WO2011091167A2 - Ciblage du récepteur de l'acide lysophosphatidique pour sclérodermie et autres maladies fibrosantes - Google Patents

Ciblage du récepteur de l'acide lysophosphatidique pour sclérodermie et autres maladies fibrosantes Download PDF

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WO2011091167A2
WO2011091167A2 PCT/US2011/021927 US2011021927W WO2011091167A2 WO 2011091167 A2 WO2011091167 A2 WO 2011091167A2 US 2011021927 W US2011021927 W US 2011021927W WO 2011091167 A2 WO2011091167 A2 WO 2011091167A2
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fibrosis
receptor
lpa
mice
peritoneal
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PCT/US2011/021927
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WO2011091167A3 (fr
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Andrew M. Tager
Andrew D. Luster
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The General Hospital Corporation
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Publication of WO2011091167A3 publication Critical patent/WO2011091167A3/fr

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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is related to the field of fibrosis.
  • the invention provides for a role of the LPAl receptor in the development and/or maintenance of fibrotic diseases involving tissues including, but not limited to, epidermal, dermal peritoneal, renal, hepatic, cardiac, lymphoid, ocular, bone cortex, and bone marrow.
  • Genetic deletion of the LPAl receptor and/or pharmacological antagonism with selective receptor inhibitors prevents and/or treats fibrosis symptoms (i.e., for example, increased tissue thickness and/or collagen content).
  • Empirical analysis shows that the LPA2 receptor system does not play a role in most fibrotic diseases, thereby suggesting that LPA signaling specifically through LPAl may provide clinically useful targets for the prevention and treatment of fibrosis.
  • Tissue injury initiates a complex series of host wound-healing responses. If successful, these responses restore normal tissue stmcture and function. If not successful, these responses can lead to tissue fibrosis and loss of function.
  • tissue fibrosis Aberrant wound- healing responses to pulmonary injury are thought to contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF).
  • IPF idiopathic pulmonary fibrosis
  • Selman et al. "Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy” Ann Intern Med 134:136-151 (2001).
  • IPF and other fibrotic lung diseases are associated with high morbidity and mortality, and are generally refractory to currently available pharmacological therapies. Better identification of the mediators linking lung injury and pulmonary fibrosis is needed to recognize new therapeutic targets for these important diseases.
  • SSc Systemic sclerosis
  • Characterization of the mediators driving these processes will identify a set of rational targets for therapies aiming to reverse, or at least slow the progression of fibrosis. What is needed in the art is a selective receptor antagonist that prevents the development, expression and maintenance of dermal fibrotic diseases, such as systemic sclerosis.
  • the present invention is related to the field of fibrosis.
  • the invention provides for a role of the LPAl receptor in the development and/or maintenance of fibrotic diseases involving tissues including, but not limited to, epidermal, dermal, peritoneal, renal, hepatic, cardiac, lymphoid, ocular, bone cortex, and bone marrow.
  • Genetic deletion of the LPAl receptor and/or pharmacological antagomsm with selective receptor inhibitors prevents and/or treats fibrosis symptoms (i.e., for example, increased tissue thickness and/or collagen content).
  • Empirical analysis shows that the LPA2 receptor system does not play a role in most fibrotic diseases, thereby suggesting that LPA signaling specifically through LPAl may provide clinically useful targets for the prevention and treatment of fibrosis.
  • the present invention contemplates a method, comprising: a) providing: i) a subject at risk for development of a fibrosis; ii) a compound comprising an affinity for a lysophosphatidic acid receptor; and b) administering said compound to said subject under conditions such that said fibrosis is prevented or reduced.
  • the lysophosphatidic acid receptor is a lysophosphatidic acid 1 receptor.
  • the compound comprises a lysophosphatidic acid receptor inhibitor.
  • the receptor inhibitor is selected from the group consisting of a small organic molecule, an amino acid sequence, a nucleic acid sequence, and an antisense sequence.
  • the antisense sequence comprises a lysophosphatidic acid receptor 1 antisense sequence.
  • the small organic molecule is AM095.
  • the fibrosis comprises a tissue selected from the group consisting of epidermal, dermal, peritoneal, renal, hepatic, cardiac, lymphoid, ocular, bone cortex, and bone marrow. In one embodiment, the fibrosis comprises post-transplantation fibrosis.
  • the fibrosis comprises multi-organ fibrosis, hi one embodiment, the fibrosis is caused by an injury selected from the group consisting of toxin exposure, surgical procedure injury, infection, and accidental injury, hi one embodiment, the fibrosis is caused by a medical condition selected from the group consisting of cirrhosis, viral hepatitis, schistosomiasis, alcoholism, nephrosclerosis, end stage kidney disease, hypertension, diabetes, cardiomyopathy, heart attack, myocarditis, hypertrophic cardiomyopathy, macular degeneration, retinal retinopathy, vitreal retinopathy, keloids, hypertrophic scars, inflammatory bowel disease, ulcerative colitis, Chron's disease, acquired immunity deficiency syndrome, filariasis, cancer, myleofibrosis, allograft rejection, graft versus host disease, surgical complications, drug exposure, radiation exposure, contracture, pain, and infertility, i one embodiment, the fibrosis comprises symptoms, drug exposure
  • the administering is selected from the group consisting of topical, oral, parenteral, pulmonary, anal, vaginal, ocular, and intranasal.
  • the present invention contemplates a method, comprising: a) providing: i) a subject comprising a fibrosis; ii) a compound comprising an affinity for a lysophosphatidic acid receptor; b) administering said compound to said subject under conditions such that said fibrosis is reduced.
  • the lysophosphatidic acid receptor is a lysophosphatidic acid 1 receptor
  • the compound comprises a lysophosphatidic acid receptor inhibitor.
  • the receptor inhibitor is selected from the group consisting of a small organic molecule, an amino acid sequence, a nucleic acid sequence, and an antisense sequence.
  • the antisense sequence comprises a lysophosphatidic acid receptor 1 antisense sequence, i one embodiment, the small organic molecule is AM095.
  • the fibrosis comprises a tissue selected from the group consisting of epidermal, dermal, peritoneal, renal, hepatic, cardiac, lymphoid, ocular, bone cortex, and bone marrow.
  • the fibrosis comprises post-transplantation fibrosis, hi one embodiment, the fibrosis comprises multi-organ fibrosis.
  • the fibrosis is caused by an injury selected from the group consisting of toxin exposure, surgical procedure injury, infection, and accidental injury.
  • the fibrosis is caused by a medical condition selected from the group consisting of cirrhosis, viral hepatitis, schistosomiasis, alcoholism, nephrosclerosis, end stage kidney disease, hypertension, diabetes, cardiomyopathy, heart attack, myocarditis, hypertrophic cardiomyopathy, macular degeneration, retinal retinopathy, vitreal retinopathy, keloids, hypertrophic scars, inflammatory bowel disease, ulcerative colitis, Chron's disease, acquired immunity deficiency syndrome, filariasis, cancer, myleofibrosis, allograft rejection, graft versus host disease, surgical complications, drug exposure, radiation exposure, contracture, pain, and infertility.
  • a medical condition selected from the group consisting of cirrhosis, viral hepatitis, schistosomiasis, alcoholism, nephrosclerosis, end stage kidney disease, hypertension, diabetes, cardiomyopathy, heart attack, myocarditis,
  • the fibrosis comprises symptoms selected from the group consisting of increased tissue thickness, increased collagen content, increased hydroxyproline content, increased a-SMA + myofibroblasts and phosphoSmad2 + cells.
  • the composition further comprises at least one additional drug.
  • the additional drug comprises an antiproliferative drug.
  • the administering is selected from the group consisting of topical, oral, parenteral, pulmonary, anal, vaginal, ocular, and intranasal.
  • the present invention contemplates a method, comprising: a) providing; i) an isolated lysophosphatidic acid receptor, wherein said receptor is derived from a fibroblast; and ii) a test compound capable of an interaction with said receptor; b) contacting said receptor with said test compound; and c) detecting said interaction of said receptor with said test compound.
  • the fibroblast is derived from a tissue selected from the group consisting of epidermal, dermal, peritoneal, renal, hepatic, cardiac, lymphoid, ocular, bone cortex, and bone marrow.
  • the test compound is selected from the group consisting of an amino acid sequence, a nucleic acid sequence, a small organic molecule.
  • the small organic molecule is AM095.
  • the nucleic acid sequence is an antisense sequence.
  • the amino acid sequence is an antibody.
  • the present invention contemplates a method, comprising: a) providing; i) a cell culture comprising a lysophosphatidic acid receptor; ii) a test compound capable of binding to said receptor portion; and iii) a portion of a lysophosphatidic acid molecule; b) contacting said lysophosphatidic acid molecule portion with said cell culture in the presence of said test compound; and c) detecting a response of said cell culture, wherein said response identifies said test compound as a lysophosphatidic acid receptor inhibitor.
  • the lysophosphatidic acid receptor is a lysophosphatidic acid 1 receptor.
  • the cell culture is selected from the group consisting of an epidermal cell culture, a peritoneal cell culture, a renal cell culture, a hepatic cell culture, a cardiac cell culture, a lymphoid cell culture, an ocular cell culture, a bone cortical cell culture and a bone marrow cell culture.
  • the test compound is selected from the group consisting of an amino acid sequence, a nucleic acid sequence, a small organic molecule.
  • the small organic molecule is AM095.
  • the nucleic acid sequence is an antisense sequence.
  • the amino acid sequence is an antibody.
  • the present invention contemplates a kit, comprising: a) a first container comprising at least one small organic molecule capable of binding to an LPA1 receptor; b) a second container comprising a pharmaceutically acceptable formulation; and c) a set of instructions for administering said at least one small organic molecule and said formulation to a patient exhibiting at least one symptom of a fibrosis.
  • the small organic molecule is AM095.
  • the present invention contemplates a kit, comprising: a) a first container comprising an LPA1 antisense sequence; b) a second container comprising a pharmaceutically acceptable formulation; and c) a set of instructions for administering said antisense sequence and said formulation to a patient exhibiting at least one symptom of a fibrosis.
  • the antisense sequence is complementary to an LPA1 deoxyribonucleic acid coding region sequence.
  • the present invention contemplates a kit, comprising: a) a first container comprising at least one antibody specifically directed to an LPA1 receptor; b) a second container comprising a pharmaceutically acceptable formulation; and c) a set of instructions for administering said at least one antibody and said formulation to a patient exhibiting at least one symptom of a fibrosis.
  • the present invention contemplates a method, comprising: a) providing: i) a subject at risk for an epidermal or dermal injury, wherein said injury is capable of resulting in fibrosis; ii) a compound comprising an affinity for a lysophosphatidic acid receptor; b) administering said compound to said subject before said injury, under conditions such that said fibrosis is prevented or reduced, i one embodiment, the lysophosphatidic acid receptor is a lysophosphatidic acid 1 receptor.
  • the compound comprises a lysophosphatidic acid receptor inhibitor
  • the receptor inhibitor includes, but is not limited to a small organic molecule, an amino acid sequence, a nucleic acid sequence, and an antisense sequence.
  • the antisense sequence comprises a lysophosphatidic acid receptor 1 antisense sequence.
  • the small organic molecule is AM095.
  • the injury comprises a skin injury. In one embodiment, the skin injury comprises systemic sclerosis.
  • the epidermal or dermal injury includes, but is not limited to, toxin exposure, surgical procedure injury, infection, and accidental injury
  • the fibrosis comprises symptoms including, but not limited to, increased tissue thickness, increased collagen content, increased hydroxyproline content, increased a-SMA + myofibroblasts and phosphoSmad2 + cells.
  • the composition further comprises at least one additional drug, hi one embodiment, the additional drug comprises an antiproliferative drug, hi one embodiment, the administering includes, but is not limited to, topical, oral, parenteral, pulmonary, anal, vaginal, ocular, and intranasal.
  • the present invention contemplates a method, comprising: a) providing: i) a subject comprising an epidermal or dermal injury, wherein said injury resulted in a fibrosis; ii) a compound comprising an affinity for a lysophosphatidic acid receptor; b) administering said compound to said subject after said injury, under conditions such that said fibrosis is reduced, hi one embodiment, the lysophosphatidic acid receptor is a
  • the compound comprises a
  • the receptor inhibitor includes, but is not limited to, a small organic molecule, an amino acid sequence, a nucleic acid sequence, and an antisense sequence.
  • the antisense sequence comprises a lysophosphatidic acid receptor 1 antisense sequence.
  • the small organic molecule is AM095.
  • the injury comprises a skin injury.
  • the skin injury comprises systemic sclerosis.
  • the epidermal or dermal injury includes, but is not limited to, toxin exposure, surgical procedure injury, infection, and accidental injury.
  • the fibrosis comprises symptoms including, but not limited to, increased tissue thickness, increased collagen content, increased hydroxyproline content, increased a-SMA + myofibroblasts and phosphoSmad2 + cells.
  • the composition further comprises at least one additional drug.
  • the additional drug comprises an antiproliferative drug, hi one embodiment, the administering includes, but is not limited to, topical, oral, parenteral, pulmonary, anal, vaginal, ocular, and intranasal.
  • the present invention contemplates a method, comprising: a) providing; i) an isolated lysophosphatidic acid receptor, wherein said receptor is derived from a fibroblast; and ii) a test compound capable of an interaction with said receptor; b) contacting said receptor with said test compound; and c) detecting said interaction of said receptor with said test compound.
  • the fibroblast is derived from an epidermal tissue.
  • the test compound includes, but is not limited to, an amino acid sequence, a nucleic acid sequence, or a small organic molecule.
  • the small organic molecule is AM095.
  • the nucleic acid sequence is an antisense sequence.
  • the amino acid sequence is an antibody.
  • the present invention contemplates a method, comprising: a) providing; i) a cell culture comprising a lysophosphatidic acid receptor; ii) a test compound capable of binding to said receptor portion; and iii) a portion of a lysophosphatidic acid molecule; b) contacting said lysophosphatidic acid molecule portion with said cell culture in the presence of said test compound; and c) detecting a response of said cell culture, wherein said response identifies said test compound as a lysophosphatidic acid receptor inhibitor.
  • the lysophosphatidic acid receptor is a lysophosphatidic acid 1 receptor.
  • the cell culture is an epidermal cell culture.
  • the test compound includes, but is not limited to, an amino acid sequence, a nucleic acid sequence, or a small organic molecule.
  • the small organic molecule is AM095.
  • the nucleic acid sequence is an antisense sequence
  • the amino acid sequence is an antibody.
  • the present invention contemplates a kit, comprising: a) a first container comprising at least one small organic molecule capable of binding to an LPA1 receptor; b) a second container comprising a pharmaceutically acceptable formulation; and c) a set of instructions for administering said at least one small organic molecule and said formulation to a patient exhibiting at least one symptom of a dermal fibrosis.
  • the small organic molecule is AM095.
  • the present invention contemplates a kit, comprising: a) a first container comprising an LPA1 antisense sequence; b) a second container comprising a pharmaceutically acceptable formulation; and c) a set of instructions for administering said antisense sequence and said formulation to a patient exhibiting at least one symptom of a dermal fibrosis.
  • the antisense sequence is complementary to an LPA1 deoxyribonucleic acid coding region sequence.
