WO2006135660A2 - Therapies involving lymphotoxin beta receptor - Google Patents

Therapies involving lymphotoxin beta receptor Download PDF

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Publication number
WO2006135660A2
WO2006135660A2 PCT/US2006/022241 US2006022241W WO2006135660A2 WO 2006135660 A2 WO2006135660 A2 WO 2006135660A2 US 2006022241 W US2006022241 W US 2006022241W WO 2006135660 A2 WO2006135660 A2 WO 2006135660A2
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Prior art keywords
ltβr
liver
mice
light
modulator
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PCT/US2006/022241
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French (fr)
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WO2006135660A3 (en
Inventor
Yang-Xin Fu
James C. Lo
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University Of Chicago
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Publication of WO2006135660A3 publication Critical patent/WO2006135660A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

  • the present invention relates generally to the fields of molecular biology and medicine. More particularly, it concerns methods involving the modulation of lymphotoxin beta receptor (LT ⁇ R) for the prevention and/or treatment of diseases (e.g., cardiac diseases, liver diseases) and compositions for use in such methods.
  • diseases e.g., cardiac diseases, liver diseases
  • Atherosclerosis is the major cause of death in western societies and there are a variety of important risk factors including dyslipidaemia, increased body fat mass and smoking.
  • lipid parameters triglycerides and high-density lipoprotein (HDL) cholesterol are the most important predictors of coronary heart disease.
  • HDL high-density lipoprotein
  • Coronary heart disease is regarded as a low-grade chronic inflammatory process.
  • leukocytes During the early stages of atherogenesis and following endothelial injury, local and recruited leukocytes release various inflammatory mediators, bind to the endothelium and migrate into the lesion. Leukocytes are required for both the formation of these so called “plaques” and endothelial cell dysfunction. The rupturing of these plaques is frequently the cause of myocardial infarction (MI) and stroke.
  • MI myocardial infarction
  • triglycerides and cholesterol can be very useful for the treatment and prevention of cardiovascular disease.
  • a relationship has been proposed between dyslipidaemia and numbers of blood leukocytes, another inflammatory marker that predicts chronic heart disease mortality and morbidity.
  • Increased levels of triglycerides have been shown to result in activation of leukocytes, resulting in proliferation and increased production of cytokines.
  • Leukocyte activation is suggestive of a proinflammatory and proatherogenic condition, most likely indicating an increased capacity of these cells to adhere to the endothelium.
  • liver regeneration there exists a need for methods and therapies to promote liver regeneration.
  • diseases including cirrhosis of the liver and viral infections of the liver ⁇ e.g., hepatitis) result in significant damage to the liver and would benefit from therapies that promote liver regeneration.
  • the inability of the host to restore liver mass is often the cause of chronic liver diseases, which cause significant morbidity and mortality worldwide.
  • liver transplantation methods to promote liver regeneration could be used in therapies involving liver transplantation. Organ shortages continue to press the need for liver transplantation alternatives. The ability to increase the renewal of liver would make split liver transplants for living adult donor and even cellular transplants more feasible.
  • lymphotoxin beta receptor and certain soluble forms of LT ⁇ R are described in WO9703687, WO9413808, and U.S. Patent 6,669,941.
  • the LT ⁇ R has been described as being involved with lymphocyte-mediated diseases and ThI cell-mediated immune responses.
  • LT ⁇ R is expressed on nonlymphoycytes and non-hemapoietic cells (including hepatocytes).
  • LT ⁇ R has two ligands, lymphotoxin and LIGHT (Fu and Chaplin, 1999; Mauri et al, 1998).
  • LIGHT is a recently described TNF family member that can modulate LT ⁇ R.
  • LIGHT is detected on activated T cells.
  • LIGHT has two receptors: LT ⁇ R and the herpes virus entry mediator receptor (HVEM) which is expressed on all hemapoietic cells (Force et al, 1995; Mauri et al, 1998). Both LIGHT and LT expressed on activated T cells is often detected at an inflammatory site and can contribute to enhanced autoimmunity (Gommerman and Browning, 2003; Tamada et al, 2000; Wang and Fu, 2003; Wang et al, 2001).
  • HVEM herpes virus entry mediator receptor
  • the present invention overcomes deficiencies in the art by providing methods and compositions for the treatment and/or prevention of a variety of medical conditions. For example, in certain embodiments these methods and compositions are useful, for promoting liver regeneration, for treating and/or preventing a cardiovascular disease (or cardiovascular damage), or a liver disease (or liver damage) by modulating LT ⁇ R.
  • LT ⁇ R blockers e.g., certain forms of soluble LT ⁇ R
  • serum cholesterol e.g., LDL, VLDL
  • Certain aspects of the present invention are related to the use of LT ⁇ R agonists to promote liver regeneration.
  • An aspect of the present invention relates to a method of treating a cardiovascular disease, treating a liver disease, promoting liver regeneration, or altering lipid levels; comprising administering a LT ⁇ R modulator to a subject.
  • the subject may be a mammal, such as a human.
  • the LT ⁇ R modulator may be an LT ⁇ R blocker, such as LT ⁇ R-Ig.
  • the LT ⁇ R blocker may be an anti-LIGHT antibody or an anti-LT antibody. In certain embodiments, both an anti-LIGHT antibody or an anti-LT antibody are administered to the subject.
  • the LT ⁇ R modulator may be an antibody, such as a humanized antibody.
  • the LT ⁇ R blocker may be comprised in a pharmaceutically acceptable carrier or excipient.
  • the present invention may also be used to treat a patient who does not have, but is at risk of a cardiovascular disease; for example, the present invention may be used to reduce serum LDL and VLDL in a patient in order to decrease the risk of a cardiovascular disease.
  • these methods and compositions may be used to treat or prevent any medical condition which is amenable to such treatment or prevention.
  • the method further comprises administering an additional pharmacological therapeutic agent and/or a surgery to the subject.
  • the LT ⁇ R modulator may be administered once or repeatedly to a subject. Where the LT ⁇ R modulator is administered repeatedly to a subject the duration between administrations may vary from minutes to weeks.
  • a LT ⁇ R modulator e.g., a soluble LT ⁇ R fusion protein, LT ⁇ R-Ig
  • the duration of time between administration of the LT ⁇ R modulator may be about 12-24 h of each other or within about 6- 12 h of each other.
  • the LT ⁇ R modulator e.g., a soluble LT ⁇ R, LT ⁇ R-Ig
  • the dose may be from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 niicrogram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • the LT ⁇ R modulator ⁇ e.g., a soluble LT ⁇ R, LT ⁇ R-Ig
  • the LT ⁇ R modulator may be comprised in a pharmaceutical composition, which may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be suitable for such routes of administration as injection.
  • the LT ⁇ R modulator can be administered intravenously; however, additional administration routes may be used including intradermally, transdermally, intraarterially, orally, inhalation ⁇ e.g., aerosol inhalation), injection, infusion, continuous infusion, via a catheter, via a lavage, in cremes, in lipid compositions ⁇ e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.
  • the invention contemplates any pharmaceutical formulation comprising a LT ⁇ R modulator that may be of use in the methods disclosed herein.
  • Such formulation may take any form know to those of skill in the art and/or disclosed herein that will deliver an appropriate dosage in an appropriate regime.
  • the method comprises treating a cardiovascular disease (e.g., hypertriglyceridemia, hypercholesterolemia, atherosclerosis, artery atherosclerosis restenosis, stenosis, thrombosis, aneurism, embolus, hypertension, stroke, critical stenosis or myocardiac infarction).
  • a cardiovascular disease e.g., hypertriglyceridemia, hypercholesterolemia, atherosclerosis, artery atherosclerosis restenosis, stenosis, thrombosis, aneurism, embolus, hypertension, stroke, critical stenosis or myocardiac infarction.
  • the cardiovascular disease is artery atherosclerosis.
  • the LT ⁇ R blocker may be LT ⁇ R-Ig.
  • the method may comprise altering lipid levels in the subject.
  • cholesterol is reduced, triglycerides are reduced, LDL is -educed, VLDL is reduced, or HDL is increased in the blood of
  • the LT ⁇ R nodulator may be a LT ⁇ R blocker, such as LT ⁇ R-Ig.
  • the method .iirther comprises administering an additional pharmacological therapeutic agent or a surgery :o the subject.
  • the methods comprise treating a liver disease or promoting liver regeneration.
  • the LT ⁇ R modulator may be an LT ⁇ R agonist, such as LIGHT or a LT ⁇ R agonistic antibody.
  • Another aspect of the present invention involves a method of promoting liver regeneration in vitro, comprising contacting a liver cell with a LT ⁇ R agonist.
  • the LT ⁇ R agonist may be LIGHT or a LT ⁇ R agonistic antibody.
  • the liver cell may be comprised in liver tissue.
  • the liver cell may be used for tissue engineering.
  • the liver cell may be subsequently implanted into a subject.
  • the subject maybe a mammal, such as a human.
  • the LT ⁇ R modulator may be an antibody, such as a humanized antibody.
  • the pharmaceutical composition may further comprise an additional therapeutic agent.
  • the additional agent may be effective, either alone or in combination with the LT ⁇ R modulator to treat or prevent a medical condition, inculding but not limited to cardiovascular disease and/or a liver disease, promote liver regeneration, and/or alter a lipid level, in a subject.
  • the LT ⁇ R modulator is an LT ⁇ R blocker, sue as LT ⁇ R-Ig.
  • the LT ⁇ R-Ig may be comprised at a concentration of from about 0.2 mg/kg to about 2 mg/kg.
  • the LT ⁇ R blocker may be an anti-LIGHT antibody and/or an anti-LT antibody.
  • patient or “subject” can include an animal.
  • Preferred animals are mammals, including but not limited to humans, pigs, cats, dogs, rodents, horses, cattle, sheep, goats and cows.
  • Preferred patients and subjects are humans.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • FIG. 1 A T cell derived TNF superfamily member, LIGHT, has a profound effect on the liver in an LT ⁇ R dependent fashion.
  • Transgenic mice expressing /c/c-LIGHT have a dramatically increased liver weight relative to total body weight compared to wild type mice.
  • FIG. 2 Increased mortality of LT ⁇ R and lympho toxin deficient mice after partial hepatectomy. Wild type mice (close triangle) survive partial hepatectomy while mice deficient in either the LT ⁇ R (open triangle) or L Ta ligand (closed diamond) show increased mortality.
  • FIGS. 3A-B LT ⁇ R and lymphotoxin deficient mice show evidence of liver damage and decreased DNA synthesis following partial hepatectomy.
  • FIG. 3A Following partial hepatectomy serum aminotransferase levels are significantly elevated in either the LT ⁇ R or LTa deficient mice compared to similarly treated wild type mice at 48 hours.
  • FIG. 3B The amount of DNA synthesis following partial hepatectomy was determined using a 2 hr pulse with BrdU. BrdU incorporation into actively synthesized hepatocyte DNA was determined using immunohistochemical staining and counting the number of positively stained nuclei in 20 high power fields. There was significantly more active DNA synthesis in the wild type mice compared to LT ⁇ R-/-, LT ⁇ -/- or sham treated mice.
  • FIG. 4 LT ⁇ R deficient mice have markedly decreased percentage of intrahepatic NKT cells and show a marginal increase in NKT cells following partial hepatectomy. Following partial hepatectomy in wild type mice the percentage of CD4+ T-cells and CD4+ CDId tetramer + cells, the major subset of NKT cells, increases significantly ("54.2", as shown) compared to before partial hepatectomy ("29.7", as shown). Similarly, the percentage of CD3+ CDId tetramer positive cells (NKT cells) significantly increases in the liver of wild type mice following partial hepatectomy ("63.9", as shown) compared to before partial hepatectomy ("35.8", as shown).
  • FIGS. 5A-B NKT cell deficient CD1d -/- mice have suppressed hepatocyte proliferation in vivo and in vitro post partial hepatectomy.
  • CD1d -/- mice and their wild type control mice were subject 70% partial hepatectomy.
  • the hepatocyte proliferation was determined by 2 hour pulse BrdU labeling both in vivo and in vitro at different time points.
  • FIG. 6 LTbR-Ig treatment reduces total cholesterol and triglycerides.
  • FIG. 7 LTbR-Ig treatment increases HDL and reduces VLDL.
  • 5 adult LDLR deficient mice from each group were treated with either control or LTbR-Ig.
  • the sera were collected one month and 2 month for HPLC to determine VLDL, LDL, and HDL.
  • FIGS. 8A-D T lymphocyte-derived LIGHT promotes dyslipidemia. Plasma total cholesterol and triglycerides were measured in WT and LIGHT Tg mice fed a chow diet or WTD for two weeks.
  • FIG. 8A and FIG. 8B WT and LIGHT Tg mice on a normal chow diet were assayed for plasma total cholesterol (FIG. 8A) and triglycerides (FIG. 8B).
  • FIG. 8C and FIG. 8D WT and LIGHT Tg mice on WTD were assayed for plasma total cholesterol (FIG. 8C) and triglycerides (FIG. 8D).
  • Each individual symbol represents values from WT (triangles) and LIGHT Tg (circles) mice and the means are plotted as columns. Statistical significance was determined by a two-tailed Student's t-test comparing WT and LIGHT Tg mice (*P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001).
  • FIGS. 9A-D LIGHT-dependent dyslipidemia is mediated by the LT ⁇ R and not HVEM.
  • Plasma total cholesterol and triglycerides were measured in WT, LIGHT Tg, LT ⁇ R-' ' , HVEMT' ' , LIGHT Tg/LT ⁇ R ⁇ ' ⁇ , and LIGHT TgZHVEAT' ' mice.
  • FIG. 9A and FIG. 9B WT, LIGHT Tg, HVEM ⁇ ' ⁇ , and LIGHT Tg/HVEAT' ' mice on WTD were assayed for plasma total cholesterol (FIG. 9A) and triglycerides (FIG. 9B).
  • FIGS. 10A-B Dysregulated LIGHT expression on T cells repro grams hepatic gene expression.
  • FIG. 10A Hepatic lipase expression levels of WT and LIGHT Tg mice. Data shown represent the means of three independent microarray experiments.
  • FIG. 10B LIGHT-mediated repression of hepatic lipase is dependent on the LT ⁇ R. Relative mRNA levels of hepatic lipase were quantified by real-time PCR analysis and normalized to gapdh levels. Relative values of hepatic lipase from mice of the indicated genotypes are shown. Results represent the means (columns) and standard errors. Statistical significance between Tg and Tg/ LT ⁇ R -/- was determined by the Mann- Whitney test (P ⁇ 0.05).
  • FIGS. 11A-H LT ⁇ R signaling controls hepatic lipase expression on hepatocytes and a soluble LT ⁇ R antagonist reverses dyslipidemia in a murine model of atherosclerosis.
  • FIG. 11A and FIG. 11B LDL-R-deficient mice were fed WTD and injected with control or a soluble decoy receptor LT ⁇ R-Ig weekly. The plasma levels of total cholesterol (FIG. 11A) and triglycerides (FIG. 11B) were analyzed. Each symbol represents a data point from an individual mouse. The means are shown as solid lines. (FIG.
  • FIG. 11C Plasma levels of apolipoprotein cholesterols in LDL-RT -/- mice treated with control or LT ⁇ R- Ig. Results represent the means (columns) and standard errors (FIG. HE) Plasma total cholesterol levels of lethally irradiated LDL-R-deficient mice reconstituted with bone marrow from WT, LT ⁇ -/- , and LIGHT -/- mice fed WTD for nine weeks after reconstitution. (FIG.
  • FIG. 11F Hepatic lipase expression levels were quantified by real-time PCR from livers of LDL- R -/- mice on WTD treated with control or LT ⁇ R-Ig. Results represent the means (columns) and standard errors (FIG. 11G) Hepatic lipase mRNA levels of liver samples from (E) were quantified by real-time PCR analysis. (FIG. 11H) Immortalized hepatocytes cell line was stimulated with recombinant LIGHT protein and hepatic lipase expression levels were determined by real-time PCR. Statistical significance was determined by a two-tailed Student's t-test (*P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001). DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • the present invention overcomes deficiencies in the art by providing methods, in certain embodiments, for promoting liver regeneration, for treating and/or preventing a cardiovascular disease (or cardiovascular damage), or a liver disease (or liver damage) by modulating LT ⁇ R.
  • LT ⁇ R blockers e.g., certain forms of soluble LT ⁇ R
  • serum cholesterol e.g., LDL, VLDL
  • Certain aspects of the present invention are related to the use of LT ⁇ R agonists to promote liver regeneration.
  • LT ⁇ R modulators includes LT ⁇ R blockers and LT ⁇ R agonists.
  • LT ⁇ R agonists may be full or partial agonists, such as, for example recombinant LIGHT or an agonistic antibody of LT ⁇ R.
  • LT ⁇ R binds to several ligands, such as lymphotoxin and LIGHT, both TNFSF members.
  • LT or LIGHT can deliver the signaling via LT ⁇ R on stromal cells leading to upregulation of chemokines and cytokines.
  • LTbR-Ig is a soluble form that can block LT ⁇ R ligand activities.
  • LT ⁇ R blockers may be used with the present invention to treat and/or prevent a cardiovascular disease, such as atherosclerosis.
  • LT ⁇ R blockers include, but are not limited to, certain soluble forms of LT ⁇ R (e.g., LT ⁇ R-Ig) and antagonistic antibodies to LT ⁇ R.
  • Certain LT ⁇ R blockers, such as soluble forms of LT ⁇ R, are described, for example, in WO9703687A1, WO9413808A3, and U.S. Patent 6669941.
  • An antibody (or other compound) directed against LIGHT or LT may also be used as a LT ⁇ R blocker; in certain embodiments, both an anti-LIGHT antibody and an anti-LT antibody are administered to a subject to enhance the inhibition of LT ⁇ R.
  • the antibodies are preferably humanized. It is envisioned that these and other LT ⁇ R blockers which are currently known or may be subsequently discovered may be used with the present invention.
  • recombinant LIGHT or an agonistic antibody of LT ⁇ R may be used as an LT ⁇ R agonist.
  • Recombinant LIGHT is described, for example, in U.S. Application 10/865,623 and in Duhen et al. (2004).
  • Recombinant LIGHT may be mutated or non-mutated (i.e., "wild-type"). These compounds may be particularly useful in certain embodiments of the present invention involving promotion of liver regeneration.
  • Agonistic antibodies are preferably humanized.
  • a LT ⁇ R blocker may be used to decrease levels (e.g., serum levels) of a lipid, decrease triglyceride levels, decrease cholesterol, decrease LDL, decrease VLDL, and/or increase HDL in a subject (e.g., a human patient).
  • a LT ⁇ R blocker e.g., LT ⁇ R-Ig
  • the present invention provides, in certain embodiments, methods of treatment and/or prevention of cardiovascular diseases (e.g., atherosclerosis).
  • cardiovascular diseases e.g., atherosclerosis
  • the present invention may also be used to treat a patient who does not have, but is at risk of a cardiovascular disease; for example, the present invention may be used to reduce serum LDL and VLDL in a patient in order to decrease the risk of a cardiovascular disease.
  • Cardiovascular disease includes, but is not limited to, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, restenosis, stenosis, thrombosis, aneurism, embolus, hypertension, stroke, and myocardiac infarction.
  • Atherosclerosis is a serious problem clinically and may result in a critical stenosis, thrombosis, aneurism, or embolus.
  • the cardiovascular disease results from increased inflammation in a patient.
  • Certain embodiments of the present invention are directed towards the treatment of a liver disease.
  • Liver diseases include, but are not limited to, cirrhosis of the liver, viral infection of the liver (e.g., hepatitis infection), liver transplantation, acute liver injury and hepatic failure. Liver diseases also include a disease which results in an impaired ability for the liver to adequately regenerate. Further, the present invention also may be used for the treatment of trauma to the liver (e.g., physical trauma or exposure to a toxin). It is anticipated that the present invention may be used to treat these and other liver diseases.
  • trauma e.g., physical trauma or exposure to a toxin
  • the present invention provides, in certain embodiments, a method for stimulating liver regeneration by stimulation or enhancement of the growth and/or function of NKT cells.
  • NKT cells may be used or modulated in vitro or in vivo to enhance liver regeneration.
  • NKT cells may be added to an in vitro cell culture that includes liver tissue, hi certain embodiments, a modulator of NKT cells may be added to an in vitro cell culture or administered to a subject (e.g., a human patient) in vivo to stimulate NKT cell function.
  • compositions of the present invention comprise an effective amount of one or more LT ⁇ R modulator or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one LT ⁇ R modulator or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional ca ⁇ ier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
  • the LT ⁇ R modulator may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, ntranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, nucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, ⁇ ontinuous infusion, localized perfusion bathing target cells directly, via a catheter, via a avage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
  • the LT ⁇ R modulator may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
  • the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens ⁇ e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • preservatives such as various antibacterial and antifungal agents, including but not limited to parabens ⁇ e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
  • the composition is combined or mixed thoroughly with a semi-solid or solid carrier.
  • the mixing can be carried out in any convenient manner such as grinding.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
  • stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
  • the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include LT ⁇ R modulator, one or more lipids, and an aqueous solvent.
  • lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those of skill in the art, and as the term "lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic ⁇ i.e., designed or produced by man). However, a lipid is usually a biological substance.
  • Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • neutral fats phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • lipids are also encompassed by the compositions and methods of the present invention.
  • the LT ⁇ R modulator may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
  • the dispersion may or may not result in the formation of liposomes.
  • the actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above, [n certain embodiments, the LT ⁇ R modulator ⁇ e.g., a soluble LT ⁇ R, LT ⁇ R-Ig) may be administered to a subject at a dose of from about 0.2 mg/kg to about 2 mg/kg.
  • the LT ⁇ R modulator is formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al, 1997; Hwang et al, 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety).
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, com starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as,
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001.
  • the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial jnterocytes and Peyer's patch M cells.
  • a syrup of elixir may contain the active compound iucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and lavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a buccal tablet, oral spray, or sublingual orally- administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • a LT ⁇ R modulator may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered for sxample, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,753,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by ⁇ eference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts nay be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • the active compound LT ⁇ R modulator may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
  • topical i.e., transdermal
  • mucosal administration intranasal, vaginal, etc.
  • inhalation inhalation
  • compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder.
  • Ointments include all oleaginous, adsorption, emulsion and water-solubility based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
  • compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture.
  • Transdermal administration of the present invention may also comprise the use of a "patch".
  • the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
  • the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety).
  • the delivery of drugs using intranasal microparticle resins Takenaga et al, 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts.
  • transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
  • aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant.
  • the typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers.
  • Suitable containers will vary according to the pressure requirements of the propellant.
  • Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.
