JP2013100326A - ψεRACK PEPTIDE COMPOSITION AND METHOD FOR PROTECTION AGAINST TISSUE DAMAGE DUE TO ISCHEMIA - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for protecting tissue from damage due to an ischemic event, a method for administering an εPKC peptide agonist for inducing ischemic preconditioning, and a method for ameliorating tissue damage due to ischemic event.SOLUTION: The composition for reducing injury to organs in vivo exposed to ischemic or hypoxic condition, comprises ψεRACK peptide and a pharmaceutical vehicle. The composition containing ψεRACK peptide is used for a preparation for treating organs in vivo exposed to an ischemia or a hypoxic condition.

Description

(Background of the Invention)
The present invention relates to a method of protecting cells and tissues from damage caused by ischemic events. The method of the present invention comprises administering a peptide agonist of protein kinase C, more specifically administering a pseudo-epsilon RACK (ψεRACK) peptide.

  (References)

（発明の背景）
プロテインキナーゼＣ（ＰＫＣ）は、細胞増殖、遺伝子発現の調節、およびイオンチャネル活性の調節を含む、種々の細胞機能に関与するシグナル伝達において重要な酵素である。ＰＫＣファミリーのアイソザイムとして、少なくとも１１の異なるプロテインキナーゼが挙げられ、これらはそれらの相同性およびアクチベーターに対する感受性に基づき少なくとも３つのサブファミリーのメンバーに分類され得る。古典的なサブファミリーつまりｃＰＫＣサブファミリー（α、β_Ｉ、β_ＩＩ、およびγＰＫＣ）は、アイソザイム固有の領域（可変領域またはＶ領域）により間隔を空けられた４つの相同ドメイン（Ｃ１、Ｃ２、Ｃ３、およびＣ４）を含み、活性化のためにカルシウム、ホスファチジルセリン（ＰＳ）、およびジアシルグリセロール（ＤＧ）またはホルボールエステルを必要とする。新規のサブファミリーつまりｎＰＫＣサブファミリーのメンバー（δ、ε、η、およびθＰＫＣ）はＣ２相同ドメインを欠き、活性化のためにカルシウムを必要としない。最後に、変則的なサブファミリーつまりαＰＫＣサブファミリーのメンバー（ζおよびλ／ιＰＫＣ）はＣ２相同ドメインとＣ１相同ドメインの半分とを欠き、ＤＧ、ホルボールエステル、およびカルシウムに非感受性である。

(Background of the Invention)
Protein kinase C (PKC) is an important enzyme in signal transduction involved in various cellular functions, including cell proliferation, regulation of gene expression, and regulation of ion channel activity. PKC family isozymes include at least 11 different protein kinases, which can be classified into members of at least three subfamilies based on their homology and sensitivity to activators. The classical or cPKC subfamily (α, β _I , β _II , and γPKC) is divided into four homologous domains (C1, C2, C3) spaced by isozyme-specific regions (variable or V regions). And C4) and requires calcium, phosphatidylserine (PS), and diacylglycerol (DG) or phorbol ester for activation. A new subfamily, members of the nPKC subfamily (δ, ε, η, and θPKC) lack the C2 homology domain and do not require calcium for activation. Finally, the anomalous subfamily or members of the αPKC subfamily (ζ and λ / ιPKC) lack the C2 homology domain and half of the C1 homology domain and are insensitive to DG, phorbol esters, and calcium.

  Studies on the subcellular distribution of PKC isozymes indicate that activation of PKC causes its redistribution in cells (also called translocation), whereby activated PKC isozymes are converted into plasma membranes, cytoskeletal elements, It has been demonstrated to bind to the nucleus and other subcellular fractions (Saito et al., 1989; Papadopoulos and Hall, 1989; Mochly-Rosen et al., 1990).

