EP1359883A4 - (psi)(epsilon)rack peptide composition and method for protection against tissue damage due to ischemia - Google Patents
(psi)(epsilon)rack peptide composition and method for protection against tissue damage due to ischemiaInfo
- Publication number
- EP1359883A4 EP1359883A4 EP01273811A EP01273811A EP1359883A4 EP 1359883 A4 EP1359883 A4 EP 1359883A4 EP 01273811 A EP01273811 A EP 01273811A EP 01273811 A EP01273811 A EP 01273811A EP 1359883 A4 EP1359883 A4 EP 1359883A4
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- European Patent Office
- Prior art keywords
- peptide
- ψεrack
- seq
- tissue
- ischemic
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- 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 relates to a method of protecting cells and tissues from damage due to an ischemic event.
- the method involves administering a peptide agonist of protein kinase C, and more specifically, administering a pseudo-epsilon RACK ( ⁇ RACK) peptide.
- ⁇ RACK pseudo-epsilon RACK
- PKC Protein kinase C
- the PKC family of isozymes includes at least 11 different protein kinases which can be divided into at least three subfamilies based on their homology and sensitivity to activators.
- cPKC subfamily Members of the classical or cPKC subfamily, ⁇ , ⁇ *, ⁇ ⁇ and ⁇ PKC, contain four homologous domains (CI, C2, C3 and C4) inter-spaced with isozyme-unique (variable or V) regions, and require calcium, phosphatidylserine (PS), and diacylglycerol (DG) or phorbol esters for activation.
- PS phosphatidylserine
- DG diacylglycerol
- phorbol esters Members of the novel or nPKC subfamily, ⁇ , ⁇ , ⁇ , and ⁇ PKC, lack the C2 homologous domain and do not require calcium for activation.
- members of the atypical or ⁇ PKC subfamily, ⁇ and ⁇ /vPKC lack both the C2 and one half of the CI homologous domains and are insensitive to DG, phorbol esters and calcium.
- PKC isozymes demonstrate that activation of PKC results in its redistribution in the cells (also termed translocation), such that activated PKC isozymes associate with the plasma membrane, cytoskeletal elements, nuclei, and other subcellular compartments (Saito, et al, 1989; Papadopoulos and Hall, 1989; Mochly-Rosen, et al, 1990).
- ⁇ PKC binds to cross-striated structures (possibly the contractile elements) and cell-cell contacts following activation or after addition of exogenous activated ⁇ PKC to fixed cells (Mochly-Rosen, et al., 1990; Disatnik, et al, 1994).
- RACKs Receptors for Activated C-Kinase
- RACKs are thought to function by selectively anchoring activated PKC isozymes to their respective subcellular sites. RACKs bind only fully activated PKC, but RACKs are not necessarily substrates of the enzyme nor is the binding to RACKs mediated via the catalytic domain of the kinase (Mochly-Rosen, et al, 1991). Translocation of PKC reflects binding of the activated enzyme to RACKs anchored to the cell particulate fraction and the binding to RACKs is required for PKC to produce its cellular responses (Mochly-Rosen, 1995).
- PKC Translocation of PKC is required for proper function of PKC isozymes.
- Peptides that mimic either the PKC -binding site on RACKs (Mochly-Rosen, 1991a; Mochly-Rosen, 1995) or the RACK-binding site on PKC (Ron, et al, 1995; Johnson, et al, 1996) are isozyme-specific translocation inhibitors of PKC that selectively inhibit the function of the enzyme in vivo.
- an eight amino acid peptide derived from ⁇ PKC peptide ⁇ Vl-2; SEQ ID NO:l, Glu Ala Val Ser Leu Lys Pro Thr
- U.S. Patent No. 6,165,977 an eight amino acid peptide derived from ⁇ PKC (peptide ⁇ Vl-2; SEQ ID NO:l, Glu Ala Val Ser Leu Lys Pro Thr) is described in U.S. Patent No. 6,165,977.
- the peptide contains a part of the RACK-binding site on ⁇ PKC and selectively inhibits specific ⁇ PKC -mediated functions in cardiac myocytes.
- PKC and more specifically ⁇ PCK have been shown to be involved in cardiac preconditioning to provide protection from ischemic injury.
- Prolonged ischemia causes irreversible myocardium damage primarily due to death of cells at the infarct site.
