of the composition.

The composition may further comprise at least one pharmaceutically acceptable excipient, such as pluronic, cellulose, alginate, acrylate, hyaluronic acid, polyethylene glycol, injectable excipients, and chitosan. The fucan can be administered orally, directly to the site of disease, by intraocular, intraperitoneal, intramuscular, intraarticular, intralesional, subcutaneous, intravaginal, rectal, or local injection to the site of disease, or in other desired ways.

In another aspect, the method comprises treating arthritis, psoriasis, or angiogenic eye disease comprising administering a therapeutically effective amount of fucan to the site of the disease.

In another aspect, the present invention provides a pharmaceutical composition comprising a polymeric dosage form of fucan comprising a therapeutically effective amount of fucan and at least one pharmaceutically acceptable excipient selected from the group consisting of pluronics, alginates, acrylates, hyaluronic acid, polyethylene glycol, injectable excipients, and chitosan. The polymer dosage form may be a film, paste, microsphere, spray, lotion, liquid formulation or implant or other form as desired. The pharmaceutical composition may also comprise a therapeutically effective amount of at least one other drug, such as antisense oligonucleotides, ribozymes, and oligonucleotide RNA inhibitors.

The composition can be used as desired in the manufacture of a medicament for treating adhesions (e.g., surgical adhesions), arthritis, psoriasis, or other diseases. Also provided is a method of producing a medicament capable of alleviating a symptom associated with at least one of adhesions, arthritis, and psoriasis in a patient, the method comprising combining a pharmaceutically effective amount of a fucan, such as fucoidan, a pharmaceutically acceptable excipient or buffer.

These and other aspects, features and embodiments are set forth in this application, including the following "detailed description" and figures. Also, various references discussing certain systems, apparatus, methods, and other information are provided herein, including in the cross-reference to related applications; all such references and all their teachings and disclosures are incorporated herein by reference in their entirety, regardless of where in the present application the reference appears.

Brief Description of Drawings

FIG. 1 is a graph of the inhibition of cell proliferation by fucoidan in synovial cells and smooth muscle cells after 48 hours of contact.

FIG. 2 is a graph of fucoidan inhibition of Phorbol Myristate (PMA) induced neutrophil activation.

FIG. 3 is a photograph depicting the inhibition of collagenase and stromelysin expression by fucoidan at a concentration of 0.5% (w/v) without excessive inhibition of proteoglycan expression.

Figure 4 is a graph of the release of fucoidan from an ethylene vinyl acetate film.

Figure 5 is a graph of fucoidan release from polycaprolactone paste.

Detailed Description

The present invention includes compositions and methods comprising methods of inhibiting cell proliferation, inflammatory responses and angiogenesis with sulfated polysaccharides known as fucoidans and useful for treating surgical adhesions, arthritis and psoriasis. It appears that fucoidans such as fucoidan can inhibit neutrophil activation, inhibit the release of inflammatory enzymes from arthritis-associated cells, and inhibit angiogenesis in chicken membranes and surgical adhesions. Since all cells contain fucose-binding receptors, in some embodiments, the delivery of fucoidan directly to the disease site provides substantially continuous contact of the target tissue with the fucoidan (e.g., fucoidan) through controlled release from the polymeric dosage form. Because fucoidan has a variety of in vivo effects (particularly affecting blood thrombin and complement), site-directed controlled release of fucoidan is an alternative to systemic administration, which can reduce blood toxicity.

The following will first discuss fucoidans, adhesions, arthritis, and psoriasis generally, then discuss some embodiments of the invention, and then provide some examples.

General background discussion of fucoidan, adhesions, arthritis, and psoriasis

Fucosan

Fucoidans (including fucoidans) are high molecular weight sulfated polysaccharides extracted from brown seaweeds. These compounds have been reported to have a variety of in vivo and in vitro inhibitory effects, including antithrombin, antiproliferative, anticomplementary, anticancer, and anti-neutrophil migration effects. Fucoidan can block various binding events on the cell surface, including cell-cell binding by integrin-selectin molecules, or by binding to thrombin or complement in the blood or to fucose receptors on the cell surface.

These activities are believed to determine anti-inflammatory properties, as these activities prevent subsequent invasion of lymphocytes or neutrophils into the tissue compartment following inflammation, for example, by inhibiting binding of these cells to vascular endothelial cells. Patankar, m.s. et al, j.biol.chem.268: 21770 and 21776 (1993); brandley, b.k., et al, j.cell biol.105: 991-997(1987). Recent studies have also shown that fucoidan inhibits the proliferation of vascular smooth muscle cells, Logeart, d, et al, eur.j.cell biol.74: 376-. Fucoidan has also been shown to be slowly internalized in cells after binding to the surface of endothelial cells and smooth muscle cells. Glabe, c.g., et al, j.cell Science 61: 475-; logeart, d, et al, eur.j.cell biol.74: 376-384(1997).