The intrinsic cellular function of different PKC isozymes appears to be determined by their subcellular location. For example, activated β _I PKC is found inside the nucleus, and activated β _II PKC is found around the nucleus and in the cell periphery of cardiomyocytes (Disatnik et al., 1994). Furthermore, in the same cells, εPKC binds to striated structures (probably contractile elements) and cell-cell contacts after activation or addition of exogenous activated εPKC to fixed cells (Mochly-Rosen et al., 1990; Disatnik et al., 1994). Localization of different PKC isozymes to different regions of the cell appears to be due to binding of the activated isozyme to a specific anchor molecule called a receptor for activated C-kinase (RACK).

  RACK is believed to function by selectively immobilizing activated PKC isozymes at each subcellular site. Although RACK binds only to fully activated PKC, RACK is not necessarily a substrate for this enzyme, and binding to RACK is not necessarily mediated through the catalytic domain of this kinase (Mochly -Rosen et al., 1991). PKC translocation reflects the binding of this activated enzyme to RACK immobilized in the cell particle fraction, and binding to RACK is required for PKC to produce its cellular response (Mochly-Rosen). 1995). Inhibition of PKC binding to RACK in vivo inhibits PKC translocation and PKC-mediated functions (Johnson et al., 1996; Ron et al., 1995; Smith and Mochly-Rosen, 1992).

CDNA clones encoding RACK1 and RACK2 have been identified (US Pat. No. 5,519,003; Ron et al., 1994; Csukai et al., 1995). Both are homologs of the β subunit of the G protein, which is a receptor for another translocated protein kinase (β-adrenergic receptor kinase, βARK) (Pitcher et al., 1992). Similar to Gβ, RACK1 and RACK2 have 7 WD40 repeats (Ron et al., 1994; Csukai et al., 1995). Recent data suggests that RACK1 is a β _II PKC-specific RACK (Stebbins et al., 2001) and RACK2 is specific for activated εPKC (Csukai et al., 1997).

  PKC translocation is required for proper functioning of the PKC isozyme. Peptides that mimic either the PKC binding site on RACK (Mochly-Rosen, 1991a; Mochly-Rosen, 1995) or the RACK binding site on PKC (Ron et al., 1995; Johnson et al., 1996) Isozyme-specific translocation inhibitor of PKC that selectively inhibits enzyme function. For example, an 8-amino acid peptide derived from εPKC (peptide εV1-2; SEQ ID NO: 1, Glu Ala Val Ser Leu Lys Pro Thr) is described in US Pat. No. 6,165,977. This peptide contains part of the RACK binding site on εPKC and selectively inhibits specific εPKC-mediated functions in cardiomyocytes.

  Recently, PKC, more specifically εPCK, has been shown to be involved in cardiac preconditioning to provide protection against ischemic damage. Long-term ischemia causes irreversible myocardial damage primarily due to cell death at the infarct site. Studies in isolated cardiomyocytes in animal models, isolated heart preparations and cultures show that short-term myocardial ischemia reduces such tissue damage in subsequent long-term ischemia Have been demonstrated (Liu, Y. et al., 1995, 1996; Hu et al., 1995; Brew et al., 1995; Schultz et al., 1996). This protection occurs naturally after angina and is referred to as preconditioning and can be mimicked by various non-specific PKC agonists (Mitchell et al., 1993; Mitchell et al., 1995; Murry et al., 1986; Speedy-Dick et al. 1994). Activation of both δPKC and εPKC occurs after preconditioning (Gray et al., 1997), but εPKC activation is required for protection of cardiomyocytes from ischemia-induced cell death ( US Pat. No. 6,165,977).

  In recent studies, cardioprotection from ischemia when εPKC-selective peptide agonists are administered intracellularly to isolated neonatal and adult cardiomyocytes and when produced intracellularly in transgenic mice in vivo. (Dorn G. et al., 1999). In this study, εPKC peptide agonists were administered intracellularly to cells isolated in vitro by experimental techniques or gene transfection appropriate for the cellular level. Unfortunately, none of these techniques are appropriate for human treatment or likely to succeed. Furthermore, it is not clear from this study whether εPKC can be delivered extracellularly to all tissues or intact organs in vivo to achieve a therapeutic effect.

