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WO2010020786A1 - Protection d'organe - Google Patents

Protection d'organe Download PDF

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Publication number
WO2010020786A1
WO2010020786A1 PCT/GB2009/002039 GB2009002039W WO2010020786A1 WO 2010020786 A1 WO2010020786 A1 WO 2010020786A1 GB 2009002039 W GB2009002039 W GB 2009002039W WO 2010020786 A1 WO2010020786 A1 WO 2010020786A1
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alkyl
aryl
camp
cells
compound
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PCT/GB2009/002039
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English (en)
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Leo Sebastian Price
Bob Van De Water
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Universiteit Leiden
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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

Definitions

  • the present invention provides compositions, medicaments and methods for use in preventing and/or alleviating the damaging effects of hypoxia and/or reperfusion injury.
  • Exchange pjoteins directly activated by cAMP are a family of guanine nucleotide exchange factors for Rap GTPases and are activated by direct binding of cAMP.
  • Rap proteins are members of the Ras family of GTPases and include Rapl and Rap2 which are known to control a number of cellular functions, including cell-cell and cell-matrix adhesion.
  • reperfusion injury refers to damage caused by reperfusion of a hypoxic organ with blood.
  • the precise mechanisms by which reperfusion injury occurs are varied and complex but are known to involve the release of inflammatory factors and free radicals.
  • hypoxia related damage and reperfusion (or ischaemic reperfusion) injury is the primary cause of damage to transplanted kidneys and results from the state of hypoxia that develops during cold storage of the donor organ. After periods of cold storage, reperfusion with oxygenated blood (i.e. following connection of the donor organ to the recipients circulation), can lead to inflammation and the induction of oxidative stress pathways.
  • An essential part of any organ transplantation process is an effective storage protocol which preserves the organ and minimises damage, ensuring the organ is maintained in an optimal morphological and biochemical state from the time of retrieval to the time of transplantation and offers means not only to avoid the insults of initial cold ischaemia, but also the damage which may occur following reperfusion.
  • hypoxic conditions and sudden reperfusion lead to cell damage may include, for example, trauma (particularly head trauma) or stroke.
  • trauma particularly head trauma
  • stroke the cells of a particular tissue (for example, brain tissue) may be deprived of oxygen and/or nutirents by, for example, a blood clot which blocks or reduces the flow of blood to that tissue.
  • a blood clot which blocks or reduces the flow of blood to that tissue.
  • the resulting hypoxia leads to cell death which, depending on the particular tissue involved, may have serious implications for the patient.
  • the damage caused by the limited availability of oxygen and/or nutrients may be further compounded by damage resulting from reperfusion.
  • the mechanisms involved in reperfusion injury occuring as a result of trauma or stroke are as decribed above. It should be noted that owing to the nature of the condition, the damage resulting from localised ischaemic events (such as stroke or trauma) may be referred to as acute cellular degeneration.
  • compositions, medicaments, and methods capable of eliminating, reducing and/or alleviating the damage caused by the development of hypoxic conditions within organs and/or reperfusion of organs.
  • the present invention concerns exchange proteins directly activated by cAMP (Epacs) and the finding that these are highly expressed in a number of organs. More specifically, the inventors have discovered that compounds which are capable of modulating Epacs activity, may be useful in treating and/or preventing ischaemic events.
  • Epacs exchange proteins directly activated by cAMP
  • the present invention provides a use of a compound capable of modulating the activity of Epacs for treating or preventing ischaemia induced injury.
  • the present invention provides a use of a compound capable of modulating the activity of Epacs for the manufacture of a medicament for treating or preventing ischaemia induced injury.
  • said compound is specific for modulating the activity of Epacs, that is it may not substantially effect the activity of other proteins or molecules, such as protein kinase A (PKA).
  • PKA protein kinase A
  • ischaemia induced injury encompasses diseases and/or conditions which may consist of, begin with, or involve, an ischaemic event or the development of ischaemia. Ischaemic events may, in turn, comprise the development of hypoxic conditions within, for example, cells, tissues and/or organs.
  • diseases and/or conditions which may be regarded as ischaemia induced injuries are those known in the art as “reperfusion injury” and/or “ischaemic reperfusion injury” where reperfusion of a hypoxic tissue and/or organ may lead to damage caused by inflammatory mediators and/or the formation of free radicals.
  • ischaemia induced injury may include injury resulting from conditions such as stroke, coma and/or trauma (particularly head trauma) whereby, as a result of, for example, the development of a clot or other such blockage stemming the flow of blood to a tissue/organ, cells of that tissue/organ are deprived of oxygen and/or nutrients, become hypoxic and begin to die or undergo apoptosis.
