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WO2003092672A1 - Procede d'inhibition du rejet de matiere transplantee - Google Patents

Procede d'inhibition du rejet de matiere transplantee Download PDF

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Publication number
WO2003092672A1
WO2003092672A1 PCT/AU2003/000523 AU0300523W WO03092672A1 WO 2003092672 A1 WO2003092672 A1 WO 2003092672A1 AU 0300523 W AU0300523 W AU 0300523W WO 03092672 A1 WO03092672 A1 WO 03092672A1
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Prior art keywords
acid
alkyl
substituted fatty
fatty acid
group
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PCT/AU2003/000523
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English (en)
Inventor
Ravi Krishnan
Peter D. Collin
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Queen Elizabeth Hospital Research Foundation Inc
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Queen Elizabeth Hospital Research Foundation Inc
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Filing date
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Priority claimed from AUPS2124A external-priority patent/AUPS212402A0/en
Priority claimed from AU2003900961A external-priority patent/AU2003900961A0/en
Application filed by Queen Elizabeth Hospital Research Foundation Inc filed Critical Queen Elizabeth Hospital Research Foundation Inc
Priority to AU2003227120A priority Critical patent/AU2003227120A1/en
Publication of WO2003092672A1 publication Critical patent/WO2003092672A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the present invention relates to methods and compositions for inhibiting the rejection of transplanted material.
  • Transplantation is the introduction of foreign material into a recipient organism so as to treat conditions involving the loss of some biological function in the recipient organism.
  • transplantation is considered to be the introduction of biological material such as organs, tissues, cells and biological fluids into a recipient organism.
  • biological material such as organs, tissues, cells and biological fluids into a recipient organism.
  • non-biological materials may also be transplanted to treat conditions involving the loss of some important biological function in the recipient organism.
  • biological material For the transplantation of biological material, there are three types of biological material that may be transplanted into a recipient: (i) syngeneic transplants involving transplantation of biological material between isogenic recipients; (ii) allogeneic transplants involving the transplantation of biological material from one organism to a recipient organism of the same species; and (iii) xenogeneic transplants involving the transplantation of biological material from different species. Allogeneic transplants in particular are of considerable medical importance, especially for conditions involving irreversible organ failure in a subject.
  • Allogeneic and xenogeneic transplants are almost always destroyed by immunological processes unless some preventive action is taken to impair the immunological process.
  • the main barrier to allogeneic and xenogeneic transplantation is acute rejection of the transplanted material.
  • Rejection of transplanted material involves the coordinated activation and proliferation of alloreactive T lymphocytes and antigen-presenting cells such as monocyte-macrophages, dendritic cells, and B cells.
  • a broad array of effector mechanisms participate in the destruction of the transplanted material. Through the release of cytokines and cell-to-cell interactions, a diverse assembly of lymphocytes and other proinflammatory leukocytes participate in the rejection of the transplanted material.
  • lymphocytes In the case of acute rejection of allogeneic transplants (allograft rejection), the main effectors of rejection are lymphocytes rather than antibodies.
  • the activation and proliferation of lymphocytes are key steps in the mechanism of allograft rejection and the mechanism involving the rejection of xenogeneic transplants.
  • the ability of an agent to inhibit lymphocyte activation and/or proliferation in vitro is a powerful indicator that the agent may have efficacy for the inhibition of rejection of transplanted biological material.
  • immunosuppressive drugs such as cyclosporin A, FK506 and rapamycin are used to inhibit rejection.
  • cytotoxic drugs including azathioprine, cyclophosphamide and methotrexate.
  • Steroids such as prednisolone, and monoclonal antibodies such as anti-CD3 and anti-CD25 monoclonal antibodies, may also be used to inhibit rejection.
  • immunosuppressive and/or cytotoxic drugs form the standard regime for inhibiting the rejection of transplanted material. It appears that these agents may all function in some way to prevent rejection by preventing lymphocyte activation, proliferation and/or cytokine production.
  • graft versus host disease GVHD
  • Donor lymphocytes proliferate and become activated in response to antigenic differences in the recipient's molecules.
  • GVHD may also be prevented by the use of immunosuppressive drugs.
  • the present invention relates to the identification of a class of agents that inhibit the rejection of transplanted material.
  • the present invention relates to methods of inhibiting the rejection of transplanted material and pharmaceutical compositions suitable for inhibiting the rejection of transplanted biological material.
  • the present invention provides a method of inhibiting leukocyte proliferation in a biological system, the method including the step of administering to the biological system an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting leukocyte proliferation and the alkyl-substituted fatty acid has the following chemical formula: R
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the present invention also provides a method of inhibiting leukocyte activation in a biological system, the method including the step of administering to the biological system an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting leukocyte activation and the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the present invention also provides a method of reducing the amount of an agent administered to a biological system to achieve a desired level of inhibition of leukocyte proliferation, the method including the step of administering to the biological system an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the present invention also provides a method of reducing the amount of an agent administered to a biological system to achieve a desired level of inhibition of leukocyte activation, the method including the step of administering to the biological system an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the present invention also provides a method of inhibiting rejection of transplanted material in a subject, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting rejection of transplanted material and the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the present invention further provides a method of inhibiting rejection of a corneal transplant in a subject, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting rejection of a corneal transplant and the alkyl-substituted fatty acid has the following chemical formula:
  • the present invention also provides a method of reducing the amount of an agent administered to a subject to achieve a desired level of inhibition of rejection of transplanted material, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • the present invention also provides a method of inhibiting graft versus host disease in a subject, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl- substituted fatty acid is capable of inhibiting graft versus host disease and the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the present invention also provides a method of reducing the amount of an agent administered to a subject to achieve a desired level of inhibition of graft versus host disease, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl- substituted fatty acid has the following chemical formula: R
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the present invention also provides a method of down regulating the expression of a cell adhesion molecule on a leukocyte, the method including the step of administering to the leukocyte an effective amount of an alkyl- substituted fatty acid, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the present invention also provides a method of down regulating the cell surface expression of a molecule on a dendritic cell involved in T-lymphocyte stimulation, the method including the step of administering to the dendritic cell an effective amount of an alkyl-substituted fatty acid, wherein the alkyl- substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the present invention also provides a method for inhibiting the maturation of a dendritic cell, the method including the step of administering to the dendritic cell an effective amount of an alkyl-substituted fatty acid, wherein the alkyl- substituted fatty acid has the following chemical formula:
  • the present invention also provides a method of inhibiting proliferation and/or stimulation of a lymphocyte mediated by a dendritic cell, the method including the step of administering to the dendritic cell an effective amount of an alkyl- substituted fatty acid, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the present invention further provides a pharmaceutical composition including an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting leukocyte proliferation and/or rejection of transplanted biological material and the alkyl-substituted fatty acid has the following chemical formula:
  • the present invention also provides a pharmaceutical composition including an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting rejection of a corneal transplant and the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the present invention also provides a pharmaceutical composition including an alkyl-substituted fatty acid and immunosuppressant, wherein the alkyl- substituted fatty acid has the following chemical formula: R
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the present invention arises out of studies into the ability of alkyl-substituted fatty acids to inhibit the activation and proliferation of leukocytes in an in vitro rejection assay.
  • the applicant has surprisingly found that alkyl- substituted fatty acids have the capacity to inhibit the activation and proliferation of leukocytes in an in vitro rejection assay.
  • the alkyl-substituted fatty acid 12-methyltetradecanoic inhibits the rejection of a donor heart transplanted heterotopically into a mouse.
  • alkyl-substituted fatty acid as used throughout the specification is to be understood to mean any branched fatty acid that may be described by the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms.
  • x is equal to or greater than 0, y is equal to or greater than 0, and x + y is between 0 and 46.
  • x or y is equal to or greater than 2, at least one CH 2 -CH 2 group in (CH 2 ) X and/or
  • alkyl-substituted fatty acid includes in its scope any salts of the carboxylic acid, or any derivatives of the compounds according to the above chemical formula that are functionally equivalent to the compounds in terms of their ability to inhibit leukocyte proliferation and/or activation, or inhibit rejection.
  • inhibitor as used throughout the specification is to be understood to mean a reduction in the progress of a process, including the start, continuation or termination of a process. Such processes include for example the activation or proliferation of leukocytes or the rejection of transplanted biological material.
  • transplanted material is rendered dysfunctional by an immunological response.
  • the term also includes within its scope any steps in the process preceding the destruction of the transplanted material, including for example, the accumulation of leukocytes in the transplanted material before destruction of the transplanted material.
  • the term includes hyperacute rejection, acute rejection and chronic rejection of transplanted material.
  • transplanted material as used throughout the specification is to be understood to mean any material capable of transplantation into a recipient organism, such as any part of an organ, tissue, group of cells or other transplantable biological material, or any other non-biological material that when transplanted into a recipient organism " invokes an immune response to the transplanted material.
  • biological system as used throughout the specification is to be understood to mean any multi-cellular system and includes isolated groups of cells to whole organisms.
  • the biological system may be cells in culture, cells isolated from a subject, a tissue or organ, or an entire human or animal subject suffering the effects of rejection of transplanted material.
  • immunosuppressant as used throughout the specification is to be understood to mean any agent that can modify the immune response and/or surveillance, such that the response of immune cells towards alloantigens, autoantigens, xenoantigens or inflammatory mediators is reduced.
  • Figure 1 shows the ability of various concentrations of 12-MTA to inhibit a 2-way MLR and also shows the ability of 12-MTA to inhibit a 2-way MLR in combination with a sub-optimal concentration of cyclosporin A.
  • Figure 2 shows the ability of various concentrations of 16-MHA to inhibit a 2- way MLR and also shows the ability of 12-MHA to inhibit a 2-way MLR in combination with a sub-optimal concentration of cyclosporin A.
  • Figure 3 shows the ability of various concentrations of 12-MTA to inhibit a murine 2-way MLR and also shows the ability of 12-MTA to inhibit a murine 2- way MLR in combination with a sub-optimal concentration of cyclosporin A.
  • Figure 4 shows the ability of various alkyl-substituted fatty acids at 200 ⁇ M concentration to inhibit an allogeneic MLR.
  • Figure 5 shows the ability of various concentrations of alkyl-substituted fatty acids to inhibit mitogen induced lymphocyte proliferation.
  • Figure 6 shows the ability of 12-MTA to inhibit the proliferation of concanavalin- A stimulated lymphocyte proliferation.
  • Figure 7 shows dot plot analysis of the ability of 12-MTA and 16-MHA to inhibit CD4 and CD8 T cell proliferation.
  • Figure 8 shows the ability of 12-MTA and 16-MHA to inhibit the expression of dendritic cell maturation markers.
  • Figure 9 shows the ability of 12-MTA to down regulate the cell adhesion molecules LFA-1 and VLA-4.
  • Figure 10 shows the effect of 12-MTA and 16-MHA to reduce concanalavin-A mediated lymphocyte clustering.
  • the present invention provides a method of inhibiting leukocyte proliferation in a biological system, the method including the step of administering to the biological system an effective amount of an alkyl- substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting leukocyte proliferation and the alkyl-substituted fatty acid has the following chemical formula: R
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the leukocytes may be any leukocytes, including leukocytes that are undergoing proliferation in response to one or more antigenic stimuli on transplanted material, or leukocytes that have the capacity to undergo proliferation in response to one or more antigenic stimuli on transplanted material.