  • the present invention contemplates a kit, comprising: a) a first container comprising at least one antibody specifically directed to an LPA1 receptor; b) a second container comprising a pharmaceutically acceptable formulation; and c) a set of instructions for administering said at least one antibody and said formulation to a patient exhibiting at least one symptom of a dermal fibrosis.
  • the present invention contemplates a method, comprising: a) providing: i) a subject at risk for a peritoneal injury, wherein said injury is capable of resulting in fibrosis; ii) a compound comprising an affinity for a lysophosphatidic acid receptor; b) administering said compound to said subject before said injury, under conditions such that said fibrosis is prevented or reduced, hi one embodiment, the lysophosphatidic acid receptor is a lysophosphatidic acid 1 receptor. In one embodiment, the compound comprises a lysophosphatidic acid receptor inhibitor.
  • the receptor inhibitor includes, but is not limited to a small organic molecule, an amino acid sequence, a nucleic acid sequence, and an antisense sequence.
  • the antisense sequence comprises a lysophosphatidic acid receptor 1 antisense sequence.
  • the small organic molecule is AM095.
  • the injury comprises an intestinal injury.
  • the intestinal injury comprises peritonitis, i one embodiment, the peritoneal injury includes, but is not limited to, peritoneal dialysis fluid exposure, toxin exposure, surgical procedure injury, infection, and accidental injury.
  • the fibrosis comprises symptoms including, but not limited to, increased peritoneal thickness, increased collagen content, increased hydroxyproline content, increased a-SMA +
  • the composition further comprises at least one additional drug.
  • the additional drug comprises an antiproliferative drug.
  • the administering includes, but is not limited to, topical, oral, parenteral, pulmonary, anal, vaginal, ocular, and intranasal.
  • the present invention contemplates a method, comprising: a) providing: i) a subject comprising a peritoneal injury, wherein said injury resulted in a fibrosis; ii) a compound comprising an affinity for a lysophosphatidic acid receptor; b) administering said compound to said subject after said injury, under conditions such that said fibrosis is reduced.
  • the lysophosphatidic acid receptor is a
  • the compound comprises a lysophosphatidic acid receptor inhibitor.
  • the receptor inhibitor includes, but is not limited to, a small organic molecule, an amino acid sequence, a nucleic acid sequence, and an antisense sequence.
  • the antisense sequence comprises a lysophosphatidic acid receptor 1 antisense sequence.
  • the small organic molecule is AM095.
  • the injury comprises an intestinal injury. In one embodiment, the intestinal injury comprises peritonitis.
  • the peritoneal injury includes, but is not limited to, peritoneal dialysis fluid exposure, toxin exposure, surgical procedure injury, infection, and accidental injury
  • the fibrosis comprises symptoms including, but not limited to, increased peritoneal thickness, increased collagen content, increased hydroxyproline content, increased a-SMA+ myofibroblasts and phosphoSmad2+ cells
  • the composition further comprises at least one additional drug.
  • the additional drug comprises an antiproliferative drug.
  • the administering includes, but is not limited to, topical, oral, parenteral, pulmonary, anal, vaginal, ocular, and intranasal.
  • the present invention contemplates a composition comprising a lysophosphatidic acid receptor inhibitor, wherein said inhibitor is AM095.
  • the composition further comprises a pharmaceutically acceptable formulation.
  • the formulation comprises a controlled release formulation.
  • the formulation includes, but is not limited to, a cream, a gel or a liquid.
  • fibrosis refers to any medical condition marked by increase of interstitial fibrous tissue.
  • skin fibrosis is characterized by a scarring or thickening of an epidermal layer tissue (i.e., for example, skin).
  • Fibrosis symptoms include, but are not limited to, increased tissue thickness, increased collagen content, increased hydroxyproline content, increased a-SMA + myofibroblasts and phosphoSmad2 + cells.
  • inhibitory compound refers to any compound capable of interacting with (i.e., for example, attaching, binding etc) to a binding partner (i.e., for example, an LPA1 receptor) under conditions such that the binding partner becomes unresponsive to its natural ligands.
  • Inhibitory compounds may include, but are not limited to, small organic molecules, amino acid sequences such as antibodies, proteins, and/or peptides, or nucleic acid sequences such as antisense sequences.
  • an inhibitory compound may include, but is not limited to, a small organic molecule such as AM095.
  • lysophosphatidic acid receptor refers to any protein capable of binding lysophosphatidic acid (LP A).
  • LP A receptor may reside in the cell membrane and respond to circulating levels of LP A in order to mediate various physiological responses. The type of response depends upon LPA receptor subtype (i.e., for example, LPA1, LPA2, LP A3, LPA4, or LPA5).
  • injury denotes a bodily disruption of the normal integrity of any tissue structure.
  • the term is intended to encompass surgery.
  • the term is intended to encompass irritation, inflammation, infection, and/or the development of fibrosis.
  • the term is intended to encompass wounds including, but not limited to, contused wounds, incised wounds, lacerated wounds, nonpenetrating wounds (i. e., wounds in which there is no disruption of the skin but there is injury to underlying structures), open wounds, penetrating wound, perforating wounds, puncture wounds, septic wounds, subcutaneous wounds, bum injuries etc.
  • Conditions related to wounds or sores which may be successfully treated according to the invention are skin diseases.
  • peritoneal injury refers to any effect on any abdominal tissue (i.e., for example, peritoneal lining, stomach, intestine, bowel, bladder etc.) that impairs its functional and/or structural integrity.
  • abdominal tissue i.e., for example, peritoneal lining, stomach, intestine, bowel, bladder etc.
  • injury may be a result of, but not limited to, exposure to toxins, surgical procedures, or accident.
  • tissue injury refers to any effect on epidermal tissue that impairs its functional and/or structural integrity.
  • injury may be a result of, but not limited to, toxin exposure, surgical procedures, or accident.
  • binding refers to any interaction between at least two compounds and/or molecules. Such binding may be reversible or irreversible. Such binding may be, but is not limited to, non-covalent binding, covalent bonding, ionic bonding, Van de Waal forces or friction, and the like.
  • Attachment refers to any interaction between a medium (or carrier) and a drug. Attachment may be reversible or irreversible. Such attachment includes, but is not limited to, covalent bonding, ionic bonding, Van der Waals forces or friction, and the like.
  • a drug is attached to a medium (or carrier) if it is impregnated, incorporated, coated, in suspension with, in solution with, mixed with, etc.
  • a medium refers to any material, or combination of materials, which serve as a carrier or vehicle for delivering of a drug or compound to a treatment point (e.g., wound, surgical site etc.).
  • a treatment point e.g., wound, surgical site etc.
  • a medium can comprise a carrier, wherein said carrier is attached to a drug or compound and said medium facilitates delivery of said carrier to a treatment point.
  • a carrier may comprise an attached drug or compound wherein said carrier facilitates delivery of said drug or compound to a treatment point.
  • a medium is selected from the group including, but not limited to, foams, gels (including, but not limited to, hydrogels), xerogels, microparticles (i.e., microspheres, liposomes, microcapsules etc.), bioadhesives, or liquids. Such combinations of microparticles may be combined with hydrogels, bioadhesives, foams or liquids.
  • Any medium contemplated herein may comprise a controlled release formulation.
  • a medium constitutes a drug delivery system that provides a controlled and sustained release of drugs over a period of time lasting approximately from 1 day to 6 months.
  • drug refers to any pharmacologically active substance capable of being administered which achieves a desired effect.
  • Drugs or compounds can be synthetic or naturally occurring, non-peptide, proteins or peptides, oligonucleotides or nucleotides, polysaccharides or sugars.
  • administered or “administering" a drug or compound, as used herein, refers to any method of providing a drug or compound to a patient such that the drug or compound has its intended effect on the patient.
  • one method of administering is by an indirect mechanism using a medical device such as, but not limited to a catheter, applicator gun, syringe etc.
  • a second exemplary method of administering is by a direct mechanism such as, local tissue administration (i. e., for example, extravascular placement), oral ingestion, transdermal patch, topical, inhalation, suppository etc.
  • affinity refers to any attractive force between substances or particles that causes them to enter into and remain in chemical combination.
  • an inhibitor compound that has a high affinity for a receptor will provide greater efficacy in preventing the receptor from interacting with its natural ligands, than an inhibitor with a low affinity.
  • an effective amount refers to a particular amount of a pharmaceutical composition comprising a therapeutic agent (i.e., for example, an LPA1 receptor inhibitor) that achieves a clinically beneficial result.
  • a therapeutic agent i.e., for example, an LPA1 receptor inhibitor
  • derived from refers to any source of a compound or sequence.
  • a compound or sequence may be derived from an organism or particular species.
  • a compound or sequence may be derived from a larger complex or sequence.
  • test compound refers to any compound or molecule considered a candidate as an inhibitory compound.
  • protein refers to any of numerous naturally occurring extremely complex substances (as an enzyme or antibody) that comprise amino acid residues joined by peptide bonds, contain elements including, but not limited to, carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general, a protein comprises amino acids having an order of magnitude within the hundreds.
  • peptide refers to any of various amides that are deri ved from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another and are usually obtained by partial hydrolysis of proteins, hi general, a peptide comprises amino acids having an order of magnitude with the tens.
  • pharmaceutically refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable formulation” as used herein, includes any and all solvents, or a dispersion medium including, but not limited to, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils, coatings, isotonic and absorption delaying agents, liposome, commercially available cleansers, and the like. Supplementary bioactive ingredients also can be incorporated into such carriers.
  • purified may refer to a peptide composition that has been subjected to treatment (i.e., for example, fractionation) to remove various other components, and which composition substantially retains its expressed biological activity.
  • substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition (i.e., for example, weight/weight and/or weight/volume).
  • purified to homogeneity is used to include compositions that have been purified to
  • a purified composition is not intended to mean that some trace impurities may remain.
  • substantially purified refers to molecules, such as an amino acid sequence, that is removed from their natural environment, isolated or separated, and are at least 60%o free, preferably 75% free, and more preferably 90%> free from other components with which they are naturally associated.
  • An “isolated polypeptide” is therefore a
  • amino acid sequence and “polypeptide sequence” as used herein, are interchangeable and to refer to a sequence of amino acids.
  • portion when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein.
  • the fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
  • antibody refers to any immunoglobulin evoked in animals by any immunogen (antigen) and/or those produced by hybridoma technology. It is desired that the antibody demonstrates specificity to epitopes contained in the immunogen.
  • polyclonal antibody refers to immunoglobulin produced from more than a single clone of cells; in contrast “monoclonal antibody” refers to immunoglobulin produced from a single clone of cells.
  • the terms "specific binding” or “specifically binding” when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., for example, an antigenic determinant or epitope) on a protein; in other words an antibody is recognizing and binding to a specific protein structure rather than to proteins in general.
  • an antibody is specific for epitope "A”
  • the presence of a protein containing epitope A (or free, unlabelled A) in a reaction containing labeled "A” and the antibody will reduce the amount of labeled A bound to the antibody.
  • small organic molecule refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size from approximately 10 Da up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • antisense is used in reference to any first nucleic acid sequence which is complementary to, and disrupts the function of, a second nucleic acid sequence.
  • Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a coding strand. Once introduced into a cell, this transcribed strand combines with natural mRNA produced by the cell to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant pheno types may be generated.
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
  • the designation (-) i.e., “negative” is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.
  • sample as used herein is used in its broadest sense and includes environmental and biological samples.
  • Environmental samples include material from the environment such as soil and water.
  • Biological samples may be animal, including, human, fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g., milk), and solid foods (e.g., vegetables).
  • a biological sample suspected of containing nucleic acid encoding a LP A receptor protein may comprise a cell, tissue extract, body fluid,
  • a "variant" of a protein is defined as an amino acid sequence which differs by one or more amino acids from a polypeptide sequence or any homolog of the polypeptide sequence.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
  • a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs including, but not limited to, DNAStar ® software.
  • a “deletion” is defined as a change in an amino acid sequence in which one or more amino acid residues, respectively, are absent as compared to the natural sequence.
  • An "insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more amino acid residues, respectively, as compared to the natural sequence.
  • substitution results from the replacement of one or more amino acids by different amino acids, respectively, as compared to the natural sequence.
  • derivative refers to any chemical modification of an amino acid. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group.
  • a nucleic acid derivative would encode a polypeptide which retains essential biological characteristics.
  • LPA1 receptor biological activity refers to any molecule having structural, regulatory or biochemical functions.
  • LPA1 receptor biological activity may be determined, for example, by restoration of wild-type growth in cells lacking an LPA1 receptor (i.e., for example, LPA1 receptor protein null cells and/or "knock out" cells).
  • Cells lacking LPA1 receptors may be produced by many methods (i.e., for example, point mutation and frame- shift mutation). Complementation is achieved by transfecting cells which lack LPA1 receptors with an expression vector which expresses LPA1 receptor protein, a derivative thereof, or a portion thereof.
  • immunologically active defines the capability of a natural, recombinant or synthetic peptide, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and/or to bind with specific antibodies.
  • antigenic determinant refers to that portion of a molecule that is recognized by a particular antibody (i.e., an epitope).
  • a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants.
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • immunogen refers to any substance capable of generating antibodies when introduced into an animal.
  • an immunogen must contain at least one epitope (the specific biochemical unit capable of causing an immune response), and generally contains many more. Proteins are most frequently used as immunogens, but lipid and nucleic acid moieties complexed with proteins may also act as immunogens. The latter complexes are often useful when smaller molecules with few epitopes do not stimulate a satisfactory immune response by themselves.
  • the terms “complementary” or “complementarity” are used in reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “C-A-G-T,” is complementary to the sequence “G-T-C-A.” Complementarity can be “partial” or “total.”
  • the term “gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA.
  • the sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences.
  • the sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3 1 non-translated sequences.
  • the term “gene” encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene which are transcribed into heterogeneous nuclear RNA (bnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5 Or 3' to the non-translated sequences present on the mRNA transcript).
  • the 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene.
  • the 3' flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.
  • Figure 1 presents exemplary data showing that LPAl knock-out (KO) mice are protected from bleomycin (BLM)-induced dermal fibrosis.
  • Data are from one of two experiments with similar results, each with n >5 wild-type (WT) and LPAl KO mice for both treatment groups.
  • Data are expressed as mean dermal thickness ⁇ SEM or mean hydroxyproline content/6 mm punch biopsy skin sample ⁇ SEM, respectively.
  • Figure 1A H&E (upper panels) and Masson's trichrome staining (lower panels) of the skin of WT and LPAl KO mice following PBS or BLM challenge. Magnification x 100; bar, 100 ⁇ .
  • Figure IB Dermal thickness measured in 5 locations/HPF, in 10 HPF/skin sample, *p ⁇ 0.001, BLM-challenged WT vs. LPAl KO mice; p ⁇ 0.02, WT- BLM vs. WT-PBS mice.
  • Figure 1C Skin hydroxyproline content, *p ⁇ 0.001, BLM-challenged WT vs.
  • LPAl KO mice p ⁇ 0.01, WT-BLM vs. WT-PBS mice.
  • Figure 2 presents exemplary data showing that LPA2 KO mice are not protected from bleomycin-induced dermal fibrosis. Data are from one of two experiments with similar results, each with n >5 WT and LPA2 KO mice for both treatment groups. Data expressed as mean dermal thickness ⁇ SEM or mean hydroxyproline content/6 mm punch biopsy skin sample ⁇ SEM, respectively.