Riou, d, et al, Anticancer res, 16 (3A): 1213 and 1218 (1996); itoh, h, Anticancer res, 13 (6A): 2045-; nishiro, t, et al, Thromb. Res., 62: 765-773 (1991); blondin, c, et al, mol.immunol., 31: 247, 253 (1994); patankar, m.s. et al, j.biol.chem., 268: 21770-21776(1993). Fucoidan extracted from various seaweeds is sold as a health food in japan. Fucoidan is also proposed as a cosmetic or skin care product. JP 01031707 and JP 01085905. Fucoidan is reported to be a potential anticancer agent. Riou.d., Anticancer res.16: 3a 1213-18 (1996); itoh, h., et al, Anticancer res, 15: 5b 1937-47 (1995). Fucoidan is reported to not inhibit angiogenesis in vitro. Soeda, S. et al, Biochim. Biophysica Acta (1): 127-134(2000). Similarly, fucoidan was found to stimulate serum-induced HUVE cell proliferation (in vitro), suggesting a possible pro-angiogenic effect (although possibly inhibited when fibroblast growth factor is present). Giraux, j, current, eur.j.cell biol.774: 352-9(1998). Studies have also shown that fucoidan inhibits endothelial cell monolayer binding. Glabe, c.g., j.cell Science, 61: 475-490(1983). Since the cells that make up the capillaries are endothelial cells, the report indicates that certain aspects of cell adhesion can be inhibited in vitro, but these data fail to demonstrate any in vivo anti-angiogenic effect of fucoidan. Fucoidan has been reported to inhibit the binding of helicobacter (helicobacter) to gastric cells, suggesting its anti-gastric ulcer effect. Shibat, h.j., nutr.sci.vitaminol.45: 325-336(1999).

Other sulfated polysaccharides, including branched and straight chain types, have been reported to have different anticoagulant activities. Pereira, m.s., j.biol.chem.12: 7656-67(1999). Dextran sulfate and its derivatives have been reported to inhibit cancer cell growth (Bittoun, P., Carbohydrate Res. (3-4): 247-255(1999)) and have anticoagulant effects (Mauray, S., J.Biomat. Sci.Poly. 9: 373-87 (1998)). Sulfated polysaccharides have been proposed as antiviral agents for use against, for example, AIDS. EP 00293826; JP 01313433.

Adhesion of the components

Adhesion formation is a complex process in which normally separated tissues in the body grow together. Surgical adhesions (also called post-surgical adhesions) arise from the development of wounds from an abnormal wound healing response of the tissue and occur in all abdominal surgery patients greater than 2/3. Ellis, h, surg, gynecol, obstet.133: 497 (1971); wiebel, M-a. and Majno, g., am.j.surg.126: 345(1973). The consequences of these adhesions vary depending on the surgical site involved. Problems can include pain, infertility, ileus, and even an increased risk of death after cardiac surgery. diZerega, g.s., prog.clin.biol.res.381: 1-18 (1993); diZerega, g.s., fertil. steril.61: 219, 235 (1994); dobell, a.r., Jain, a.k., ann.thorac.surg.37: 273-278(1984).

The process of adhesion formation initially involves the establishment of a fibrin framework and normal tissue repair. The normal repair process allows fibrinolysis and mesothelial repair. However, in surgical adhesion formation, as fibroblasts proliferate into a network and angiogenesis occurs, the fibrin matrix matures, resulting in the establishment of organized adhesions within 3 to 5 days. Buckman, r.f. et al, j.surg.res.21: 67-76 (1976); raferty, a.t., j.antat.129: 659-664(1979).

The inflammatory process includes neutrophil activation in the injured tissue, fibrin deposition and binding of adjacent tissue, macrophage infiltration, fibroblast proliferation to the area, collagen deposition, angiogenesis and establishment of permanent adhesion tissue. Today, prophylactic treatment includes prevention of fibrin deposition, reduction of inflammation (steroidal and non-steroidal anti-inflammatory drugs) and removal of fibrin deposition.

