WO2002078684A2 - Carbon monoxide generating compounds for treatment of vascular, inflammatory and immune disorders - Google Patents
Carbon monoxide generating compounds for treatment of vascular, inflammatory and immune disorders Download PDFInfo
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- WO2002078684A2 WO2002078684A2 PCT/US2002/010115 US0210115W WO02078684A2 WO 2002078684 A2 WO2002078684 A2 WO 2002078684A2 US 0210115 W US0210115 W US 0210115W WO 02078684 A2 WO02078684 A2 WO 02078684A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
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Definitions
- the immune system is an extraordinarily complex combination of cells and compositions that protects a mammalian host against a wide variety of pathogens, while surveiling the body against deleterious aberrations, such as neoplasia.
- One branch of the immune system involves the cells that carry out immune system functions, including both (a) lymphocytes, such as the bone marrow-derived B- lymphocytes, the thymus-derived T lymphocytes and natural-killer (NK) cells, and (b) the mononuclear phagocytes, including both monocytes and macrophages.
- lymphocytes such as the bone marrow-derived B- lymphocytes, the thymus-derived T lymphocytes and natural-killer (NK) cells
- NK natural-killer
- lymphocytes are primarily associated with specific immune responses, due to their ability to specifically recognize and distinguish antigenic determinants, the mononuclear phagocytes are most often involved in the general removal of foreign microbes through phagocytosis as well as the production and secretion of cytokines as induced either directly by a microbe itself or in response to antigen-stimulated T lymphocytes.
- the functions of lymphocytic cells and the mononuclear phagocytes are highly interconnected and essential for proper immune system function.
- Cytokines such as the various interferons, interleukins, tumor necrosis factors, chemokines, hematopoietic growth factors and migration inhibition factors are a diverse group of proteins that are produced by a wide variety of different cells types of the immune system. Most importantly, cytokines are produced and/or responded to by various lymphocytes and mononuclear phagocytes in response to various stimuli. For the most part, cytokines are produced during the effector phases of both natural and specific immunity and serve to mediate and regulate both immune and inflammatory responses. Cytokines, like other polypeptide hormones, initiate their action by binding to specific receptors on the surface of target cells, their activation often resulting in an ___3a ⁇ _ ⁇ matory response.
- cytokine-mediated icdlammatory response functions to adversely affect the health of the host, such as inflammatory responses associated with such maladies as septic shock, rheumatoid arthritis, Crohn's disease, colitis, and the like.
- Another incidence is where there is a failure on the part of CTLs in that they attack cells where the MHC and associated peptide are both endogenous, as occurs in autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM).
- IDDM insulin-dependent diabetes mellitus
- An additional incidence is associated with transplantation, where one rarely has an identical match between the donor and recipient of the MHC antigens.
- Immunosuppression has become a general approach in situations where activation of CTLs is undesired.
- immunosuppressants such as cyclosporin
- A, FK506, and the like have numerous undesirable side effects. Additionally, various approaches have been employed for controlling or inhibiting inflammatory responses, however, many of these approaches also have one or more undesirable effects. There is, therefore, substantial interest in identifying new agents which can act to inhibit the activation of lymphocytic cells, particularly CTLs, while having less of a universal immunosuppressive effect on the immune system and fewer side effects, so as to leave the host with a substantial proportion of the immune system for protection against adventitious infection. There is also a substantial interest in identifying new agents that function to control or inhibit adverse inflammatory reactions.
- Heme oxygenases are the rate-limiting enzymes that catalyze the conversion of heme to biliverdin, carbon monoxide (CO) and free iron, the first step in the oxidative conversion of heme to bilirubin.
- HO-2 is the constitutive isoform present under physiological conditions
- HO-1 is the inducible isoformthat provides protection against oxidative injury.
- great interest has been placed on the role of HO-1 in cellular responses to oxidative stress and insult, including ischemic and immunogenic effects. Upregulation or inducement of HO-1 expression has been found to produce a variety of potent anti-inflammatory and immunosuppressive effects, including prolongation of allograft survival and alleviation of graft versus host disease.
- the heme oxygenase pathway also plays a critical role in regulating and maintaining vascular tone to ensure adequate tissue oxygenation and perfusion.
- Vascular cells respond to an environment of oxidative stress by inducing endogenous antioxidant defense mechanisms.
- the main intracellular regulator under physiologic conditions is endothelial-derived nitric oxide (NO), which maintains normal vascular tone through its regulation of cyclic guanosine 3', 5'-monophosphate (cGMP) levels in vascular smooth muscle cells (VSMC) by guanylate cyclase activation.
- NO endothelial-derived nitric oxide
- cGMP cyclic guanosine 3', 5'-monophosphate
- VSMC vascular smooth muscle cells
- induction of heme oxygenase may provide an important secondary line of antioxidant defense through generation of the antioxidant bilirubin and the vasodilator CO.
- CO has been identified as a dilator of VSMC via a cGMP-mecha ism, and has been shown to suppress endothelin-1 (ET-1) and platelet-derived gi'owth factor-B gene expression in endothelial cells and subsequently inhibit the proliferation of smooth muscle cells.
- CO also has endothelial cell-independent effects on VSMC proliferation through its suppression of E2F-1 gene expression, a transcription factor implicated in the control of cell cycle progression. Morita et al, J. Biol. Chem. 272(52):32804-9 (1997).
- endogenous CO generated by the heme oxygenase pathway also protects against excessive VSMC proliferation, a main event in the pathogenesis of many cardiovascular diseases including atherosclerosis, intimal hyperplasia and pulmonary hypertension.
- VSMC proliferation and accumulation is also implicated in neointimal development elicited by arterial injury, such as denudation caused by balloon injury.
- Balloon injury induces the production of several vasoactive factors, including ET-1, and exposes the VSMC layer directly to red blood cells in the blood stream, which may change the shear stress and redox state in the vascular wall.
- ET-1 vasoactive factors
- CO inhibits neointimal formation and thus serves a critical protective function for arterial injury as well.
- the present invention provides methods and compositions for treating vascular, inflammatory and immune diseases using carbon monoxide generating compounds, which are capable of being metabolized into carbon monoxide in vivo.
- the carbon monoxide generating compound is methylene chloride (CH 2 C1 2 ), which is metabolized in vivo into CO and CO 2 .
- the invention provides a pharmaceutical composition for the treatment of vascular, inflammatory and immune disorders in a mammal, comprising a carbon monoxide generating compound capable of increasing the carboxyhemoglobin level in said mammal.
- the carbon monoxide generating compound comprises methylene chloride.