  • Figure 2A H&E (upper panels) and Masson's trichrome staining (lower panels) of the skin of WT and LPA2 KO mice following PBS or BLM challenge. Magnification xlOO; bar, 100 tm.
  • Figure 2B Dermal thickness measured in 5 locations/HPF, in 10 HPF/skin sample.
  • BLM-challenged WT and LPA2 KO mice were not significantly different (NS); p ⁇ 0.002, WT-BLM vs. WT-PBS; pO.001, LPA2 KO-BLM vs. LPA2 KO-PBS mice.
  • Figure 3(C) Skin hydroxyproline content, BLM-challenged WT vs. LPA2 KO mice were not significantly different (NS); p ⁇ 0.003, WT-BLM vs. WT-PBS, p ⁇ 0.002, LPA2 KO-BLM vs. LPA2 KO-PBS mice.
  • Figure 3 presents exemplary data showing bleomycin-induced accumulation of - SMA + myofibroblasts and phosphoSmad2 + cells is diminished in LPAl KO mice.
  • Figure 3 A Skin of WT and LPAl KO mice following PBS- or BLM- challenge stained with anti-a-SMA antibody (magnification x400).
  • Figure 3C Skin of WT and LPAl KO mice following PBS or BLM challenge, stained with anti-phosphoSmad2 (pSmad2) antibody (x400).
  • Figure 4 presents one embodiment of an LPAl antagonist and related exemplary pharmacokinetic data.
  • Figure 4A Chemical structure of the selective LPAl antagonist, AM095 (Sodium ⁇ 4'-[3 -methyl-4-((R)- 1 -phenyl-ethoxycarbonylamino)- isoxazol-5-yl]-biphenyl-4-yl ⁇ -acetate).
  • Figure 4B AM095 inhibition of LPA-induced calcium flux in CHO cells recombinantly expressing human or mouse LPAl .
  • Figure 5 presents exemplary data showing that pharmacological LPAl receptor antagonism attenuates bleomycin-induced fibrosis.
  • Figure 5 A H&E (left panels) and Masson's trichrome staining (right panels) of the skin of PBS- or BLM challenged C57B1/6 mice treated with vehicle or AM095 in a 'preventive' regimen. Magnification xlOO; bar, ⁇ .
  • FIG. 5B Dermal thickness of PBS- or BLM-challenged C57B1/6 mice treated with vehicle, 'preventive' AM095, or 'therapeutic' AM095 begun 7
  • N 5 mice/group, data expressed as mean hydroxyproline/skin sample ⁇ SEM (*p ⁇ 0.0005, **p ⁇ 0.02 and ***p ⁇ 0.02, vehicle-treated vs. preventive AM095-, therapeutic AM095 #1- and therapeutic AM095 #2- treated BLM challenged mice, respectively).
  • Figure 6 presents one embodiment of a human LPAl nucleotide sequence (SEQ ID NO: 1
  • Figure 7 presents one embodiment of a human LPAl amino acid sequence (SEQ ID NO:2) (Accession No. NM_057159).
  • Figure 8 presents one embodiment of a human LPAl nucleotide sequence (SEQ ID NO:3) (Accession No. NM_001401).
  • Figure 9 presents one embodiment of a human LPAl amino acid sequence (SEQ ID NO-.4) (Accession No. NM_00-1401).
  • Figure 10 presents one embodiment of a human LPAl nucleotide sequence
  • Figure 11 presents one embodiment of a human LPAl amino acid sequence (SEQ ID NO: 1
  • Figure 12 presents one embodiment of a mouse LPAl nucleotide sequence (SEQ ID NO:7) (Accession No. NM_010336).
  • Figure 13 presents one embodiment of a mouse LPAl amino acid sequence (SEQ ID NO:8) (Accession No. NM_010336).
  • Figure 14 presents exemplary data showing that LPAl KO mice are protected from CG-induced peritoneal fibrosis following Masson's trichrome staining of the peritoneum (magnification 200x). Data presented are expressed as mean ⁇ SEM. *P ⁇ 0.05 comparing PBS-challenged WT and LPAl KO mice.
  • Figure 14 A PBS -challenged WT mice.
  • Figure 14B CG-challenged WT mice.
  • Figure 15 A CG-challenged WT mice stained with anti-aSMA antibody/peroxidase (magnification 400x)
  • Figure 15B CG-challenged LPAl KO mice stained with anti-aSMA antibody/peroxidase (magnification 400x).
  • Figure 15C Counts of the numbers of aSMA + cells in 10 randomly selected non-overlapping high power fields (HPF) per peritoneal section confirmed the protection of LPAl KO mice from CG-induced increases in peritoneal aSMA + myofibroblasts.
  • Figure 15D Increased peritoneal CTGF expression induced by CG in WT mice was reduced in LPAl KO mice.
  • Figure 16 presents exemplary data showing that LPA-induced CTGF expression is attenuated by a ROCK inhibitor and actin polymerization inhibitor in peritoneal mesothelial cells.
  • Figure 16 A Time-course of CTGF upregulation following LP A stimulation.
  • Figure 16B Dose-response of CTGF upregulation following LP A stimulation.
  • Figure 16C Pre-treatment with Y27632 (ROCK inhibitor; 0.5 ⁇ ) or latrunculin B (actin polymerization inhibitor; lmg/ml) suppressed the expression of CTGF in peritoneal mesothelial cells.
  • Y27632 ROCK inhibitor; 0.5 ⁇
  • latrunculin B actin polymerization inhibitor
  • the present invention is related to the field of fibrosis.
  • the invention provides for a role of the LPAl receptor in the development and/or maintenance of fibrotic diseases involving tissues including, but not limited to, epidermal, dermal, peritoneal, renal, hepatic, cardiac, lymphoid, ocular, bone cortex, and bone marrow.
  • Genetic deletion of the LPAl receptor and/or pharmacological antagonism with selective receptor inhibitors prevents and/or treats fibrosis symptoms (i.e., for example, increased tissue thickness and/or collagen content).
  • Empirical analysis shows that the LPA2 receptor system does not play a role in most fibrotic diseases, thereby suggesting that LPA signaling specifically through LPAl may provide clinically useful targets for the prevention and treatment of fibrosis.
  • LPA Lysophosphatidic Acid
  • Lysophosphatidic acid has potent fibroblast chemoattractant properties.
  • LPA is believed to be a lipid mediator that may provide signals through specific GPCRs.
  • LPAl high-affinity LPA receptors
  • P2Y5 may represent a low affinity receptor that is likely to join the LPA receptor family as LPA6.
  • Choi et al. "LPA receptors: subtypes and biological actions" Annu Rev Pharmacol Toxicol 50:157-186. Recently, LPA-LPA1 signaling has been implicated in the pathogenesis of pulmonary fibrosis.
  • LPA- LPA2 signaling has also been implicated in pulmonary fibrosis.
  • LPA-LPA2 signaling can induce ⁇ integrin-mediated activation of latent TGF- ⁇ by lung epithelial cells in culture.
  • Xu et al. "Lysophosphatidic acid induces ⁇ 6 integrin-mediated TGF-beta activation via the LPA2 receptor and the small G protein G alpha(q)" Am J Pathol
  • TGF- ⁇ activation by ⁇ 6 integrin may be involved in the development of bleomycin-induced lung fibrosis. Munger et al., "The integrin ⁇ 6 binds and activates latent TGF- ⁇ : a mechanism for regulating pulmonary inflammation and fibrosis" Cell 96(3):319-128 (1999).
  • LP A levels may be increased in the skin during the development of dermal fibrosis.
  • dermal fibrosis resulting from insults including but not limited to injury, toxicity (e.g., drug exposure such as bleomycin) or disease (e.g., scleroderma and/or systemic sclerosis).
  • the present invention contemplates methods that modulate LPA actions through its receptors to regulate the development of fibrosis. While the data presented herein is directed to two different organs in two different diseases, these are merely exemplary of the broader application of LPA and its receptors in fibrotic diseases, which can affect all organ systems. These organ systems, and the fibrotic diseases that affect them, include but are not limited to, liver, kidney, heart, ocular, dermal, intestinal, lymphatic, bone marrow, post-transplantation, or multi-organ fibrosis.
  • the present invention contemplates a method for preventing and/or treating liver fibrosis.
  • the liver fibrosis comprises cirrhosis.
  • the preventing and/or treating comprises modulation of the LPA1 receptor system.
  • the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the cirrhosis is caused by factors including but are not limited to viral hepatitis, schistosomiasis, and/or alcoholism.
  • the present invention contemplates a method for preventing and/or treating kidney fibrosis.
  • the kidney fibrosis comprises nephroclerosis.
  • the kidney fibrosis comprises end stage kidney disease.
  • the preventing and/or treating comprises modulation of the LPA1 receptor system.
  • the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the nephrosclerosis and/or end stage kidney disease is caused by factors including but are not limited to hypertension and/or diabetes.
  • the present invention contemplates a method for preventing and/or treating cardiac fibrosis, one embodiment, the cardiac fibrosis comprises heart fibrosis, hi one embodiment, the cardiac fibrosis comprises cardiomyopathy.
  • the preventing and/or treating comprises modulation of the LPA1 receptor system.
  • the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the cardiomyopathy is caused by factors including but are not limited to heart attack, hypertension, myocarditits, and/or genetic diseases of hypertrophic cardiomyop athy .
  • the present invention contemplates a method for preventing and/or treating ocular fibrosis, h one embodiment, the ocular fibrosis comprises eye fibrosis. In one embodiment, the ocular fibrosis is caused by macular degeneration. In one
  • the ocular fibrosis is caused by retinal retinopathy. In one embodiment, the ocular fibrosis is caused by vitreal retinopathy, hi one embodiment, the preventing and/or treating comprises modulation of the LPA1 receptor system. In one embodiment, the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the present invention contemplates a method for preventing and/or treating skin fibrosis.
  • the skin fibrosis is caused by keloids.
  • the skin fibrosis is caused by hypertrophic scars, hi one embodiment, the preventing and/or treating comprises modulation of the LPA1 receptor system.
  • the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the present invention contemplates a method for preventing and/or treating intestinal fibrosis.
  • the intestinal fibrosis is caused by inflammatory bowel disease.
  • the inflammatory bowel disease is caused by Chron's disease.
  • the inflammatory bowel disease is caused by ulcerative colitis.
  • the preventing and/or treating comprises modulation of the LPA1 receptor system.
  • the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the present invention contemplates a method for preventing and/or treating lympatic fibrosis, hi one embodiment, the lymphtic fibrosis comprises at least one lymph node. In one embodiment, the lymphatic fibrosis comprises at least one lymph duct. In one embodiment, the intestinal fibrosis is caused by aquired immunity deficiency syndrome. In one embodiment, the intestinal fibrosis is caused by filariasis. In one embodiment, the intestinal fibrosis is caused by cancer. In one embodiment, the preventing and/or treating comprises modulation of the LPA1 receptor system. In one embodiment, the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the present invention contemplates a method for preventing and/or treating bone tissue fibrosis.
  • the bone tissue fibrosis comprises bone marrow.
  • the bone tissue fibrosis comprises bone cortex.
  • the bone tissue fibrosis is caused by cancer.
  • the bone tissue fibrosis is caused by myelofibrosis.
  • the preventing and/or treating comprises modulation of the LPA1 receptor system.
  • the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the present invention contemplates a method for preventing and/or treating post organ transplant fibrosis
  • the post organ transplant fibrosis comprises solid organ allograft rejection.
  • the allograft rejection comprises chronic rejection.
  • the organ transplant includes, but is not limited to, lung, heart, kidney or liver.
  • the post organ transplant fibrosis comprises post bone marrow transplant graft versus host disease.
  • the preventing and/or treating comprises modulation of the LPA1 receptor system.
  • the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the present invention contemplates a method for preventing and/or treating multi-organ fibrosis.
  • the multi-organ fibrosis comprises surgical complications.
  • the multi-organ fibrosis comprises a drug- induced fibrosis.
  • the multi-organ fibrosis comprises a radiation-induced fibrosis.
  • the mult-organ fibrosis comprises a mechanical injury-induced fibrosis.
  • the surgical complications comprise intra-organ scar tissue.
  • the surgical complications comprise contracture.
  • the surgical complications comprise pain.
  • the surgical complications comprise infertility.
  • the drug-induced fibrosis comprises a
  • the drug-induced fibrosis comprises gadolimium.
  • the organ transplant includes, but is not limited to, lung, heart, kidney or liver.
  • the preventing and/or treating comprises modulation of the LPA1 receptor system.
  • the modulation comprises an LPA1 receptor inhibitor such as AM095.
  • the present invention contemplates that fibrotic diseases are mediated by LPA.
  • LPA is believed to be a major lipid growth factor in the blood that exerts diverse biological effects on many cell types and tissues. Nguyen et al., "Protein kinase A inhibits lysophosphatidic acid induction of serum response factor via alterations in the actin cytoskeleton" Cell Signal 16: 1141-1151 (2004). Recently, LPA-LPA1 signaling has been implicated in the pathogenesis of IPF, a disease characterized by progressive and usually fatal lung fibrosis.
  • mice lacking LPA1 were protected from fibrosis and mortality in a mouse model of this disease, demonstrating reduced fibroblast recruitment and vascular leak in response to fibrogenic lung injury. It was further found that LPA signaling through LPA1 may contribute to the excessive accumulation of fibroblasts in the lungs of IPF patients. LPA has also been reported to stimulate fibroblasts to produce various pro-fibrotic genes such as CTGF. Heusinger-Ribeiro et al., "Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP" J Am Soc Nephrol 12: 1853-1861 (2001).
  • CTGF is highly expressed during development of various fibrotic disorders and a recent study suggests that CTGF may be involved in myofibroblast accumulation into fibrotic lesion. Liu et al., "CCN2 is required for bleomycin-induced skin fibrosis in mice” Arthritis Rheum 63 :239-46.
  • the present invention contemplates a method where inhibition of the LPA-LPA1 pathway prevents and/or treats peritoneal fibrosis through regulation of CTGF production.
  • the LPA-LPA1 pathway inhibition comprises a therapeutic strategy for fibrotic diseases. LPA levels were observed to be increased in broncho alveolar lavage (BAL) fluid from IPF patients. Further, parameters of peritoneal function including peritoneal high transport rate as well as ultrafiltration failure have been shown to be associated with peritoneal fibrosis.
  • BAL broncho alveolar lavage
  • Fibroblast cells are believed to be a source of collagen and other extracellular matrix components generated in dermal fibrotic conditions (i.e., for example, scleroderma and/or systemic sclerosis). Further, dermal fibrosis may be associated with the differentiation of fibroblasts into myofibroblasts. Such differentiation may be characterized by the acquisition of smooth muscle cell features, including but not limited to: i) the expression of -smooth muscle actin ( -SMA); and ii) the ability to secrete increased levels of matrix components, including collagen.
  • -SMA smooth muscle actin
  • LPA Wound Repair Regen 13:7-12 (2005).
  • LPA is currently believed to mediate several processes leading to fibroblast accumulation, including recruitment, proliferation, and protection from apoptosis.