Interventional measures to prevent post-operative adhesion formation include the use of water flotation (hydroplotation) techniques or barrier devices. The water-floating method involves instillation of large volumes of polymer solutions such as dextran (adhesion research group, Fertil. Steril.40: 612-619(1983)) or carboxymethyl cellulose (Elkins, T.E., et al, Fertil. Steril.41: 926-928(1984)) into the surgical space to keep the organs separated. Regeneration of cellulose by oxidation (interceded)^TM) Synthetic barrier membranes, polytetrafluoroethylene (Gore-tex surgical membrane) and modified hyaluronic acid/carboxymethylcellulose (HA/CMC) compositions (Seprafilm) were prepared^TM) The prepared fully resorbable films are also used to reduce the formation of post-operative adhesions in animals and humans. Burns, j.w. et al, eur.j.surg.suppl.577: 40-48 (1997); burns, j.w., et al, fertil. steril.66: 814- & ltSUB & gt 821 (1996); becker, j.m., et al, j.am.col.surg.183: 297-306(1996). The success of these HA/CMC membranes may be due to their ability to separate tissues during peritoneal wound repair, when adhesions form. It was found that 3-5 days after application (this period of time matched the time course of post-operative adhesion formation) the film formed a clear, adherent thin layer on the injured tissue. Ellis, h., br.j.surg.50: 10-16(1963). Intraperitoneal administration of an anti-inflammatory agent such as dexamethasone or a corticosteroid produced marginal inhibition of adhesion formation. diZerega, g.s.fertil.steril.61: 219, 235 (1994); hocker, m.ann.chip.gynecol.76: 306-313(1987).

Arthritis (arthritis)

Arthritis (e.g. rheumatoid arthritis, RA) is a debilitating chronic inflammatory disease that affects almost 2% of the world population. The symptoms are characterized by pain, swelling, synovial cell proliferation (pannus formation), angiogenesis and destruction of joint tissue. In advanced stages, the disease often damages critical organs and can be fatal. The disease involves multiple members of the immune system (macrophages/monocytes, neutrophils, B cells and T cells), complex cytokine interactions and synovial cell dysfunction and proliferation. Early active treatment with disease-modifying antirheumatic drugs (DMARDS) such as methotrexate in combination with cyclosporine or azathioprine is now recommended. Arthritis and rheumatism, 39 (5): 713-722(1996).

Crystal-induced arthritis affects approximately 1% of the population and is characterized by crystal-induced macrophage and neutrophil activation in the joints, followed by severe pain for many days. The progression of the disease results in shorter intervals of labor and an unacceptable increase in patient morbidity. NSAIDs are commonly used to treat the symptoms of the disease. A more detailed discussion of the pathophysiology of this and other forms of inflammatory Arthritis is found in Arthritis and related diseases written by McCarty et al (Arthritis and AlliedConsortions), Lea and Febiger, Philadelphia 1495 (1985).

Psoriasis vulgaris

Psoriasis is a common chronic inflammatory skin disease characterized by elevated, thickened, scaly lesions that are prone to itching, burning, stinging and bleeding. More than 2% of americans suffer from psoriasis and patients often suffer from concomitant arthritic symptoms. The etiology of the disease is unknown and today there is no cure for the disease. Evidence supports that it is an autoimmune disease. Other features of the disease are neutrophil activation, cell proliferation and angiogenesis.

Skin cells can follow two growth pathways: normal growth and wound healing. In normal growth, cells are produced in the basal layer and move up through the epidermis to the skin surface. Dead cells are shed from the surface at the same rate as the next novacells. During wound healing, accelerated growth and repair are initiated, leading to rapid renewal of skin cells, increased blood supply and inflammation. In some aspects, psoriasis is an exaggerated wound healing process. If the skin sheds skin cells (keratinocytes) less rapidly than those cells do, buildup will occur. This can lead to scale damage and angiogenesis (to increase blood supply). Meanwhile, lymphocytes, neutrophils and macrophages can cause pain, swelling and inflammation. Current drug therapy includes the use of steroidal and non-steroidal anti-inflammatory agents to treat inflammatory conditions. Methotrexate and cyclosporin, which have marginal efficacy, are also used. The cost of psoriasis treatment today in the united states is over 30 billion dollars per year.

General discussion

The present invention provides fucoidan (including derivatives and analogs thereof) for treating or preventing surgical adhesions, rheumatoid arthritis, and psoriasis (where treatment as used herein includes treatment of existing diseases and inhibition of underlying diseases). As illustrated in the examples below, fucoidan (particularly fucoidan) inhibits cell proliferation, inflammatory responses/events and angiogenesis including, for example, in surgical adhesions.

In one embodiment, fucoidan (e.g., fucoidan) is used to inhibit or prevent angiogenesis. In another embodiment, fucoidan (e.g., fucoidan) is used to inhibit or prevent inflammatory cell activation, such that cells that elicit an inflammatory response at the site of disease are inhibited. This is important, for example, because many diseases such as osteoarthritis are not necessarily associated with the accumulation of inflammatory cells at the site of the disease. Thus, the use of fucoidan can inhibit or prevent long-term activation of resident macrophages, neutrophils, and other inflammation-causing cells, which leads to the chronic deleterious effects of the disease. In this context, the activity of these fucoidans is applied to surgical adhesions, arthritis and psoriasis.