  • a LT ⁇ R modulator e.g., a LT ⁇ R blocker or recombinant LIGHT
  • an agent effective in the treatment of vascular or cardiovascular disease or disorder it is contemplated that a conventional therapy or agent, including but not limited to, a pharmacological therapeutic agent, a surgical therapeutic agent ⁇ e.g., a surgical procedure) or a combination thereof, may be combined with LT ⁇ R modulator administration.
  • a therapeutic benefit comprises reduced hypertension in a vascular tissue, or reduced restenosis following vascular or cardiovascular intervention, such as occurs during a medical or surgical procedure).
  • a therapeutic method of the present invention may comprise administration of a LT ⁇ R modulator of the present invention in combination with another therapeutic agent.
  • This process may involve contacting the cell(s) with an agent(s) and the LT ⁇ R modulator at the same time or within a period of time wherein separate administration of the LT ⁇ R modulator and an agent to a cell, tissue or organism produces a desired therapeutic benefit.
  • agent(s) and the LT ⁇ R modulator at the same time or within a period of time wherein separate administration of the LT ⁇ R modulator and an agent to a cell, tissue or organism produces a desired therapeutic benefit.
  • the cell, tissue or organism may be contacted (e.g., by administration) with a single composition or pharmacological formulation that includes both a LT ⁇ R modulator and one or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes a LT ⁇ R modulator and the other includes one or more agents.
  • the LT ⁇ R modulator may precede, be co-current with and/or follow the other agent(s) by intervals ranging from minutes to weeks.
  • the LT ⁇ R modulator, and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the LT ⁇ R modulator and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism.
  • one or more agents may be administered within of from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 2 hours
  • compositions of a LT ⁇ R modulator may follow general protocols for the administration of vascular or cardiovascular therapeutics, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary. In particular embodiments, it is contemplated that various additional agents may be applied in any combination with the present invention.
  • Non-limiting examples of a pharmacological therapeutic agent that may be used in the present invention include an antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, a blood coagulant, an antiarrhythmic agent, an antihypertensive agent, a vasopressor, a treatment agent for congestive heart failure, an antianginal agent, an antibacterial agent or a combination thereof.
  • an agent that lowers the concentration of one of more blood lipids and/or lipoproteins may be combined with administration of a LT ⁇ R modulator for cardiovascular therapy, particularly in treatment of athersclerosis and thickenings or blockages of vascular tissues.
  • an antihyperlipoproteinemic agent may comprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acid sequesterant, a HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroid hormone or thyroid hormone analog, a miscellaneous agent or a combination thereof.
  • Non-limiting examples of aryloxyalkanoic/fibric acid derivatives include beclobrate, enzaf ⁇ brate, binifibrate, ciprofibrate, clinofibrate, clofibrate (atromide-S), clofibric acid, etof ⁇ brate, fenofibrate, gemfibrozil (lobid), nicof ⁇ brate, pirifibrate, ronif ⁇ brate, simfibrate and theofibrate.
  • Non-limiting examples of resins/bile acid sequesterants include cholestyramine (cholybar, questran), colestipol (colestid) and polidexide.
  • HMG CoA reductase inhibitors include lovastatin (mevacor), pravastatin (pravochol) or simvastatin (zocor).
  • Non-limiting examples of nicotinic acid derivatives include nicotinate, acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid.
  • Non-limiting examples of thyroid hormones and analogs thereof include etoroxate, thyropropic acid and thyroxine.
  • miscellaneous antihyperlipoproteinemics include acifran, azacosterol, benfluorex, ⁇ -benzalbutyramide, carnitine, chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium, 5,8, 11, 14, 17-eicosapentaenoic acid, eritadenine, jrazabol, meglutol, melinamide, mytatrienediol, ornithine, ⁇ -oryzanol, pantethine, entaerythritol tetraacetate, ⁇ -phenylbutyramide, pirozadil, probucol (lorelco), ⁇ -sitosterol, ⁇ ltosilic acid-piperazine salt, tiadenol, triparanol and xenbucin.
  • Non-limiting examples of an antiarterioscl erotic include pyridinol carbamate.
  • administration of an agent that aids in the removal or revention of blood clots may be combined with administration of a LT ⁇ R modulator for ardiovascular therapy, particularly in treatment of atherosclerosis and vasculature ⁇ e.g., rterial) blockages.
  • a LT ⁇ R modulator for ardiovascular therapy particularly in treatment of atherosclerosis and vasculature ⁇ e.g., rterial
  • antithrombotic and/or fibrinolytic agents aclude anticoagulants, anticoagulant antagonists, antiplatelet agents, thrombolytic agents, thrombolytic agent antagonists or combinations thereof.
  • antithrombotic agents that can be administered orally, such s, for example, aspirin and wafarin (Coumadin), are preferred.
  • a non-limiting example of an anticoagulant include acenocoumarol, ancrod, inisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextran sulfate sodium, licumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol, fluindione, heparin, ⁇ rudin, lyapolate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, ihosvitin, picotamide, tioclomarol and warfarin.
  • Non-limiting examples of antiplatelet agents include aspirin, a dextran, dipyridamole persantin), heparin, sulfinpyrano ⁇ e (anturane) and ticlopidine (ticlid).
  • Non-limiting examples of thrombolytic agents include tissue plasminogen activator activase), plasmin, pro-urokinase, urokinase (abbokinase) streptokinase (streptase), mistreplase/ APSAC (eminase).
  • tissue plasminogen activator activase tissue plasminogen activator activase
  • plasmin pro-urokinase
  • urokinase abbrevity
  • streptokinase streptokinase
  • mistreplase/ APSAC eminase
  • an agent that may enhance blood coagulation may be used.
  • a blood coagulation promoting agent include thrombolytic agent antagonists and anticoagulant antagonists.
  • Non-limiting examples of anticoagulant antagonists include protamine and vitamin Kl.
  • Non-limiting examples of thrombolytic agent antagonists include aminocaproic acid (amicar) and tranexamic acid (amstat).
  • Non-limiting examples of antithrombotics include anagrelide, argatroban, cilstazol, daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.
  • antiarrhythmic agents include Class I antiarrythmic agents (sodium channel blockers), Class II antiarrythmic agents (beta-adrenergic blockers), Class II antiarrythmic agents (repolarization prolonging drugs), Class IV antiarrhythmic agents (calcium channel blockers) and miscellaneous antiarrythmic agents.
  • Non-limiting examples of sodium channel blockers include Class IA, Class IB and Class IC antiarrhythmic agents.
  • Class IA antiarrhythmic agents include disppyramide (norpace), procainamide (pronestyl) and quinidine (quinidex).
  • Class IB antiarrhythmic agents include lidocaine (xylocaine), tocainide (tonocard) and mexiletine (mexitil).
  • Class IC antiarrhythmic agents include encainide (enkaid) and flecainide (tambocor).
  • Non-limiting examples of a beta blocker otherwise known as a ⁇ -adrenergic blocker, a ⁇ -adrenergic antagonist or a Class II antiarrhythmic agent, include acebutolol (sectral), alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol (brevibloc), indenolol, la
  • the beta blocker comprises an aryloxypropanolamine derivative.
  • aryloxypropanolamine derivatives include acebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol
  • Non-limiting examples of an agent that prolong repolarization also known as a Class III antiarrhythmic agent, include amiodarone (cordarone) and sotalol (brittce).
  • Non-limiting examples of a calcium channel blocker include an arylalkylamine (e.g., bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline, verapamil), a dihydropyridine derivative (felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) a piperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) or a micellaneous calcium channel blocker such as bencyclane, etafenone, magnesium, mibefradil or perhexiline.
  • a calcium channel blocker comprises a long-acting dihydropyridine (nifedipine- type) calcium antagonist.
  • miscellaneous antiarrhymic agents include adenosine (adenocard), digoxin (lanoxin), acecainide, ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid, cifenline, disopyranide, hydroquinidine, indecainide, ipatropium bromide, lidocaine, lorajmine, lorcainide, meobentine, moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidine polygalacturonate, quinidine sulfate and viquidil.
  • antihypertensive agents include sympatholytic, alpha/beta blockers, alpha blockers, anti-angiotensin II agents, beta blockers, calcium channel blockers, vasodilators and miscellaneous antihypertensives.
  • an alpha blocker also known as an ⁇ -adrenergic blocker or an ⁇ -adrenergic antagonist
  • an alpha blocker include amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin and yohimbine.
  • an alpha blocker may comprise a quinazoline derivative.
  • quinazoline derivatives include alfuzosin, bunazosin, doxazosin, prazosin, terazosin and trimazosin.
  • an antihypertensive agent is both an alpha and beta adrenergic antagonist.
  • alpha/beta blocker comprise labetalol (normodyne, trandate).
  • Non-limiting examples of anti-angiotension II agents include include angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists.
  • Non-limiting examples of angiotension converting enzyme inhibitors (ACE inhibitors) include alacepril, enalapril (vasotec), captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril, perindopril, quinapril and ramipril.
  • angiotensin II receptor blocker also known as an angiotension II receptor antagonist, an ANG receptor blocker or an ANG-II type-1 receptor blocker (ARBS)
  • angiocandesartan eprosartan, irbesartan, losartan and valsartan.
  • Non-limiting examples of a sympatholytic include a centrally acting sympatholytic or a peripherially acting sympatholytic.
  • Non-limiting examples of a centrally acting sympatholytic also known as an central nervous system (CNS) sympatholytic, include clonidine (catapres), guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet).
  • Non-limiting examples of a peripherally acting sympatholytic include a ganglion blocking agent, an adrenergic neuron blocking agent, a ⁇ -adrenergic blocking agent or a alphal- adrenergic blocking agent.
  • Non-limiting examples of a ganglion blocking agent include mecamylamine (inversine) and trimethaphan (arfonad).
  • Non-limiting of an adrenergic neuron blocking agent include guanethidine (ismelin) and reserpine (serpasil).
  • Non-limiting examples of a ⁇ -adrenergic blocker include acenitolol (sectral), atenolol (tenormin), betaxolol (kerlone), carteolol (cartrol), labetalol (normodyne, trandate), metoprolol (lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken), propranolol (inderal) and timolol (blocadren).
  • Non-limiting examples of alphal -adrenergic blocker include prazosin (minipress), doxazocin (cardura) and terazosin (hytrin).
  • a cardiovasculator therapeutic agent may comprise a vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or a peripheral vasodilator).
  • a vasodilator comprises a coronary vasodilator.
  • Non-limiting examples of a coronary vasodilator include amotriphene, bendazol, benfurodil hemisuccinate, benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane, etafenone, fendiline, floredil, ganglefene, herestrol bis( ⁇ -diethylaminoethyl ether), hexobendine, itramin tosylate, khellin, lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin, pentaerythritol tetranitrate, pentrinitrol, perhexiline, pimefylline, trapidil, tricromyl, trimeta
  • a vasodilator may comprise a chronic therapy vasodilator or a hypertensive emergency vasodilator.
  • a chronic therapy vasodilator include hydralazine (apresoline) and minoxidil (loniten).
  • a hypertensive emergency vasodilator include nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten) and verapamil. 6. Miscellaneous Antihypertensives
  • miscellaneous antihypertensives include ajmaline, ⁇ - aminobutyric acid, bufeniode, cicletainine, ciclosidomine, a cryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4- pyridyl ketone thiosemicarbazone, muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, a protoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodium nitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase and urapidil.
  • an antihypertensive may comprise an arylethanolamine derivative, a benzothiadiazine derivative, a iV-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative, a guanidine derivative, a hydrazines/phthalazine, an imidazole derivative, a quanternary ammonium compound, a reserpine derivative or a sulfonamide derivative.
  • Non-limiting examples of arylethanolamine derivatives include amosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol and sulfinalol.
  • Non-limiting examples of benzothiadiazine derivatives include althizide, bendrofiumethiazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane, metolazone, parafiutizide, polythizide, tetrachlormethiazide and trichlormethiazide.
  • Non-limiting examples of iV-carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapril and ramipril.
  • Non-limiting examples of dihydropyridine derivatives include amlodipine, felodipine, isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine and nitrendipine. e. Guanidine Derivatives
  • Non-limiting examples of guanidine derivatives include bethanidine, debrisoquin, guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan.
  • Non-limiting examples of hydrazines/phthalazines include budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine and todralazine.
  • Non-limiting examples of imidazole derivatives include clonidine, lofexidine, phentolamine, tiamenidine and tolonidine.
  • quantemary ammonium compounds include azamethonium bromide, chlorisondamine chloride, hexamethonium, pentacynium bis(methylsulfate), pentamethonium bromide, pentolinium tartrate, phenactropinium chloride and trimethidinium methosulfate.
  • Non-limiting examples of reserpine derivatives include bietaserpine, deserpidine, rescinnamine, reserpine and syrosingopine.
  • Non-limiting examples of sulfonamide derivatives include ambuside, clopamide, furosemide, indapamide, quinethazone, tripamide and xipamide.
  • Vasopressors generally are used to increase blood pressure during shock, which may occur during a surgical procedure.
  • a vasopressor also known as an antihypotensive, include amezinium methyl sulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin, gepefrine, metaraminol, midodrine, norepinephrine, pholedrine and synephrine.
  • agents for the treatment of congestive heart failure include anti-angiotension II agents, afterload-preload reduction treatment, diuretics and inotropic agents.
  • an animal patient that can not tolerate an angiotensin antagonist may be treated with a combination therapy.
  • Such therapy may combine adminstration of hydralazine (apresoline) and isosorbide dinitrate (isordil, sorbitrate).
  • Non-limiting examples of a diuretic include a thiazide or benzothiadiazine derivative (e.g., althiazide, bendroflumethazide, benzthiazide, benzylliydrochloro thiazide, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide), an organomercurial ⁇ e.g., chlormerodrin, meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mer
  • Non-limiting examples of a positive intropic agent also known as a cardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline, amrinone, benfurodil hemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin, cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine, dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine, prenalterol, proscillaridine, resibufogenin, scillaren, sci
  • an intropic agent is a cardiac glycoside, a beta-adrenergic agonist or a phosphodiesterase inhibitor.
  • a cardiac glycoside includes digoxin (lanoxin) and digitoxin (crystodigin).
  • Non-limiting examples of a ⁇ -adrenergic agonist include albuterol, bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex), dopamine (intropin), dopexamine, ephedrine, etafedrine, ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol and xamoterol.
  • Antianginal agents may comprise organonitrates, calcium channel blockers, beta blockers and combinations thereof.
  • Non-limiting examples of organonitrates also known as nitrovasodilators, include nitroglycerin (nitro-bid, nitrostat), isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol, vaporole).
  • Antibacterials are generally used to reduce or prevent infection.
  • Non-limiting examples of antibacterials include antibiotic antibacterials, synthetic antibacterials, leprostatic antibacterials rickettsia antibacterials, tuberculostatic antibacterial or a combination thereof.
  • Non-limiting examples of antibiotic antibacterials include an aminoglycoside (e.g., amikacin, apramycin, arbekacin, a bambermycin, butirosin, dibekacin, dihydrostreptomycin, a fortimicin, gentamicin, isepamicin, kanamycin, micronomicin, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, streptonicozid, tobramycin), an amphenol (e.g., azidamfenicol, chloramphenicol, chloramphenicol palmitate, chloramphenicol pantothenate, florfenicol, thiamphenicol), an ansamycin (e.g., rifamide, rifampin, rifamycin, rifaximin), a ⁇ -lactam (e.g.
  • Non-limiting examples of a carbapenem ⁇ -lactam include imipenem.
  • Non-limiting examples of a cephalosporin ⁇ -lactam include cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime proxetil, cefroxadine, cefsulodin, ceftazidime, cefteram, cftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephacetrile sodium, cephalexin, cephaloglycin, cephaloridine, cephalosporin C, cephalothin, cephapir
  • Non-limiting examples of a cephamycin ⁇ -lactam include cefbuperazone, cefmetazole, cefminox, cefotetan and cefoxitin.
  • Non-limiting examples of a monobactam ⁇ -lactam include aztreonam, carumonam and tigemonam.
  • Non-limiting examples of a oxacephem ⁇ -lactam include flomoxef and moxolactam.
  • Non-limiting examples of a penicillin ⁇ -lactam include amidinocillin, amdinocillin pivoxil, amoxicillin, ampicillin, apalcillin, aspoxicillin, azidocillin, azlocillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, carbenicillin, carfecillin sodium, carindacillin, clometocillin, cloxacillin, cyclacillin, dicloxacillin diphenicillin sodium, epicillin, fenbenicillin, floxacillin, hetacillin, lenampicillin, metampicillin, methicillin sodium, mezlocillin, nafcillin sodium, mezlocillin, nafcillin sodium, oxacillin, penamecillin, penethamate hydridide, penicillin G benethiamine, penicillin G benzathine, penicillin G benzhydrylamine
  • Non-limiting examples of synthetic antibacterials include 2,4-diaminopyrimidines (e.g., brodimoprim, tetroxoprim, trimethoprim), nitrofurans (e.g., furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nirurpirinol, nifurprazine, nifurtoinol, nitrofurantion), quinolones and quinone analogs (e.g., amifoxacin, cinoxacin, ciprofloxacin, difloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, miloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid, piromidic acid, rosoxacin, temafloxacin,
  • Non-limiting examples of leprostatic antibacterials include acedapsone, acetosulfone sodium, clofazimine, dapsone, diathymosulfone, glucosulfone sodium, hydnocarpic acid, solasulfone, succisulfone and sulfoxone sodium.
  • Non-limiting examples of rickettsia antibacterials also known as antirickettsials, include p-aminobenzoic acid, chloramphenicol, chloramphenicol palmitate, chloramphenicol pantothenate and tetracycline.
  • tuberculostatic antibacterials include ⁇ -aminosalicylic acid, ⁇ -aminosalicylic acid hydrazine, benzoylpas, 5-bromosalicylhydroxamic acid, capreomycin, clofazimine, cyacetacide, cycloserine, dihydrostrptomycin, enviomycin, ethambutol, ethionamide, 4'-formylsuccinanilic acid thiosemicarbazone, furonazide, glyconiazide, isobutol, isoniazide, isoniazid methanesulfonate, morphazinamide, openingazide, parsiniazide, phenyl aminosalicylate, protionamide, pyrazinamide, rifampin, salinazide, streptomycin, subathizone, sulfoniazide, thiacetazone, tiocarlide
  • a therapeutic agent may comprise a surgery of some type, which includes, for example, preventative, diagnostic or staging, curative and palliative surgery.
  • Surgery and in particular a curative surgery, may be used in conjunction with other therapies, such as the present invention and one or more other agents.
  • Such surgical therapeutic agents for vascular and cardiovascular diseases and disorders are well known to those of skill in the art, and may comprise, but are not limited to, performing surgery on an organism, providing a cardiovascular mechanical prostheses, angioplasty, coronary artery reperfusion, catheter ablation, providing an implantable cardioverter defibrillator to the subject, mechanical circulatory support or a combination thereof.
  • a mechanical circulatory support that may be used in the present invention comprise an intra-aortic balloon counterpulsation, left ventricular assist device or combination thereof.
  • Further treatment of the area of surgery may be accomplished by perfusion, direct injection, systemic injection or local application of the area with at least one additional therapeutic agent (e.g., a LT ⁇ R modulator of the invention, a pharmacological therapeutic agent), as would be known to one of skill in the art or described herein.
  • at least one additional therapeutic agent e.g., a LT ⁇ R modulator of the invention, a pharmacological therapeutic agent
  • liver maintains its unique ability to undergo self-renewal throughout life (Michalopoulos and DeFrances, 1997). Acute viral and toxic insults lead to loss of hepatic mass and continued host survival requires proper restoration of hepatic mass. The inability of the host to restore liver mass is the basis for chronic liver diseases, which cause significant morbidity and mortality worldwide. Controlling the ability of the liver to regulate its mass has broad implications beyond chronic liver disease. Organ shortages continue to press the need for liver transplantation alternatives. The ability to increase the renewal of liver would make split liver transplants for living adult donor and even cellular transplants more feasible. Further understanding of liver growth control mechanisms offers a possible solution to both chronic liver disease and liver transplantation.
  • the partial hepatectomy is a particularly useful model for beginning to understand how the liver regenerates because the liver's synchronized response facilitates study and the liver tightly controls its size (Diehl, 2002).
  • Diehl, 2002 the cellular and molecular mechanisms that control the process are not well defined.
  • the understanding of liver regeneration has surrounded the cytokine and growth factor network involved in the process (Galun and Axelrod, 2002).
  • TNF alpha TNF alpha
  • IL 6 interleukin 6
  • HGF hepatocytes growth factor
  • EGF epidermal growth factor
  • TGF alpha transforming growth factor alpha
  • the liver is a complex organ consisting of multiple cell types.
  • Non-parenchymal cells ⁇ i.e. non-hepatocytes
  • non-hepatocytes which make up a significant cellular component of the liver, also respond to partial hepatectomy and appear to be important in maintaining intercellular relationships during liver regeneration.
  • hepatocytes There is already evidence indicating that the cellular mechanisms driving hepatocyte renewal depends on cells from both the bone marrow and from within the liver itself (Wang et al, 2003; Petersen et al, 1999; Vassilopoulos et al., 2003).
  • LIGHT is a newly described TNF family member that is detected on activated T cells.
  • LIGHT has two receptors, the lymphotoxin beta receptor (LT ⁇ R) which is expressed on non- lymphocytes and non-hemapoietic cells (including hepatocytes) and the herpes virus entry mediator receptor (HVEM) which is expressed on all hemapoietic cells (Force et al. 1995; Mauri et al, 1998).
  • LIGHT performs a unique role bridging hemapoietic and non-hemapoietic signaling.
  • LIGHT expression on activated T cells can signal to other hemapoietic cells via the HVEM receptor and to non-hemapoietic cells through the LT ⁇ R.
  • LT ⁇ R-/- Transgenic mice expressing LIGHT on T cells show a marked increase in liver mass even though liver size is ordinarily tightly controlled. Furthermore, LT ⁇ R deficient mice (LT ⁇ R-/-) also show an impaired ability to regenerate their liver after partial hepatectomy suggesting that the LT ⁇ R pathway is required for optimal liver regeneration. The inventors also extend theses observations to show that T cells are also required for liver regeneration following partial hepatectomy. The inventors have further found that a deficiency of natural killer T lymphocytes (NKT), a cell type particularly numerous in the wild type liver and severely decreased in LT ⁇ R-/- and lymphotoxin alpha deficient mice (LTa-/-), appears to be responsible for supporting hepatocytes division. Therefore, this study establishes a key interaction between T cell derived cytokines, T cells and hepatocytes during liver regeneration.
  • NKT natural killer T lymphocytes
  • mice and Reagents Mice carrying a transgene for LIGHT have previously been reported (Wang et al, 2001).