(Summary of the Invention)
Accordingly, it is an object of the present invention to provide a method for protecting tissue from damage due to ischemic events.

  It is a further object of the invention to provide a method of administering an εPKC peptide agonist for the induction of ischemic preconditioning.

  It is yet another object of the present invention to provide a method for mitigating tissue damage caused by ischemic events.

  Accordingly, in one aspect, the invention encompasses a method of reducing damage to cells exposed to ischemia or hypoxia by administering a ψεRACK peptide to the cells. In one embodiment, the peptide is administered before the cells are exposed to ischemia or hypoxia. For example, the peptide is administered over a period of about 1-180 minutes before the cells are exposed to ischemia or hypoxia. In another embodiment, the peptide is administered after the cell has been exposed to ischemia or hypoxia. For example, the peptide is administered over a period of about 1-180 minutes after the cells are exposed to ischemia or hypoxia. In another embodiment, the peptide is administered to cells during ischemia or hypoxia.

  In one embodiment, the ψεRACK peptide has the 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 ψεRACK peptide is a moiety effective to facilitate transport across the cell membrane (eg, Tat-derived peptide (SEQ ID NO: 5), Antennapedia carrier peptide (SEQ ID NO: 3), or polyarginine. Peptide).

  The peptide can be administered by a route selected from the group consisting of intravenous, parenteral, subcutaneous, inhalation, intranasal, sublingual, mucosal, and transdermal.

  In another aspect, the invention includes a method of reducing damage to tissue exposed to ischemia or hypoxia by administering a ψεRACK peptide to the tissue as described above.

(Short description of the sequence)
SEQ ID NO: 1 is the prior art of εPKC octapeptide (US Pat. No. 6,165,977).

  SEQ ID NO: 2 is the ψεRACK octapeptide.

  SEQ ID NO: 3 is a carrier peptide derived from Drosophila Antennapedia homeodomain.

  SEQ ID NO: 4 is a scrambled ψεRACK octapeptide.

  SEQ ID NO: 5 is a carrier peptide derived from Tat.

  SEQ ID NO: 6 is a variant of SEQ ID NO: 2.

  SEQ ID NO: 7 is a variant 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 variant of SEQ ID NO: 2.

  SEQ ID NO: 11 is a variant of SEQ ID NO: 2.

  SEQ ID NO: 12 is a variant of SEQ ID NO: 2.

  SEQ ID NO: 13 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 14 is a variant of SEQ ID NO: 2.

  SEQ ID NO: 15 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 16 is a variant of SEQ ID NO: 2.

  SEQ ID NO: 17 is a variant of SEQ ID NO: 2.

SEQ ID NO: 18 is a fragment of SEQ ID NO: 2.
The application may further provide:
(Item 1) A composition for reducing damage to cells or organs exposed to ischemic or hypoxic conditions,
A composition comprising a ψεRACK peptide.
(Item 2) The composition according to item 1, wherein the ψεRACK peptide is administered before the cell or organ is exposed to the ischemic state or the hypoxic state. Composition.
(Item 3) The composition according to item 1, wherein the ψεRACK peptide is administered after the cell or organ is exposed to the ischemic state or the hypoxic state. object.
(Item 4) The composition according to item 2 or item 3, wherein the ψεRACK peptide is administered in a period of about 1 minute to 180 minutes.
(Item 5) The composition according to Item 1, wherein the ψεRACK peptide is administered during exposure of the cell or organ to the ischemic state or the hypoxic state. Composition.
(Item 6) The composition according to any one of items 1 to 5, wherein the peptide has a sequence identified as SEQ ID NO: 2 or SEQ ID NO: 6 to SEQ ID NO: 18. object.
(Item 7) The composition according to item 6, wherein the ψεRACK peptide is linked to a moiety effective to facilitate transport across the cell membrane.
(Item 8) The composition according to Item 7, wherein the part is a Tat-derived peptide (SEQ ID NO: 5), Antennapedia carrier peptide (SEQ ID NO: 3), or polyarginine peptide.
(Item 9) The composition according to any one of Items 1 to 8, wherein the peptide is an intravenous route, parenteral route, subcutaneous route, inhalation route, intranasal route, sublingual A composition characterized in that it is administered by a route selected from the group consisting of a route, a mucosal route, and a transdermal route.
(Item 10) The composition according to any one of Items 1 to 8, wherein the peptide is administered to a tissue as a whole organ ex vivo or in vivo. ,Composition.
(Item 11) The composition according to item 10, wherein the organ is selected from heart, lung, liver, brain, and kidney.
(Item 12) The composition according to item 10, wherein the peptide is administered by infusion into an intact heart through a coronary artery.