- Studies in animal models, isolated heart preparations and isolated cardiac myocytes in culture have demonstrated that short bouts of ischemia of cardiac muscle reduce such tissue damage in subsequent prolonged ischemia (Liu, Y., et al, 1995, 1996; Hu, et al, 1995; Brew, et al, 1995; Schultz, et al, 1996).
- an ⁇ PKC-selective peptide agonist was shown to provide cardio- protection from ischemia when administered intracellulary to isolated neonatal and adult cardiomyocytes and when produced intracellulary in vivo in transgenic mice (Dorn G. et al, 1999).
- a ⁇ PKC peptide agonist was administered intracellulary to isolated cells in vitro by laboratory techniques suitable at the cellular level or by genetic transfection. Unfortunately, neither of these techniques are suitable or likely to be successful for human therapy. Moreover, it is unknown from this work whether or not the ⁇ PKC peptide could be delivered extracellulary to whole tissue or intact organs in vivo to achieve a therapeutic effect.
- the invention includes a method of reducing injury to a cell exposed to an ischemic or an hypoxic condition by administering to the cell a ⁇ RACK peptide.
- the peptide is administered prior to exposing the cell to the ischemic or hypoxic condition.
- the peptide administered for a period of time of between about 1-180 minutes prior to exposing the cell to ischemia or hypoxia.
- the peptide is administered after the cell is exposed to an ischemic or hypoxic condition.
- the peptide is administered for between about 1-180 minutes after the cell is exposed to an ischemic or hypoxic condition.
- the peptide is administered during to the cell during the period of ischemia or hypoxia.
- the ⁇ RACK peptide has a sequence identified as SEQ ID NO: 2. In other embodiments, the peptide has a sequence selected from the group consisting of SEQ ID NOS:6-18. In yet another embodiment, the ⁇ RACK peptide is linked to a moiety effective to facilitate transport across a cell membrane, such as a Tat-derived peptide (SEQ ID NO: 5), an Antennapedia carrier peptide (SEQ ID NO: 3), or a polyarginine peptide.
- the peptide can be administered by a route selected from the group consisting or intraveneous, parenteral, subcutaneous, inhalation, intranasal, sublingual, mucosal, and transdermal.
- the invention includes a method of reducing injury to tissue exposed to an ischemic or an hypoxic condition by administering to the tissue a ⁇ RACK peptide, as described above.
- Fig. 1A shows creatine phosphokinase (CPK) release as a function of time during reverse perfiision in ex vivo rat hearts treated with ⁇ RACK (open circles) or with scrambled ⁇ RACK (control, open squares) prior to an ischemic event.
- the treated hearts were compared to hearts subjected to the ischemic period but left untreated (closed triangles) and to hearts maintained under normoxia conditions (no ischemia, no peptide treatment; closed squares) as controls.
- Fig. IB shows the total amount of CPK released in the study described in Fig. 1A during 30 minutes of reperfusion.
- Fig. 2 A shows the functional recovery of a working heart perfused with ⁇ RACK prior to global ischemia, where the left ventricle developed pressure (LVP, in mmHg), its first derivative (dP/dt, in mmHg/sec), and the coronary perfiision pressure (PP, in mmHg) are shown.
- LVP left ventricle developed pressure
- dP/dt first derivative
- PP coronary perfiision pressure
- Fig. 2B is a scan similar to Fig. 2 A for a working heart perfused with scrambled ⁇ RACK prior to ischemia.
- Fig. 3 A shows CPK release as a function of time following ischemic insult in ex vivo rat hearts treated for the first 20 minutes of reperfusion with ⁇ RACK (open circles) and in hearts left untreated after ischemia (solid triangles).
- Fig. 3B is a bar graph showing the total CPK release during a 60 minute reperfusion period following an ischemic insult to whole rat hearts (i) treated ex vivo with ⁇ RACK for 20 minutes after the ischemic insult, or (ii) left untreated.
- Figs. 4A-4B are computer generated photos of pig heart slices taken from pigs five days after an in vivo treatment with ⁇ RACK (Fig. 4A) or with scrambled ⁇ RACK peptide as a control (Fig. 4B) during the initial 10 minutes of a 30 minute ischemic insult.
- Fig. 4C is a bar graph showing the total infarct area as a percent of area at risk, measured in grams of cardiac tissue, for pigs treated with ⁇ RACK peptide or with scrambled ⁇ RACK peptide (control).
