US20050202010A1 - Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor - Google Patents

Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor Download PDF

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US20050202010A1
US20050202010A1 US10/927,494 US92749404A US2005202010A1 US 20050202010 A1 US20050202010 A1 US 20050202010A1 US 92749404 A US92749404 A US 92749404A US 2005202010 A1 US2005202010 A1 US 2005202010A1
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mif
subject
treating
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Brett Giroir
Monte Willis
Vidal De La Cruz
Thais Sielecki-Dzurdz
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Cytokine Pharmasciences Inc
University of Texas System
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Cytokine Pharmasciences Inc
University of Texas System
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Priority to US10/927,494 priority Critical patent/US20050202010A1/en
Priority to CN2004800247776A priority patent/CN1972713B/zh
Priority to CN201110370579.7A priority patent/CN102499984B/zh
Priority to PCT/US2004/027945 priority patent/WO2005020919A2/en
Priority to CA2537928A priority patent/CA2537928C/en
Priority to PL04782427T priority patent/PL1658037T3/pl
Priority to ES04782427.1T priority patent/ES2599032T3/es
Priority to EP16170803.7A priority patent/EP3078386A3/en
Priority to JP2006524893A priority patent/JP4891769B2/ja
Priority to DK04782427.1T priority patent/DK1658037T3/en
Priority to MXPA06001828A priority patent/MXPA06001828A/es
Priority to HUE04782427A priority patent/HUE029888T2/en
Priority to PT47824271T priority patent/PT1658037T/pt
Priority to AU2004268017A priority patent/AU2004268017B2/en
Priority to EP04782427.1A priority patent/EP1658037B1/en
Priority to BRPI0413404-4A priority patent/BRPI0413404A/pt
Assigned to BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM, CYTOKINE PHARMASCIENCES, INC. reassignment BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SIELECKI-DZURDZ, THAIS M., DE LA CRUZ JR., VIDAL F, GIROIR, BRETT P., WILLIS, MONTE S.
Publication of US20050202010A1 publication Critical patent/US20050202010A1/en
Priority to MX2011013436A priority patent/MX340217B/es
Priority to US11/932,909 priority patent/US8747843B2/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF TEXAS SW MEDICAL CENTER AT DALLAS
Priority to JP2010039451A priority patent/JP5905659B2/ja
Priority to JP2011133728A priority patent/JP2011251966A/ja
Priority to US14/281,870 priority patent/US20150017179A1/en
Priority to CY20161100837T priority patent/CY1117925T1/el
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the invention generally relates to pathology and physiology in vertebrate species involving cytokines and other cellular signaling mechanisms, and also diagnostic assays involving cytokines and other cellular signaling mechanisms.
  • Other aspects of the invention relate to macrophage migration inhibitory factor (MIF) as a myocardial depressant factor and as a mediator of endotoxin-induced cardiac dysfunction in vivo.
  • MIF macrophage migration inhibitory factor
  • Other aspects of the invention relate to mediating and/or inhibiting the production or activity of MIF, and compounds, compositions, methods of treating and preventing cardiac dysfunction, sepsis, burn injury or other conditions related to burns.
  • Other aspects of the invention relate to the MIF release from the heart, liver, and spleen and the role of TNF receptor I/II signaling after LPS challenge.
  • Other aspects of the invention relate to TNF receptor I/II signaling independent release of MIF into the serum.
  • Other aspects of the invention relate to the expression of CD74 on cardiomyocyte
  • Macrophage migration inhibitory factor is a pluripotent, pro-inflammatory cytokine whose mechanisms of action have been scrutinized over the past four decades.
  • the current understanding in the art relating to MIF includes studies directed to its crystallization as a trimer, its physiologically relevant oligomerization state; its putative membrane receptor(s); and the physiologic relevance of its intracellular enzymatic activity as a tautomerase and oxidoreductase.
  • MIF has an important role in diseases as diverse as rheumatoid arthritis
  • M. Leech, et al. “Macrophage Migration Inhibitory Factor in Rheumatoid Arthritis: Evidence of Proinflammatory Function and Regulation by Glucocorticoids”, Arthritis Rheum, 42, 1601-1608 (1999)
  • M. Leech, et al. “Involvement of Macrophage Migration Inhibitory Factor in the Evolution of Rat Adjuvant Arthritis”, Arthritis Rheum., 41, 910-917 (1998), A.
  • MIF is a Pituitary-Derived Cytokine that Potentiates Lethal Endotoxaemia”, Nature, 365, 756-759 (1993), M. Bozza, et al., “Targeted Disruption of Migration Inhibitory Factor Gene Reveals Its Critical Role in Sepsis”, J. Exp. Med. 189, 341-346 (1999), T. Calandra, et al., “MIF as a Glucocorticoid-Induced Modulator of Cytokine Production”, Nature, 377, 68-71 (1995), T.
  • MIF is increased in the plasma of animals and humans, and the blockade of MIF activity by monoclonal or polyclonal antibodies results in a marked improvement in the survival of animals with experimentally induced sepsis (M. Bozza, et al., “Targeted Disruption of Migration Inhibitory Factor Gene Reveals Its Critical Role in Sepsis”, J. Exp. Med. 189, 341-346 (1999), T. Calandra, et al., “Protection from Septic Shock by Neutralization of Macrophage Migration Inhibitory Factor”, Nat. Med., 6, 164-170 (2000)).
  • Blockade of MIF activity has been demonstrated with a number of inhibitors.
  • Blockade of MIF enzymatic activity has been demonstrated with diverse chemical compounds as shown in U.S. patent application Ser. No. 10/226,299, filed Aug. 23, 2002, now pending. See also, for instance, U.S. Pat. No. 6,492,428.
  • Antibodies have also been used to blockade MIF activity as shown in U.S. Pat. No. 6,030,615.
  • MIF expression can also be inhibited using antisense technology as disclosed in U.S. patent application Ser. No. 08/738,947, filed Oct. 24, 1996, now pending, or U.S. Pat. No. 6,268,151 which further demonstrates pharmaceutical formulations that can be used with all the above-mentioned MIF inhibitors.
  • LPS Lipopolysaccharide depresses intrinsic myocardial contractility and is thought to be important in myocardial dysfunction that occurs in sepsis and septic shock
  • A. M. Lefer “Mechanisms of cardiodepression in endotoxin shock”, Circ Shock Suppl 1:1-8 (1979), J. E. Parrillo, et al., “A circulating myocardial depressant substance in humans with septic shock. Septic shock patients with a reduced ejection fraction have a circulating factor that depresses in vitro myocartdial cell performance”, J. Clin. Invest. 76:1539-1553 (1985), J. M.
  • Macrophage migration inhibitory factor is a caridac-derived myocardial depressant factor
  • S. Krishnagopalan, et al. “Myocardial dysfunction in the patient with sepsis”, Curr. Opin. Crit. Care 8:376-388 (2002)).
  • MIF macrophage migration inhibitory factor
  • Macrophage migration inhibitory factor is involved in the pathogenesis of several diseases, including sepsis. MIF opposes the anti-inflammatory effects of glucocorticoids, and also significantly alters tissue metabolism. Although MIF appears to be ubiquitously expressed, there are currently no publications indicating whether MIF is expressed in the myocardium in vivo, or whether release of MIF from the myocardium or other tissues during sepsis could adversely affect cardiac performance.
  • mice were protected when the antibodies were given as late as 8 h after the onset of infection (T. Calandra, et al., “Protection from septic shock by neutralization of macrophage migration inhibitory factor”, Nat. Med., 6, 164-170 (2000)).
  • MIF has a number of properties that make it unique among cytokines. MIF is released preformed from numerous cell types including lymphocytes, macrophages, and the anterior pituitary (J. Bernhagen, et al., “Regulation of the Immune Response by Macrophage Migration Inhibitory Factor: Biological and Structural Features”, J. Mol. Med., 76, 151-161 (1998), T. Calandra, et al., “Macrophage Migration Inhibitory Factor (MIF): A Glucocorticoid Counter-Regulator Within the Immune System”, Crit. Rev. Immunol., 17, 77-88 (1997), S. C.
  • MIF Macrophage Migration Inhibitory Factor
  • MIF has at least 2 catalytic activities that are distinct: tautomerase and oxidoreductase activity.
  • MIF-related diseases such as sepsis, acute respiratory distress syndrome (ARDS), asthma, atopic dermatitis, rheumatoid arthritis, nephropathy, and cancer
  • ARDS acute respiratory distress syndrome
  • asthma asthma
  • atopic dermatitis rheumatoid arthritis
  • nephropathy nephropathy
  • cancer nephropathy
  • MIF Macrophages expressing anti-sense MIF cDNA (leading to less endogenous MIF) secrete/express significantly less TNF- ⁇ , IL-6, and NO, while NF- ⁇ B activity is decreased in response to LPS (44). Therefore, it appears that MIF may directly interact with the LPS signaling pathway (H. Lue, et al., “Macrophage Migration Inhibitory Factor (MIF): Mechanisms of Action and Role in Disease”, Microbes Infect., 4, 449-460 (2002)).
  • MIF Macrophage Migration Inhibitory Factor
  • MIF knockout mice as demonstrated in U.S. patent application Ser. No. ______ (Attorney Docket No. 9551-095-27) filed Dec. 19, 2002, which are resistant to lethal doses of LPS, have lower circulating plasma levels of TNF- ⁇ compared to wild-type mice at baseline.
  • LPS challenge Upon LPS challenge, they demonstrate diminished circulating TNF- ⁇ concentrations, increased nitric oxide (NO) concentrations, and unchanged IL-6 and IL-12 concentrations (M. Bozza, et al., “Targeted Disruption of Migration Inhibitory Factor Gene Reveals Its Critical Role in Sepsis”, J. Exp. Med. 189, 341-346 (1999)).
  • MIF appears to promote pro-inflammatory cytokines
  • the effects of MIF have been shown to act in a TNF- ⁇ -independent manner.
  • CLP was performed in TNF- ⁇ knock out mice
  • a 60% survival rate was seen in mice administered anti-MIF antibodies compared to a 0% survival rate in wild-type mice (T. Calandra, et al., “Protection from Septic Shock by Neutralization of Macrophage Migration Inhibitory Factor”, Nat. Med., 6, 164-170 (2000)).
  • Such dysfunctions include, but are not limited to, mycarditis, endocarditis, pericarditis, rheumatic heart disease, myocardial infarction, arrythmia, fibrillation, cardiogenic shock, ischemia, hypertrophy, cardiomyopathy, angina, heart murmur or palpitation, heart attack or failure, and any of the symptoms or defects associated with congenital heart diseases generally.
  • Macrophage migration inhibitory factor is a expressed in many organs including the heart and has been linked with a delayed cardiac dysfunction in a murine model of endotoxicosis (Garner, et al., “Macrophage Migration Inhibitory Factor is A Cardiac-Derived Myocardial Depressant Factor”, Am. J. Physiol. Heart Circ. Physiol, 258, H2500-H2509 (2003)).
  • Burn injury results in cardiac injury and contractile dysfunction involving decreased cardiac output, shock, and left ventricular failure (J. T. Murphy, et al., “Evaluation of Troponin-I as An Indicator of Cardiac Dysfunction After Thermal Injury, 45, 700-704, (1998), E. M. Reynolds, et al., “Left Ventricular Failure Complicating Severe Pediatric Burn Injuries”, J. Pediatr. Surg. 30, 264-269; discussion 269-270 (1995), W. C. Shoemaker, et al., k′′Burn Pathophysiology In Man. I. Sequential Hemodynamic Alterations, J. Surg. Res., 14, 64-73 (1973), R. R.
  • MIF macrophage migration inhibitory factor
  • MIF has been shown to play a key role in ARDS (K. N. Lai, et al., “Role For Macrophage Migration Inhibitory Factor in Acute Respiratory Distress Syndrome”, J. Pathol., 199, 496-508 (2003)), a common complication of burn injury (M. Bhatia, et al., “Role of Inflammatory Mediators in the Pathophysiology of Acute Respiratory Distress Syndrome”, J. Pathol., 202, 145-156 (2004)).
  • U.S. Pat. No. 6,030,615 relates to methods and compositions for treating a disease caused by cytokine-mediated toxicity.
  • U.S. Pat. No. 6,420,188 relates to methods and compositions for antagonizing MIF activity and methods of treating various diseases based on this activity.
  • U.S. Pat. No. 6,599,938 relates to methods and compositions for antagonizing MIF activity and methods of treating various diseases based on this activity.
  • U.S. Pat. No. 6,645,493 relates to compositions and methods for inhibiting the release and/or biological activity of MIF.
  • One embodiment of the present invention relates to a pharmaceutical composition effective for at least one selected from the group including treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, and a combination thereof, which includes:
  • Another embodiment of the present invention relates to a pharmaceutical composition effective for at least one selected from the group including treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, and a combination thereof, which includes:
  • Another embodiment of the present invention relates to a pharmaceutical composition effective for at least one selected from the group including treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, and a combination thereof, which includes:
  • Another embodiment of the present invention relates to a method for treating and/or preventing cardiac dysfunction in a subject, the method including:
  • Another embodiment of the present invention relates to a method for treating and/or preventing burn injury-associated cardiac dysfunction in a subject, the method including:
  • Another embodiment of the present invention relates to a method for treating and/or preventing cardiac dysfunction in a subject, the method including:
  • Another embodiment of the present invention relates to a method for improving cardiac function in a subject following acute myocardial infarction, the method including:
  • Another embodiment of the present invention relates to a method for identifying an MIF inhibitor, the method including:
  • Another embodiment of the present invention relates to a method for treating and/or preventing cardiac dysfunction in a subject following acute myocardial infarction, the method including:
  • Another embodiment of the present invention relates to a method for treating and/or preventing cardiac dysfunction in a subject, the method including:
  • Another embodiment of the present invention relates to a method for treating and/or preventing burn injury-associated cardiac dysfunction in a subject, the method including:
  • Another embodiment of the present invention relates to a method for treating and/or preventing cardiac dysfunction in a subject, the method including:
  • Another embodiment of the present invention relates to a method for improving cardiac function in a subject following acute myocardial infarction, the method including:
  • Another embodiment of the present invention relates to a method for treating and/or preventing cardiac dysfunction in a subject following acute myocardial infarction, the method including:
  • Another embodiment of the invention relates to a method for at least one selected from the group including treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, and a combination thereof, which includes administering to said subject an effective amount of at least one selected from the group including a small molecule MIF inhibitor, salt thereof, prodrug thereof, and a combination thereof.
  • Another embodiment of the invention relates to a method for at least one selected from the group including treating or preventing cardiac dysfunction in a subject in need thereof, treating or preventing irregularity in myocardial activity in a subject in need thereof, treating or preventing depression in myocardial activity in a subject in need thereof, treating or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating or preventing cardiodepression in a subject in need thereof, and a combination thereof, which includes administering to a subject in need thereof an effective amount of at least one anti-TNFR antibody; and
  • FIG. 1 MIF protein release is detected within 12 h of LPS challenge in cardiac tissue. Each data point is the mean (+/ ⁇ standard error) of 3 independent Western blot experiments. A representative Western blot is shown below the graph. *p ⁇ 0.05
  • FIG. 2 LPS challenge does not upregulate MIF mRNA in cardiac tissue.