- the invention provides a pharmaceutical composition for increasing the carboxyhemoglobin level in a mammal, comprising methylene chloride in a pharmaceutically acceptable vehicle. Also provided is a method for increasing the carboxyhemoglobin level in a mammal, comprising the administration of a carbon monoxide generating compound such as methylene chloride to said mammal in an amount sufficient to increase the blood carboxyhemoglobin level to between about 1 and 10 %, more preferably between about 2 and 9%, most preferably between about 3 and 8%, generally between about 3 and 10%.
- a carbon monoxide generating compound such as methylene chloride
- the present invention provides methods and compositions for modulating inflammatory and immune processes throughout the body.
- the subject compounds are capable of modulating the activity of various iirimune system cells, inhibiting the production of pro-inflammatory cytokines and enhancing production of anti-inflammatory cytokines by cells capable of producing such cytokines, thereby being effective in the treatment of conditions associated with adverse inflammatory responses.
- Methods for extending the survival of an organ transplant in a recipient comprise administering to said recipient a carbon monoxide generating compound that functions to modulate the immune response against the transplanted organ, whereby the survival time of the organ transplant in the recipient is extended.
- Administration of the carbon monoxide generating compounds of the present invention may be ex vivo of an organ to be transplanted or in vivo by any convenient means, including parental, systemic or localized administration, in sufficient amount to substantially inhibit lymphocyte activation and the inflammatory process through modulation of anti- and pro-inflammatory cytokine production.
- the subject compounds are capable of regulating vascular tone, inhibiting VSMC proliferation and protecting against oxidative stress and hypoxia, which have profound effects on vascular tone, endothelial permeability and coagulating function.
- the subject carbon-monoxide generating compounds will find use in treating vascular proliferative diseases and other disorders associated with HO-1 induction in response to oxidative stress.
- methods for inhibiting neointimal formation and improving the outcome of invasive vascular procedures comprising administering to a patient undergoing a procedure requiring or involving arterial injury such as balloon angioplasty a carbon monoxide generating compound that functions to protect against neointimal development.
- the subject carbon- monoxide generating compounds are employed to prevent atherogenesis, either in response to a specific oxidative event in the vasculature or prophylactically in patients at higher risk, such as, e.g., those with high levels of low-density lipoproteins (LDL) thought to be involved in atherogenesis.
- Administration of the carbon monoxide generating compounds of the present invention may be by any convenient means, including parental, systemic or localized administration, in sufficient amount to substantially inhibit VSMC proliferation and modulate the vascular response to oxidative stress.
- FIGS. 1A, IB & 1C are graphs of the levels of serum TNF- ⁇ , carboxyhemoglobin and O 2 Hb in mice treated with LPS with or without 500 ppm gaseous CO.
- Figure 2 is a graph of the effect of methylene chloride administration on LPS- induced TNF-C. production.
- Figure 3 is a graph of the effect of methylene chloride administration on blood carboxyhemoglobin levels.
- Figure 4 is a graph of the effect of exogenous CO on portal vein resistance in an ex vivo rat liver model of cold ischemia followed by reperfusion.
- Figure 5 is a graph showing the effect of exogenous CO on bile production in an ex vivo rat liver model of cold ischemia followed by reperfusion.
- Figure 6 is a graph showing the effect of exogenous CO on neutrophil activity as measured by a myeloperoxidase assay in an ex vivo rat liver model of cold ischemia followed by reperfusion.
- Figure 7 is a graph showing the effect of exogenous CO on COHb levels in an ex vivo rat liver model of cold ischemia followed by reperfusion.
- Figure 8 is a graph showing is a graph showing the effect of exogenous CO on bile production in an ex vivo rat liver model of cold ischemia followed by reperfusion, with and without the addition of L-NAME (an inducible NO inhibitor) or LY-83583 (a cGMP analogue) or pretreatment with ZnPP, an HO-1 inhibitor.
- Figure 9 is a graph showing the effect of exogenous CO on bile production in an ex vivo rat liver model of cold ischemia followed by reperfusion, with and without the addition of SB203580, a p38 MAPK inhibitor.
- Figure 10 is a graph showing in vitro cytotoxicity to Fas-bearing YAC-1 target cells after exposure to Yac-1 and Hela cells transfected with Ad-CD95 + Ad-HO-1 (filled bars) and AD-CD95 + Ad- ⁇ -gal (open bars).
- Figure 11 is a graph showing a pharxnacokinetic study of systemic carboxyhemoglobin (COHb) levels after oral methylene chloride administration in a rat aorta model.
- COHb carboxyhemoglobin
- Figure 12 is a chart showing computer-assisted morphometry of intima thickness in syngeneic and allogeneic rat aortic grafts at day 30 after transplantation, when treated with control (Addl324), Ad-HO-1 or methylene chloride.
- Figure 13 is a graph illustrating alloantibody levels in recipients of aortic allografts treated with AdHO-1 or CO.
- Figure 14 is a chart showing the arthritic score in control and MC-treated rats in a rat collagen-arthritis model.
- the carbon monoxide generating compounds of the subject invention find use for regulating vascular tone, inhibiting VSMC proliferation and protecting against oxidative stress, thereby being useful for treating various disorders such as atherogenesis, restenosis, pressure or volume overload of the heart, hypertension, subarachnoidal hemorrhage, neointima formation and development, vasoconstriction, edema in the lung, and thrombus formation in the venous circulation.
- methods for inhibiting neointimal formation and improving the outcome of invasive vascular procedures comprising administering to a patient undergoing a procedure involving arterial injury such as balloon angioplasty a carbon monoxide generating compound that functions to protect against neointimal development.
- the subject carbon- monoxide generating compounds are employed to prevent atherogenesis, either in response to a specific oxidative event or prophylactically in patients at higher risk, such as, e.g., those having high levels of low-density lipoproteins (LDL) thought to be involved in atherogensis.
- LDL low-density lipoproteins
- the carbon monoxide generating compounds of the subject invention find use for inhibiting the production of inflammatoiy cytokines and enhancing the production of anti- inflammatory cytokines, including TNFoc, interferons such as interferon- ⁇ , interleukins such as IL-1, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, MlPl ⁇ , chemokines, hematopoietic growth factors and the like, thereby being useful for inhibiting inflammatory responses associated with various disorders such as rheumatoid arthritis, septic shock, Crohn's disease, colitis, multiple sclerosis, granulomatous inflammation, hepatitis, allergic reactions, autoimmune diseases, ischemic/ reperfusion injury, and the like, and delaying the onset of IDDM in a patient at risk for developing IDDM, both in vitr
- the subject compounds find use in treating rheumatoid arthritis, improving the outcome of organ transplantation (e.g, kidney, liver, heart, etc.) and preventing ischemia/reperfusion injury.
- organ transplantation e.g, kidney, liver, heart, etc.
- the above-described carbon monoxide generating compounds will function both in vivo and in vitro to modulate inflamation and/or the immune response in a host or sample, respectively, into which they are introduced.