- Fig. 5 is a graph showing the ejection fraction, as measured by left ventricurogram in pigs at three time points: (1) before occlusion of left anterior descending artery by balloon catheter (before occlusion); (2) immediately after reperfusion with ⁇ RACK (post ⁇ RACK); and (3) before sacrifice five days later (5 days post), for animals treated with ⁇ RACK (solid triangles) and for control animals treated with a scrambled peptide (open circles).
- SEQ ID NO: l is a prior art (U.S. Patent No. 6,165,977) ⁇ PKC octapeptide.
- SEQ ID NO:2 is a ⁇ RACK octapeptide
- SEQ ID NO:3 is the Drosophila Antennapedia homeodomain-derived carrier peptide.
- SEQ ID NO:4 is a scrambled ⁇ RACK octapeptide.
- SEQ ID NO: 5 is a Tat-derived carrier peptide.
- SEQ ID NO:6 is a modification of SEQ ID NO:2.
- SEQ ID NO:7 is a modification of SEQ ID NO:2.
- SEQ ID NO:8 is a modification of SEQ ID NO:2.
- SEQ ID NO:9 is a modification of SEQ ID NO:2.
- SEQ ID NO: 10 is a modification of SEQ ID NO:2.
- SEQ ID NO:l 1 is a modification of SEQ ID NO:2.
- SEQ ID NO: 12 is a modification of SEQ ID NO:2.
- SEQ ID NO: 13 is a modification of SEQ ID NO:2.
- SEQ ID NO: 14 is a modification of SEQ ID NO:2.
- SEQ ID NO: 15 is a modification of SEQ ID NO:2.
- SEQ ID NO: 16 is a modification of SEQ ID NO:2.
- SEQ ID NO: 17 is a modification of SEQ ID NO:2.
- SEQ ID NO: 18 is a fragment of SEQ ID NO:2.
- tissue refers to a group of similarly specialized cells that perform a common function. Tissues compose the organs and structural components of living organisms. As used herein, tissue is intended to include an organ composed of a given tissue and to the cells, individually or collectively, that compose the tissue.
- Ischemia or an "ischemic event” refers to an insufficient supply of blood to a specific cell, tissue or organ. A consequence of decreased blood supply is an inadequate supply of oxygen to the organ or tissue (hypoxia). Prolonged hypoxia may result in injury to the affected organ or tissue.
- oxia refers to a virtually complete absence of oxygen in the organ or tissue, which, if prolonged, may result in death of the cell, organ or tissue.
- Oxypoxia or a “hypoxic condition” intends a condition under which a cell, organ or tissue receives an inadequate supply of oxygen.
- Ischemic injury refers to cellular and/or molecular damage to an organ or tissue or cell as a result of a period of ischemia.
- Hypoxic injury refers to damage to a cell, organ, or tissue due to a period of inadequate oxygen supply.
- Reperfusion refers to return of fluid flow into a tissue after a period of no-flow or reduced flow.
- fluid or blood returns to the heart through a supply line, such as the coronary arteries in vivo, after removal of an occlusion to the fluid or blood supply.
- Treating a disease refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease.
- Constant amino acid substitutions are substitutions which do not result in a significant change in the activity (e.g., ⁇ PKC-agonist activity or ⁇ RACK-agonist activity) or tertiary structure of a selected polypeptide. Such substitutions typically involve replacing a selected amino acid residue with a different residue having similar physico- chemical properties. For example, substitution of Glu for Asp is considered a conservative substitution since both are similarly-sized negatively-charged amino acids. Groupings of amino acids by physico-chemical properties are known to those of skill in the art. 5 With respect to a specific sequence, “conservative substitutions thereof refers to sequences that differ from the specific sequence by having conservative amino acid substitutions at one or more positions.
- Peptide and “polypeptide” are used interchangeably herein and refer to a compound made up of a chain of amino acid residues linked by peptide bonds. Unless l o otherwise indicated, the sequence for peptides is given in the order from the amino termiums to the carboxyl terminus.
- first peptide or polypeptide When a first peptide or polypeptide is said to "correspond” or to be “homologous” to a second peptide or polypeptide fragment, it means that the peptide or fragments have a similarity in amino acid residues if they have an alignment score of >5 (in standard
- the two sequences (or parts thereof) are more preferably homologous if their amino acids are greater than or equal to 50%, more preferably 70%, still more
- a polypeptide sequence or fragment is "derived" from another polypeptide sequence or fragment when it has an identical sequence of amino acid residues as a region of the another sequence or fragment.