  • Each data point in the graph is the mean (+/ ⁇ standard error) of 3 independent Northern blot experiments. A representative Northern blot is shown below the graph. No significant differences between time points were identified (p>0.05).
  • FIG. 3 The presence of MIF in the heart, liver, and spleen before and after LPS challenge.
  • Preformed MIF in the heart, liver, and spleen decreases 12 h after LPS challenge (B, E, and H) and is replenished after 24 h (C, F, and 1) as demonstrated by immunohistochemistry.
  • FIG. 4 Cardiac function determined by Langendorff preparation post-rMIF challenge in C57BL/6J mice and endotoxin-resistant C3H/HeJ mice demonstrates rMIF mediates cardiac dysfunction in an LPS-independent mechanism. Data represents the average of 7 (C3H/HeJ) to 10 (C57BL/6J) independent Langendorff experiments per group. *p ⁇ 0.05.
  • FIG. 5 Echocardiographic assessment of the effects of LPS and LPS plus anti-MIF antibody administration on cardiac function. Representative M-mode echocardiograms in wild-type mice at baseline and 8 h after LPS administration, A and B, respectively. C and D show, respectively, representative echocardiograms in LPS plus anti-MIF treated mice at 8 and 48 h. A significant protection in cardiac function (FS %) is observed in LPS challenged mice when anti-MIF anti-bodies are given pre-treatment (E). Data represents the average of 9 cardiac cycles from 3 mice monitored at multiple time points. *p ⁇ 0.05.
  • FIG. 6 Burn model demonstrating inhibition of MIF with anti-MIF antibody following LPS Challenge.
  • the burn data demonstrates inhibition of MIF with the anti-MIF antibody and restores cardiac function following burning.
  • FIG. 7 A graphical representation demonstrating MIF release rate from the heart following thermal trauma. Macrophage migration inhibitory factor (MIF) is constitutively expressed in cardiac tissue and released maximally 8 hours post-burn injury. Each data point represents the mean density in arbitrary units (A.U.)/mm 2 ⁇ SE of 3 independent Western blot experiments. A representative Western blot is shown below the graph. A One Way ANOVA and a multiple comparison procedure using the Tukey method were employed to determine statistical significance compared to the control group (*p ⁇ 0.05).
  • MIF Macrophage migration inhibitory factor
  • FIG. 8 Immunochemistry staining slides demonstrating MIF presence in various tissue samples. Insert G-MIF, constitutively present in the heart, liver, spleen, lung, and kidney is decreased after burn injury. Preformed MIF in the heart, kidney, and spleen (A, E, 1) decreases 8 hours after burn injury (B, F, J), except for liver (M,N) and increases at 24 hours in heart and kidney (C, G), but not heart or liver (K,O) as demonstrated by immunohistochemistry.
  • a negative control secondary antibody without the primary anti-MIF antibody
  • FIG. 9 Graphical representations of concentration change of three different cytokines over time following thermal trauma.
  • FIG. 9 -Serum concentrations of MIF (ng/ml), IL-12 (pg/ml), and IL-6 (pg/ml) following burn injury (A-C, respectively).
  • Data are expressed as the mean ⁇ SE of six C57BL/6J mice as determined by ELISA and were statistically analyzed using a One Way-ANOVA with a multiple comparison procedure employing the Bonferroni method to determine significance between groups (*p ⁇ 0.05 compared to baseline).
  • FIG. 10 Graphical representation and representative Northern Blot showing MIF mRNA upregulation following thermal trauma. Burn injury upregulates MIF mRNA expression in cardiac tissue significantly by 8 hours. MIF and ⁇ -actin mRNAs were detected using 32 P radiolabeled probes complementary to MIF and ⁇ -actin mRNAs. Each data point represents the mean density in arbitrary units (A.U.)/mm 2 ⁇ SE of 3 independent Northern blot experiments. A representative Northern blot is shown below the graph. Normalized MIF was determined by multiplying the MIF density by the relative ⁇ -actin density present. A One Way-ANOVA and a multiple comparison procedure using the Tukey method were employed to determine statistical significance compared to baseline (*p ⁇ 0.05).
  • FIG. 11 Graphical representations of Coronary Flow Rates calculated three different ways comparing control measure of flow rate with untreated thermal trauma versus thermal trauma treated with Anti-MIF. Cardiac function determination by Langendorff preparation 18 hours after burn injury as a function of coronary flow (A) and Ca 2+ . Cardiac function is expressed as the mean ⁇ SE of 25 independent Langendorff experiments (11 sham, 9 burn injury, 5 burn injury after anti-MIF treatment). Separate analyses were performed for each LVP, +dP/dt max , and ⁇ dP/dt max as a function of treatment group and coronary flow rate using a Repeated Measures ANOVA and a multiple comparison procedure employing the Bonferroni method to determine significant differences between groups (*p ⁇ 0.05).
  • FIG. 12 Echocardiographic and graphical representation of the effects of anti-MIF antibody therapy after burn injury demonstrating the cardioprotective effects of MIF blockade. Echocardiographic assessment of the effects of anti-MIF antibody therapy after burn injury demonstrates the cardioprotective effects of MIF blockade. Representative M-mode echocardiograms in wild-type mice at baseline and 8 hours after burn injury, A and B, respectively. C and D depict representative echocardiograms in burn injury plus anti-MIF treated mice at 4 and 48 hours, respectively. A significant recovery of cardiac function (FS %) is observed in burn injury mice given anti-MIF anti-bodies pre-burn injury (E). Data from each group represent the mean ⁇ SE of 9 cardiac cycles from 3 mice monitored at multiple time points.
  • Cardiac function determined by echocardiography is expressed as fractional shortening % (LVED ⁇ LVES/LVED ⁇ 100) ⁇ SE and was analyzed using a One Way Repeated Measures-ANOVA and a multiple comparison procedure employing the Tukey Test to determine significant differences between specific groups.
  • FIG. 13 Serum MIF concentration (fold increase from baseline) following a 4 mg/kg endotoxin challenge in: (A) wild type mice, (B) TNFR ⁇ / ⁇ mice, and (C) wild type mice pre-treated (60 minutes) with Enbrel®. Data are expressed a fraction of the baseline levels of MIF (mean+/ ⁇ standard error) of: (A) 6 C57BL/6J mice, (B) 6 TNFR ⁇ / ⁇ mice, and (C) 3 C57BL/6J (C) pre-treated with Enbrel®. Serum levels were determined by ELISA and were statistically analyzed using a One Way-ANOVA with a multiple comparison procedure employing the Bonferroni method to determine significance between groups (*p ⁇ 0.05 compared to baseline).
  • FIG. 14 MIF protein release is not detected after LPS challenge in cardiac tissue. Each data point is the mean (+/ ⁇ standard error) of 3 independent Western blot experiments. A representative Western blot is shown below the graph. *p ⁇ 0.05
  • FIG. 15 The presence of MIF in the heart, liver, and spleen before and after LPS challenge in TNFR ⁇ / ⁇ mice.
  • Preformed MIF in the heart, liver, and spleen (A, D, G) does not decrease 12 hours after LPS challenge (B, F, J) which is seen in wild type mice as demonstrated by immunohistochemistry.
  • FIG. 16 LPS challenge does not upregulate MIF mRNA in cardiac tissue in TNFR ⁇ / ⁇ mice.
  • Each data point in the graph is the mean (+/ ⁇ standard error) of 3 independent Northern blot experiments. A representative Northern blot is shown below the graph. No significant differences between time points were identified (p>0.05).
  • FIG. 17 Cardiac function determined by Langendorff preparation post-rMIF challenge in C57BL/6J, B6/129S, and TNFR ⁇ / ⁇ mice demonstrates rMIF mediates cardiac dysfunction independent of TNF- ⁇ signaling ex vivo. Data represents the average of 6 (C57BL/6J), 4 (B6/129S), and 4 (TNFR ⁇ / ⁇ ) independent Langendorff experiments per group. *p ⁇ 0.05.
  • FIG. 18 Echocardiographic assessment of the effects of LPS and LPS plus anti-MIF antibody administration on cardiac function in TNFR ⁇ / ⁇ mice. Representative M-mode echocardiograms in wild-type mice at baseline and 4 h after LPS administration, A and B, respectively. C and D show, respectively, representative echocardiograms in LPS plus anti-MIF treated mice at 4 and 48 hours. A significant protection in cardiac function (FS %) is observed in LPS challenged mice when anti-MIF anti-bodies are given pre-treatment (E). Data represents the average of 9 cardiac cycles from 3 mice monitored at multiple time points. *p ⁇ 0.05.
  • FIG. 19 Serum cytokine determination in wild type and TNFR ⁇ / ⁇ mice after LPS challenge.
  • FIG. 20 Serum cytokine determination in wild type after LPS challenge with or without anti-MIF pre-treatment (90 minutes). Shown are the modulated cytokines: (A) IFN- ⁇ and (B) IL-10 as assayed on the Luminex platform. Data are expressed as the mean+/ ⁇ standard error of serum cytokine concentrations from 3 independent experimental mice at each time point.
  • FIG. 21 Compares cardiac function (fractional shortening) in post LAD ligation with LAD only and anti-MIF+LAD.
  • FIG. 22 Shows the effect of anti-MIF therapy pre-LAD with LAD only and anti-MIF+LAD.
  • FIG. 23 Presents cardiac function data 48 hours post-LAD for several treatment groups.
  • FIG. 24 Shows the serum troponin concentration 48 hrs post-LAD with pre- and delayed anti-MIF treatment.
  • FIG. 25 Shows the serum troponin I and MIF concentrations through two weeks post ligation.
  • FIG. 26 Shows the organ CD74 constitutive expression and heart CD74 post-LPS challenge expression.
  • FIG. 28 Shows post LPS challenge serum sCD74 release.
  • FIG. 29 Shows the CD74 series gel. See also Table 5.
  • FIG. 30 Shows the coronary flow rate v. LVP, +dP/dt max, and ⁇ dP/dt max in CD74 KO Mouse.
  • FIG. 31 Shows the hours post ligation v. fractional shortening for control, small molecule MIF inhibitor+DMSO/MI and DMSO/MI.
  • One preferred embodiment of the invention relates to a method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor.
  • MIF macrophage migration inhibitory factor
  • One preferred embodiment of the invention relates to a method of treatment and/or prevention of cardiac dysfunction associated with burn injury.
  • One preferred embodiment of the invention relates to the modulation of MIF as therapy for myocardial infarction.
  • One preferred embodiment of the invention relates to the modulation of TNF- ⁇ in cardiac dysfunction.
  • One preferred embodiment of the invention relates to a method of cardioprotection by inhibition of CD74.
  • One preferred embodiment of the invention relates to the modulation of MIF with anti-MIF antibodies.
  • One preferred embodiment of the invention relates to a bioassay for identifying agents that inhibit MIF activity.
  • One preferred embodiment of the invention relates to the inhibition of MIF with anti-MIF antibody and concomitant restoration of (post-burning) cardiac function.
  • One preferred embodiment of the invention relates to a method for the improvement of burn-injury-associated cardiac depression by the administration of anti-MIF antibodies.
  • One preferred embodiment of the invention relates to the improvement of cardiac function following acute myocardial infarction by the administration of anti-MIF antibody.
  • One preferred embodiment of the invention relates to the observation that MIF release from the heart, liver and spleen is dependent upon TNF- ⁇ receptor I/II signaling, and thus TNF- ⁇ may be a therapeutic target.
  • One preferred embodiment of the invention relates to the neutralization of TNF- ⁇ with recombinant human TNFR:Fc.
  • One preferred embodiment of the invention relates to the neutralization of MIF activity with one or more anti-MIF antibodies.
  • One preferred embodiment of the invention relates to the discovery of TNF- ⁇ as an upstream mediator of MIF.
  • CD74 an MIF receptor
  • One preferred embodiment of the invention relates to the inhibition of MIF and improving cardioprotection by inhibiting the CD74 receptor with one or more anti-CD74 monoclonal neutralizing antibodies.
  • One preferred object of the invention relates to a method for treating and/or preventing cardiac dysfunction, such as an irregularity or depression in myocardial activity.
  • the method preferably includes administering an effective amount of a composition comprising a macrophage migration inhibitory factor (MIF) inhibitor.
  • MIF macrophage migration inhibitory factor
  • the inhibitor can be an antibody.
  • the inhibitor can affect a particular MIF activity including an enzymatic activity, such as tautomerase activity or oxidoreductase activity.
  • One preferred embodiment of the invention relates to an assay for identifying agents that inhibit MIF activity.
  • the assay preferably involves both a myocyte, either in vitro or in vivo, and MIF in the presence and in the absence of an agent that may inhibit MIF activity.
  • the assay analyzes myocyte activity, for example, using such tools as immunochemistry or echocardiography, based on the presence of MIF and a potential inhibitor.
  • One preferred embodiment of the invention relates to a method of using this assay to identify an agent that inhibits MIF activity comprising placing a myocyte and MIF in the presence of an agent that may inhibit MIF activity, and determining the effect on myocyte activity.
  • the myocyte may be in vitro or in vivo and the effect may be measured utilizing immunochemistry or echocardiography.
  • MIF is an inducer of myocardial dysfunction, which is known to contribute significantly to the morbidity and mortality of sepsis in humans.
  • sepsis associated cardiac dysfunction is characterized by biventricular dilatation, decreased systolic contractility, and diminished diastolic relaxation. While not wishing to be bound by theory, it is believed that its pathogenesis is multifactorial, with systemic and myocardial derived cytokines such as tumor necrosis factor-alpha (TNF- ⁇ ) involved in inducing its onset.
  • TNF- ⁇ tumor necrosis factor-alpha
  • One embodiment of the present application is directed to identifying whether MIF or other cardiac derived proteins mediate, by paracrine or autocrine mechanisms, myocardial dysfunction in sepsis and other cardiac diseases.
  • Screening microarray analysis of cardiac gene expression in mice suggests that MIF is expressed in the heart, and is differentially regulated after lipopolysaccharide (LPS)-challenge.
  • LPS lipopolysaccharide
  • the following examples were constructed to verify whether MIF was expressed by cardiomyocytes in vivo, whether this expression was altered by endotoxin challenge and that MIF had a physiologically important effect on cardiac function.
  • Several of the examples herein demonstrate cardiac MIF expression in vivo, and determine that MIF depresses cardiac function in a sublethal endotoxin challenge in vivo.
  • MIF is constitutively expressed in the normal myocardium, and is released by cardiomyocytes following endotoxin challenge, with cardiac tissue levels reaching a nadir 12 hours after challenge.
  • Evidence supporting a delayed release is seen in the present application by western blot and immunohistochemistry demonstrating significant release at 12 h from cardiac and spleen tissue, and supported indirectly by the delayed onset of cardiac protection beginning at eight hours, and continuing thereafter.
  • Treatment of LPS challenged mice with anti-MIF monoclonal antibodies significantly improves in vivo cardiac function as evidenced by improvement in left ventricular shortening fraction.
  • Microarray data on cardiac gene expression highlights that MIF is also expressed in cardiac tissue.
  • MIF perfusion directly depresses cardiac function in vitro; and moreover, treatment with either of two independent monoclonal antibodies directed against MIF mitigates late myocardial depression.
  • Another preferred embodiment of the invention relates to a method for treating and/or preventing burn injury associated conditions, including but not limited to cardiac dysfunction, such as an irregularity or depression in myocardial activity.