- the modulation will generally be exemplified by an inhibition of the expression of pro-iriflammatory cytokines and/or an increase in the production of anti-inflammatory cytokines. Reliable and sensitive assays for determining the expression levels of such cytokines are well known and commercially available from such sources as BioSource International, Inc. in Camarillo, California.
- carbon monoxide generating compounds compounds capable of metabolic conversion into carbon monoxide and other biocompatible breakdown products.
- the carbon monoxide generating compound is methylene chloride (MC), which is metabolized exclusively into CO and CO 2 via the cytochrome P-450 oxidative system.
- MC methylene chloride
- Carbon monoxide generated by the metabolism of the subject compounds, e.g., methylene chloride will bind in vivo to hemoglobin so as to increase the patient's carboxyhemoglobin (COHb) level to a therapeutic range.
- the carbon monoxide generating compound is administered to a patient in an amount sufficient to increase the patient's systemic (i.e., blood) COHb level to about 1 - 10%, more preferably 2 - 9%, most preferably 3 - 8%, usually 3 - 10%.
- Monitoring of the resulting COHb levels may be readily accomplished using sensitive assays known and available to the skilled artisan, for systemic monitoring as well as for monitoring in individual tissues or organs. See, e.g., Wong et al, Trans. Am. Gin. Gimatol Assoc. l l l(l):61-75 (2000).
- the subject carbon monoxide generating compounds may be formulated in a variety of ways, depending upon the nature and purpose of administration, the specific inflammatory disease being treated, the particular generating compound, the number of adrninistrations, the inclusion or use of other drugs, and the like, and such may be determined empirically by those skilled in the art.
- the formulation will generally be in a physiologically acceptable form, and may include various carriers or solvents such as water, deionized water, phosphate buffered saline, aqueous ethanol, glucose, propylene glycol, vegetable oils, olive oil or the like.
- the subject carbon monoxide generating compounds may be formulated in a slow release formulation, where the subject compounds may be encapsulated in a wide variety of carriers, may be administered as capsules, or as a prodrug.
- the formulations may also include bacterial agents, stabilizers, buffers, or the like.
- the subject carbon monoxide generators may also find use in adjunctive therapy with other antiinflammatory compounds (e.g., steroids, non-steroidal antiinflammatory agents (NSAIDS), monoclonal antibodies such as Remicade®, cytokine antagonists or inhibitors such as Enbrel® (TNF inhibitor), and the like) or immunosuppressive drugs (e.g., cyclosporine, Prograf® (FK-506), mycophenolate, monoclonal antibodies such as Simulect®, Zenapax®, or other biologies such as
- antiinflammatory compounds e.g., steroids, non-steroidal antiinflammatory agents (NSAIDS), monoclonal antibodies such as Remicade®, cytokine antagonists or inhibitors such as Enbrel® (TNF inhibitor), and the like
- immunosuppressive drugs e.g., cyclosporine, Prograf® (FK-506), mycophenolate, monoclonal antibodies such as Simulect®, Zenapax®, or other biologies such as
- Thymoglobulin®, Lymphoglobuline®, and the like where reduced amounts of the drug may be used, generally reducing the amount employed by at least 25%, more usually at least 40% or more, from the therapeutic dosage for the indication.
- the subject compounds may also be advantageously combined with other agents that may be employed in the treatment of the specific disease indications discussed herein (e.g. antibiotics, anti-metabolites or other cytotoxic agents, human leukocyte antigens, cyclooxygenase inhibitors, lipid-altering agents, ACE inhibitors or other vasodilators, sulfasalazine and related compounds, and the like).
- the subject generator compounds may be administered either in vivo, ex vivo or in vitro, and may be taken parenterally or orally, generally being administered intravascularly, subcutaneously, intravenously or intramuscularly. In vivo delivery also includes, but is not limited to, direct injection via catheter or by other means of perfusion into a vessel, organ or tissue involved in or affected by an adverse proliferative, inflammatory or immune response.
- the subject compounds may be administered intravascularly at a location proximal to a transplanted organ or inflamed tissue, for example, or administered systemically.
- One of ordinary skill in the art will recognize the advantages and disadvantages of each mode of delivery, and will be able to determine a satisfactory means of delivery and delivery regimen without undue experimentation.
- the amount administered will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the host, the manner of administration, the number of administrations and the interval between administrations, and the like, all of which may be determined empirically by those skilled in the art. Applying these factors, the dosage will generally be in the range of about 5-500 mg/kg. When administered parenterally, the total amount of the subject carbon monoxide generating compound per day will generally be in the range of about 1-500 mg/kg, more usually in the range of about 1-
- the dose may be in a single bolus or may be divided up and administered in portions to provide the desired level of carbon monoxide in the host over a period of time, and will be adjusted based on the metabolic conversion rate of the subject compound.
- methylene chloride for example, only about 50-80%) of the compound is converted into carbon monoxide.
- administration of 10-500 mg/kg methylene chloride will typically result in about 3-165 mg/kg CO in vivo.
- Information relating to the pharmacokinetics and metabolism of such compounds is known in the art and available to the skilled artisan for empirically determining the proper dosages. See, e.g., Angelo et al, J. Pharmacokinetics and Biqpharmaceutics 12(4):413-435
- Methylene chloride is a particularly preferred embodiment herein in that it has a near linear dose-response relationship, thus providing the skilled artisan with control over the degree of COHb formation so as to maintain COHb levels within the desired therapeutic range.
- MC provides a considerable advantage over other therapeutic modalities in that the predictability of its dose-response relationship enables maintenance of a therapeutic level of COHb while avoiding the toxicities associated with severe CO poisoning, e.g., carboxyhemglobinemia.
- MC will preferably be orally administered in an amount between about 1 - 100 mg/kg, more preferably between about 1 - 80 mg/kg, most preferably between about 1- 60 mg/kg, generally between about 1- 30 mg/kg.
- the carbon monoxide generating compounds described herein also find use for inhibiting the activation of immune system cells, either by themselves or in conjunction with other immunosuppressant agents, particularly in extending the lifetime of transplants.
- the present invention provides a method for prolonging the acceptance of transplants in a mammalian host, which employs the aciministration of a carbon monoxide generating prior to, concomitant with, subsequent to or a combination thereof with the transplant.
- a particular regimen is employed for administration, where a single bolus or plurality of doses may be administered to the recipient and/or donor before, concomitant with, or subsequent to the implanting of the organ in the recipient.
- the particular protocol will depend upon the nature of the organ, whether the donor, recipient or organ is being treated, the particular carbon monoxide generating compound which is employed, and the use of other immunosuppressants.