FIG. 1A shows the release of creatine phosphokinase (CPK) as a function of time during reverse perfusion in ex vivo rat hearts treated with ψεRACK (◯) or scrambled ψεRACK (control, □) prior to ischemic events. . Treated hearts were compared to hearts that were subjected to an ischemic period but remained untreated (▲) and hearts maintained under normoxia (no ischemia, no peptide treatment, ■) as controls . FIG. 1B shows the total amount of CPK released during 30 minutes of reperfusion in the study described in FIG. 1A. FIG. 2A shows the functional recovery of a working heart perfused with ψεRACK prior to global ischemia (left ventricular pressure (LVP, mmHg), its first derivative (dP / dt, mmHg / sec). ), And coronary perfusion pressure (PP, mmHg). The right panel shows an enlargement of the same trace before (baseline) and after reperfusion. FIG. 2B is a scan similar to FIG. 2A for an active heart perfused with scrambled ψεRACK prior to ischemia. FIG. 3A shows CPK as a function of time after ischemic injury in ex vivo borate hearts treated with ψεRACK during the first 20 minutes of reperfusion (◯) and hearts not treated after ischemia (▲). Indicates release. FIG. 3B shows total CPK release during 60 minutes of reperfusion after ischemic injury to (i) whole rat heart ex vivo treated with ψεRACK or (ii) whole rat heart not treated with ψεRACK for 20 minutes after ischemic injury. It is a bar graph which shows. FIG. 4A is a computer generated photograph of a pig heart section taken from a pig 5 days after in vivo treatment with ψεRACK during the first 10 minutes of a 30 minute ischemic injury. FIG. 4B is a computer generated photograph of a pig heart section taken from a pig 5 days after in vivo treatment with a scrambled ψεRACK peptide as a control during the first 10 minutes of a 30 minute ischemic injury. FIG. 4C is a bar graph showing total infarct area as a percentage of dangerous area measured in grams of heart tissue for pigs treated with ψεRACK peptide or scrambled ψεRACK peptide (control). FIG. 5 shows three time points: (1) before occlusion of the left anterior descending artery with a balloon catheter (before occlusion); (2) immediately after reperfusion with ψεRACK (after ψεRACK); and (3) 5 days after treatment before sacrifice. FIG. 5 is a graph showing the ejection fraction measured by porcine left ventricular imaging (after 5 days) (animal treated with ψεRACK (▲) and control animal treated with scrambled peptide (◯)).

(Detailed description of the invention)
(I. Definition)
As used herein, “tissue” refers to a group of similarly specialized cells that perform a common function. Tissue constitutes the organs and structural components of living organisms. As used herein, a tissue is intended to include an organ composed of a given tissue and the cells that make up the tissue, either individually or collectively.
“Ischemia” or “ischemic phenomenon” refers to an inadequate supply of blood to a particular cell, tissue, or organ. The result of the reduced blood supply is an inadequate supply of oxygen to the organ or tissue (hypoxia). Long-term hypoxemia can cause damage to affected organs or tissues.

  “Anoxia” refers to a substantially complete lack of oxygen in an organ or tissue and, if prolonged, can cause cell, organ, or tissue death.

  “Hypoxia” or “hypoxia” refers to a condition in 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.

  “Anoxic damage” refers to damage to cells, organs, or tissues due to inadequate oxygen supply for a period of time.