  • conditions such as "stroke", trauma and/or coma may be temporary and thus the ischaemia induced injury may be an acute event resulting in localised cell death or degeneration.
  • reperfusion of the hypoxic tissue may result in further damage through reperfusion injury.
  • ischaemia induced injury encompasses reperfusion injury.
  • prevention may relate to the prophylactic use and/or administration of the medicaments and/or treatment regimes provided by this invention.
  • treatment may be held to encompass the act of curing a disease and/or resolving a condition, it may also more generally relate to alleviating and/or reducing the symptoms and/or effects of that disease and/or condition.
  • Epac is a cAMP-dependent exchange factor (activator) of the small GTPase protein "Rap” and, without wishing to be bound by theory, it is thought that compounds capable of modulating Epac activity are useful in the treatment of ischaemia induced injury (such as reperfusion injury) as they modulate Epac-Rap signalling which in turn may prevent disruption of the actin cytoskeleton as well as cell detachment and apoptosis.
  • ischaemia induced injury such as reperfusion injury
  • the compound capable of modulating the activity of Epacs and which may be used in any of the medicaments, methods and/or compositions described herein is a cAMP analogue.
  • An exemplary compound useful in the present invention has the structural formula given as Formula (I) below:
  • Ri can be independently H, halogen, azido, alkyl, aryl, amino-alkyl, amido- aryl , OH, O-alkyl, O-aryl, SH, S-alkyl, s-aryl, SeH, Se-alkyl, Se-aryl, amino, NH- alkyl, NH-aryl, N-bisalkyl, N-bisaryl, cycloalkylamino;
  • R 2 can be independently H, halogen, azido, O-alkyl, S-alkyl, Se-alkyl, NH- alkyl, N-bisalkyl, alkyl-carbamoyl, cycloalkylamino, silyl;
  • R 3 can be independently H, halogen, OH, azido, amino-alkyl, amido-aryl, O- alkyl, O-aryl, SH, A-alkyl, S-aryl, amino, NH-alkyl, NH-aryl, N-bisalkyl, N-bisaryl, NH-alkyl-carbamoyl, cycloalkylamino; and wherein
  • R 5 is O(H), S(H), amino, H, alkyl, O-alkyl, O-aryl, S-alkyl, S-aryl, NH-alkyl, NH-aryl, N-bisalkyl, N-bisaryl; or
  • R 4 is O (H), S (H), amino, H, alkyl,O-alkyl,O-aryl, S- alkyl, S-aryl, NH-alkyl, NH-aryl, N-bisalkyl, N-bisaryl; and
  • R 5 is O(H) or S(H) ; and pharmaceutically acceptable salts, esters, and/or solvates thereof.
  • the compound capable of activating Epacs is specific and does so without activating other targets of cAMP.
  • the most prominent target of cAMP is protein kinase A (PKA) and concentrations of cAMP analogues that activate PKA can have serious side effects, particularly when administered systemically.
  • PKA protein kinase A
  • concentrations of cAMP analogues that activate PKA can have serious side effects, particularly when administered systemically.
  • the compounds capable of activating of activating Epacs provided by this invention do not activate PKA.
  • a preferred embodiment of the invention may involve the use of the cAMP analogue 8CPT2'-O-Me-cAMP (otherwise referred to as 007).
  • exemplary compounds useful in the compositions, medicaments and methods described herein are detailed in WO03/104250.
  • One of skill in the art will appreciate that other compounds capable of modulating the activity of Epacs, and in particular those which are capable of modulating Epac-Rap signalling, may also be useful in the treatment and/or prevention of ischaemia induced injury.
  • 007 mimetic compounds, structural analogues and the like may be useful in this way.
  • the compound capable of activating Epacs is an 8CPT2'- O-Me-cAMP modified to include an acetoxymethyl group.
  • Such a compound may otherwise be known as 8CPT2'-O-Me-cAMP-AM (or "007-AM").
  • Compounds of this type are particularly advantageous as they are more easily taken up by cells.
  • the present invention provides a method of treating or preventing an ischaemia induced injury, said method comprising the step of administering to a patient, a pharmaceutically effective amount of a compound capable of modulating the activity of Epacs.
  • the medicaments and treatment regimes provided by this invention treat or prevent ischaemia induced injury
  • a patient who has received or will receive a donor organ or organs may be administered a medicament or subjected to a treatment regime provided by this invention prior to undergoing surgery or receiving the donor organ and/or for a period thereafter.
  • the donor organ may be protected against the damaging effects caused by reperfusion with the recipient's blood.
  • the present invention may find particular application in the field of organ storage and/or organ preservation.