  • the present invention contemplates a method for decreasing dermal fibrosis by reducing fibroblast cell accumulation and differentiation into
  • myofibroblast cells Although it is not necessary to understand the mechanism of an invention, it is believed that LPA may be implicated by the data presented herein showing decreased dermal fibrosis in bleomycin-challenged LPA1 KO mice. Myofibroblast cells have been reported to predominate in areas of increased collagen deposition in scleroderma lesional skin. Sappino et al., "Smooth muscle differentiation in scleroderma fibroblastic cells" Am J Pathol 137:585-591 (1990); and Kissin et al.,
  • Myofibroblasts and hyalinized collagen as markers of skin disease in systemic sclerosis Arthritis Rheum 54:3655-3660 (2006). Further, the quantity of myofibroblast cells in lesional skin may correlate with the extent and severity of patients' skin fibrosis, as assessed by modified Rodnan skin scores (MRSS) and durometer measurements. Kissin et al.,
  • TGF- ⁇ is believed to be a regulator of both physiological and pathological fibrosis. Sonnylal et al., "Postnatal induction of transforming growth factor beta signaling in fibroblasts of mice recapitulates clinical, histologic, and biochemical features of scleroderma” Arthritis Rheum 56:334-344 (2007). For example, in fibroblasts, TGF- ⁇ has been reported to function as a fibrogenic stimulus by enhancing collagen synthesis, proliferation, migration, adhesion and differentiation into myofibroblasts.
  • TGF- ⁇ signaling confers protection against dermal fibrosis in commonly used mouse models of scleroderma, including the bleomycin challenge.
  • Lakos et al. "Targeted disruption of TGF-betalSmad3 signaling modulates skin fibrosis in a mouse model of scleroderma” Am J Pathol 165:203-217 (2004).
  • the Smad signal transduction pathways may mediate several TGF- ⁇ responses in fibroblasts wherein phosphorylation of Smad2 may mediate TGF- ⁇ activation.
  • Peritoneal dialysis is believed to be a life-sustaining therapy used by >100,000 patients with end-stage renal diseases worldwide, accounting for approximately 10 to 15% of the dialysis population.
  • Devuyst et al. "The pathophysiology of the peritoneal membrane” J Am Soc Nephrol 21 : 1077-1085.
  • Long-term exposure to hyperosmotic, hyperglycemic and acidic dialysis solutions has been reported to cause peritoneal injury, however, which can limit the effectiveness of this method of dialysis.
  • chronic peritoneal damage which involves approximately 50% of all PD patients, is characterized functionally by decreased ultrafiltration capacity, and histopathologically by submesothelial fibrosis.
  • Kidney Int 78:611-8 What is needed are more effective therapies to treat and/or prevent the fibrotic complications of PD. Although it is not necessary to understand the mechanism of an invention, it is believed that the present invention solves at least one problem in developing such more effective therapies by showing the involvment of the LPA1 receptor system in peritoneal fibrosis. In one embodiment, the present invention contemplates molecular mediators that interact with the LPA1 receptor system that can modulate the peritoneal fibrosis process.
  • peritoneal fibrosis is attributed to a combination of biocompatible factors in dialysate, including but not limited to high osmolarity, high glucose, advanced glycation/glucose degradation products, uremic inflammation or acute peritonitis with inflammation.
  • a pharmacological clinical target for treating peritoneal fibrosis comprises an LPA1 receptor.
  • LP A idiopathic pulmonary fibrosis
  • IPF idiopathic pulmonary fibrosis
  • LPA1 -deficient mice are protected from peritoneal fibrosis induced by intraperitoneal injections of chlorhexidine gluconate (CG).
  • CG chlorhexidine gluconate
  • WT wild type mice
  • increases in peritoneal thickness and collagen induced by CG were markedly attenuated in LPA1 KO mice.
  • Increases in the numbers of a- smooth muscle actin ( -SMA)-positive peritoneal mesothelial cells and myofibroblasts induced by CG in WT mice were also greatly reduced in LPA1 KO mice, as induction of the pro-fibrotic mediator connective tissue growth factor (CTGF).
  • CGF pro-fibrotic mediator connective tissue growth factor
  • CTGF induction has recently been implicated in the pathogenesis of PD-induced fibrosis in humans.
  • Mizutani et al. "Connective tissue growth factor (CTGF/CCN2) is increased in peritoneal dialysis patients with high peritoneal solute transport rate" American Journal Of Physiology 298:F721-F733.
  • LPA-LPAl signaling contribute to the development of peritoneal fibrosis in both mouse models and in human disease.
  • the present invention contemplates a method to prevent and/or treat toxin-induced peritoneal fibrosis.
  • the toxin comprises
  • Symptoms of toxin-induced peritoneal fibrosis includes, but is not limited to, increased peritoneal thickness, increased collagen content, accumulation of oc-SMA + myofibroblasts, and up-regulated peritoneal CTGF expression.
  • the preventing and/or treating of peritoneal fibrosis comprises a pharmacological LPA1 receptor inhibitor.
  • the pharmacological inhibitor is AM095.
  • the preventing and/or treating reduces the accumulation of collagen-producing cells such as fibroblasts, myofibroblasts and mesothelial cell-derived (myo)fibroblasts.
  • the numbers of these cells maybe evaluated after toxin exposure by using transgenic mice that express enhanced green fluorescent protein (GFP) in cells producing collagen type I, al (coll- GFP mice).
  • GFP green fluorescent protein
  • Peritoneal mesothelial cells are more than just structural cells. For example, it is now believed that peritoneal mesothelial cells are metabolically active and participate in both peritoneal homeostasis and peritoneal responses to injury. It has been reported that peritoneal mesothelial cells may participate in the development of peritoneal fibrosis by providng a source of pro-fibrotic molecules, including but not limited to cytokines, growth factors, matrix proteins, or intracellular adhesion molecules.
  • the present invention contemplates a m ethod for modulating the expression of pro-fibrotic molecules by peritoneal mesothelial cells, i one embodiment, the method inhibits LPA-LPA1 signaling thereby reducing the expression of pro-fibrotic molecules.
  • the pro- fibrotic molecule comprises CTGF. In one embodiment, the method reorganizes the actin cytoskeleton.
  • peritoneal fibrosis symptoms may be associated with functional and/or biochemical markers of peritoneal fibrosis including but not limited to: i) increased levels of LPA in the plasma or spent dialysis effluent; ii) increased activity levels of the LPA- generating enzyme autotaxin in the plasma, spent dialysis effluent; or iii) increased peritoneal mesothelial cells recovered from spent dialysis effluent.
  • D/P creat i n i ne , D 4 /Do g iucose, and ultrafiltration capacity may be assessed in plasma or spent dialysis effluent as measures of peritoneal function, and compared with putative fibrosis biomarker levels such as the procollagen peptides PICP or ⁇ .
  • the present invention contemplates a method for modulating LPA-LPAl signaling contribution to fibrotic disease prevention and/or treatment.
  • toxin-induced fibrosis including bleomycin-induced and CG-induced fibrosis express symptoms including, but not limited to, accumulation of fibroblasts/myofibroblasts and/or peritoneal expression of CTGF.
  • a pharmacological inhibitor of LPA-LPAl signaling i.e., for example, AM095 reproduces the LPAl KO phenotype in CG- challenged WT mice.
  • ColI-GFP mice treated by AM095 will reveal that LPA-LPAl signaling contributes to the accumulation of fibroblasts and myofibroblasts in CG-induced peritoneal fibrosis.
  • LPA is believed to be a bioactive phospholipid that signals through LPA receptors, which are G protein-coupled receptors (GPCRs).
  • GPCRs G protein-coupled receptors
  • mammals at least seven LPA GPCRs have been identified and can be subdivided into two groups depending on their primary structure.
  • LPA GPCRs LPA GPCRs
  • LPA-LPA2 signaling has been reported to be involved in a5p6-mediated transforming growth factor (TGF)-P activation in lung epithelial cells.
  • LPAl KO mice were protected from the increase in peritoneal thickness and collagen that were produced in a bleomycin-induced dermal fibrosis model or a CG-induced peritoneal fibrosis model.
  • the increases in the number of peritoneal aSMA-positive myofibroblasts as well as peritoneal CTGF expression induced by CG in WT mice were abrogated in LPAl KO mice. Accumulation of fibroblast cells, as well as myofibroblast cells, has been recognized as a hallmark for progressive organ fibrosis.
  • fibroblasts GFP +
  • myofibroblast aSMA +
  • mesothelial cells cytokeratin +
  • toxin-challenged mice were treated with an LPA1 selective receptor inhibitor (AM095) to determine the effect of LPA-LPA1 signaling on cell type- specific involvement in fibrosis prevention and/or treatment.
  • scleroderma is a potentially fatal autoimmune disease of unknown etiology, characterized by progressive multi-organ fibrosis that is refractory to current therapies. Fibrogenesis in SSc is thought to result from tissue injury followed by dysregulated wound healing. Abraham et al., "Scleroderma: from cell and molecular mechanisms to disease models" Trends in
  • the present invention contemplates a method for preventing and/or treating dermal fibrosis.
  • the preventing and/or treating comprises inhibiting a lysophosphatidic acid (LPA) receptor.
  • the receptor is an LPA1 receptor.
  • LPA-LPA1 signaling has recently been implicated in other fibrotic diseases including: i) pulmonary fibrosis (Tager et al, "The lysophosphatidic acid receptor LPA(l) links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak” Nat Med 14(l):45-54 (2008)); ii) renal tubulointerstitial fibrosis (Pradere et al., "LPA1 receptor activation promotes renal interstitial fibrosis” J Am Soc Nephrol 18(12):3110-3118 (2007)); and iii) hepatic fibrosis (Watanabe et al., "Plasma lysophosphatidic acid level and serum autotaxin activity are increased in liver injury in rats in relation to its severity" Life Sci 81(12):1009-1015 (2007).
  • LPA1 pathway in the development of lung, kidney, liver and/or skin fibrosis.
  • therapeutic agents i.e., for example, LPAl receptor inhibitors
  • the first efficacious LPAl -selective antagonists i.e., for example, AM095 has been shown herein using a dermal fibrosis bleomycin model.
  • LPA may be involved in SSc pathogenesis because arachidonoyl (20:4)-LPA levels have been reported to be significantly higher in SSc patients' serum versus controls.
  • Tokumura et al. "Elevated serum levels of arachidonoyl-lysophosphatidic acid and sphingosine 1-phosphate in systemic sclerosis" Int J Med Sci 6(4):168-176 (2009).
  • LPA is increasingly becoming of more interest in the pathogenesis of SSc, and Tokumura et al. speculated that LPA might be a marker for SSc.
  • LPA The metabolic origin of LPA in the skin, as well as the mechanism by which LPA is increased in the skin after dermal injury, however remained unknown.
  • Previous studies have evaluated LPA in various skin disorders, for example, an investigation of multiple bullous dermatoses showed that both the enzymatic pathways required for LPA synthesis and cells expressing LPAl are present in injured skin. Further, the injured human skin was shown to contain increased amounts of both LPA and cells expressing LPAi.
  • Mazereeuw-Hautier et al. "Production of lysophosphatidic acid in blister fluid: involvement of a lysophospholipase D activity" J Invest Dermatol 125:421-427 (2005).
  • LPA also has been found to stimulate migration of fibroblasts and cancer cells.
  • the present invention contemplates a method for regulating dermal fibrosis comprising LPA signaling.
  • the LPA signaling is mediated through an LPAi receptor. In one embodiment, the LPA signaling is mediated through an LPA 2 receptor.
  • the bleomycin model of scleroderma provides a sufficient research model for dermal fibrosis with which to screen for, and identify clinically useful targets that modulate the regulation of dermal fibrosis.
  • LPA-LPAl signaling contributes to dermal fibrosis.
  • WT mice bleomycin-challenged LPAl KO mice fail to develop increased numbers of dermal myofibroblasts and of dermal cells with nuclear Smad2 phosphorylation.
  • LPA-LPAl signaling may play a role in scleroderma fibrogenesis by modulating: i) myofibroblast accumulation; and/or ii) TGF-p-Smad signaling pathway activation.
  • LPA is also believed to mediate some of its activity through the LPA2 G protein- coupled transmembrane spanning receptor.
  • LPA2 receptor implicated the LPA2 receptor in promoting TGF- ⁇ activation and mediating fibrosis in lung injury.
  • LPA was shown to induce a5p6-mediated TGF- ⁇ activation via the LPA2 receptor.
  • Xu et al. "Lysophosphatidic acid induces alphavbeta6 mtegrrn- mediated TGF-beta activation via the LPA2 receptor and the small G protein G alpha(q)" Am J Pathol 174:1264-1279 (2009).
  • a bleomycin-induced fibrosis challenge usually involves repeated subcutaneous injections and results in dermal fibrosis resembling scleroderma.
  • Yamamoto et al "Animal Model of Sclerotic Skin. I: Local Injections of Bleomycin Induce Sclerotic Skin Mimicking Scleroderma” J Invest Dermatol 112(4):456-462 (1999). Collagen deposition and both fibroblast and myofibroblast accumulation are also reported to occur.
  • Wu et al. "hi perspective: murine models of scleroderma” Curr Rheumatol Rep 10(3):173-182 (2008).
  • the present invention contemplates a method for treating dermal fibrosis with an LPAl inhibitor.
  • the LPAl inhibitor is AM095.
  • LPA2 KO mice were not protected from bleomycin-induced dermal fibrosis using an LPA receptor inhibitor.
  • mice wild type (WT) and LPAl- and LPA2-deficient (LPAl KO and LPA2 KO) mice were injected subcutaneously with bleomycin or PBS once daily for twenty-eight days (e.g., 28 doses). Dermal thickness, collagen content, and numbers of oc-smooth muscle actin (aSMA) + or phosphoSmad2 + cells were determined in bleomycin- and PBS-injected skin.
  • WT wild type
  • LPAl KO and LPA2-deficient mice e.g. 28 doses.
  • dermal thickness, collagen content, and numbers of oc-smooth muscle actin (aSMA) + or phosphoSmad2 + cells were determined in bleomycin- and PBS-injected skin.
  • a novel selective LPAl antagonist AM095, or vehicle alone was administered by oral gavage to C57B1/6 mice challenged with 28 daily injections of bleomycin or PBS.
  • AM095 or vehicle treatments were initiated concurrently with, or 7 or 14 days after the onset of bleomycin and PBS injections, and continued to the end of the experiments.
  • Dermal thickness and collagen content were determined in injected skin.
  • LPAl KO mice were markedly resistant to bleomycin-induced increases in dermal thickness and collagen, whereas LPA2 KO mice were as susceptible as WTs. Bleomycin-induced increases in dermal SMA + and phosphoSmad2 + cells were abrogated in LPAl KO mice.
  • Myofibroblast cells are believed to predominate in areas of increased collagen deposition in scleroderma lesional skin, whereas the number of myofibroblasts correlates with fibrosis severity.
  • Sappino et al. "Smooth muscle differentiation in scleroderma fibroblastic cells” Am J Pathol 137(3):585-591 (1990); and Kissin et al., "Myofibroblasts and hyalinized collagen as markers of skin disease in systemic sclerosis" Arthritis Rheum
  • H&E hemotoxylin and eosin
  • the hydroxyproline content of bleomycin-challenged WT mice was significantly greater than that of bleomycin challenged KO mice.
  • LPA2 may mediate pulmonary fibrosis.
  • LPA1 may mediate dermal fibrosis.