  “Reperfusion” refers to the return of fluid flow to tissue after a period of time when there has been no flow or reduced flow. For example, in cardiac reperfusion, fluid or blood returns to the heart through a supply line (eg, an in vivo coronary artery) after removal of an obstruction to the fluid or blood supply.

  “Treating a disease” refers to administering an effective therapeutic agent to reduce symptoms of the disease and / or to reduce the severity of the disease.

  A “conservative amino acid substitution” is one in which the activity (eg, εPKC agonist or ψεRACK agonist activity) or tertiary structure of a selected polypeptide does not change significantly. Such substitution typically involves the replacement of a selected amino acid residue with a different residue having similar physicochemical properties. For example, the Asp to Glu substitution is considered a conservative substitution because both are negatively charged amino acids of similar size. The grouping of amino acids by physicochemical properties is known to those skilled in the art.

  With respect to a particular sequence, “there are conservative substitutions” refers to sequences that differ from the particular sequence by having conservative amino acid substitutions at one or more positions.

  “Peptide” and “polypeptide” are used interchangeably herein and refer to a compound consisting of a chain of amino acid residues linked by peptide bonds. Unless otherwise indicated, peptide sequences are presented in order from the amino terminus to the carboxyl terminus.

  When a first peptide or polypeptide is said to “correspond” or “homologous” to a second peptide or polypeptide fragment, it means that the peptide or fragment is a program ALIGN (mutation gap matrix). And using gap penalties of 6 or more) means having similarity in amino acid residues when having an alignment score greater than 5 (in units of standard deviation) (Dayhoff, MO, ATLAS OF (PROTEIN SEQUENCE AND STRUCTURE (1972) Vol. 5, National Biomedical Research Foundation, pages 101 to 110, and Appendix 2, pages 1 to 10 for this volume) . More preferably, the two sequences (or portions thereof) are more than 50%, more preferably more than 70%, more preferably more than 80% when their amino acids are optimally aligned using the ALIGN program described above. If they are more than 1% identical, they are homologous.

  A polypeptide sequence or fragment is “derived from” another polypeptide sequence or fragment if it has the same sequence of amino acid residues as the region of the other sequence or fragment.

  An “εPKC agonist peptide” or “εPKC specific agonist peptide” is understood to mean a peptide of about 4 and about 30, preferably between about 5 and about 15 amino acids in length, derived from εPKC. In one embodiment, the εPKC agonist peptide is a region between about 70 and about 120 amino acids of εPKC, preferably a region between about 80 and about 100 amino acids, more preferably about 85 and about 92 amino acids. Derived from the area between.

  Abbreviations: “PKC”, protein kinase C; “RACK”, receptor for activated C-kinase.

  Abbreviations for amino acid residues are standard three letter and / or one letter symbols used in the art to refer to one of the 20 common L-amino acids.

(II. Administration of ψεRACK peptide agonist)
In one aspect, the invention administers a peptide capable of activating a signaling protein (eg, PKC) activated in vivo by binding to a cognate polypeptide (eg, receptor protein (RACK)). Provides a method of protecting cells, tissues, or organs from damage caused by ischemic events or hypoxia. The region of homology between the PKC signaling peptide and its RACK is called the “pseudo-RACK” sequence (ψ-RACK; Ron et al., 1994, 1995) and is typically the corresponding PKC-binding region of RACK. And has a similar sequence. The ψ-RACK sequence that acts as an εPKC-specific agonist peptide is identified herein as SEQ ID NO: 2. This peptide (referred to herein as ψεRACK) is an εRACK-specific agonist peptide and induces translocation of εPKC. Previous studies have not shown whether the ψεRACK peptide itself can be administered throughout the organ in vivo or ex vivo to induce εPCK translocation and confer protection against ischemia, so the effect of ψεRACK Attention has been focused on identifying drugs and peptide mimetics that mimic. In studies conducted in support of the present invention, ψεRACK was administered throughout the heart ex vivo and in vivo before and after exposure to ischemic conditions. This peptide reduced the extent of ischemic damage as described herein.