  • the present invention may be useful in the preparation of compositions known to those skilled in this field as "organ preservation solutions" which are formulated so as to provide a medium in which organs can be stored for prolonged periods of time without becoming hypoxic or incurring substantial damage.
  • organ preservation solutions which are formulated so as to provide a medium in which organs can be stored for prolonged periods of time without becoming hypoxic or incurring substantial damage.
  • the storage of an organ using an organ preservation solution is particularly preferable as the organ may maintain the morphological and biochemical characteristics of the organ at the time of retrieval.
  • organ may be taken to refer to whole organs or to samples, portions or biopsies thereof.
  • organ may be taken to encompass tissues, cells and/or components of the circulatory system such as veins or arteries which may be grafted to a patient.
  • the subject is a human subject, but other non-human organisms may also receive transplants and as such the compositions of the present invention may also be used in the storage/preservation of non-human animal organs.
  • Organs which may be protected from ischaemia induced injury may include, for example, the kidney, liver, heart, lung, pancreas and the like and the organ may be obtained, although not necessarily so, from a cadaver.
  • the human or animal body from which the organ is retrieved shall be referred to hereinafter as the "donor".
  • organ storage In the field of organ transplantation, correct organ storage is essential. Once an organ has been retrieved or removed from a donor, it may be necessary to place the organ into cold storage so as to minimise morphological/biochemical changes and/or cell death. During cold storage, an organ may become hypoxic and susceptible to damage caused by reperfusion with the recipient's blood (i.e. reperfusion injury). As such, there is a drive to provide improved storage conditions.
  • the fourth aspect of this invention provides a composition for use as an organ preservation solution, wherein the composition comprises a compound capable of modulating the activity of Epacs.
  • the present inventors have determined that a number of organs (particularly the kidney and the epithelial tubules cells thereof) exhibit high levels of Epac expression and as such are particularly susceptible to the effects of the compounds described herein which, insofar as they modulate Epac activity, may prevent disruption of the actin cytoskeleton, cell detachment and apoptosis - processes which occur during ischaemia induced injury.
  • administering may encompass the act of immersing an organ in a composition according to the fourth aspect of this invention. Additionally, or alternatively, the term “administering” may involve perfusing the organ with a composition according to the fourth aspect of this invention and using procedures and apparatus well known in the art.
  • a compound capable of modulating the activity of Epacs for example a compound having structural formula (I), a functionally active analogue and/or derivative thereof, 8CPT2'-O-Me- cAMP or cAMP mimetic compounds
  • the term “administering” may encompass the act of immersing an organ in a composition according to the fourth aspect of this invention. Additionally, or alternatively, the term “administering” may involve perfusing the organ with a composition according to the fourth aspect of this invention and using procedures and apparatus well known in the art.
  • the organ should be administered a composition according to this invention, as soon as possible after removal from the donor. In this way, it may be possible to ensure that immediately prior to being transplanted, the organ retains the morphological and biochemical features present at the time of retrieval from the donor.
  • organ storage is best performed at a reduced temperature such as 2 - 10 0 C and that prior to use the organ may be immersed and/or reperfused with a solution at or close to body temperature, such as about 37°C.
  • a (in vitro) method of protecting an organ against ischaemia induced injury comprising the step of administering a composition according to the fourth aspect of this invention to an organ.
  • the composition is in the form of a solution which may further comprise one or more other components.
  • compositions provided by the fourth aspect of this invention may comprise other components such as, for example, those used in solutions such as University of Wisconsin (UW) solution (see US 4,798,824 and 4,879,283, for example), Celsior and Belzer MPS solutions.
  • Other components may include, for example, sources of nutrition and/or acid-base buffers to help maintain the pH of the solution.
  • Typical buffers may be based on phosphates, such as KH 2 PO 4 .
  • the composition may comprise other compounds such as, compounds providing or acting as a source of potassium and/or sodium. Such compounds may be particularly useful when the solution is required to have a certain osmolality.
  • Other components may include, or be selected from, for example, starch; hydroxyethyl starch; lactobionic acid; sodium and/or potassium gluconate; glucose; CaCl 2 ; potassium phosphate; EDTA or other metal chelating agent such as chelex magnesium sulphate; raffinose; dextran; recombinant albumin; further agents which protect against ischaemic insult, such as adenosine; antioxidants, such as allopurinol and/or pentafraction.
  • Additional optional components may include antibiotics, such as penicillin; insulin (to aid in glucose uptake) and/or antiinflammatories such as dexanethasone.
  • the composition and/or those other components to be added thereto may be in solid form such that they may be reconstituted in a suitable solution (for example water) prior (preferably immediately prior) to use.
  • a suitable solution for example water
  • the composition in its solid form may further comprise the one or more other components mentioned above or, additionally, or alternatively, all or some of these may be present in the solution in which the composition is to be reconstituted.