- ⁇ PKC agonist peptide or " ⁇ PKC specific agonist peptide” is understood to mean a peptide between about 4 and about 30, preferably between about 5 and about 15, amino acids in length that is derived from ⁇ PKC.
- the ⁇ PKC agonist peptide is derived from the region of ⁇ PKC between about amino acids 70 and 120, preferably between about amino acids 80 and 100, more preferably between about amino
- PLC protein kinase C
- RACK receptor for activated C-kinase.
- amino acid residues are the standard 3-letter and/or 1 -letter codes used in the art to refer to one of the 20 common L-amino acids.
- the invention provides a method of protecting a cell, a tissue, or an organ from damage due to an ischemic event or a hypoxic condition by administering a peptide capable of activating signaling proteins, such as PKC, that are activated in vivo by binding to a cognate polypeptide such as a receptor protein (RACK).
- a peptide capable of activating signaling proteins such as PKC
- RACK receptor protein
- Regions of homology between the PKC signaling peptide and its RACK are termed "pseudo-RACK” sequences ( ⁇ -RACK; Ron et al, 1994, 1995) and typically have a sequence similar to the PKC- binding region of the corresponding RACK.
- a ⁇ -RACK sequence that acts as an ⁇ PKC specific agonist peptide is identified herein as SEQ ID NO:2.
- ⁇ RACK This peptide, referred to herein as ⁇ RACK, is an ⁇ PCK specific agonist peptide and induces translocation of ⁇ PKC.
- ⁇ RACK This peptide, referred to herein as ⁇ RACK, is an ⁇ PCK specific agonist peptide and induces translocation of ⁇ PKC.
- studies have focussed on identifying drugs and peptidomimetics that mimic the action of ⁇ RACK, as it was unknown if the peptide itself could be administered in vivo or ex vivo to a whole organ to induce translocation of ⁇ PCK to confer protection from ischemia.
- ⁇ RACK was administered ex vivo and in vivo to whole hearts prior to and after exposure to an ischemic condition. The peptide reduced the extent of ischemic injury, as will now be described.
- a peptide having the sequence identified herein as SEQ ID NO: 2 was synthesized and purified as described in the Methods section.
- a scrambled ⁇ RACK peptide (SEQ ID NO: 2
- peptides were conjugated to a carrier peptide, such as Drosophila Antennapedia homeodomain (SEQ ID NO:3). It will be appreciated that administration of the native peptide, that is the peptide unmodified by attachment to a carrier, is also contemplated.
- a carrier peptide such as Drosophila Antennapedia homeodomain (SEQ ID NO:3). It will be appreciated that administration of the native peptide, that is the peptide unmodified by attachment to a carrier, is also contemplated.
- Carrier peptides other than Drosophila Antennapedia are also contemplated, and other exemplary carrier peptides include Tat-derived peptide (SEQ ID NO:5, Fawell et al , 1994, Vives et al, 1997) or a polyarginine peptide (Mitchell et al , 2000; Rolhbard et al, 2000), or other like carries described in the art (Lindgren et al, 2000; Schwarlze et al, 2000). Animals were anesthetized as described in Example 1 and their hearts were rapidly removed and cannulated for perfusion on a Langendorff apparatus. Hemodynamic parameters were monitored until stabilized, typically for 10-20 minutes.
- ⁇ RACK peptide SEQ ID NO:2
- SEQ ID NO:4 a scrambled ⁇ RACK peptide
- ischemia-induced cell damage was determined by measuring the activity of creatine phosphokinase (absorbance at 520 nm) in the perfusate. Creatine phosphokinase is a cytosolic enzyme in cardiac myocytes and its presence in the perfusate is proportional to the number of cardiomyocytes damaged by the ischemia. The results are shown in Figs. 1 A-1B.