  • the method preferably includes administering an effective amount of a composition comprising a macrophage migration inhibitory factor (MIF) inhibitor.
  • MIF macrophage migration inhibitory factor
  • the inhibitor can inhibit MIF activity and/or MIF production.
  • the inhibitor is preferably an antibody or protein.
  • the inhibitor can affect a particular MIF activity including an enzymatic activity, such as tautomerase activity or oxidoreductase activity.
  • the inhibitor can inhibit or block MIF activity or MIF production in myocardial tissue.
  • the inhibitor can also inhibit or prevent MIF release, such as inhibitors of the ABC transporter.
  • Another preferred embodiment of the invention relates to an assay for identifying agents that inhibit MIF activity or production.
  • the assay would involve a myocyte, either in vitro or in vivo, and possibly MIF in the presence and in the absence of an agent that inhibits MIF activity or MIF production.
  • the assay would analyze myocyte activity, using such tools as immunochemistry or echocardiography, based on the presence of MIF and a potential inhibitor.
  • One embodiment of the invention utilizes the role of MIF in burn associated cardiac dysfunction in methods of prevention/treatment and diagnostic assays.
  • a murine burn injury model (40% TBSA)
  • the present inventors identified that constitutive cardiac MIF significantly decreased (2.1 fold) 8 hours after burn injury as determined by western blot analysis. Serum MIF was maximal at 4 hours after burn injury (2.2 fold increase). These patterns are consistent with MIF release from pre-formed cytoplasmic stores of cardiac and systemic origin following burn injury.
  • mice were pre-treated with anti-MIF neutralizing monoclonal antibodies.
  • mice with burn injury alone demonstrated a depressed left ventricular fractional shortening percentage (FS %) of 38.6+/ ⁇ 1.8% (Sham FS % 56.0+/ ⁇ 2.6%).
  • Anti-MIF treated mice demonstrated a delayed improved cardiac function after burn injury, with complete recovery of function by 24 hours. This demonstrates that the cytokine MIF mediates late burn injury associated cardiac dysfunction, and also demonstrates that MIF is a pharmacologic target for the treatment of burn injury associated cardiac dysfunction as well as other MIF mediated complications such as ARDS associated with burn injury.
  • LPS LPS mediates the associated cardiac dysfunction through its interaction with the Tlr-4 (toll-like receptor 4) and its interaction with IRAK-1.
  • the source of LPS is believed to be gut-derived due to several potential insults associated with burn injury. These include intestinal ischemia, bacterial translocation, and increased intestinal permeability. While not wishing to be bound by theory, it is thought that the production and release of inflammatory factors becomes systemic through gut associated lymphoid tissues.
  • MIF mediated late cardiac depressant effects in vivo. It is also determined that recombinant MIF induces an immediate cardiac depression ex vivo by Langendorff assay in an LPS-independent manner.
  • one embodiment of the present invention suitably has several advantages compared to observations made in a model of endotoxicosis. First, MIF is released at an earlier time point in the burn model (4 hours, FIG. 7 ) compared to the LPS challenge (8 hours) and this increase in systemic MIF concentrations is significantly higher (2.2 fold increase ( FIG. 9 ) vs. 1.5 fold).
  • the degree of cardiac dysfunction was not as great in the burn injury model compared to the endotoxicosis model as measured by echocardiography.
  • the present inventors desirably allows fractional shortening percent (an estimate of cardiac output) to be decreased 38% from baseline by four hours (56.2 FS % ⁇ 34.8 FS %/56.2 FS %) compared to 53.7% in the endotoxicosis model (67.2 FS % ⁇ 31.1 FS %/67.2 FS %) at four hours.
  • MIF inhibition in accordance with one embodiment of the present invention results in complete cardiac protection by 24 hours ( FIG.
  • TNF- ⁇ , IL-1 ⁇ , IL-6, and IL-10 are secreted by cardiomyocytes and TNF- ⁇ and IL-1 ⁇ are the primary mediators of the myocardial depression.
  • the present invention contemplates the role these play in early cardiac dysfunction occurring before MIF mediated cardiac dysfunction. Since MIF is released locally from the heart just prior (8 hours) to the protective effects of anti-MIF treatment (12 hours) seen by echocardiography, the present invention contemplates that MIF plays a significant role in the cardiac dysfunction seen at later time points in this model (12-48 hours after burn injury).
  • MIF cardiac malondialdehyde
  • the cardiac release of pre-formed MIF may be initiated with increases in oxidative stress in the heart which signals the release of MIF ( FIGS. 7 and 8 ) and upregulates its transcription ( FIG. 10 ) in order to replenish the stores in cardiomyocytes.
  • MIF cytokines
  • MIF cytokines
  • cytokines have tightly controlled expression that is upregulated after stimulation.
  • MIF exists preformed in substantial amounts and its expression relies not only on de novo protein synthesis, but also from pre-existing stores which are controlled by secretory mechanisms involving ABC transporters.
  • the MIF gene does not encode for an N-terminal signal sequence whose role is to translocate it to the endoplasmic reticulum.
  • MIF is located predominantly in the cytosol in small vesicles and the nucleus which are pinched off and released to the outside of cells. Necrotic cell damage therefore leads to a release of the pre-stored MIF.
  • MIF release may be directly released from necrotic cells of the skin, since MIF has been identified in the skin and localized to the basal layers of the epidermis.
  • MIF is upregulated and plays a pivotal role in the pathophysiology of the disease.
  • Total body UV B exposure in vivo has been shown to increase MIF production, suggesting its involvement in tissue injuries.
  • Injured epidermis and cultured fibroblasts also increase the expression of MIF which contributes positively to the wound healing process.
  • Systemic levels of MIF may increase more quickly and dramatically (2.2 fold by 4 hours in the burn injury model vs. 1.5 fold increase by 8 hours in the endotoxicosis model) in this burn injury model compared to the endotoxicosis model due to factors involving MIF released from burn injured skin.
  • MIF has been shown has been hypothesized to play a role in ARDS and lung complications of sepsis.
  • Anti-MIF therapy has been shown to decrease pulmonary neutrophil accumulation in acute lung injury associated with sepsis.
  • MIF is expressed in alveolar capillary endothelium and infiltrating macrophages from ARDS patients.
  • MIF expression has been shown to form an amplifying loop with TNF- ⁇ effectively linking severe inflammation to these two cytokines in ARDS. Since ARDS is an important and common complication of burn injury, the present invention contemplates anti-MIF therapies that are useful in other than cardiac protective indications, and seriously affect outcomes.
  • MIF is unique among cytokines because it has multiple enzymatic activities including oxidoreductase and tautomerase activity. Inhibition of its tautomerase activity has been shown to counteract known MIF activities such as its glucocorticoid override activities. Pharmacological inhibitors of MIF tautomerase activity have been developed for diseases anti-MIF therapies have been effective such as sepsis, asthma, atopic dermatitis, and acute respiratory syndrome (ARDS).
  • ARDS acute respiratory syndrome
  • the cytokine MIF plays a significant role in the late cardiac dysfunction associated with burn injury.
  • MIF itself is a direct cardiac depressant and has a delayed release from the heart.
  • the delayed release of MIF and development of inhibitors that potentially inhibit the activity of MIF make MIF a potential target for diseases such as burn injury associated with morbidity and mortality related to its cardio-pulmonary effects.
  • the present inventors have found that MIF release from the heart, liver, and spleen is dependent upon TNF receptor I/II signaling after LPS challenge. Additionally, the present inventors identify TNF receptor I/II signaling independent release into the serum of MIF. Without TNF receptor signaling, MIF levels appear slightly delayed (12-24 hours compared to 8 hours in wild type) and slightly increased (1.7-2.3 fold baseline compared to 1.5 increase in wild type mice). Moreover, the TNF receptor independent MIF release in TNF receptor I/II deficient mice (TNFR ⁇ / ⁇ ) is sufficient to mediate cardiac dysfunction by at least 24 hours after LPS challenge despite the lack of MIF release from tissues which has been previously identified in wild type mice.
  • the cytokine MIF is constitutively expressed in numerous cell types including lymphocytes, macrophages, and the anterior pituitary. Many tissues also contain MIF including the heart, lung, liver, adrenal, spleen, kidney, skin, muscle, thymus, skin, and testes. The mechanism of secretion has recently been described in LPS stimulated monocytes. Inhibitors of classical protein secretion such as monensin or brefelding A do not inhibit the secretion of MIF, suggesting a non-classical protein export route. When inhibitors of ABCA1 (ATP binding cassette A1) transporters (glyburide and probenicide) were given, MIF secretion did not occur.
  • ABCA1 ATP binding cassette A1
  • MIF is located predominantly in the cytosol in small vesicles and the nucleus which are pinched off and released to the outside of cells.
  • This non-classical, vesicle-mediated secretory pathway has been shown to be a mechanism of secretion of other important inflammatory mediators such as HMGB1, which has been shown to play a significant role in inflammatory diseases and specifically sepsis.
  • HMGB1 important inflammatory mediators
  • the dependence of MIF secretion on TNF- ⁇ signaling in several tissues is described for the first time.
  • MIF has numerous biological activities including glucocorticoid antagonist properties, catalytic properties which are regulated through the coactivator JAB1/CSN5 and the cell surface protein CD74/Ii chain. Specific secretion of MIF results after inflammatory stimuli such as endotoxin (LPS) and tumor necrosis factor, as well as hormones such as ACTH, and angiotensin II. In addition to immune cells, endocrine cells and some epithelial cells secrete MIF. Secretion is due to an enhancement of MIF expression and de novo synthesis as well as an induction of the release from pre-existing stores; both of which have been previously demonstrated in the heart.
  • Cardiac MIF has been reported to be released maximally at 12 hours after LPS challenge in wild type mice. Serum MIF levels in wild type mice after LPS challenge maximally release at 8 hours, corresponding to early protection of cardiac dysfunction.
  • TNF- ⁇ signaling is inhibited after LPS challenge by either Enbrel®(D pretreatment or in TNFR ⁇ / ⁇ mice, the MIF levels peak later and slightly higher, indicating that TNF- ⁇ has some control over serum MIF levels, but does not inhibit MIF release.
  • Enbrel® D pretreatment or in TNFR ⁇ / ⁇ mice
  • TNF- ⁇ signaling has been studied by other investigators, specifically in relationship to the cytokine IL-18 in endotoxemia models.
  • IL-18 levels in the heart are not significantly changed, while wild type mice demonstrated significant increases in IL-18 levels.
  • IL-18 is neutralized, this study demonstrated that the LPS induced cardiac dysfunction is reduced and that IL-18 appears to have downstream effects on tissue TNF- ⁇ , IL-1 ⁇ , as well as ICAM-1/VCAM-1 levels. While this study focused on the myocardial production and release of IL-18 in the myocardium, non-cardiac sources of IL-18 were not investigated. This study is believed to be similar to that of the present inventors in the TNR- ⁇ dependence on tissue (cardiac) production/release of IL-18.
  • mice deficient in IL-6 have augmented expression of IL-1 ⁇ and TNF- ⁇ after LPS challenge and the present inventors contemplate that cardiac IL-6 suppresses the expression of proinflammatory mediators including itself by a negative feedback mechanism.
  • TNF- ⁇ signaling occurs through 2 receptors, TNF- ⁇ receptor 1 and 2. These two pathways have divergent signaling pathways. The interaction of TNF- ⁇ and receptor 1 activates several signal transduction pathways including NF-KB, which the TNF- ⁇ receptor does not.
  • TNF- ⁇ receptor 1 activates several signal transduction pathways including NF-KB, which the TNF- ⁇ receptor does not.
  • the present inventors challenged mice in which IkB overexpression in the heart resulted in nearly complete NF-kB inhibition. By western analysis, the present inventors demonstrated that no release occurred in the same manner as the TNFR ⁇ / ⁇ mice at all time points tested (data not shown, identical to FIG. 2A ). These mice have circulating TNF- ⁇ equivalent to wild types (since NF-kB inhibition is cardiac specific).
  • mice express MIF in the serum similar to wild type mice. Accordingly, the present invention contemplates that the TNF receptor 1 may mediate the tissue release seen in wild type mice from the heart. Since the phenotype of this heart is completely protected after LPS challenge by echo during the first 48 hours (data not shown), the present inventors contemplate that circulating MIF requires upstream NF-kB mediated proteins to be signal (TNF- ⁇ , IL-1 ⁇ ) or that MIF mediates its effects by NF-kB itself.
  • IFN- ⁇ was signficantlly suppressed.
  • the expression of iNOS in cardiac myocytes have been shown to expressed when TNF- ⁇ and LPS are given with IFN- ⁇ , but NOT without IFN- ⁇ . Since iNOS itself plays a role in the regulatory pathways of LPS associated cardiac dysfunction, these cytokine pathways are complex and likely interact closely.
  • MIF has been shown to be physiologically relevant in a model of live polymicrobial peritonitis (CLP).
  • CLP live polymicrobial peritonitis
  • MIF release is delayed and partially unaffected by blocking of important upstream mediators such as TNF- ⁇ , it may represent one good therapeutic target.
  • the inhibition of MIF's tautomerase activity that mediate some of its biological functions may be intervened pharmacologically.
  • recent non-classical secretory mechanisms of release also are potential target for therapy.
  • HMGB1 significant targets in sepsis that are known
  • undescribed targets still exist. Therefore, it is important to understand the effects of therapeutic invention of all of these mediators.
  • MIF plays a significant role in LPS induced cardiac dysfunction which is believed to contribute to myocardial dysfunction during sepsis.
  • TNF- ⁇ is thought to be an important sentinel cytokine in LPS induced cardiac dysfunction
  • the effects of blocking TNF- ⁇ signaling pathways in vivo on MIF induced cardiac dysfunction have been investigated. Serum concentrations and the temporal distribution of MIF was slightly increased and delayed by the inhibition of TNF- ⁇ signaling (maximally increased 12-24 hours (1.7-2.3 fold baseline with TNF- ⁇ signaling inhibition vs. 8 hours in wild type (1.5 fold increase).
  • Macrophage migration inhibitory factor is pluripotent cytokine with direct and significantly deleterious effects on heart function during sepsis (severe infections).
  • MIF Macrophage migration inhibitory factor
  • One embodiment of the present invention demonstrates in a mouse model that the inhibition of MIF activity can profoundly improve cardiac function following acute myocardial infarction (See FIGS. 21-25 ). This improvement is evident within hours, and lasts for the duration of the experiment (1 week). It is highly likely that modulation of MIF will decrease infarct size and other pathologic parameters. The degree of improvement in cardiac function is remarkable, and substantially in excess by at least 10 fold compared to other immune targets such as TNF-alpha.
  • One embodiment of the present invention solves the problems of acute and chronic heart dysfunction following acute myocardial infarction.
  • the present invention makes it possible to provide a unique class of therapies in that it modulates an immune mediator, i.e., MIF.
  • MIF an immune mediator
  • the present invention may directly decreases infarct size.
  • modulation of MIF activity in accordance with the present invention would minimize the need for intra-aortic balloon pumps and other mechanical devices.
  • Suitable monoclonal anti-MIF antibodies are obtained from Cytokine PharmaScience, Inc., King of Prussia, Pa.