In one embodiment of the invention, fucoidan (including derivatives and analogs thereof) can be formulated in a controlled release formulation to provide a sustained effective concentration of the drug at the site of disease. In another embodiment, the fucoidan is used to treat surgical adhesions. Embodiments are provided herein. These examples demonstrate the inhibitory effect of fucoidan against primary chondrocytes from neonatal cartilage (cells involved in rheumatoid arthritis). This suggests that the drug may be useful as an anti-arthritic. In particular, the ability of fucoidan to significantly inhibit the production of collagenase and stromelysin provides a therapeutic approach when the release of collagenase and stromelysin and/or other metalloproteinases leads to medical problems.

In other embodiments, fucoidan and other therapeutic agents may be used in combination to obtain better efficacy and low toxicity against the disease process. For example, in the treatment of surgical adhesions, strong antiproliferative drugs (such as doxorubicin, camptothecin, etoposide, mitoxantrone, methotrexate, menadione, plumbagin, juglone, β -lapachone, cyclosporine, sulfasalazine, steroids, rapamycin, retinoids, paclitaxel, docetaxel, colchicine, and other microtubule inhibitors) and their other analogs and derivatives may have deleterious toxicity at drug concentrations required to inhibit the adhesion process in the absence of fucoidan, but they may be combined with fucoidan (such as fucoidan) at low concentrations to achieve the desired effect.

In another embodiment it is proposed that the fucan itself may be in the form of a medicament. For example, fucoidan can be made into a thin film that can be placed directly on the surface of a surgical wound area, so that the slow dissolution of fucoidan exposes the tissue to sustained and effective drug concentrations. In practice, the formulation may act as a controlled release drug delivery system for other drugs (such as paclitaxel) either by themselves (as the active agent) or may be placed in the formulation. The fucoidan can also be formulated into tablet, capsule, microsphere, paste, gel, powder, aerosol, or administered orally or rectally as solid or solution.

Generally, the fucan can be administered alone or as part of a composition, and can be applied or injected as a paste, gel, spray, microgranule, film, solution, liquid, lotion, cream, or implant. Routes of administration and sites of administration include oral administration, systemic administration, intraocular, subcutaneous, intraperitoneal, intramuscular, intraarticular, intralesional, intravaginal, rectal, or topical administration (e.g., in a patch). In some cases, these pathways may also be sites of action for suggested formulations of fucoidan or fucoidan pharmaceutical compositions. The therapeutically effective amount of fucan can be delivered as part of the composition and can comprise about 5% to 50%, 20-80%, 80% -100% (w/v) of the composition. The fucan can be provided in a suitable container, which can then be provided in a kit and provided with a label, preferably approved by a corresponding governmental regulatory agency, such as the U.S. food and drug administration.

For the treatment of adhesions, the fucan or fucan-containing composition can be applied directly to the disease or surgical site as a solution, microgranule, suspension, film, paste, gel, spray, liquid formulation, lotion, implant, or other desired form. Adhesions can also be treated by systemic delivery of fucoidan by intravenous, subcutaneous, intramuscular, intraperitoneal, oral, or other desired routes of administration. For the treatment of arthritis, the fucan or the fucan-containing composition can be injected directly into the joint as a paste, gel, spray, liquid formulation, lotion, solution, suspension or other desired form. The fucoidan can be delivered systemically by intravenous, intramuscular, intraperitoneal, subcutaneous, or oral routes of administration to treat or prevent arthritis.

In some aspects, the invention provides for treating angiogenic diseases of the eye. For example, diabetic retinopathy is a potentially blinding complication of diabetes mellitus, which destroys blood vessels in the retina and then grows new blood vessels (angiogenesis) resulting in blurred vision or retinal destruction. Macular degeneration, caused by the invasion of new blood vessels beneath the retina, is the leading cause of blindness in the United states and Europe, with 200,000 new cases per year in the United states, of which only 15% are treatable with current laser therapy. In some embodiments, the invention provides pharmaceutical routes for treating these diseases using the methods and compositions discussed herein that are suitable for use in the eye. For example, the fucan can be applied directly to the surface of the eye or injected into the eye. Improvements in these systems include chemical crosslinking to slow the rate of dissolution of the dosage form or mixing with other excipients such as pluronic, alginates, acrylates, cellulose, hyaluronic acid, polyethylene glycol, chitosan (including their analogs and derivatives) and a variety of other pharmaceutically acceptable formulations.

In yet another embodiment, the fucan and a positively charged excipient (e.g., chitosan or poly-l-lysine) form a charged hydrogel. Drugs (e.g., antisense oligonucleotides, ribozymes, and oligonucleotide RNA inhibitors) can be incorporated into such gels for application to the disease site. Alternatively, such a gel containing the drug or the drug dissolved in the fucan solution may be dried and ground into particles. These particles can be applied to the disease site as a controlled release formulation, or the particles can act as a transfection agent, as the surface of the bound fucan is taken up into the cell. The use of these particles is further facilitated by the use of excipients such as pluronic, cellulose, alginates, acrylates, hyaluronic acid, polyethylene glycol, chitosan, injectable excipients (including their analogs and derivatives) and various polymer-based carriers, which are pharmaceutically acceptable excipients.