  • FIG. 1 007 Caspase activation is a critical event in the induction of cell death by apoptosis. Caspase activation induced by cisplatin treatment is inhibited by treatment with 007 in primary mouse kidney epithelial cells, demonstrating that 007 protects kidney cells from damage. Cell cycle analysis is an alternative method of measuring cell death by apoptosis. Cell cycle analysis also demonstrates that 007- treatment protects against cisplatin-induced injury.
  • FIG. 2 Microscopy images of control isolated primary proximal tubule cells and cells treated with cisplatin in the presence and absence of 007 demonstrate that 007 treatment protects against cisplatin-induced cell damage. Note rounded, detached and fewer cells in cisplatin-treated cells without 007 (top right) compared to cells treated with 007 (bottom right).
  • FIG. 3 Intra-renal injection of 007 during ischaemia induced injury significantly reduces injury as determined by measurement of plasma urea levels. Plasma samples were taken 24 hours after ischemic insult, which consisted of clamping the renal artery for 25 minutes to restrict blood flow. Reduced ischemic injury in animals treated with 007 was confirmed histologically by a pathologist (not shown).
  • FIG. 4 007 was injected via the tail vein in mice. After 30 minutes, mice were sacrificed, kidneys were isolated and frozen in liquid nitrogen. Kidneys were homogenised in lysis buffer and active Rap (the target of the 007-Epac complex) was precipitated using a probe specific for the active (GTP-Rap) form. Precipitated protein was resolved by SDS-PAGE and following Western blotting, identified by anti Rapl antibodies. Equivalent amounts of total protein (containing active + inactive Rap) was similarly analysed (lower panel). The results show that Rapl is specifically activated by intravenous 007 injection compared to saline vehicle. Similar results were also obtained with intravenous injection of 20 mg/kg 007 (not shown).
  • Figure 5 Assay of proportion of proximal tubule epithelial monolayer remaining after chemical hypoxia in the presence and absence of 007.
  • Confluent cells in 6-well dishes were treated with serum-free medium alone (control) or serum-free medium containing deoxyglucose + antimycin A (anoxia) for 60 minutes in the presence ('007') or absence ('no 007') of 007. Cells were washed to remove detached cells and the proportion of remaining attached cells was evaluated.
  • Figure 6 Effect of 007 on morphological changes induced by hypoxia by oil immersion.
  • Cells were incubated with (C) 50 ⁇ M 007 or (A+B) vehicle for 30 minutes and then subjected to (B+C) in vitro hypoxia by mineral oil submersion or incubated with (A) serum-free medium for 60 minutes.
  • Cells were fixed directly after hypoxia and the F actin cytoskeleton was stained using rhodamine-conjugated phalloidin. Magnification: 100-fold.
  • IM-PTEC cells express Epacl and respond to 8-pCPT-2'-O-Me- cAMP by activation of Rap: (a) Conditionally immortalized IM-PTEC were cultured under permissive (per) or restrictive (res) conditions and analyzed for expression of Epacl by Western blotting. Tubulin (tub) was used as a loading control, (b) IM-PTEC were exposed to vehicle (saline) as control, 50 ⁇ M 8-pCPT-2'-O-Me-cAMP (007), lO ⁇ M forskolin or 2.5 ⁇ M 8-pCPT-2'-O-Me-cAMP-AM (007-AM).
  • IM-PTEC were cultured on glass coverslips and stained for (c) Epacl (red) and nuclei (blue), (d) IM-PTEC stained with secondary antibodies only.
  • FIG. 9 8-pCPT-2'-O-Me-cAMP reduces redistribution of beta-catenin during hypoxia: IM-PTEC were treated with 2.5 ⁇ M 8-pCPT-2'-O-Me-cAMP-AM (007-AM) or vehicle for 30 minutes and subjected to 60 minutes of hypoxia or maintained under normal culture conditions. After 60 minutes, cells were fixed directly. Cells were stained for beta-catenin (green), ZO-I (red), f-actin (purple) and counterstained with Hoechst to visualize nuclei. Original magnification: 60Ox.
  • Figure 10 Protection of focal adhesion complexes during hypoxia by 8-pCPT- 2'-O-Me-cAMP: IM-PTEC cells were treated with 2.5 ⁇ M 8-pCPT-2'-O-Me-cAMP- AM (007-AM) or vehicle for 30 minutes and subjected to 60 minutes of hypoxia or maintained under normal culture conditions. After 60 minutes cells were fixed and stained for phosph- (pY118) paxillin (green), talin (blue), f-actin (red). Original magnification.