  • LPA2 KO mice in bleomycin-induced dermal fibrosis.
  • LPA1 KO mice the data demonstrate that LPA2 KO mice were not resistant to dermal fibrosis development.
  • LPA and LPA1 may contribute to dermal fibrosis, but other processes are also implicated in scleroderma, including but not limited to, accumulation of, or increase in, myofibroblast cells and/or activation of the TGF-p-Smad signaling pathway. It has been reported that SSc fibrogenesis may be associated with fibroblast cell differentiation into myofibroblast cells, which secrete increased amounts of extracellular matrix components, including collagen. Desmouliere et al., "Tissue repair, contraction, and the myofibroblast” Wound Repair Regen 13(1):7-12 (2005). Further, myofibroblast cell differentiation may be characterized by the acquisition of smooth muscle cell features, including -SMA expression. Abraham et al., "New developments in fibroblast and myofibroblast biology: implications for fibrosis and scleroderma" Curr Rheumatol Rep 9(2):136-143 (2007).
  • TGF- ⁇ may play a role in scleroderma fibrogenesis. It has been previously reported that: i) fibroblast-specific expression of a constitutively- active TGF- ⁇ receptor is sufficient to recapitulate many features of scleroderma in mice, including dermal fibrosis.
  • TGF- ⁇ targets such as gene expression induced by treating normal fibroblasts with TGF- ⁇ . Whitfield et al., "Systemic and cell type- specific gene expression patterns in scleroderma skin” Proc Natl Acad Sci USA
  • TGF- ⁇ activity may be primarily regulated through the post-translational activation of latent TGF- ⁇ complexes.
  • Munger et al "Latent transforming growth factor- beta: structural features and mechanisms of activation” Kidney Int 51(5):1376-1382 (1997); and Annes et al., "Making sense of latent TGFbeta activation” J Cell Sci 116(Pt 2) :217-224 (2003).
  • LPA1 would be the receptor most likely to mediate LPA-induced TGF- ⁇ activation in the skin.
  • activation of TGF- ⁇ by the epithelial cell-restricted ⁇ 5 ⁇ 6 integrin is involved in the development of lung fibrosis in several animal models, including the bleomycin model of pulmonary fibrosis.
  • Munger et al. "The integrin alpha v beta 6 binds and activates latent TGF beta 1 : a mechanism for regulating pulmonary inflammation and fibrosis" Cell 96(3):319-328 (1999).
  • TGF- ⁇ may drive fibroblast activation and differentiation into
  • myofibroblasts and therefore may be dependent on the activation of TGF- ⁇ by adjacent epithelial cells in a paracrine fashion.
  • activation of TGF- ⁇ by fibroblasts themselves contributes to fibroblast activation and differentiation into myofibroblasts in an autocrine fashion.
  • the present invention contemplates a method for LPAl modulation of TGF- ⁇ activation during the development of dermal fibrosis.
  • the modulation comprises ⁇ 5 ⁇ 5 and ⁇ 5 ⁇ 3 integrins.
  • the integrins are expressed by skin fibroblasts.
  • TGF- ⁇ Dermal Smad2 Phosphorylation And The LPAl Receptor Myofibroblast differentiation and synthesis of matrix proteins have been reported to involve TGF- ⁇ .
  • Hinz B. "Formation and function of the myofibroblast during tissue repair” J Invest Dermatol 127(3):526-537 (2007); and Werner et al., "Regulation of wound healing by growth factors and cytokines” Physiol Rev 83(3):835-870 (2003).
  • TGF- ⁇ is thought to play a role in SSc fibrogenesis.
  • TGF- ⁇ receptors when bound by active TGF- ⁇ , TGF- ⁇ receptors transmit signals through phosphorylation of cytoplasmic Smad proteins, which translocate to the nucleus and act as transcription factors.
  • Smad transcription factors Genes Dev 19(23):2783-2810 (2005).
  • LPA may mediate TGF- ⁇ activation and signaling. Although it is not necessary to understand the mechanism of an invention, it is believed that that reduced TGF- ⁇ pathway activity contributes to the protection of LPAl KO mice from dermal fibrosis. Whether the development of dermal fibrosis involved a relationship between LPA-LPA1 signaling and TGF- ⁇ signaling pathway activation was assessed by comparing the number of cells with nuclear Smad2 phosphorylation in the dermis of bleomycin- and PBS -challenged WT and LPAl KO mice. An antibody specific for phospho-Smad2 was used to characterize phospho-Smad2 expression in the dermis using immunohistochemistry.
  • LPA and LPAl may contribute to the activation of the TGF- ⁇ -Smad signaling pathway during the development of dermal fibrosis.
  • the reduced number of nuclear phosphoSmad2 + cells in bleomycin-challenged LPAl KO mice could be at least partially attributable to reductions in the numbers of fibroblasts and myofibroblasts accumulating in the dermis of these mice. Reductions in fibroblast and myofibroblast numbers would reduce the number of cells present in the dermis that are able to respond to TGF- ⁇ by Smad phosphorylation.
  • the present invention contemplates a method for reorganizing an actin cytoskeleton.
  • the reorganizing comprises LPA-LPA1 signaling in connection with the expression of CTGF.
  • CTGF a peritoneal mesothelial cell
  • a peritoneal mesothelial cell has been reported to be the first cell in contact with dialysis fluid and may play a role in fibrosis development.
  • Yung et al. "Peritoneal mesothelial cell culture and biology" Perit Dial int 26:162-173 (2006).
  • Recent study revealed that CTGF mRNA and protein expression were detected in peritoneal mesothelial cells in human peritoneal specimen with advanced fibrosis.
  • CTGF/CCN2 Connective tissue growth factor
  • actin polymerization has been demonstrated to liberate myocardin-related transcriptional factor (MRTF) to enter the nucleus, and then activate nuclear transcriptional factor, serum response factor (SRF), to modulate gene expression.
  • MRTF myocardin-related transcriptional factor
  • SRF serum response factor
  • CTGF promoter has SRF-binding site, and CTGF is one of SRF target genes.
  • Muehlich et al. "Actin-dependent regulation of connective tissue growth factor” Am J Physiol Cell Physiol 292:C1732-C1738 (2007).
  • the present invention contemplates a composition comprising a targeting agent to the actin-MRTF-SRF circuit.
  • PD-related factors including but not limited to high glucose, glucose degradation products, and inflammation have been suggested to be involved in peritoneal fibrosis, however, identification of therapeutic target is required to treat patients with peritoneal fibrosis. In addition, identifying patients at high risk is of concern for clinician as to when, and if, PD patients should be switched to hemodialysis or transplantation.
  • the present invention contemplates a method to identify a high risk peritoneal fibrosis patient comprising modulation of the LPA-LPAl axis.
  • LPA levels have been reported to be elevated in the biological fluid such as blood or ascites of patients with various diseases including ovarian cancer. Jeon et al., "Cancer-derived lysophosphatidic acid stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells" Stem Cells 26:789-797 (2008).
  • LPA levels were found to be increased in bronchoalveolar lavage (BAL) fluid from IPF patients.
  • BAL bronchoalveolar lavage
  • LPA lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak
  • LPA is produced as a result of hydrolysis of membrane phospholipids by different phopho lipases, such as lysophospholipase D, which is identical to autotaxin.
  • Lisophosphatidic acid is a lipid mediator with wide range of biological activities.
  • the method comprises determining plasma or spent dialysis effluent levels of LPA, CTGF, procollagen peptides and autotaxin activity as well as the parameters of peritoneal function, and then estimate the correlation of those values.
  • the method further comprises; i) determining the expression of LPA receptors as well as autotaxin; and ii) measuring basal and LPA-treated CTGF expression levels using HPMCs from dialysis effluent.
  • the present invention contemplates that LPA-LPA1 signaling upregulation contributes to the development of peritoneal fibrosis in PD patients.
  • LPA concentration and autotaxin activity are increased in spent dialysate effluent. Although it is not necessary to understand the mechanism of an invention, it is believed that these values correlate with the levels of CTGF concentration and procollagen peptides in dialysate and inversely correlate with ultrafiltration capacity.
  • Dialysate and plasma samples obtained fro PD patients are analyzed to determine whether LPA-LPA1 signaling upregulation is associated with peritoneal fibrosis in PD patients.
  • Accumulating evidence demonstrate that parameters of peritoneal membrane function test such as ultrafiltration capacity can predict most patients at high risk of developing peritoneal fibrosis.
  • Lambie et al. "The peritoneal osmotic conductance is low well before the diagnosis of encapsulating peritoneal sclerosis is made" Kidney Int 78:611- 618.
  • recent study revealed positive correlation of dialysate CTGF concentration with a marker for peritoneal solute transport rate.
  • LP A concentration and autotaxin activity in dialysate correlate with the levels of CTGF concentration and procollagen peptides in dialysate and peritoneal function such as peritoneal solute transport rate and ultrafiltration capacity.
  • peritoneal mesothelial cells in the pathogenesis of peritoneal fibrosis.
  • An expression profile of LP A receptors will be determined as well as autotaxin, and then basal and LPA-treated CTGF expression levels by human peritoneal mesothelial cells isolated from dialysis effluent are measured.
  • Peritoneal dialysate samples will be collected from overnight dwelled samples. Each dialysate sample will be centrifuged at 3000 g at 4°C for 20 minutes and the supematants will be transferred to siliconized low-binding Eppendorff tubes and stored at -20°C until further use. Blood samples will be collected from all subjects in heparin-treated tubes (BD) and centrifuged at 2000 g for 10 minutes. LP A concentration will be determined by electrospray ionization mass spectrometry. Tager et al., "The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak" Nature Medicine 14:45-54 (2008).
  • Enzyme-linked immunosorbent assay will be performed to determine CTGF protein (Uscn Life Science Inc.). Autotaxin activity will be estimated by measuring the amount of choline released following the addition of lysophosphatidylcholine. Enzyme-linked immunosorbent assay will estimate the levels of procollagen type I C- terminal propeptide (PICP) (Takara Bio Inc.). Radioimmunoassay can examine the levels of procollagen type ⁇ N-terminal propeptide ( ⁇ ) (Orion Diagnostica Inc.).
  • PICP procollagen type I C- terminal propeptide
  • Radioimmunoassay can examine the levels of procollagen type ⁇ N-terminal propeptide ( ⁇ ) (Orion Diagnostica Inc.).
  • RNA preparation from cultured mesothelial cells will be done using Rneasy Mini kit (Quiagen). Then, quantitative realtime PCR (QPCR) analysis using an Mx4000 Multiplex Quantitative PCR System (Stratagene) will be performed.
  • QPCR quantitative realtime PCR
  • Protein will be extracted using NP40 buffer (Invitrogen).
  • LPA is a major lipid growth factor in the blood. Nguyen et al., "Protein kinase A inhibits lysophosphatidic acid induction of serum response factor via alterations in the actin cytoskeleton" Cell Signal 16: 1141-1151 (2004). Therefore, it is expected that LPA concentrations are higher in patients with high peritoneal transport rate than those in patients with low peritoneal transport rate. In addition, ultrafiltration failure has been reported to reflect the extent of peritoneal fibrosis.
  • LP A and LPAl contributions to the development of peritoneal fibrosis were assessed by evaluating the accumulation of myofibroblasts and one of pro-fibrotic genes, CTGF expression, in WT and LPAl KO mice following intraperitoneal injection of CG.
  • Myofibroblasts were identified in peritoneum sections by staining with an antibody against oc-smooth muscle actin (ocSMA).
  • ocSMA oc-smooth muscle actin
  • peritoneal mesothelial cells provide a source of pro-fibrotic molecules, including but not limited to numerous cytokines, growth factors, matrix proteins, and intracellular adhesion molecules. Consequently, experiments were performed to assess the role of LPA-LPA1 signaling in the biology of peritoneal mesothelial cell, especially CTGF expression.
  • LPA stimulation up-regulated CTGF expression in peritoneal mesothelial cells in both a time-course and dose-dependent manner. See, Figure 16A and Figure 16B, resepectively. Accumulating evidence demonstrates that LPA may induce cell contraction through actin polymerization, whereas the small GTPase RhoA-Rho kinase (ROCK) cascade may be involved in actin polymerization in various cells, such as fibroblasts (8). The effect of RhoA- ROCK cascade was assessed on LPA-induced CTGF expression in peritoneal mesothelial cells.
  • ROCK small GTPase RhoA-Rho kinase
  • the present invention contemplates that LPA-LPA1 signaling contributes to the expression of CTGF through MRTF-SRF axis in peritoneal mesothelial cells. In one embodiment, the inhibition of MRTF-SRF axis reduces CTGF expression.
  • transforming growth factor (TGF) ⁇ is one of pro-fibrotic factors which mediate its action partly through the expression of CTGF.
  • TGFbRII TGF- ⁇ receptor type II
  • TGFpRI TGF- ⁇ receptor type I
  • CTGF expression may be estimated by LPA stimulation using TGF RJJ-deficient peritoneal mesothelial cells.
  • peritoneal mesothelial cells from WT mice ii) peritoneal mesothelial cells from LPA1 -deficient mice
  • mice iv) peritoneal mesothelial cells from type I procollagen-producing cell-specific TGFbRII-deficient mice
  • murine peritoneal mesothelial cells will be isolated by digestion of harvested parietal peritoneum, and used between their second and sixth passages.
  • Nakav et al. "Blocking adenosine A2A receptor reduces peritoneal fibrosis in two independent experimental models" Nephrol Dial Transplant 24:2392-2399 (2009).
  • Peritoneal mesothelial cells constitutively produce type I procollagen therefore, type I procollagen-producing cell- specific TGFpRII-deficient mice can be used to harvest TGFpRII-deficient peritoneal mesothelial cells.
  • Type I procollagen-producing cell-specific TGFpRII-deficient mice will be achieved by administration of tamoxifen (lmg/50ml for 5 days), starting between 7-14 days old to compound mutant C57BL/6 mice harbouring the Cre-ERT trans gene and floxed TGF/3RU alleles.
  • Cre-ERT encodes a ligand-dependent Cre- recombinase linked to a promoter subcloned from the mouse proa2(I)collagen (colla2) gene (37).
  • Postnatal deletion of TGF/3RH will be confirmed by sequence specific PCR, and Western blotting and immuno staining of cultured mesothelial cells for TGF/3RH. Hoyles et al., "An Essential Role for Resident Fibroblasts in Experimental Lung Fibrosis Is Defined by Lineage- Specific Deletion of High- Affinity Type II Transforming Growth Factor ⁇ beta ⁇ Receptor" Am J Respir Crit Care Med 183:249-261.
  • RhoA activation assay Quantification of RlioA activation will be performed by pull-down assay using GTP-RhoA-binding domain of rhotekin (Cytoskeleton inc.).
  • LPA-LPA1 signaling contributes to nuclear translocation of MRTF
  • localization of MRTF can be determined by immunocytochemistry.
  • An antibody for MRTF- A (kind gift from Dr. Gerszten) may be used to detect endogenous MRTF -A.
  • transient transfection of an expression plasmid encoding HA-tagged MRTF-A (kind gift from Dr. Gerszten) may be performed with the FuGENE 6 transfection reagent (Roche) to clearly confirm the localization of MRTF-A.