  • Another embodiment of the present invention relates to inhibition of CD74 to protect cardiodysfunction associated with severe disease such as sepsis, trauma, acute MI, and congestive heart failure. While CD74 has been described on circulating immune cells and antigen presenting cells (in association with MHC Class II), until the present invention, the presence of CD74 in the heart (as well as other organs) has not been reported. More importantly, the function role of CD74 on cardiac function in physiological or disease processes has not previously been demonstrated. While CD74 has been shown to mediate MIF activity in vitro, this has not been confirmed independently and is restricted to fibroblasts and leukocytes.
  • Suitable anti-human CD74 antibodies are available, for example, from BD Biosciences (product catalog numbers 555538 (Clone M-B741; Format Purified; Isotype Mouse IgG 2a , ⁇ ; W.S. No V CD74.4; Reactivity Human) and 555612 (Clone LN2; Format Purified; Isotype Mouse IgG 1 , K; W.S. No V CD74.3; Reactivity Human).
  • the present invention makes it possible to use anti-cytokine therapy (including anti-MIF) in cardiac diseases. Preliminary experiments using anti-TNF therapy in sepsis models did not work. Additionally, MIF, the putative cytokine blocked by CD74 inhibition, is a cytokine that occurs later, and in our model of acute MI is increased for several weeks after the insult, allowing for intervention during any of that time.
  • cytokine therapy can enhance performance and potentially lower the morbidity associated with each.
  • MI reperfusion
  • MI and sepsis inotropes
  • both acute and chronic cardiogenic impairment may be attenuated and improve survival/outcomes.
  • MIF is secreted from cardiomyocytes following LPS challenge, and directly mediates a late onset (>6 hours) cardiac dysfunction.
  • CD74 was determined to be the MIF receptor, exerting effects via ERK1/2 intracellular signaling pathways.
  • the present inventors challenged: 1) wild type mice (C57BL/6) with LPS; 2) wild type mice pre-treated with anti-CD74 monocolonal neutralizing antibodies; and challenged with LPS, and 3) CD74 knock-out mice with LPS (4 mg/kg). Serial echocardiography was performed and fractional shortening (FS %) was determined.
  • Another embodiment of the present invention relates to the inhibition of MIF activity by use of one or more soluble MIF receptor or MIF receptor antagonist.
  • soluble MIF receptor or MIF receptor antagonist As an example, with anti-TNF ⁇ therapies, REMICADETM or INFLIXIMABTM (antibody TNF ⁇ ) and ENBRELTM or ETANERCEPTTM (soluble TNF-receptor) are suitable.
  • This method includes administering one or more of the soluble MIF receptor and/or MIF receptor antagonist in an effective amount for treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof to a subject in need thereof.
  • Another embodiment of the present invention relates to the use of small molecule MIF inhibitors (sometimes called “MIF antagonists” or “isoxazoline compounds”) in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof.
  • MIF antagonists sometimes called “MIF antagonists” or “isoxazoline compounds”
  • the use of the superscript on a substituent is to identify a substituent name (e.g., “R 2 ” is used to indicate an R 2 -named substituent), while the use of a subscript is used to enumerate the number of times a substituent occurs at that molecular site (e.g., “R 2 “or “(R) 2 ” both are used to indicate two substituents simply named as “R”).
  • a suitable small molecule MIF inhibitor for use in the methods herein has the following Formula I: wherein:
  • the compound of Formula I is a p-hydroxyphenyl-isoxazoline-containing compound, wherein each of R, R 1-4 , X and Y is H or —CH 2 -A, and Z is OR. More preferably, the compound of Formula I is an ester of (R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic, particularly the acid methyl ester thereof (sometimes identified as “ISO-1” or “CPSI” or “CPSI-26” herein) which is also known as p-hydroxyphenol-isoxazoline methyl ester.
  • the compound is an ester of 2- ⁇ 3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl ⁇ -3-phenyl-propinoic acid, particularly the methyl ester thereof (identified as “ISO-2”).
  • the present invention also relates to the pharmaceutically acceptable acid addition salts of the compounds of general Formulas I, II, or III.
  • the acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds of this invention are those which form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, glutamate, L-lactate, L-tartrate, tosylate, mesylate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1′-
  • the invention also relates to base addition salts of the small molecule MIF inhibitors.
  • the chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of those compounds of general Formulas I, II, or III that are acidic in nature are those that form non-toxic base salts with such compounds.
  • Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines.
  • the compounds and prodrugs of the small molecule MIF inhibitors can exist in several tautomeric forms, and geometric isomers and mixtures thereof. All such tautomeric forms are included within the scope of the present invention. Tautomers exist as mixtures of tautomers in solution. In solid form, usually one tautomer predominates. Even though one tautomer may be described, the present invention includes all tautomers of the small molecule MIF inhibitors.
  • the present invention also includes atropisomers of the small molecule MIF inhibitors.
  • Atropisomers refer to compounds of the small molecule MIF inhibitors that can be separated into rotationally restricted isomers.
  • the small molecule MIF inhibitors may contain olefin-like double bonds. When such bonds are present, the small molecule MIF inhibitors exist as cis and trans configurations and as mixtures thereof.
  • a “suitable substituent” is intended to mean a chemically and pharmaceutically acceptable functional group i.e., a moiety that does not negate the inhibitory activity of the small molecule MIF inhibitors. Such suitable substituents may be routinely selected by those skilled in the art.
  • substituents include, but are not limited to halo groups, perfluoroalkyl groups, perfluoroalkoxy groups, alkyl groups, alkenyl groups, alkynyl groups, hydroxy groups, oxo groups, mercapto groups, alkylthio groups, alkoxy groups, aryl or heteroaryl groups, aryloxy or heteroaryloxy groups, aralkyl or heteroaralkyl groups, aralkoxy or heteroaralkoxy groups, HO—(C ⁇ O)— groups, amino groups, alkyl- and dialkylamino groups, carbamoyl groups, alkylcarbonyl groups, alkoxycarbonyl groups, alkylaminocarbonyl groups dialkylamino carbonyl groups, arylcarbonyl groups, aryloxycarbonyl groups, alkylsulfonyl groups, arylsulfonyl groups and the like.
  • Preferred small molecule MIF inhibitors may be found in U.S. provisional application 60/556,440, filed Mar. 26, 2004, U.S. provisional application 60/296,478, filed Jun. 8, 2001; and U.S. application Ser. No. 10/164,630, filed Jun. 10, 2002, the entire contents of each of which is hereby incorporated by reference for all purposes.
  • an effective amount of one or more small molecule MIF inhibitors and/or salts thereof is administered as active ingredient to a subject in need thereof.
  • Combinations of small molecule MIF inhibitors are also possible.
  • the small molecule MIF inhibitor compounds can also be administered in form of their pharmaceutically active salts and/or prodrugs as appropriate. Combinations of salts and/or prodrugs are possible, as are combinations of salt-forms and non-salt-forms of the small molecule MIF inhibitor.
  • Another embodiment of the present invention relates to pharmaceutical compositions suitable in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof, which includes one or more small molecule MIF inhibitors and/or salts thereof as active ingredient and at least one pharmaceutically acceptable carrier, excipient, adjuvent and/or diluent.
  • Another embodiment of the present invention relates to the administration, to a subject in need thereof, of an effective amount of a composition which includes at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof and at least one anti-MIF antibody in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof.
  • a composition which includes at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof and at least one anti-MIF antibody in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardi
  • Another embodiment of the present invention relates to pharmaceutical compositions suitable in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof, which includes an effective amount of a combination of at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof and at least one anti-MIF antibody, and at least one pharmaceutically acceptable carrier, excipient, adjuvent and/or diluent.
  • Another embodiment of the present invention relates to the administration, to a subject in need thereof, of an effective amount of a composition which includes a combination of at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof, at least one anti-TNFR antibody and at least one anti-MIF antibody in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof.
  • a composition which includes a combination of at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof, at least one anti-TNFR antibody and at least one anti-MIF antibody in treating and
  • Another embodiment of the present invention relates to pharmaceutical compositions suitable in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof, which includes an effective amount of a combination of at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof, at least one anti-TNFR antibody and at least one anti-MIF antibody, and at least one pharmaceutically acceptable carrier, excipient, adjuvent and/or diluent.
  • Another embodiment of the present invention relates to the administration, to a subject in need thereof, of an effective amount of a composition which includes a combination of at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof, at least one anti-CD-74 antibody and at least one anti-MIF antibody in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof.
  • a composition which includes a combination of at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof, at least one anti-CD-74 antibody and at least one anti-MIF antibody in
  • Another embodiment of the present invention relates to pharmaceutical compositions suitable in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof, which includes an effective amount of a combination of at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof, at least one anti-CD-74 antibody and at least one anti-MIF antibody, and at least one pharmaceutically acceptable carrier, excipient, adjuvent and/or diluent.
  • Another embodiment of the present invention relates to the administration, to a subject in need thereof, of an effective amount of a composition which includes a combination of at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof and at least one anti-CD-74 antibody in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof.
  • Another embodiment of the present invention relates to pharmaceutical compositions suitable in treating and/or preventing cardiac dysfunction in a subject in need thereof, treating and/or preventing irregularity in myocardial activity in a subject in need thereof, treating and/or preventing depression in myocardial activity in a subject in need thereof, treating and/or preventing burn-injury associated cardiac dysfunction in a subject in need thereof, treating and/or preventing cardiac dysfunction following acute myocardial infarction in a subject in need thereof, treating and/or preventing cardiodepression in a subject in need thereof, or a combination thereof, which includes an effective amount of a combination of at least one small molecule MIF inhibitor, salt thereof, and/or prodrug thereof, at least one anti-CD-74 antibody, and at least one pharmaceutically acceptable carrier, excipient, adjuvent and/or diluent.
  • the present inventors have found that inhibiting MIF and/or neutralizing MIF tautomerase activity provides an anti-cytokine/inflammation therapy against cardiac diseases. Inhibition of MIF improves cardiac function after myocardial infarction and makes it possible to help with the acute sequelae of myocardial infarctions, such as reducing cardiac dysfunction early in acute myocardial infarction and reducing the associated high morbidity and mortality.
  • One advantage the present invention has over current technologies is that unlike therapeutic interventions in cardiac disease that focus on inotropes, the present invention, by treating and/or preventing the cause of the cardiac dysfunction, both acute and chronic cardiogenic impairment may be attenuated and improve survival/outcomes.
  • One embodiment of the present invention relates to pharmaceutical compositions comprising at least one compound of the present invention as an active ingredient (and/or salt and/or prodrug thereof) and at least one pharmaceutically acceptable carrier, excipient, adjuvent and/or diluent.
  • compositions of the present invention may be prepared in any conventional solid or liquid carrier or diluent and optionally any conventional pharmaceutically-made adjuvant at suitable dosage level in a known way.
  • the preferred preparations are in administrable form which is suitable for oral application. These administrable forms, for example, include pills, tablets, film tablets, coated tablets, capsules, powders and deposits.
  • the compounds of the present invention and/or pharmaceutical preparations containing said compounds may be administered by any appropriate means, including but not limited to injection (intravenous, intraperitoneal, intramuscular, subcutaneous) by absorption through epithelial or mucocutaneous linings (oral mucosa, rectal and vaginal epithelial linings, nasopharyngial mucosa, intestinal mucosa); orally, rectally, transdermally, topically, intradermally, intragastrally, intracutanly, intravaginally, intravasally, intranasally, intrabuccally, percutanly, sublingually, or any other means available within the pharmaceutical arts.
  • compositions of the present invention containing at least one compound of the present invention or pharmaceutically acceptable salts thereof as an active ingredient, will typically be administered in admixture with suitable carrier materials suitably selected with respect to the intended form of administration, i.e. oral tablets, capsules (either solid-filled, semi-solid filled or liquid filled), powders for constitution, oral gels, elixirs, dispersible granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices.
  • suitable carrier materials suitably selected with respect to the intended form of administration, i.e. oral tablets, capsules (either solid-filled, semi-solid filled or liquid filled), powders for constitution, oral gels, elixirs, dispersible granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices.
  • compositions may be comprised of from about 5 to about 95 percent by weight of the active ingredient, which range includes all values and subranges therebetween, including 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, and 95% by weight.
  • Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethyl-cellulose, polyethylene glycol and waxes.
  • lubricants there may be mentioned for use in these dosage forms, boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Disintegrants include starch, methylcellulose, guar gum and the like. Sweetening and flavoring agents and preservatives may also be included where appropriate.
  • the compounds or compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimize the therapeutic effects, i.e. antihistaminic activity and the like.
  • Suitable dosage forms for sustained release include layered tablets containing layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.
  • Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water, ethanolic, water-ethanol or water-propylene glycol solutions for parenteral injections or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.
  • Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
  • a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
  • the compounds of the present invention may also be deliverable transdermally.
  • the transdermal compositions may take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
  • capsule refers to a special container or enclosure made of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredients.
  • Hard shell capsules are typically made of blends of relatively high gel strength bone and pork skin gelatins.
  • the capsule itself may contain small amounts of dyes, opaquing agents, plasticizers and preservatives.
  • Powders for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended in water or juices.
  • Suitable diluents are substances that usually make up the major portion of the composition or dosage form.
  • Suitable diluents include sugars such as lactose, sucrose, mannitol and sorbitol, starches derived from wheat, corn rice and potato, and celluloses such as microcrystalline cellulose.
  • the amount of diluent in the composition can range from about 5 to about 95% by weight of the total composition, preferably from about 25 to about 75%, more preferably from about 30 to about 60% by weight.
  • disintegrants refers to materials added to the composition to help it break apart (disintegrate) and release the medicaments.
  • Suitable disintegrants include starches, “cold water soluble” modified starches such as sodium carboxymethyl starch, natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar, cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, microcrystalline celluloses and cross-linked microcrystalline celluloses such as sodium croscarmellose, alginates such as alginic acid and sodium alginate, clays such as bentonites, and effervescent mixtures.
  • the amount of disintegrant in the composition can range from about 2 to about 20% by weight of the composition, more preferably from about 5 to about 10% by weight.
  • Glidents are materials that prevent caking and improve the flow characteristics of granulations, so that flow is smooth and uniform.
  • Suitable glidents include silicon dioxide and talc.
  • the amount of glident in the composition can range from about 0.1% to about 5% by weight of the total composition, preferably from about 0.5 to about 2% by weight.
  • Coloring agents are excipients that provide coloration to the composition or the dosage form. Such excipients can include food grade dyes and food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide. The amount of the coloring agent can vary from about 0.1 to about 5% by weight of the composition, preferably from about 0.1 to about 1%.
  • a suitable composition comprising at least one compound of the invention may be a solution of the compound in a suitable liquid pharmaceutical carrier or any other formulation such as tablets, pills, film tablets, coated tablets, dragees, capsules, powders and deposits, gels, syrups, slurries, suspensions, emulsions, and the like.
  • treating and/or preventing refers to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which the term applies, or one or more symptoms of the disorder or condition.
  • treatment refers to the act of treating and/or preventing as the term is defined above.
  • the treated or administered subject is a human subject and more preferably a human subject in need of treatment.
  • ⁇ ективное amount or “therapeutically effective amount” means an amount sufficient to cause any observable or measurable difference and preferably improvement in a subject's condition or indication, and preferably that condition or indication sought to be treated.