For transfection and use of fucoidan drugs containing nucleic acid sequences, the drug frontier known as gene therapy is limited by drug delivery problems, since cellular uptake of gene fragments or nucleic acid strands, such as oligonucleotides (including ribozymes, antisense nucleotides, and oligonucleotide RNA inhibitors), is inhibited due to the charge and large molecular weight of these compounds. Recently, it has been proposed to use microparticles containing genes or nucleic acids (e.g., calcium phosphate) as transfection agents so that they bind to the cell surface and are taken up by endocytosis or endocytosis, resulting in entry of the genes or nucleic acids into the cell. Most cells contain fucose receptors on the membrane surface. The invention provides application of fucosan as a nucleic acid chain transfection agent. In one embodiment, the nucleic acid strands may be incorporated or encapsulated in the fucoidan microparticle agent, and the particles may be chemically cross-linked to inhibit their dissolution prior to application to the target cellular site.

The fucoidan, either alone or in combination with other drugs, can be combined with other materials implanted in the body. These materials include, but are not limited to, various medical devices such as catheters, shunts, membranes, stents, sponges, fillers, artificial replacement joints and portions thereof, and other orthopedic related implants. These implants may contain or be coated with fucoidan, which may be used alone or in combination with other drugs and excipients.

Unless otherwise indicated, except in the claims, the use of "or" includes "and vice versa. Non-limiting terms are not to be construed as limiting unless otherwise indicated herein or otherwise clearly contradicted by context. (e.g., "including," "having," and "containing" generally mean "including, but not limited to,") unless otherwise indicated or clearly contradicted by context, the singular forms (including in the claims) such as "a," "an," and "the" include plural referents.

The scope of the present systems and methods, etc., includes both method-plus-function and step-plus-function concepts. However, if the term "method" is not specifically recited in the claims, the term set forth in this application should not be construed as a relationship representing "method plus function" in the claims, and when the term "method" is specifically recited in the claims, the terms should be construed as a relationship representing "method plus function" in the claims. Similarly, if the term "step" is not specifically recited in the claims, then that term as set forth in this application should not be construed in the claims as a relationship representing "step plus function" in the method claims, and when the term "step" is specifically recited in the claims, then that term should be construed in the claims as a relationship representing "step plus function".

Examples

Example 1: effect of fucoidan on in vitro proliferation of synoviocytes and smooth muscle cells

Proliferation was measured using a dimethylthiazoldiphenyltetrazolium bromide (MTT) proliferation/cytotoxicity assay.

The first day, 1500-. Place the plate at 37 ℃ CO₂An incubator. The following day fucoidan was added at various concentrations. The first column (blank) and the second column (untreated column) were used without fucoidan for control. The cells were exposed for 48 hours. In the late phase of the exposure period, 50. mu.l of dimethyl thiazole diphenyl tetrazole bromide salt (MTT) in medium was added and incubated at 37 ℃ for 4 hours. Then, the medium was aspirated and 200. mu.l of dimethyl sulfoxide (DMSO) was added. The plate was shaken for 30 minutes and the absorbance read at 562 nm. The measured optical density was converted to cell number using a standard optical density map of known cell number and the viability of the cells was expressed as% growth (% of this value compared to control cells).

As shown in figure 1, fucoidan induced concentration-dependent inhibition of cell proliferation in both synovial cells and smooth muscle cells after 48 hours of exposure. Inhibitory concentrations (IC50) with 50% effect on proliferation were 15. mu.M and 6. mu.M, respectively.

Example 2: effect of fucoidan on Phorbol Myristate (PMA) -induced neutrophil chemiluminescence

In this experiment freshly prepared human neutrophils were incubated with 0.5% (w/v) fucoidan before stimulating the cells with PMA. Stimulation (or activation) of the cells results in the production of superoxide anions, which can be measured by light emission (chemiluminescence). Thus, inhibition of neutrophil function was measured by inhibition of chemiluminescence. Hanks Buffered Saline Solution (HBSS) at ph7.4 was used throughout the study. Unless otherwise noted, all chemicals were purchased from Sigma Chemical Co, (st. All experiments were performed at 37 ℃. Neutrophils were prepared from freshly collected human citrate buffered whole blood. Briefly, 400ml of blood was mixed with 80ml of 4% dextran T500(Pharmacia LKB, Biotechnology AB Uppsala, Sweden) dissolved in HBSS and left for 1 hour. Plasma was collected continuously and 5ml was added to 5ml Ficoll Paque (Pharmacia) in a 15ml polypropylene tube (Corning, NY). After centrifugation at 500 Xg for 30 minutes, the neutrophil pellet was washed and freed from erythrocytes by 20 seconds of hypotonic shock. Neutrophils were resuspended in HBSS, kept on ice and used for experiments within 3 hours. The viability and purity of neutrophils is always greater than 90%.