  • Cells cultured under normoxic conditions either with or without prior 8-pCPT-2'-O-Me-cAMP-AM exposure display thick focal adhesion complexes near the site of the cell-cell junction but also fibrillar adhesions which associate with actin fibers.
  • Cells subjected to hypoxia lose the vast majority of their focal adhesion complexes and fibrillar adhesions.
  • Cells treated with 8-pCPT-2'-O-Me-cAMP-AM and subjected to 60 minutes of hypoxia maintain smaller focal adhesions at the site of cell-cell junctions and small fibrillar adhesions.
  • FIG. 11 Intrarenal administration of 8-pCPT-2'-O-Me-cAMP activates Rapl and reduces renal failure during IR injury: Kidney sections were stained for (a and b) Epacl expression or (c) labeled with secondary antibodies only, (d) GTP-Rapl pull down analyses demonstrated that treatment with 8-pCPT-2'-O-Me-cAMP (007) induced Rap activation.
  • FIG. 11 is a representative western blot image with samples from saline (-) and 8-pCPT-2'-O-Me-cAMP (+) treated kidneys. Rap activation was quantified by densitometric analysis (upper part) and expressed as the amount of active Rapl over total Rapl.
  • FIG. 12 Tubular epithelial cell injury following ischemia is reduced after 8- pCPT-2'-O-Me-cAMP treatment:
  • Treatment with 8-pCPT-2'-O-Me-cAMP (007) significantly reduced clusterin- ⁇ expression (right hand micrographs and black bars in bar chart). Data are expressed as mean ⁇ SEM. Original magnification: 20Ox.
  • mice monoclonal 5D3 was used for western blot analysis (11) and the rabbit polyclonal 2293 for immunostainings (9).
  • a goat-anti- Rapl-IgG, goat-anti-clusterin (M- 18) and a rabbit-anti-beta actin-IgG were purchased from Santa Cruz Biotech (San Cruz, CA).
  • the rabbit-anti-ZO-1-IgG was from Zymed (Burlington, NC)
  • the antibody to pY118-paxillin was from Cell Signaling (Danvers, MA), to beta-catenin from BD Biosciences (San Jose, CA).
  • the mouse-anti-talin and mouse-anti-tubulin antibodies were purchased from Sigma (St.Louis, MO).
  • the mouse-anti-HIFla antibody was from Abeam (Cambridge, UK). Secondary antibodies conjugated to HRP were from Jackson Immunoresearch (Newmarket, UK); antibodies conjugated to Alexa-488 and Cy3 and rhodamine + Alexa-644 conjugated phalloidin were from Invitrogen (Breda, The Netherlands). Forskolin was purchased from Calbiochem (Nottingham, UK); 8-pCPT-2'-O-Me-cAMP and 8-pCPT-2'-O-Me- cAMP-AM (12) were from BIOLOG (Bremen, Germany). Animals and experimental IR model
  • Renal tissue was fixed in 4% formaldehyde for 24 hours and embedded in paraffin in a routine fashion. Four ⁇ m thick sections were cut and used for all stainings. To determine tubular damage, sections were pretreated with ⁇ -amylase (Sigma), stained with periodic acid-Schiff reagens (PAS/D) and counterstained using hematoxylin. To determine renal function, plasma samples were analyzed using a Reflotron and urea-specific test strips (Roche Diagnostics, Almere, The Netherlands).
  • tissue sections were dewaxed and blocked with normal goat serum (Jackson). Primary antibodies were labeled with HRP-conjugated secondary antibody. Visualization was performed using 3,3'-diaminobenzidine and sections were counterstained with hematoxylin. Sections were imaged using a Leica DM6000B light microscope (Rijswijk, The Netherlands). Immunoblotting
  • Lysates were centrifuged and the supernatants were incubated with Gluthation Sepharose 4B (Roche) beads coated with RaIGDS-RBD fusion protein as described previously (14). Samples were then used for Rapl immunoblotting as described below.
  • TEC were isolated from Immorto mice as described previously (13) labeled with antibodies to neprilsyin/CDlO and aquaporin 4 combined, as markers for proximal tubular epithelium (15, 16) and sorted by flow cytometry on a FacsAria cell sorter (BD Biosciences).
  • HK-2 medium DMEM/F12 medium (Invitrogen) with 5% fetal bovine serum (Hyclone, Etten-Leur, The Netherlands), 5 ⁇ g/ml insulin and transferrin, 5 ng/ml sodium selenite (Roche), 20 ng/m tri-iodo- thyrionine (Sigma Aldrich), 50 ng/ml hydrocortisone (Sigma Aldrich) and 5 ng/ml prostaglandin El (Sigma Aldrich) with L-glutamine and antibiotics (both from Invitrogen) and mouse interferon- ⁇ (IFN- ⁇ , 1 ng/ml, R&D)) at 33 0 C in 5% CO2 and 95% air.