- Administration may begin within 14 days prior to the transplant, preferably within about 3 days, and desirably will include the day prior to the transplant and most preferably, the same day as and/or the day after the transplantion. Administration may be on consecutive days or non-consecutive days, generally any gap fewer than 10 days. In a preferred embodiment, administration concomitant with the transplant or on the same day is employed, and in a particularly preferred embodiment administration will begin on the same day as the transplant or the day before, and may be continued until the transplant is stabilized, generally not exceeding twelve months, more usually not exceeding four to twelve weeks. However, after implantation, the subject compounds may be administered as needed, depending upon the response of the recipient to the organ or cells.
- the subject compounds may be administered chronically, as long as the implant is present in the host.
- the carbon monoxide generating compound may also be administered to the donor, usually within three days of the removal of the organ, more usually not later than the day prior to removal of the organ, desirably within about 12 hours of the removal of the organ.
- the subject carbon monoxide generating compounds may be used with a wide variety of hosts, particularly primates, more particularly humans, or with domestic animals, and the like.
- the subject carbon monoxide generating compositions may be used in conjunction with the transplantation of a wide variety of organs, such as kidney, heart, liver, spleen, bone marrow, pancreas, lung, islet of langerhans, etc.
- the graft life will be extended for at least three days beyond what could normally be anticipated in the absence of the subject carbon monoxide compounds, more usually at least five days. This can be useful in areas where xenogeneic grafts have been used awaiting an allogeneic graft, to allow for reduced amounts of immunosuppressants or avoid using immunosuppressants altogether.
- the subject compounds may be used for allogeneic, as well as xenogeneic, grafts.
- EXAMPLE 1 Exogenous CO Administration To examine the effect of gaseous CO on the immune system, C57/BL6 mice (B6, Jackson Laboratory, Bar Harbor, ME) were first exposed to 500 ppm CO in air (Praxair, Danbury, CT) for one hour in a sealed chamber before injection of lipopolysaccharide (LPS) (0.3 mg/kg, i.v., Sigma, St Louis, MO). After injection, they were exposed to another hour in the CO chamber. Blood samples were collected one hour after LPS injection (through the aortic artery) and the COHb level in whole blood was measured by a whole blood AVOXimeter 4000 (A-NOX Systems, San Antonio, TX). Serum samples were separated and were kept at -80°C until analysis. Serum
- LPS lipopolysaccharide
- T ⁇ F- ⁇ was measured by sandwich ELISA (Biosource, Camarillo, CA).
- EXAMPLE 2 Methylene Chloride as a Carbon Monoxide Generating Compound
- MC methylene chloride
- Different concentrations of MC were prepared by using olive oil as a solvent. Mice were treated with MC at 5 mg/kg., 50 mg/kg, and 500 mg kg, p.o., one hour before LPS administration.
- mice treated with MC at 5 mg/kg 3720 ⁇ 1666 pg/ml
- LPS-treated controls 5090.7 ⁇ 1595 pg/ml
- MC at 50 mg/kg and MC at 500 mg/kg had a significant increase in COHb levels (5.48 ⁇ 0.7% COHb, p ⁇ 0.05 and 13.92 ⁇ 1.7% COHb, p ⁇ 0.05) compared to untreated mice.
- mice treated with gaseous CO had a higher level of COHb than mice treated with the inhibitory dosages of MC. This may be due to the fact that the majority of the inhaled CO is captured by pulmonary hemoglobin rather than directed to the target tissue, the liver, and thus leads to a higher COHb level. Conversely, orally administered MC, which is absorbed through the GI tract, is metabolized in liver. Therefore, most of the released CO is centrally located within the liver rather than being bound to COHb. Thus the data indicates that CO generating compounds can be the choice vehicle to deliver potentially therapeutic CO into inflammatory areas in order to inhibit unregulated immune responses. CO generating compounds can be a family of immunosuppressive drug candidates which control allograft rejection and autoimmune diseases.
- Ischemia/reperfusion (I/R) insult is an antigen-independent component of the harvesting injury in orthotopic liver transplantation, and remains one of the major ' limitations of this procedure. Farmer et al, Transplantation Reviews 14(2): 106-116 (2000). The extent of liver damage due to I/R ranges from reversible changes with elevation of liver enzymes to severe injury resulting in cell death and ultimate liver failure. Previous studies have shown that upregulation of HO-1 can protect liver and heart cells from the oxidative stress caused by ischemic and reperfusion insult. Kato et al, Am. J. Transplant. 1:121-28 (2001); Katori et al, Transplantation (in press). To better understand the mechanism of HO-1 mediated protection against I/R injury, this study was designed to test the effects of HO byproduct CO on cold I/R injury in an ex- vivo isolated perfusion rat liver model.
- Isolated perfusion liver apparatus An isolated perfusion liver apparatus was used, as described in Amersi et al, supra, and Maulik et al, Circulation 94:398-406 (1996).
- syngeneic rat blood obtained for each experiment from four donor animals, was diluted to a hematocrit of 15% with Krebs Ringer Bicarbonate Buffer (mM: NaCl 118, KH 2 POF 1, MgSO4 0.9, CaCl 2 2.5, dextrose 11.1, and NaHCO 2 25), and maintained at pH of 7.4.
- Krebs Ringer Bicarbonate Buffer mM: NaCl 118, KH 2 POF 1, MgSO4 0.9, CaCl 2 2.5, dextrose 11.1, and NaHCO 2 25
- the perfusate was pumped from a heated reservoir that warmed the perfusate to 37°C through silastic tubing oxygenator connected to a flow meter that measured portal vein blood flow (Cole Palmer Instruments, Chicago, IL).
- Portal pressure was kept constant via a pressure monometer connected to a T fitting in the portal vein canula.
- the outflow cannula in the inferior vena cava drained into an outflow reservoir.
- pH, temperature and oxygenation were kept constant.
- MPO Myeloperoxidase assay.
- MPO is a naturally occurring constituent of neutrophils and is used as a marker for neutrophil infiltration.
- Frozen tissue samples were thawed and suspended in an iced solution of 0.5% hexadecyltrimethyl- ammonium (Sigma) and 50 mMol potassium phosphate buffer solution (Sigma) with pH adjusted to 5. Samples are homogenized for 30 sec, centrifuged at 15,000 rpm for 15 min at 4°C. 0.1 ml of the supernatant was then mixed in solution of hydrogen peroxide-sodium acetate and tetramethyl benzidine (Sigma).
- the change in absorbance at 460 nm was measured with a Beckman DU spectrophotometer (Beckman Institute, Fullerton, CA).
- the quantity of enzyme degrading 1 ⁇ Mol peroxide per minute at 25°C per gram of tissue was defined as one unit of MPO activity.