  • the leukocytes are animal or human leukocytes. Most preferably, the leukocytes are human leukocytes.
  • the leukocytes are undergoing (or capable of undergoing) proliferation in response to rejection of transplanted biological material, including hyperacute, acute or chronic rejection of allogeneic or xenogeneic transplanted biological material, or leukocytes undergoing (or capable of undergoing) proliferation in response to graft versus host disease. More preferably, the leukocytes are undergoing (or capable of undergoing) proliferation in response to allogeneic transplanted material. Most preferably, the leukocytes are undergoing (or capable of undergoing) proliferation in response to acute rejection of allogeneic transplanted biological material.
  • the leukocytes are lymphocytes or dendritic cells. Most preferably, the lymphocytes are T lymphocytes.
  • the biological system may be any system that includes leukocytes that have the capacity to proliferate.
  • the biological system is a human or animal subject that includes leukocytes that have the capacity to proliferate. More preferably, the biological system is a human or animal subject that includes the proliferation of leukocytes associated with the transplantation of foreign material. More preferably, the biological system is a human or animal subject that includes the proliferation of leukocytes associated with the transplantation of biological material. More preferably, the biological system is a human or animal subject suffering from the effects of rejection of allogeneic or xenogeneic transplanted biological material.
  • the biological system is a human or animal subject suffering from acute rejection of allogeneic transplanted biological material.
  • the transplanted biological material may be all or part of organs or tissues derived from skin, heart, lung, heart-lung, liver, kidney, cornea, blood, bone marrow, brain, spleen, pancreas, pancreatic islet, or stem-cells.
  • alkyl-substituted fatty acid in the various forms of the present invention is any branched fatty acid that may be described by the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms.
  • x is equal to or greater than 0, y is equal to or greater than 0, and x + y is between 0 and 46.
  • x or y is equal to or greater than 2, at least one CH 2 -CH 2 group in (CH 2 ) X and/or
  • the alkyl group (R) in the alkyl-substituted fatty acid is located on the first carbon atom directly adjacent to the terminal alkyl carbon atom, or on the second carbon removed from the terminal alkyl carbon atom.
  • the alkyl group in the alkyl-substituted fatty is a methyl or ethyl group. Most preferably, the alkyl group is a methyl group.
  • the alkyl-substituted fatty acid is a saturated alkyl-substituted fatty acid. More preferably, the saturated alkyl-substituted fatty acid is a derivative of undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, or eicosanoic acid. Most preferably, the saturated alkyl-substituted fatty acid is a derivative of tetradecanoic acid.
  • the saturated alkyl-substituted fatty acid is 18-methylnonadecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 16- methylheptadecanoic acid, 15-methylheptadecanoic acid, 15- methylhexadecanoic acid, 14-methylhexadecanoic acid, 14- methylpentadecanoic acid, 13-methylpentadecanoic acid, 13- methyltetradecanoic acid, 12-methyltetradecanoic acid, 12-methyltridecanoic acid, 11 -methyltridecanoic acid, 11 -methyldodecanoic acid, 10- methyldodecanoic acid, or any combination of these alkyl-substituted fatty acids.
  • the alkyl-substituted fatty acid is 12- methyltetradecanoic, 13-methyltetradecanoic acid, 17-methyloctadecanoic acid, 16-methylheptadecanoic acid, 10-methyloctadecanoic acid, 10- methyldodecanoic acid or any combination of these alkyl-substituted fatty acids.
  • the alkyl-substituted fatty acid is 12-methyltetradecanoic acid.
  • the unsaturated alkyl- substituted unsaturated fatty acids are preferably derivatives of undecenoic acid, dodecenoic acid, tridecenoic acid, tetradecenoic acid, pentadecenoic acid, hexadecenoic acid, heptadecenoic acid, octadecenoic acid, nonadecenoic acid or eicosenoic acid.
  • the amount of alkyl-substituted fatty acid to be administered in the various forms of the present invention is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits a pharmacologically useful or therapeutic effect.
  • an effective amount of alkyl-substituted fatty acid may be appropriately chosen, depending upon the extent of leukocyte proliferation or activation to be inhibited, the type of rejection occurring, the type of biological material transplanted, the age and body weight of the subject, and the frequency of administration.
  • the effective amount of alkyl-substituted fatty acid administered in the various forms of the invention is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl-substituted fatty acid administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the administration of alkyl-substituted fatty acid in the various forms of the present invention may be within any time suitable to produce the desired effect.
  • the administration may be within a suitable time to inhibit proliferation or activation of leukocytes at the site of transplantation.
  • the administration may also be within a suitable time to inhibit rejection of transplanted biological or non-biological material, including prior to the transplantation, prior to rejection occurring, and/or during the course of rejection or post rejection.
  • the administration of alkyl-substituted fatty acid to a subject may first occur at least one day prior to the transplant operation. Daily administration of the alkyl-substituted fatty acid to the subject may then continue for at least two to three weeks following the transplant.
  • the post-transplant administration period may vary from subject to subject and will depend upon the material being transplanted as well as other factors.
  • alkyl-substituted fatty acid in the various forms of the present invention may be administered under suitable means known in the art including administration orally, parenterally, topically or by any other suitable means, and therefore transit time of the drug must be taken into account.
  • alkyl-substituted fatty acid in the various forms of the present invention may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into consideration the particular physical and chemical characteristics of the alkyl-substituted fatty acid to be administered.
  • pharmaceutically acceptable additives including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into consideration the particular physical and chemical characteristics of the alkyl-substituted fatty acid to be administered.
  • the alkyl-substituted fatty acid can be prepared into a variety of pharmaceutical preparations in the form of, e.g., an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a gel, etc., and these preparations can be administered as intramuscular or subcutaneous injection or as injection to the organ, or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc.
  • the composition may be administered in the form of oral preparations (for example solid preparations such as tablets, capsules, granules or powders; liquid preparations such as syrup, emulsions or suspensions).
  • Compositions containing the alkyl- substituted fatty acid may also contain a preservative, stabiliser, dispersing agent, pH controller or isotonic agent.
  • suitable preservatives are glycerin, propylene glycol, phenol or benzyl alcohol.
  • suitable stabilisers are dextran, gelatin, -tocopherol acetate or alpha-thioglycerin.
  • suitable dispersing agents include polyoxyethylene (20), sorbitan mono-oleate (Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylene hydrogenated castor oil 60.
  • suitable pH controllers include hydrochloric acid, sodium hydroxide and the like.
  • suitable isotonic agents are glucose, D-sorbitol or D-mannitol.
  • alkyl-substituted fatty acid in the various forms of the invention may be in the form of a composition containing a pharmaceutically acceptable carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavorant or sweetener, taking into consideration the physical and chemical properties of the particular alkyl- substituted fatty acid.
  • composition may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrastemal, and intracranial injection or infusion techniques.
  • the composition When administered parenterally, the composition will normally be in a unit dosage, sterile injectable form (solution, suspension or emulsion) which is preferably isotonic with the blood of the recipient with a pharmaceutically acceptable carrier.
  • sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally- acceptable diluents or solvents, for example, as solutions in 1 ,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution.
  • sterile, fixed oils are conventionally employed as solvents or suspending mediums.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides, corn, cottonseed, peanut, and sesame oil.
  • Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables.
  • These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.
  • the carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example anti-oxidants, buffers and preservatives.
  • additives such as substances that enhance solubility, isotonicity, and chemical stability, for example anti-oxidants, buffers and preservatives.
  • the composition When administered orally, the composition will usually be formulated into unit dosage forms such as tablets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art.
  • Such formulations typically include a solid, semisolid, or liquid carrier.
  • Exemplary carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.
  • a tablet may be made by compressing or moulding the active ingredient optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free- flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.
  • the administration of alkyl-substituted fatty acid in the various forms of the present invention may also utilize controlled release technology.
  • the alkyl- substituted fatty acid may also be administered as a sustained-release pharmaceutical.
  • the composition may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty acid esters such as ethyl oleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof (weight average molecular weight: ca.
  • the alkyl-substituted fatty acid may be incorporated into a hydrophobic polymer matrix for controlled release over a period of days.
  • the composition of the invention may then be moulded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the alkyl-substituted fatty acid over a prolonged period of time without the need for frequent re-dosing.
  • Such controlled release films are well known to the art.
  • Other examples of polymers commonly employed for this purpose that may be used include nondegradable ethylene-vinyl acetate copolymer a degradable lactic acid-glycolic acid copolymers which may be used externally or internally.
  • hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.
  • the carrier may also be a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time release characteristics and release kinetics.
  • the composition for administration may then be moulded into a solid implant suitable for providing efficacious concentrations of the alkyl- substituted fatty acid over a prolonged period of time without the need for frequent re-dosing.
  • the alkyl-substituted fatty acid can be incorporated into the biodegradable polymer or polymer mixture in any suitable manner known to one of ordinary skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be moulded into a solid implant.
  • alkyl-substituted fatty acids to inhibit the activation and proliferation of leukocytes is markedly improved in the presence of immunosuppressants.
  • the ability of 12- methyltetradecanoic acid or 16-methylheptadecanoic acid to inhibit the activation and proliferation of leukocytes is further markedly improved in the presence of cyclosporin A or rapamycin.
  • alkyl-substituted fatty acid 12-MTA synergistically acts with cyclosporin A in a 2-way mixed leukocyte reaction, demonstrating that an immunosuppressant may be used at a lower concentration in combination with an alkyl-substituted fatty acid to inhibit leukocyte proliferation/activation or the inhibition of rejection of transplanted material.
  • the administration of alkyl-substituted fatty acid in the various forms of the present invention may further include the administration of an immunosuppressant.
  • the immunosuppressant is an immunosuppressive agent that inhibits T-lymphocyte activation and/or proliferation. More preferably, the immunosuppressant is an immunosuppressive agent that inhibits the activation and/or proliferation of T- lymphocytes via the modulation of antigen presenting cells that initiate and promote the rejection response.
  • the immunosuppressant is selected from one or more of the group consisting of cyclosporin A, rapamycin, tacrilomus, corticosteriods, mycophenolate mofetil, mizoribine, brequinar sodium, 15-deoxyspergualin, rapamycin, FK506, prednisone, azathioprine cyclophos-phamide, antilymphocyte antibodies, antithymocyte antibodies and muromonab-CD3, fusion proteins to co-stimulatory molecules, or monoclonal antibodies to co- stimulatory molecules.
  • the immunosuppressant is cyclosporin A or rapamycin.
  • the present invention provides a method of inhibiting leukocyte proliferation in a biological system, the method including the step of administering to the biological system an effective amount of cyclosporin A and 12-methyltetradecanoic acid, 17-methyloctadecanoic acid, 10- methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the present invention provides a method of inhibiting leukocyte proliferation in a biological system, the method including the step of administering to the biological system an effective amount of rapamycin and 12- methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • An effective amount of the immunosuppressant may be appropriately chosen, depending upon the amount of alkyl-substituted fatty acid in the composition, the extent of leukocyte proliferation to be inhibited, the type of rejection occurring, the type of material transplanted, the age and body weight of the subject or patient, and the frequency of administration.