  • Six to ten week old male C57B/L6J, TCRbeta-/-delta-/-, (Stock # 002122) TRC delta-/- (Stock # 002120), and RAGl-/- (Stock # 002216) were purchased from The Jackson Laboratory (Bar Harbor, ME).
  • LTa-/- mice were a gift from David Chaplin (Washington University, St. Louis MS) and backcross to B6 background for 13 generations.
  • LT ⁇ R-/- mice were generated by Klaus Pfeffer (Futterer et al., 1998) and backcrossed to B6 background for 5-7 generation and HVEM-/- mice were recently generated in Klaus Pfeffer' s laboratory (Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Kunststoff, Germany) and backcrossed to B6 for 5 generations and genetic targeting was confirmed by southern blot analysis and antibody staining.
  • CD IdI-/- mice were generated at University of Chicago (Chen et al., 1997). All of mice were maintained in University of Chicago and handled according to NIH guideline and approved by the Institutional Animal Care and Use Committee.
  • Bone marrow transplantation was performed by lethally irradiating recipients with 900 rads followed by i.v. injection of 3 million bone marrow cells which were isolated by flushing the donors femur and humerus with sterile PBS.
  • Partial hepatectomy The procedure was performed as originally described by Higgins and Anderson (1931). In brief, the mice were anesthetized with ketamine and xylzaline (100 mg/kg/10 mg/kg IP), the liver was exposed through a midline incision, the median and left lobes were mobilized and delivered after ligature with a single silk 4-0 suture. Sham procedures consist of anesthetized mice in which the peritoneum was opened and the liver is manipulated but not resected. Serum aspartate transaminase activity was determined by the method of Karmen using a Elan Diagnostics (SmithField, RI) analyzer according to the manufactures protocols in the University of Chicago Animal Research Core laboratory. Histology and BrdU labeling.
  • Tissue section were fixed in 70% alcohol followed by buffered formalin and processed either for routine hemotoxylin and eosin staining or for BrdU immunohistochemical studies as per the manufactures instructions (Zymed San Francisco, CA).
  • the liver was perfusion in situ with collagenase and > 85% viable hepatocytes were purified from 3 mice after differential centrifugation.
  • Hepatocytes were seeded onto type I collagen coated coverslips in alpha-MEM (Invitrogen, Carlsbad, CA) Brdu incorporation was measured by incubation of the cells with (BrdU) (final dilution 1 :50, Zymed Laboratory Inc., South San Francisco, CA) in alpha MEM medium containing 125 mM Hepes, 200 mM L-glutamine, 0.13 mM L- proline, insulin (5 ⁇ g/ml), transferrin (5 ⁇ g/ml) and selenium (5 ng/ml), hydrocortisone (1 microM) and epidermal growth factor (50 ng/ml). BrdU incorporation was scored in 10 hpf after staining the coverslips according to the manufacturer's instructions (Zymed)
  • livers were pressed through a 200# mesh screen and centrifuged 5 min at 800xg. Pellets were suspended in 35% Percoll and 100 unit heparin/ml and centrifuged at 800xg for 20 minutes. The pellet was lysed of RBC and mononuclear cells were counted and prepared for staining. Lymphocytes were stained with CD3-FITC (clone 2Cl IBD Pharmingen Franklin Lakes, NJ) and CDId alpha-gal-cer loaded tetramer-PE and analyzed by FACS (Becton Dickson FACS scan II).
  • CD3-FITC clone 2Cl IBD Pharmingen Franklin Lakes, NJ
  • CDId alpha-gal-cer loaded tetramer-PE and analyzed by FACS (Becton Dickson FACS scan II).
  • liver maintains its unique ability to undergo self-renewal throughout life with strict control of cell turnover (Michalopoulos and DeFrances, 1997).
  • TNF superfamily ligand LIGHT TNSF 14
  • Transgenic mice expressing /c/c-LIGHT on T cells have a dramatically increased liver weight relative to total body weight (FIG. 1). It suggests that a T cell derived factor can be sufficient for controlling liver homeostasis.
  • Liver histology reveals markedly enlarged hepatocytes and frequent mitotic figures in Tg LIGHT mice compared to age matched wild type livers.
  • LIGHT has two known putative receptors, the [ymphotoxin beta receptor (LT ⁇ R) and herpes virus entry mediator (HVEM), the LIGHT transgenic mice were crossed to mice deficient in each of these receptors.
  • LIGHT has two known putative receptors, the [ymphotoxin beta receptor (LT ⁇ R) and herpes virus entry mediator (HVEM), the LIGHT transgenic mice were crossed to mice deficient in each of these receptors.
  • LIGHT has two known putative receptors
  • HVEM herpes virus entry mediator
  • the wild type mice receiving LIGHT transgenic bone marrow also displayed an enlarged liver with abnormal liver histology compared to wild type bone marrow transplant controls. Furthermore, a previous study showed a human hepatocyte cell line expresses mRNA for the LT ⁇ R, and that LIGHT pretreatment prevents TNF-mediated hepatocyte apoptosis (Matsui et al., 2002). This series of experiments hint that T cells acting through the LT ⁇ R expressing cells contribute to liver homeostasis.
  • Tg LIGHT mice crossed to LIGHT's other putative receptor, HVEM did not have an effect on the development of abnormal liver histology.
  • the Tg LIGHT x HVEM-/- mice still have markedly enlarged hepatocytes.
  • Bone marrow from Tg LIGHT mice transplanted into lethally irradiated wild type mice also results in an enlarged liver. Wild type bone marrow transplanted into wild type mice results in normal liver histology while Tg LIGHT bone marrow transplanted into wild type mice results in the same liver histology seen in Tg LIGHT mice.
  • LT ⁇ R signaling by T cells may be essential for liver regeneration
  • LT ⁇ R Since the LT ⁇ R appeared to mediate liver proliferation in response to a T cell derived cytokine, the inventors wanted to better understand how the LT ⁇ R might contribute to restoration of liver mass following partial resection. Partial hepatectomy, in which 70% of the liver is removed, is regarded as the preferred method to study liver regeneration (Higgins and Anderson, 1931; Fausto and Campbell, 2003). LT ⁇ R deficient mice were challenged with a 70% hepatectomy. Wild type mice showed no significant increase in morbidity or mortality following 70% hepatectomy while there was a dramatically increased morbidity and mortality of LT ⁇ R -/- after 70% hepatectomy (FIG. 2).
  • mice deficient in lymphotoxin (LT ⁇ -/- ), another lymphocyte derived ligand that signals through the LT ⁇ R, also displayed a significant decrease in survival following 70% hepatectomy (FIG. 2).
  • LT ⁇ R -/- and LT ⁇ -/- mice showed evidence of liver damage at 48 hours following partial hepatectomy with significantly elevated serum aminotransferase levels compared to similarly treated wild type mice (FIG. 3A). Histological examination of the livers also showed large areas of necrosis in the LT ⁇ R -/- or LT ⁇ -/- mice at 48 hours following partial hepatectomy compared to wild type mice. Large areas of necrosis (pale hepatocytes outlines without a nucleus) were observed in the LT ⁇ R or LTa deficient mice at 48 hours following partial hepatectomy compared to wild type mice.
  • NK T cells are critical for liver regeneration
  • T-cell derived cytokines such as LIGHT and lymphotoxin
  • T-cell derived cytokines can effect the liver.
  • lymphocytes are essential for controlling liver regeneration
  • RAGl -/- mice that lack B, T and NKT cells were tested for their ability to undergo liver regeneration following partial hepatectomy and were found to be defective (Table 1).
  • T cell receptor deficient (TCR ⁇ -/- ⁇ -/- ) mice that lack both T and NKT cells all display reduced survival following partial hepatectomy (Table 1).
  • mice that only lack a small subset of T cells TCR ⁇ -/-
  • TCR ⁇ -/- TCR ⁇ T cells
  • a T cell defect common to LT ⁇ R -/- and LT ⁇ -/- mice is a marked decrease in the percentage and total number of intrahepatic NKT cells while the total number of other T cell types remains normal (Elewaut et ah, 2000).
  • NK T cells are particularly rich in the liver, and it has been shown that NK T cells increase in total number and percentage following partial hepatectomy in wild type mice (Minagawa et al, 2000). Twelve hours following partial hepatectomy there is a sharp increase in NKT cells (CD3 + CDId tetramer + ) and (CD4 + CDId tetramer) (FIG. 4).
  • the LT ⁇ R -/- mice have a decreased percentage, total number of NKT cells (CD3 + CD1d tetramer + ) and an impaired increase in the percentage of NKT cells following partial hepatectomy.
  • This series of experiments demonstrates lymphotoxin deficient mice have reduced numbers of intrahepatic NKT cells before and after partial hepatectomy. It supports the idea that NKT cells, an important component of the innate immune response, might be critical for liver regeneration.
  • LT ⁇ R deficient mice have a markedly decreased percentage of intrahepatic NKT cells and show a marginal increase in NKT cells following partial hepatectomy.
  • the percentage of CD4+ T-cells and CD4+ CDId tetramer + cells increases significantly compared to before partial hepatectomy.
  • the percentage of CD3+ CDId tetramer positive cells (NKT cells) significantly increases in the liver of wild type mice following partial hepatectomy compared to before partial hepatectomy.
  • the percentage of CD3+ CDId+ NKT cells is significantly decreased in LT ⁇ R deficient mice prior to partial hepatectomy with a marginal increase in the percentage of NKT cells following partial hepatectomy.
  • mice which are deficient in NKT cells or depleted wild type mice of NKT cells. Since most NKT cells in the liver express CD4 (FIG. 4), wild type mice were depleted of CD4+ cells with 150 to 300 micrograms of anti-CD4 (GKl .5) 7 days prior to the procedure. The survival of these mice was significantly reduced following partial hepatectomy (Table 1).
  • mice depleted of NKl.1+ cells which depletes NK and NKT
  • NK and NKT anti NKl.1
  • wild type mice depleted of NKT cells displayed significantly reduced survival following partial hepatectomy (Table 1).
  • the inventors further confirmed this finding using mice completely deficient in CDl dependent NKT cells (Chen et al, 1997). Mice deficient in CDId, which lack a major subset of NKT cells, have smaller liver/body weights at baseline and also display reduced survival following partial hepatectomy (Table 1).
  • livers proliferative activity at 24 and 36 h following partial hepatectomy shows that induction of hepatocyte DNA synthesis is inhibited consistently in CD1d - /- mice.
  • time of peak proliferative activity in normal mice 100 fold fewer hepatocytes have entered S phase in CD1d - /- mice than controls (FIG. 5A).
  • Liver histology reveals significantly decreased hepatocyte proliferation presented by BrdU+ brown nuclei in NKT cell deficient CD1d - /- mice comparing to wild type control mice 36 hour post partial hepatectomy.
  • hepatocytes in CD1d - /- mice show a decreased ability to proliferate ex-vivo following 70% hepatectomy (Fig. 5B).
  • the maintenance of hepatic mass in the adult is tightly controlled.
  • the cellular and molecular mechanisms that govern this process are unclear. Massive hepatomegaly in Tg LIGHT mice suggests that up-regulation of LIGHT on T cells disrupts the mechanisms that normally balance hepatocyte proliferation and hepatocyte death, leading to net accumulation of hepatocytes.
  • lymphotoxin (LT) deficient mice which is expressed on activated T cells and is another ligand for LT ⁇ R.
  • LT ⁇ R lymphotoxin
  • the LT/LT ⁇ R pathway uniquely bridges the hematopoietic system, responsible for the expression on LIGHT and lymphotoxin, to the non- hematopoietic system, which expresses the LT ⁇ R.
  • cytokine and growth factor production are not the only mechanisms functioning during liver regeneration. Understanding of how the hematopoietic and non-hematopoietic cells interact will be critical in order to apply this model to clinical situations. The inventors realization of this connection has further allowed us to address the role of T cells in liver regeneration.
  • the liver contains a large number of resident T cells that have access to hepatocytes through fenestrated endothelial cells without having to cross a basement membrane (Maher, 2001).
  • the role of residual T cells in liver homeostasis and function has been speculated. It was noticed that CD4+ NKT cells, 80% NKT cells inside liver, are often activated and increased inside the liver after partial hepatectomy but its role has not been well elucidated (Minagawa et ah, 2000). NKT cells appear to partially contribute to liver inflammation (Kato et ah, 2004; Ito et al, 2003). This study has clearly demonstrated that T cells are essential for liver regeneration.
  • NKT cells are essential for the process.
  • NKT cells a dominant T cell population inside liver, are important population for innate immunity and contain massive cytokines and ligands similar to those within activated T cells, which allow them to rapidly release large amounts of cytokines and. activating ligands following tissue injury.
  • the inventors show that depleting mice of CD4+, or NKl.1+ cells aborts this component of the regenerative response and impairs survival after partial hepatectomy.
  • CDId ' ' ' mice that cannot support normal NKT cell development have depleted NKT cell populations and exhibit somewhat atrophic livers at baseline. Following partial hepatectomy, CDId 1' display significantly decreased induction of hepatocyte DNA synthesis and increased mortality following partial hepatectomy. Taken together, these data support the concept that NKT cells provide trophic signals that are necessary for hepatocytes to proliferate.
  • LT ⁇ R-Ig LDL receptor deficient mice have been powerful models to study how lipid level is controlled. The mice will develop hypercholesterolemia, triglycerimia and increased VLDL and HDL 1-2 weeks after feeding with fatty food, eventually leading to severe atherosclerosis in 2-3 months. In contrast to control Ig, LT ⁇ R-Ig treated group much reduced triglycerides and cholesterols as well as reduced VLDL and increased HDL ⁇ e.g., FIG. 6, FIG. 7): Therefore, treating patients suffering hyperlipidemia and atherosclerosis with LT ⁇ R modifier may be a new treatment.
  • Atherosclerosis is a chronic inflammatory response to hyperlipidemia and associated high incidence of cardiovascular and brain vascular diseases. Both innate and adaptive immunity influence the progression of this inflammation. Most studies in this field focus on the inflammation lesions and how to reduce inflammation-mediated lesion on artery. The effect of the immune system on atherosclerosis is rather complex. Few studies focus on the lipid profile. Whether and which cytokines are involved in the regulation of lipid profile is unclear (Wang and Paigen, 2005).
  • Atherosclerosis is the major cause of death in the world. Fasting and postprandial hyperlipidemia are important risk factors for coronary heart disease (CHD). Recent developments have undoubtedly indicated that inflammation is pathophysiologically closely linked to atherogenesis and its clinical consequences. Inflammatory markers such as C- reactive protein (CRP), leukocyte count and complement component 3 (C3) have been linked to CHD and to hyperlipidemia and several other CHD risk factors. Increases in these markers may result from activation of endothelial cells (CRP, leucocytes, C3), disturbances in adipose tissue fatty acid metabolism (CRP, C3), or from direct effects of CHD risk factors (leucocytes).
  • CRP C- reactive protein
  • C3 complement component 3
  • lipoproteins, triglycerides, fatty acids and glucose can activate endothelial cells, most probably as a result of the production of reactive oxygen species (van Oostrom et al, 2004). Similar mechanisms may also lead to leukocyte activation. Increases in triglycerides, fatty acids and glucose are common disturbances in the metabolic syndrome and are most prominent in the postprandial phase. People are in a postprandial state most of the day, and this phase is proatherogenic. Inhibition of the activation of leucocytes, endothelial cells, or both, is an interesting target for intervention, as activation is obligatory for adherence of leucocytes to the endothelium, thereby initiating atherogenesis.
  • Potential interventions include the use of unsaturated long-chain fatty acids, polyphenols, antioxidants, angiotensin converting enzyme inhibitors and high-dose aspirin, which have direct antiinflammatory and antiatherogenic effects.
  • C-reactive protein is a predictor of future risk for cardiovascular disease.
  • LT- alpha is a proinflammatory cytokine that plays an important role in the pathogenesis of atherosclerosis in mice.
  • MI Myocardial infarction
  • LTA cytokine lymphotoxin-alpha
  • LT ⁇ R-Ig The generation of LT ⁇ R-Ig fusion.
  • cDNA encoding murine LT ⁇ R extracellular domain was generated by RT-PCR using the sense primer (5'- AAAGGCCGCCATGGGCCT-3') (SEQ ID NO:1) and the antisense primer 5'- TTAAGCTTCAGTAGCATTGCTCCTGGCT-3') (SEQ ID NO:2)from mouse lung mRNA, digested by Ncol/Hindlll and then fused to a IL-3 leader sequence in p30242 vector.
  • the fusion fragment was then subcloned into pX58 vector containing IE- 175 promoter and Fc portion of human IgGl.
  • the construct was then transfected into BHK/VP16 cells and the mouse LT ⁇ R-human Ig was purified by Protein A column. This LT ⁇ R-Ig fusion is referred to as "LT ⁇ R-Ig" herein.
  • LDL receptor deficient mice with LT ⁇ R-Ig.
  • the adult hosts were i.p. inoculated with 100 ⁇ g of LT ⁇ R-Ig, once a week for 8 weeks.
  • the sera were collected once a month and subjected to HPLC for lipid profile and total cholesterol and triglycerides were also monitored.
  • LT ⁇ R-Ig treatment reduces total cholesterol and triglycerides (FIG. 6).
  • 5 adult LDLR deficient mice from each group were treated with either control or LT ⁇ R-Ig.
  • the sera were collected one month and 2 month.
  • TC means Total cholesterol and TG means total triglycerides.
  • the tissues (arteries) were collected one week later.
  • LT ⁇ R-Ig treatment increases HDL and reduces VLDL (FIG. 7).
  • 5 adult LDLR deficient mice from each group were treated with either control or LT ⁇ R-Ig.
  • the sera were collected one month and 2 month for HPLC to determine VLDL, LDL, and HDL.
  • LT ⁇ R-Ig treatment limits hyperlipidemia and improves HDL levels (Table T).
  • LDLR-/- mice were injected with Control or LT ⁇ R IgG for 8 weeks while on high cholesterol/high fat diet. Animals were sacrificed 1 week after injections were terminated.
  • T cell-derived proinflammatory ligands in LT/LIGHT regulate the expression of genes that control hepatic lipid metabolism.
  • T cells intimately contact hepatocytes but their role in hepatocyte-mediated lipid homeostasis is unclear.
  • Lymphotoxin (LT) and LIGHT which are TNF superfamily ligands for LT ⁇ R expressed on lymphocytes, are identified as critical players in controlling key lipid regulatory enzymes in hepatocytes leading to the development of dyslipidemia.
  • Dysregulated LIGHT expression on T cells resulted in hypertriglyceridemia and hypercholesterolemia.
  • disruption of LT/LIGHT with a soluble LT ⁇ R decoy protein in LDL-R-deficient mice corrected the dyslipidemia.
  • TNFSF tumor necrosis factor superfamily
  • lymph node aplasia impaired splenic microarchitecture, autoimmunity, viral infection, and grossly impaired immunoglobulin responses.
  • Lck LIGHT transgenic mice develop systemic inflammation and hepatomegaly (Wang, 2001; Shaikh, 2001). Lymphocytes intimately contact hepatocytes, raising the possibility that some T cell derived ligands, such as LT/LIGHT, expressed on activated T cells, may regulate various liver functions.
  • LT/LIGHT have been shown to impart signals necessary for liver regeneration as well as hepatitis (Anders et al, 2005; Anand et al, 2006). Given the observed effects of LT/LIGHT on the liver and that lipid homeostasis is tightly regulated by the liver, the lipid profiles of lck LIGHT Tg and wild-type mice were examined.
  • the LIGHT Tg mice displayed hypercholesterolemia (P ⁇ 0.001) on a normal chow diet (FIG. 8A).
  • the hyperlipidemia phenotype was exaggerated when the mice were placed on a high fat, high cholesterol western type diet (WTD).
  • LIGHT Tg mice developed hypertriglyceridemia (P ⁇ 0.01) along with worsening hypercholesterolemia (P ⁇ 0.01) on WTD (FIG. 8C and FIG. 8D).
  • the lipid profile of LIGHT Tg mice was examined by FPLC. LIGHT Tg mice displayed an increase in VLDL and LDL but not HDL, further supporting a role in the pathogenesis of dyslipidemia.
  • LIGHT Tg mice were crossed with mice lacking the two defined receptors for LIGHT, the Herpes virus entry mediator (HVEM) and LT ⁇ R.
  • HVEM Herpes virus entry mediator
  • LT ⁇ R LT ⁇ R
  • the LIGHT-mediated dyslipideniia was still present in LIGHT Tg/HVEM -/- (FIG. 9A and FIG. 9B) mice but largely corrected in LIGHT Tg/LT ⁇ lC -/- mice (FIG. 9C and FIG. 9D).
  • LIGHT Tg/LT ⁇ R -/- mice had a trend of reduction in cholesterol compared to LIGHT Tg mice (P ⁇ 0.05) and more impressively a complete normalization in the levels of triglycerides (P ⁇ 0.01) (FIG.
  • LIGHT-LT ⁇ R but not LIGHT-HVEM interactions are essential for the dyslipidemia in LIGHT Tg mice.
  • Two likely target organs that are known to regulate lipid homeostasis are the liver and intestine.
  • the feces of LIGHT Tg mice appeared grossly similar to that of WT mice on both the normal chow and high fat diet, suggesting that gastrointestinal and hepatobiliary excretion was not a major mechanism for the dyslipidemia.
  • the hepatic gene expression profile of wild-type and LIGHT Tg mice was analyzed by Affymetrix microarray analysis.
  • the inventors identified 385 out of more than 45,000 genes that were altered by five- fold or more.
  • the inventors further subdivided these genes into pathways, with particular interest paid to genes that regulate lipid and cholesterol metabolism.
  • genes with known lipid and cholesterol regulatory functions only three genes were identified that were upregulated by two-fold or more and two that were downregulated by two-fold or more in the Tg. Strikingly, only two genes in the lipid and cholesterol metabolism pathways were significantly altered five-fold or more in the Tg livers.
  • hepatic lipase was decreased by more than five-fold in LIGHT Tg mice (FIG. 10A).
  • Hepatic triglyceride lipase hydrolyzes triglycerides and phospholipids and is a major regulator of triglycerides, lipoprotein metabolism in the liver (Santamarina-Fojo, 2004 #13).
  • Inactivation of the hepatic lipase gene results in dyslipidemia whereas overexpression of hepatic lipase acts to substantially decrease triglycerides and apolipoprotein cholesterols, especially those containing apoB.
  • the results of the gene array studies were confirmed using semi-quantitative RT-PCR and quantitative real-time PCR analyses (FIG. 10B).
  • Wild-type and LIGHT Tg mice were fed WTD for two weeks, sacrificed, liver RNA were isolated and subjected to Affymetrix microarray analysis. A heat map for genes involved in lipid and cholesterol metabolism is shown. The genes shown below in Table 3 represent those that were changed by 1.2-fold or more and statistically significant (P ⁇ 0.05).