(A. Administration of ψεRACK peptide agonist before ischemia)
The peptide having the sequence identified herein as SEQ ID NO: 2 was synthesized and purified as described in the methods section. A scrambled ψεRACK peptide (SEQ ID NO: 4) was also prepared for use as a comparison or negative control for the ψεRACK peptide. In some studies, these peptides were conjugated to a carrier peptide (eg, Drosophila Antennapedia homeodomain (SEQ ID NO: 3)). It will be appreciated that administration of a native peptide, which is a peptide that has not been modified by attachment to a carrier, is also contemplated. Carrier peptides other than Drosophila Antennapedia are also contemplated, and examples of other carrier peptides include Tat-derived peptide (SEQ ID NO: 5, Fawell et al., 1994, Vives et al., 1997) or polyarginine peptide (Mitchell et al., 2000; Rolhard) 2000), or other similar carriers described in the art (Lindgren et al., 2000; Schwarze et al., 2000).

  As described in Example 1, the animals were anesthetized and their hearts were quickly removed and cannulated for perfusion with the Langendorff device. Hemodynamic parameters were monitored until stabilized (typically 10-20 minutes). After equilibration, ψεRACK peptide (SEQ ID NO: 2) or scrambled ψεRACK peptide (SEQ ID NO: 4) was delivered to the heart via the coronary artery for 20 minutes using 0.5 μM peptide.

  To induce global ischemia, flow to the heart was interrupted for 45 minutes. The heart was then reperfused for 30 minutes. During reperfusion, ischemia-induced cell damage was determined by measuring the activity of creatine phosphokinase (absorbance at 520 nm) during perfusion. Creatine phosphokinase is a cytosolic enzyme in cardiomyocytes, and its presence in the perfusate is proportional to the number of cardiomyocytes damaged by ischemia. The results are shown in FIGS.

  FIG. 1A shows the ex vivo heart treated with 500 nM (0.5 μM) ψεRACK (◯) or 0.5 μM scrambled ψεRACK (□) as a function of time during 30 minutes of reperfusion before ischemic events. 1 shows creatine phosphokinase (CPK) release. As controls, CPK release is also shown for hearts that were subjected to an ischemic period but were not treated with peptides (A) and hearts maintained in normoxia (no ischemia, no peptide treatment; ■). Hearts treated with ψεRACK prior to ischemia have CPK release close to that of control hearts maintained in normoxia. In contrast, hearts treated with scrambled ψεRACK have significant release of CPK, indicating significant cell damage.

  FIG. 1B is a bar graph showing total CPK release during the reperfusion period for hearts treated with ψεRACK and scrambled ψεRACK. Also shown is total CPK release from the heart exposed to ischemic events but remaining untreated.

  1A-1B show that hearts treated with ψεRACK prior to an ischemic event provide protection from damage caused by the subsequent ischemic event. Accordingly, in one embodiment, the present invention contemplates a method of reducing or preventing damage to tissue exposed to ischemia or hypoxia by administering an amount of a ψεRACK agonist peptide to the tissue. The peptide may be administered 1 to 180 minutes before the ischemic event, more preferably about 1 to 120 minutes before the ischemic event, more preferably about 1 to 60 minutes before the ischemic event. In another embodiment, a period of less than about 180 minutes, more preferably less than 120 minutes, and even more preferably less than about 60 minutes separates the cessation of peptide delivery from the ischemic event.

  In another study in support of the present invention, as described in Example 1, the functional recovery of the ex vivo heart after ischemic events was measured by constant pulsation (3.3 Hz) and constant coronary flow ( Evaluated by monitoring constant volume left ventricular pressure (LVP) at a coronary flow (10 mL / min). Prior to the ischemic event, the heart was treated with 500 nM ψεRACK (SEQ ID NO: 2) or scrambled ψεRACK peptide (SEQ ID NO: 4). After 30 minutes of global ischemia, the heart was monitored during a 30 minute reperfusion period. The results are shown in FIGS.