  • this agent is administered so that the concentration at the desired site of action is in the range from 10 nM to 2 ⁇ M. More preferably, cyclosporin A is administered so that the concentration at the desired site of action is in the range from 10 nM to 100 nM. In the case of administration of rapamycin, preferably this agent is administered so that the concentration at the desired site of action is in the range from 0.1 nM to 30 nM. More preferably, rapamycin is administered so that the concentration at the desired site of action is in the range from 0.1 nM to 10 nM.
  • the administration of immunosuppressant may be within any time suitable to produce the desired effect of inhibiting leukocyte proliferation in conjunction with the alkyl-substituted fatty acid.
  • the immunosuppressant may be administered orally, parenterally or by any other suitable means and therefore transit time of the drug must be taken into account.
  • the administration of the immunosuppressant may occur at the same time and in the same manner as the administration of the alkyl-substituted fatty acid.
  • the administration of the immunosuppressant may occur at a pharmacologically appropriate time before or after administration of the alkyl- substituted fatty acid.
  • the administration of the immunosuppressant in the various forms of the present invention may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents.
  • pharmaceutically acceptable additives including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents.
  • the inhibition of the proliferation of leukocytes in the biological system may be determined by a suitable method known in the art, such as a mixed leukocyte reaction (MLR) for in vitro measurement.
  • MLR mixed leukocyte reaction
  • methods such as the use of histological analysis, flow cytometry, a MLR derived from the subject, or cytokine analysis may be employed.
  • Determination of the ability of an alkyl-substituted fatty acid to inhibit proliferation of leukocytes may be by any suitable assay of measuring leukocyte proliferation that is well known in the art.
  • a human mixed leukocyte reaction may be used.
  • human dendritic cells are used as stimulators to allogeneic leukocytes as responders.
  • Leukocyte proliferation may be measured, for example, by tritiated thymine uptake.
  • the ability of the alkyl- substituted fatty acid (ie the test fatty acid) to inhibit proliferation in such an assay may then be tested by contacting the leukocytes with the test fatty acid and determining the extent of inhibition of proliferation that occurs at any particular concentration of the test fatty acid.
  • test fatty acid in determining the ability of a test fatty acid to inhibit the proliferation of leukocytes, the test fatty acid will be delivered at a concentration and in form that are suitable to the particular physical and chemical characteristics of the test fatty acid.
  • the present invention also provides a method of inhibiting leukocyte activation in a biological system, the method including the step of administering to the biological system an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting leukocyte activation and the alkyl-substituted fatty acid has the following chemical formula:
  • the leukocytes may be any leukocytes, including leukocytes that are undergoing activation in response to one or more antigenic stimuli on transplanted material, or leukocytes that have the capacity to undergo activation in response to one or more antigenic stimuli on transplanted material.
  • the leukocytes are animal or human leukocytes. Most preferably, the leukocytes are human leukocytes.
  • the leukocytes are undergoing (or capable of undergoing) activation in response to rejection of transplanted biological material, including hyperacute, acute or chronic rejection of allogeneic or xenogeneic transplanted biological material, or leukocytes undergoing (or capable of undergoing) activation in response to graft versus host disease. More preferably, the leukocytes are undergoing (or capable of undergoing) activation in response to allogeneic transplanted biological material. Most preferably, the leukocytes are undergoing (or capable of undergoing) ⁇ activation in response to acute rejection of allogeneic transplanted biological material.
  • the leukocytes are lymphocytes or dendritic cells. More preferably, the lymphocytes are T lymphocytes.
  • the inhibition of leukocyte activation by the alkyl-substituted fatty acid may include a mechanism of inhibition of leukocyte activation that involves down regulation of the expression of the cell adhesion molecules LFA-1 (leukocyte function antigen-1) and/or VLA-4 (very late antigen-4).
  • LFA-1 leukocyte function antigen-1
  • VLA-4 very late antigen-4
  • the biological system may be any system that includes leukocytes that have the capacity to be activated.
  • the biological system is a human or animal subject that include leukocytes that have the capacity to be activated.
  • the biological system is a human or animal subject that includes the activation of leukocytes associated with the transplantation of foreign material.
  • the biological system is a human or animal subject that includes the activation of leukocytes associated with the transplantation of biological material.
  • the biological system is a human or animal subject suffering from the effects of rejection of allogeneic or xenogeneic transplanted biological material.
  • the biological system is a human or animal subject suffering from acute rejection of allogeneic transplanted biological material.
  • the transplanted biological material may be all or part of organs or tissues derived from skin, heart, lung, heart-lung, liver, kidney, cornea, blood, bone marrow, brain, spleen, pancreas, pancreatic islet, or stem-cells.
  • the present invention provides a method of inhibiting leukocyte activation in a biological system, the method including the step of administering to the biological system an effective amount of cyclosporin A and 12-methyltetradecanoic acid, 17-methyloctadecanoic acid, 10- methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the present invention provides a method of inhibiting leukocyte activation in a biological system, the method including the step of administering to the biological system an effective amount of rapamycin and 12- methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • An effective amount of the immunosuppressant may be appropriately chosen, depending upon the amount of alkyl-substituted fatty acid in the composition, the extent of leukocyte activation to be inhibited, the type of rejection occurring, the type of biological material transplanted, the age and body weight of the subject or patient, and the frequency of administration.
  • this agent is administered so that the concentration at the desired site of action is in the range from 10 nM to 2 ⁇ M. More preferably, cyclosporin A is administered so that the concentration at the desired site of action is in the range from 10 nM to 100 nM. In the case of administration of rapamycin, preferably this agent is administered so that the concentration at the desired site of action is in the range from 0.1 nM to 30 nM. More preferably, rapamycin is administered so that the concentration at the desired site of action is in the range from 0.1 nM to 10 nM.
  • the administration of immunosuppressant may be within any time suitable to produce the desired effect of inhibiting leukocyte activation in conjunction with the alkyl-substituted fatty acid.
  • the immunosuppressant may be administered orally, parenterally, topically or by any other suitable means and therefore transit time of the drug must be taken into account.
  • the administration of the immunosuppressant may occur at the same time and in the same manner as the administration of the alkyl-substituted fatty acid.
  • the administration of the immunosuppressant may occur at a pharmacologically appropriate time before of after administration of the alkyl-substituted fatty acid.
  • the inhibition of the activation of leukocytes in the biological system may be determined by a suitable method known in the art, such as a mixed leukocyte reaction for in vitro measurement.
  • a suitable method known in the art such as a mixed leukocyte reaction for in vitro measurement.
  • the use of markers of activation, the measurement of the levels of cytokine mRNA, cytokine ELISA or ELISPOT assay may all be used for the measurement of leukocyte activation.
  • Determination of the ability of an alkyl-substituted fatty acid to inhibit activation of leukocytes may be by any suitable assay of measuring leukocyte activation that is known in the art.
  • a human mixed leukocyte reaction may be used.
  • human dendritic cells are used as stimulators to allogeneic leukocytes as responders.
  • Leukocyte activation may be measured, for example, by an induction in the mRNA levels of interleukin-2, interleukin-12 or ⁇ -interferon in the leukocytes, and/or the expression of cell surface markers (eg LFA-1 , VLA-4) that are associated with activation.
  • cell surface markers eg LFA-1 , VLA-4
  • the ability of the alkyl-substituted fatty acid (ie the test fatty acid) to inhibit activation in such an assay may then be tested by contacting the leukocytes with the test fatty acid and determining the extent of inhibition of activation that occurs at any particular concentration of the test fatty acid.
  • test fatty acid in determining the ability of a test fatty acid to inhibit the activation of leukocytes, the test fatty acid will be delivered at a concentration and in form that are suitable to the particular physical and chemical characteristics of the test fatty acid.
  • the present invention also provides a method of reducing the amount of an agent administered to a biological system to achieve a desired level of inhibition of leukocyte proliferation, the method including the step of administering to the biological system an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • the amount of an agent administered to a biological system to inhibit leukocyte proliferation may be reduced by also administering an alkyl- substituted fatty acid.
  • an alkyl- substituted fatty acid For example, the amount of cyclosporin A or rapamycin required to achieve a desired level of inhibition of leukocyte proliferation may be reduced in the presence of an alkyl-substituted fatty acid.
  • the leukocytes may be any leukocytes, including leukocytes that are undergoing proliferation in response to one or more antigenic stimuli on transplanted material, or leukocytes that have the capacity to undergo proliferation in response to one or more antigenic stimuli on transplanted material.
  • the leukocytes are animal or human leukocytes. Most preferably, the leukocytes are human leukocytes.
  • the leukocytes are undergoing (or capable of undergoing) proliferation in response to rejection of transplanted biological material, including hyperacute, acute or chronic rejection of allogeneic or xenogeneic transplanted biological material, or leukocytes undergoing (or capable of undergoing) proliferation in response to graft versus host disease. More preferably, the leukocytes are undergoing (or capable of undergoing) proliferation in response to rejection of allogeneic transplanted biological material. Most preferably, the leukocytes are undergoing (or capable of undergoing) proliferation in response to acute rejection of allogeneic transplanted biological material.
  • the leukocytes are lymphocytes. More preferably, the lymphocytes are T lymphocytes.
  • the biological system may be any system that includes leukocytes that have the capacity to proliferate.
  • the biological system is a human or animal subject that include leukocytes that have the capacity to proliferate.
  • the biological system is a human or animal subject that includes the proliferation of leukocytes associated with the transplantation of foreign material.
  • the biological system is a human or animal subject that includes the proliferation of leukocytes associated with the transplantation of biological material.
  • the biological system is a human or animal subject suffering from the effects of rejection of allogeneic or xenogeneic transplanted biological material.
  • the biological system is a human or animal subject suffering from acute rejection of allogeneic transplanted biological material.
  • the transplanted biological material may be all or part of organs or tissues derived from skin, heart, lung, heart-lung, liver, kidney, cornea, blood, bone marrow, brain, spleen, pancreas, pancreatic islet, or stem-cells.
  • the effective amount of alkyl-substituted fatty acid to be administered is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits a pharmacologically useful effect to reduce the amount of agent normally administered to achieve a desired level of inhibition of leukocyte proliferation.
  • the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl-substituted fatty acid administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the administration of alkyl-substituted fatty acid may be within any time suitable to produce the desired effect of reducing the amount of an agent administered to a biological system necessary to achieve a desired level of inhibition of leukocyte proliferation in the biological system.
  • the alkyl-substituted fatty acid may be administered orally, parenterally, topically or by any other suitable means, and therefore transit time of the drug must be taken into account.
  • agents capable of inhibiting leukocyte proliferation include cyclosporin A, rapamycin, tacrilomus, corticosteriods, mycophenolate mofetil, mizoribine, brequinar sodium, 15-deoxyspergualin, rapamycin, FK506, prednisone, azathioprine cyclophos-phamide, antilymphocyte antibodies, antithymocyte antibodies and muromonab-CD3, fusion proteins to co- stimulatory molecules, or monoclonal antibodies to co-stimulatory molecules.