- Protein was extracted from liver samples with PBSTDS buffer (50 mM Tris, 150 mM NaCl, 0.1%SDS, 1% sodium deoxycholate, and 1% triton X- 100, pH 7.2). Proteins (30 ⁇ g/ sample) in SDS-loading buffer (50 mM Tris, pH 7.6,
- HO-1 Enzymatic Activity Livers were homogenized on ice in a Tris-HCl lysis buffer (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Samples were frozen in small aliquots until use. Homogenates (100 ⁇ l) were mixed with 0.8 mM
- NADPH 0.8 mM glucose-6-phosphate 1.0 unit G-60P dehydrogenase, 1 mM MgCl 2 and 10 ml purified rat liver biliverdin reductase at 4°C.
- the reaction was initiated by the addition of hemin (final concentration 0.25 mM).
- the reaction mixture was incubated at 37°C in the dark for 15 min. At the end of incubation period, any insoluble material was removed by centrifugation and supernatants were analyzed for bilirubin concentration. An extinction coefficient of 40 mM "1 cm '1 at A 460-530 was used to calculate the amount of bilirubin formed.
- Controls included naive samples in the absence of the NADPH generating system and all the ingredients of the reaction mixture in the absence of graft homogenates.
- Biliverdin reductase was purified from rat liver, as described in Browne and Ultrich, Mol Pharmacol. 32:497-504 (1987).
- ELISAfor HO-1 protein expression Livers were homogenized on ice in a Tris-HCl lysis buffer (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Flat-bottom microtiter 96-well plates (Nunc) were coated with 7 ⁇ g/ml anti-HO-1 mAb (OSA-111, Stressgen, Canada) in PBS for 18 h at room temperature. Unbound Ab was removed by washing (wash buffer: 0.05% Tween 20 in 50 mM phosphate buffer, pH 7.5) and remaining binding sites were blocked by incubation with a 5% BSA/PBS solution (1 h).
- HO-1 Recombinant HO-1 (SPP-730) and tissue homogenate were diluted in assay diluent (0.5% BSA/0.05% Tween 20/PBS) and incubated in anti-HO-1 mAb coated wells for 1 h at room temperature. Subsequently, plates were washed three times with wash buffer and incubated with rabbit anti-HO-1 polyclonal antibody (SPA-895, Stressgen; diluted 1:1000 in assay diluent) for 30 min at room temperature. Bound rabbit IgG was detected with a donkey anti-rabbit (gG-HRP conjugate (711-
- Portal vein resistance is affected by sinusoidal congestion and hepatocyte injury. Addition of CO to the perfusate significantly decreased (p ⁇ 0.001) portal resistance (rnn_H 2 0/mir_ / ml) throughout the 2 h reperfusion period, as compared with controls (Fig. 4). Further, as shown in Fig. 5, CO-treated livers produced significantly more bile (ml g tissue weight), as compared with livers perfused with blood exposed to air alone (p ⁇ 0.005).
- the MPO assay was employed to determine neutrophil activity in liver tissue at the conclusion of 2 h of reperfusion. As shown in Fig. 6, control livers demonstrated a significant increase in MPO activity (4.0 U/mg), as compared with livers that were perfused with CO ( 1.3 ⁇ 0.2; p ⁇ 0.04).
- LY-83583 a cGMP analog that interferes with the action of the nucleotide was added to the perfusate with CO at the time of reperfusion. After 2 h of reperfusion, inhibition of cGMP after adjunctive use of LY-83583 had no significant effects on hepatic function, as compared with livers perfused with CO alone. Although portal blood resistance was slightly increased in the groups perfused with LY-83683
- Exogenous CO can substitute for endogenous HO-1 in preventing hepatic I/R injury.
- depression of endogenous HO-1 activity affects the ability of exogenous CO to protect against I/R injury, we administered ZnPP
- livers pretreated with ZnPP exhibited similar functional features as those in the group perfused with CO alone, i.e. decreased portal vein resistance (Fig. 8), increased bile production (Fig. 9), and improved hepatocyte function, as measured by sGOT levels
- ELISA-assisted detection of HO- 1 protein expression in liver samples revealed markedly diminished HO-1 content in the ZnPP pretreatment group (0.85 ⁇ 0.62) as compared with CO only (7.51 ⁇ 2.13; p ⁇ 0.01) or air only (1.28 ⁇ 1.46; p ⁇ 0.01) perfusion groups (data not shown).
- HO-1 and iNOS Western Blot analysis showed that CO-mediated cytoprotective effects against hepatic I/R injury correlated with upregulation of HO-1 expression.
- HO-1 protein was accentuated ca. 3-fold at 2 h after perfusion with CO, CO + L-NAME, and CO + LY-83583, when compared to the control group (air alone) and the group treated with ZnPP (data not shown).
- Analysis of iNOS expression using Western Blot resulted in no detectable bands in the CO, CO + L-NAME, CO + LY83583, and the CO + ZnPP treated groups; however a low density band was detected in the control livers after 2 h of reperfusion with air alone.
- CO prevents hepatic I/R injury through the activation ofp38 MAPK.
- CO prevents endothelial cell apoptosis via the activation of p38 MAPK transduction pathway (Brouard et al. , supra)
- Livers treated with SB203580, a pyridinyl imidazol p38 MAPK inhibitor, in the presence of CO showed a significant increase in portal vein resistance (p ⁇ 0.025) and produced significantly less bile (p ⁇ 0.01), as compared with livers perfused with CO alone after 2 h of reperfusion (Fig. 8 and Fig. 9, respectively).
- CO-mediated cytoprotective effects were nitric oxide or cGMP -independent, but p38 mitogen activated protein kinase (MAPK)-dependent.
- MPK mitogen activated protein kinase
- CO could substitute for endogenous HO-1 in preventing hepatic I/R injury through the activation of p38 MAPK.
- CO administration has potential therapeutic application in preventing hepatic I/R injury and expanding the liver donor pool for transplant recipients.
- EXAMPLE 4 Methylene Chloride Administration Prevents Apoptosis and Extends Liver Allograft Survival
- Apoptosis, or programmed cell death, is critical for the homeostasis of the immune system, and plays a central role in the destructive phase of acute allograft rejection by cytotoxic T lymphocytes (CTLs).
- CTLs can utilize a variety of mechanisms to lyse target cells, including the CD95/FAS system. Ju et al, Proc. Natl Acad. Sci. USA 91:4185-89 (1994). This study investigated the effects of CO as a downstream mediator of HO-1 in preventing CD95/ FAS-mediated apoptosis and prolonging allogeneic OLT survival.
- Ad-HO-1 recombinant adenovirus (Ad) encoding Fas ligand (Ad-CD95), heme oxygenase 1 (Ad-HO-1) and ⁇ -galactosidase reporter gene (Ad- ⁇ -gal).