  • an “acute” condition e.g. acute myocardial infarction
  • a “chronic” condition e.g., chronic congestive heart failure
  • antibody suitably includes antibody-derived fragment(s), Fab, Fab fragment(s), Fab 2 , CDR-derived regions, antibody-derived peptides, and/or single chain antibodies) as is known to those of ordinary skill in this art. Fab is preferred.
  • cardiac dysfunction may suitably include one or more indications selected from the group including cardiac dysfunction, irregularity in myocardial activity, depression in myocardial activity, burn-injury associated cardiac dysfunction, cardiac dysfunction following acute myocardial infarction, cardiodepression, and a combination thereof. These terms are understood by a physician of ordinary skill in this art.
  • a pharmaceutical composition for the treatment and prevention of cardiac dysfunction in a subject including:
  • Goat anti-hMIF IgG and rhMIF were reconstituted in PBS and 0.1% BSA in PBS respectively, aliquoted, and stored at ⁇ 20° C. until use.
  • Rabbit anti-goat IgG-HRP BioRad Corp., Hercules, Calif. stored at 4° C. until use.
  • C57BL/6J and C3H/HeJ mice were obtained at 6-10 weeks of age (Jackson Labs, Bar Harbor, Me.).
  • Adult Sprague-Dawley rats (Harlan Laboratories, Houston, Tex.) weighing 325-360 g were used in this study.
  • Commercial chow and tap water were made available ad libitum. All animal protocols were reviewed and approved by the University of Texas Southwestern Medical Center Institutional Animal Care Advisory Committee and were in compliance with the rules governing animal use as published by the NIH.
  • C57BL/6J mice were injected i.p. with 4 mg/kg E. coli 0111:B4 LPS (Sigma-Aldrich Corp., St.
  • mice were used as controls.
  • Two anti-MIF antibodies (III.D.9 and XIV.15.5, Rockland Immunochemicals, Inc., Gilbertsville, Pa.) and their isotype control (HB-49, Rockland Immunochemicals, Inc., Gilbertsville, Pa.) were injected (100 ⁇ g in 200 ⁇ l PBS) i.p. 90 m before the LPS challenge in the echocardiogram studies.
  • Whole hearts were removed and snap frozen in liquid nitrogen and stored at ⁇ 80° C. or fixed in 10% neutral-buffered formalin for 24 h and placed in 70% ethanol for immunohistochemistry.
  • the gel was transferred to PVDF membranes (NEN, Boston, Mass.) using a semi-dry transfer apparatus (Bio-Rad, Hercules, Calif.) at 15 V for 15 m.
  • Membranes were blocked with TBS/0.1% Tween-20 (TBS-T) with 0.5% nonfat dry milk for 30 in and incubated with goat anti-hMIF IgG (1:750) in TBS/0.1% Tween-20/5% nonfat milk overnight at 4° C.
  • TBS-T TBS/0.1% Tween-20
  • goat anti-hMIF IgG 1:750
  • TBS/0.1% Tween-20/5% nonfat milk overnight at 4° C.
  • the membranes were washed 3 times for 10 m in TBS-T, incubated with rabbit anti-goat IgG-HRP (1:1000) for 1 h at RT, and washed 4 times for 10 m with TBS-T.
  • the membranes were exposed to 5 ml of a mixture of luminol plus hydrogen peroxide under alkaline conditions (SuperSignal West Pico, Pierce, Rockford, Ill.) for 5 min and the resulting chemiluminescent reaction was detected by Kodak X-OMAT AR Film (Eastman Kodak Co., Rochester, N.Y.).
  • RNA Extraction was extracted with Trizol (Invitrogen, Carlsbad, Calif.) from hearts thawed on ice according to the manufacturer's protocol and quantified by spectrophotometry.
  • a MIF specific Northern probe was prepared by isolating DNA (DNeasy Tissue Kit, Qiagen, Valencia, Calif.) from the MIF plasmid (Research Genetics, Huntsville, Ala.) and subsequently cutting it with ECOR1 and NOT I restriction enzymes (Fisher Scientific, Pittsburgh, Pa.).
  • the resultant DNA was resolved on a 1.2% agarose gel, purified (GenElute Agarose Spin Columns, Supelco, Bellefonte, Pa.), labeled with 5 ⁇ l 32 P-dCTP (3000 Ci/mmol)(PerkinElmer, Boston, Mass.) using Ready-To-Go Labeling Beads (Amersham Pharmacia, Piscatany, N.J.), and purified in ProbeQuant Microcolumns (Amersham Pharmacia, Piscatany, N.J.) according to manufacturer's protocols.
  • the membranes were washed twice for 30 m in 2 ⁇ SSC/0.1% SDS at 46° C., and washed twice for 30 m in 0.2 ⁇ SSC/0.1% SDS at 46° C., and detected by Kodak X-OMAT AR Film (Eastman Kodak Co., Rochester, N.Y.). The same membranes were then probed with radiolabeled ⁇ -actin to ensure equal loading of protein.
  • Tissue was fixed in neutral buffered formalin and processed to paraffin and subsequently immunostained at RT on a BioTek Solutions TechmateTM 1000 automated immunostainer (Ventana Medical Systems, Arlington, Ariz.) using the Ultra-streptavidin biotin system with horseradish peroxidase and diaminobenzidine (DAB) chromogen (Signet Laboratories, Dedham, M A).
  • Optimum primary antibody dilutions were predetermined using known positive control tissues (rat post-LPS challenge). Paraffin sections were cut at 3 ⁇ m on a rotary microtome, mounted on positively charged glass slides (POP100 capillary gap slides, Ventana Medical Systems, Arlington, Ariz.) and air-dried overnight.
  • Sections were then deparaffinized in xylene and ethanol, quenched with fresh 3% hydrogen peroxide for 10 m to inhibit endogenous tissue peroxidase activity, and rinsed with deionized water. Sections were incubated in unlabeled blocking serum for 15 m to block nonspecific binding of the secondary antibody and then incubated for 25 m with either rabbit anti-MIF (1:400, Torrey Pines BioLabs, Inc., Houston, Tex.) diluted in 1% citrate buffer (BioPath, Oklahoma City, Okla.), or with buffer alone as a negative reagent control.
  • the aorta was cannulated with PE50 tubing, the heart perfused in a retrograde manner through the aortic root with prefiltered, oxygenated Krebs-Hanseleit Buffer at a constant flow rate of 1.5 ml/m (T 37° C.) and a recirculating volume of 100 ml.
  • the heart was placed in a water-jacketed chamber to maintain constant temperature and humidity.
  • PE60 intratnedic polyethylene tubing was connected to a Statham pressure transducer inserted into the left ventricle (LV) to measure LV pressure. Temperature was monitored using a 27 G thertnistor needle inserted into the LV muscle.
  • LV pressure and its first derivative (dP/dt), heart rate, and coronary perfusion were measured simultaneously with a multichannel Grass 7D polygraph (Grass Instruments, Quincy, M A). Cardiac function for all hearts was determined by plotting peak systolic LV pressure and ⁇ dP/dt,Ia, values against changes in coronary flow rate. Hearts were perfused with or without 20 ng/ml RMIF added to the perfusate.
  • Echocardiograms to assess systolic function were performed using M-mode measurements. Mice were anesthestized with 5% isofluorane with 2.5 L/m O 2 for 20 seconds (until unconscience) followed by 2% isofluorane and O 2 for an average of 12-15 m. Hair was removed from the thorax and upper abdomen using Nair® hair remover after sitting for 3 m using gauze. Echocardiography measurements were obtained on anesthetized mice approximately 5-8 m after induction to allow any transient cardiac depression to pass. These transient, minimal changes in cardiac function detected by echocardiography have been reported using inhaled isofluorane, although FS (%) has been reported to be stable.
  • Cardiac echocardiography was performed using a Hewlett-Packard Sonos 5500 (Agilent Technologies; Edmonton, Alberta, Canada) with a frame rate of 300-500 frames/s in a random and blinded manner.
  • a 12 MHz linear transducer was placed on the left hemithorax interfaced with a layer of US transmission gel (Aquasonic 100, Parker Laboratories; Fairfield, N.J.).
  • the two dimensional parastemal short-axis imaging plane guided LV M-mode tracings close to the papillary muscle level. Depth was set at a minimum of 2 cm with a sweep speed of 150 m/s. Tracings were printed on a Sony color printer (UP-5200, Sony).
  • End diastole was defined as the maximal LV diastolic dimension
  • end systole was defined as the peak of posterior wall motion.
  • Systolic function was calculated from LV dimensions as fractional shortening (FS) as follows: FS (%): LVED ⁇ LVES/LVED ⁇ 100, as shown in FIG. 5A .
  • Northern and Western data are expressed as mean ⁇ standard error and statistically analyzed using a One Way Analysis of Variance. Determination of significance between experimental and control groups was performed using the Tukey method (p ⁇ 0.05). Cardiac function determined by the Langendorff preparation is expressed as mean ⁇ standard error and separate analyses were performed for each of LVP, +dP/dt max , and ⁇ dP/dt max , as a function of treatment group and coronary flow rate using a Repeated Measures Analysis of Variance. A multiple comparison procedure employing the Bonferroni method was used to determine significant differences between groups (p ⁇ 0.05).
  • Cardiac function determined by echocardiogram is expressed by fractional shortening % (LVED ⁇ LVES/LVEP ⁇ 100.) ⁇ standard deviation and analyzed using a One Way Repeated Measures Analysis of Variance. Additional comparisons were performed using the Tukey Test to determine significant differences between specific groups (p ⁇ 0.05). All statistical analyses were performed using SigmaStat 2.03 (SPSS Inc., Chicago, Ill.) and Microsoft Excel (Microsoft Corp., Seattle, Wash.).
  • MIF protein is constituitively expressed by cardiac myocytes in vivo and is released in response to LPS Challenge.
  • Both immunochemistry and Western analysis performed on cardiac tissue documented the presence of MIF in cardiac cells, including ventricular and atrial myocytes, under baseline control conditions ( FIGS. 1 and 2 ).
  • both immunochemistry and immunoblot analysis document a significant decrease in cardiac tissue MIF following endotoxin. This decrease was most profound (75% decrease) at 12 h, but returned to near baseline control levels by 24 h.
  • This expression pattern in the heart is similar to that witnessed in the liver and spleen ( FIG. 2 ), and consistent with the hypothesis that MIF is released from preformed stores within tissue following LPS challenge.
  • RNA obtained from the hearts of either control mice or from LPS challenged mice at given time points indicates that MIF mRNA is constitutively expressed in control mice, and that after LPS challenge, no significant change in MIF mRNA concentration is detectable in whole heart preparations ( FIG. 3 ).
  • MIF induces systolic and diastolic cardiac dysfunction.
  • rMIF recombinant MIF
  • Responses to MIF were determined in hearts from both C57BL/6J mice, and C3H/HeJ mice.
  • C3H/HeJ mice are resistant to endotoxin (41-43), therefore controlling for the possibility that any depression observed might be due to trace endotoxin in the perfusate.
  • Table 2 illustrates the responses of both mouse strains to retrograde aortic perfusion at 1.5 ml/m with control perfusate or perfusate containing 20 ng/ml recombinant MIF.
  • Perfusion with MIF led to a significant decrease in LVP, +dP/dt max , and ⁇ dP/dt max in both mouse strains.
  • FIG. 4 illustrates the effect of MIF over a range of coronary flow rates. There is a step-wise increase in contractile performance in all hearts regardless of experimental group assignment. Comparison of the MIF exposed hearts with control hearts revealed a downward shift in the function curves, indicating significant systolic and diastolic depression in response to 20 ng/ml rMIF (p ⁇ 0.05).
  • Anti-MIF antibodies improve LPS-induced cardiac depression in vivo.
  • serial echocardiography M-mode was performed on LPS challenged mice which had been pre-treated (90 minutes prior) with either anti-MIF monoclonal antibodies, an isotype control antibody, or no treatment ( FIG. 5 ).
  • FS fractional shortening %
  • mice injected with either anti-MIF monoclonal antibody demonstrated statistically significant recovery of FS % compared to LPS challenged groups receiving either no treatment or isotype antibody control ( FIG.
  • Antibodies and cytokines A polyclonal rabbit anti-rat MIF IgG (Torrey Pines BioLabs, Inc., Houston, Tex.) was used for western immunoblot and immunohistochemistry. This antibody has previously been shown to cross react with murine MIF and was prepared as previously described (23).
  • a polyclonal goat anti-rabbit IgG-HRP BioRad Corp., Hercules, Calif. was used as a secondary antibody for western immunoblots and was stored at 4° C.
  • MIF IgG1 antibodies Two monoclonal mouse anti-mouse (and human) MIF IgG1 antibodies (XIV.15.5 and III.D.9, gift of Cytokine PharmaSciences, Inc.) and a monoclonal mouse IgG1 isotype control antibody (HB-49, gift from Cytokine PharmaSciences, Inc.) were used in the echocardiographic studies. In vivo neutralization of MIF activity by both the XIV.15.5 and III.D.9 clones have been previously demonstrated.
  • mice Male C57BL/6J mice ages 6-10 weeks (Jackson Labs, Bar Harbor, Me.) were maintained in a specific pathogen free environment. Commercial chow and tap water were made available ad libitum. All animal protocols were reviewed and approved by the University of Texas Southwestern Medical Center Institutional Animal Care Advisory Committee and were in compliance with the rules governing animal use as published by the NIH. Mice were subjected to a 40% TBSA burn injury. Briefly, mice were anesthetized with isoflourane (1-2%) with 2.5 L/minute oxygen to effect. Hair was then removed from their back and sides using a surgical prep blade and 70% ethanol. Brass probes heated to 100° C.
  • mice received anesthesia and were shaven but not given the burn injury.
  • Intraperitoneal injection of Lactated Ringer's with Buprenex (2 cc LR+0.2 cc Buprenex ( 0.05 mg/kg)) was given after the burn injury after the anesthesia was removed (with oxygen continued).
  • Mice were then placed in individual cages under a heat lamp for approximately 1 hour and on a heating pad for the duration of the study and monitored closely. Mice were sacrificed at time points indicated in the figures by CO 2 asphyxiation followed by cervical dislocation.
  • Monoclonal anti-MIF antibodies (III.D.9 and XIV.15.5) or an isotypic control (HB-49) were injected (100 ⁇ g in 200 ⁇ l PBS) intraperitoneally 90 minutes prior to burn injury in the echocardiogram studies.
  • Whole hearts were removed, snap frozen in liquid nitrogen, and stored at ⁇ 80° C.
  • hearts were fixed in 10% neutral-buffered formalin for 24 hours and were then placed in 70% ethanol until they were processed for immunohistochemistry.
  • Whole blood was collected by retro-orbital bleeding and serum collected and stored.
  • Prestained SDS-PAGE standards were run with each gel in order to determine the approximate M.W. of detected bands.
  • the gel was transferred to a PVDF membrane (NEN, Boston, Mass.) using a mini transblot transfer apparatus (Bio-Rad, Hercules, Calif.) at 100 V for 70 minutes and cooled with ice packs.
  • the membrane was re-wet with methanol, washed a minimum of 3 times with 100+ ml water, and blocked (5% nonfat dry milk (Bio-Rad)/TBS/0.1% Tween-20 (TBS-T) overnight at 4° C.