  • mice develop a severe form of hyperlipidemia soon after initiation of a high fat, high cholesterol western type diet (WTD) that is difficult to be corrected (Reardon et al, 2001). Recent evidence have suggested a role for lymphocytes in hyperlipidemia, but the mechanism is unknown (Reardon et al, 2003).
  • WTD high fat, high cholesterol western type diet
  • the mice were fed WTD and injected weekly with a low dose of LT ⁇ R-Ig and the plasma concentrations of triglycerides and total cholesterol were determined.
  • LDL-R -/- mice treated with LT ⁇ R-Ig showed a substantial reduction in :otal cholesterol (P ⁇ 0.001) and triglycerides (P ⁇ 0.01) (FIG. 11A and FIG. 11B).
  • lipid levels decreased as early as four weeks.
  • FPLC analyses demonstrated that LT ⁇ R-Ig treatment dramatically reduced levels of VLDLc (P ⁇ 0.01) and LDLc (P ⁇ 0.05) without perturbing the levels of HDLc (FIG. HC and FIG. HD).
  • LDL-R-def ⁇ cient mice that were the recipients of LIGHT -/- donor bone marrow, exhibited a mild and statistically significant reduction (P ⁇ 0.05) in total cholesterol (FIG. HE).
  • P 0.01
  • LT may be the more dominant LT ⁇ R ligand in the pathogenesis of dyslipidemia in LDL-R-deficient mice and that blockade of both LT and LIGHT, which was achieved with the use of LT ⁇ R-Ig, may be necessary to completely alleviate the phenotype of dyslipidemia.
  • the inventors had earlier observed a decrease in hepatic lipase in the dyslipidemic animals.
  • the inventors sought to determine if blocking LT/LIGHT interactions with LT ⁇ R-Ig in LDL-R -/- mice may increase the expression of hepatic lipase and help to alleviate the dyslipidemia.
  • the real time quantitative PCR analyses revealed an increase in hepatic lipase of the livers of mice treated with LT ⁇ R-Ig (P ⁇ 0.05) (FIG. 1 IF). To dissect the contribution of LT and LIGHT on the regulation of hepatic lipase expression, hepatic lipase transcription was tested in the radiation bone marrow chimeric mice.
  • LT or LIGHT may signal via the LT ⁇ R on hepatocytes to inhibit the expression of hepatic lipase.
  • hepatocytes were stimulated with LIGHT and measured hepatic lipase transcripts. LIGHT stimulation depressed the expression of hepatic lipase (P ⁇ 0.05) (FIG. HH).
  • LT/LIGHT/LT ⁇ R signaling can be used to reverse the dyslipidemia that is a prerequisite in many cardiovascular disease states such as peripheral vascular disease and coronary heart disease.
  • LT ⁇ R blockade on the development of atherosclerosis are currently under investigation.
  • lymphocytes closely contact hepatocytes and could physically provide direct ligands to communicate with hepatocytes.
  • Activated lymphocytes express both LT and LIGHT, which may regulate lipid metabolism inside liver.
  • these ligands control hepatic regeneration and reduce ConA-mediated hepatitis (Anders et ah, 2005; Anand et al, 2006).
  • LT ⁇ R-Ig treatment diminishes T cell-mediated hepatitis, suggesting an essential role of T cell-derived LT/LIGHT in the regulation of liver function. Whether other liver functions are also regulated by hepatic lymphocytes remain to be determined.
  • This study provides evidence that LT/LIGHT on T cells regulate lipid homeostasis.
  • LT/LIGHT are also expressed on other hematopoietic cell types such as B, NK, NKT, and dendritic cells which may contribute to lipid metabolism.
  • organs that express the LT ⁇ R which may also play important roles in lipid biology.
  • the LT ⁇ R and its ligands may also exert effects on adipose tissue, the gastrointestinal system, the endocrine system, and other immune cells such as macrophages to maintain lipid homeostasis.
  • the contribution of these other potential target organs to proinflammatory cytokine-mediated lipid modulation will certainly be an area of active future research.
  • LT/LIGHT are expressed preferentially on activated lymphocytes
  • one expected outcome of inflammation is 1) increased LT/LIGHT expression on lymphocytes, 2) stimulation of the LT ⁇ R on hepatocytes, 3) depressed hepatic lipase gene expression, and 4) hypertriglyceridemia and hypercholesterolemia.
  • the data provide an explanation for the long-time dogma that chronic inflammation is associated with hyperlipidemia, the mechanisms of which have previously not been well defined. This may be an intended and advantageous product of inflammation an or untoward consequence of combating pathogens.
  • TNFSF is a regulator of lipid homeostasis.
  • the inventors have identified two ligands, primarily derived from T cells, of the LT ⁇ R which play a critical role in hypertriglyceridemia and hypercholesterolemia. Additionally, a new murine model in the LIGHT Tg mice is provided to study dyslipidemia. These data identify a target for LT/LIGHT in hepatic lipase to regulate lipid metabolism. Disruption of the LT/LIGHT/LT ⁇ R axis ameliorates dyslipidemia via a mechanism of increased hepatic lipase.
  • mice and treatments C57B1/6 (B6) and LDL-R -/- mice were purchased from the National Cancer Institutes or The Jackson Laboratory. Lck LIGHT Tg, LT ⁇ -/- , LIGHT -/- , and LT ⁇ R -/- mice backcrossed to B6 mice and maintained under specific pathogen-free conditions as described (Wang et al, 2001; De Togni et al, 1994; Tamada et al, 2002; Futterer et al, 1998). Mice were maintained on a standard chow diet until placed on a Western-type diet (Harland Teklad TD88137) containing 0.15% cholesterol and 21% fat for the indicated times.
  • Western-type diet Harland Teklad TD88137
  • control human IgG Sigma
  • LT ⁇ R-Ig LT ⁇ R-Ig
  • the LT ⁇ R-Ig used in this study has been previously described (Wu et al, 2001). Briefly, cDNA encoding the extracellular domain of murine LT ⁇ R was fused with the Fc portion of human IgG, transfected into BHK/VP16 cells, and the supernatant was collected.
  • Plasma total triglycerides were determined by The InfinityTM Triglycerides single liquid stable reagent from Thermo Electron Corporation (Waltham, MA). Plasma total cholesterols were determined by Cholesterol liquid reagent set from Pointe Scientfic, Inc. (Canton, MI). Plasma was collected by retro-orbital bleeding. Lipoproteins were separated by fast protein liquid chromatography on tandem Superose 6 columns, and the amount of cholesterol in the even-numbered fractions was determined and expressed as micrograms cholesterol per milliliter of plasma. The area under each lipoprotein peak was quantified by computer digitizer (SigmaScan) and expressed as percent total cholesterol.
  • RNA and real-time PCR were extracted by TRIZOLCg ) Reagent from Invitrogen and cleaned up using RNeasy spin column from QIAGEN.
  • RNA were digested with DNase I and reverse transcribed using random primers with AMV Reverse Transcriptase (Promega).
  • Each cDNA sample was amplified for hepatic lipase or glyceraldehyde phosphodehydrogenase (gapdh) using the Taqman Universal PCR master mixture (PE Applied Biosystems) and run on the SmartCycler (Cepheid).
  • hepatic lipase For hepatic lipase, the following primers were used: forward, 5'-CCCTGCGGGCCCTATG-3' (SEQ ID N0:3); reverse, 5'-TTGGCATCATCAGGAGAAAGG-3' (SEQ ID N0:4); probe, 5'-FAM (6- carboxyfluorescein)-TTGAGGGAACGTCCCCCAACGA-TAMRA (N,N,N',N'-tetramethyl- 6-carboxyrhodamine)-3' (SEQ ID N0:5).
  • forward 5'-CCCTGCGGGCCCTATG-3' (SEQ ID N0:3)
  • reverse 5'-TTGGCATCATCAGGAGAAAGG-3'
  • probe 5'-FAM (6- carboxyfluorescein)-TTGAGGGAACGTCCCCCAACGA-TAMRA (N,N,N',N'-tetramethyl- 6-carboxyrhodamine)-3' (SEQ ID N0:
  • the following primers were used: forward, 5'-AACCACGAGAAATATGACAACTCACT-3' (SEQ ID N0:6); reverse, 5'- GGCATGGACTGTGGTCATGA-3' (SEQ ID N0:7); probe, 5'-TET (tetra-chloro-6- carboxyfluorescein)-TGCATCCTGCACCACCAACTGCTTAG-TAMRA-3' (SEQ ID N0:8).
  • the concentration of the target gene was determined using the comparative Cj (threshold cycle number at a cross-point between amplification plot and threshold) method and normalized to GAPDH.
  • Bone marrow chimeras Mice were lethally irradiated with 1000 rads and adoptively transferred intravenously with 2-3 x 10 6 bone marrow cells of the indicated genotype. Bactrim was added to the drinking water for two weeks following irradiation. Four weeks after irradiation and reconstitution, mice were placed on WTD.
  • hepatocytes were cultured at 33° C in media with interferon- ⁇ (R&D) and then allowed to differentiate at 39° C for two days in media without interferon- ⁇ . Hepatocytes were stimulated with soluble murine LIGHT protein (R&D) at 50 ng/mL.
  • R&D soluble murine LIGHT protein
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038 and 1570-1580, 1990. Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, pp. 1289-1329,

Abstract

The present invention provides, in certain embodiments, methods involving the administration of LTβR modulators, such as solubilized LTβR to block its ligands activities, for the prevention and/or treatment of diseases including cardiac diseases and/or liver diseases.

Description

DESCRIPTION
THERAPIES INVOLVING LYMPHOTOXIN BETA RECEPTOR
BACKGROUND OF THE INVENTION
The government owns rights in the present invention pursuant to grant number ROl- HD37104 and R01-DK58897 from the National Institutes of Health.
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and medicine. More particularly, it concerns methods involving the modulation of lymphotoxin beta receptor (LTβR) for the prevention and/or treatment of diseases (e.g., cardiac diseases, liver diseases) and compositions for use in such methods.
2. Description of Related Art
Atherosclerosis is the major cause of death in western societies and there are a variety of important risk factors including dyslipidaemia, increased body fat mass and smoking. Of the lipid parameters, triglycerides and high-density lipoprotein (HDL) cholesterol are the most important predictors of coronary heart disease. Although the liver plays a significant role in lipid metabolism, limited studies have focused on liver-mediated lipid homeostasis.
There remains a need for improved treatments for cardiovascular diseases such as atherosclerosis. Coronary heart disease is regarded as a low-grade chronic inflammatory process. During the early stages of atherogenesis and following endothelial injury, local and recruited leukocytes release various inflammatory mediators, bind to the endothelium and migrate into the lesion. Leukocytes are required for both the formation of these so called "plaques" and endothelial cell dysfunction. The rupturing of these plaques is frequently the cause of myocardial infarction (MI) and stroke.
Therapies that can reduce blood levels of triglycerides and cholesterol (e.g., LDL) can be very useful for the treatment and prevention of cardiovascular disease. A relationship has been proposed between dyslipidaemia and numbers of blood leukocytes, another inflammatory marker that predicts chronic heart disease mortality and morbidity. Increased levels of triglycerides have been shown to result in activation of leukocytes, resulting in proliferation and increased production of cytokines. Leukocyte activation is suggestive of a proinflammatory and proatherogenic condition, most likely indicating an increased capacity of these cells to adhere to the endothelium.
Additionally, there exists a need for methods and therapies to promote liver regeneration. Several diseases including cirrhosis of the liver and viral infections of the liver {e.g., hepatitis) result in significant damage to the liver and would benefit from therapies that promote liver regeneration. The inability of the host to restore liver mass is often the cause of chronic liver diseases, which cause significant morbidity and mortality worldwide.
Further, methods to promote liver regeneration could be used in therapies involving liver transplantation. Organ shortages continue to press the need for liver transplantation alternatives. The ability to increase the renewal of liver would make split liver transplants for living adult donor and even cellular transplants more feasible.
The lymphotoxin beta receptor (LTβR) and certain soluble forms of LTβR are described in WO9703687, WO9413808, and U.S. Patent 6,669,941. The LTβR has been described as being involved with lymphocyte-mediated diseases and ThI cell-mediated immune responses. LTβR is expressed on nonlymphoycytes and non-hemapoietic cells (including hepatocytes).
LTβR has two ligands, lymphotoxin and LIGHT (Fu and Chaplin, 1999; Mauri et al, 1998). LIGHT is a recently described TNF family member that can modulate LTβR. LIGHT is detected on activated T cells. LIGHT has two receptors: LTβR and the herpes virus entry mediator receptor (HVEM) which is expressed on all hemapoietic cells (Force et al, 1995; Mauri et al, 1998). Both LIGHT and LT expressed on activated T cells is often detected at an inflammatory site and can contribute to enhanced autoimmunity (Gommerman and Browning, 2003; Tamada et al, 2000; Wang and Fu, 2003; Wang et al, 2001).
SUMMARY OF THE INVENTION
The present invention overcomes deficiencies in the art by providing methods and compositions for the treatment and/or prevention of a variety of medical conditions. For example, in certain embodiments these methods and compositions are useful, for promoting liver regeneration, for treating and/or preventing a cardiovascular disease (or cardiovascular damage), or a liver disease (or liver damage) by modulating LTβR. The inventors have discovered that certain LTβR blockers (e.g., certain forms of soluble LTβR) decrease serum cholesterol (e.g., LDL, VLDL) and triglycerides. Certain aspects of the present invention are related to the use of LTβR agonists to promote liver regeneration.
An aspect of the present invention relates to a method of treating a cardiovascular disease, treating a liver disease, promoting liver regeneration, or altering lipid levels; comprising administering a LTβR modulator to a subject. The subject may be a mammal, such as a human. The LTβR modulator may be an LTβR blocker, such as LTβR-Ig. The LTβR blocker may be an anti-LIGHT antibody or an anti-LT antibody. In certain embodiments, both an anti-LIGHT antibody or an anti-LT antibody are administered to the subject. The LTβR modulator may be an antibody, such as a humanized antibody. The LTβR blocker may be comprised in a pharmaceutically acceptable carrier or excipient. The present invention may also be used to treat a patient who does not have, but is at risk of a cardiovascular disease; for example, the present invention may be used to reduce serum LDL and VLDL in a patient in order to decrease the risk of a cardiovascular disease. Of course, these methods and compositions may be used to treat or prevent any medical condition which is amenable to such treatment or prevention. In certain embodiments, the method further comprises administering an additional pharmacological therapeutic agent and/or a surgery to the subject.
In the methods of the invention, the LTβR modulator may be administered once or repeatedly to a subject. Where the LTβR modulator is administered repeatedly to a subject the duration between administrations may vary from minutes to weeks. In certain embodiments, a LTβR modulator (e.g., a soluble LTβR fusion protein, LTβR-Ig) may be administered continuously to a subject, for example via an intravenous drip to a human patient. For repeated administration of the LTβR modulator, the duration of time between administration of the LTβR modulator may be about 12-24 h of each other or within about 6- 12 h of each other. In some situations, it may be desirable to allow from several d (e.g., 2, 3, 4, 5, 6 or 7) to several wk (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) to lapse between the respective administrations of the LTβR modulator. In certain embodiments, the LTβR modulator (e.g., a soluble LTβR, LTβR-Ig) may be administered to a subject once per week or twice per month.
Various doses of the LTβR modulator may be administered to a subject (e.g., a human patient). For example, the dose may be from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 niicrogram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above. In certain embodiments, the LTβR modulator {e.g., a soluble LTβR, LTβR-Ig) may be administered to a subject at a dose of from about 0.2 mg/kg to about 2 mg/kg.
The LTβR modulator may be comprised in a pharmaceutical composition, which may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be suitable for such routes of administration as injection. The LTβR modulator can be administered intravenously; however, additional administration routes may be used including intradermally, transdermally, intraarterially, orally, inhalation {e.g., aerosol inhalation), injection, infusion, continuous infusion, via a catheter, via a lavage, in cremes, in lipid compositions {e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Of course, the invention contemplates any pharmaceutical formulation comprising a LTβR modulator that may be of use in the methods disclosed herein. Such formulation may take any form know to those of skill in the art and/or disclosed herein that will deliver an appropriate dosage in an appropriate regime.
In certain embodiments, the method comprises treating a cardiovascular disease (e.g., hypertriglyceridemia, hypercholesterolemia, atherosclerosis, artery atherosclerosis restenosis, stenosis, thrombosis, aneurism, embolus, hypertension, stroke, critical stenosis or myocardiac infarction). In certain embodiments, the cardiovascular disease is artery atherosclerosis. The LTβR blocker may be LTβR-Ig. The method may comprise altering lipid levels in the subject. In certain embodiments, cholesterol is reduced, triglycerides are reduced, LDL is -educed, VLDL is reduced, or HDL is increased in the blood of the subject. The LTβR nodulator may be a LTβR blocker, such as LTβR-Ig. In certain embodiments, the method .iirther comprises administering an additional pharmacological therapeutic agent or a surgery :o the subject.
In certain the methods comprise treating a liver disease or promoting liver regeneration. The LTβR modulator may be an LTβR agonist, such as LIGHT or a LTβR agonistic antibody.
Another aspect of the present invention involves a method of promoting liver regeneration in vitro, comprising contacting a liver cell with a LTβR agonist. The LTβR agonist may be LIGHT or a LTβR agonistic antibody. The liver cell may be comprised in liver tissue. The liver cell may be used for tissue engineering. The liver cell may be subsequently implanted into a subject. The subject maybe a mammal, such as a human.
Another aspect of the present invention relates to a pharmaceutical composition comprising a LTβR modulator. The LTβR modulator may be an antibody, such as a humanized antibody. The pharmaceutical composition may further comprise an additional therapeutic agent. The additional agent may be effective, either alone or in combination with the LTβR modulator to treat or prevent a medical condition, inculding but not limited to cardiovascular disease and/or a liver disease, promote liver regeneration, and/or alter a lipid level, in a subject. In certain embodiments, the LTβR modulator is an LTβR blocker, sue as LTβR-Ig. The LTβR-Ig may be comprised at a concentration of from about 0.2 mg/kg to about 2 mg/kg. The LTβR blocker may be an anti-LIGHT antibody and/or an anti-LT antibody.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
The term "about" or "approximately" are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.
The term "effective," as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.
The terms "patient" or "subject" can include an animal. Preferred animals are mammals, including but not limited to humans, pigs, cats, dogs, rodents, horses, cattle, sheep, goats and cows. Preferred patients and subjects are humans.
The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."
The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."
As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the examples, while indicating specific embodiments of the invention, are given by way of illustration only. Additionally, it is contemplated that changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1: A T cell derived TNF superfamily member, LIGHT, has a profound effect on the liver in an LTβR dependent fashion. Transgenic mice expressing /c/c-LIGHT have a dramatically increased liver weight relative to total body weight compared to wild type mice.
FIG. 2: Increased mortality of LTβR and lympho toxin deficient mice after partial hepatectomy. Wild type mice (close triangle) survive partial hepatectomy while mice deficient in either the LTβR (open triangle) or L Ta ligand (closed diamond) show increased mortality.
FIGS. 3A-B: LTβR and lymphotoxin deficient mice show evidence of liver damage and decreased DNA synthesis following partial hepatectomy. FIG. 3A, Following partial hepatectomy serum aminotransferase levels are significantly elevated in either the LTβR or LTa deficient mice compared to similarly treated wild type mice at 48 hours. FIG. 3B, The amount of DNA synthesis following partial hepatectomy was determined using a 2 hr pulse with BrdU. BrdU incorporation into actively synthesized hepatocyte DNA was determined using immunohistochemical staining and counting the number of positively stained nuclei in 20 high power fields. There was significantly more active DNA synthesis in the wild type mice compared to LTβR-/-, LT α-/- or sham treated mice.
FIG. 4: LTβR deficient mice have markedly decreased percentage of intrahepatic NKT cells and show a marginal increase in NKT cells following partial hepatectomy. Following partial hepatectomy in wild type mice the percentage of CD4+ T-cells and CD4+ CDId tetramer + cells, the major subset of NKT cells, increases significantly ("54.2", as shown) compared to before partial hepatectomy ("29.7", as shown). Similarly, the percentage of CD3+ CDId tetramer positive cells (NKT cells) significantly increases in the liver of wild type mice following partial hepatectomy ("63.9", as shown) compared to before partial hepatectomy ("35.8", as shown). On the other hand, the percentage of CD3+ CDId+ NKT cells is significantly decreased in LTβR deficient mice prior to partial hepatectomy ("7.04", as shown) with a marginal increase in the percentage of NKT cells following partial tiepatectomy ("11.6", as shown). FIGS. 5A-B: NKT cell deficient CD1d-/- mice have suppressed hepatocyte proliferation in vivo and in vitro post partial hepatectomy. CD1d-/- mice and their wild type control mice were subject 70% partial hepatectomy. The hepatocyte proliferation was determined by 2 hour pulse BrdU labeling both in vivo and in vitro at different time points. (FIG. 5A) In vivo BrdU labeling shows significantly decreased BrdU+ nuclei in CD1d-/- mice 36 hour post PH. (FIG. 5B) In vitro hepatocytes BrdU labeling also shows decreased BrdU incorporation in hepatocytes from CD1d-/- mice after PH.
FIG. 6: LTbR-Ig treatment reduces total cholesterol and triglycerides. LDL receptor KO mice (8 weeks old) were fed with fatty food for two months and blood was collected at 4 and 8 weeks for lipid profile study. On day on of fatty food, one group (n=5) of mice was treated with LTbR-Ig, a soluble LT beta receptor (100 ug/injection). Total cholesterol (TC) and triglycerides (TG) level were compared. The mean of group was presented.
FIG. 7: LTbR-Ig treatment increases HDL and reduces VLDL. 5 adult LDLR deficient mice from each group were treated with either control or LTbR-Ig. The sera were collected one month and 2 month for HPLC to determine VLDL, LDL, and HDL.
FIGS. 8A-D. T lymphocyte-derived LIGHT promotes dyslipidemia. Plasma total cholesterol and triglycerides were measured in WT and LIGHT Tg mice fed a chow diet or WTD for two weeks. (FIG. 8A and FIG. 8B) WT and LIGHT Tg mice on a normal chow diet were assayed for plasma total cholesterol (FIG. 8A) and triglycerides (FIG. 8B). (FIG. 8C and FIG. 8D) WT and LIGHT Tg mice on WTD were assayed for plasma total cholesterol (FIG. 8C) and triglycerides (FIG. 8D). Each individual symbol represents values from WT (triangles) and LIGHT Tg (circles) mice and the means are plotted as columns. Statistical significance was determined by a two-tailed Student's t-test comparing WT and LIGHT Tg mice (*P < 0.05, **P < 0.01, ***P < 0.001).