- Ad-HO-1 was generated, as described in Shibahara et al, Proc. Nat 'I Acad. Sci. USA
- Ad-HO-1 or Ad- ⁇ -gal were added and incubated for 1 hr with 100 ⁇ l of DMEM without serum. The medium was then removed and changed to 100 ⁇ l of DMEM with 2% FBS for incubation 36-48 hr. After removing medium and washing cells three times, 10 ⁇ l of MTT (5 mg/ml, Sigma Chemical, St. Louis, MO) was added to each well and incubated for 4 hr. After removal of medium, 100 ⁇ l of isopropyl alcohol with 0.01% HCl was added. An enzyme-linked immunosorbent assay reader was used at OD of 550. The percent of cytotoxicity was calculated as: 1 - OD experimentaJ/OD control x 100%.
- Ad-HO-1 transduction in OUT model Male Dark-Agouti (DA; RT1 3 ) and Lewis (LEW; RT1') rats of 10-16 weeks of age were purchased from Harlan Sprague
- Transplant 1 121-28 (2001). Ex-vivo gene transfer into liver grafts was performed during cold preservation (4°C) via perfusion of the portal vein with 2 ml of cold lactated Ringer's solution containing 5x10 10 pfu (plaque-forming unit) of Ad-HO-1. Control grafts were perfused with 5xl0 10 pfu of Ad- ⁇ -gal. Animals were followed for survival. Separate groups of recipients were sacrificed at day 3, 7 and 10 post- transplant, OLTs were harvested for histological evaluation, whereas blood samples were collected for measurement of sGOT levels.
- Liver allografts were harvested at day 3, 7 and 10 post-transplant. The tissue was sliced into small pieces, preserved in 10% neutral-buffered formalin, cut into 5- ⁇ m section, and stained with hematoxylin and eosin (H&E) by standard methods. In vivo detection of apoptosis. A commercial in situ histochemical assay
- Ad-HO-1 gene transfer prevents CD95 /Fas-mediated apoptosis in vitro.
- Cytotoxicity assay and TUNEL staining were used to analyze the effects of Ad-based HO-1 overexpression in vitro.
- CD-95-mediated cytotoxicity to Fas-bearing YAC-1 target cells was consistently diminished in Ad-CD95 + Ad-HO-1 transfected group, as compared with Ad-CD95 + Ad- ⁇ -gal control. Indeed, at MOI of 5, 10 and 20, the cell death rate of 39%, 49%, and 76.5% in controls was significantly QXO.001) higher as compared with 4.5%, 7% and 14% cell death in Ad-CD95 + Ad- HO-1 group.
- Ad-HO-1 gene transfer prolongs allogeneic OLT survival, ameliorates histological signs of acute rejection, and improves hepatic function.
- Untreated LEW rats died within 10 days following orthotopic transplantation of DA livers (see Table 1 below).
- MST mean survival time
- the survival of Ad-HO-1 transfected OLTs increased significantly to > 32 ⁇ 42 days, with 2 out of 12 livers maintained for >120 days (Table 1).
- the X-gal positive staining after Ad- ⁇ -gal transfection was ca.
- OLTs in Ad- ⁇ -gal control group showed progressive signs of severe acute rejection, with necrosis, hemorrhage, and less than 25% of the hepatic parenchyma viable by day 10 post-transplant.
- the corresponding OLT samples in the Ad-HO-1 group exhibited mild to moderate rejection, with dense inflammatory infiltrate, but more than 90% of parenchyma preserved.
- sGOT levels were decreased in AD-HO-1 gene transfer group (412 ⁇ 105), as compared with Ad- ⁇ -Gal controls (1208 ⁇ 611; p ⁇ 0.05).
- Ad-HO-1 gene transfer prevents apoptosis and upregulate the expression of anti-apoptotic molecules in allogeneic OLTs.
- liver allografts in the Ad- ⁇ - gal group showed hepatocellular apoptosis with dense nuclear margination (64 ⁇ 25 of TUNEL+ cells/field).
- the number of apoptotic cells in allogeneic OLTs that underwent AD-HO-1 gene transfer remained within background levels (0.8 ⁇ 0.7 of TUNEL+ cells/field; p ⁇ 0.05). Upregulation of endogenous CO prolongs allogeneic OLT survival.
- MST ⁇ SD >47 ⁇ 46 days
- OLTs in untreated recipients showed severe acute rejection, with dense inflammatory infiltrate, portal/central veins showing necrotizing endothelitis, and less than 10% of the hepatic parenchyma viable.
- OLTs harvested from methylene chloride-treated hosts showed a mild to moderate inflammatory infiltrate and central vein endothelitis, indicating mild to moderate rejection, and more than 80% of parenchyma well preserved.
- Ad-HO-1 gene therapy prevented CD95/Fas- mediated apoptosis in vitro, while enhanced in vivo HO-1 expression via Ad-HO-1 gene therapy significantly prolonged animal survival after allogeneic OLT, decreased histological severity of acute rejection and preserved hepatocyte architecture, and also improved OLT function as measured by sGOT levels.
- daily feedings of OLT recipients with methylene chloride alone and with no other immunosuppression uniformly prevented ca. 10 day acute OLT rejection and significantly prolonged animal survival, with ca. 50% of rat recipients surviving >3 weeks. Elevated levels of CoHb following methylene chloride administration (from ca. 1.3% in untreated rats to ca.
- CO can also ameliorate graft rejection by depressing the fibrynolytic axis (Fujita et al), inhibiting platelet aggregation (Brune and Ullrich, Mol Pharmacol 32:497-504 (1987) and/or promoting vasodilation (Motterlini et al, Cir. Res. 83:568-77 (1998).
- Chronic rejection is characterized by allograft arteriosclerosis, a diffuse, progressive narrowing of the graft vessels due to intima hyperplasia. Libby and Roper, Immunity 14:387-97 (2001). Both I/R injury and immune responses against incompatible MHC antigens expressed by endothelial cells are viewed as the initiating causes of the disease. Endothelial cell destruction and/or activation as well as leukocyte infiltration of the intima and the adventitia lead to abnormal proliferation and migration of VSMCs from their normal position in the media to the intima subendothelial space, and to abnormal vasoconstriction. Id. Unlike acute rejection, there has been little progress in reducing the rate of chronic rejection in the last decades and there is an urgent need for new treatment strategies.
- HO-1 has been shown to suppress inflammation in pathological situations relevant to chronic rejection such as ischaemia reperfusion injury (Amersi et al, supra), atherosclerosis (Ishikawa et al, Cir. Res. 88:506-604 (2001)), neointima formation following arterial injury (Togane et al, Am. J. Physiol. Heart Circ. Physiol. 278:623-32 (2000)) as well as xenogeneic (Soares et al. and Sato et al, supra) and allogeneic graft rejection (Woo et al.