PMA was added after incubating the cells with fucoidan at various concentrations for 15 minutes at 37 ℃.

At a cell concentration of 5X 10 at 0.5. mu.M PMA⁶Chemiluminescence studies were performed in individual cells/ml HBSS. To the tube was added 10. mu.L of luminol in 25% DMSO in HBSS to a final concentration of 1mM, and the sample was mixed to initiate neutrophil activation. Chemiluminescence was detected at 37 ℃ with an LKB luminometer (Model 1250) with shaking immediately prior to measurement. Control tubes contained cells, fucoidan, and luminol.

As shown in figure 2, fucoidan strongly inhibited PMA-induced activation of neutrophils. Data were from three independent PMA-neutrophil incubations. These data demonstrate the anti-inflammatory effect of fucoidan.

Example 3: effect of fucoidan on IL-1 induced expression of collagenase and stromelysin genes in chondrocytes

In this assay, the RNA levels of two metalloproteinases, collagenase and stromelysin, are measured. Overexpression of these genes results in the two enzymes being attenuated from articular cartilageSecreted in the cell and the overexpression of these two genes may represent part of the pathophysiology of rheumatoid arthritis. Drugs that inhibit the overexpression of collagenase and stromelysin are potential anti-arthritic drugs. If the drug also significantly inhibits proteoglycan gene expression, its anti-arthritic potential is reduced. Proteoglycan gene expression is part of the normal physiology of chondrocytes. The primary chondrocyte culture was freshly isolated from calf cartilage. Cells were plated in 100X 20mm dishes (2.5X 10)⁶Ml) and incubated overnight at 37 ℃ in Ham's F12 medium containing 5% Fetal Bovine Serum (FBS). Cells were starved overnight with serum-free medium. With a concentration of 10^-6M、10^-7M and 10^-8M camptothecin pre-treated cells for 6 hours. IL-1(20ng/ml) was then added to each plate and incubation of the plates was continued for 18 hours. Total RNA was isolated by the acidified guanidinium isothiocyanate method and electrophoresed on a denaturing gel. Denatured RNA samples (15. mu.g) were analyzed by electrophoresis in 1% denatured gel, transferred to nylon membranes, and separately applied³²A P-labeled collagenase cDNA probe,³²P-labeled stromelysin cDNA probe,³²P-labeled proteoglycan cDNA probes and³²p-labeled glyceraldehyde phosphate dehydrogenase (PAGDH) cDNA. PAGDH levels serve as an internal standard to ensure substantially equal loading. The results of the experiment on the X-ray film were scanned and analyzed by HP ScanJet.

As shown in fig. 3, fucoidan completely inhibited the expression of collagenase and stromelysin at a concentration of 0.5% (w/v) without excessive inhibition of proteoglycan expression. At a concentration of 0.1% (w/v), there was a strong inhibition of collagenase and stromelysin expression without any inhibition of proteoglycan expression. These results demonstrate the anti-inflammatory effect of fucoidan.

Example 4: effect of fucoidan on angiogenesis in the chorioallantoic membrane of chick embryos (CAM assay)

Fertilized eggs were obtained from a local hatchery and placed in an incubator with an automatic rotator and left to shell or open after 3.5 days at 37 ℃.

Several sheets of sterile wax paper were placed over the window created in the air chamber to prevent contamination and dehydration of the egg contents. These 4cm x 4cm sheets were sterilized by spraying them with 70% ethanol and dried on a laminar flow hood hyperstatic table. After 3 days the eggs were manually rotated in the incubator so that their tips were facing upwards for 5-10 minutes to detach the egg contents from the inner membranes. The whole egg shell was swept with 70% ethanol and a Kimwipe to clean and disinfect the outside of the egg. In the laminar flow hood hyperstatic station, the egg is forced up at its blunt end and a hole is made in the egg's blunt end by carefully breaking the shell through the end of the tweezers. The eggshell residue was gently removed with forceps to form a hole at the blunt end. The circular hole has a diameter of 2 to 3cm and does not damage the inner membrane. Once the hole was created in the shell, the inner egg shell membrane (which contained the egg contents) was gently torn off and removed with forceps, taking care not to destroy the chorioallantoic membrane (CAM) (which contained the egg yolk and developed the chick embryo).