  • FuGENE 6 transfection reagent FuGENE 6 transfection reagent
  • cultured peritoneal mesothelial cells will be co-transfected with a plasmid that encodes firefly luciferase under the control of a basal promoter and contains three tandem SRF binding elements upstream of the promoter (pSRE-luc; Clontech) and with a plasmid that constitutively encodes renilla luciferase under the control of a strong thymidine kinase promoter (pRL-TK; Promega) using FuGENE 6 transfection reagent (Roche).
  • LPA stimulation will not fail to do those changes in mesothehal cells from LPA 1 -deficient mice. It is expected that LPA-induced MRTF-SRF-CTGF pathway will be abrogated in mesothehal cells from MRTF-deficient mice. In addition, it is expected that LPA-induced RhoA-MRTF- SRF-CTGF pathway will be partly suppressed in mesothehal cells from type I procollagen- producing cell-specific TGFbRII-deficient mice.
  • LPA1 KO mice were protected from the increase in peritoneal thickness and collagen that were produced in CG-induced peritoneal fibrosis model.
  • the increases in the number of peritoneal aSMA + myofibroblasts as well as peritoneal CTGF expression induced by CG in WT mice were also abrogated in LPA1 KO mice.
  • the present invention contemplates a method for reproducing the LPA1 KO phenotype by administering an LPA1 receptor pharmacological inhibitor in CG- challenged WT mice.
  • the pharmacological inhibitor is AM095.
  • oral administration of AM095 is performed after the onset of peritoneal fibrosis to estimate the possibility for therapeutical application of LPA-LPA1 signaling blockade to peritoneal fibrosis. Accumulation of collagen-producing cells such as fibroblasts as well as myofibroblasts has been recognized as a hallmark for progressive organ fibrosis. Sonnylal et al., "Selective expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosis" Arthritis Rheum 62:1523-32.
  • fibroblasts GFP +
  • myofibroblast ASMA +
  • mesothelial cells cytokeratin +
  • CG- induced coll- GFP mice are also treated with AM095 to determine the effect of LPA-LPA1 signaling on cell type-specific involvement in peritoneal fibrosis.
  • WT mice and colI-GFP mice in C57B1/6 background are used wherein peritoneal fibrosis is induced by intraperitoneal injection of 0.1% CG dissolved in 15% ethanol/phosphate buffered saline (PBS) every other day, over a period of 21 days.
  • PBS ethanol/phosphate buffered saline
  • Yoshio et al. "TNP-470, an angiogenesis inhibitor, suppresses the progression of peritoneal fibrosis in mouse experimental model" Kidney Int 66:1677-16785 (2004).
  • Age- and sex-matched control mice of each genotype will be injected with the same volume of PBS alone on the same schedule.
  • the selective LPA1 antagonist AM095 is dissolved in sterile water, and a dose of 30 mg/kg per mouse, or sterile water alone (vehicle), is administered by oral gavage to WT mice and colI-GFP mice, twice daily.
  • AM095 is administered from the onset of CG challenge in a 'preventive' regimen, or beginning 10 days after the onset of CG challenge in a 'therapeutic' regimen.
  • peritoneal samples will be obtained at the completion of the experiment as described above.
  • Fibroblast Accumulation And Myofibroblast Differentiation Fibroblasts, myofibroblasts as well as mesothelial cells are enumerated in lesional peritoneum of mice sacrificed after 0 and 21days of CG or PBS injections. Fibroblasts are identified as GFP + cells with anti-GFP antibody (Abeam) in peritoneal sections.
  • Myofibroblasts will be identified in peritoneal sections with anti-ctSMA antibody (Sigma). Peritoneal mesothelial cells will be identified with anti-E-cadherin antibody (Abeam). In addition, GFP + /E-cadherin- cells on peritoneal surface are identified as fibroblasts derived from mesothelial cells, and SMA + /E-cadherin- cells on peritoneal surface will be identified as myofibroblasts derived from mesothelial cells.
  • Azole compounds have been reported to modify the physiological activity of lysophosphatidic acid by an LPA receptor antagonistic action. Yamamoto et al. "Novel azole compound", United States Patent Application Number 2006/0194850 (filed February 3, 2006). These azole compounds were not differentiated as to whether the compounds are specific for LPA 1; LPA 2 or both. As a result, these azole LPA receptor antagonists were suggested to be agents for the prophylaxis or treatment of fibroblast cell proliferation during lung fibrosis. Yamamoto et al., however, provided no data demonstrating that any LPA receptor antagonist was actually effective treating any fibroblast proliferative-related disorders, much less dermal fibrosis.
  • Lysophosphatidic acid analogs have been used as agonists or antagonists of LPA ls LPA 2 , and LPA 3 receptors. Lynch et al. "Lysophosphatidic acid receptor agonists and antagonists", United States Patent Number 7,169,818. These analog LPA compounds are 2- substituted ethanolamide derivatives exhibiting differing degrees of size, hydrophobicity, and stereochemistry. The parent N-acyl ethanolamine phosphate is nearly indistinguishable from LPA in stereochemical interactions with both the LPAi and the LPA 2 receptors. Further, some LPA antagonists have different affinities (and therefore efficacies) between the three different receptor subtypes. These LPA analogs are suggested for the treatment of diseases characterized by cell hyperproliferation.
  • LPAi and LPA 2 regulation has been suggested to inhibit the development of nasal polyps, a form of cellular hyperproliferation.
  • the LPAi and LPA 2 receptors were observed to be constitutively expressed in lung and nasal polyp-derived epithelial cells when exposed to LPA. It was suggested that cellular hyperproliferation resulting from LPAi and LPA 2 mRNA expression may be mediated by a variety of signaling cytochemicals. Barekzi et al. does not teach any LPA antagonists for reducing the development of dermal fibrosis.
  • the LPA receptor antagonist Kil6425 inhibits LPA-induced responses mediated by LPAi ⁇ LPA 3 » LPA 2 , without appreciable effects on cellular responses mediated by closely related lipid receptors, such as sphingosine 1-phosphate receptors.
  • Ohta et al. "Kil6425, a subtype-selective antagonist for EDG-family lysophosphatidic acid receptors" Mol
  • VPC 12249 is another specific LPA antagonist that inhibits LPA-induced responses mediated by LPAi ⁇ LPA 3 » LPA 2 .
  • LPA 2-substituted lysophosphatidic acid
  • the present invention contemplates evaluated selective LPAl antagonists to determine whether LPAl might be a therapeutic target (i.e., for example, a clinically useful target) to treat dermal fibrosis.
  • LPAl a therapeutic target
  • one selective LPAl antagonist evaluated was AM095 (Amira Pharmaceuticals, sodium ⁇ 4'-[3-methyl-4-((R)-l-phenyl- ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl ⁇ -acetate).
  • AM095 Anamic Biologicals, sodium ⁇ 4'-[3-methyl-4-((R)-l-phenyl- ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl ⁇ -acetate.
  • Figure 4A Verification assays showed that AM095 inhibited LPA-induced calcium flux of CHO cells stably transfected with human or mouse LPAl . See, Figure
  • the IC 50 for AM095 antagonism of LPA-induced calcium flux of human or mouse LPAl-transfected CHO cells was 0.025 and 0.023 mM, respectively.
  • the IC 5 0 for AM095 antagonism of LPA-induced calcium flux was >5 mM for CHO, human embryonic kidney (HEK), or B 103 rat neuroblastoma cells transfected with one of the other four established human or mouse LPA receptors, demonstrating AM095's selectivity for LPAl. See, Table 1.
  • Table 1 Inhibition by AM095 of LP A- stimulated intracellular calcium release from cells recombinantly expressing LPAi -5 .
  • the present invention contemplates a method for preventing and/or treating dermal fibrosis by administering a small organic molecule.
  • the small organic molecule is an LPAI receptor antagonist.
  • the LPAI receptor antagonist is AM095.
  • Bleomycin challenge increased the dermal thickness of vehicle-treated mice by 82%, but by only 25%, 12% or 32% in the preventive, therapeutic #1 or therapeutic #2 AM095-treated mice, respectively.
  • Preventive pharmacological inhibition of LPAI therefore attenuated the bleomycin-induced increase in dermal thickness by 70%
  • therapeutic pharmacological inhibition of LPAI begun on day 7 or 14 attenuated the bleomycin-induced increase in dermal thickness by 85% or 61%, respectively.
  • bleomycin challenge increased the skin hydroxyproline content of vehicle-treated mice by 117%, but by only 56%, 79% or 86%o in the preventive, therapeutic #1 or therapeutic #2 AM095-treated mice, respectively.
  • Figure 5C See, Figure 5C.
  • the present invention contemplates a nucleic acid comprising a sequence at least partially complementary with the LPAi receptor coding region.
  • the nucleic acid comprises an antisense sequence.
  • the coding region comprises deoxyribonucleic acid (DNA).
  • the coding region comprises ribonucleic acid (RNA).
  • the ribonucleic acid is messenger RNA (mRNA).
  • the present invention contemplates a method comprising administering an antisense sequence to a fibrosis patient under conditions such that the LPAi receptor population is reduced.
  • the antisense sequence is at least partially complementary to at least a portion of an LPAi receptor DNA.
  • the antisense sequence is partially complementary to at least a portion of an LPAi receptor mRNA.
  • an LPAI antisense inhibitor will inhibit LPA-induced increases in a-SMA + myofibroblasts and phosphoSmad2 + cells. It is further believed that an LP A ! antisense inhibitor will reduce the development of dermal fibrosis.
  • the present invention contemplates a peptide capable of attaching to an LPAI receptor under conditions such that dermal fibrosis development is reduced.
  • the peptide comprises the reverse amino acid sequence of a portion of the LPA1 receptor.
  • the portion encodes a binding pocket of the LPA1 receptor.
  • detection of LP Ai receptor expression comprises measuring the expression of corresponding mRNA in a biological sample (i.e., for example, a blood sample).
  • mRNA expression may be measured by any suitable method, including but not limited to, those disclosed below.
  • RNA is detection by Northern blot analysis.
  • Northern blot analysis involves the separation of RNA and hybridization of a complementary labeled probe.
  • RNA expression is detected by enzymatic cleavage of specific structures (INVADER assay, Third Wave Technologies; See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557; and 5,994,069; each of which is herein incorporated by reference).
  • the INVADER assay detects specific nucleic acid (e.g., RNA) sequences by using structure-specific enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes.
  • RNA is detected by hybridization to an oligonucleotide probe.
  • a variety of hybridization assays using a variety of technologies for hybridization and detection are available.
  • TaqMan assay PE Biosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each of which is herein incorporated by reference
  • the assay is performed during a PCR reaction.
  • the TaqMan assay exploits the 5 '-3' exonuclease activity of the AMPLITAQ GOLD DNA polymerase.
  • oligonucleotide with a 5'-reporter dye e.g., a fluorescent dye
  • a 3'-quencher dye is included in the PCR reaction.
  • the 5 -3' nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probe between the reporter and the quencher dye.
  • the separation of the reporter dye from the quencher dye results in an increase of fluorescence.
  • the signal accumulates with each cycle of PCR and can be monitored with a fluorimeter.
  • RNA reverse-transcriptase PCR
  • RNA is enzymatically converted to complementary DNA or "cDNA" using a reverse transcriptase enzyme.
  • the cDNA is then used as a template for a PCR reaction.
  • PCR products can be detected by any suitable method, including but not limited to, gel electrophoresis and staining with a DNA specific stain or hybridization to a labeled probe.
  • PA ⁇ receptor gene expression may be detected by measuring the expression of a protein or polypeptide. Protein expression may be detected by any suitable method. In some embodiments, proteins are detected by immunohistochemistry. In other embodiments, proteins are detected by their binding to an antibody raised against the protein. The generation of antibodies is described below.
  • Antibody binding may be detected by many different techniques including, but not limited to, (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),
  • “sandwich” immunoassays immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays, etc.
  • agglutination assays e.g., gel agglutination assays, hemagglutination assays, etc.
  • complement fixation assays immunofluorescence assays
  • protein A assays protein A assays
  • Immunoelectrophoresis assays etc.
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody, hi a further embodiment, the secondary antibody is labeled.
  • an automated detection assay is utilized.
  • Methods for the automation of immunoassays include those described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by reference.
  • the analysis and presentation of results is also automated.
  • software that generates a prognosis based on the presence or absence of a series of proteins corresponding to cancer markers is utilized.
  • a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of a given marker or markers) into data of predictive value for a clinician.
  • a clinician can access the predictive data using any suitable means.
  • the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data.
  • the data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.
  • the present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, wherein the information is provided to medical personal and/or subjects.
  • a sample e.g., a biopsy or a serum or urine sample
  • a profiling service e.g., clinical lab at a medical facility, genomic profiling business, etc.
  • any part of the world e.g., in a country different than the country where the subject resides or where the information is ultimately used
  • the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a urine sample) and directly send it to a profiling center.
  • the sample comprises previously determined biological information
  • the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems).
  • the profiling service Once received by the profiling service, the sample is processed and a profile is produced (i.e., expression data), specific for the diagnostic or prognostic information desired for the subject.
  • the profile data is then prepared in a format suitable for interpretation by a treating clinician.
  • the prepared format may represent a diagnosis or risk assessment (e.g., likelihood of a virus infection) for the subject, along with recommendations for particular treatment options.
  • the data may be displayed to the clinician by any suitable method.
  • the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.
  • the information is first analyzed at the point of care or at a regional facility.
  • the raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient.
  • the central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis.
  • the central processing facility can then control the fate of the data following treatment of the subject.
  • the central facility can provide data to the clinician, the subject, or researchers.
  • the subject is able to directly access the data using the electronic communication system.
  • the subject may chose further intervention or counseling based on the results.
  • the data is used for research use.
  • the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease.
  • the present invention provides isolated antibodies (i.e., for example, polyclonal or monoclonal).
  • the present invention provides monoclonal antibodies that specifically bind to an isolated polypeptide comprised of at least five amino acid residues of the receptor proteins described herein (e.g., LPA1). These antibodies find use in the treatment methods described above.
  • An antibody against a protein of the present invention may be any monoclonal or polyclonal antibody, as long as it can recognize the protein.
  • Antibodies can be produced by using a protein of the present invention as the antigen according to a conventional antibody or antiserum preparation process.
  • the present invention contemplates the use of both monoclonal and polyclonal antibodies. Any suitable method may be used to generate the antibodies used in the methods and compositions of the present invention, including but not limited to, those disclosed herein.
  • a monoclonal antibody protein, as such, or together with a suitable carrier or diluent is administered to an animal (e.g., a mammal) under conditions that permit the production of antibodies.
  • complete or incomplete Freund's adjuvant may be administered.
  • the protein is administered once every 2 weeks to 6 weeks, in total, about 2 times to about 10 times.
  • Animals suitable for use in such methods include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc.
  • an individual animal whose antibody titer has been confirmed e.g., a mouse
  • 2 days to 5 days after the final immunization, its spleen or lymph node is harvested and antibody-producing cells contained therein are fused with myeloma cells to prepare the desired monoclonal antibody producer hybridoma.
  • Measurement of the antibody titer in antiserum can be carried out, for example, by reacting the labeled protein, as described hereinafter and antiserum and then measuring the activity of the labeling agent bound to the antibody.
  • the cell fusion can be carried out according to known methods, for example, the method described by Koehler and Milstein (Nature 256:495 [1975]).
  • a fusion promoter for example, polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.