  • DMEM/F12 medium Invitrogen
  • fetal bovine serum Hyclone, Etten-Leur, The Netherlands
  • 5 ⁇ g/ml insulin and transferrin 5 ng/ml sodium selenite
  • Cells were grown in flasks at restrictive conditions for 7 days, passed to the appropriate assay plates at high density and cultured for an additional two days. Cells were briefly serum-starved in DMEM/F12 for 2 hours. Before being subjected to hypoxia, cells were pre-treated with 50 ⁇ M 8-pCPT-2'-O-Me-cAMP, 2.5 ⁇ M 8-pCPT- 2'-O-Me-cAMP-AM, lO ⁇ M forskolin or vehicle for 30 minutes. Hypoxia was induced by submersion of the monolayer in paraffin oil (Bufa, Uitgeest, The Netherlands) for 60 minutes as described previously (13).
  • paraffin oil Bufa, Uitgeest, The Netherlands
  • IM-PTEC cells were cultured under restrictive conditions for 7 days prior to testing. Cells were briefly serum-starved in DMEM/F12 for 2 hours. Hypoxia was induced chemically by treatment for 60 minutes with deoxyglucose (1OmM) and antimycin A (10 uM) in the presence or absence of 007 (100 ⁇ M). Cells were washed twice to remove dead and floating cells and the remaining cells were determined by Bradford assay.
  • IM-PTEC cells were cultured as for chemical hypoxia. Cells were incubated with 50 ⁇ M 007 or vehicle for 30 minutes. The medium was then removed and a layer of mineral oil was placed over the cells for 60 minutes to deprive the cells of atmospheric oxygen. Cells were then fixed and stained with rhodamine phalloidin to visualise the actin cytoskeleton and photographed using a Nikon inverted epifluorescence microscope. Cisplatin treatment
  • IM-PTEC cells were cultured under restrictive conditions for 7 days prior to testing. Cells were then treated with 25 ⁇ M cisplatin for 24 hours in HK-2 medium in the presence or absence of 007 (lOO ⁇ M). cells were then photographed and processed for cell cycle analysis and analysis of caspase activation as previously described (Imamdi et al, JPET 2004; 311 :892-903). Cell and cryosection immunofluorescence staining
  • Formaldehyde fixed cells on glass coverslips were permeabilized and blocked in PBS containing 0.05% Triton X-100 (Sigma) and 0.5% bovine serum albumin (TBP). All antibodies were diluted in TBP. Ten ⁇ m cryosections were used for immunostainings. Sections were dried to air and fixed in 4% buffered formaldehyde. Sections were permeabilized in 0.2% Triton X-100 in PBS and blocked with 5% normal horse serum (Jackson) in PBS with 0.05% Triton X-100. All antibodies were diluted in PBS with 0.05% Triton X-100. Cells and sections were counterstained with Hoechst 33342 dye.
  • IM-PTEC were cultured in 24 well plates, exposed to 8-pCPT-2'-O-Me- cAMP, 8-pCPT-2'-O-Me-cAMP -AM and forskolin and subjected to hypoxia in duplo as described above.
  • Cells were fixed and stained with rhodamin-phalloidin at 15 minutes intervals. Plates were imaged using a BD Pathway 855 high-content bioimager (BD Biosciences) using a long- working distance objective lens (2Ox magnification). Six images per well were made. Phalloidin staining was analyzed using Image-Pro Plus v ⁇ .l analysis software (MediaCybernetics, Gleichen, Germany) by quantification of the fluorescent signal per field. Data from control cells was set at 0 and 1 representing minimal and maximal monolayer disruption respectively. Values from stimulated cells where expressed accordingly.
  • Clusterin expression was quantified by using five 2Ox magnifications per stained cryosection. Clusterin expression was expressed as the percentage of the area with positive signal per total field in the corticomedullary region of the kidney using Image-Pro Plus v ⁇ .l analysis software. Epithelial barrier function measurement
  • Epithelial barrier function was determined using the electric cell-substrate impedance sensing (ECIS) method on an ECIS 1600R using 8Wl OE electrode array slides (Applied Biophysics, Troy, NY). All measurements were performed using 400Hz frequency. Cells were subjected to pre-stimulation and hypoxia as described above. After 60 minutes, an equal volume of DMEM/F12 medium was added to the cells submerged in paraffin oil, enabling re-continuation of the ECIS measurement. Barrier function was determined before and during pre-stimulation and directly after recovery from hypoxia. Statistical analyses
  • Results are expressed as mean ⁇ standard error of the mean (SEM). Data were tested for normality using the Kolmogorov-Smirnow test and analyzed using an unpaired t test. Tubular injury scores were analyzed using the non-parametric Mann- Whitney U Test. Values of P ⁇ 0.05 were considered statistically significant. All statistical analyses were performed using Graphpad Prism4 (GraphPad Software, San Diego, California, USA). Results
  • proximal tubule cells Treatment of proximal tubule cells with cisplatin induces cellular damage via induction of reactive oxygen, DNA modification and other unknown mechanisms, leading to apoptosis.