- HO-1 may protect from chronic rejection by acting on immune and non-immune components of the disease.
- CO was shown to suppress the pro-inflammatory phenotype associated with monocyte macrophage activation (Otterbein et al, supra), to protect a variety of cell types from undergoing apoptosis (Petrache et al, Am. J. Physiol. Lung Cell. Mol. Physiol. 278:L312-319 (2000); Brouard et al, supra), to suppress xenograft rejection (Sato et al, supra) and to depress fibrinolysis (Fujita et al, supra).
- the present example demonstrates the effects of CO delivery in a well- characterized and widely used model of chronic aorta allograft rejection (Libby, supra) using the carbon monoxide generating compound methylene chloride.
- the descending thoracic aortas were harvested, perfused with saline and anastomosed to the recipient's abdominal aorta below the renal arteries and above the aortic bifurcation. Anastomosis was performed in a termino-lateral fashion and the recipient abdominal aorta was ligated between the two-graft anastomosis. Grafted aortas were harvested 30 days after transplantation.
- aorta segment was fixed with 10% formaldehyde for morphometric evaluation and another segment was embedded in OCT compound (Tissue Tek, Miles Laboratories, Elkhart, IN) and frozen in liquid nitrogen for immunohistological analysis.
- OCT compound Tissue Tek, Miles Laboratories, Elkhart, IN
- MC Sigma, St. Louis, MO
- This dose saturates the cytochrome P- 450 oxidative system, and yields maximal COHb values of 10% COHb in venous or aortic blood.
- COHb levels were evaluated in heparinized venous blood using the VOXimeter sensor (A- VOX Systems, San Antonio, TX) and expressed as the percentage of total hemoglobin.
- Bicarbonates, soluble CO 2 , total CO 2 and pH were measured using standard clinical biochemistry techniques (Laboratory of
- AdHO-1 adenovirus coding for HO-1
- AdHO-1 contains an expression cassette with the human CMV promoter and the human HO-1 cDNA fused to a Flag sequence in its 3' end.
- Donor aortas were harvested, recombinant adenoviruses (10 10 IP in 200 ⁇ l of DMEM supplemented with 1% FCS) were infused into the lumen and both extremities were ligated. Aortas were then incubated for 45 min at 37°C 5% CO 2 , flushed with DMEM to remove non- incorporated adenoviruses and transplanted into recipients. Histology and morphometric analysis. After formaldehyde fixation, aorta segments were embedded in paraffin and 5 ⁇ m sections were stained with hematoxylin-eosin-saffron (HES). Microscopic images were collected using a color camera.
- HES hematoxylin-eosin-saffron
- mice M2 mouse Mab anti-Flag (clone M2) (Sigma, St. Louis, MO) or a mouse Mab anti- ⁇ -tubulin (Calbiochem, San Diego, CA). They were then washed and incubated (2 h, room temperature) with a HRP -labeled anti-rabbit or anti-mouse IgG antibody (Jackson Immunoresearch, West Grove, PA) and detected with enhanced chemoluminiscence (Amersham, Arlington Heights, IL) using x-ray films.
- HRP -labeled anti-rabbit or anti-mouse IgG antibody Jackson Immunoresearch, West Grove, PA
- Immunohistological analysis was performed on cryostat sections as previously described in detail. Guillot et al, J. Immunol 164:5258-68 (2000). Immunohistological analysis of infiltrating leukocytes was performed at day 30 after transplantation using the following mouse Mab: a mixture of two anti- leukocyte CD45 Mabs (OX1 AND OX30), anti-monocyte/ macrophage CD68 (EDI), anti- ⁇ TCR (R.7.3), anti-CD4 (W3/25), anti-CD8 ⁇ chain (OX8), anti-monomorphic class II MHC antigens (OX6), anti-CD25 (OX39) (all fromECACC, Wiltshire, UK), anti-CD54 (ICAM-1) (Seikagaku America Inc., Rockville, MA), anti-CD86 (B7.2) (Pharmingen, Franklin Lakes, NJ) and an irrelevant mouse Mab (3G8, anti-human CD 16). VSMCs were detected using a mouse anti- leukocyte CD
- Donor LEW.1 W splenocyts were incubated with heat-inactivated serum from ELW.1 A recipients, serially diluted in PBS. Cells were then washed and simultaneously incubated with FITC-coupled donkey anti-rat IgG
- HO-1 after adenovirus-mediated gene transfer and CO release after MC administration.
- the expression of HO-1 following infection with AdHO-1 was confirmed in cultured rat ECs and in aortas. Untreated rat ECs and Addl324- transduced cells showed low levels of endogeneous HO-1 expression whereas Ad-HO-
- HO-1 transduced ECs displayed strong expression of HO-1 as detected by Western blot with anti-HO-1 and anti-Flag antibodies. Due to the presence of the Flag peptide, HO- 1 expressed following AdHO-1 -transfection has a higher molecular weight than endogenous HO-1 (33 vs. 32 kDa).
- the anti-Flag antibody displayed a band of the expected molecular weight only in AdHO-1 transduced EC despite a non-specific cross-reactivity in control cells.
- the enzymatic activity of HO-1 (the generation of bilirubin) was augmented in cells transduced with AdHO-1 compared to Addl324 transduced cells.
- HO-1 was confirmed by immunohistology in aortas transduced with AdHO-1 using anti-HO-1 and anti-Flag antibodies. Expression of the HO-1 -Flag molecule was absent in control adenovirus-treated tissue and anti-Flag antibodies. HO- 1 expression by the endothelium was also detected in AdHO- 1 but not Addl 324- transduced aortas by immunohistology on whole aorta fragments, using a previously described technique (Merrick et al , supra). HO-1 expression was detected up to day 10 in transplanted aortas and was absent at day 15. These results indicate that HO-1 vectorized by AdHO-1 was expressed following gene transfer into ECs and aorta.
- CD68+ macrophages and to a lesser extent T lymphocytes (Table 2).
- the T cell population contained more CD4+ cells than CD8+ cells (Table 2).
- non- codant adeno virus-treated allogeneic aortas showed VSMCs in the intima and a reduced number in the media (Table 2) .
- Table 2
- Frequency of stained cells was graded as: -, not present; +, low; ++, moderate; +++ frequent
- Syngeneic aortas showed weak labeling of ICAM-1 on the endothelium, no labeling for B7.2 and MHC class II antigens and low to moderate labeling in the media and adventitia for IP 10, TGF ⁇ l and iNOS (Table 3).
- Control- adenovirus-treated allogeneic aortas showed large numbers of cells in the intima and adventitia strongly expressing ICAM-I, B7.2 and MHC class II antigens (Table 3).
- IP 10, TGF ⁇ l and iNOS expression was also increased in the intima and adventitia and additionally in the media of control adenovirus-treated aortas, as demonstrated in Table 3 below.