FIGS. 9A-D. LIGHT-dependent dyslipidemia is mediated by the LTβR and not HVEM. Plasma total cholesterol and triglycerides were measured in WT, LIGHT Tg, LTβR-'', HVEMT'', LIGHT Tg/LTβR~'~, and LIGHT TgZHVEAT'' mice. (FIG. 9A and FIG. 9B) WT, LIGHT Tg, HVEM~'~, and LIGHT Tg/HVEAT'' mice on WTD were assayed for plasma total cholesterol (FIG. 9A) and triglycerides (FIG. 9B). (FIG. 9C and FIG. 9D) WT, LIGHT Tg, LTβR-/-, and LIGHT Tg/ LTβR-/- mice on WTD were assayed for plasma total cholesterol (FIG. 9C) and triglycerides (FIG. 9D). The means are represented as a solid line. All other individual symbols represent values from mice of the indicated genotype. Statistical significance was determined by a two-tailed Student's t-test comparing LIGHT Tg and LIGHT Tg/ LTβR-/- mice (*P < 0.05).
FIGS. 10A-B. Dysregulated LIGHT expression on T cells repro grams hepatic gene expression. (FIG. 10A) Hepatic lipase expression levels of WT and LIGHT Tg mice. Data shown represent the means of three independent microarray experiments. (FIG. 10B) LIGHT-mediated repression of hepatic lipase is dependent on the LTβR. Relative mRNA levels of hepatic lipase were quantified by real-time PCR analysis and normalized to gapdh levels. Relative values of hepatic lipase from mice of the indicated genotypes are shown. Results represent the means (columns) and standard errors. Statistical significance between Tg and Tg/ LTβR-/- was determined by the Mann- Whitney test (P < 0.05).
FIGS. 11A-H. LTβR signaling controls hepatic lipase expression on hepatocytes and a soluble LTβR antagonist reverses dyslipidemia in a murine model of atherosclerosis. (FIG. 11A and FIG. 11B) LDL-R-deficient mice were fed WTD and injected with control or a soluble decoy receptor LTβR-Ig weekly. The plasma levels of total cholesterol (FIG. 11A) and triglycerides (FIG. 11B) were analyzed. Each symbol represents a data point from an individual mouse. The means are shown as solid lines. (FIG. 11C) FPLC analysis of plasma from LDL-R-/- mice on WTD for 12 weeks treated with control or LTβR-Ig. (FIG. 11D) Plasma levels of apolipoprotein cholesterols in LDL-RT-/- mice treated with control or LTβR- Ig. Results represent the means (columns) and standard errors (FIG. HE) Plasma total cholesterol levels of lethally irradiated LDL-R-deficient mice reconstituted with bone marrow from WT, LT α-/-, and LIGHT-/- mice fed WTD for nine weeks after reconstitution. (FIG. 11F) Hepatic lipase expression levels were quantified by real-time PCR from livers of LDL- R-/- mice on WTD treated with control or LTβR-Ig. Results represent the means (columns) and standard errors (FIG. 11G) Hepatic lipase mRNA levels of liver samples from (E) were quantified by real-time PCR analysis. (FIG. 11H) Immortalized hepatocytes cell line was stimulated with recombinant LIGHT protein and hepatic lipase expression levels were determined by real-time PCR. Statistical significance was determined by a two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention overcomes deficiencies in the art by providing methods, in certain embodiments, for promoting liver regeneration, for treating and/or preventing a cardiovascular disease (or cardiovascular damage), or a liver disease (or liver damage) by modulating LTβR. The inventors have discovered that certain LTβR blockers (e.g., certain forms of soluble LTβR) decreases serum cholesterol (e.g., LDL, VLDL) and triglycerides. Certain aspects of the present invention are related to the use of LTβR agonists to promote liver regeneration.
I. LYMPHOTOXIN BETA RECEPTOR MODULATORS
"LTβR modulators", as used herein, includes LTβR blockers and LTβR agonists. LTβR agonists may be full or partial agonists, such as, for example recombinant LIGHT or an agonistic antibody of LTβR. Indeed, LTβR binds to several ligands, such as lymphotoxin and LIGHT, both TNFSF members. LT or LIGHT can deliver the signaling via LTβR on stromal cells leading to upregulation of chemokines and cytokines. LTbR-Ig is a soluble form that can block LTβR ligand activities.
LTβR blockers may be used with the present invention to treat and/or prevent a cardiovascular disease, such as atherosclerosis. LTβR blockers include, but are not limited to, certain soluble forms of LTβR (e.g., LTβR-Ig) and antagonistic antibodies to LTβR. Certain LTβR blockers, such as soluble forms of LTβR, are described, for example, in WO9703687A1, WO9413808A3, and U.S. Patent 6669941. An antibody (or other compound) directed against LIGHT or LT may also be used as a LTβR blocker; in certain embodiments, both an anti-LIGHT antibody and an anti-LT antibody are administered to a subject to enhance the inhibition of LTβR. The antibodies are preferably humanized. It is envisioned that these and other LTβR blockers which are currently known or may be subsequently discovered may be used with the present invention.
In certain embodiments, recombinant LIGHT or an agonistic antibody of LTβR may be used as an LTβR agonist. Recombinant LIGHT is described, for example, in U.S. Application 10/865,623 and in Duhen et al. (2004). Recombinant LIGHT may be mutated or non-mutated (i.e., "wild-type"). These compounds may be particularly useful in certain embodiments of the present invention involving promotion of liver regeneration. Agonistic antibodies are preferably humanized. In certain embodiments, a LTβR blocker may be used to decrease levels (e.g., serum levels) of a lipid, decrease triglyceride levels, decrease cholesterol, decrease LDL, decrease VLDL, and/or increase HDL in a subject (e.g., a human patient). For example, in certain embodiments, a LTβR blocker (e.g., LTβR-Ig) may be administered to a subject to treat hyperlipidemia.
II. TREATMENT OF CARDIOVASCULAR DISEASE
The present invention provides, in certain embodiments, methods of treatment and/or prevention of cardiovascular diseases (e.g., atherosclerosis). The present invention may also be used to treat a patient who does not have, but is at risk of a cardiovascular disease; for example, the present invention may be used to reduce serum LDL and VLDL in a patient in order to decrease the risk of a cardiovascular disease.
"Cardiovascular disease", as used herein includes, but is not limited to, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, restenosis, stenosis, thrombosis, aneurism, embolus, hypertension, stroke, and myocardiac infarction. Atherosclerosis is a serious problem clinically and may result in a critical stenosis, thrombosis, aneurism, or embolus. In certain embodiments, the cardiovascular disease results from increased inflammation in a patient.
III. LIVER DISEASE AND REGENERATION
Certain embodiments of the present invention are directed towards the treatment of a liver disease. Liver diseases include, but are not limited to, cirrhosis of the liver, viral infection of the liver (e.g., hepatitis infection), liver transplantation, acute liver injury and hepatic failure. Liver diseases also include a disease which results in an impaired ability for the liver to adequately regenerate. Further, the present invention also may be used for the treatment of trauma to the liver (e.g., physical trauma or exposure to a toxin). It is anticipated that the present invention may be used to treat these and other liver diseases.
The present invention provides, in certain embodiments, a method for stimulating liver regeneration by stimulation or enhancement of the growth and/or function of NKT cells. NKT cells may be used or modulated in vitro or in vivo to enhance liver regeneration. In certain embodiments, NKT cells may be added to an in vitro cell culture that includes liver tissue, hi certain embodiments, a modulator of NKT cells may be added to an in vitro cell culture or administered to a subject (e.g., a human patient) in vivo to stimulate NKT cell function.
IV. PHARMACEUTICAL PREPARATIONS
Pharmaceutical compositions of the present invention comprise an effective amount of one or more LTβR modulator or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one LTβR modulator or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional caπier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
The LTβR modulator may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, ntranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, nucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, ϊontinuous infusion, localized perfusion bathing target cells directly, via a catheter, via a avage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
The LTβR modulator may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
Further in accordance with the present invention, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens {e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art. In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
In further embodiments, the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include LTβR modulator, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those of skill in the art, and as the term "lipid" is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic {i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.
One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the LTβR modulator may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.
The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above, [n certain embodiments, the LTβR modulator {e.g., a soluble LTβR, LTβR-Ig) may be administered to a subject at a dose of from about 0.2 mg/kg to about 2 mg/kg. 1. Alimentary Compositions and Formulations
In preferred embodiments of the present invention, the LTβR modulator is formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al, 1997; Hwang et al, 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, com starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial jnterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound iucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and lavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a buccal tablet, oral spray, or sublingual orally- administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
Additional formulations which are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
2. Parenteral Compositions and Formulations
In further embodiments, a LTβR modulator may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for sxample, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,753,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by ■eference in its entirety).
Solutions of the active compounds as free base or pharmacologically acceptable salts nay be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
3. Miscellaneous Pharmaceutical Compositions and Formulations
In other preferred embodiments of the invention, the active compound LTβR modulator may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-solubility based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a "patch". For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al, 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.
V. COMBINATION THERAPIES
In order to increase the effectiveness of a LTβR modulator {e.g., a LTβR blocker or recombinant LIGHT), it may be desirable to combine these compositions and methods of the invention with an agent effective in the treatment of vascular or cardiovascular disease or disorder. In some embodiments, it is contemplated that a conventional therapy or agent, including but not limited to, a pharmacological therapeutic agent, a surgical therapeutic agent {e.g., a surgical procedure) or a combination thereof, may be combined with LTβR modulator administration. In a non-limiting example, a therapeutic benefit comprises reduced hypertension in a vascular tissue, or reduced restenosis following vascular or cardiovascular intervention, such as occurs during a medical or surgical procedure). Thus, in certain embodiment, a therapeutic method of the present invention may comprise administration of a LTβR modulator of the present invention in combination with another therapeutic agent.
This process may involve contacting the cell(s) with an agent(s) and the LTβR modulator at the same time or within a period of time wherein separate administration of the LTβR modulator and an agent to a cell, tissue or organism produces a desired therapeutic benefit. The terms "contacted" and "exposed," when applied to a cell, tissue or organism, are used herein to describe the process by which a therapeutic construct of a LTβR modulator and/or therapeutic agent are delivered to a target cell, tissue or organism or are placed in direct juxtaposition with the target cell, tissue or organism. The cell, tissue or organism may be contacted (e.g., by administration) with a single composition or pharmacological formulation that includes both a LTβR modulator and one or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes a LTβR modulator and the other includes one or more agents.
The LTβR modulator may precede, be co-current with and/or follow the other agent(s) by intervals ranging from minutes to weeks. In embodiments where the LTβR modulator, and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the LTβR modulator and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e. within less than about a minute) as the LTβR modulator. In other aspects, one or more agents may be administered within of from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months, and any range derivable therein, prior to and/or after administering the LTβR modulator. Various combination regimens of the LTβR modulator and one or more agents may be employed. Non-limiting examples of such combinations are shown below, wherein a composition LTβR modulator is "A" and an agent is "B":
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
Administration of the composition of a LTβR modulator to a cell, tissue or organism may follow general protocols for the administration of vascular or cardiovascular therapeutics, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary. In particular embodiments, it is contemplated that various additional agents may be applied in any combination with the present invention.
1. Pharmacological Therapeutic Agents
Pharmacological therapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art (see for example, the "Physicians Desk Reference", Goodman & Gilman's "The Pharmacological Basis of Therapeutics", "Remington's Pharmaceutical Sciences", and "The Merck Index, Eleventh Edition", incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject, and such individual determinations are within the skill of those of ordinary skill in the art.
Non-limiting examples of a pharmacological therapeutic agent that may be used in the present invention include an antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, a blood coagulant, an antiarrhythmic agent, an antihypertensive agent, a vasopressor, a treatment agent for congestive heart failure, an antianginal agent, an antibacterial agent or a combination thereof.
A. Antihyperlipoproteinemics
In certain embodiments, administration of an agent that lowers the concentration of one of more blood lipids and/or lipoproteins, known herein as an "antihyperlipoproteinemic," may be combined with administration of a LTβR modulator for cardiovascular therapy, particularly in treatment of athersclerosis and thickenings or blockages of vascular tissues. In certain aspects, an antihyperlipoproteinemic agent may comprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acid sequesterant, a HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroid hormone or thyroid hormone analog, a miscellaneous agent or a combination thereof.
1. Aryloxyalkanoic Acid/Fibric Acid Derivatives
Non-limiting examples of aryloxyalkanoic/fibric acid derivatives include beclobrate, enzafϊbrate, binifibrate, ciprofibrate, clinofibrate, clofibrate (atromide-S), clofibric acid, etofϊbrate, fenofibrate, gemfibrozil (lobid), nicofϊbrate, pirifibrate, ronifϊbrate, simfibrate and theofibrate.
2. Resins/Bile Acid Sequester ants
Non-limiting examples of resins/bile acid sequesterants include cholestyramine (cholybar, questran), colestipol (colestid) and polidexide.
3. HMG CoA Reductase Inhibitors
Non-limiting examples of HMG CoA reductase inhibitors include lovastatin (mevacor), pravastatin (pravochol) or simvastatin (zocor).
4. Nicotinic Acid Derivatives
Non-limiting examples of nicotinic acid derivatives include nicotinate, acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid.
5. Thryroid Hormones and Analogs
Non-limiting examples of thyroid hormones and analogs thereof include etoroxate, thyropropic acid and thyroxine.
6. Miscellaneous Antihyperlipoproteinemics
Non-limiting examples of miscellaneous antihyperlipoproteinemics include acifran, azacosterol, benfluorex, β-benzalbutyramide, carnitine, chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium, 5,8, 11, 14, 17-eicosapentaenoic acid, eritadenine, jrazabol, meglutol, melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine, entaerythritol tetraacetate, α-phenylbutyramide, pirozadil, probucol (lorelco), β-sitosterol, αltosilic acid-piperazine salt, tiadenol, triparanol and xenbucin.
B. Antiarteriosclerotics
Non-limiting examples of an antiarterioscl erotic include pyridinol carbamate.
C. Antithrombotic/Fibrinolytic Agents
In certain embodiments, administration of an agent that aids in the removal or revention of blood clots may be combined with administration of a LTβR modulator for ardiovascular therapy, particularly in treatment of atherosclerosis and vasculature {e.g., rterial) blockages. Non-limiting examples of antithrombotic and/or fibrinolytic agents aclude anticoagulants, anticoagulant antagonists, antiplatelet agents, thrombolytic agents, thrombolytic agent antagonists or combinations thereof.
In certain embodiments, antithrombotic agents that can be administered orally, such s, for example, aspirin and wafarin (Coumadin), are preferred.
1. Anticoagulants
A non-limiting example of an anticoagulant include acenocoumarol, ancrod, inisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextran sulfate sodium, licumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol, fluindione, heparin, ύrudin, lyapolate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, ihosvitin, picotamide, tioclomarol and warfarin.
2. Antiplatelet Agents
Non-limiting examples of antiplatelet agents include aspirin, a dextran, dipyridamole persantin), heparin, sulfinpyranoήe (anturane) and ticlopidine (ticlid).
3. Thrombolytic Agents
Non-limiting examples of thrombolytic agents include tissue plasminogen activator activase), plasmin, pro-urokinase, urokinase (abbokinase) streptokinase (streptase), mistreplase/ APSAC (eminase). D. Blood Coagulants
In certain embodiments wherein a patient is suffering from a hemorrhage or an increased likelihood of hemorrhaging, an agent that may enhance blood coagulation may be used. Non-limiting examples of a blood coagulation promoting agent include thrombolytic agent antagonists and anticoagulant antagonists.
1. Anticoagulant Antagonists
Non-limiting examples of anticoagulant antagonists include protamine and vitamin Kl.
2. Thrombolytic Agent Antagonists and Antithrombotics
Non-limiting examples of thrombolytic agent antagonists include aminocaproic acid (amicar) and tranexamic acid (amstat). Non-limiting examples of antithrombotics include anagrelide, argatroban, cilstazol, daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.
E. Antiarrhythmic Agents
Non-limiting examples of antiarrhythmic agents include Class I antiarrythmic agents (sodium channel blockers), Class II antiarrythmic agents (beta-adrenergic blockers), Class II antiarrythmic agents (repolarization prolonging drugs), Class IV antiarrhythmic agents (calcium channel blockers) and miscellaneous antiarrythmic agents.
1. Sodium Channel Blockers
Non-limiting examples of sodium channel blockers include Class IA, Class IB and Class IC antiarrhythmic agents. Non-limiting examples of Class IA antiarrhythmic agents include disppyramide (norpace), procainamide (pronestyl) and quinidine (quinidex). Non- limiting examples of Class IB antiarrhythmic agents include lidocaine (xylocaine), tocainide (tonocard) and mexiletine (mexitil). Non-limiting examples of Class IC antiarrhythmic agents include encainide (enkaid) and flecainide (tambocor).
2. Beta Blockers
Non-limiting examples of a beta blocker, otherwise known as a β-adrenergic blocker, a β-adrenergic antagonist or a Class II antiarrhythmic agent, include acebutolol (sectral), alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol, propanolol (inderal), sotalol (betapace), sulfinalol, talinolol, tertatolol, timolol, toliprolol and xibinolol. In certain aspects, the beta blocker comprises an aryloxypropanolamine derivative. Non-limiting examples of aryloxypropanolamine derivatives include acebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol and toliprolol.
3. Repolarization Prolonging Agents
Non-limiting examples of an agent that prolong repolarization, also known as a Class III antiarrhythmic agent, include amiodarone (cordarone) and sotalol (betapace).
4. Calcium Channel Blockers/ Antagonist
Non-limiting examples of a calcium channel blocker, otherwise known as a Class IV antiarrythmic agent, include an arylalkylamine (e.g., bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline, verapamil), a dihydropyridine derivative (felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) a piperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) or a micellaneous calcium channel blocker such as bencyclane, etafenone, magnesium, mibefradil or perhexiline. In certain embodiments a calcium channel blocker comprises a long-acting dihydropyridine (nifedipine- type) calcium antagonist.
5. Miscellaneous Antiarrhythmic Agents
Non-limiting examples of miscellaneous antiarrhymic agents include adenosine (adenocard), digoxin (lanoxin), acecainide, ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid, cifenline, disopyranide, hydroquinidine, indecainide, ipatropium bromide, lidocaine, lorajmine, lorcainide, meobentine, moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidine polygalacturonate, quinidine sulfate and viquidil.
F. Antihypertensive Agents
Non-limiting examples of antihypertensive agents include sympatholytic, alpha/beta blockers, alpha blockers, anti-angiotensin II agents, beta blockers, calcium channel blockers, vasodilators and miscellaneous antihypertensives.
1. Alpha Blockers
Non-limiting examples of an alpha blocker, also known as an α-adrenergic blocker or an α-adrenergic antagonist, include amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin and yohimbine. In certain embodiments, an alpha blocker may comprise a quinazoline derivative. Non-limiting examples of quinazoline derivatives include alfuzosin, bunazosin, doxazosin, prazosin, terazosin and trimazosin.
2. Alpha/Beta Blockers
In certain embodiments, an antihypertensive agent is both an alpha and beta adrenergic antagonist. Non-limiting examples of an alpha/beta blocker comprise labetalol (normodyne, trandate).
3. Anti- Angiotensin II Agents
Non-limiting examples of anti-angiotension II agents include include angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists. Non-limiting examples of angiotension converting enzyme inhibitors (ACE inhibitors) include alacepril, enalapril (vasotec), captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril, perindopril, quinapril and ramipril. Non-limiting examples of an angiotensin II receptor blocker, also known as an angiotension II receptor antagonist, an ANG receptor blocker or an ANG-II type-1 receptor blocker (ARBS), include angiocandesartan, eprosartan, irbesartan, losartan and valsartan. 4. Sympatholytics
Non-limiting examples of a sympatholytic include a centrally acting sympatholytic or a peripherially acting sympatholytic. Non-limiting examples of a centrally acting sympatholytic, also known as an central nervous system (CNS) sympatholytic, include clonidine (catapres), guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet). Non-limiting examples of a peripherally acting sympatholytic include a ganglion blocking agent, an adrenergic neuron blocking agent, a β-adrenergic blocking agent or a alphal- adrenergic blocking agent. Non-limiting examples of a ganglion blocking agent include mecamylamine (inversine) and trimethaphan (arfonad). Non-limiting of an adrenergic neuron blocking agent include guanethidine (ismelin) and reserpine (serpasil). Non-limiting examples of a β-adrenergic blocker include acenitolol (sectral), atenolol (tenormin), betaxolol (kerlone), carteolol (cartrol), labetalol (normodyne, trandate), metoprolol (lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken), propranolol (inderal) and timolol (blocadren). Non-limiting examples of alphal -adrenergic blocker include prazosin (minipress), doxazocin (cardura) and terazosin (hytrin).
5. Vasodilators ,
In certain embodiments a cardiovasculator therapeutic agent may comprise a vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or a peripheral vasodilator). In certain preferred embodiments, a vasodilator comprises a coronary vasodilator. Non-limiting examples of a coronary vasodilator include amotriphene, bendazol, benfurodil hemisuccinate, benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane, etafenone, fendiline, floredil, ganglefene, herestrol bis(β-diethylaminoethyl ether), hexobendine, itramin tosylate, khellin, lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin, pentaerythritol tetranitrate, pentrinitrol, perhexiline, pimefylline, trapidil, tricromyl, trimetazidine, trolnitrate phosphate and visnadine.
In certain aspects, a vasodilator may comprise a chronic therapy vasodilator or a hypertensive emergency vasodilator. Non-limiting examples of a chronic therapy vasodilator include hydralazine (apresoline) and minoxidil (loniten). Non-limiting examples of a hypertensive emergency vasodilator include nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten) and verapamil. 6. Miscellaneous Antihypertensives
Non-limiting examples of miscellaneous antihypertensives include ajmaline, γ- aminobutyric acid, bufeniode, cicletainine, ciclosidomine, a cryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4- pyridyl ketone thiosemicarbazone, muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, a protoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodium nitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase and urapidil.