  • Simultaneous treatment of cells with 007 attenuates cisplatin- induced injury as measured by cell cycle analysis and caspase activation. See figure 1.
  • Intra venous administration of 007 also induces activation of Rap in kidney tissue.
  • ischemia-reperfusion injury results in apoptosis of tubular epithelial cells and detachment and subsequent loss into the urine.
  • An in vitro model of this is chemical-induced anoxia whereby ATP production and oxygen metabolism are inhibited chemically.
  • Chemical-induced anoxia of immortalized mouse PTECs resulted in cell rounding and detachment of 60% of cells (see upper panel of figure 4). The detachment of epithelial cells was largely prevented by simultaneous incubation with 007.
  • Epac-Rap signaling can be induced in conditionally immortalized proximal tubular epithelial cells (IM-PTEC).
  • Conditionally immortalized PTECs were used to model renal epithelium. Culturing these cells at restrictive conditions resulted in a complete loss of SV40 expression (data not shown) and was accompanied by the acquisition of an epithelial phenotype, associated with characteristic localization of the tight junction protein zona occludens-1 (ZO-I) (figure IF). Interestingly, loss of SV40 expression was associated with upregulation of Epac expression (figure Ia). Epac expression by IM- PTEC was also confirmed by immunofluorescence staining (figure Id). Therefore, for subsequent experiments, cells cultured under restrictive (SV40 negative) conditions were used.
  • IM-PTEC cells were exposed to 8-pCPT-2'-O-Me-cAMP, forskolin and 8-pCPT-2'- O-Me-cAMP-AM (figure Ic).
  • a Rap pull down analysis was performed to determine the level of active, GTP-bound Rapl.
  • 8-pCPT-2'-O-Me-cAMP induced a modest increase in active Rapl.
  • 8-pCPT-2'-O-Me-cAMP-AM which has a higher uptake in cells (ref), showed increased Rapl activation, similar to that of forskolin.
  • Epac-Rap activation by 8-pCPT-2'-O-Me-cAMP-AM reduces monolayer disruption and protects the tubular barrier function during in vitro hypoxia
  • hypoxia affects the actin cytoskeleton in TECs, causes a disruption of the epithelial monolayer and, during prolonged hypoxia, results in detachment of cells.
  • Previous experiments showed an effect of Epac-Rap signalling on both cell-cell and cell-extracellular matrix adhesions (refs). We therefore tested whether activation of Epac-Rap signalling could prevent hypoxia-induced damage to the epithelial monolayer.
  • Cells in 96-well plates were exposed to 8-pCPT-2'-O-Me-cAMP, 8- pCPT-2'-O-Me-cAMP-AM, forskolin or vehicle for 30 minutes prior to induction of hypoxia.
  • Intrarenal administration of 8-pCPT-2'-O-Me-cAMP induces activation of Rapl and protects against loss of renal function during IR injury
  • Epac-Rap pathway As a therapeutic target in ischemia-reperfusion injury and the potential of 8-pCPT-2'-O-Me-cAMP as a prototype drug, we tested the effect of 8-pCPT-2'-O-Me-cAMP administration in a mouse model for IR injury.
  • Epac is expressed by the tubular epithelium in most segments of the nephron.
  • mice were treated with 8-pCPT-2'-O-Me-cAMP by intrarenal administration.
  • kidneys of mice were clamped, injected with 8-pCPT-2'-O-Me-cAMP or saline (vehicle) and collected 30 minutes after injection.
  • Rapl -GTP pull down analysis performed on tissue cryosections showed that injection of 8-pCPT-2'-O-Me-cAMP led to a significant increase in whole kidney Rapl activation (figure 5d).
  • Clusterin (apolipoprotein J) is thought to be involved in cellular stress as a regulator of apoptosis (18). It has been shown to be an early cellular marker of tubular epithelial damage (19) and can be used as a urinary marker for tubular injury (20). Immunostainings for clusterin- ⁇ showed low reactivity on tissue sections of sham operated animals (figure 6a). Ischemic tissue showed increased clusterin- ⁇ expression which was predominantly present inside the tubular lumen although some cells in the epithelial lining also stained positive.