  • myeloma cells examples include NS-1, P3U1, SP2/0, AP-1 and the like.
  • the proportion of the number of antibody producer cells (spleen cells) and the number of myeloma cells to be used is preferably about 1 : 1 to about 20:1.
  • PEG preferably PEG 1000- PEG 6000
  • Cell fusion can be carried out efficiently by incubating a mixture of both cells at about 20°C to about 40°C, preferably about 30°C to about 37°C for about 1 minute to 10 minutes.
  • a hybridoma producing the antibody e.g., against a tumor antigen or autoantibody of the present invention
  • a supernatant of the hybridoma is added to a solid phase (e.g., microplate) to which antibody is adsorbed directly or together with a carrier and then an anti-immunoglobulin antibody (if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used) or Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.
  • a solid phase e.g., microplate
  • an anti-immunoglobulin antibody if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used
  • Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.
  • a supernatant of the hybridoma is added to a solid phase to which an anti-immunoglobulin antibody or Protein A is adsorbed and then the protein labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.
  • Selection of the monoclonal antibody can be carried out according to any known method or its modification. Normally, a medium for animal cells to which HAT
  • RPMI 1640 medium containing 1% to 20%, preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetal bovine serum, a serum free medium for cultivation of a hybridoma (SFM-101, Nissui Seiyaku) and the like can be used.
  • the cultivation is carried out at 20°C to 40°C, preferably 37°C for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% C02 gas.
  • the antibody titer of the supernatant of a hybridoma culture can be measured according to the same manner as described above with respect to the antibody titer of the anti-protein in the antiserum.
  • Separation and purification of a monoclonal antibody can be carried out according to the same manner as those of conventional polyclonal antibodies such as separation and purification of immunoglobulins, for example, salting-out, alcoholic precipitation, isoelectric point precipitation,
  • electrophoresis adsorption and desorption with ion exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method wherein only an antibody is collected with an active adsorbent such as an antigen-binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.
  • ion exchangers e.g., DEAE
  • ultracentrifugation e.g., ultracentrifugation
  • gel filtration e.g., gel filtration, or a specific purification method wherein only an antibody is collected with an active adsorbent such as an antigen-binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.
  • Polyclonal antibodies may be prepared by any known method or modifications of these methods including obtaining antibodies from patients. For example, a complex of an immunogen (an antigen against the protein) and a carrier protein is prepared and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation. A material containing the antibody against is recovered from the immunized animal and the antibody is separated and purified.
  • an immunogen an antigen against the protein
  • a carrier protein is prepared and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation.
  • a material containing the antibody against is recovered from the immunized animal and the antibody is separated and purified.
  • any carrier protein and any mixing proportion of the carrier and a hapten can be employed as long as an antibody against the hapten, which is crosslinked on the carrier and used for immunization, is produced efficiently.
  • bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. may be coupled to an hapten in a weight ratio of about 0.1 part to about 20 parts, preferably, about 1 part to about 5 parts per 1 part of the hapten.
  • various condensing agents can be used for coupling of a hapten and a carrier.
  • glutaraldehyde, carbodiimide, maleimide activated ester, activated ester reagents containing thiol group or dithiopyridyl group, and the like find use with the present invention.
  • the condensation product as such or together with a suitable carrier or diluent is administered to a site of an animal that permits the antibody production.
  • complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 3 times to about 10 times.
  • the polyclonal antibody is recovered from blood, ascites and the like, of an animal immunized by the above method.
  • the antibody titer in the antiserum can be measured according to the same manner as that described above with respect to the supernatant of the hybridoma culture. Separation and purification of the antibody can be carried out according to the same separation and purification method of immunoglobulin as that described with respect to the above monoclonal antibody.
  • the protein used herein as the immunogen is not limited to any particular type of immunogen.
  • a protein expressed on a fibroblast and/or leukocyte can be used as the immunogen.
  • fragments of the protein may be used. Fragments may be obtained by any methods including, but not limited to expressing a fragment of the gene, enzymatic processing of the protein, chemical synthesis, and the like.
  • the present invention provides drug screening assays (e.g., to screen for LPA1 inhibitor drugs).
  • the present invention provides screening methods for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to an LPA receptor and have an inhibitory (or stimulatory) effect on, for example, increased dermal thickness, increased collagen content, increased hydroxyproline content, increased a- SMA + myofibroblasts, increased phosphoSmad2 + cells, and fibroblast migration and/or recruitment.
  • modulators i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to an LPA receptor and have an inhibitory (or stimulatory) effect on, for example, increased dermal thickness, increased collagen content, increased hydroxyproline content, increased a- SMA + myofibroblasts, increased
  • the invention provides assays for screening candidate or test compounds that are substrates of an LPA receptor or a portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of an LPA receptor or portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in
  • combinatorial library methods including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al., J Med. Chem. 37: 2678 85 (1994); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one- bead one-compound” library method; and synthetic library methods using affinity
  • a screening assay comprises a cell-based assay in which a cell that expresses an LP A receptor or portion thereof is contacted with a test compound, and the ability of the test compound to inhibit fibrosis is determined. Determining the ability of the test compound to modulate fibrosis development can be accomplished by monitoring, for example, changes in dermal thickness.
  • the cell for example, can be of mammalian origin.
  • test compound to modulate LP A receptor binding to an inhibitory compound. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, can be determined by detecting the labeled compound, e.g., substrate, in a complex.
  • the LPA receptor is coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to binding to the substrate.
  • a radioisotope or enzymatic label to monitor the ability of a test compound to binding to the substrate.
  • compounds e.g., substrates
  • compounds can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a microphysiometer can be used to detect the interaction of a compound with an LPA receptor without the labeling of either the compound or the receptor. McConnell et al., Science 257:1906-1912 (1992).
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • a cell-free screening assay in which an LPA receptor or portion thereof is contacted with a test compound and the ability of the test compound to bind to the receptor or portion thereof is evaluated.
  • a portion of the LPA receptor to be used in assays of the present invention include fragments that participate in interactions with substrates or other proteins, e.g., fragments with high surface probability scores.
  • Cell-free screening assays involve preparing a reaction mixture of the receptor and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.
  • Interactions between two (or more) molecules can be detected, e.g., using
  • FRET fluorescence energy transfer
  • a fluorophore label may be selected such that a first donor molecule's emitted fluorescent energy will be absorbed by a fluorescent label on a second, "acceptor" molecule, which in turn is able to fluoresce due to the absorbed energy.
  • the "donor" protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the "acceptor” molecule label may be differentiated from that of the "donor". Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the "acceptor" molecule label in the assay should be maximal. An FRET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).
  • determining the ability of an LPA receptor to bind to a test compound can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, Anal. Chem. 63:2338 2345 [1991] and Szabo et al. Curr. Opin. Struct. Biol. 5:699 705 [1995]).
  • BIA Biomolecular Interaction Analysis
  • the LPA receptor or the test compound is anchored onto a solid phase. These complexes are anchored on the solid phase can be detected at the end of the reaction.
  • the receptor can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.
  • the complexes can be dissociated from the matrix, and the level of test compound binding to the receptor determined using standard techniques.
  • Other techniques for immobilizing either receptor proteins or a target compound on matrices include using conjugation of biotin and streptavidin.
  • Biotinylated virus induced marker protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, EL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non- immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-IgG antibody).
  • Screening assays may be performed utilizing antibodies reactive with LPA receptors or test compounds which do not interfere with binding of the receptor and test compound.
  • Such antibodies can be derivatized to the wells of the plate, and unbound target or virus induced markers protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the receptor or test compound, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the receptor or test compound.
  • cell free screening assays can be conducted in a liquid phase, h such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including, but not limited to: differential centrifugation (Rivas et al., Trends Biochem Sci 18:284-287 (1993)); chromatography (i.e., for example, gel filtration chromatography, ion-exchange chromatography); electrophoresis (Ausubel et al., eds. In: Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (Ausubel et al., eds. In: Current Protocols in Molecular Biology 1999, J. Wiley: New York). Such resins and chromatographic techniques have been reported.
  • fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.
  • a screening assay may also comprise contacting receptor proteins or portion thereof with a known compound that binds a receptor to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a marker protein, wherein determining the ability of the test compound to interact with a marker protein includes determining the ability of the test compound to preferentially bind to markers or biologically active portion thereof, or to modulate the activity of a receptor, as compared to the known compound.
  • the present invention contemplates detecting novel agents as identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., an LPA1 receptor inhibitory agent, an LPA1 specific antibody, or an LPA1 marker-binding partner) in an appropriate animal model (such as those described herein) to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be, e.g., used for treatments as described herein.
  • an agent identified as described herein e.g., an LPA1 receptor inhibitory agent, an LPA1 specific antibody, or an LPA1 marker-binding partner
  • novel agents identified by the above-described screening assays can be, e.g., used for treatments as described herein.
  • the present invention further provides pharmaceutical compositions (e.g., comprising the small organic compounds and/or antibody compounds described above).
  • pharmaceutical compositions e.g., comprising the small organic compounds and/or antibody compounds described above.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.
  • Thickeners flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • the pharmaceutical formulations of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • the compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models or based on the examples described herein.
  • dosage is from 0.01 /xg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly.
  • the treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • the present invention contemplates several drug delivery systems that provide for roughly uniform distribution and have controllable rates of compound release (i.e., for example, an LPA1 receptor inhibitor compound).
  • compound release i.e., for example, an LPA1 receptor inhibitor compound.
  • a variety of different media are described below that are useful in creating drug delivery systems. It is not intended that any one medium or carrier is limiting to the present invention. Note that any medium or carrier may be combined with another medium or carrier; for example, in one embodiment a polymer microparticle carrier attached to a compound maybe combined with a gel medium.
  • Carriers or mediums contemplated by this invention comprise a material selected from the group comprising gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including, but not limited to, 2-hydroxyethyl methacrylate, poly(ortho esters), cyanoacrylates, gelatin- resorcin-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.
  • the present invention contemplates a medical device comprising several components including, but not limited to, a reservoir comprising an LPA1 receptor inhibitor, a catheter, a sprayer or a tube, i one embodiment, said medical device administers either an internal or external spray to a patient, h another embodiment, said medical device administers either an internal or external gel to a patient.
  • the present invention contemplates a medium comprising a microparticle.
  • microparticles comprise liposomes, nanop articles, microspheres, nanospheres, microcapsules, and nanocapsules.
  • some microparticles Preferably, some microparticles
  • poly(lactide-co-glycolide) aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, psuedo-poly(amino acids), polyhydroxybutrate-related copolymers, polyanhydrides, polymethylmethacrylate, poly(ethylene oxide), lecithin and phospholipids.
  • poly(lactide-co-glycolide) aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, psuedo-poly(amino acids), polyhydroxybutrate-related copolymers, polyanhydrides, polymethylmethacrylate, poly(ethylene
  • the present invention contemplates liposomes capable of attaching and releasing LPA 1 receptor inhibitor compounds.
  • Liposomes are microscopic spherical lipid bilayers surrounding an aqueous core that are made from amphophilic molecules such as phospholipids.
  • one liposome embodiment comprises an inhibitor compound trapped between hydrophobic tails of a phospholipid micelle.
  • Water soluble drugs can be entrapped in the core and lipid-soluble drugs can be dissolved in the shell-like bilayer. Liposomes have a special characteristic in that they enable water soluble and water insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers.
  • Liposomes may form spontaneously by forcefully mixing phosopholipids in aqueous media. Water soluble compounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, therefore, these compounds are trapped within the aqueous liposomal center. The liposome wall, being a phospholipid membrane, holds fat soluble materials such as oils. Liposomes provide controlled release of incorporated compounds. In addition, liposomes can be coated with water soluble polymers, such as polyethylene glycol to increase the pharmacokinetic half-life.
  • One embodiment of the present invention contemplates an ultra high-shear technology to refme liposome production, resulting in stable, unilamellar (single layer) liposomes having specifically designed structural characteristics. These unique properties of liposomes, allow the simultaneous storage of normally immiscible compounds and the capability of their controlled release.
  • the present invention contemplates cationic and anionic liposomes, as well as liposomes having neutral lipids comprising an LPA receptor inhibitor.
  • cationic liposomes comprise negatively-charged materials by mixing the materials and fatty acid liposomal components and allowing them to charge-associate.
  • the choice of a cationic or anionic liposome depends upon the desired pH of the final liposome mixture. Examples of cationic liposomes include lipofectin, lipofectamine, and lipofectace.
  • the present invention contemplates a medium comprising liposomes that provide controlled release of LPA1 inhibitor compounds.
  • liposomes that are capable of controlled release i) are biodegradable and non-toxic; ii) carry both water and oil soluble compounds; iii) solubilize recalcitrant compounds; iv) prevent compound oxidation; v) promote protein stabilization; vi) control hydration; vii) control compound release by variations in bilayer composition such as, but not limited to, fatty acid chain length, fatty acid lipid composition, relative amounts of saturated and unsaturated fatty acids, and physical configuration; viii) have solvent dependency; iv) have pH-dependency and v) have temperature dependency.
  • compositions of liposomes are broadly categorized into two classifications.
  • Conventional liposomes are generally mixtures of stabilized natural lecithin (PC) that may comprise synthetic identical-chain phospholipids that may or may not contain glycolipids.
  • PC stabilized natural lecithin
  • Special liposomes may comprise: i) bipolar fatty acids; ii) the ability to attach antibodies for tissue-targeted therapies; iii) coated with materials such as, but not limited to lipoprotein and carbohydrate; iv) multiple encapsulation and v) emulsion compatibility.
  • Liposomes may be easily made in the laboratory by methods such as, but not limited to, sonication and vibration.
  • compound-delivery liposomes are commercially available. For example, Collaborative Laboratories, Inc. are known to manufacture custom designed liposomes for specific delivery requirements.
  • Microspheres and microcapsules are useful due to their ability to maintain a generally uniform distribution, provide stable controlled compound release and are economical to produce and dispense.
  • an associated delivery gel or the compound-impregnated gel is clear or, alternatively, said gel is colored for easy visualization by medical personnel.
  • microspheres, microcapsules and microparticles i.e., measured in terms of micrometers
  • nanospheres, nanocapsules and nanoparticles i.e., measured in terms of nanometers.
  • micro/nanosphere, micro/nanocapsule and micro/nanoparticle are used interchangeably, as will the discussion herein.
  • Microspheres are obtainable commercially (Prolease” 8 ', Alkerme's: Cambridge, Mass.).
  • a freeze dried LPA receptor inhibitor medium is homogenized in a suitable solvent and sprayed to manufacture microspheres in the range of 20 to 90 ⁇ . Techniques are then followed that maintain sustained release integrity during phases of purification, encapsulation and storage. Scott et al., "Improving Protein Therapeutics With Sustained Release Formulations” Nature Biotechnology 16:153-157 (1998).
  • Modification of the microsphere composition by the use of biodegradable polymers can provide an ability to control the rate of LPAi receptor inhibitor release.
  • Miller et al. "Degradation Rates of Oral Resorbable Implants ⁇ Polylactates and Polyglycolates: Rate Modification and Changes in PLA/PGA Copolymer Ratios" J. Biomed. Mater. Res., Vol. 11:711-719 (1977).