  • the present invention provides a method of reducing the amount of cyclosporin A administered to a biological system to achieve a desired level of inhibition of leukocyte proliferation, the method including the step of administering to the biological system an effective amount of 12- methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the present invention provides a method of reducing the amount of rapamycin administered to a biological system to achieve a desired level of inhibition of leukocyte proliferation, the method including the step of administering to the biological system an effective amount of 12- methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the amount of the agent necessary to achieve a desired level of inhibition of leukocyte proliferation will be empirically determined by a method known in the art, and as such will also depend upon the desired level of leukocyte proliferation to be inhibited, the type of rejection occurring, the type of material transplanted, the age and body weight of the subject or patient, and the frequency of administration.
  • the administration of the agent necessary to achieve a desired level of inhibition of leukocyte proliferation will be in a suitable form and within a suitable time to produce the desired effect of inhibiting the proliferation of leukocytes to the desired level.
  • the alkyl-substituted fatty acid may be administered orally, parenterally, topically or by any other suitable means and therefore transit time of the drug must be taken into account.
  • the administration of the alkyl-substituted fatty acid may occur at the same time and in the same manner as the administration of the agent capable of inhibiting leukocyte proliferation in the biological system.
  • the administration of the alkyl-substituted fatty acid may be separate to the administration of the agent capable of inhibiting leukocyte proliferation in the biological system, and occur at a pharmacologically appropriate time before or after administration of the agent.
  • the present invention also provides a method of reducing the amount of an agent administered to a biological system to achieve a desired level of inhibition of leukocyte activation, the method including the step of administering to the biological system an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • the amount of an agent administered to a biological system to inhibit leukocyte activation may be reduced by also administering an alkyl- substituted fatty acid.
  • an alkyl- substituted fatty acid For example, the amount of cyclosporin A or rapamycin required to achieve a desired level of inhibition of leukocyte activation may be reduced in the presence of an alkyl-substituted fatty acid.
  • the leukocytes may be any leukocytes, including leukocytes that are undergoing activation in response to one or more antigenic stimuli on transplanted material, or leukocytes that have the capacity to undergo activation in response to one or more antigenic stimuli on transplanted material.
  • the leukocytes are animal or human leukocytes. Most preferably, the leukocytes are human leukocytes.
  • the leukocytes are undergoing (or capable of undergoing) activation in response to rejection of transplanted biological material, including hyperacute, acute or chronic rejection of allogeneic or xenogeneic transplanted biological material, or leukocytes undergoing (or capable of undergoing) activation in response to graft versus host disease. More preferably, the leukocytes are undergoing (or capable of undergoing) activation in response to allogeneic transplanted material. Most preferably, the leukocytes are undergoing (or capable of undergoing) activation in response to acute rejection of allogeneic transplanted biological material.
  • the leukocytes are lymphocytes or dendritic cells. More preferably, the lymphocytes are T lymphocytes.
  • the reduction in the amount of the agent administered to the biological system by administering an effective amount of the alkyl-substituted fatty acid may include a mechanism of inhibition of leukocyte activation that involves down regulation of the expression of the cell adhesion molecules LFA-1 (leukocyte function antigen-1) and/or VLA-4 (very late antigen-4) by the alkyl-substituted fatty acid.
  • LFA-1 leukocyte function antigen-1
  • VLA-4 very late antigen-4
  • the biological system may be any system that includes leukocytes that have the capacity to be activated.
  • the biological system is a human or animal subject that include leukocytes that have the capacity to be activated.
  • the biological system is a human or animal subject that includes the activation of leukocytes associated with the transplantation of foreign material.
  • the biological system is a human or animal subject that includes the activation of leukocytes associated with the transplantation of biological material.
  • the biological system is a human or animal subject suffering from the effects of rejection of allogeneic or xenogeneic transplanted biological material.
  • the biological system is a human or animal subject suffering from acute rejection of allogeneic transplanted biological material.
  • the transplanted biological material may be all or part of organs or tissues derived from skin, heart, lung, heart-lung, liver, kidney, cornea, blood, bone marrow, brain, spleen, pancreas, pancreatic islet, or stem-cells.
  • the effective amount of alkyl-substituted fatty acid to be administered is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits a pharmacologically useful effect to reduce the amount of agent normally administered to achieve a desired level of inhibition of leukocyte activation.
  • the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl-substituted fatty acid administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the administration of alkyl-substituted fatty acid may be within any time suitable to produce the desired effect of reducing the amount of an agent administered to a biological system to achieve a desired level of inhibition of leukocyte activation in the biological system.
  • the alkyl- substituted fatty acid may be administered orally, parenterally, topically or by any other suitable means, and therefore transit time of the drug must be taken into account.
  • agents capable of inhibiting leukocyte activation include cyclosporin A, rapamycin, tacrilomus, corticosteriods, mycophenolate mofetil, mizoribine, brequinar sodium, 15-deoxyspergualin, rapamycin, FK506, prednisone, azathioprine cyclophos-phamide, antilymphocyte antibodies, antithymocyte antibodies and muromonab-CD3, fusion proteins to co- stimulatory molecules, or monoclonal antibodies to co-stimulatory molecules.
  • the present invention provides a method of reducing the amount of cyclosporin A administered to a biological system to achieve a desired level of inhibition of leukocyte activation, the method including the step of administering to the biological system an effective amount of 12- methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the present invention provides a method of reducing the amount of rapamycin administered to a biological system to achieve a desired level of inhibition of leukocyte activation, the method including the step of administering to the biological system an effective amount of 12- methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the amount of the agent necessary to achieve a desired level of inhibition of leukocyte activation will be empirically determined by a method known in the art, and as such will also depend upon the desired level of leukocyte activation to be inhibited, the type of rejection occurring, the type of biological material transplanted, the age and body weight of the subject or patient, and the frequency of administration.
  • the administration of the agent necessary to achieve a desired level of inhibition of leukocyte activation will be in a suitable form and within a suitable time to produce the desired effect of inhibiting the activation of leukocytes to the desired level.
  • the alkyl-substituted fatty acid may be administered orally, parenterally, topically or by any other suitable means and therefore transit time of the drug must be taken into account.
  • the administration of the alkyl-substituted fatty acid may occur at the same time and in the same manner as the administration of the agent capable of inhibiting leukocyte activation in the biological system.
  • the administration of the alkyl-substituted fatty acid may be separate to the administration of the ⁇ agent capable of inhibiting leukocyte activation in the biological system, and occur at a pharmacologically appropriate time before or after administration of the agent.
  • the present invention also provides a method of inhibiting rejection of transplanted material in a subject, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting rejection of transplanted material and the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the rejection of the transplanted material may be rejection of allogeneic transplanted material, xenogeneic transplanted material or non-biological transplanted material. In the case of allogeneic rejection, the transplanted biological material will be derived from the same species as the recipient of the material.
  • the transplanted biological material will be derived from a different species as the recipient.
  • the rejection of the allogeneic or xenogeneic transplanted material may be hyperacute rejection, acute rejection or chronic rejection of the transplanted material.
  • the transplanted biological material may be any material suitable for transplantation, including biological material such as all or part of organs or tissues derived from skin, heart, lung, heart-lung, liver, kidney, cornea, blood, bone marrow, brain, spleen, pancreas, pancreatic islet, or stem-cells.
  • the origin of the transplanted biological material may be from any suitable donor organism.
  • the transplanted biological material is derived from a human or animal. Most preferably, the transplanted biological material is derived from a human.
  • the subject is any recipient of transplanted material.
  • the subject is an animal or human.
  • the subject is a human.
  • the inhibition of rejection by the alkyl-substituted fatty acid may include a mechanism of inhibiting leukocyte activation that involves down regulation of the expression of the cell adhesion molecules LFA-1 (leukocyte function antigen-1) and/or VLA-4 (very late antigen-4).
  • LFA-1 leukocyte function antigen-1
  • VLA-4 very late antigen-4
  • the inhibition of rejection by the alkyl-substituted fatty acid may also include a mechanism of down regulating the expression of one or more of CD83, CD1a and CD80 on dendritic cells.
  • the alkyl-substituted fatty acid is administered to a subject at a dose greater than 100 mg/kg body weight of the subject. More preferably, the alkyl- substituted fatty acid is administered to a subject at a dose of equal to or greater than 200 mg/kg body weight of the subject.
  • the alkyl-substituted fatty acid is preferably dissolved in autologous serum before administration.
  • a suitable concentration for dissolving the alkyl-substituted fatty acid in serum is 10 mg/ml.
  • Determination of the ability of an alkyl-substituted fatty acid to inhibit rejection of transplanted material may be by any suitable assay of measuring rejection that is known in the art. For example, animal studies using cardiac or kidney allograft transplantation may be performed. In this case, the transplanted material may be transplanted to a suitable recipient animal and the ability of a test fatty acid to inhibit rejection may be determined a suitable time after transplantation. Examples of methods for determining the extent of rejection include assays of organ function (eg palpable heart beats for cardiac transplants; creatine for kidney transplants), histological assays and morbidity/mortality rates.
  • organ function eg palpable heart beats for cardiac transplants; creatine for kidney transplants
  • an alkyl-substituted fatty acid ie the test fatty acid
  • the ability of an alkyl-substituted fatty acid (ie the test fatty acid) to inhibit rejection in such assays may be tested by exposing the recipient of the transplanted material to the test fatty acid and determining the extent of inhibition of rejection that occurs at any particular concentration of the test fatty acid.
  • the test fatty acid may be delivered to the transplanted material by any appropriate method, including administration orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, or via an implanted reservoir.
  • test fatty acid in determining the ability of a test fatty acid to inhibit rejection, the test fatty acid will be delivered at a concentration and in form that are suitable to the particular physical and chemical characteristics of the test fatty acid.
  • the present invention provides a method of inhibiting rejection of a corneal transplant in a subject, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • the administration of the alkyl-substituted fatty acid will include topical administration to the cornea.
  • the alkyl-substituted fatty acid may be prepared as an emulsion in unpreserved paraffin and lanolin ophthalmic ointment base, and the composition applied topically to the cornea.
  • the present invention also provides a method of reducing the amount of an agent administered to a subject to achieve a desired level of inhibition of rejection of transplanted material, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • the amount of an agent administered to the subject to inhibit rejection of transplanted material may be reduced by also administering an alkyl-substituted fatty acid.
  • an agent administered to the subject to inhibit rejection of transplanted material may be reduced in the presence of an alkyl-substituted fatty acid.
  • the rejection of the transplanted biological material may be rejection of allogeneic transplanted biological material, xenogeneic transplanted biological material or non-biological transplanted material.
  • allogeneic rejection the transplanted biological material will be derived from the same species as the recipient of the material.
  • xenogeneic rejection the transplanted biological material will be derived from a different species as the recipient.
  • the rejection of the allogenic transplanted material or the xenogeneic transplanted material may be hyperacute rejection, acute rejection or chronic rejection of the transplanted material.
  • the transplanted biological material may be any material suitable for transplantation, including biological material such as all or part of organs or tissues derived from skin, heart, lung, heart-lung, liver, kidney, cornea, blood, bone marrow, brain, spleen, pancreas, pancreatic islet, or stem-cells.
  • the origin of the transplanted biological material may be from any suitable donor organism.