The wells were then covered with sterile parafilm wax paper by gently stretching the parafilm and placing it around the wells. The eggs were then placed on egg racks in an incubator (37 ℃) and placed in a manner that prevented the eggs from turning. After 6 days each egg was removed one by one from the incubator (blunt side up) and the parafilm covering the window was removed to directly contact the CAM produced from the hindgut of the embryo. Fucoidan-loaded PCL pellets were prepared by melting poly (e-caprolactone) (PCL) at 60 ℃ and physically mixing the fucoidan into the PCL and hardening the pellets by cooling to room temperature. Fucoidan pellets were placed on a growing CAM capillary bed. The egg contents were then resealed with parafilm and placed back in the 37 ℃ incubator. After 2 days, the analysis of the CAM vasculature was recorded (48 hours after placing the drug on the CAM capillary bed). The effect of the drug on the CAM was evaluated on a avascular scale that classified the drug effect as a 0, 1, 2 or 3 scale. The assignment of the avascular grade is described as follows:

0 has no anti-angiogenic activity

1 micropipe reduction

2 small avascular zone of drug pellet size (2 mm diameter)

3 avascular zone of 4-5mm diameter

As shown in table 1, fucoidan strongly inhibited angiogenesis in the CAM. Low concentrations of 2% W/W fucoidan in the PCL inhibited angiogenesis in 4 CAM fractions or in 2 CAM fractions, respectively.

Table 1: anti-angiogenic activity of fucoidan. The numbers in each column show the number of eggs that do not, partially or maximally inhibit angiogenesis (CAM number).

Drug concentration	Anti-angiogenic activity
					Wu (0)	Moiety (1-2)	Maximum (3)
Fucoidan 2.0%	-	4	2
				Fucoidan 5.0%	-	1	4
Fucoidan 15.0%	-	2	3
				Fucoidan 30.0%	-	2	1
Control	11	-	-

These data demonstrate the anti-angiogenic activity of fucoidan and indicate that the polymeric sustained release formulation of fucoidan is an effective method of delivering therapeutically effective concentrations of drug without causing undue toxicity.

Example 5: encapsulation of fucoidan in ethylene vinyl acetate film and polycaprolactone paste

5mg of fucoidan (Sigma) and 45mg of ethylene vinyl acetate (EVA, molecular weight about 50k, Polysciences) were dissolved/suspended in 1ml of dichloromethane. Pipette 200. mu.l of the solution onto a 1cm diameter Teflon disc and dry overnight (solvent evaporated) to form an elastic film to give about 10mg film thickness of about 100. mu.m.

A portion of 5mg of the films were placed in 20ml glass tubes with a lid containing 10ml of phosphate buffer (PHS, pH 7.4) to determine the release rate of the drug from these films. The tube was covered and placed in an orbital shaker (37 ℃). The tubes were removed at specific times and analyzed for the amount of drug released by absorption spectroscopy. The release profile of fucoidan (figure 4) is characterized by an initial burst of drug release followed by a slow sustained release. This dosage form of fucoidan represents a biocompatible, biodegradable, injectable pharmaceutical formulation that releases the drug in a controlled manner.

PCL paste: fucoidan was mixed into polycaprolactone (PCL, Birmingham polymers, molecular weight 54K) by spatula milling at 60 deg.C to give a concentration of 10% (w/w). The mixture was taken up in a 1ml plastic syringe and cooled. The formulation can be injected through an 18 gauge needle at 56 ℃.

To measure drug release from the PCL paste, a 10mg aliquot of the molten paste was injected into the bottom of a 15ml glass tube, cooled and fixed. To each tube add 15ml PBS and the tube is covered, and in 37 degrees C oven top and bottom. The tubes were removed at specific times and analyzed for the amount of drug released by absorption spectroscopy. The release profile of fucoidan is shown in FIG. 5. The release profile of fucoidan is an initial burst of drug release followed by a slow sustained release. This dosage form of fucoidan represents a biocompatible, biodegradable, injectable pharmaceutical formulation that releases the drug in a controlled manner.

Example 6: film agent loaded with fucoidan for treating rat surgical adhesion

The effect of fucoidan on surgical adhesion was studied using a rat caecum lateral wall model of surgical adhesion. In this model, 16 rats were divided into two groups of 8 rats each. Immediately after surgical trauma, rats were treated with or without fucoidan-containing cross-linked Hyaluronic Acid (HA) membrane (control).

Materials and methods pharmaceutical grade sodium hyaluronate was obtained from Lifecore Scientific. All solvents were HPLC grade and obtained from Fisher. Plastic petri dishes were obtained from Fisher Scientific. Ethyl-3- (dimethylamino) carbodiimide (EDAC) and fucoidan were obtained from Sigma (st.

Preparation of film preparation a film loaded with fucoidan was prepared by preparing an aqueous solution of 0.6% w/v fucoidan, 0.4% w/v sodium hyaluronate and 0.15% w/v glycerol. Control films (without fucoidan) were prepared by preparing a solution or mixture of 0.4% w/v sodium hyaluronate and 0.15% w/v glycerin in water. Each solution was pipetted 4g into separate plastic petri dishes with a diameter of 2.5cm and dried at 60 ℃ for 24 hours to give fucoidan-loaded and control films. The crosslinker EDAC was contained at 4mM (final concentration). Each dried film was then carefully removed from the petri dish with a scalpel.