  • the membrane was then incubated with the primary rabbit anti-MIF (1:1250 dilution) for 2 hours at room temperature in 5% milk/TBS-T and washed once for 15 minutes in TBS-T, followed by five washes (5 minutes each) in TBS-T. It was then incubated for 1 hour with a HRP conjugated goat anti-rabbit antibody in TBS-T (1:5000) at room temperature, washed twice for 15 minutes, followed by five additional washes (5 minutes each) in TBS-T.
  • ECL reagent SuperSignal West Pico, Pierce, Rockford, Ill.
  • chemiluminescent reaction was detected by Kodak X-OMAT AR Film (Eastman Kodak Co., Rochester, N.Y.).
  • the quantification of the single band density with the approximate molecular weight of MIF (12.5 kD) was determined using Quantity One software (Bio-Rad, Hercules, Calif., Ver. 4.4.0, Build 36) following conversion of radiographic film to TIFF files (8 bit grayscale) using a Scanjet 3400c (Hewlett Packard, Palo Alto, Calif.) and reported in arbitrary units (A.U.)/mm 2 .
  • Tissue was fixed in neutral buffered formalin, processed to paraffin, and subsequently immunostained at room temperature on a BioTek Solutions TechmateTM1000 automated immunostainer (Ventana Medical Systems, Arlington, Ariz.) using the Ultra-streptavidin biotin system with horseradish peroxidase and diaminobenzidine (DAB) chromogen (Signet Laboratories, Dedham, Mass.).
  • Optimum primary antibody concentrations were predetermined using known positive control tissues (LPS challenged rat). Paraffin sections were cut at 3 ⁇ m on a rotary microtome, mounted on positively charged glass slides (POP100 capillary gap slides, Ventana Medical Systems, Arlington, Ariz.), and air-dried overnight.
  • Sections were then deparaffinized in xylene and ethanol, quenched with fresh 3% hydrogen peroxide for 10 minutes to inhibit endogenous tissue peroxidase activity, and rinsed with de-ionized water. Sections were incubated in unlabeled blocking serum for 15 minutes to block nonspecific binding of the secondary antibody and then incubated for 25 minutes with either the polyclonal rabbit anti-rat MIF IgG (1:400, Torrey Pines BioLabs, Inc., Houston, Tex.) diluted in 1% citrate buffer (BioPath, Oklahoma City, Okla.) or with buffer alone as a negative reagent control. A negative reagent control was run for each time point and for each organ.
  • TMB 3, 3′, 5, 5′-tetramethylbenzidene
  • Plasma inflammatory cytokine (IL-1 ⁇ , IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN- ⁇ , TNF- ⁇ , and GM-CSF) concentrations were determined using the Mouse Cytokine Ten-Plex Antibody Bead Kit (Biosource International, Inc., Camarillo, Calif.) on a Luminex xMAP” system (Luminex Corp., Austin, Tex.) according to the manufacturer's instructions. The plate was loaded onto the Luminex XYPTM platform, the instrument set to remove 50 ⁇ l, and the total event set to equal the 100 per bead set.
  • At least 100 events (most >200) for each cytokine were collected in each sample in order to determine statistically significant results.
  • Data was collected using the LuminexTM Data Collector Software (Luminex Corp., Austin, Tex.). The concentrations of the lot specific reconstituted standards used in each run were entered into the software and the analyte concentrations for unknown samples were then extrapolated from the cytokine specific standard curve using MasterPlexTM QT software (Version 1.2.8.58, Mirai Bio, Inc., Alameda, Calif.). Final concentrations were multiplied by 2 in order to account for the initial dilution factor. No samples were detected that were higher than the standards curves for any cytokine.
  • RNA isolation was prepared from an MIF containing plasmid (Image Clone I.D. 634910, Research Genetics, Huntsville, Ala.) isolated using Genelute HP Plasmid MidiPrep kit (Sigma, St. Louis, Mo.).
  • the fragment was prepared by an EcoR1 and Not1 digestion (Fisher Scientific, Pittsburgh, Pa.) and gel purified and isolated on a 1.2% agarose gel using GenElute Agarose Spin Columns (Supelco, Bellefonte, Pa.).
  • GenElute Agarose Spin Columns GenElute Agarose Spin Columns (Supelco, Bellefonte, Pa.).
  • the ⁇ -actin probe DNA fragment was purchased from Ambion (Austin, Tex.).
  • Both MIF and ⁇ -actin probes were labeled with 5 ⁇ l [ ⁇ - 32 P]dATP (3000 Ci/mmol, 10 mCi/ml) (PerkinElmer, Boston, Mass.) using Strip-EZTM DNA probe synthesis kit (Ambion, Austin, Tex.) and purified in ProbeQuant Microcolumns (Amersham Pharmacia, Piscatany, N.J.) according to manufacturers' protocols.
  • RNA (10 ⁇ g) was resolved on 1.2% agarose gels at 100 volts for 1 hour and transferred to a Hybond-N+ membrane (Amersham Pharmacia, Buckingham, England) at 1.5 amps for 70 minutes on a transfer electrophoresis unit (TransPhor PowerLid, Hoefer Scientific Instruments, San Francisco). RNA was linked to the membrane for approximately 2 minutes using a GS Gene Linker (Bio-Rad, Hercules, Calif.). The membrane was prehybridized in a hybridization oven (Sorvall Life Science, Inc., Greensboro, N.C.) in Perfect-Hyb Plus (Sigma, St. Louis, Mo.) for 1 hour at 68° C.
  • Sheared, denatured salmon or herring testis DNA (100 ⁇ g/ml) was then added for 1 hour, followed by the addition of approximately 0.1 ⁇ g probe labeled at >5 ⁇ 10 8 cpm/ ⁇ g.
  • the blot was then hybridized for 12 hours at 68° C. in the hybridization oven followed by washing at 68° C. in 2 ⁇ SSC, 0.1% SDS.
  • the membrane was washed for 1 hour, the buffer was exchanged, and then the membrane was washed for an additional hour at 68° C.
  • the membrane was wrapped in Saran wrap, and mRNA was detected by Kodak X-OMAT AR Film after 24 hours (Eastman Kodak Co., Rochester, N.Y.).
  • the aorta was cannulated with PE50 tubing, the heart perfused in a retrograde manner through the aortic root with pre-filtered, oxygenated Krebs-Henseleit Buffer at a constant flow rate of 1.5 ml/minute (T 37° C., 100 ml recirculating volume).
  • the heart was placed in a water-jacketed chamber to maintain constant temperature and humidity.
  • LV pressure and its first derivative (dP/dt), heart rate, and coronary perfusion were measured simultaneously with a multi-channel Grass 7D polygraph (Grass Instruments, Quincy, Mass.). Ventricular performance as a function of coronary perfusion was determined for all hearts by plotting peak systolic LV pressure and ⁇ dP/dt max values against incremental increases in coronary flow rate.
  • Echocardiograms to assess systolic function were performed using M-mode measurements. Mice were anesthestized with 5% isofluorane with 2.5 L/m O 2 for 20 seconds (until unconscience) followed by 2% isofluorane and O 2 for an average of 12-15 minutes. Hair was removed from the thorax and upper abdomen using Nair® hair remover and gauze after sitting for 3 minutes. Echocardiographic measurements were obtained on anesthetized mice approximately 5-8 minutes after induction. Echocardiography was performed using a Hewlett-Packard Sonos 5500 (Agilent Technologies; Edmonton, Alberta, Canada) with a frame rate of 300-500 frames/second in a random and blinded manner.
  • a 12 MHz linear transducer was placed on the left hemithorax interfaced with a layer of US transmission gel (Aquasonic 100, Parker Laboratories; Fairfield, N.J.).
  • Northern and Western data are expressed as mean ⁇ standard error (SE) and statistically analyzed using a One Way-Analysis of Variance (ANOVA).
  • SE mean ⁇ standard error
  • ANOVA One Way-Analysis of Variance
  • a multiple comparison procedure was employed using the Tukey method to determine statistical significance between groups.
  • Cardiac function determined by the Langendorff preparation (including stabilization data) is expressed as the mean ⁇ SE and separate analyses were performed for each LVP, +dP/dt max , and ⁇ dP/dt max as a function of treatment group and coronary flow rate using a Repeated Measures-ANOVA.
  • a multiple comparison procedure employing the Bonferroni method was used to determine significant differences between groups.
  • Serum MIF levels are expressed as the mean ⁇ SE and were statistically analyzed using a One Way-ANOVA, with a multiple comparison procedure employing the Bonferroni method to determine significance between groups. Cardiac function determined by M-mode echocardiography is expressed as fractional shortening % ⁇ SE and analyzed using a One Way Repeated Measures-ANOVA. Additional comparisons were performed using the Tukey Test to determine significant differences between specific groups. Statistical significance for all analyses was defined as p ⁇ 0.05. All statistical analyses were performed using SigmaStat 2.03 (SPSS Inc., Chicago, Ill.) and Microsoft Excel (Microsoft Corp., Seattle, Wash.).
  • MIF protein is constitutively expressed by cardiac myocytes in vivo and is released in response to burn injury.
  • the cytokine macrophage migration inhibitory factor (MIF) is present in both ventricular and atrial myocytes at baseline as demonstrated by western and immunohistochemistry ( FIGS. 7 and 8 ).
  • a significant decrease (2.1 fold) was identified at 8 hours with tissue concentrations of MIF returning to baseline levels by 12 hours ( FIG. 7 ).
  • This expression pattern was paralleled in liver, spleen, and the lung after burn injury ( FIG. 8 ) and is consistent with the hypothesis that MIF is released in response mediators of burn injury.
  • Systemic MIF and IL-6 levels are increased after burn injury.
  • Maximum systemic release of MIF (2.2 fold increase) was identified in serum at 4 hours and returned to baseline levels by 8 hours ( FIG. 9 ).
  • Maximum serum IL-6 levels were identified at 12 hours which returned to baseline levels by 48 hours.
  • Serum IL-12 levels decreased after burn injury and were minimum at 24 hours and returned to baseline by 48 hours. No other cytokines tested (as listed in the materials and methods) were detected in the serum.
  • MIF mRNA in the heart significantly increases in the heart by 8 hours after burn injury.
  • the levels of MIF mRNA were detected by Northern analysis from total RNA isolated from hearts of either sham mice or mice at 4, 8, 12, 24, and 48 hours following burn injury ( FIG. 10 ).
  • MIF mRNA is constitutively expressed in the heart, and significant increases in transcription initially occur at 8 hours, which are upregulated for the rest of the time course examined (48 hours) ( FIG. 10 ).
  • Anti-MIF antibodies improve LPS-induced cardiac depression ex vivo.
  • the responses of hearts to retrograde aortic perfusion at 1.5 ml/minute from mice undergoing the sham operation, burn injury, or burn injury with pre-treatment of anti-MIF antibodies were determined using a Langendorff analysis of heart function. Significant decreases in LVP, +dP/dt max , ⁇ dP/dT max , DR, dP40, TPP, RT90 and Time to Max ⁇ dP/dt were identified in mice 18 hours after undergoing burn injury (TABLE 3). Mice pre-treated with anti-MIF (Clone III.D.9) undergoing burn injury were completely protected by 18 hours (Table 3), while mice treated with the isotype control did not differ significantly from burn injury alone (data not shown).
  • FIG. 11 illustrates the function of hearts over a range of coronary flow rates from sham mice, burn injury mice, and burn injury mice pre-treated with anti-MIF antibodies 18 hours after the burn injury or sham procedure.
  • Increases in coronary flow resulted in incremental increases in contractile performance in all hearts (groups) tested.
  • Mice undergoing burn injury demonstrated a downward shift in the LVP, +dP/dt max , and ⁇ dP/dT max function curves demonstrating significant systolic and diastolic dysfunction ( FIG. 11 ). This dysfunction, however, was not present when anti-MIF antibodies were given where no significant differences to sham mice were identified ( FIG. 11 ).
  • Anti-MIF monoclonal antibody therapy improves burn injury associated cardiac depression in vivo.
  • Serial echocardiography was (M-mode) was performed on mice receiving burn injury, and mice pre-treated 90 minutes prior before burn injury with either of two anti-MIF antibodies, an isotype control, or no treatment ( FIG. 12 ).
  • FS % fractional shortening percentage of all burn injury treated mice were similarly depressed 21.4 FS (56.2 FS %-34.8 FS %), irrespective of anti-MIF treatment.
  • mice injected with either of the two monoclonal anti-MIF antibodies demonstrated statistically significant recovery of FS % compared to burn injury mice receiving either no treatment of an isotype antibody control ( FIG. 12 ).
  • mice receiving isotype control antibodies did not demonstrate significant differences from animals undergoing burn injury alone, indicating specificity of the anti-MIF antibody effects.
  • Antibodies and cytokines A polyclonal rabbit anti-rat MIF IgG (Torrey Pines BioLabs, Inc., Houston, Tex.) which cross reacts with murine MIF was used for western immunoblot and immunohistochemistry. A polyclonal goat anti-rabbit IgG-HRP (BioRad Corp., Hercules, Calif.) was used as a secondary antibody for western immunoblots.
  • mice were injected intraperitoneally with 4 mg/kg E. coli 0111:B4 LPS (Sigma-Aldrich Corp., St. Louis, Mo.) and sacrificed by CO 2 asphyxiation and subsequent cervical dislocation. Uninjected mice were used as controls.
  • the two anti-MIF antibodies (III.D.9 and XIV.15.5, gift of Cytokine PharmaSciences, Inc., King of Prussia, Pa.) and their isotype control (HB-49, gift from Cytokine PharmaSciences, Inc.) were injected (100 ⁇ g in 200 ⁇ g PBS) intraperitoneally 90 minutes before the LPS challenge in the echocardiogram studies.
  • Enbrel® (rhTNFR:Fc) was injected intraperitoneally (5 mg/kg or 300 ⁇ g in 0.5 ml PBS) 75 minutes prior to LPS challenge in wild type mice.
  • the stop reagent was added to each well, gently mixed, and the ELISA was read on an ELISA plate reader (EL 312e Microplate Reader, Bio-Tek Instruments, Winooski, Vt.) at 450 nm (630 nm background) within 30 minutes of completion of the assay.
  • ELISA plate reader EL 312e Microplate Reader, Bio-Tek Instruments, Winooski, Vt.
  • Prestained SDS-PAGE standards (Kaleidoscope Broad range, Bio-Rad Laboratories, Inc., Hercules, Calif.) were run (10 ⁇ l/lane) with each gel in order to determine the approximate M.W. of detected bands.
  • the gel was transferred to a PVDF membrane (NEN, Boston, Mass.) using the Mini Transblot® electrophoretic transfer cell (Bio-Rad, Hercules, Calif.) at 100 V for 70 minutes and cooled with ice packs.
  • the membrane was re-wet with methanol, washed a minimum of 3 times with 100 ml water, and placed in block (5% nonfat dry milk (Bio-Rad)/TBS/0.1% Tween-20 (TBS-T) overnight at 4° C.
  • the membrane was then incubated with the primary rabbit anti-MIF (1:1250 dilution) for 2 hours at room temperature in 5% milk/TBS-T.
  • the membrane was washed 1 time for 15 minutes in TBS-T, followed by five washes (5 minutes each).
  • the membrane was then incubated for 1 hour with a HRP conjugated goat anti-rabbit antibody (diluted 1:5000) in TBS-T at room temperature.
  • the membrane was then washed twice for 15 minutes, followed by five additional washes (5 minutes each).