  • the transplanted biological material is derived from a human or animal. Most preferably, the transplanted biological material is derived from a human.
  • the subject is any recipient of the transplanted material.
  • the subject is an animal or human.
  • the recipient is a human.
  • the reduction in the amount of an agent administered to the subject to inhibit rejection by administering an effective amount of the alkyl-substituted fatty acid may include a mechanism of inhibition of leukocyte activation that involves down regulation of the expression of the cell adhesion molecules LFA-1 (leukocyte function antigen-1) and/or VLA-4 (very late antigen-4) by the alkyl- substituted fatty acid.
  • LFA-1 leukocyte function antigen-1
  • VLA-4 very late antigen-4
  • the reduction in the amount of the agent administered to the subject to inhibit rejection by administering an effective amount of the alkyl-substituted fatty acid may also include a mechanism of down regulating the expression of one or more of CD83, CD1a and CD80 on dendritic cells.
  • the effective amount of alkyl-substituted fatty acid to be administered is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits a pharmacologically useful effect to reduce the amount of agent normally administered to achieve a desired level of inhibition of rejection of transplanted material.
  • the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl-substituted fatty acid administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the administration of alkyl-substituted fatty acid may be within any time suitable to produce the desired effect of reducing the amount of an agent administered to a subject necessary to achieve a desired level of inhibition of rejection of transplanted material.
  • the alkyl-substituted fatty acid may be administered orally, parenterally, topically or by any other suitable means, and therefore transit time of the drug must be taken into account.
  • the administration may include topical administration.
  • agents capable of inhibiting the rejection of transplanted material include cyclosporin A, rapamycin, tacrilomus, corticosteriods, mycophenolate mofetil, mizoribine, brequinar sodium, 15-deoxyspergualin, rapamycin, FK506, prednisone, azathioprine cyclophos-phamide, antilymphocyte antibodies, antithymocyte antibodies and muromonab-CD3, fusion proteins to co- stimulatory molecules, or monoclonal antibodies to co-stimulatory molecules.
  • the present invention provides a method of reducing the amount of cyclosporin A administered to a subject to achieve a desired level of inhibition of rejection of transplanted material, the method including the step of administering to the subject an effective amount of 12-methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the present invention provides a method of reducing the amount of rapamycin administered to a subject to achieve a desired level of inhibition of rejection of transplanted material, the method including the step of administering to the subject an effective amount of 12-methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the amount of the agent necessary to achieve a desired level of inhibition of rejection of transplanted material will be empirically determined by a method known in the art, and as such will also depend upon the type of rejection occurring, the type of material transplanted, the age and body weight of the subject or patient, and the frequency of administration.
  • the administration of the agent necessary to achieve a desired level of inhibition of rejection of transplanted material will be in a suitable form and within a suitable time to produce the desired effect of inhibiting the rejection of transplanted material to the desired level.
  • the alkyl-substituted fatty acid may be administered orally, parenterally, topically or by any other suitable means and therefore transit time of the drug must be taken into account.
  • the administration of the alkyl-substituted fatty acid may occur at the same time and in the same manner as the administration of the agent capable of inhibiting the rejection of transplanted material.
  • the administration of the alkyl-substituted fatty acid may be separate to the administration of the agent capable of inhibiting rejection, and occur at a pharmacologically appropriate time before or after administration of the agent.
  • the present invention also provides a method of inhibiting graft versus host disease in a subject, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl- substituted fatty acid is capable of inhibiting graft versus host disease and the alkyl-substituted fatty acid has the following chemical formula: R
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the graft versus host disease may occur upon the transplantation of any suitable biological material into a host that is immunosuppressed for some reason.
  • the graft versus host disease occurs in an animal or human subject.
  • the graft versus host disease occurs in a human subject.
  • the graft versus host disease may be due to allogeneic transplanted material or xenogeneic transplanted material.
  • the transplanted biological material that gives rise to the graft versus host disease may be any biological material suitable for transplantation, including all or part of organs or tissues derived from skin, heart, lung, heart-lung, liver, kidney, cornea, blood, bone marrow, brain, spleen, pancreas, pancreatic islet, or stem-cells.
  • the origin of the transplanted biological material may be from any suitable donor organism.
  • the transplanted biological material is derived from a human or animal. Most preferably, the transplanted biological material is derived from a human.
  • the reduction in the amount of an agent administered to the subject to inhibit graft versus host disease by administering an effective amount of the alkyl- substituted fatty acid may include a mechanism of inhibition of leukocyte activation that involves down regulation of the expression of the cell adhesion molecules LFA-1 (leukocyte function antigen-1) and/or VLA-4 (very late antigen- 4) by the alkyl-substituted fatty acid.
  • LFA-1 leukocyte function antigen-1
  • VLA-4 very late antigen- 4
  • the reduction in the amount of the agent administered to the subject to inhibit graft versus host disease by administering an effective amount of the alkyl- substituted fatty acid may also include a mechanism of down regulating the expression of one or more of CD83, CD1a and CD80 on dendritic cells.
  • Determination of the ability of an alkyl-substituted fatty acid to inhibit graft versus host disease may be by any suitable assay of measuring graft versus host disease that is known in the art.
  • the transplanted material may be grafted to a recipient animal that is immunosuppressed by the action of drugs suitable for this purpose and the ability of a test fatty acid to inhibit graft versus host disease may be determined at an appropriate time after transplantation. Examples of methods for determining the extent of graft versus host disease include histological assays and morbidity/mortality rates.
  • test fatty acid alkyl-substituted fatty acid
  • the ability of an alkyl-substituted fatty acid (ie the test fatty acid) to inhibit graft versus host disease in such assays may be tested by administering to the subject with the transplanted biological material with the test fatty acid and determining the extent of inhibition of graft versus host disease that occurs at any particular concentration of the test fatty acid.
  • the test fatty acid may be delivered to the subject by any appropriate method, including administration orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir.
  • the test fatty acid in determining the ability of a test fatty acid to inhibit graft versus host disease, the test fatty acid will be delivered at a concentration and in form that are suitable to the particular physical and chemical characteristics of the test fatty acid.
  • the present invention also provides a method of reducing the amount of an agent administered to a subject to achieve a desired level of inhibition of graft versus host disease, the method including the step of administering to the subject an effective amount of an alkyl-substituted fatty acid, wherein the alkyl- substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms
  • x is equal to or greater than
  • y is equal to or greater than
  • x + y is between 0 and 46 for saturated alkyl- substituted fatty acids
  • x or y is equal to or greater than 2
  • the graft versus host disease may occur upon the transplantation of any suitable biological material into a host that is immunosuppressed for some reason.
  • the graft versus host disease occurs in an animal or human subject.
  • the graft versus host disease occurs in a human subject.
  • the graft versus host disease may be due to allogeneic transplanted material or xenogeneic transplanted material.
  • the transplanted biological material that gives rise to the graft versus host disease may be any biological material suitable for transplantation, including all or part of organs or tissues derived from skin, heart, lung, heart-lung, liver, kidney, cornea, blood, bone marrow, brain, spleen, pancreas, pancreatic islet, or stem-cells.
  • the origin of the transplanted biological material may be from any suitable donor organism.
  • the transplanted biological material is derived from a human or animal. Most preferably, the transplanted biological material is derived from a human.
  • the reduction in the amount of an agent administered to the subject to inhibit graft versus host disease by administering an effective amount of the alkyl- substituted fatty acid may include a mechanism of inhibition of leukocyte activation that involves down regulation of the expression of the cell adhesion molecules LFA-1 (leukocyte function antigen-1) and/or VLA-4 (very late antigen- 4) by the alkyl-substituted fatty acid.
  • LFA-1 leukocyte function antigen-1
  • VLA-4 very late antigen- 4
  • the reduction in the amount of the agent administered to the subject to inhibit graft versus host disease by administering an effective amount of the alkyl- substituted fatty acid may also include a mechanism of down regulating the expression of one or more of CD83, CD1a and CD80 on dendritic cells.
  • the effective amount of alkyl-substituted fatty acid to be administered is not particularly limited, so long as it is within such an amount that generally exhibits a pharmacologically useful effect to reduce the amount of agent normally administered to achieve a desired level of inhibition of graft versus host disease.
  • the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl-substituted fatty acid administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the administration of alkyl-substituted fatty acid may be within any time suitable to produce the desired effect of reducing the amount of an agent administered to a subject necessary to achieve a desired level of inhibition of graft versus host disease.
  • the alkyl-substituted fatty acid may be administered orally, parenterally or by any other suitable means, and therefore transit time of the drug must be taken into account.
  • agents capable of inhibiting graft versus host disease include cyclosporin A, rapamycin, tacrilomus, corticosteriods, mycophenolate mofetil, mizoribine, brequinar sodium, 15-deoxyspergualin, rapamycin, FK506, prednisone, azathioprine cyclophos-phamide, antilymphocyte antibodies, antithymocyte antibodies and muromonab-CD3, fusion proteins to co- stimulatory molecules, or monoclonal antibodies to co-stimulatory molecules.
  • the present invention provides a method of reducing the amount of cyclosporin A administered to a subject to achieve a desired level of inhibition of graft versus host disease, the method including the step of administering to the subject an effective amount of 12-methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the present invention provides a method of reducing the amount of rapamycin administered _to a subject to achieve a desired level of inhibition of graft versus host disease, the method including the step of administering to the subject an effective amount of 12-methyltetradecanoic acid, 17-methyloctadecanoic acid, 10-methyloctadecanoic acid, 10-methyldodecanoic acid, 16-methylheptadecanoic acid, or any combination of these fatty acids.
  • the amount of the agent necessary to achieve a desired level of inhibition of graft versus host disease will be empirically determined by a method known in the art, and as such will also depend upon the extent of GVHD occurring, the type of biological material transplanted, the age and body weight of the subject or patient, and the frequency of administration.
  • the administration of the agent necessary to achieve a desired level of inhibition of graft versus host disease will be in a suitable form and within a suitable time to produce the desired effect of inhibiting the graft versus host disease to the desired level.
  • the alkyl-substituted fatty acid may be administered orally, parenterally, topically or by any other suitable means and therefore transit time of the drug must be taken into account.
  • the administration of the alkyl-substituted fatty acid may occur at the same time and in the same manner as the administration of the agent capable of inhibiting the graft versus host disease.
  • the administration of the alkyl-substituted fatty acid may be separate to the administration of the agent capable of inhibiting graft versus host disease, and occur at a pharmacologically appropriate time before or after administration of the agent.
  • the present invention also provides a method of down regulating the expression of a cell adhesion molecule on a leukocyte, the method including the step of administering to the leukocyte an effective amount of an alkyl- substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of down regulating the expression of a cell adhesion molecule on a leukocyte and the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the leukocyte may be any leukocyte, including a leukocyte that is undergoing proliferation and/or activation in response to one or more antigenic stimuli, or a leukocyte that has the capacity to undergo proliferation and/or activation in response to one or more antigenic stimuli.
  • the leukocyte is a leukocyte that is undergoing proliferation and/or activation in response to one or more antigenic stimuli on transplanted biological material.
  • the leukocytes are animal or human leukocytes. Most preferably, the leukocytes are human leukocytes.