- Frequency of stained cells was graded as: -, not present; 4-, low; 4-4-, moderate; 4-H- frequent
- AdHO- 1 -treated aortas displayed a reduced number of cells expressing ICAM- 1, B7.2, MHC class II antigens. IP10, TGF ⁇ l and iNOS were expressed with less intensity compared to control adenovirus-treated aortas (Table 3). CO-treatment through MC administration moderately reduced the expression of ICAM-1, B7.2 and MHC class II molecules (Table 3). IP 10 expression was reduced in the media and adventitia but not in the intima whereas TGF ⁇ 1 and iNOS expression was reduced in the intima but not in the media or adventitia (Table 3). IL-2 receptor (CD25) and IFN ⁇ were detected in rare and dispersed cells of allografts without differences between the experimental groups and were not detected in syngeneic grafts (data not shown).
- Ad-HO-1 effect could be explained by the production of biliverdin and bilirubin within Ad-HO-ltransduced EC, as well as iron depletion, thus inhibiting EC activation and therefore leukocyte adhesion and tissue infiltration. Simultaneously,
- CO diffusing from Ad-HO-ltransduced EC could act not only on adjacent EC and macrophages but also on VSMCs, inhibiting their apoptosis, proliferation and activation.
- the transient expression of HO-1 mediated by Ad-HO-1 which is undetectable at day 15 after transplantation, may explain a more efficient effect on the early leukocyte infiltration phase and a less pronounced effect on later VSMC proliferation.
- methylene chloride therapy was administered continuously throughout the experiment and could have inhibited VSMC proliferation more effectively than leukocyte infiltration.
- CO delivery may also produce higher levels of CO in the arterial wall compared to Ad-HO-1 gene transfer. Analysis of alloantibody levels in recipients with grafts treated with AdHO-1- and after CO delivery.
- Alloantibodies are produced in secondary lymphoid organs and reflect CD4-dependent alloreactivity. Alloantibodies have been implicated in the development of chronic rejection in certain but not all models (Libby and Pober, supra). A predominance of anti-donor MHC class II alloantibodies has been previously described associated with long-term allograft survival (Cuturi et al, Eur. J.
- HO-1 gene transfer or CO therapy mainly act through local immunosuppressive effects on effector mechanisms.
- both adenovirus-mediated HO-1 gene transfer into the endothelium of the aorta and CO delivery resulted in a significant reduction in intima thickness compared to control non-coding adenovirus-treated aortas.
- Aortas transduced with Ad-HO-1 or treated with CO showed a reduction in the number of macrophages, T cells and CD4+ cells as well as in the expression of adhesion molecules, costimulatory molecules and cytokines, with the gene transfer displaying a more pronounced effect than the CO treatment.
- CO inhibited VSMC accumulation in the intima and preserved the vascular media more efficiently than Ad-HO-1 treatment Based on the observation that CO therapy using methylene chloride revealed an inhibition of chronic rejection similar to that obtained with AdHO-1, the above results suggest that CO can mediate protective effects associated with increased expression of HO-1.
- CIA Rat Collagen- Arthritis Model Collagen-induced arthritis
- type II collagen in Freund's incomplete adjuvant susceptible rats develop polyarthritis with histologic changes of pannus formation and bone/cartilage erosion.
- humoral and cellular responses to collagen type II occur in CIA as well as rheumatoid arthritis.
- CIA is a useful and accepted animal model for rheumatoid arthritis that serves as an in vivo system for the exploration of inflammatory synovitis etiologies and for the investigation of potentially new therapeutic interventions.
- methylene chloride as a carbon-monoxide generating compound in this disease model
- female Lewis rats weighing between 120 and 150 g were injected intradermally with 0.5 mg native chicken collagen type II solubilized in 0.1M acetic acid and emulsified in incomplete Freund's adjuvant.
- animals were divided into three groups.
- a blinded analysis of bone erosion by X-ray confirmed the therapeutic effect of methylene chloride therapy.
- the X-ray score for limbs from vehicle treated animals was 4.8+/-0.7.
- Methylene chloride therapy with 100 mg/kg/day resulted in a score of 2.7+/-0.8, therapy with 500 mg/kg/day in a score of 1.8+/-0.7. This difference was statistically significant (p ⁇ 0.05).
- the carotid is incised with Vannas scissors proximal to the ligature.
- a curved flexible wire (0.35 mm/0.014 in diameter) is introduced into the external carotid and passed three times along the wall of the common carotid while being rotated. Upon removal of the wire the proximal carotid ligature is tied and the skin is reopposed with 6-0 silk.
- Methylene chloride (25, 100, 400 mg/kg/day) is administered intraperitoneally or orally starting on day -1 until day 28.
- a control group is treated with vehicle.
- animals are sacrificed and after incision, the right and left common, external, and internal carotids are ligated. After sternotomy, common carotids are dissected further to the aortic arch.
- a 27-gauge needle is placed in the left ventricle and a systemic perfusion with phosphate buffered paraformaldehyde (100 M, 4% wt/vol, pH 7.3) is performed at 100 mmHg via the left ventricular cannula.
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- 2002-04-01 IL IL15818202A patent/IL158182A0/en unknown
- 2002-04-01 EP EP02757910A patent/EP1381354A2/en not_active Withdrawn
- 2002-04-01 RU RU2003131675/15A patent/RU2003131675A/en not_active Application Discontinuation
- 2002-04-01 CA CA002442457A patent/CA2442457A1/en not_active Abandoned
- 2002-04-01 MX MXPA03008820A patent/MXPA03008820A/en unknown
- 2002-04-01 WO PCT/US2002/010115 patent/WO2002078684A2/en not_active Application Discontinuation
- 2002-04-01 US US10/115,276 patent/US20030068387A1/en not_active Abandoned
- 2002-04-01 JP JP2002576950A patent/JP2004526739A/en active Pending
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US9062089B2 (en) | 2011-07-21 | 2015-06-23 | Alfama, Inc. | Ruthenium carbon monoxide releasing molecules and uses thereof |
US9611286B2 (en) | 2011-07-21 | 2017-04-04 | Alfama, Inc. | Ruthenium carbon monoxide releasing molecules and uses thereof |
Also Published As
Publication number | Publication date |
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JP2004526739A (en) | 2004-09-02 |
US20030068387A1 (en) | 2003-04-10 |
CA2442457A1 (en) | 2002-10-10 |
IL158182A0 (en) | 2004-03-28 |
WO2002078684A3 (en) | 2002-12-19 |
RU2003131675A (en) | 2005-03-10 |
MXPA03008820A (en) | 2004-07-30 |
EP1381354A2 (en) | 2004-01-21 |
CN1507348A (en) | 2004-06-23 |
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