In certain aspects, an antihypertensive may comprise an arylethanolamine derivative, a benzothiadiazine derivative, a iV-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative, a guanidine derivative, a hydrazines/phthalazine, an imidazole derivative, a quanternary ammonium compound, a reserpine derivative or a sulfonamide derivative.
a. Arylethanolamine Derivatives
Non-limiting examples of arylethanolamine derivatives include amosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol and sulfinalol.
b. Benzothiadiazine Derivatives
Non-limiting examples of benzothiadiazine derivatives include althizide, bendrofiumethiazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane, metolazone, parafiutizide, polythizide, tetrachlormethiazide and trichlormethiazide.
c. iV-carboxyalkyl(peptide/lactam) Derivatives
Non-limiting examples of iV-carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapril and ramipril.
d. Dihydropyridine Derivatives
Non-limiting examples of dihydropyridine derivatives include amlodipine, felodipine, isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine and nitrendipine. e. Guanidine Derivatives
Non-limiting examples of guanidine derivatives include bethanidine, debrisoquin, guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan.
f. Hydrazines/Phthalazines
Non-limiting examples of hydrazines/phthalazines include budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine and todralazine.
g. Imidazole Derivatives
Non-limiting examples of imidazole derivatives include clonidine, lofexidine, phentolamine, tiamenidine and tolonidine.
h. Quanternary Ammonium Compounds
Non-limiting examples of quantemary ammonium compounds include azamethonium bromide, chlorisondamine chloride, hexamethonium, pentacynium bis(methylsulfate), pentamethonium bromide, pentolinium tartrate, phenactropinium chloride and trimethidinium methosulfate.
i. Reserpine Derivatives
Non-limiting examples of reserpine derivatives include bietaserpine, deserpidine, rescinnamine, reserpine and syrosingopine.
j. Suflonamide Derivatives
Non-limiting examples of sulfonamide derivatives include ambuside, clopamide, furosemide, indapamide, quinethazone, tripamide and xipamide.
G. Vasopressors
Vasopressors generally are used to increase blood pressure during shock, which may occur during a surgical procedure. Non-limiting examples of a vasopressor, also known as an antihypotensive, include amezinium methyl sulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin, gepefrine, metaraminol, midodrine, norepinephrine, pholedrine and synephrine. H. Treatment Agents for Congestive Heart Failure
Non-limiting examples of agents for the treatment of congestive heart failure include anti-angiotension II agents, afterload-preload reduction treatment, diuretics and inotropic agents.
1. Afterload-Preload Reduction
In certain embodiments, an animal patient that can not tolerate an angiotensin antagonist may be treated with a combination therapy. Such therapy may combine adminstration of hydralazine (apresoline) and isosorbide dinitrate (isordil, sorbitrate).
2. Diuretics
Non-limiting examples of a diuretic include a thiazide or benzothiadiazine derivative (e.g., althiazide, bendroflumethazide, benzthiazide, benzylliydrochloro thiazide, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide), an organomercurial {e.g., chlormerodrin, meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurous chloride, mersalyl), a pteridine (e.g., furterene, triamterene), purines (e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom, protheobromine, theobromine), steroids including aldosterone antagonists (e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative (e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide, chloraminophenamide, clofenamide, clopamide, clorexolone, diphenylmethane-4,4'-disulfonamide, disulfamide, ethoxzolamide, furosemide, indapamide, mefruside, methazolamide, piretanide, quinethazone, torasemide, tripamide, xipamide), a uracil (e.g., aminometradine, amisometradine), a potassium sparing antagonist (e.g., amiloride, triamterene)or a miscellaneous diuretic such as aminozine, arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine, isosorbide, mannitol, metochalcone, muzolimine, perhexiline, ticrnafen and urea.
3. Intropic Agents
Non-limiting examples of a positive intropic agent, also known as a cardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline, amrinone, benfurodil hemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin, cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine, dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine, prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin, strphanthin, sulmazole, theobromine and xamoterol.
In particular aspects, an intropic agent is a cardiac glycoside, a beta-adrenergic agonist or a phosphodiesterase inhibitor. Non-limiting examples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin (crystodigin). Non-limiting examples of a β-adrenergic agonist include albuterol, bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex), dopamine (intropin), dopexamine, ephedrine, etafedrine, ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol and xamoterol. Non-limiting examples of a phosphodiesterase inhibitor include amrinone (inocor).
I. Antianginal Agents
Antianginal agents may comprise organonitrates, calcium channel blockers, beta blockers and combinations thereof.
Non-limiting examples of organonitrates, also known as nitrovasodilators, include nitroglycerin (nitro-bid, nitrostat), isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol, vaporole).
J. Antibacterials
Antibacterials are generally used to reduce or prevent infection. Non-limiting examples of antibacterials include antibiotic antibacterials, synthetic antibacterials, leprostatic antibacterials rickettsia antibacterials, tuberculostatic antibacterial or a combination thereof.
1. Antibiotic Antibacterials
Non-limiting examples of antibiotic antibacterials include an aminoglycoside (e.g., amikacin, apramycin, arbekacin, a bambermycin, butirosin, dibekacin, dihydrostreptomycin, a fortimicin, gentamicin, isepamicin, kanamycin, micronomicin, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, streptonicozid, tobramycin), an amphenol (e.g., azidamfenicol, chloramphenicol, chloramphenicol palmitate, chloramphenicol pantothenate, florfenicol, thiamphenicol), an ansamycin (e.g., rifamide, rifampin, rifamycin, rifaximin), a β-lactam (e.g., a carbapenem, a cephalosporin, a cephamycin, a monobactam, an oxacephem, a penicillin), a lincosamide (e.g., clindamycin, lincomycin), a macrolide (e.g., azithromycin, carbomycin, clarithromycin, erythromycin acistrate, erythromycin estolate, erthromycin glucoheptonate, erythromycin lactobionate, erythromycin lactobionate, erythromycin propionate, erythromycin stearate, josamycin, leucomycin, midecamycin, miokamycin, oleandomycin primycin, primycin, rokitamycin, rosaramicin, roxithromycin, spiramycin, troleandomycin), polypeptides (e.g., amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, fusagungine, a gramicidin, a gramicidin S, mikamycin, polymyxin, polymyxin B-Methanesulfonic acid, pristinamycin, ristoceitin, teicoplanin, thiostrepton, tuberactinomycin, tyrocidine, tyrotliricin, vancomycin, viomycin, viomycin pantothenate, virginiamycin, zinc bacitracin), tetracycline (e.g., apicycline, chlortetracycline, clomocycline, demeclocycline, doxycycline, guamecycline, lymecycline, meclocycline, methacycline, minocycline, oxytetracycline, penimepicycline, pipacycline, rolitetracycline, sancycline, senociclin, tetracycline) or a micellaneous antibiotic antibacterial (e.g., cycloserin, mupirocin, tuberin).
Non-limiting examples of a carbapenem β-lactam include imipenem. Non-limiting examples of a cephalosporin β-lactam include cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime proxetil, cefroxadine, cefsulodin, ceftazidime, cefteram, cftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephacetrile sodium, cephalexin, cephaloglycin, cephaloridine, cephalosporin C, cephalothin, cephapirin sodium, cephradine and pivcefalexin. Non-limiting examples of a cephamycin β-lactam include cefbuperazone, cefmetazole, cefminox, cefotetan and cefoxitin. Non-limiting examples of a monobactam β-lactam include aztreonam, carumonam and tigemonam. Non-limiting examples of a oxacephem β-lactam include flomoxef and moxolactam. Non-limiting examples of a penicillin β-lactam include amidinocillin, amdinocillin pivoxil, amoxicillin, ampicillin, apalcillin, aspoxicillin, azidocillin, azlocillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, carbenicillin, carfecillin sodium, carindacillin, clometocillin, cloxacillin, cyclacillin, dicloxacillin diphenicillin sodium, epicillin, fenbenicillin, floxacillin, hetacillin, lenampicillin, metampicillin, methicillin sodium, mezlocillin, nafcillin sodium, mezlocillin, nafcillin sodium, oxacillin, penamecillin, penethamate hydridide, penicillin G benethiamine, penicillin G benzathine, penicillin G benzhydrylamine, penicillin G calcium, penicillin G hydrabamine, penicillin G potassium, penicillin G procaine, penicillin N, penicillin O, penicillin V, penicillin V benzathine, penicillin V hhdrabamiiie, penimepicycline, phenethicillin potassium, piperacillin, pivampicillin, propicillin, quinacillin, sulbenicillin, talampicillin, temocillin and ticarcillin.
2. Synthetic Antibacterials
Non-limiting examples of synthetic antibacterials include 2,4-diaminopyrimidines (e.g., brodimoprim, tetroxoprim, trimethoprim), nitrofurans (e.g., furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nirurpirinol, nifurprazine, nifurtoinol, nitrofurantion), quinolones and quinone analogs (e.g., amifoxacin, cinoxacin, ciprofloxacin, difloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, miloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid, piromidic acid, rosoxacin, temafloxacin, tosulfoxacin), sulfonamides (e.g., acetyl sulfamehtoxypraxine, acetyl sulfisoxazole, azosulfamide, benzylsulfamide, choramine-B, chloramine-T, dichloramine T, formosulfathiazole, N2-formylsulfisomidine, N4-β-D-glucosylsulfanilamide, mafenide, 4'- (methylsulfanoyl)sulfanilamide, p-nitrosulfathiazole, phthalysulfacetamide, phthalylsulfathiazole, salazosulfadimidine, succinylsulfathiazole, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfachrysoidine, sulfacytine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfadoxine, sulfaethidole, sulfaguanidine, sulfaguanol, sulfalene, sulfaloxic acid, sulfamerazine, sulfameter, sulfamethazine, sulfamethizole, sulfamethomidine, sulfamethoxazole, sulfamethoxypyridazine, sulfametrole, sulfamidochrysoidine, sulfamoxole, sulfanilamide, sulfanilamidomethanesulfonic acid triethanolamine salt, 4-sulfanilamidosalicylic acid, N4-sulfanilylsulfanilamide, sulfanilylurea, N-sulfanilyl-3,4-xylamide, sulfanitran, sulfaperine, sulfaphenazole, sulfaproxyline, sulfapyrazine, sulfapyridine, sulfasomizole, sulfasymazine, sulfathiazole, sulfathiourea, sulfatolamide, sulfisomidine, sulfisoxazole), sulfones (acedapsone, acediasulfone, acetosulfone sodium, dapsone, diathymosulfone, glucosulfone sodium, solasulfone, succisulfone, sulfanilic acid, p-sulfanilylbenzylamine, p,p, -sulfonyldianiline- N,N'diagalactoside, sulfoxone sodium, thiazolsulfone), and miscellaneous synthetic antibacterials (e.g., clofoctol, hexedine, methenamine, methenamine anhydromethylene- citrate, methenamine hippurate, methenamine mandelate, methenamine sulfosalicylate, nitroxoline, xibornol).
3. Liprostatic Antibacterials
Non-limiting examples of leprostatic antibacterials include acedapsone, acetosulfone sodium, clofazimine, dapsone, diathymosulfone, glucosulfone sodium, hydnocarpic acid, solasulfone, succisulfone and sulfoxone sodium.
4. Rickettsia Antibacterials
Non-limiting examples of rickettsia antibacterials, also known as antirickettsials, include p-aminobenzoic acid, chloramphenicol, chloramphenicol palmitate, chloramphenicol pantothenate and tetracycline.
5. Tuberculostatic Antibacterials
Non-limiting examples of tuberculostatic antibacterials include ^-aminosalicylic acid, ^-aminosalicylic acid hydrazine, benzoylpas, 5-bromosalicylhydroxamic acid, capreomycin, clofazimine, cyacetacide, cycloserine, dihydrostrptomycin, enviomycin, ethambutol, ethionamide, 4'-formylsuccinanilic acid thiosemicarbazone, furonazide, glyconiazide, isobutol, isoniazide, isoniazid methanesulfonate, morphazinamide, opiniazide, parsiniazide, phenyl aminosalicylate, protionamide, pyrazinamide, rifampin, salinazide, streptomycin, subathizone, sulfoniazide, thiacetazone, tiocarlide, tuberactinomycin, tubercidin, tuberin verazide, viomycin and vicmycin pantothenate.
2. Surgical Therapeutic Agents
In certain aspects, a therapeutic agent may comprise a surgery of some type, which includes, for example, preventative, diagnostic or staging, curative and palliative surgery. Surgery, and in particular a curative surgery, may be used in conjunction with other therapies, such as the present invention and one or more other agents.
Such surgical therapeutic agents for vascular and cardiovascular diseases and disorders are well known to those of skill in the art, and may comprise, but are not limited to, performing surgery on an organism, providing a cardiovascular mechanical prostheses, angioplasty, coronary artery reperfusion, catheter ablation, providing an implantable cardioverter defibrillator to the subject, mechanical circulatory support or a combination thereof. Non-limiting examples of a mechanical circulatory support that may be used in the present invention comprise an intra-aortic balloon counterpulsation, left ventricular assist device or combination thereof.
Further treatment of the area of surgery may be accomplished by perfusion, direct injection, systemic injection or local application of the area with at least one additional therapeutic agent (e.g., a LTβR modulator of the invention, a pharmacological therapeutic agent), as would be known to one of skill in the art or described herein.
VI. EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1
Regulation of Liver Homeostasis
The liver maintains its unique ability to undergo self-renewal throughout life (Michalopoulos and DeFrances, 1997). Acute viral and toxic insults lead to loss of hepatic mass and continued host survival requires proper restoration of hepatic mass. The inability of the host to restore liver mass is the basis for chronic liver diseases, which cause significant morbidity and mortality worldwide. Controlling the ability of the liver to regulate its mass has broad implications beyond chronic liver disease. Organ shortages continue to press the need for liver transplantation alternatives. The ability to increase the renewal of liver would make split liver transplants for living adult donor and even cellular transplants more feasible. Further understanding of liver growth control mechanisms offers a possible solution to both chronic liver disease and liver transplantation. The partial hepatectomy is a particularly useful model for beginning to understand how the liver regenerates because the liver's synchronized response facilitates study and the liver tightly controls its size (Diehl, 2002). However, the cellular and molecular mechanisms that control the process are not well defined. The understanding of liver regeneration has surrounded the cytokine and growth factor network involved in the process (Galun and Axelrod, 2002). From these studies it appears as though TNF alpha (TNF) and interleukin 6 (IL 6) are important cytokines that set the replicative processes in motion and that growth factors such as hepatocytes growth factor (HGF), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) help hepatocytes progress through the cell cycle (Cressman et al., 1996; Akerman et al, 1992). While exposure to cytokines and growth factors appears to be important for proper liver regeneration, it is clear that the cellular events that lead to production of cytokines and growth factors is critical.
The liver is a complex organ consisting of multiple cell types. Non-parenchymal cells {i.e. non-hepatocytes), which make up a significant cellular component of the liver, also respond to partial hepatectomy and appear to be important in maintaining intercellular relationships during liver regeneration (Malik et ah, 2002). There is already evidence indicating that the cellular mechanisms driving hepatocyte renewal depends on cells from both the bone marrow and from within the liver itself (Wang et al, 2003; Petersen et al, 1999; Vassilopoulos et al., 2003). These studies indicate that deeper understanding of liver's regenerative processes must take into account the contribution of the different cell types that comprise the liver.
LIGHT is a newly described TNF family member that is detected on activated T cells. LIGHT has two receptors, the lymphotoxin beta receptor (LTβR) which is expressed on non- lymphocytes and non-hemapoietic cells (including hepatocytes) and the herpes virus entry mediator receptor (HVEM) which is expressed on all hemapoietic cells (Force et al. 1995; Mauri et al, 1998).. Therefore, LIGHT performs a unique role bridging hemapoietic and non-hemapoietic signaling. LIGHT expression on activated T cells can signal to other hemapoietic cells via the HVEM receptor and to non-hemapoietic cells through the LTβR. Transgenic mice expressing LIGHT on T cells show a marked increase in liver mass even though liver size is ordinarily tightly controlled. Furthermore, LTβR deficient mice (LTβR-/-) also show an impaired ability to regenerate their liver after partial hepatectomy suggesting that the LTβR pathway is required for optimal liver regeneration. The inventors also extend theses observations to show that T cells are also required for liver regeneration following partial hepatectomy. The inventors have further found that a deficiency of natural killer T lymphocytes (NKT), a cell type particularly numerous in the wild type liver and severely decreased in LTβR-/- and lymphotoxin alpha deficient mice (LTa-/-), appears to be responsible for supporting hepatocytes division. Therefore, this study establishes a key interaction between T cell derived cytokines, T cells and hepatocytes during liver regeneration.
Materials and Methods
Mice and Reagents. Mice carrying a transgene for LIGHT have previously been reported (Wang et al, 2001). Six to ten week old male C57B/L6J, TCRbeta-/-delta-/-, (Stock # 002122) TRC delta-/- (Stock # 002120), and RAGl-/- (Stock # 002216) were purchased from The Jackson Laboratory (Bar Harbor, ME). LTa-/- mice were a gift from David Chaplin (Washington University, St. Louis MS) and backcross to B6 background for 13 generations. LTβR-/- mice were generated by Klaus Pfeffer (Futterer et al., 1998) and backcrossed to B6 background for 5-7 generation and HVEM-/- mice were recently generated in Klaus Pfeffer' s laboratory (Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Germany) and backcrossed to B6 for 5 generations and genetic targeting was confirmed by southern blot analysis and antibody staining. CD IdI-/- mice were generated at University of Chicago (Chen et al., 1997). All of mice were maintained in University of Chicago and handled according to NIH guideline and approved by the Institutional Animal Care and Use Committee. Cellular depletions were accomplished with anti-CD4 (GKl.5) or ant-NKl.l (PKl 36). Bone marrow transplantation was performed by lethally irradiating recipients with 900 rads followed by i.v. injection of 3 million bone marrow cells which were isolated by flushing the donors femur and humerus with sterile PBS.
Partial hepatectomy. The procedure was performed as originally described by Higgins and Anderson (1931). In brief, the mice were anesthetized with ketamine and xylzaline (100 mg/kg/10 mg/kg IP), the liver was exposed through a midline incision, the median and left lobes were mobilized and delivered after ligature with a single silk 4-0 suture. Sham procedures consist of anesthetized mice in which the peritoneum was opened and the liver is manipulated but not resected. Serum aspartate transaminase activity was determined by the method of Karmen using a Elan Diagnostics (SmithField, RI) analyzer according to the manufactures protocols in the University of Chicago Animal Research Core laboratory. Histology and BrdU labeling. Tissue section were fixed in 70% alcohol followed by buffered formalin and processed either for routine hemotoxylin and eosin staining or for BrdU immunohistochemical studies as per the manufactures instructions (Zymed San Francisco, CA). For ex-vivo culture of hepatocytes following hepatectomy, the liver was perfusion in situ with collagenase and > 85% viable hepatocytes were purified from 3 mice after differential centrifugation. Hepatocytes were seeded onto type I collagen coated coverslips in alpha-MEM (Invitrogen, Carlsbad, CA) Brdu incorporation was measured by incubation of the cells with (BrdU) (final dilution 1 :50, Zymed Laboratory Inc., South San Francisco, CA) in alpha MEM medium containing 125 mM Hepes, 200 mM L-glutamine, 0.13 mM L- proline, insulin (5 μg/ml), transferrin (5 μg/ml) and selenium (5 ng/ml), hydrocortisone (1 microM) and epidermal growth factor (50 ng/ml). BrdU incorporation was scored in 10 hpf after staining the coverslips according to the manufacturer's instructions (Zymed)
Isolation of intrahepatic lymphocytes. After perfusion with phosphate buffered saline, livers were pressed through a 200# mesh screen and centrifuged 5 min at 800xg. Pellets were suspended in 35% Percoll and 100 unit heparin/ml and centrifuged at 800xg for 20 minutes. The pellet was lysed of RBC and mononuclear cells were counted and prepared for staining. Lymphocytes were stained with CD3-FITC (clone 2Cl IBD Pharmingen Franklin Lakes, NJ) and CDId alpha-gal-cer loaded tetramer-PE and analyzed by FACS (Becton Dickson FACS scan II).
Increase of T cell derived LIGHT leads to hepatomegaly in a LTβR-dependent fashion.
The liver maintains its unique ability to undergo self-renewal throughout life with strict control of cell turnover (Michalopoulos and DeFrances, 1997). The role of T cells in liver homeostasis is unclear. Much to the inventors surprise, a TNF superfamily ligand LIGHT (TNSF 14) expressed only on T-cells has a dramatic impact upon liver homeostasis. Transgenic mice expressing /c/c-LIGHT on T cells have a dramatically increased liver weight relative to total body weight (FIG. 1). It suggests that a T cell derived factor can be sufficient for controlling liver homeostasis. Liver histology reveals markedly enlarged hepatocytes and frequent mitotic figures in Tg LIGHT mice compared to age matched wild type livers. Furthermore, in 6 to 8 month only transgenic mice, spontaneous develop hepatocellular carcinomas in 10% of mice. Since LIGHT has two known putative receptors, the [ymphotoxin beta receptor (LTβR) and herpes virus entry mediator (HVEM), the LIGHT transgenic mice were crossed to mice deficient in each of these receptors. The dramatic effect of LIGHT on liver size and histology was removed when the Tg LIGHT mice were crossed with LTβR-/- mice but not HVEM-/- mice. The dramatic effects of LIGHT on the liver could be transferred through bone marrow transplanted from Tg LIGHT mice into lethally irradiated wild type mice. The wild type mice receiving LIGHT transgenic bone marrow also displayed an enlarged liver with abnormal liver histology compared to wild type bone marrow transplant controls. Furthermore, a previous study showed a human hepatocyte cell line expresses mRNA for the LTβR, and that LIGHT pretreatment prevents TNF-mediated hepatocyte apoptosis (Matsui et al., 2002). This series of experiments hint that T cells acting through the LTβR expressing cells contribute to liver homeostasis.
Liver histology revealed markedly enlarged hepatocytes and frequent mitotic figures in Tg LIGHT mice compared to wild type livers. The liver histology and size return to normal in Tg LIGHT mice crossed with mice deficient in the lymphotoxin beta receptor ( LTβR-/-), one of LIGHT's putative receptors. On the contrary, Tg LIGHT mice crossed to LIGHT's other putative receptor, HVEM, did not have an effect on the development of abnormal liver histology. The Tg LIGHT x HVEM-/- mice still have markedly enlarged hepatocytes. Bone marrow from Tg LIGHT mice transplanted into lethally irradiated wild type mice also results in an enlarged liver. Wild type bone marrow transplanted into wild type mice results in normal liver histology while Tg LIGHT bone marrow transplanted into wild type mice results in the same liver histology seen in Tg LIGHT mice.
LTβR signaling by T cells may be essential for liver regeneration
Since the LTβR appeared to mediate liver proliferation in response to a T cell derived cytokine, the inventors wanted to better understand how the LTβR might contribute to restoration of liver mass following partial resection. Partial hepatectomy, in which 70% of the liver is removed, is regarded as the preferred method to study liver regeneration (Higgins and Anderson, 1931; Fausto and Campbell, 2003). LTβR deficient mice were challenged with a 70% hepatectomy. Wild type mice showed no significant increase in morbidity or mortality following 70% hepatectomy while there was a dramatically increased morbidity and mortality of LTβR-/- after 70% hepatectomy (FIG. 2). Furthermore, mice deficient in lymphotoxin (LT α -/-), another lymphocyte derived ligand that signals through the LTβR, also displayed a significant decrease in survival following 70% hepatectomy (FIG. 2). This series of experiments indicated the LTβR was an important receptor for hepatic homeostasis by interacting with lymphocyte derived ligands. It raises the intriguing possibility that lymphocytes can play a critical and unexpected role in liver homeostasis.