  FIG. 2A shows the results for a heart treated with ψεRACK peptide prior to global ischemia, and FIG. 2B shows the results for a heart treated with scrambled ψεRACK before ischemia. In the comparison of baseline and reperfusion levels in treated and untreated hearts, a significant reduction in ischemic damage is seen with administration of the pre-ischemic ψεRACK peptide. This is shown in particular by comparing the recovery of left ventricular pressure (LVP) in hearts pretreated with ψεRACK and those in hearts pretreated with scrambled peptides. A 4-fold improvement in recovery of both LVPs and their first derivative (dP / dt) was achieved by pretreatment with ψεRACK. Furthermore, ψεRACK decreased the elevated LVP target diastolic pressure and coronary perfusion pressure (PP).

  Thus, these studies (FIGS. 1 and 2) show that cell damage to tissues due to ischemia or hypoxia is reduced by administering ψεRACK prior to ischemic or hypoxic events. . In this study, this ψεRACK is administered to the organ through the coronary artery for a period prior to exposure to ischemia and / or hypoxia. This period, as well as the dose of peptide administered, can vary considerably as discussed in more detail below.

(B. Administration of ψεRACK peptide agonist following ischemia)
In another study conducted in support of the present invention, ψεRACK was administered to the ex vivo heart after an extended ischemic period, and ψεRACK was effective in providing protection from ischemic damage. It was. As described in Example 2, the entire rat heart was perfused on a Langendorff apparatus. After a 30 minute equilibration period, global ischemia was induced by stopping fluid flow for 45 minutes. The heart was then reperfused for 20 minutes with or without the ψεRACK peptide, followed by 40 minutes without peptide. During the 60 minutes following ischemia (20 minutes peptide reperfusion + 40 minutes perfusion), CPK activity in this perfusate was analyzed. The results are shown in FIGS.

  FIG. 3A shows CPK release as a function of time after ischemia for hearts treated with ψεRACK (（) and untreated after ischemia (▲). FIG. 3B is a bar graph showing total CPK release during 60 minutes of perfusion for peptide-treated and untreated ex vivo hearts. These data indicate that subsequent administration of ψεRACK peptide to tissue previously exposed to ischemia or hypoxia is effective to reduce cell damage. FIG. 3B shows that there is approximately one-half total CPK release for tissues treated with the ψεRACK peptide.

(C. Administration of ψεRACK peptide in vivo)
In another study in support of the present invention, the ability of the ψεRACK peptide to protect tissue from damage due to ischemic or hypoxic events was assessed by administering the peptide in vivo. As detailed in Example 3, ψεRACK peptide (SEQ ID NO: 2) or scrambled ψεRACK peptide (SEQ ID NO: 4) was administered to adult pigs during the first 10 minutes of the 30 minute ischemic injury. Administered. After 5 days, the heart was analyzed for tissue damage. The results are shown in FIGS.

  4A-4B are computer-generated photographs of porcine heart sections obtained from pigs treated in vivo using ψεRACK (FIG. 4A) or using a scrambled ψεRACK peptide as a control (FIG. 4B). It is. Allowing the heart to determine the area at risk of ischemic injury (the area in the arrow in FIG. 4B, mainly in the middle wall) and the infarcted area (the white area in FIG. 4B), Staining was performed using a double-staining technique (Example 3). As seen in FIG. 4B, the control heart treated with the scrambled ψεRACK peptide has a large infarcted site within the risk site (borderline indicated by arrows). In contrast, pigs that received the ψεRACK peptide (FIG. 4A) have significantly reduced infarcted sites. Measurements of surface area, region weight, and total tissue weight of risk and infarct regions showed that the control heart had an average of 36.5 ± 0.3% infarct in the risk site, In contrast, hearts treated with the ψεRACK peptide averaged 14.9 ±. Shown to have 6% infarct (p <0.005).

  FIG. 4C is a bar graph showing infarcted sites as a percentage of risk sites measured in grams of heart tissue. As can be seen, the rate of infarct was reduced by more than one-half for animals treated with ψRACK. Thus, delivery of the ψεRACK peptide in vivo before or during an ischemic event is effective to reduce the rate of infarct by at least one-half.