Sterilization the films were wrapped in 5cm by 5cm weighing paper (Fisher scientific) and heat sealed in plastic wrap. The film was then finally sterilized with gamma irradiation from a cobalt-60 radioactive source and exposed to 2.5Mrad of radiation and the sealed tube was cooled on ice.

Animal studies surgical trauma induction was as follows: 16 mature Sprague Dawley rats (each weighing 350g of 225-. Only animals that were generally normal (i.e., clean and wrinkle-free coat, bright and clear eyes, active posture) were used for the study. Animals were randomly assigned to one of the two groups, weighed and anesthetized with a single injection of ketamine hydrochloride (6mg/kg) in the large muscle of the thigh. The abdomen was shaved and washed with alcohol. A4 cm incision was made in the skin starting at about 2cm from the tail of the white line, while the muscles were held with forceps. The cecum is rubbed 4 times over the abdominal and dorsal surfaces with a mechanical rubbing device that allows the operator to independently and controllably rub over the fixation area. The adhesion of the cecum is estimated and scored according to a predefined scoring system:

0 ═ no adhesion

Thin adhesions with easily identifiable planes

Slight sticking with freely separable flat surface

Moderate blocking with difficult to separate planes

4-tight adhesion with indivisible planes

(class 1 adhesions are the lowest level of identifiable adhesions (thin adhesions with identifiable flat surfaces)).

After cecal rubbing, animals in group 1 received no treatment. Animals in group 2 received the fucoidan-HA film discussed above. The film was wrapped around the cecum. The incision was then sutured with 3.0Dexon suture. On day 7 post-surgery, animals were euthanized and evaluated for the presence of grade 2 (or higher) post-operative adhesions. Grade 2 blocking is defined as slight blocking with a freely separable plane.

As a result:

table 2.

Group of	The adhesion is more than or equal to 2 percent	Mean incidence + -SEM	No adhesion%
				Control	75	1.4±0.4	25
Film agent loaded with fucoidan	38	0.5±0.4	50

The membrane only surrounds about half of the cecum

No abnormality was found at necropsy (no residual substance, no ascites, no abnormal healing on the cecum or on the midline of the incision)

The results demonstrate that the fucoidan-loaded film is effective in inhibiting the formation of adhesions because the average incidence of adhesions is reduced and the percentage of non-adherent rats is increased in the rats treated with fucoidan. A film loaded with fucoidan that completely surrounds the cecum may be more effective in inhibiting adhesion formation.

From the foregoing, it will be appreciated that, although specific embodiments have been discussed herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the disclosure. Accordingly, the systems and methods, etc., which include such modifications and all permutations and combinations of the subject matter, are not to be limited except by the appended claims.

Claims (17)

1. Use of fucoidan for the preparation of a medicament for treating surgical adhesions at a site of an animal having an underlying disease.

2. The use of claim 1, wherein the disease site is a surgical site and the medicament is prepared as a composition for direct delivery to the disease site.

3. Use according to claim 1, wherein the medicament is formulated for oral administration.

4. The use according to claim 1, wherein the medicament is prepared in the form of an injection.

5. The use of claim 1, wherein the medicament is formulated for intraocular, subcutaneous, intraperitoneal, intramuscular, intraarticular, intralesional, intravaginal, rectal, or topical administration.

6. Use according to claim 1, wherein the fucan is fucoidan.

7. Use according to claim 1, wherein the medicament is formulated as a controlled release polymer dosage form.

8. The use of claim 7, wherein said polymeric dosage form comprises a film, patch, paste, microsphere, implant, gel, spray or liquid formulation.

9. Use according to claim 1, wherein the medicament is in the form of a pharmaceutical composition comprising at least one cream, a paste, an injectable excipient and a polymer.

10. Use according to claim 1, wherein the fucan is delivered as part of a pharmaceutical composition and the fucan comprises from 0.1% to 35% w/v of the pharmaceutical composition.

11. Use according to claim 1, wherein the fucan is delivered as part of a pharmaceutical composition and the fucan comprises 80% to 100% w/v of the pharmaceutical composition.

12. Use according to claim 1, wherein the fucan is delivered as part of a pharmaceutical composition and the fucan comprises from 5% to 50% w/v of the pharmaceutical composition.

13. Use according to claim 1, wherein the fucan is delivered as part of a pharmaceutical composition and the fucan comprises from 20% to 80% w/v of the pharmaceutical composition.

14. The use according to claim 1, wherein the fucan is part of a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.

15. The use of claim 14, wherein the pharmaceutically acceptable excipient is selected from the group consisting of pluronic, cellulose, alginates, acrylates, hyaluronic acid, polyethylene glycol, and chitosan.

16. The use according to claim 1, wherein the fucan is prepared for direct delivery to the site of disease.

17. The use according to claim 1, wherein the animal is a human.