  • the leukocytes are undergoing (or capable of undergoing) proliferation and/or activation in response to rejection of transplanted biological material, including hyperacute, acute or chronic rejection of allogeneic or xenogeneic transplanted biological material, or leukocytes undergoing (or capable of undergoing) proliferation and/or activation in response to graft versus host disease. More preferably, the leukocytes are undergoing (or capable of undergoing) proliferation and/or activation in response to allogeneic transplanted material. Most preferably, the leukocytes are undergoing (or capable of undergoing) proliferation and/or activation in response to acute rejection of allogeneic transplanted biological material.
  • the leukocytes are lymphocytes or dendritic cells. More preferably, the lymphocytes are T lymphocytes.
  • the leukocyte may be present in any biological system that includes leukocytes that have the capacity to proliferate and/or be activated.
  • the leukocyte may be an isolated leukocyte in culture, be a leukocyte present as part of a group of cells isolated from a subject, be a leukocyte present in a tissue or organ, or be a leukocyte present in an entire human or animal subject.
  • the cell adhesion molecule is a molecule involved in an interaction with a T-lymphocyte. More preferably, the cell adhesion molecule is a molecule involved in a homotypic or heterotypic interaction between T-lymphocytes.
  • the cell adhesion molecule is LFA-1 (leukocyte function antigen-1) or VLA-4 (very late antigen-4).
  • the amount of alkyl-substituted fatty acid to be administered to the leukocyte is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits the effect of down-regulating the expression of a cell adhesion molecule on the leukocyte.
  • an effective amount of the alkyl-substituted fatty acid may be appropriately chosen, depending upon the extent of down-regulation of the expression of the cell adhesion molecule on the leukocyte to be achieved.
  • the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl-substituted fatty acid administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the alkyl- substituted fatty acid may be administered by introducing the alkyl-substituted fatty acid into the culture medium.
  • the administration of the alkyl-substituted fatty acid may be within any time suitable and in a suitable form to produce the desired effect of down- regulating the expression of the cell adhesion molecule.
  • the administration may be within a suitable time to down-regulate expression of cell adhesion molecules on leukoctyes at the site of transplantation.
  • alkyl-substituted fatty acid may be by a suitable means known in the art, including administration orally, parenterally or by any other suitable means, and therefore transit time of the drug must be taken into account.
  • the down-regulation of the expression of the cell adhesion molecule on a leukocyte may be determined by a suitable method known in the art, including the use of flow cytometry with appropriate antibodies.
  • the present invention also provides a method of down regulating the cell surface expression of a molecule on a dendritic cell involved in T-lymphocyte stimulation, the method including the step of administering to the dendritic cell an effective amount of an alkyl-substituted fatty acid, wherein the alkyl- substituted fatty acid is capable of down regulating the cell surface expression of a molecule on a dendritic cell involved in T-lymphocyte stimulation and the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the dendritic cell may be any dendritic cell, including a dendritic cell that is undergoing activation and/or maturation in response to one or more antigenic stimuli, or a dendritic cell that has the capacity to undergo activation and/or maturation in response to one or more antigenic stimuli.
  • the dendritic cell is undergoing activation and/or maturation in response to one or more antigenic stimuli on transplanted biological material.
  • the dendritic cell is a human or animal dendritic cell. Most preferably, the dendritic cell is a human dendritic cell.
  • the dendritic cell is undergoing (or capable of undergoing) activation and/or maturation in response to rejection of transplanted biological material, including hyperacute, acute or chronic rejection of allogeneic or xenogeneic transplanted biological material, or a dendritic cell undergoing (or capable of undergoing) activation and/or maturation in response to graft versus host disease. More preferably, the dendritic cell is undergoing (or capable of undergoing) activation and/or maturation in response to allogeneic transplanted material. Most preferably, the dendritic cell is undergoing (or capable of undergoing) activation and/or maturation in response to acute rejection of allogeneic transplanted biological material.
  • the dendritic cell may be present in any biological system that includes dendritic cells that have the capacity to be activated and/or undergo maturation.
  • the dendritic cell may be an isolated dendritic cell in culture, be a dendritic cell present as part of a group of cells isolated from a subject, be a dendritic cell present in a tissue or organ, or be a dendritic cell present in an entire human or animal subject.
  • the molecule expressed on the surface of the dendritic cell may be any molecule that is involved in stimulation of a T-lymphocyte.
  • the molecule expressed on the surface of the dendritic cell involved in T-lymphocyte stimulation is CD83, CD1a or CD80.
  • the amount of alkyl-substituted fatty acid to be administered to the dendritic cell is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits the effect of down-regulating the cell surface expression of a molecule involved in T-lymphocyte stimulation.
  • an effective amount of alkyl-substituted fatty acid may be appropriately chosen, depending upon the extent of down-regulation of the expression of the cell surface molecule on the dendritic cell to be achieved.
  • the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl-substituted fatty acid administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the alkyl- substituted fatty acid may be administered by introducing the alkyl-substituted fatty acid into the culture medium.
  • the administration of the alkyl-substituted fatty acid may be within any time suitable and in a suitable form to produce the desired effect of down-regulating the cell surface expression of a molecule involved in T- lymphocyte stimulation.
  • the administration may be within a suitable time to down-regulate expression of the cell surface molecule at the site of transplantation.
  • alkyl-substituted fatty acid may be by a suitable means known in the art, including administration orally, parenterally or by any other suitable means, and therefore transit time of the drug must be taken into account.
  • the down-regulation of the expression of the cell surface molecule on a dendritic cell may be determined by a suitable method known in the art, including the use of flow cytometry with appropriate antibodies.
  • the present invention also provides a method for inhibiting the maturation of a dendritic cell, the method including the step of administering to the dendritic cell an effective amount of an alkyl-substituted fatty acid, wherein the alkyl- substituted fatty acid is capable of inhibiting the maturation of a dendritic cell and the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the dendritic cell may be any dendritic cell, including a dendritic cell that is undergoing maturation in response to one or more antigenic stimuli, or a dendritic cell that has the capacity to undergo maturation in response to one or more antigenic stimuli.
  • the dendritic cell is undergoing maturation in response to one or more antigenic stimuli on transplanted biological material.
  • the dendritic cell is a human or animal dendritic cell. Most preferably, the dendritic cell is a human dendritic cell.
  • the dendritic cell is undergoing (or capable of undergoing) maturation in response to rejection of transplanted biological material, including hyperacute, acute or chronic rejection of allogeneic or xenogeneic transplanted biological material, or a dendritic cell undergoing (or capable of undergoing) maturation in response to graft versus host disease. More preferably, the dendritic cell is undergoing (or capable of undergoing) maturation in response to allogeneic transplanted biological material. Most preferably, the dendritic cell is undergoing (or capable of undergoing) maturation in response to acute rejection of allogeneic transplanted biological material.
  • the dendritic cell may be present in any biological system that includes dendritic cells that have the capacity to undergo maturation.
  • the dendritic cell may be an isolated dendritic cell in culture, be a dendritic cell present as part of a group of cells isolated from a subject, be a dendritic cell present in a tissue or organ, or be a dendritic cell present in an entire human or animal subject.
  • the amount of alkyl-substituted fatty acid to be administered to the dendritic cell is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits the effect of inhibiting the maturation of the dendritic cell.
  • an effective amount of alkyl-substituted fatty acid may be appropriately chosen, depending upon the extent of inhibition of dendritic cell maturation to be achieved.
  • the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl- substituted fatty acid administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the alkyl- substituted fatty acid may be administered by introducing the alkyl-substituted fatty acid into the culture medium.
  • the administration of the alkyl-substituted fatty acid may be within any time suitable and in a suitable form to produce the desired effect of inhibiting maturation of the dendritic cell.
  • the administration may be within a suitable time to inhibit maturation of the dendritic cell at the site of transplantation.
  • alkyl-substituted fatty acid may be by a suitable means known in the art, including administration orally, parenterally or by any other suitable means, and therefore transit time of the drug must be taken into account.
  • the inhibition of dendritic cell maturation may be determined by a suitable method known in the art, including the use of flow cytometry with appropriate antibodies, such as antibodies to CD1a, CD83 and CD80.
  • the present invention also provides a method of inhibiting proliferation and/or stimulation of a lymphocyte mediated by a dendritic cell, the method including the step of administering to the dendritic cell an effective amount of an alkyl- substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting proliferation and/or stimulation of a lymphocyte mediated by a dendritic cell and the alkyl-substituted fatty acid has the following chemical formula:
  • the dendritic cell may be any dendritic cell that has the capacity to mediate the proliferation and/or stimulation of a lymphocyte in response to an antigenic stimulus.
  • the dendritic cell is a dendritic cell that is involved in mediating the proliferation and/or stimulation of a lymphocyte in response to the transplantation of biological material in a subject, including hyperacute, acute or chronic rejection of the transplanted biological material.
  • the dendritic cell is a dendritic cell that is involved in mediating the proliferation and/or stimulation of a lymphocyte in response to the transplantation of allogeneic or xenogeneic transplanted biological material, or a dendritic cell that is involved in mediating the proliferation and/or stimulation of a lymphocyte in response to graft versus host disease. More preferably, the dendritic cell is a dendritic cell that is involved in mediating the proliferation and/or stimulation of a lymphocyte in response to the transplantation of allogeneic transplanted biological material. Most preferably, the dendritic cell is a dendritic cell that is involved in mediating the proliferation_and/or stimulation of a lymphocyte in response to acute rejection of allogeneic transplanted biological material.
  • the dendritic cell is a human or animal dendritic cell. Most preferably, the dendritic cell is a human dendritic cell.
  • the dendritic cell may be present in any biological system that includes dendritic cells that have the capacity to mediate proliferation and/or stimulation of a lymphocyte.
  • the dendritic cell may be an isolated dendritic cell in culture, be a dendritic cell present as. part of a group of cells isolated from a subject, be a dendritic cell present in a tissue or organ, or be a dendritic cell present in an entire human or animal subject.
  • the lymphocyte may be any lymphocyte that is capable of proliferation and/or stimulation in response to the action of a dendritic cell.
  • the lymphocyte is a T-lymphocyte.
  • the amount of alkyl-substituted fatty acid to be administered to the dendritic cell is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits the effect of inhibiting the proliferation and/or stimulation of a lymphocyte.
  • an effective amount of alkyl-substituted fatty acid may be appropriately chosen, depending upon the extent of inhibition of proliferation and/or stimulation of the lymphocyte to be achieved.
  • the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid administered is such that it results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl- substituted fatty acid administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the alkyl- substituted fatty acid may be administered by introducing the alkyl-substituted fatty acid into the culture medium.
  • the administration of the alkyl-substituted fatty acid may be within any time suitable and in a suitable form to produce the desired effect of inhibiting the proliferation and/or stimulation mediated by the dendritic cell.
  • the administration of alkyl-substituted fatty acid may be by a suitable means known in the art, including administration orally, parenterally or by any other suitable means, and therefore transit time of the drug must be taken into account.
  • the inhibition of proliferation and/or stimulation of the lymphocyte by the dendritic cell may be may be determined by a suitable method known in the art, including flow cytometry for VLA-4, LFA-1 , CD4, CD8, 3[H] proliferation assays, or cytokine assays.