LTβR-/- and LT α-/- mice showed evidence of liver damage at 48 hours following partial hepatectomy with significantly elevated serum aminotransferase levels compared to similarly treated wild type mice (FIG. 3A). Histological examination of the livers also showed large areas of necrosis in the LTβR-/- or LT α-/- mice at 48 hours following partial hepatectomy compared to wild type mice. Large areas of necrosis (pale hepatocytes outlines without a nucleus) were observed in the LTβR or LTa deficient mice at 48 hours following partial hepatectomy compared to wild type mice. Not only was there significant liver cell death in the mice deficient in lymphotoxin signaling, but there was also a significant decrease in hepatocyte DNA synthesis. Mice were treated with a thymidine analog, bromodeoxyuridine (BrdU), 48 hours following 70% hepatectomy or a sham procedure. DNA synthesis following 70% hepatectomy was determined by counting the number of BrdU positive hepatocyte nuclei following immunohistochemical staining. There was significantly more DNA synthesis in the wild type mice compared to either LTβR-/- or LT α-/- mice (FIG. 2C). As expected, sham procedures did not induce DNA synthesis. This indicated that the lymphotoxin receptor as well as the lymphotoxin ligand was important for the liver's ability to regenerate hepatic mass.
NK T cells are critical for liver regeneration
These results suggest that T-cell derived cytokines, such as LIGHT and lymphotoxin, can effect the liver. To test whether lymphocytes are essential for controlling liver regeneration, mice lacking different subsets of lymphocytes were used. Recombinase deficient (RAGl-/-) mice that lack B, T and NKT cells were tested for their ability to undergo liver regeneration following partial hepatectomy and were found to be defective (Table 1). To further confirm the role of T cells, T cell receptor deficient (TCRβ-/- δ-/-) mice that lack both T and NKT cells all display reduced survival following partial hepatectomy (Table 1). On the other hand, mice that only lack a small subset of T cells, γδ T cells (TCR δ-/-), fully survive partial hepatectomy (Table 1). The preliminary data showed that TCR deficient mice reconstituted with T cells from wild type mice fully survive partial hepatectomy. This suggests that T cells play a sufficient role in liver regeneration.
Figure imgf000043_0001
A T cell defect common to LT βR-/- and LT α -/- mice is a marked decrease in the percentage and total number of intrahepatic NKT cells while the total number of other T cell types remains normal (Elewaut et ah, 2000). NK T cells are particularly rich in the liver, and it has been shown that NK T cells increase in total number and percentage following partial hepatectomy in wild type mice (Minagawa et al, 2000). Twelve hours following partial hepatectomy there is a sharp increase in NKT cells (CD3+ CDId tetramer+) and (CD4+ CDId tetramer) (FIG. 4). The LT βR-/- mice have a decreased percentage, total number of NKT cells (CD3+ CD1d tetramer+) and an impaired increase in the percentage of NKT cells following partial hepatectomy. This series of experiments demonstrates lymphotoxin deficient mice have reduced numbers of intrahepatic NKT cells before and after partial hepatectomy. It supports the idea that NKT cells, an important component of the innate immune response, might be critical for liver regeneration.
LTβR deficient mice have a markedly decreased percentage of intrahepatic NKT cells and show a marginal increase in NKT cells following partial hepatectomy. Following partial hepatectomy in wild type mice the percentage of CD4+ T-cells and CD4+ CDId tetramer + cells, the major subset of NKT cells, increases significantly compared to before partial hepatectomy. Similarly, the percentage of CD3+ CDId tetramer positive cells (NKT cells) significantly increases in the liver of wild type mice following partial hepatectomy compared to before partial hepatectomy. On the other hand, the percentage of CD3+ CDId+ NKT cells is significantly decreased in LTβR deficient mice prior to partial hepatectomy with a marginal increase in the percentage of NKT cells following partial hepatectomy.
In order to test if the observed correlation between the decrease in NKT cells seen in LTβR-/- mice could be independently confirmed, the inventors checked other mice, which are deficient in NKT cells or depleted wild type mice of NKT cells. Since most NKT cells in the liver express CD4 (FIG. 4), wild type mice were depleted of CD4+ cells with 150 to 300 micrograms of anti-CD4 (GKl .5) 7 days prior to the procedure. The survival of these mice was significantly reduced following partial hepatectomy (Table 1). As an additional approach to deplete NK T cells, another set of wild type mice were depleted of NKl.1+ cells (which depletes NK and NKT) prior to partial hepatectomy with 300 μg of anti NKl.1 (clone PKl 36). In either case, wild type mice depleted of NKT cells displayed significantly reduced survival following partial hepatectomy (Table 1). The inventors further confirmed this finding using mice completely deficient in CDl dependent NKT cells (Chen et al, 1997). Mice deficient in CDId, which lack a major subset of NKT cells, have smaller liver/body weights at baseline and also display reduced survival following partial hepatectomy (Table 1). Careful examination of the livers proliferative activity at 24 and 36 h following partial hepatectomy shows that induction of hepatocyte DNA synthesis is inhibited consistently in CD1d- /- mice. For example, by 36 h post partial hepatectomy, the time of peak proliferative activity in normal mice, 100 fold fewer hepatocytes have entered S phase in CD1d- /- mice than controls (FIG. 5A). Liver histology reveals significantly decreased hepatocyte proliferation presented by BrdU+ brown nuclei in NKT cell deficient CD1d- /- mice comparing to wild type control mice 36 hour post partial hepatectomy. Furthermore, the hepatocytes in CD1d- /- mice show a decreased ability to proliferate ex-vivo following 70% hepatectomy (Fig. 5B). This indicates that NKT cells create a critical milieu in the liver, which allows hepatocytes to undergo cell division. Therefore, the data have clearly revealed an under appreciated role of NKT cells in the promoting liver regeneration. The maintenance of hepatic mass in the adult is tightly controlled. However, the cellular and molecular mechanisms that govern this process are unclear. Massive hepatomegaly in Tg LIGHT mice suggests that up-regulation of LIGHT on T cells disrupts the mechanisms that normally balance hepatocyte proliferation and hepatocyte death, leading to net accumulation of hepatocytes. The latter action of LIGHT is dependent upon T cells and the expression of the LTβR since crossing Tg LIGHT mice to LTβR-/- mice returns the liver to normal size. Evidence that LTβR-/- mice develop extensive areas of hepatocyte death when challenged with partial hepatectomy supports the concept that LTβR-mediated microenvironment transduces viability signals in hepatocytes (Matsui et al., 2002). Moreover, hepatocytes that survive partial hepatectomy in LTβR-/- mice are inhibited from entering the replicative phases of the cell cycle. This combination of increased liver damage and a reduced regenerative response to liver injury induces significant partial hepatectomy mortality in mice that cannot activate the LTβR. A similar defect was observed in lymphotoxin (LT) deficient mice which is expressed on activated T cells and is another ligand for LTβR. Interestingly, there is a 10 fold increase in LIGHT and 5 fold increase in LT mRNA at 12 hours following partial hepatectomy in the non-parenchymal cells of the liver. Together, these data suggest that LTβR signaling by T cells may be critical for liver homeostasis.
Acute and chronic insults by pathogens and toxin lead to loss of hepatic mass and continued host survival requires proper regeneration of hepatic mass. Much of the attention surrounding liver growth has come from proinflammatory cytokines, such as TNF and IL 6 (Cressman et al, 1996; Akerman et al., 1992). In addition to macrophages, many cell types, hematopoietic and non-hematopoietic cells, can produce cytokines such as TNF and IL 6. For years, the actual cellular components controlling these cytokines contribution to liver regeneration have not been identified. This study has revealed for the first time that LT/LTβR pathway is essential for liver regeneration. The LT/LTβR pathway uniquely bridges the hematopoietic system, responsible for the expression on LIGHT and lymphotoxin, to the non- hematopoietic system, which expresses the LTβR. Clearly, cytokine and growth factor production are not the only mechanisms functioning during liver regeneration. Understanding of how the hematopoietic and non-hematopoietic cells interact will be critical in order to apply this model to clinical situations. The inventors realization of this connection has further allowed us to address the role of T cells in liver regeneration. Unlike many tissues, such as kidney and brain, the liver contains a large number of resident T cells that have access to hepatocytes through fenestrated endothelial cells without having to cross a basement membrane (Maher, 2001). The role of residual T cells in liver homeostasis and function has been speculated. It was noticed that CD4+ NKT cells, 80% NKT cells inside liver, are often activated and increased inside the liver after partial hepatectomy but its role has not been well elucidated (Minagawa et ah, 2000). NKT cells appear to partially contribute to liver inflammation (Kato et ah, 2004; Ito et al, 2003). This study has clearly demonstrated that T cells are essential for liver regeneration. Furthermore, the inventors have identified a subset of T cells, NKT cells, are essential for the process. NKT cells, a dominant T cell population inside liver, are important population for innate immunity and contain massive cytokines and ligands similar to those within activated T cells, which allow them to rapidly release large amounts of cytokines and. activating ligands following tissue injury. The lack of NKT cells in LT βR ' ' mice, supports the idea that NKT cells may contribute to the process of liver regeneration. Furthermore, the inventors show that depleting mice of CD4+, or NKl.1+ cells aborts this component of the regenerative response and impairs survival after partial hepatectomy. Moreover, CDId''' mice that cannot support normal NKT cell development have depleted NKT cell populations and exhibit somewhat atrophic livers at baseline. Following partial hepatectomy, CDId1' display significantly decreased induction of hepatocyte DNA synthesis and increased mortality following partial hepatectomy. Taken together, these data support the concept that NKT cells provide trophic signals that are necessary for hepatocytes to proliferate.
This possibility is further supported by studies of cultured hepatocytes from CDId1' mice. Unlike primary hepatocytes from wild type mice, hepatocytes from CDId'' mice after partial hepatectomy fail to enter S phase when incubated with growth factors. Conversely, the inventors have found that that increasing NKT cells in healthy, control mice leads to dramatic increases in the "basal" proliferative activity of hepatocytes. Although additional studies are necessary to determine if NKT cells interact directly with hepatocytes or act via more indirect mechanisms to promote hepatocyte proliferation, these results prove that the T cells can play a critical role in regulating hepatic homeostasis during adulthood. It will be interesting to determine whether some causes of hepatomegaly may also be due to abnormal activation of T cells. Interestingly, the inventors have noticed enlargement of the liver in MRL/lpr mice, which have a dramatic accumulation of activated T cells in the liver. Understanding of liver regeneration will lead to better diagnosis and treatment of liver diseases as well as improve liver transplantation in the future. Understanding the mechanisms that control hepatocytes survival has broad implications in acute and chronic liver diseases. Furthermore, these findings raise the possibility that T cells might play a distinct role in the biology and regeneration of other tissues.
EXAMPLE 2 Alteration of Lipid Levels by LTβR-Ig Administration
In this Example, the inventors have observed a dramatic reduction of various lipid levels, reduced cholesterol, triglycerides, VLDL, and increased HDL, reduced atherosclerosis only after a short-term treatment of host with low dose (100 μg) of LTβR-Ig. LDL receptor deficient mice have been powerful models to study how lipid level is controlled. The mice will develop hypercholesterolemia, triglycerimia and increased VLDL and HDL 1-2 weeks after feeding with fatty food, eventually leading to severe atherosclerosis in 2-3 months. In contrast to control Ig, LTβR-Ig treated group much reduced triglycerides and cholesterols as well as reduced VLDL and increased HDL {e.g., FIG. 6, FIG. 7): Therefore, treating patients suffering hyperlipidemia and atherosclerosis with LTβR modifier may be a new treatment.
Atherosclerosis is a chronic inflammatory response to hyperlipidemia and associated high incidence of cardiovascular and brain vascular diseases. Both innate and adaptive immunity influence the progression of this inflammation. Most studies in this field focus on the inflammation lesions and how to reduce inflammation-mediated lesion on artery. The effect of the immune system on atherosclerosis is rather complex. Few studies focus on the lipid profile. Whether and which cytokines are involved in the regulation of lipid profile is unclear (Wang and Paigen, 2005).
Atherosclerosis is the major cause of death in the world. Fasting and postprandial hyperlipidemia are important risk factors for coronary heart disease (CHD). Recent developments have undoubtedly indicated that inflammation is pathophysiologically closely linked to atherogenesis and its clinical consequences. Inflammatory markers such as C- reactive protein (CRP), leukocyte count and complement component 3 (C3) have been linked to CHD and to hyperlipidemia and several other CHD risk factors. Increases in these markers may result from activation of endothelial cells (CRP, leucocytes, C3), disturbances in adipose tissue fatty acid metabolism (CRP, C3), or from direct effects of CHD risk factors (leucocytes). It has been shown that lipoproteins, triglycerides, fatty acids and glucose can activate endothelial cells, most probably as a result of the production of reactive oxygen species (van Oostrom et al, 2004). Similar mechanisms may also lead to leukocyte activation. Increases in triglycerides, fatty acids and glucose are common disturbances in the metabolic syndrome and are most prominent in the postprandial phase. People are in a postprandial state most of the day, and this phase is proatherogenic. Inhibition of the activation of leucocytes, endothelial cells, or both, is an interesting target for intervention, as activation is obligatory for adherence of leucocytes to the endothelium, thereby initiating atherogenesis. Potential interventions include the use of unsaturated long-chain fatty acids, polyphenols, antioxidants, angiotensin converting enzyme inhibitors and high-dose aspirin, which have direct antiinflammatory and antiatherogenic effects.
C-reactive protein (CRP) is a predictor of future risk for cardiovascular disease. LT- alpha is a proinflammatory cytokine that plays an important role in the pathogenesis of atherosclerosis in mice. Recent studies have examined the association between gene polymorphism of the LT-alpha coding gene, LTA A252G, and CRP based on a case-control study (Suzuki et al, 2004). After adjusting for the effect of sex, age, BMI, WBC, Hb, and HbAIc, the LTA 252G allele was found to be associated with high CRP levels (odds ratio = 1.93, P = 0.007) by multiple logistic regression analysis. It is possible that CRP levels are influenced not only by environmental factors but also by the polymorphism of LTA or other genes in the same haplotype block.
Myocardial infarction (MI) has become one of the leading causes of death in the world. Its pathogenesis includes chronic formation of plaque inside the vessel wall of the coronary artery and acute rupture of the artery, implicating a number of inflammation- mediating molecules, such as the cytokine lymphotoxin-alpha (LTA). Functional variations in LTA are associated with susceptibility to MI (Ozaki et al, 2004). Recent case-control association study in a Japanese population showed that a single nucleotide polymorphism in LGALS2 encoding galectin-2 is significantly associated with susceptibility to ML This genetic substitution affects the transcriptional level of galectin-2 in vitro, potentially leading to altered secretion of LTA, which would then affect the degree of inflammation; however, its relevance to other populations remains to be clarified. Smooth muscle cells and macrophages n the human atherosclerotic lesions expressed both galectin-2 and LTA. Recent findings suggest a link between the LTA cascade and the pathogenesis of MI. LTβR has two ligands, lymphotoxin and LIGHT (Fu and Chaplin, 1999; Mauri et al, 1998). Both LIGHT and LT expressed on activated T cells and often detected at inflammatory site and contribute to enhanced autoimmunity (Gommerman and Browning, 2003; Tamada et al, 2000; Wang and Fu, 2003; Wang et al, 2001). The above data demonstrates that LTβR- Ig treatment can greatly reduce lipid level in the blood, which may be critical for reduction of atherosclerosis.
Methods
The generation of LTβR-Ig fusion. In brief, cDNA encoding murine LTβR extracellular domain was generated by RT-PCR using the sense primer (5'- AAAGGCCGCCATGGGCCT-3') (SEQ ID NO:1) and the antisense primer 5'- TTAAGCTTCAGTAGCATTGCTCCTGGCT-3') (SEQ ID NO:2)from mouse lung mRNA, digested by Ncol/Hindlll and then fused to a IL-3 leader sequence in p30242 vector. The fusion fragment was then subcloned into pX58 vector containing IE- 175 promoter and Fc portion of human IgGl. The construct was then transfected into BHK/VP16 cells and the mouse LTβR-human Ig was purified by Protein A column. This LTβR-Ig fusion is referred to as "LTβR-Ig" herein.
The treatment of LDL receptor deficient mice with LTβR-Ig. The adult hosts were i.p. inoculated with 100 μg of LTβR-Ig, once a week for 8 weeks. The sera were collected once a month and subjected to HPLC for lipid profile and total cholesterol and triglycerides were also monitored.
Results
LTβR-Ig treatment reduces total cholesterol and triglycerides (FIG. 6). In these studies, 5 adult LDLR deficient mice from each group were treated with either control or LTβR-Ig. The sera were collected one month and 2 month. TC means Total cholesterol and TG means total triglycerides. The tissues (arteries) were collected one week later.
LTβR-Ig treatment increases HDL and reduces VLDL (FIG. 7). 5 adult LDLR deficient mice from each group were treated with either control or LTβR-Ig. The sera were collected one month and 2 month for HPLC to determine VLDL, LDL, and HDL.
LTβR-Ig treatment limits hyperlipidemia and improves HDL levels (Table T). In these studies, LDLR-/- mice were injected with Control or LTβR IgG for 8 weeks while on high cholesterol/high fat diet. Animals were sacrificed 1 week after injections were terminated.
Figure imgf000050_0001
EXAMPLE 3 The LT/LIGHT axis in lipid homeostasis
This example demonstrates that T cell-derived proinflammatory ligands in LT/LIGHT regulate the expression of genes that control hepatic lipid metabolism. T cells intimately contact hepatocytes but their role in hepatocyte-mediated lipid homeostasis is unclear. Lymphotoxin (LT) and LIGHT, which are TNF superfamily ligands for LTβR expressed on lymphocytes, are identified as critical players in controlling key lipid regulatory enzymes in hepatocytes leading to the development of dyslipidemia. Dysregulated LIGHT expression on T cells resulted in hypertriglyceridemia and hypercholesterolemia. Furthermore, disruption of LT/LIGHT with a soluble LTβR decoy protein in LDL-R-deficient mice corrected the dyslipidemia. This example demonstrates that T cell-derived proinflammatory ligands in LT/LIGHT regulate the expression of genes that control hepatic lipid metabolism. Atherosclerosis is a combination of dyslipidemia and inflammatory-mediated pathology of the vasculature. Systemic inflammation is associated with hyperlipidemia but its mechanism is unclear. The tumor necrosis factor superfamily (TNFSF) is involved in a wide array of biological processes, including immune function, cell death and survival, atherosclerosis, but its role in lipid metabolism is not well defined (Locksley, 2001). LT, LIGHT and LTβR belong to the TNFSF of proteins. Recent studies have identified them to play key roles in secondary lymphoid tissue development, tolerance, and lymphocyte activation. Their disruption leads to a wide array of phenotypes including lymph node aplasia, impaired splenic microarchitecture, autoimmunity, viral infection, and grossly impaired immunoglobulin responses. (Fu and Chaplin, 1999; Gommerman and Browning, 2003; Ware, 2005).
Recent evidence have linked LT to the development of atherosclerosis and myocardial infarctions (Schreyer et al, 2002; Ozaki et al, 2004). The contribution of LT to lipid homeostasis, a putative obligatory precursor to atherogenesis, is not known. Lck LIGHT transgenic (Tg) mice develop systemic inflammation and hepatomegaly (Wang, 2001; Shaikh, 2001). Lymphocytes intimately contact hepatocytes, raising the possibility that some T cell derived ligands, such as LT/LIGHT, expressed on activated T cells, may regulate various liver functions. LT/LIGHT have been shown to impart signals necessary for liver regeneration as well as hepatitis (Anders et al, 2005; Anand et al, 2006). Given the observed effects of LT/LIGHT on the liver and that lipid homeostasis is tightly regulated by the liver, the lipid profiles of lck LIGHT Tg and wild-type mice were examined. The LIGHT Tg mice displayed hypercholesterolemia (P < 0.001) on a normal chow diet (FIG. 8A). The LIGHT Tg mice also developed an elevation in triglycerides compared to wild-type mice (P = 0.02) (FIG. 8B). The hyperlipidemia phenotype was exaggerated when the mice were placed on a high fat, high cholesterol western type diet (WTD). LIGHT Tg mice developed hypertriglyceridemia (P < 0.01) along with worsening hypercholesterolemia (P < 0.01) on WTD (FIG. 8C and FIG. 8D). The lipid profile of LIGHT Tg mice was examined by FPLC. LIGHT Tg mice displayed an increase in VLDL and LDL but not HDL, further supporting a role in the pathogenesis of dyslipidemia. These results have revealed that a TNFSF member, and specifically LIGHT expressed on T lymphocytes, can unexpectedly regulate lipid homeostasis.
To further interrogate the mechanism of the LIGHT-mediated dyslipidemia, LIGHT Tg mice were crossed with mice lacking the two defined receptors for LIGHT, the Herpes virus entry mediator (HVEM) and LTβR. The LIGHT-mediated dyslipideniia was still present in LIGHT Tg/HVEM-/- (FIG. 9A and FIG. 9B) mice but largely corrected in LIGHT Tg/LTβlC-/- mice (FIG. 9C and FIG. 9D). LIGHT Tg/LTβR-/- mice had a trend of reduction in cholesterol compared to LIGHT Tg mice (P < 0.05) and more impressively a complete normalization in the levels of triglycerides (P < 0.01) (FIG. 9C and FIG. 9D). These data suggest that LIGHT-LTβR but not LIGHT-HVEM interactions are essential for the dyslipidemia in LIGHT Tg mice. Two likely target organs that are known to regulate lipid homeostasis are the liver and intestine. The feces of LIGHT Tg mice appeared grossly similar to that of WT mice on both the normal chow and high fat diet, suggesting that gastrointestinal and hepatobiliary excretion was not a major mechanism for the dyslipidemia.