  • a sustained or controlled release microsphere preparation is prepared using an in-water drying method, where an organic solvent solution of a biodegradable polymer metal salt is first prepared. Subsequently, a dissolved or dispersed solution of an LPAi receptor inhibitor is added to the biodegradable polymer metal salt solution.
  • the weight ratio of an LPA receptor inhibitor to the biodegradable polymer metal salt may for example be about 1 : 100000 to about 1 : 1, preferably about 1 :20000 to about 1 : 500 and more preferably about 1 : 10000 to about 1 :500.
  • the organic solvent solution containing the biodegradable polymer metal salt and inhibitor is poured into an aqueous phase to prepare an oil/water emulsion. The solvent in the oil phase is then evaporated off to provide microspheres. Finally, these microspheres are then recovered, washed and lyophilized. Thereafter, the microspheres may be heated under reduced pressure to remove the residual water and organic solvent.
  • the present invention contemplates a medium comprising a microsphere or microcapsule capable of delivering a controlled release of a compound for a duration of approximately between 1 day and 6 months.
  • Controlled release microcapsules may be produced by using known encapsulation techniques such as centrifugal extrusion, pan coating and air suspension. Using techniques well known in the state of the art, these microspheres/microcapsules can be engineered to achieve particular release rates.
  • Oliosphere ® Macromed
  • These particular microsphere's are available in uniform sizes ranging between 5 - 500 /mi and composed of biocompatible and biodegradable polymers. It is well known in the art that specific polymer compositions of a microsphere control the drug release rate such that custom-designed microspheres are possible, including effective management of the burst effect.
  • ProMaxx ® (Epic Therapeutics, Inc.) is a protein-matrix drug delivery system. The system is aqueous in nature and is adaptable to standard pharmaceutical drug delivery models. In particular, ProMaxx ® are bioerodible protein microspheres that deliver both small and macromolecular drugs, and may be customized regarding both microsphere size and desired drug release characteristics.
  • a microsphere or microparticle comprises a pH sensitive encapsulation material that is stable at a pH less than the pH of the internal mesentery.
  • the typical range in the internal mesentery is pH 7.6 to pH 7.2. Consequently, the microcapsules should be maintained at a pH of less than 7.
  • the pH sensitive material can be selected based on the different pH criteria needed for the dissolution of the microcapsules. The encapsulated compound, therefore, will be selected for the pH environment in which dissolution is desired and stored in a pH preselected to maintain stability.
  • Examples of pH sensitive material useful as encapsulants are Eudragit ® L-100 or S- 100 (Rohm GMBH), hydroxypropyl methylcellulose phthalate, hydroxypropyl
  • lipids comprise the inner coating of the microcapsules.
  • these lipids may be, but are not limited to, partial esters of fatty acids and hexitiol anhydrides, and edible fats such as triglycerides.
  • lipids may be, but are not limited to, partial esters of fatty acids and hexitiol anhydrides, and edible fats such as triglycerides.
  • a microparticle contemplated by this invention comprises a gelatin, or other polymeric cation having a similar charge density to gelatin (i.e., poly-L- lysine) and is used as a complex to form a primary microparticle.
  • a primary microparticle is produced as a mixture of the following composition: i) Gelatin (60 bloom, type A from porcine skin), ii) chondroitin 4-sulfate (0.005% - 0.1%), iii) glutaraldehyde (25%, grade 1), and iv) l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC
  • hydrochloride hydrochloride
  • ultra-pure sucrose Sigma Chemical Co., St. Louis, Mo.
  • the source of gelatin is not thought to be critical; it can be from bovine, porcine, human, or other animal source.
  • the polymeric cation is between 19,000-30,000 daltons. Chondroitin sulfate is then added to the complex with sodium sulfate, or ethanol as a coacervation agent.
  • a compound i.e., for example, an LPA1 receptor inhibitor
  • a compound is directly bound to the surface of the microparticle or is indirectly attached using a "bridge" or "spacer".
  • the amino groups of the gelatin lysine groups are easily derivatized to provide sites for direct coupling of a compound.
  • spacers i.e., linking molecules and derivatizing moieties on targeting ligands
  • avidin-biotin are also useful to indirectly couple targeting ligands to the microparticles.
  • Stability of the microparticle is controlled by the amount of glutaraldehyde- spacer crosslinking induced by the EDC hydrochloride.
  • a controlled release medium is also empirically determined by the final density of glutaraldehyde-spacer crosslinks.
  • the present invention provides kits for the detection, use, and characterization of LPAi receptor inhibitors.
  • the inhibitors are nucleic acid sequences.
  • the inhibitors are amino acid sequences.
  • the inhibitors are small organic molecules.
  • the kits contain antibodies specific for an LPA receptor protein, in addition to detection reagents and buffers, h other embodiments, the kits contain reagents specific for the detection of mRNA or cDNA (e.g., oligonucleotide probes or primers).
  • the kits contain all of the components necessary to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.
  • kits for the practice of the methods of this invention.
  • the kits preferably include one or more containers containing a DNA detection method of this invention.
  • the kit can optionally include a normal cell culture to be utilized as a control (i.e., for example, a fibroblast cell culture).
  • the kit can optionally include an LPA1 receptor inhibitor as contemplated herein.
  • the kit can optionally include nucleic acids capable of hybridizing to an LPA1 gene region (i.e., for example, PCR primers and/or antisense sequences).
  • the kit can optionally include enzymes capable of performing PCR (i.e., for example, DNA polymerase, Taq polymerase and/or restriction enzymes).
  • the kit can optionally include a pharmaceutically acceptable excipient and/or a delivery vehicle (e.g., a liposome).
  • a pharmaceutically acceptable excipient and/or a delivery vehicle e.g., a liposome
  • the reagents may be provided suspended in the excipient and/or delivery vehicle or may be provided as a separate component which can be later combined with the excipient and/or delivery vehicle.
  • the kit may optionally contain additional therapeutics to be co-administered with the LPA1 receptor inhibitor.
  • kits may also optionally include appropriate systems (e.g. opaque containers) or stabilizers (e.g. antioxidants) to prevent degradation of the reagents by light or other adverse conditions.
  • appropriate systems e.g. opaque containers
  • stabilizers e.g. antioxidants
  • kits may optionally include instructional materials containing directions (i.e., protocols) providing for the use of the reagents in the diagnosis, detection, and/or treatment of a fibrosis (i.e., for example, a dermal or peritoneal fibrosis) within a mammal, a particular the disease can include any one or more of the disorders described herein.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention.
  • Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
  • Such media may include addresses to internet sites that provide such
  • Jerold Chun demonstrate impaired suckling in neonatal pups due to defective olfaction, which leads to increased neonatal mortality, and reduced body size in survivors. Survivors also sometimes demonstrate craniofacial dysmorphism characterized by shorter snouts and more widely spaced eyes, but this symptom was not observed in the animals used herein. Contos et al., "Requirement for the IpAl lysophosphatidic acid receptor gene in normal suckling behavior" Proc Natl Acad Sci USA 97(24): 13384-13389 (2000).
  • LPA2 KO mice used offspring of mice homozygous for the mutant LPA2 allele in the BALB/c genetic background and WT BALB/c mice (Charles River Laboratories).
  • LPA2 KO mice (The Scripps Research Institute; Dr. Jerold Chun) were born at the expected frequency and displayed no obvious phenotypic
  • Bleomycin (Gensia Sicor) was dissolved in PBS at 10 mg/ml and sterilized by filtration. Bleomycin or PBS (100 ml) was injected subcutaneously into two locations on the shaved backs of LPAl KO, LPA2 KO or WT mice, once per day for twenty-eight days (e.g., 28 doses). Mice were then sacrificed and full thickness 6 mm punch biopsies were obtained from each injection site. One skin sample was fixed in 10% fonrialin and embedded in paraffin for histology and iimiiunohistochemistry studies; the other was frozen immediately at -80°C for hydroxyproline analysis.
  • Full thickness 6mm punch biopsies were obtained from all injected skin sites. For each mouse, one 6mm punch biopsy was fixed in 10% formalin solution and embedded in paraffin, and the other 6mm punch biopsy was frozen immediately at -80C for subsequent hydroxyproline assay.
  • Hydroxyproline content was determined as a measure of skin collagen using a previously reported standard protocol.
  • Tager et al. "Inhibition of pulmonary fibrosis by the chemoldne IP-10/CXCLlO" Am JRespir Cell Mol Biol 31(4):395-404 (2004). Briefly, skin samples were homogenized in physiological buffered saline (PBS) and hydrolyzed overnight in 6N HC1 at 120°C. A 25 ml aliquot was desiccated, resuspended in 25 ml H 2 0 and added to 0.5 ml of 1.4% chloramine T (Sigma), 10% n-propranolol, and 0.5 M sodium acetate, pH 6.0.
  • Human and mouse LPA1 and human LP A3 receptors were stably expressed in CHO cells (Invitrogen) and cultured in F12 media with 10% FBS and 1 mg/ml hygromycin B.
  • Mouse LP A3 was stably expressed in HEK cells (Invitrogen) and cultured in DMEM with 10% FBS and 200 ⁇ g/ml hygromycin B.
  • Human and mouse LPA2 and LPA5 and human LPA4 were transiently expressed in rat neuroblastoma B 103 cells using LipofectamineTM 2000 (Invitrogen) as per the manufacturer instructions.
  • LPA receptor-transfected cells were plated in 96-well Poly-D-Lysine-coated black- wall clear-bottom plates (BD BioCoat) at 20,000-40,000 cells/well and cultured overnight in complete media. Cells were then washed with PBS and cultured in serum- free media either overnight (for stably expressing cells) or 4 hours (for transient transfectants) prior to dye loading. On the day of the assay, cells were loaded for 1 hour at 37°C with 100 ⁇ FLIPR ® Calcium 4 dye (Molecular Devices) in HBSS supplemented with 20 mM HEPES, 2 mM probenecid and 0.3% FFA-HSA.
  • FLIPR ® Calcium 4 dye Molecular Devices
  • Test compounds 25 ⁇ 1% DMSO were added to each well and incubated at RT for 30 minutes.
  • LPA 50 ⁇ of 5X stock solutions prepared in HBSS with 20 mM HEPES and 0.3% fatty acid-free HSA was added after 15 seconds of baseline measurement.
  • Final concentrations of LP A used were dependent on the receptor expressed: LPAl and LP A3 assays used 10 nM LP A, LPA2 and LPA5 assays used 30 nM LP A and LPA4 assay used 300 nM LP A.
  • Intracellular calcium mobilization was measured using the FLEXstation HI
  • C57B1/6 mice were administered the selective LPAl antagonist AM095 by oral gavage (30 mg/kg) at time 0 and 8h.
  • Blood samples were collected by cardiac puncture under anesthesia in sodium EDTA tubes at 0, 4, 8, 9, 12 and 24h. Plasma samples were stored at -40°C prior to analysis of AM095 concentrations by liquid chromatography/mass spectrometry (LC-MS/MS).
  • AM095 Known amounts of AM095 were added to thawed mouse plasma to yield a concentration range from 0.8 to 4,000 ng/ml. Plasma samples were precipitated using acetonitrile containing the internal standard buspirone. The analyte mixture ( ⁇ 10 ml) was injected using a Leap PAL autosampler. Calibration curves were constructed by plotting the peak-area ratio of analyzed peaks against known concentrations. The lower limit of quantitation was 1 ng/ml. The data were subjected to linear regression analysis with l/x2 weighting. The pharmacokinetic parameters of AM095 were calculated by non- compartmental analysis using WinNonlin Professional (Pharsight). C max and time to C max (Tmax) were obtained directly from the measured data.
  • the selective LPAl antagonist AM095 was dissolved in sterile water. A dose of 30 mg/kg per mouse, or sterile water alone (vehicle), was administered by oral gavage to C57B1/6 mice, twice daily on weekdays and once daily on weekends. AM095 was administered from the onset of bleomycin challenge in a 'preventive' regimen, or beginning either 7 or 14 days after the onset of bleomycin challenge in two 'therapeutic' regimens.
  • bleomycin or PBS was injected subcutaneously for 28 consecutive days, and skin samples were obtained at the completion of the experiment as described above.

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Abstract

Selon l'invention, la signalisation par le LPA par l'intermédiaire du LPA1, mais non du LPA2, s'est révélée être impliquée dans le développement de la fibrose dermique induite par la bléomycine, ayant une incidence tant sur l'accumulation de myofibroblastes que sur la signalisation par TGF-β-Smad. En outre, une délétion génétique, une inhibition pharmacologique préventive et une inhibition pharmacologique thérapeutique du LPA1 a protégé des souris d'une fibrose dermique. Par exemple, un antagoniste sélectif du récepteur LPA1, AMO95, atténue la fibrose dermique quand elle est déclenchée après l'apparition d'une lésion tissulaire dans un traitement thérapeutique. Par conséquent, l'antagonisme du LPA1 peut être efficace dans le traitement de patients ayant une fibrose déjà existante, comme il serait nécessaire pour des médicaments anti-fibrosants utiles sur le plan clinique. L'inhibition du LPA-LPA1 a le potentiel de représenter une nouvelle stratégie thérapeutique efficace de la fibrose dermique, et ce LPA1 représente une cible viable pour une intervention pharmacologique.
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Cited By (3)

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WO2014104372A1 (fr) 2012-12-28 2014-07-03 宇部興産株式会社 Composé hétérocyclique substitué par un halogène
KR20170016988A (ko) 2014-06-27 2017-02-14 우베 고산 가부시키가이샤 할로겐 치환 헤테로환 화합물의 염
CN116236486A (zh) * 2023-03-30 2023-06-09 复旦大学附属中山医院 髓过氧化物酶抑制剂在制备心脏保护药物中的应用

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WO2008112201A2 (fr) * 2007-03-12 2008-09-18 The General Hospital Corporation Récepteur d'acide lysophosphatidique ciblant une maladie pulmonaire

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WO2008112201A2 (fr) * 2007-03-12 2008-09-18 The General Hospital Corporation Récepteur d'acide lysophosphatidique ciblant une maladie pulmonaire

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014104372A1 (fr) 2012-12-28 2014-07-03 宇部興産株式会社 Composé hétérocyclique substitué par un halogène
KR20150100756A (ko) 2012-12-28 2015-09-02 우베 고산 가부시키가이샤 할로겐 치환 헤테로환 화합물
US10000463B2 (en) 2012-12-28 2018-06-19 Ube Industries, Ltd. Halogen-substituted heterocyclic compound
EP3360869A1 (fr) 2012-12-28 2018-08-15 Ube Industries, Ltd. Composé hétérocyclique à substitution halogène pour le traitment des maladies associées avec lpa
US10597375B2 (en) 2012-12-28 2020-03-24 Ube Industries, Ltd. Halogen-substituted heterocyclic compound
KR20170016988A (ko) 2014-06-27 2017-02-14 우베 고산 가부시키가이샤 할로겐 치환 헤테로환 화합물의 염
US10023554B2 (en) 2014-06-27 2018-07-17 Ube Industries, Ltd. Halogen-substituted heterocyclic compound salt
CN116236486A (zh) * 2023-03-30 2023-06-09 复旦大学附属中山医院 髓过氧化物酶抑制剂在制备心脏保护药物中的应用
CN116236486B (zh) * 2023-03-30 2024-03-22 复旦大学附属中山医院 髓过氧化物酶抑制剂在制备心脏保护药物中的应用

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