  • the present invention also provides a pharmaceutical composition including an alkyl-substituted fatty acid, wherein the alkyl-substituted fatty acid is capable of inhibiting leukocyte proliferation and/or rejection of transplanted biological material and the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the amount of alkyl-substituted fatty acid to be used in the pharmaceutical composition is not particularly limited, so long as it is within such an amount and is such a form that generally exhibits a pharmacologically therapeutic effect when the composition is administered to a subject.
  • the amount of alkyl-substituted fatty acid in the pharmaceutical composition may be appropriately chosen, depending upon the extent of leukocyte proliferation or activation to be inhibited, the type of rejection or graft versus host disease to be treated, the age and body weight of the subject or patient, and the frequency of administration.
  • the effective amount of alkyl- substituted fatty acid in the composition is such that when administered results in a concentration of the compound at the desired site of action in the range from 50 nM to 5 mM. More preferably, the effective amount of alkyl-substituted fatty acid in the composition is such that when administered results in a concentration of the compound at the desired site of action in the range from 50 nM to 1 mM. Most preferably, the effective amount of alkyl-substituted fatty acid in the composition is such that when administered results in a concentration of the compound at the desired site of action in the range from 25 ⁇ M to 500 ⁇ M.
  • the pharmaceutical composition may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into account the physical and chemical properties of the alkyl-substituted fatty acid.
  • pharmaceutically acceptable additives including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into account the physical and chemical properties of the alkyl-substituted fatty acid.
  • the alkyl-substituted fatty acid can be prepared into a variety of pharmaceutical preparations in the form of, e.g., an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a gel, etc., for administration as intramuscular or subcutaneous injection or as injection to the organ, or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc.
  • the composition of the present invention can also be administered in the form of oral preparations (for example solid preparations such as tablets, capsules, granules or powders; liquid preparations such as syrup, emulsions or suspensions).
  • Compositions containing alkyl- substituted fatty acid may also contain a preservative, stabiliser, dispersing agent, pH controller or isotonic agent.
  • suitable preservatives are glycerin, propylene glycol, phenol or benzyl alcohol.
  • suitable stabilisers are dextran, gelatin, -tocopherol acetate or alpha-thioglycerin.
  • suitable dispersing agents include polyoxyethylene (20), sorbitan mono-oleate (Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylene hydrogenated castor oil 60.
  • suitable pH controllers include hydrochloric acid, sodium hydroxide and the like.
  • suitable isotonic agents are glucose, D-sorbitol or D-mannitol.
  • the composition When administered orally, the composition will usually be formulated into unit dosage forms such as tablets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art.
  • Such formulations typically include a solid, semisolid, or liquid carrier.
  • Exemplary carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.
  • a tablet may be made by compressing or moulding the active ingredient optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free- flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.
  • compositions may utilize controlled release or sustained release technology.
  • the composition may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty acid esters such as ethyl oleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof (weight average molecular weight: ca. 80,000 to 2,000,000), carboxymethylcellulose sodium (weight average molecular weight: ca.
  • hydroxypropylcellulose viscosity in 2% aqueous solution: 3 to 4,000 cps
  • atherocollagen weight average molecular weight: ca. 300,000
  • polyethylene glycol weight average molecular weight: ca. 400 to 20,000
  • polyethylene oxide weight average molecular weight: ca. 100,000 to 9,000,000
  • hydroxypropylmethylcellulose viscosity in 1% aqueous solution: 4 to 100,000 cSt
  • methylcellulose viscosity in 2% aqueous solution: 15 to 8,000 cSt
  • polyvinyl alcohol viscosity: 2 to 100 cSt
  • polyvinylpyrrolidone weight average molecular weight: 25,000 to 1 ,200,000.
  • the alkyl-substituted fatty acid may be incorporated into a hydrophobic polymer matrix for controlled release over a period of days.
  • the composition of the invention may then be moulded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the alkyl-substituted fatty acid over a prolonged period of time without the need for frequent re-dosing.
  • Such controlled release films are well known to the art.
  • Other examples of polymers commonly employed for this purpose that may be used include nondegradable ethylene-vinyl acetate copolymer a degradable lactic acid-glycolic acid copolymers which may be used externally or internally.
  • Certain hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.
  • the carrier may also be a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time release characteristics and release kinetics.
  • the composition may then be moulded into a solid implant suitable for providing efficacious concentrations of the alkyl-substituted fatty acid over a prolonged period of time without the need for frequent re-dosing.
  • the alkyl-substituted fatty acid can be incorporated into the ' biodegradable polymer or polymer mixture in any suitable manner known to one of ordinary skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be moulded into a solid implant.
  • the present invention provides a pharmaceutical composition including an alkyl-substituted fatty acid, wherein the alkyl- substituted fatty acid is capable of inhibiting rejection of a corneal transplant and the alkyl-substituted fatty acid has the following chemical formula:
  • the present invention provides the use of an alkyl-substituted fatty acid for the preparation of a medicament for inhibiting leukocyte proliferation and/or rejection of transplanted material, wherein the alkyl- substituted fatty acid has the following chemical formula:
  • the pharmaceutical composition according to the various forms of the present invention may further include the administration of an immunosuppressant.
  • the immunosuppressant is an immunosuppressive agent that inhibits T-lymphocyte activation and proliferation or an immunosuppressive agent that inhibits the activation and proliferation of T-lymphocytes via the modulation of antigen presenting cells that initiate and promote the rejection response.
  • the present invention also provides a pharmaceutical composition including an alkyl-substituted fatty acid and an immunosuppressant, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • R is an alkyl group of 1 to 6 carbon atoms; x is equal to or greater than 0, y is equal to or greater than
  • the immunosuppressant is selected from one or more of the group consisting of cyclosporin A, tacrilomus, corticosteriods, mycophenolate mofetil, rapamycin, mizoribine, brequinar sodium, 15-deoxyspergualin, rapamycin, FK506, prednisone, azathioprine cyclophos-phamide, antilymphocyte antibodies, antithymocyte antibodies and muromonab-CD3, fusion proteins to co-stimulatory molecules, or monoclonal antibodies to co-stimulatory molecules.
  • the immunosuppressant is cyclosporin A and/or rapamycin.
  • a dose of the immunosuppressant in the composition may be appropriately chosen, depending upon the amount of alkyl-substituted fatty acid in the composition, the extent of leukocyte proliferation and/or activation to be inhibited, the type of rejection occurring or the type of graft versus host disease to be treated, the age and body weight of the subject or patient, and the frequency of administration.
  • this agent is present in the composition such that when administered to a subject the concentration of the agent at the site of action is in the range from 10 nM to 2 ⁇ M. More preferably, this agent is present in the composition such that when administered to a subject the concentration of the agent at the site of action is in the range from 10 nM to 100 nM.
  • this agent is present in the composition such that when administered to a subject the concentration of the agent at the site of action is in the range from 0.1 nM to 30 nM. More preferably, this agent is present in the composition such that when administered to a subject the concentration of the agent at the site of action is in the range from 0.1 nM to 10 nM.
  • the composition may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, or any other additive that aids in the control of the release of the alkyl-substituted fatty acid or the immunosuppressant or aid in the delivery of the alkyl-substituted fatty acid or the immunosuppressant to a subject.
  • pharmaceutically acceptable additives including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, or any other additive that aids in the control of the release of the alkyl-substituted fatty acid or the immunosuppressant or aid in the delivery of the alkyl-substituted fatty acid or the immunosuppressant to a subject.
  • the present invention provides the use of an alkyl-substituted fatty acid and an immunosuppressant for the preparation of a medicament for inhibiting leukocyte proliferation and/or rejection of transplanted material, wherein the alkyl-substituted fatty acid has the following chemical formula:
  • 12-methyltetradecanoic acid and other alkyl-substituted fatty acids were obtained from Sigma Chemicals.
  • alkyl-substituted fatty acids were prepared in a similar manner.
  • Human Dendritic cells were prepared by differentiating monocytes by treatment with IL-4 and GMCSF for 5 days in culture and then for a further two days with TNF-alpha. Human dendritic cells are potent stimulators of allogeneic lymphocytes. A mixed lymphocyte culture comprising of mature DC and unrelated lymphocytes will induce lymphocyte proliferation. The lymphocyte proliferation can then be assayed by tritiated thymidine uptake. A stimulator (dendritic cells) to responder (lymphocytes) ratio of 1 :1000, 1 :100, and 1 :10 generates proliferation in a dose response manner (ie more dendritic cells - more proliferation).
  • Table 1 shows the results of the MLR and the effect of the addition of 100 ⁇ M 12-methyltetradecaoic acid to the MLR on the proliferation of lymphocytes. As can be seen, 12-MTA caused a significant inhibition of lymphocyte proliferation at all stimulator to responder ratios.
  • a two-way MLR was performed using lymphocyes from two unrelated individuals to assess the inhibition of lymphocyte proliferation by 12- methyltetradecanoic acid in the presence of cyclosporin A or rapamycin.
  • the data is shown in Table 2.
  • the effect of 12-methyltetradecanoic acid and 12- methyltetradecanoic acid in combination with cyclosporin A and rapamycin on the MLR is reported as % inhibition with respect to untreated MLR.
  • 12-methyltetradecanoic acid inhibited lymphocyte proliferation in a dose dependent manner.
  • cyclosporin A an inhibitor of the phosphatase activity of calcineurin
  • rapamycin which acts to block signal transduction mediated by IL2 and other costimulatory pathways
  • Cyclosporin A augments the ability of 12-methyltetradecanoic acid to inhibit lymphocyte proliferation
  • rapamycin also augments the ability of 12-methyltetradecanoic acid to inhibit lymphocyte proliferation, particular at higher concentrations of 12-methyltetradecanoic acid.
  • Table 3 shoes the data from a 2-way MLR performed with mouse splenocytes from two allogeneic strains of mice (BALB/c and CBA).
  • a dose response for the inhibition with 12-MTA was shown at a concentration of 25 ⁇ M to 200 ⁇ M.
  • the combination of 50 ⁇ M 12-MTA with 10 nM cyclosporin A demonstrated a greater inhibition (83%) of the murine MLR compared with 50 ⁇ M 12-MTA (54%) and 10 nM cyclosporin A (65%) alone.
  • Table 3 12-MTA and cyclosporin A show synergistic inhibition of MLR
  • cyclosporin A may be used in lower concentrations when used in combination with an alkyl-substituted fatty acid.
  • a two-way mixed lymphocyte reaction was performed using human peripheral blood mononuclear cells (PBMNC) from unrelated donors. Cells from each individual (1 x 10 5 cells in 50 ⁇ l) were mixed into each round-bottomed well of a 96-well plate. Control wells did not contain alkyl-substituted fatty acid and test wells contained alkyl-substituted fatty acid at concentrations of 25, 50, 100, 200 and 400 ⁇ M. Four replicate wells were performed for each test.
  • PBMNC peripheral blood mononuclear cells
  • the data shown in Table 4 shows a dose response inhibitory profile for the various alkyl-substituted fatty acids.