- Fig. 1A shows the creatine phosphokinase (CPK) release as a function of time during the 30 minute reperfusion of ex vivo hearts treated with 500 nM (0.5 ⁇ M) ⁇ RACK (open circles) or with 0.5 ⁇ M scrambled ⁇ RACK (open squares) prior to the ischemic event. Also shown is the CPK release for hearts subjected to the ischemic period but untreated with a peptide (closed triangles) and to hearts maintained under normoxia conditions (no ischemia, no peptide treatment; closed squares), as controls. The hearts treated with ⁇ RACK prior to ischemia have a release of CPK close to the control hearts maintained under normoxia conditions. In contrast, hearts treated with scrambled ⁇ RACK have significant release of CPK, indicating significant cell damage.
- CPK creatine phosphokinase
- Fig. IB is a bar graph showing the total CPK release during the reperfusion period for the hearts treated with ⁇ RACK and with scrambled ⁇ RACK. The total CPK release from hearts exposed to the ischemic event but left untreated are also shown.
- Figs. 1A-1B show that hearts treated with ⁇ RACK prior to an ischemic event provides protection from damage resulting from a subsequent ischemic event.
- the invention contemplates a method of reducing or preventing injury to a tissue exposed to an ischemic or hypoxic event by administering to the tissue an amount of ⁇ RACK agonist peptide.
- the peptide can be administered from between 1-180 minutes prior to the ischemic event, more preferably from about 1-120 minutes prior to the ischemic event, more preferably from about 1-60 minutes prior to the ischemic event.
- Figs. 2A-2B show the results for the hearts treated with ⁇ RACK peptide prior to global ischemia
- Fig. 2B shows the results for hearts treated with scrambled ⁇ RACK prior to ischemia.
- ⁇ RACK ⁇ RACK Peptide Agonist Subsequent to Ischemia
- ⁇ RACK was administered to hearts ex vivo after a prolonged ischemic period and was effective to provide protection from ischemic injury.
- whole rat hearts were perfused on a Langendorff apparatus. After a 30 minute equilibration period, global ischemia was induced by stopping fluid flow for 45 minutes. The hearts were then reperfused with or without ⁇ RACK peptide for 20 minutes, followed by 40 minutes perfiision without peptide.
- the CPK activity in the perfusate was analyzed. The results are shown in Figs. 3A-3B.
- Fig. 3 A shows the CPK release as a function of time following ischemia for hearts treated with ⁇ RACK (open circles) and for hearts left untreated after ischemia (solid triangles).
- Fig. 3B is a bar graph showing the total CPK release during the 60 minute perfiision period for the peptide treated and untreated ex vivo hearts. The data shows that subsequent administration of ⁇ RACK peptide to tissue previously exposed to an ischemic or hypoxic condition is effective to reduce the cellular damage.
- Fig. 3B shows there was a nearly 2-fold lower total CPK release for tissue treated with ⁇ RACK peptide.
- ⁇ RACK peptide In another study in support of the invention, the ability of ⁇ RACK peptide to protect tissue from damage due to an ischemic or hypoxic event was evaluated by administering the peptide in vivo.
- ⁇ RACK peptide SEQ ID NO: 2
- scrambled ⁇ RACK peptide SEQ ID NO:4
- Figs 4A-4C The results are shown in Figs 4A-4C.
- Figs. 4A-4B are computer-generated photos of pig heart slices taken from the pigs treated in vivo with ⁇ RACK (Fig.
- Fig. 4C is a bar graph showing the infarct area as a percent of area at risk, measured in grams of cardiac tissue. As seen, the percent infarct was reduced by more than 2-fold for the animals treated with ⁇ RACK peptide. Accordingly, delivery of a ⁇ RACK peptide in vivo prior to or during an ischemic event is effective to reduce the percentage of infarct by at least 2-fold.
- Blood samples and tissue samples of lung, liver, brain, gut, kidney, etc. were collected from the animals and analyzed at a pathology lab. All samples were normal and no inflammation or tissue abnormalities were observed.
- LNG left ventricurogram
- EF Ejection fraction
- Ejection fraction is a measure of how well the heart is functioning, with a higher ejection fraction indicative of a better functioning heart.
- An ejection fraction of less than 50% in a short period of time can suggest progression into a state of heart failure.
- animals treated with ⁇ RACK closed triangles
- had a higher ejection fraction after occlusion compared to the control animals treated with a scrambled peptide open circles
- the peptide reduces or prevents damage to the cells and tissue due to ischemia.
- animals treated with ⁇ RACK had an ejection fraction on par with that measured prior to ischemia and about 10% higher than the untreated animals.