  • kidney tissue from mice treated with 8-pCPT-2'-O-Me-cAMP had a lower level of clusterin- ⁇ expression during IR injury than mice from the control group suggesting that treatment with 8-pCPT-2'-O-Me-cAMP reduces tubular epithelial cell stress during IR injury.
  • Beta catenin is a vital component of adherens junctions, and its cytoplasmic localization has been found to correlate with tubular epithelial dedifferentiation and represents an early risk factor for epithelial to mesenchymal transition in kidney allografts (21, 22). Tissue from both groups of sham operated animals showed a distinct lateral membrane staining pattern (figure 6b). Sections from saline treated ischemic kidneys showed an irregular and more cytoplasmic staining pattern. In contrast to this, localization of beta catenin in 8-pCPT-2'-O-Me-cAMP treated ischemic kidneys resembled the pattern found in sham operated controls. References 1. Abuelo, JG: Normotensive ischemic acute renal failure. N Engl J Med, 357: 797-
  • Lameire, NH & Vanholder, R Pathophysiology of ischaemic acute renal failure.
  • Murer, H & Hernando, N Regulation of sodium -proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC). Proc Natl Acad Sci USA, 103: 803-8, 2006.
  • enkephalinase membrane metalloendopeptidase, E.C. 3.4.24.11
  • rat organs Detection using a monoclonal antibody. Lab Invest, 58: 210-7, 1988.
  • Integrity of cell-cell contacts is a critical regulator of TGF-beta 1 -induced epithelial-to-myo fibroblast transition: role for beta-catenin. Am J Pathol, 165: 1955-67, 2004.
  • Zager, RA Adenine nucleotide changes in kidney, liver, and small intestine during different forms of ischemic injury. Circ Res, 68: 185-96, 1991.
  • Rapl GTPase inhibits leukocyte transmigration by promoting endothelial barrier function. J Biol Chem, 280: 11675-82, 2005.

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Abstract

La présente invention porte sur des compositions, des médicaments et des procédés d'utilisation dans la prévention et/ou le soulagement des effets dommageables d'une hypoxie et/ou d'une lésion par reperfusion.
PCT/GB2009/002039 2008-08-21 2009-08-20 Protection d'organe WO2010020786A1 (fr)

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US8772474B2 (en) 2010-12-22 2014-07-08 Alios Biopharma, Inc. Cyclic nucleotide analogs
US8871737B2 (en) 2010-09-22 2014-10-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US8916538B2 (en) 2012-03-21 2014-12-23 Vertex Pharmaceuticals Incorporated Solid forms of a thiophosphoramidate nucleotide prodrug
US8980865B2 (en) 2011-12-22 2015-03-17 Alios Biopharma, Inc. Substituted nucleotide analogs
US9012427B2 (en) 2012-03-22 2015-04-21 Alios Biopharma, Inc. Pharmaceutical combinations comprising a thionucleotide analog
US9073960B2 (en) 2011-12-22 2015-07-07 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US9441007B2 (en) 2012-03-21 2016-09-13 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
USRE48171E1 (en) 2012-03-21 2020-08-25 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8871737B2 (en) 2010-09-22 2014-10-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US9278990B2 (en) 2010-09-22 2016-03-08 Alios Biopharma, Inc. Substituted nucleotide analogs
US9365605B2 (en) 2010-12-22 2016-06-14 Alios Biopharma, Inc. Cyclic nucleotide analogs
US8772474B2 (en) 2010-12-22 2014-07-08 Alios Biopharma, Inc. Cyclic nucleotide analogs
US9605018B2 (en) 2011-12-22 2017-03-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US11021509B2 (en) 2011-12-22 2021-06-01 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US8980865B2 (en) 2011-12-22 2015-03-17 Alios Biopharma, Inc. Substituted nucleotide analogs
US10464965B2 (en) 2011-12-22 2019-11-05 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US9073960B2 (en) 2011-12-22 2015-07-07 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US9394330B2 (en) 2012-03-21 2016-07-19 Alios Biopharma, Inc. Solid forms of a thiophosphoramidate nucleotide prodrug
US9441007B2 (en) 2012-03-21 2016-09-13 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US9856284B2 (en) 2012-03-21 2018-01-02 Alios Biopharma, Inc. Solid forms of a thiophosphoramidate nucleotide prodrug
US10485815B2 (en) 2012-03-21 2019-11-26 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
USRE48171E1 (en) 2012-03-21 2020-08-25 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US8916538B2 (en) 2012-03-21 2014-12-23 Vertex Pharmaceuticals Incorporated Solid forms of a thiophosphoramidate nucleotide prodrug
US9012427B2 (en) 2012-03-22 2015-04-21 Alios Biopharma, Inc. Pharmaceutical combinations comprising a thionucleotide analog

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