  • the alkyl-substituted fatty acids differed in the carbon chain lengths (C12 to C19). In general all the alkyl-substituted fatty acids inhibited the MLR response in a dose response manner with the exception of 10-methyl octadecanoic acid, which inhibited only at the highest concentration tested.
  • Alkyl- substituted fatty acids inhibit 2-way MLR - synergy with cyclosporin A
  • FIGS. 1 and 2 demonstrate the ability of the alkyl- substituted fatty acids 12-MTA (12-methyl tetradecanoic acid) and 16-MHA (16- methyl heptadecanoic acid) to inhibit a 2-way MLR in combination with a suboptimal concentration of 10ng/ml cyclosporin A using human PBMNC.
  • 12- MTA and 16-MHA were used at 400 ⁇ M or 200 ⁇ M alone, at a concentration of 200 ⁇ M in combination with 10 ng/ml cyclosporin A, or cyclosporin A at 10 ng/ml was used alone.
  • Figure 3 demonstrates 2-way MLR data obtained by mixing mouse splenocytes obtained from two allogeneic strains of mice (Balb/c and CBA).
  • 12-MTA was used alone at a concentration of 200 ⁇ M , 100 ⁇ M, 50 ⁇ M or 25 ⁇ M, 50 ⁇ M 12-MTA was used in combination with 10 ng/ml cyclosporin A, or 10 ng/mi cyclosporin A was used alone.
  • Assays were performed in round-bottomed wells of a 96-well plate where equal numbers of mouse splenocytes were mixed to produce an allogeneic mixed lymphocyte reaction. Cultures were incubated for 96 hours at 37°C in C0 2 incubator and then pulsed for 18 hours with tritiated thymidine before harvesting cells onto a filter mat that was subject to scintillation counting. A dose response for the inhibition was shown at a concentration range of 400 to 25 ⁇ M 12-MTA. The combination of 50 ⁇ M 12-MTA with 10ng/ml CsA demonstrated a synergistic inhibition of the murine MLR as was observed with the human cells.
  • dendritic cells are the most potent antigen presenting cells and are capable of provoking a strong immune response, these cells were used in in- vitro assays of alloimmune activation of lymphocytes.
  • DC Human dendritic cells
  • the adherent monocytes were cultured in the presence of rlL4 and GMCSF for 5 days and then for a further 2 days in TNF to yield mature monocyte-derived DC.
  • These DC were used as stimulators of allogeneic lymphocytes in a DC-MLR reaction as shown in Figure 4.
  • the DC (stimulators) were mixed with lymphocytes (responders) in ratios of 10 4 :10 5 (S:R 1 :10), 10 3 :10 5 (S:R 1 :100) and 10 2 :10 5 (S:R 1:1000)in 96-well round-bottomed plates.
  • the alkyl-substituted fatty acids were solubilized in absolute ethanol and added to a final working concentration of 200 ⁇ M in the DC-lymphocyte culture medium.
  • Control reactions (UT-untreated) had no additions, but had 0.2- 0.4%v/v ethanol to account for any baseline effects attributed to the ethanol in the alkyl-substituted fatty acid treatment groups.
  • Each experimental point was calculated from 5 replicate wells +/- 1 S.D.
  • the data in figure 4 demonstrates that there was a significant inhibition in lymphocyte proliferation in all alkyl-substituted fatty acid treatment groups compared to the control group.
  • 17-MODA (17-methyl octadecanoic acid)
  • 16-MHA (16-methyl heptadecanoic acid) were potent inhibitors of the DC-MLR.
  • Strong to moderate inhibition of the DC-MLR was observed for 12- MTA (12-methyl tetradecanoic acid) and 10-MDDA (10-methyl dodecanoic acid) compared to the untreated control group. Inhibition was observed in all ratios of stimulator to responder cells.
  • This data indicates that the inhibition of lymphocyte proliferation by the alkyl- substituted fatty acid may be directly affecting either the responding lymphocyte or the stimulatory DC populations or both populations of cells in the MLR.
  • Alkyl-substituted fatty acids inhibit mitogen induced lymphocyte proliferation
  • PBMNC stimulated with ConA show high proliferation as indicated by the high tritiated thymidine incorporation.
  • the addition of alkyl- substituted fatty acid demonstrated a dose response inhibition of lymphocyte proliferation. Similar to the DC-MLR experiments, the strongest inhibition was demonstrated by the addition of 17-MODA and 16-MHA. Strong inhibitions were also noted for 12-MTA and 10-MDDA.
  • alkyl-substituted fatty acids can exert a direct inhibition of lymphocyte proliferation in the absence of allostimulatory DC.
  • Alkyl-substituted fatty acids inhibit the proliferation of ConA stimulated lymphocytes in a CFSE assay
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • the experiment consisted of PBMNC labelled with CFSE that were stimulated with ConA in the presence or absence of 200 ⁇ M 12-MTA. Cells were cultured for 6 days and then examined in a Becton and Dickinson Flow cytometer.
  • Figure 6 demonstrates that the ConA-stimulated lymphocytes showed a reduction in fluorescence intensity indicating that cell proliferation occurred, with 46% of cells growth arrested. Whereas the ConA-stimulated cells that were treated with 12-MTA (200 ⁇ M) showed 93% of the cells growth arrested.
  • the unstimulated, CFSE-labelled PBMNC served as a baseline control.
  • Figure 7 shows a dot-plot analysis of CFSE-labelled PBMNCs stained with phycoerythrin conjugated anti-CD4 or anti-CD8 mAb.
  • the data demonstrates that the CD4 and CD8 subpopulation in unstimulated CFSE-labelled PBMNC remained growth arrested.
  • stimulation with ConA dispayed a decrease in CFSE signal in both CD4 and CD8 cells, indicating proliferation had occurred.
  • ConA-treated PBMNC in the presence of 12-MTA or 16-MHA showed profound inhibition of CD4 and CD8 T cell proliferation, with 16-MHA > 12-MTA.
  • the ability of alkyl-substituted fatty acids to inhibit the DC-MLR may be attributed to specific effects of the alkyl-substituted fatty acid on the antigen presenting dendritic cells.
  • Monocyte-derived dendritic cells were prepared from adherent monocytes that were cultured in IL4 and GMCSF in the presence or absence of the appropriate alkyl-substituted fatty acid from the start of culture for 5 days and then for a further 48 hours in the presence of TNF-alpha, which is a DC maturation signal. After TNF-alpha maturation, cells were stained for the DC maturation markers using mouse anti- human monoclonal antibodies against CD1a, CD83 and MHCclassll and the costimulatory molecules CD80, CD86 and CD40. The results are shown in Figure 8.
  • PBMNC were stimulated with ConA and were further treated with 12-MTA or 16-MHA at a concentration of 200 ⁇ M. After 24-48 in culture the ConA treated cells showed the formation of lymphocyte aggregates and these aggregates showed maximal increase in size at day 5. However the alkyl-substituted fatty acid treated PBMNC showed reduced cluster sizes with profound reductions observed for the 16-MHA treated cells, as shown in Figure 10.
  • the donor heart was transplanted heterotopically into the recipient abdomen according to the method described by Corry, R.J. et al. (1973) Transplantation 16(4): 343-350.
  • Donor hearts from Balb/c mice were perfused through the vena cava and aorta with cold heparinized saline before harvesting and ligation of the vena cava and pulmonary veins.
  • the donor pulmonary artery was anastomosed to the inferior vena cava of the recipient (CBA mouse), and the donor aorta was anastomosed to the recipient infra-renal abdominal aorta with a 10-0 nylon suture.
  • Cold ischemia time was 60 ⁇ 15 min.
  • Heart allograft function was monitored by daily palpation and graded (based on table shown below), and the day of heart beat cessation was regarded as the day of rejection.
  • mice that received heart transplants were either untreated, or treated with Cyclosporin A (CsA) or 12-MTA.
  • Untreated mice were essentially injected with phosphate-buffered saline.
  • CsA was administered to mice by intra-peritoneal injections at a concentration of 0.5 mg/ml at a dose of 5mg/kg body weight.
  • the 12-MTA was dissolved in neat CBA mouse serum at a concentration of 10 mg/ml and mice were administered by intra-peritoneal injections at doses of 100 to 200mg/kg body weight.
  • mice All mice were injected with the appropriate treatment on the day of transplantation.
  • the data is shown in Table 5.
  • mice rejected at a mean time of 10.2 days compared to 14 days with the CsA treated group.
  • Mice treated at a dose of 100mg/kg of 12-MTA did not show an improved graft survival.
  • mice were treated with a higher dose of 12-MTA of 200mg/kg an improved heart allograft survival of 22 days was demonstrated indicating that 12-MTA has the ability to prolong graft allograft survival.
  • Fisher F344 kidney grafts may be transplanted into Lewis recipients to examine the chronic rejection of kidney allografts as described in McGrath, J. and Shehata, M. (2001 ) Transplant Proc. 33(31:2191 -2192.
  • Donor nephrectomy may be performed on F344 rats and the kidney transplanted orthotopically into Lewis recipient.
  • the recipient rats may be subject to ipsilateral native nephrectomy prior to implantation of the graft and the contralateral native kidney left in situ.
  • Retrieved kidneys may be perfused with organ preservation solution and stored on ice during the procedure and ischaemic time kept to a minimum.
  • Animals may then be terminated at 2 months and 4 months and examined for histological changes.
  • significant vascular changes consisting of intimal proliferation, disruption of the internal elastic lamina and medial thinning will be noted.
  • Moderate glumerulosclerosis will also be evident with thickening of the basement membrane.
  • tubular atrophy and marked interstitial cellular infiltrate and interstitial sclerosis.
  • the changes will be more drastic, the majority of glomeruli likely to be sclerotic and there will be marked interstitial sclerosis.

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Abstract

L'invention concerne un procédé d'inhibition de la prolifération des leucocytes dans un système biologiques, ledit procédé consistant à administrer au système biologique, une quantité efficace d'un acide gras alkyl-substitué, ledit acide gras alkyl-substitué étant capable d'inhiber la prolifération des leucocytes.
PCT/AU2003/000523 2002-05-03 2003-05-02 Procede d'inhibition du rejet de matiere transplantee Ceased WO2003092672A1 (fr)

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AU2003227120A AU2003227120A1 (en) 2002-05-03 2003-05-02 Method of inhibiting rejection of transplanted material

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AUPS2124A AUPS212402A0 (en) 2002-05-03 2002-05-03 Method for inhibiting rejection of transplanted material
AUPS2124 2002-05-03
AU2003900961A AU2003900961A0 (en) 2003-03-03 2003-03-03 Method of inhibiting rejection of transplanted material
AU2003900961 2003-03-03

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999053086A1 (fr) * 1998-04-14 1999-10-21 Zhenhua Yang Composes anticancereux a petites molecules et leurs procedes de production
CA2297933A1 (fr) * 2000-02-07 2001-08-07 Zhenhua Yang Une methode pour ameliorer la fonction immunitaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999053086A1 (fr) * 1998-04-14 1999-10-21 Zhenhua Yang Composes anticancereux a petites molecules et leurs procedes de production
CA2297933A1 (fr) * 2000-02-07 2001-08-07 Zhenhua Yang Une methode pour ameliorer la fonction immunitaire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Derwent World Patents Index; Class B05, AN 2001-607718/70 *

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