- delivery of a ⁇ RACK peptide in vivo after ischemia is effective to reduce cell and tissue damage, as evidenced by an ejection fraction at least 10% greater than that of untreated cells or tissues.
- a " ⁇ RACK peptide” refers to the peptide represented by SEQ ID ⁇ O:2 and to derivatives and fragments of this peptide.
- Exemplary derivatives are given in SEQ ID NOS:6-18, and include the following sequences: HEADIGYD (SEQ ID NO:6); HDAPIGYE (SEQ ID NO:7); HDAPVGYE (SEQ ID NO:8); HDAPLGYE (SEQ ID NO:9); HDAPIGDY (SEQ ID NO:10); HDAPIGEY (SEQ ID NO:l 1); ADAPIGYD (SEQ ID NO: 12); HDGPIGYD (SEQ ID NO: 13); HDAAIGYD (SEQ ID NO: 14), and combinations of these modifications.
- sequence "DAPIG” (SEQ ID NO: 18) in SEQ ID NO:2 is has no more than two modifications at any residue.
- One, two, or all three of the residues outside the sequence "DAPIG” can be modified.
- AEAPVGEY (SEQ ID NO: 15) is a derivative of SEQ ID NO:2 where all three residues outside the
- DAPIG (SEQ ED NO: 18) sequence and two residues within the “DAPIG” sequence are modified.
- Other examples include HEAPIGDN (SEQ ID NO: 16) and HDGDIGYD (SEQ ID NO: 16) and HDGDIGYD (SEQ ID NO: 16).
- SEQ ID NO: 17 5 It will also be appreciated that fragments of SEQ ID NO:2 and of the modifications described above may be suitable.
- An exemplary fragment of SEQ ID NO:2 is DAPIG,
- All of these exemplary peptides may be (i) chemically synthesized or (ii) recombinantly produced in a host cell using, e.g., an expression vector containing a l o polynucleotide fragment encoding said peptide, where the polynucleotide fragment is operably linked to a promoter capable of expressing mRNA from the fragment in the host cell.
- the dose of peptide administered will vary depending on the tissue to be treated and the condition of the patient. Dosages are readily determined by those of skill in the art 15 based on animal and human studies. Typically, between 0.05-5 ⁇ M, more preferably between 0.1-2 ⁇ M, most preferably between about 0.1-1 ⁇ M peptide is administered.
- the peptide can be administered by any route suitable, as determine by the primary care provider.
- administration by intraveneous, parenteral, subcutaneous, 20 inhalation, intranasal, sublingual, mucosal, and transdermal, and the like, is contemplated.
- the route of administration will influence the dose and timing of administration, as appreciated by those of skill.
- the peptide can be administered in the form of a fusion protein or a transport protein conjugate.
- the peptide is bound to another 25 peptide by a bond other than a Cys-Cys bond.
- An amide bond from the C-terminal of one peptide to the N-terminal of the other is exemplary of a bond in a fusion protein.
- the second peptide to which the ⁇ PKC agonist/antagonist peptide is bound can be virtually any peptide selected for therapeutic purposes or for transport purposes. For example, it maybe desirable to link the ⁇ RACK peptide to a cytokine or other peptide that elicites a 30 biological response.
- the peptide is typically conjugated to a carrier peptide, such as Tat-derived transport polypeptide (Vives et al, 1997), polyarginine
- the peptide can be introduced to a cell, tissue or whole organ using a carrier or encapsulant, such as a liposome in liposome-mediated delivery.
- ⁇ RACK as well as any compound having similar activity can be used in the methods of treatment described herein.
- Other compounds, such as peptide mimetics, chemical compounds, or other peptides, can be identified by, for example, a screening method set forth in U.S. Patent No. 6,165,977, and this portion on Col. 14, line 45-Col 15, line 54 is incorporated by reference herein.
- ⁇ PKC is immobilized inside the wells of a multiwell plate by introducing a solution containing ⁇ PKC into the plate and allowing the ⁇ PKC to bind to the plastic.
- the wells may be precoated with substances that enhance attachment of ⁇ PKC and/or that decrease the level of non-specific binding.
- the plate is then incubated with a blocking solution (containing, for example bovine serum albumin) and then washed several times.
- a solution containing reporter- labelled (e.g., radiolabelled of fluorescently-tagged) peptide ⁇ RACK (SEQ ID NO:2) and, in the test wells, as opposed to the control wells, a test compound is added.