To dissect the contribution of altered liver metabolism to the dyslipidemia in LIGHT Tg mice, the hepatic gene expression profile of wild-type and LIGHT Tg mice was analyzed by Affymetrix microarray analysis. The inventors identified 385 out of more than 45,000 genes that were altered by five- fold or more. The inventors further subdivided these genes into pathways, with particular interest paid to genes that regulate lipid and cholesterol metabolism. Among genes with known lipid and cholesterol regulatory functions, only three genes were identified that were upregulated by two-fold or more and two that were downregulated by two-fold or more in the Tg. Strikingly, only two genes in the lipid and cholesterol metabolism pathways were significantly altered five-fold or more in the Tg livers. Of interest, hepatic lipase, was decreased by more than five-fold in LIGHT Tg mice (FIG. 10A). Hepatic triglyceride lipase hydrolyzes triglycerides and phospholipids and is a major regulator of triglycerides, lipoprotein metabolism in the liver (Santamarina-Fojo, 2004 #13). Inactivation of the hepatic lipase gene results in dyslipidemia whereas overexpression of hepatic lipase acts to substantially decrease triglycerides and apolipoprotein cholesterols, especially those containing apoB. The results of the gene array studies were confirmed using semi-quantitative RT-PCR and quantitative real-time PCR analyses (FIG. 10B). By real-time PCR, the expression of hepatic lipase was decreased by approximately 18-fold in the livers of LIGHT Tg mice compared to WT (P = 0.01) (FIG. 10B). Next, to determine the role of the LTβR in the LIGHT-mediated reduction in hepatic lipase transcription, the livers from LIGHT Tg/LTβR-/- were assessed. The level of hepatic lipase was significantly elevated in LIGHT Tg/LTβR-/- compared to LIGHT Tg livers (P < 0.05) (FIG. 10B). Taken together, these results suggest that LIGHT-LTβR interactions on hepatocytes inhibit the expression of hepatic lipase. Repression of hepatic lipase expression may contribute to the development of hyperlipidemia in LIGHT Tg mice.
Wild-type and LIGHT Tg mice were fed WTD for two weeks, sacrificed, liver RNA were isolated and subjected to Affymetrix microarray analysis. A heat map for genes involved in lipid and cholesterol metabolism is shown. The genes shown below in Table 3 represent those that were changed by 1.2-fold or more and statistically significant (P < 0.05).
Figure imgf000053_0001
These data point to a pathogenic role for LIGHT and LTβR in dyslipidemia. Thus a murine model of dyslipidemia and a soluble LTβR-Ig fusion protein (to disrupt LT/LIGHT/LTβR interactions) were utilized. LDLR-deficient mice develop a severe form of hyperlipidemia soon after initiation of a high fat, high cholesterol western type diet (WTD) that is difficult to be corrected (Reardon et al, 2001). Recent evidence have suggested a role for lymphocytes in hyperlipidemia, but the mechanism is unknown (Reardon et al, 2003). The mice were fed WTD and injected weekly with a low dose of LTβR-Ig and the plasma concentrations of triglycerides and total cholesterol were determined. 12 weeks after initiation of WTD, LDL-R-/- mice treated with LTβR-Ig showed a substantial reduction in :otal cholesterol (P < 0.001) and triglycerides (P < 0.01) (FIG. 11A and FIG. 11B). When a higher dose of LTβR-Ig (250 μg/week) was administrated, lipid levels decreased as early as four weeks. FPLC analyses demonstrated that LTβR-Ig treatment dramatically reduced levels of VLDLc (P < 0.01) and LDLc (P < 0.05) without perturbing the levels of HDLc (FIG. HC and FIG. HD). These results provide evidence that LTβR-Ig can be a novel and effective treatment for dyslipidemia.
As LTβR-Ig disrupts both the membrane LT and LIGHT ligands, the inventors sought to determine the relative contributions of LT and LIGHT to dyslipidemia in the LDL-R-/- mice by performing bone marrow chimera experiments. LDL-R-defϊcient mice that were the recipients of LIGHT-/- donor bone marrow, exhibited a mild and statistically significant reduction (P < 0.05) in total cholesterol (FIG. HE). In contrast, the LDL-R-deficient mice reconstituted with LT-/- bone marrow displayed approximately a 40% reduction in total cholesterol (P = 0.01) (FIG. HE). The hypertriglyceridemia phenotype was still present in singly deficient bone marrow chimeras, suggesting that the development of hypertriglyceridemia may be more permissive to either LT or LIGHT signaling. These results suggest that LT may be the more dominant LTβR ligand in the pathogenesis of dyslipidemia in LDL-R-deficient mice and that blockade of both LT and LIGHT, which was achieved with the use of LTβR-Ig, may be necessary to completely alleviate the phenotype of dyslipidemia.
In the LIGHT Tg model of dyslipidemia, the inventors had earlier observed a decrease in hepatic lipase in the dyslipidemic animals. The inventors sought to determine if blocking LT/LIGHT interactions with LTβR-Ig in LDL-R-/- mice may increase the expression of hepatic lipase and help to alleviate the dyslipidemia. The real time quantitative PCR analyses revealed an increase in hepatic lipase of the livers of mice treated with LTβR-Ig (P < 0.05) (FIG. 1 IF). To dissect the contribution of LT and LIGHT on the regulation of hepatic lipase expression, hepatic lipase transcription was tested in the radiation bone marrow chimeric mice. LDL-R-deficient mice reconstituted with bone marrow from LT-/- donors expressed higher levels of hepatic lipase (P < 0.05) (FIG. HG). These studies further support that expression of LTβR ligands on hematopoietic cells regulates hepatic lipase expression,
LT or LIGHT may signal via the LTβR on hepatocytes to inhibit the expression of hepatic lipase. To directly assess this, hepatocytes were stimulated with LIGHT and measured hepatic lipase transcripts. LIGHT stimulation depressed the expression of hepatic lipase (P < 0.05) (FIG. HH). These data support the notion that LT/LIGHT/LTβR regulates lipid metabolism via hepatic lipase gene regulation in the liver. Augmented LTβR signaling seems to decrease hepatic lipase expression, leading to the development of hypertriglyceridemia and hypercholesterolemia. Meanwhile, targeted inhibition of LT/LIGHT/LTβR signaling, as is shown in this study, can be used to reverse the dyslipidemia that is a prerequisite in many cardiovascular disease states such as peripheral vascular disease and coronary heart disease. The effects of LTβR blockade on the development of atherosclerosis are currently under investigation.
These data demonstrate that the LT/LIGHT/LTβR pathway maintains lipid homeostasis via programming of hepatic genes. The liver is a critical organ that regulates lipid metabolism. Interestingly, lymphocytes closely contact hepatocytes and could physically provide direct ligands to communicate with hepatocytes. Activated lymphocytes express both LT and LIGHT, which may regulate lipid metabolism inside liver. Recent studies show that these ligands control hepatic regeneration and reduce ConA-mediated hepatitis (Anders et ah, 2005; Anand et al, 2006). The data show that LTβR-Ig treatment diminishes T cell-mediated hepatitis, suggesting an essential role of T cell-derived LT/LIGHT in the regulation of liver function. Whether other liver functions are also regulated by hepatic lymphocytes remain to be determined. This study provides evidence that LT/LIGHT on T cells regulate lipid homeostasis. However, LT/LIGHT are also expressed on other hematopoietic cell types such as B, NK, NKT, and dendritic cells which may contribute to lipid metabolism. Likewise, there are other organs that express the LTβR which may also play important roles in lipid biology. For instance, the LTβR and its ligands may also exert effects on adipose tissue, the gastrointestinal system, the endocrine system, and other immune cells such as macrophages to maintain lipid homeostasis. The contribution of these other potential target organs to proinflammatory cytokine-mediated lipid modulation will certainly be an area of active future research.
The evolutionary origin of the physiological role of LT/LIGHT-mediated regulation of lipid homeostasis remains to be determined. As LT/LIGHT are expressed preferentially on activated lymphocytes, one expected outcome of inflammation is 1) increased LT/LIGHT expression on lymphocytes, 2) stimulation of the LTβR on hepatocytes, 3) depressed hepatic lipase gene expression, and 4) hypertriglyceridemia and hypercholesterolemia. The data provide an explanation for the long-time dogma that chronic inflammation is associated with hyperlipidemia, the mechanisms of which have previously not been well defined. This may be an intended and advantageous product of inflammation an or untoward consequence of combating pathogens.
These data demonstrate that the TNFSF is a regulator of lipid homeostasis. The inventors have identified two ligands, primarily derived from T cells, of the LTβR which play a critical role in hypertriglyceridemia and hypercholesterolemia. Additionally, a new murine model in the LIGHT Tg mice is provided to study dyslipidemia. These data identify a target for LT/LIGHT in hepatic lipase to regulate lipid metabolism. Disruption of the LT/LIGHT/LTβR axis ameliorates dyslipidemia via a mechanism of increased hepatic lipase.
Materials and Methods
Mice and treatments. C57B1/6 (B6) and LDL-R-/- mice were purchased from the National Cancer Institutes or The Jackson Laboratory. Lck LIGHT Tg, LT α-/- , LIGHT-/-, and LTβR-/- mice backcrossed to B6 mice and maintained under specific pathogen-free conditions as described (Wang et al, 2001; De Togni et al, 1994; Tamada et al, 2002; Futterer et al, 1998). Mice were maintained on a standard chow diet until placed on a Western-type diet (Harland Teklad TD88137) containing 0.15% cholesterol and 21% fat for the indicated times. For injections, 100 μg of control human IgG (Sigma) or LTβR-Ig were i.p. injected weekly until completion of the experiment. The LTβR-Ig used in this study has been previously described (Wu et al, 2001). Briefly, cDNA encoding the extracellular domain of murine LTβR was fused with the Fc portion of human IgG, transfected into BHK/VP16 cells, and the supernatant was collected.
Lipid measurements. Plasma total triglycerides were determined by The Infinity™ Triglycerides single liquid stable reagent from Thermo Electron Corporation (Waltham, MA). Plasma total cholesterols were determined by Cholesterol liquid reagent set from Pointe Scientfic, Inc. (Canton, MI). Plasma was collected by retro-orbital bleeding. Lipoproteins were separated by fast protein liquid chromatography on tandem Superose 6 columns, and the amount of cholesterol in the even-numbered fractions was determined and expressed as micrograms cholesterol per milliliter of plasma. The area under each lipoprotein peak was quantified by computer digitizer (SigmaScan) and expressed as percent total cholesterol. Lipoprotein cholesterol levels were determined from the total plasma cholesterol and the percentage of total cholesterol in each of the lipoprotein peaks. RNA and real-time PCR. Total RNA were extracted by TRIZOLCg) Reagent from Invitrogen and cleaned up using RNeasy spin column from QIAGEN. For cDNA synthesis, RNA were digested with DNase I and reverse transcribed using random primers with AMV Reverse Transcriptase (Promega). Each cDNA sample was amplified for hepatic lipase or glyceraldehyde phosphodehydrogenase (gapdh) using the Taqman Universal PCR master mixture (PE Applied Biosystems) and run on the SmartCycler (Cepheid). For hepatic lipase, the following primers were used: forward, 5'-CCCTGCGGGCCCTATG-3' (SEQ ID N0:3); reverse, 5'-TTGGCATCATCAGGAGAAAGG-3' (SEQ ID N0:4); probe, 5'-FAM (6- carboxyfluorescein)-TTGAGGGAACGTCCCCCAACGA-TAMRA (N,N,N',N'-tetramethyl- 6-carboxyrhodamine)-3' (SEQ ID N0:5). For gapdh, the following primers were used: forward, 5'-AACCACGAGAAATATGACAACTCACT-3' (SEQ ID N0:6); reverse, 5'- GGCATGGACTGTGGTCATGA-3' (SEQ ID N0:7); probe, 5'-TET (tetra-chloro-6- carboxyfluorescein)-TGCATCCTGCACCACCAACTGCTTAG-TAMRA-3' (SEQ ID N0:8). The concentration of the target gene was determined using the comparative Cj (threshold cycle number at a cross-point between amplification plot and threshold) method and normalized to GAPDH.
Affymetrix microarray and analysis. RNA was isolated and subjected to the GeneChip Mouse Genome 430 2.0 Array (Affymetrix) according to the protocol at the University of Chicago Functional Genomics Facility. Cluster analysis was performed using the default software setting (dChip) (Li, 2001 #26).
Bone marrow chimeras. Mice were lethally irradiated with 1000 rads and adoptively transferred intravenously with 2-3 x 106 bone marrow cells of the indicated genotype. Bactrim was added to the drinking water for two weeks following irradiation. Four weeks after irradiation and reconstitution, mice were placed on WTD.
Cells and culture. Conditionally immortalized hepatocytes were cultured at 33° C in media with interferon-γ (R&D) and then allowed to differentiate at 39° C for two days in media without interferon-γ. Hepatocytes were stimulated with soluble murine LIGHT protein (R&D) at 50 ng/mL.
* * * * * All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
U.S. Patent 5,399,363 U.S. Patent 5,466,468 U.S. Patent 5,466,468 U.S. Patent 5,543,158 U.S. Patent 5,580,579 U.S. Patent 5,629,001 U.S. Patent 5,641,515 U.S. Patent 5,641,515 U.S. Patent 5,725,871 U.S. Patent 5,756,353 U.S. Patent 5,780,045 U.S. Patent 5,792, 451 U.S. Patent 5,804,212 U.S. Patent 6,613,308 U.S. Patent 6,669,941 U.S. Patent 6,753,514 U.S. Patent Appln. 10/865,623
Akerman et al, Am. J. Physiol, 263:G579-585, 1992.
Anand et al, J. Hn. Invest, 116:1045, 2006.
Anders et al, J. Immunol, 175:1295, 2005.
Chen et al, Immunity, 6:459-467, 1997.
Cressman et al, Science, 274:1379-1383, 1996.
De Togni et al, Science, 264:703, 1994.
Diehl, Front Biosci, 7:e301-304, 2002.
Duhen et al, Eur. J. Immunol, 34(12):3534-3541, 2004.
Elewaut et al, J. Immunol, 165:671-679, 2000.
Fausto and Campbell, Mech. Dev., 120:117-130, 2003.
Force et al, J. Immunol, 155:5280-5288, 1995. Fu and Chaplin, Annu. Rev. Immunol, 17:399-433, 1999.
Futterer et al, Immunity, 9:59-70, 1998.
Galun and Axelrod, Biochim. Biophys. Acta, 1592:345-358, 2002.
Gommerman and Browning, Nat. Rev. Immunol, 3:642-655, 2003.
Goodman and Gilman's The Pharmacological Basis Of Therapeutics, Hardman et al. (Eds.),
10th Ed., 32:853-860; 35:891-893, 2001.
Higgins and Anderson, Archives of Pathology, 12:196-202, 1931. Hwang et al, Cr it. Rev. Ther. Drug Carrier Syst., 15(3):243-284, 1998. Ito et al. , Hepatology, 38 : 1116- 1124, 2003. Kato et al, J. Hepatol, 40:285-290, 2004. Locksley et al, Cell, 104:487, 2001. Maher, Semin. Liver Dis., 21 :417-426, 2001. Malik et al, Semin. CellDev. Biol, 13:435-431, 2002. Mathiowitz et al, Nature, 386(6623):410-414, 1997. Matsui et al, J. Biol. Chem., 277:50054-50061, 2002. Mauri et al, Immunity, 8:21-30, 1998. Michalopoulos and DeFrances, Science, 276:60-66, 1997. Minagawa et al, Hepatology, 31:907-915, 2000. Ozaki et al, Nature, 429:72-85, 2004. PCT Appln. WO9413808 PCT Appln. WO9703687 Petersen et al, Science, 284:1168-1170, 1999.
Physician's Desk Reference, 56th Ed. Medical Economics Co., Inc., NJ, 1862-1866, 2002. Reardon et al, Arterioscler. Thromb. Vase. Biol, 23:1449, 2003. Reardon et al, Curr. Opin. Lipidol, 12:167, 2001.
Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580, 1990. Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, pp. 1289-1329,
1990.
Schreyer et al, J. UoI. Chem., 277:12364, 2002. Shaikh et al, J. Immunol, 167:6330, 2001. Suzuki et al, Atherosclerosis, 176:91-94, 2004. Takenaga et al, J. Control Release, 52(l-2):81-87, 1998. Tamada et al, J. Immunol, 168:4832, 2002. Tamada et al, Nat. Med., 6:283-289, 2000. The Merck Index, Eleventh Edition van Oostrom et al, Drugs, 64(2):19-41, 2004.
Vassilopoulos et al, Nature, 422:901-904, 2003.
Wang and Fu, J. Immunol, 170:3986-3993, 2003.
Wang and Paigen, Curr. Opin. Lipidol, 16:127-137, 2005.
Wang et a/., J. Clinic Invest., 108(12):1771-1780, 2001.
Wang et al, Nature, 422:897-901, 2003.
Wu et al, J. Exp. Med., 193:1327, 2001.

Claims

1. A method of treating a cardiovascular disease, treating a liver disease, promoting liver regeneration, or altering lipid levels comprising administering a LTjSR modulator to a subject.
2. The method of claim 1 , wherein the subj ect is a mammal.
3. The method of claims 1 to 2, wherein the mammal is a human.
4. The method of any of claims 1 to 3, wherein the LTβR modulator is an antibody.
5. The method of any of claims 1 to 4, wherein the antibody is humanized.
6. The method of any of claims 1 to 5, wherein the LTβR modulator is comprised in a pharmaceutically acceptable carrier or excipient.
7. The method of any of claims 1 to 6, wherein the method further comprises administering an additional pharmacological therapeutic agent or performing surgery to the subject.
8. The method of any of claims 1 to 7 , wherein the LTβR modulator is an LTβR blocker.
9. The method of any of claims 1 to 8, wherein the LTβR blocker is LTβR-Ig.
10. The method of any of claims 1 to 9, wherein the LTβR-Ig is administered at a dose of from about 0.2 mg/kg to about 2 mg/kg.
11. The method of any of claims 1 to 10, wherein the LTβR-Ig is administered once per week or twice per month.
12. The method of any of claims 1 to 8, wherein the LTβR blocker is an anti-LIGHT antibody or an anti-LT antibody.
13. The method of claim 12, wherein both an anti-LIGHT antibody and an anti-LT antibody are administered to the subject.
14. The method of any of claims 1 to 13, wherein the method further comprises administering an additional therapeutic agent or performing surgery to the subject.
15. The method of any of claims 1 to 14, further defined as a method of treating a cardiovascular disease.
16. The method of any of claims 1 to 15, wherein the cardiovascular disease is hypertriglyceridemia, hypercholesterolemia, atherosclerosis, restenosis, stenosis, thrombosis, aneurism, embolus, hypertension, stroke, critical stenosis or myocardiac infarction.
17. The method of any of claims 1 to 16, wherein the cardiovascular disease is artery atherosclerosis.
18. The method of any of claims 1 to 17, further defined as a method of altering a lipid level in the subject.
19. The method of any of claims 1 to 18, wherein cholesterol is reduced, triglycerides are reduced, LDL is reduced, VLDL is reduced, or HDL is increased in the blood of the subject.
20. The method of any of claims 1 to 19, further defined as a method of treating a liver disease or promoting liver regeneration.
21. A method of promoting liver regeneration in vitro, comprising contacting a liver cell with a LTjSR agonist or an agonistic antibody of LT/3R.
22. The method of claim 21, wherein the liver cell is comprised in liver tissue.
23. The method of any of claims 21 to 22, wherein the liver cell is used for tissue engineering.
24. The method of any of claims 21 to 23, wherein the liver cell is implanted into a subject.
25. The method of claim 24, wherein the subject is a mammal.
26. The method of either of claim 24 or 25, wherein the mammal is a human.
27. A pharmaceutical composition comprising a LT/3R modulator.
28. The pharmaceutical composition of claim 27, wherein the LT/3R modulator is an antibody.
29. The pharmaceutical composition of pharmaceutical composition either of claims 27 or 28, wherein the antibody is humanized.
30. The pharmaceutical composition of any of claims 27 to 29, further comprising an additional therapeutic agent.
31. The pharmaceutical composition of any of claims 27 to 30, wherein the additional agent is effective, either alone or in combination with the LT/3R modulator to treat or prevent a cardiovascular disease and/or a liver disease, promote liver regeneration, and/or alter a lipid level in a subject.
32. The pharmaceutical composition of any of claims 27 to 30, wherein the LT/3R modulator is an LT/3R blocker.
33. The pharmaceutical composition of any of claims 27 to 32, wherein the LT/3R blocker is LTjSR-Ig.
34. The pharmaceutical composition of any of claims 27 to 33, wherein the LT/?R-Ig is comprised at a concentration of from about 0.2 mg/kg to about 2 mg/kg.
35. The pharmaceutical composition of any of claims 27 to 34, wherein the LT/3R blocker is an anti-LIGHT antibody or an anti-LT antibody.
36. The pharmaceutical composition of any of claims 27 to 35, comprising both an anti-
LIGHT antibody and an anti-LT antibody.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010046289A2 (en) * 2008-10-22 2010-04-29 Universität Zürich Prorektorat Mnw BLOCKERS OF LIGHT, LTα1β2 AND LTα2β1 OR ITS RECEPTOR LTβR FOR THE PREVENTION AND TREATMENT OF CHRONIC HEPATITIS AND OTHER LIVER DISEASES

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020037836A1 (en) * 2000-09-18 2002-03-28 Henriksen Dennis Bang Use of GLP for the treatment, prevention, diagnosis, and prognosis of bone-related and nutrition-related disorders
US20050037003A1 (en) * 1995-07-21 2005-02-17 Browning Jeffrey L. Methods for inhibiting lymphotoxin beta receptor signalling

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050037003A1 (en) * 1995-07-21 2005-02-17 Browning Jeffrey L. Methods for inhibiting lymphotoxin beta receptor signalling
US20020037836A1 (en) * 2000-09-18 2002-03-28 Henriksen Dennis Bang Use of GLP for the treatment, prevention, diagnosis, and prognosis of bone-related and nutrition-related disorders

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AKHURST B. ET AL.: 'Differential lymphotoxin-beta and interferon gamma signaling during mouse liver regeneration induced by chronic and acute injury' HEPATOLOGY vol. 41, no. 2, 2005, pages 327 - 335, XP003013746 *
LOWES K.N. ET AL.: 'Upregulation of lymphotoxin beta expression in liver progenitor (oval) cells in chronic hepatitis C' GUT vol. 52, no. 9, September 2003, pages 1327 - 1332, XP003013747 *
MALLAT Z. ET AL.: 'Induction of a Regulatory T Cell Type 1 Response Reduces the Development of Atherosclerosis in Apolipoprotein EKnockout Mice' CIRCULATION vol. 108, August 2003, pages 1232 - 1237, XP009028370 *
SHAIKH R.B. ET AL.: 'Constitutive Expression of LIGHT on T Cells Leads to Lymphocyte Activation, Inflammation, and Issue Destruction' THE JOURNAL OF IMMUNOLOGY vol. 167, no. 11, December 2001, pages 6330 - 6337, XP003013745 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010046289A2 (en) * 2008-10-22 2010-04-29 Universität Zürich Prorektorat Mnw BLOCKERS OF LIGHT, LTα1β2 AND LTα2β1 OR ITS RECEPTOR LTβR FOR THE PREVENTION AND TREATMENT OF CHRONIC HEPATITIS AND OTHER LIVER DISEASES
WO2010046289A3 (en) * 2008-10-22 2011-02-17 Universität Zürich Prorektorat Mnw Blockers of light, ltalpha1beta2 and ltalpha2beta1 or their receptor ltbetar for the prevention and treatment of chronic hepatitis and other liver diseases

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