  • the membrane was then developed for 5 minutes with 5 ml of ECL reagents (SuperSignal West Pico, Pierce, Rockford, Ill.), and the resulting chemiluminescent reaction was detected by Kodak X-OMAT AR Film (Eastman Kodak Co., Rochester, N.Y.).
  • Quantification of the single band density with the approximate molecular weight of MIF (12.5 kD) was determined using Quantity One software (Bio-Rad, Hercules, Calif., Ver. 4.4.0, Build 36) following conversion of radiographic film to TIFF files (8 bit grayscale) using a Scanjet 3400c (Hewlett Packard, Palo Alto, Calif.) and reported in arbitrary units (A.U.)/mm 2 .
  • Tissue was fixed in neutral buffered formalin and processed to paraffin and subsequently immunostained at room temperature on a BioTek Solutions TechmateTM1000 automated immunostainer (Ventana Medical Systems, Arlington, Ariz.) using the Ultra-streptavidin biotin system with horseradish peroxidase and diaminobenzidine (DAB) chromogen (Signet Laboratories, Dedham, Mass.). Paraffin sections were cut at 3 ⁇ m on a rotary microtome, mounted on positively charged glass slides (POP100 capillary gap slides, Ventana Medical Systems, Arlington, Ariz.) and air-dried overnight.
  • DAB diaminobenzidine
  • Sections were then deparaffinized in xylene and ethanol, quenched with fresh 3% hydrogen peroxide for 10 minutes to inhibit endogenous tissue peroxidase activity, and rinsed with deionized water. Sections were incubated in unlabeled blocking serum for 15 minutes to block nonspecific binding of the secondary antibody and then incubated for 25 minutes with either rabbit anti-MIF (1:400, Torrey Pines BioLabs, Inc., Houston, Tex.) diluted in 1% citrate buffer (BioPath, Oklahoma City, Okla.), or with buffer alone as a negative reagent control. Following washes in buffer, sections were incubated for 25 minutes with a biotinylated polyvalent secondary antibody solution (containing goat anti-rabbit antibodies).
  • RNA isolation was prepared from an MIF containing plasmid (Image Clone I.D. 634910, Research Genetics, Huntsville, Ala.) isolated using Genelute HP Plasmid MidiPrep kit (Sigma, St. Louis, MO).
  • the fragment was prepared by an EcoR1 and Not1 digestion (Fisher Scientific, Pittsburgh, Pa.) and gel purified and isolated on a 1.2% agarose gel using GenElute Agarose Spin Columns (Supelco, Bellefonte, Pa.).
  • GenElute Agarose Spin Columns GenElute Agarose Spin Columns (Supelco, Bellefonte, Pa.).
  • the ⁇ -actin probe DNA fragment was purchased from Ambion (Austin, Tex.).
  • RNA Isolated total RNA (10 ⁇ g) was combined with formaldehyde loading dye (Ambion, Inc.) at a ratio of 1:3 sample:loading dye according to the manufacturer's protocols. Each gel had a 0.24-9.5 kB RNA ladder (Invitrogen Corp.) ran in parallel with samples (10 ⁇ g). Samples and RNA ladder were placed at 65° C.
  • the membrane was prehybridized in a hybridization oven (Sorvall Life Science, Inc., Greensboro, N.C.) in Perfect-Hyb Plus (Sigma, St. Louis, Mo.) with sheared, denatured salmon sperm DNA (100 ⁇ g/ml) for 1 hour at 68° C.
  • the probes were prepared by heating to 90° C. for 10 minutes (10 ⁇ l probe with 100 ⁇ l 10 mM EDTA), followed by the addition of approximately 0.1 ⁇ g probe labeled at >5 ⁇ 10 8 cpm/ ⁇ g to the hybridization buffer.
  • the blot was then hybridized for 12 hours at 68° C. followed by washing at 68° C. in 2 ⁇ SSC, 0.1% SDS.
  • the membrane was washed for 1 hour, the buffer was exchanged, and then the membrane was washed for an additional hour at 68° C.
  • the membrane was wrapped in Saran wrap, and mRNA was detected by Kodak X-OMAT AR Film after 24 hours (Eastman Kodak Co., Rochester, N.Y.).
  • the same membrane was then re-probed in a similar manner with radiolabeled ⁇ -actin (0.1 ⁇ g probe labeled at >5 ⁇ 10 8 cpM/ ⁇ g) (Ambion, Austin, Tex.). Densitometry was performed as described above for the western blots.
  • the ⁇ -actin mRNA bands served as a control against which to normalize the MIF mRNA densitometry.
  • the aorta was cannulated with PE50 tubing, the heart perfused in a retrograde manner through the aortic root with pre-filtered, oxygenated Krebs-Henseleit Buffer at a constant flow rate of 1.5 ml/minute (constant temperature of 37° C., 100 ml recirculating volume).
  • the heart was placed in a water-jacketed chamber to maintain constant temperature and humidity.
  • LV pressure and its first derivative (dP/dt), heart rate, and coronary perfusion were measured simultaneously with a multi-channel Grass 7D polygraph (Grass Instruments, Quincy, Mass.). Ventricular performance as a function of coronary perfusion was determined for all hearts by plotting peak systolic LV pressure and ⁇ dP/dt max values against incremental increases in coronary flow rate. Hearts were perfused with or without 20 ng/ml rMIF added to the perfusate.
  • Echocardiograms to assess systolic function were performed using M-mode measurements. Mice were anesthestized with 5% isofluorane with 2.5 L/m O 2 for 20 seconds (until unconscience) followed by 2% isofluorane and O 2 for an average of 12-15 minutes. Hair was removed from the thorax and upper abdomen using Nair® hair remover and gauze after sitting for 3 minutes. Echocardiographic measurements were obtained on anesthetized mice approximately 5-8 minutes after induction.
  • Echocardiography was performed using an Acuson SequoiaTM Model C256 (Siemens Medical Solutions, USA, Inc., Mountain View, Calif.) with a frame rate of 300-500 frames/second in a random and blinded manner.
  • a 15 MHz linear transducer (15L8, Siemens Medical Solutions, USA, Inc.) was placed on the left hemithorax interfaced with a layer of ultrasound transmission gel (Aquasonic 100, Parker Laboratories; Fairfield, N.J.).
  • the two dimensional parasternal short-axis imaging plane guided LV M-mode tracings close to the papillary muscle level. Depth was set at a minimum of 2 cm with a sweep speed of 200 m/second.
  • Plasma inflammatory cytokine (IL-1 ⁇ , IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN- ⁇ , TNF- ⁇ , and GM-CSF) concentrations were determined using the Mouse Cytokine Ten-Plex Antibody Bead Kit (Biosource International, Inc., Camarillo, Calif.) on a Luminex xMAPTM system (Luminex Corp., Austin, Tex.) according to the manufacturer's instructions. The plate was loaded onto the Luminex XYPTM platform, the instrument set to remove 50 ⁇ l, and the total event set to equal the 100 per bead set (200 collected for most).
  • LuminexTM Data Collector Software Luminex Corp., Austin, Tex.
  • concentrations of the lot specific reconstituted standards used in each run were entered into the software and the analyte concentrations for unknown samples were then extrapolated from the cytokine specific standard curve using MasterPlexTM QT software (Version 1.2.8.58, Mirai Bio, Inc., Alameda, Calif.). Final concentrations were multiplied by 2 in order to account for the initial dilution factor. No samples were detected that were higher than the standards curves for any cytokine.
  • Northern and Western data are expressed as mean ⁇ standard error (SE) and statistically analyzed using a One Way-Analysis of Variance (ANOVA).
  • SE mean ⁇ standard error
  • ANOVA One Way-Analysis of Variance
  • a multiple comparison procedure was employed using the Tukey method to determine statistical significance between groups.
  • Cardiac function determined by the Langendorff preparation (including stabilization data) is expressed as the mean ⁇ SE and separate analyses were performed for each LVP, +dP/dt max , and ⁇ dP/dt max as a function of treatment group and coronary flow rate using a Repeated Measures-ANOVA.
  • a multiple comparison procedure employing the Bonferroni method was used to determine significant differences between groups.
  • Serum MIF levels are expressed as the mean ⁇ SE and were statistically analyzed using a One Way-ANOVA, with a multiple comparison procedure employing the Bonferroni method to determine significance between groups. Cardiac function determined by M-mode echocardiography is expressed as fractional shortening % ⁇ SE and analyzed using a One Way Repeated Measures-ANOVA. Additional comparisons were performed using the Tukey Test to determine significant differences between specific groups. Statistical significance for all analyses was defined as p ⁇ 0.05. All statistical analyses were performed using SigmaStat 2.03 (SPSS Inc., Chicago, Ill.) and Microsoft Excel (Microsoft Corp., Seattle, Wash.).
  • Serum MIF levels in WT mice, WT mice pre-treated with Enbrel®, and TNFR ⁇ / ⁇ mice Serum MIF levels in WT mice, WT mice pre-treated with Enbrel®, and TNFR ⁇ / ⁇ mice. Serum levels of MIF reach maximum ( ⁇ 1.5 fold baseline) at 8 hours in wild type mice after LPS challenge ( FIG. 13A ). When TNFR ⁇ / ⁇ mice are challenged with LPS, maximum serum MIF levels occur as 12 hours ( ⁇ 1.7 fold baseline)( FIG. 13B ). Maximum serum MIF levels ( ⁇ 2.3 fold baseline) were identified in wild type mice pre-treated (60 minutes) with Enbrel® and challenged with LPS at 24 hours ( FIG. 13C ).
  • Cardiac MIF is not released from the heart, spleen, or liver in TNFR ⁇ / ⁇ mice after LPS challenge. Both western and immunohistochemistry analysis performed on cardiac, liver, and spleen demonstrated that previously documented release in wild type mice after LPS challenge did not occur at any time in TNFR ⁇ / ⁇ mice or wild type mice pre-treated with Enbrel®, both of which prevent TNF- ⁇ signaling ( FIGS. 14 and 15 ).
  • MIF transcription is not modulated in TNFR ⁇ / ⁇ mice after LPS challenge. Detection of MIF mRNA from isolated total RNA from heart tissue from TNFR ⁇ / ⁇ mice challenge with LPS demonstrates that transcription of MIF is not upregulated after LPS challenge ( FIG. 16 ), which has been identified in wild type mice previously at 48 hours.
  • MIF has direct cardiodepressant effects in TNFR ⁇ / ⁇ mice to the same extent as in WT mice.
  • rMIF recombinant human MIF
  • Table 4 demonstrates that the responses of the background strain of TNFR ⁇ / ⁇ mice, C57BL/6 mice, and the TNFR ⁇ / ⁇ mice to retrograde aortic perfusion at 1.5 ml/minute with control perfusate or perfusate containing 20 ng/ml rMIF.
  • Perfusion with rMIF led to a significant decrease in LVP, +dP/dt max , ⁇ dP/dT max , and dp40 (mm Hg/sec) in both mouse strains while other parameters (time to max ⁇ dP/dt, CPP, CVR, and HR) were unaffected.
  • FIG. 18 illustrates the effect of rMIF over a range of coronary flow rates.
  • MIF neutralization by anti MIF antibodies results in complete protection at 24-48 hours after LPS challenge in TNFR ⁇ / ⁇ mice.
  • serial echocardiography M-mode was performed on LPS challenged TNFR ⁇ / ⁇ mice pretreated (90 minutes prior) with either of two anti-MIF monoclonal antibodies, an isotype control antibody, or no treatment ( FIG. 17 ).
  • FS fractional shortening percentage
  • mice treated with either of the two monoclonal anti-MIF antibodies demonstrated statistically significant recovery of FS % compared to LPS challenged group receive or LPS or LPS and the isotype antibody ( FIG. 17 ).
  • This enhanced recovery of function persisted at 48 hours where function was completely restored and LPS challenged mice receiving the isotype control were still profoundly depressed ( FIGS. 17D, 17E ).
  • the FS % of untreated control TNFR ⁇ / ⁇ mice did not change significantly indicating that cardiac function was unaffected the testing regimen.
  • Serum cytokine release in wild type mice and TNFR ⁇ / ⁇ mice Since other inflammatory cytokines have been shown to play a role in cardiac dysfunction in addition to TNF- ⁇ early after LPS challenge (i.e. IL-1 ⁇ , IL-6), we determined serum levels of an inflammatory panel in wild type and TNFR ⁇ / ⁇ mice ( FIG. 19 ). Not obvious in this figure are the release of TNF- ⁇ , IL-1 ⁇ in wild type mice because of the significant increases in these cytokines in TNFR ⁇ / ⁇ mice (31.2 (4934/158 pg/ml) fold and 94.7 (7099/75 pg/ml) fold increase over wild type at 4 hours after LPS challenge) as shown in FIGS. 19A and 19B .
  • IL-12 was increased in the TNFR ⁇ / ⁇ mice compared to wild type mice (and 1.7 (5128/2937 pg/ml) fold) ( FIG. 19C ), while IFN- ⁇ levels were decreased 3.6 (210/58 pg/ml) fold ( FIG. 19D ).
  • IL-10 and IL-6 increased similarly in wild type and TNFR ⁇ / ⁇ mice, although the delay of each of these cytokines was diminished in the TNFR ⁇ / ⁇ mice ( FIGS. 19E and 19F ).
  • IL-6 levels were 8.5 fold in the TNFRKO mice at 4 hours compared to wild type mice (7099/835 pg/ml).
  • Serum cytokine release after MIF neutralization in wild type mice is affected the modulation (increase or decrease) of serum IFN- ⁇ and IL-10 levels after LPS challenge in wild type mice ( FIG. 20 ). Specifically, the release of IFN- ⁇ peak at 8 hours after LPS challenge ( FIG. 19C ) was attenuated 3 fold (210/69 pg/ml)( FIG. 20A ). The delayed release of IL-10 in wild type mice which peaked at 48 hours was attenuated 2.9 fold (244/84 pg/ml)( FIG. 20B ) after LPS challenge in wild type mice.
  • mice C57BL/6 mice from Charles River (12-15 weeks old) were maintained on commercial chow and tap water ad libitum. All animal protocols were reviewed and approved by the University of Texas Soiled IACAC in compliance with the rules governing animal use published by NIH.
  • Coronary artery ligation Mice were anesthetized with 1-1.5% isoflurane after which coronary artery ligation was performed. Atropine (0.75 mg/kg given intramuscularly), lidocaine (1 mg/kg intramuscularly), and saline (1 ml intraperitoneally) were given pre-operatively. Ventilation was achieved using a custom mask fitted to the mouse snout and a small animal Ventilator (Harvard Apparatus, Inc., Holliston, Mass.). An incision ( ⁇ 5 mm) was made in the left thorax in the fourth intercostal space and pericardiotomy was performed to expose the left ventricle.
  • the left coronary artery was occluded using 8-0 prolene approximately 2 mm under the left auricle. Subsequently, the chest was closed in layers and the negative pressure of the chest returned by syringe evacuation. Buprenorphine (0.10 mg/kg) was given once post-operatively for pain. Sham procedures were performed identically without the coronary ligation.
  • Anti-MIF antibody A monoclonal anti-mouse MIF IgG1 antibodies (III.D.9, gift from Cytokine PharmaScience, Inc.) and a monoclonal IgG1 isotype control antibody (HB-49, gift from Cytokine PharmaScience, Inc.) were used in the echocardiographic studies. Previous studies have demonstrated in vivo neutralization of MIF activity.