- reporter- labelled e.g., radiolabelled of fluorescently-tagged peptide ⁇ RACK (SEQ ID NO:2)
- Different wells may contain different test compounds or different concentrations of the same test compound.
- Each test compound at each concentration is typically run in duplicate and each assay is typically run with negative (wells with no test compound) as well as positive (wells where the "test compound” is unlabeled peptide) controls.
- the free peptide is then washed out, and the degree of binding in the wells is assessed.
- test compound is identified as active it if decreases the binding of the peptide, i.e., if its effect on the extend of binding is above a threshold level. More specifically, if the decrease in binding is a several-fold different between the control and experimental samples, the compound would be considered as having binding activity. Typically, a 2- fold or 4-fold threshold difference in binding between the test and control samples is sought.
- Detection methods useful in such assays include antibody-based methods, direct detection of a reporter moiety incorporated into the peptide, such as a fluorescent label, and the like.
- test compounds may be screened, including other peptides, macromolecules, small molecules, chemical and/or biological mixtures, fungal extracts, bacterial extracts or algal extracts.
- the compounds can be biological or synthetic in origin.
- the present invention has application, for example, in treatment of surviving heart attack victims, as well in treatment of persons who presently die from heart disease after admission to the hospital. Delivery of the ⁇ PKC selective peptide agonist, ⁇ RACK, is 5 valuable in the management of these patients, both acutely and chronically.
- a ⁇ PKC selective agonist such as ⁇ RACK
- ⁇ RACK can replace the preconditioning effect induced by angina in these patients and offer a higher rate of myocardial salvage during future episodes of more severe ischemia.
- Additional uses of the invention include clinical situations in which the timing of ischemia is physician-controlled.
- pharmacologic enhancement of the o preconditioning response would provide a significant advantage to the patients undergoing treatment.
- each year, in the United States alone 600,000 adults and 12,000 children undergo open heart operations utilizing cardiopulmonary bypass, during which the heart is subjected to periods of controlled ischemia ranging from several minutes to well over one hour.
- cardiopulmonary bypass a cardiopulmonary bypass
- myocardial dysfunction during the 5 immediate post-operative period remains a leading cause of morbidity and mortality in these patients.
- the exact timing of the ischemic insult is known ahead of time in these patients, allowing for administration of a ⁇ RACK peptide prior to ischemia.
- ⁇ RACK will reduce myocardial damage by inducing a preconditioning response in the hours, or days, prior to surgery. 0 Similar benefits could be realized in the area of cardiac transplantation, of which there are approximately 2500 cases annually in the U.S. Prolonged graft ischemia is one of the factors limiting long-distance donor organ acquisition for such cardiac transplantation.
- ⁇ RACK peptide at the time of organ procurement could extend the time between organ harvest and implantation and reduce the risk of post-operative myocardial dysfunction.
- a ⁇ RACK peptide may employed in the treatment of a variety of ischemic and hypoxic conditions, in addition to cardiac ischemia.
- ⁇ RACK may be administered prior to, during or after an ischemic or hypoxic event to a wide variety of cells and tissues.
- examples include the kidney, the vascular endothelium, the liver, the eye, and in the central nervous system, where tissue damage to the brain and other tissues of the central nervous system may result due to stroke.
- the peptide can be administered before, during or after an ischemic or hypoxic event.
- the peptide When delivered before an ischemic insult, the peptide effectively reduces the extent of cellular damage.
- the peptide is perfused over or through the tissue for about 1-180 minutes, more preferably for about 1-120 minutes, most preferably for about 1-60 minutes, prior to the ischemic insult.
- a period of time not greater than about three hours, more preferably not greater than about 120 minutes, and most preferably not greater than about 60 minutes lapses between cessation of peptide perfiision and the ischemic or hypoxic event.
- the peptide can be delivered for a 1 minute, 2 minute, 5 minute, 10 minute, 20 minute, 30 minute, or longer, period of time prior to the ischemic insult.
- the peptide When the peptide is delivered subsequent to an ischemic event, in a preferred embodiment a period of time not greater than about two hours, more preferably not greater than one hour, and even more preferably not greater than 30 minutes lapses between the ischemic event and initiation of administration of the peptide.
- the peptide can be delivered for a 1 minute, 2 minute, 5 minute, 10 minute, 20 minute, 30 minute, or longer, period of time following the ischemic insult.
- the peptide can also be administered during an ischemic event. Particularly, during time of controlled ischemia, such as during surgery, the care provider can initiate administration of the ⁇ RACK peptide just prior to or concurrent with initiation of the ischemic event.
- Peptide Preparation ⁇ RACK (HDAPIGYD, SEQ ID NO:2) was synthesized and purified (>95%) at the Stanford Protein and Nucleic Acid Facility. Scrambled ⁇ RACK peptide (PDYHDAGI, SEQ ID NO:4) was prepared similarly. In some studies, the peptides were modified with a carrier peptide by cross-linking via an N-terminal Cys-Cys bond to the Drosophila Antennapedia homeodomain-derived peptide (C-RQIKIWFQNRRMKWKK, SEQ ID NO:3; Theodore, L. et al, 1995; Johnson, J. A. et al, 1996a) or via an N-terminal Cys-Cys bond to Tat protein-derived peptide (C-YGRKKRRQRRR, SEQ ID NO:6).
- a carrier peptide by cross-linking via an N-terminal Cys-Cys bond to the Drosophila Antennapedia
- mice or rats were anesthetized with i.p. avertin, and their hearts were rapidly removed and cannulated via the aorta for reperfusion as described in the art (Colbert et al, 1997). Care was taken to have the hearts perfused within 90 seconds of removal.
- the left ventricular pressure and real-time derivative (dPldt) were monitored via a catheter placed in the ventricular apex. Hemodynamic parameters were archived every 20 seconds throughout the procedure.
- the hearts were perfused with oxygenated Krebs-Henseleit solution comprised of, in nmol/L, NaCl 120; KC1 5.8; NaHCO 3 25; NaH 2 O 4 1.2; MgSO 4 1.2; CaCl 2 1.0; and dextrose 10, pH 7.4 at 37 C.
- the hearts were treated with ⁇ RACK peptide (S ⁇ Q ID NO:2) or with scrambled ⁇ RACK peptide (S ⁇ Q ID NO:4) for 20 minutes.
- Perfiision was maintained at a constant flow of 10 mL/min with Krebs-Henseleit solution containing 0.5 ⁇ M of the appropriate peptide.
- the Langendorff method employed used retrograde flow from the ventricle to the aorta and into the coronary arteries, bypassing the pulmonary arteries.
- Rat hearts were prepared as described in Example 1. After a 30 minute equilibration period, global ischemia was induced by interrupting fluid flow for 45 minutes. The hearts were then reper fused with 0.5 ⁇ M of ⁇ RACK peptide for 20 minutes, followed by 40 minutes of reperfusion without the peptide. As a control, some hearts were left untreated after ischemia. During the 60 minute period following ischemia, ischemia-induced cell damage was determined by monitoring the creatine phosphokinase (CPK) activity
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US27483000P | 2000-11-10 | 2000-11-10 | |
US274830P | 2000-11-10 | ||
PCT/US2001/051600 WO2002078600A2 (en) | 2000-11-10 | 2001-11-09 | γεRACK PEPTIDE COMPOSITION AND METHOD FOR PROTECTION AGAINST TISSUE DAMAGE DUE TO ISCHEMIA |
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JP (3) | JP2004519508A (en) |
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US7851587B2 (en) | 2006-10-02 | 2010-12-14 | The Board Of Trustees Of The Leland Stanford Junior University | Peptides derived from the C2 domain of epsilon PKC |
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WO1998017299A1 (en) * | 1996-10-18 | 1998-04-30 | The Board Of Trustees Of The Leland Stanford Junior University | Isozyme-specific activators of protein kinase c, methods and compositions |
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Non-Patent Citations (7)
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GRAY MARY O ET AL: "A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 49, 5 December 1997 (1997-12-05), pages 30945 - 30951, ISSN: 0021-9258 * |
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WO2002078600A2 (en) | 2002-10-10 |
JP2004519508A (en) | 2004-07-02 |
JP2008266343A (en) | 2008-11-06 |
CA2429108A1 (en) | 2002-10-10 |
WO2002078600A3 (en) | 2003-09-04 |
AU2001297950B2 (en) | 2006-07-06 |
JP2013100326A (en) | 2013-05-23 |
EP1359883A2 (en) | 2003-11-12 |
AU2001297950B8 (en) | 2006-08-03 |
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