  • FIGS. 21-25 show the results obtained in this example.
  • FIG. 21 Compares cardiac function (fractional shortening) in post LAD ligation with LAD only and anti-MIF+LAD.
  • FIG. 22 Shows the effect of anti-MIF therapy pre-LAD with LAD only and anti-MIF+LAD.
  • FIG. 21 Compares cardiac function (fractional shortening) in post LAD ligation with LAD only and anti-MIF+LAD.
  • FIG. 22 Shows the effect of anti-MIF therapy pre-LAD with LAD only and anti-MIF+LAD.
  • FIG. 23 Presents cardiac function data 48 hours post-LAD for several treatment groups.
  • FIG. 24 Shows the serum troponin concentration 48 hrs post-LAD with pre- and delayed anti-MIF treatment.
  • FIG. 25 Shows the serum troponin I and MIF concentrations through two weeks post ligation.
  • MIF is secreted from cardiomyocytes following LPS challenge, and directly mediates a late onset (>6 hours) cardiac dysfunction.
  • CD74 was recently determined to be the MIF receptor, exerting effects via ERK1/2 intracellular signaling pathways.
  • To determine if CD74 mediates MIF-induced cardiac dysfunction in sepsis we challenged: 1) wild type mice (C57BL/6) with LPS; 2) wild type mice pre-treated with anti-CD74 monocolonal neutralizing antibodies; and challenged with LPS, and 3) CD74 knock-out mice with LPS (4 mg/kg). Serial echocardiography was performed and fractional shortening (FS %) was determined.
  • Coronary artery ligation Mice were anesthetized with 1-1.5% isoflurane after which coronary artery ligation was performed. Atropine (0.075 mg/kg given intramuscularly), lidocaine (1 mg/kg intramuscularly), and saline (1 ml intraperitoneally) were given pre-operatively. Ventilation was achieved using a custom mask fitted to the mouse snout and a small animal ventilator (Harvard Apparatus, Inc., Holliston, Mass.). An incision ( ⁇ 5 mm) was made in the left thorax in the fourth intercostal space and pericardiotomy was performed to expose the left ventricle.
  • the left coronary artery was occluded using 8-0 prolene approximately 2 mm under the left auricle. Subsequently, the chest was closed in layers and the negative pressure of the chest returned by syringe evacuation.
  • An the acid methyl ester of (R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic (“ISO-1”, “CPSI-26” or p-hydroxyphenol-isoxazoline methyl ester) was given at a dosage of 200 mg/kg in 25 microliters DMSO intraperitoneally daily for two weeks. Buprenorphine (0.10 mg/kg) was given once post-operatively for pain. Sham procedures were performed identically without the coronary ligation.

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US10/927,494 2003-08-29 2004-08-27 Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor Abandoned US20050202010A1 (en)

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US10/927,494 US20050202010A1 (en) 2003-08-29 2004-08-27 Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor
HUE04782427A HUE029888T2 (en) 2003-08-29 2004-08-30 Treatment Method and Biological Test Using Macrophage Migration Factor (MIF) as a Factor for Heart Myocardial Depression
EP04782427.1A EP1658037B1 (en) 2003-08-29 2004-08-30 Method of treatment and bioassay involving macrophage migration inhibitory factor (mif) as cardiac-derived myocardial depressant factor
AU2004268017A AU2004268017B2 (en) 2003-08-29 2004-08-30 Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor
CA2537928A CA2537928C (en) 2003-08-29 2004-08-30 Method of treatment and bioassay involving macrophage migration inhibitory factor (mif) as cardiac-derived myocardial depressant factor
CN201110370579.7A CN102499984B (zh) 2003-08-29 2004-08-30 涉及作为心脏衍生的心肌抑制因子的巨噬细胞迁移抑制因子(mif)的治疗和生物测定方法
ES04782427.1T ES2599032T3 (es) 2003-08-29 2004-08-30 Método de tratamiento y bioensayo que implica el factor de inhibición de la migración de macrófagos (MIF) como factor depresor del miocardio de origen cardiaco
EP16170803.7A EP3078386A3 (en) 2003-08-29 2004-08-30 Method of treatment and bioassay involving macrophage migration inhibitory factor (mif) as cardiac-derived myocardial depressant factor
BRPI0413404-4A BRPI0413404A (pt) 2003-08-29 2004-08-30 composições farmacêuticas, métodos de tratamento ou prevenção de disfunções cardìacas, métodos de aumento da função cardìaca e método de identificação de inibidor de mif
DK04782427.1T DK1658037T3 (en) 2003-08-29 2004-08-30 Method and treatment of bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depression factor
MXPA06001828A MXPA06001828A (es) 2003-08-29 2004-08-30 Metodo para el tratamiento y bioensayo que involucra el factor de inhibicion de migracion de macrofagos ("mif"), como un factor de depresion del miocardio, derivado del corazon.
CN2004800247776A CN1972713B (zh) 2003-08-29 2004-08-30 治疗或预防心脏功能障碍的药物组合物
PT47824271T PT1658037T (pt) 2003-08-29 2004-08-30 Método de tratamento e bioensaio envolvendo o fator de inibição da migração de macrófagos (mif) como fator depressivo do miocárdio de origem cardíaca
PCT/US2004/027945 WO2005020919A2 (en) 2003-08-29 2004-08-30 Method of treatment and bioassay involving macrophage migration inhibitory factor (mif) as cardiac-derived myocardial depressant factor
PL04782427T PL1658037T3 (pl) 2003-08-29 2004-08-30 Sposób leczenia i test biologiczny z udziałem czynnika hamującego migrację makrofagów (mif) jako czynnika depresji mięśnia sercowego pochodzenia sercowego
JP2006524893A JP4891769B2 (ja) 2003-08-29 2004-08-30 マクロファージ遊走阻止因子（ｍｉｆ）を心臓由来心筋抑制因子として含む、処置方法およびバイオアッセイ
MX2011013436A MX340217B (es) 2003-08-29 2006-02-16 Metodo para el tratamietno y bioensayo que involucra el factor de inhibicion de migracion de macrofagos (mif), como un factor de depresion del miocardio, derivado del corazon.
US11/932,909 US8747843B2 (en) 2003-08-29 2007-10-31 Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor
JP2010039451A JP5905659B2 (ja) 2003-08-29 2010-02-24 マクロファージ遊走阻止因子（ｍｉｆ）を心臓由来心筋抑制因子として含む、処置方法およびバイオアッセイ
JP2011133728A JP2011251966A (ja) 2003-08-29 2011-06-15 マクロファージ遊走阻止因子（ｍｉｆ）を心臓由来心筋抑制因子として含む、処置方法およびバイオアッセイ
US14/281,870 US20150017179A1 (en) 2003-08-29 2014-05-19 Method of Treatment and Bioassay Involving Macrophage Migration Inhibitory Factor (MIF) as Cardiac-Derived Myocardial Depressant Factor
CY20161100837T CY1117925T1 (el) 2003-08-29 2016-08-25 Μεθοδος αγωγης και βιοπροσδιορισμος που σχετιζεται με παραγοντα αναστολης της μεταναστευσης των μακροφαγων (mif) ως προερχομενο απο την καρδια μυοκαρδιακο κατασταλτικο παραγοντα

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US49865903P 2003-08-29 2003-08-29
US54705604P 2004-02-25 2004-02-25
US54705704P 2004-02-25 2004-02-25
US54705404P 2004-02-25 2004-02-25
US54705904P 2004-02-25 2004-02-25
US55644004P 2004-03-26 2004-03-26
US10/927,494 US20050202010A1 (en) 2003-08-29 2004-08-27 Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor

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US11/932,909 Active 2027-03-21 US8747843B2 (en) 2003-08-29 2007-10-31 Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor
US14/281,870 Abandoned US20150017179A1 (en) 2003-08-29 2014-05-19 Method of Treatment and Bioassay Involving Macrophage Migration Inhibitory Factor (MIF) as Cardiac-Derived Myocardial Depressant Factor

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US14/281,870 Abandoned US20150017179A1 (en) 2003-08-29 2014-05-19 Method of Treatment and Bioassay Involving Macrophage Migration Inhibitory Factor (MIF) as Cardiac-Derived Myocardial Depressant Factor

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US20080241145A1 (en) * 2004-12-08 2008-10-02 Immunomedics, Inc. Methods and compositions for immunotherapy and detection of inflammatory and immune-dysregulatory disease, infectious disease, pathologic angiogenesis and cancer
WO2009117706A3 (en) * 2008-03-20 2010-01-21 Carolus Therapeutics, Inc. Methods of treatment using anti-mif antibodies
WO2010065491A2 (en) * 2008-12-01 2010-06-10 Carolus Therapeutics, Inc. Methods of treating inflammatory disorders
US20100183598A1 (en) * 2008-11-12 2010-07-22 Carolus Therapeutics, Inc. Methods of treating cardiovascular disorders
US20110070184A1 (en) * 2008-03-24 2011-03-24 Carolus Therpeutics, Inc. Methods and compositions for treating atherosclerosis and related condidtions
US9238689B2 (en) 2011-07-15 2016-01-19 Morpho Sys AG Antibodies that are cross-reactive for macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT)

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US20050202010A1 (en) 2003-08-29 2005-09-15 Giroir Brett P. Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor
MXPA06010793A (es) * 2004-03-26 2006-12-19 Cytokine Pharmasciences Inc Compuestos composiciones, procesos de elaboracion, y metodos de uso relacionados a la inhibicion del factor inhibidor de migracion de macrofago.
WO2006116688A2 (en) * 2005-04-26 2006-11-02 Yale University Mif agonists and antagonists and therapeutic uses thereof
AU2011375306A1 (en) * 2011-08-12 2014-02-27 Alfred Health Method for diagnosis, prognosis or treatment of acute coronary syndrome (ACS) comprising measurement of plasma concentration of macrophage migration inhibitory factor (MIF)
EP2748613B1 (en) 2011-10-07 2021-05-05 Baxalta GmbH Oxmif as a diagnostic marker
KR20150091504A (ko) * 2012-12-07 2015-08-11 백스터 인터내셔널 인코포레이티드 항―ｍｉｆ 항체 세포 이동 검정법
CN105087610A (zh) * 2015-09-11 2015-11-25 中国科学院海洋研究所 文蛤巨噬细胞迁移抑制因子基因及其编码蛋白和应用
JP2018007029A (ja) * 2016-07-01 2018-01-11 株式会社村田製作所 バイアス回路
EP4334469A1 (en) * 2021-05-06 2024-03-13 Abbott Molecular Inc. Compositions and methods for simple sample extraction
CN114288387A (zh) * 2022-02-17 2022-04-08 重庆医科大学 Humanin衍生物HNG在制备治疗心衰药物中的应用

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US6492428B1 (en) 2000-07-26 2002-12-10 The Picower Institute For Medical Research Compounds having MIF antagonist activity
US6420188B1 (en) 1996-02-16 2002-07-16 The Picower Institute For Medical Research Screening assay for the identification of inhibitors for macrophage migration inhibitory factor
DE69813868T2 (de) * 1997-11-05 2004-03-04 The University Of Southern California, Los Angeles Verwendung von zytokinen und mitogenen für inhibierung von pathologischen immunantworten
JP2003513065A (ja) 1999-10-29 2003-04-08 ザ・ピコワー・インスティチュート・フォー・メディカル・リサーチ Ｍｉｆアンタゴニスト活性を有する化合物
US6268151B1 (en) 2000-01-20 2001-07-31 Isis Pharmaceuticals, Inc. Antisense modulation of macrophage migration inhibitory factor expression
US20030235584A1 (en) * 2000-02-28 2003-12-25 Kloetzer William S. Method for preparing anti-MIF antibodies
CA2455915C (en) * 2001-03-29 2013-05-14 Cytokine Pharmasciences, Inc. Methods and compositions for using mhc class ii invariant chain polypeptide as a receptor for macrophage migration inhibitory factor
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Cited By (11)

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US20080241145A1 (en) * 2004-12-08 2008-10-02 Immunomedics, Inc. Methods and compositions for immunotherapy and detection of inflammatory and immune-dysregulatory disease, infectious disease, pathologic angiogenesis and cancer
US8420786B2 (en) * 2004-12-08 2013-04-16 Immunomedics, Inc. Bispecific antibody targeting a complement factor or complement regulatory protein
WO2009117706A3 (en) * 2008-03-20 2010-01-21 Carolus Therapeutics, Inc. Methods of treatment using anti-mif antibodies
EP2254597A2 (en) * 2008-03-20 2010-12-01 Carolus Therapeutics, Inc. Methods of treatment using anti-mif antibodies
US20110044988A1 (en) * 2008-03-20 2011-02-24 Carolus Therpeutics, Inc. Methods of treatment using anti-mif antibodies
EP2254597A4 (en) * 2008-03-20 2012-04-18 Carolus Therapeutics Inc TREATMENT PROCEDURE WITH ANTI-MIF ANTIBODIES
US20110070184A1 (en) * 2008-03-24 2011-03-24 Carolus Therpeutics, Inc. Methods and compositions for treating atherosclerosis and related condidtions
US20100183598A1 (en) * 2008-11-12 2010-07-22 Carolus Therapeutics, Inc. Methods of treating cardiovascular disorders
WO2010065491A2 (en) * 2008-12-01 2010-06-10 Carolus Therapeutics, Inc. Methods of treating inflammatory disorders
WO2010065491A3 (en) * 2008-12-01 2010-09-30 Carolus Therapeutics, Inc. Methods of treating inflammatory disorders
US9238689B2 (en) 2011-07-15 2016-01-19 Morpho Sys AG Antibodies that are cross-reactive for macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT)

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CN102499984A (zh) 2012-06-20
WO2005020919A2 (en) 2005-03-10
CN1972713A (zh) 2007-05-30
PT1658037T (pt) 2016-08-30
WO2005020919A3 (en) 2006-11-30
DK1658037T3 (en) 2016-08-29
EP3078386A2 (en) 2016-10-12
JP2011251966A (ja) 2011-12-15
EP1658037A4 (en) 2008-09-10
EP1658037A2 (en) 2006-05-24
MX340217B (es) 2016-06-30
JP5905659B2 (ja) 2016-04-20
ES2599032T3 (es) 2017-01-31
HUE029888T2 (en) 2017-04-28
CN102499984B (zh) 2015-02-18
US8747843B2 (en) 2014-06-10
CA2537928C (en) 2015-05-05
EP3078386A3 (en) 2017-01-11
JP4891769B2 (ja) 2012-03-07
JP2007504158A (ja) 2007-03-01
AU2004268017A1 (en) 2005-03-10
JP2010159267A (ja) 2010-07-22
MXPA06001828A (es) 2007-05-23
PL1658037T3 (pl) 2017-08-31
CY1117925T1 (el) 2017-05-17
AU2004268017B2 (en) 2011-03-17
US20080260723A1 (en) 2008-10-23
EP1658037B1 (en) 2016-05-25
CA2537928A1 (en) 2005-03-10
AU2004268017A2 (en) 2005-03-10
US20150017179A1 (en) 2015-01-15
BRPI0413404A (pt) 2006-10-17
CN1972713B (zh) 2012-02-29

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