  Blood samples and tissue samples such as lung, liver, brain, intestine, kidney were collected from the animals and analyzed in a pathology laboratory. All samples were normal and no inflammation or tissue abnormalities were observed.

  In another study, porcine (n = 5) left ventricular angiography (LVG) was performed using 6 Fr. Pig-tail catheters were used at three time points: (1) Pre-occlusion of the left anterior descending artery with a balloon catheter (pre-occlusion); (2) Reperfusion with 5 μM / 10 mL ψεRACK Immediately after (after ψεRACK); and (3) before sacrifice after 5 days (after 5 days). The LVG was recorded from two viewpoints (right diagonally forward and left diagonally forward). During maximal contraction, the ejection fraction (EF) relative to the total maximal volume present in the left ventricle (percentage of blood ejected in the beat) is analyzed by software (Plus Plus (Sanders Data Systems)) and two The average value of the viewpoint was evaluated. The ejection fraction was calculated based on the left ventricular volume. The results are shown in FIG. Ejection rate is a measure of how well the heart is functioning, and a higher ejection rate indicates a better functioning heart. An ejection fraction of less than 50% in a short period can suggest progression to a state of heart failure. As can be seen in FIG. 5, animals treated with ψεRACK (▲) had a higher ejection fraction after occlusion compared to control animals treated with scrambled peptide (◯). Suggested reducing or preventing ischemia-induced damage to cells and tissues. This is also the data point after 5 days of ischemia and treatment (animals treated with ψεRACK are equivalent to the ejection fraction measured before ischemia and about 10% higher than untreated animals, It was clear from the fact that it had an ejection fraction. Thus, delivery of ψεRACK peptide in vivo after ischemia to reduce cell and tissue damage, as indicated by ejection fraction that is at least 10% greater than that of untreated cells or tissues. It is valid.

(III. ΨεRACK peptide)
As used herein, “ψεRACK peptide” refers to the peptide represented by SEQ ID NO: 2, as well as 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); HDAPIGIYE (SEQ ID NO: 7); HDAPVGYE (SEQ ID NO: 8); HDAPIGDY (SEQ ID NO: 10); HDAPIGEY (SEQ ID NO: 11); ADAPIGID (SEQ ID NO: 12); HDGPIGYD (SEQ ID NO: 13); HDAAIGYD (SEQ ID NO: 14) and combinations of these variants.

  In one preferred embodiment, the sequence “DAPIG” (SEQ ID NO: 18) in SEQ ID NO: 2 has no more than 2 modifications at any residue. One, two, or all three of the residues outside the sequence “DAPIG” can be modified. For example, AEAPVGEY (SEQ ID NO: 15) is a derivative of SEQ ID NO: 2 in which all three residues outside the “DAPIG” (SEQ ID NO: 18) sequence and two residues within the “DAPIG” sequence have been modified. Other examples include HEAPIGDN (SEQ ID NO: 16) and HDGDIGYD (SEQ ID NO: 17).

  It will also be appreciated that SEQ ID NO: 2 and fragments of the above variants may be suitable. An exemplary fragment of SEQ ID NO: 2 is DAPIG (SEQ ID NO: 18).

  All of these exemplary peptides can be (i) chemically synthesized or (ii) recombinantly produced in a host cell using, for example, an expression vector comprising a polynucleotide fragment encoding the 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 being treated and the condition of the patient. The dosage is readily determined by those skilled in the art based on animal and human studies. Typically, between 0.05 and 5 μM, more preferably between 0.1 and 2 μM, and most preferably about 0.1 to 1 μM of peptide is administered. However, the upper and lower limits of these ranges are merely illustrative.

  The peptide can be administered by any suitable route, as determined by the principal healthcare professional. For example, administration by intravenous, parenteral, subcutaneous, inhalation, intranasal, sublingual, mucosal, transdermal, and the like is contemplated. Of course, the route of administration will affect the dose and timing of administration, as will be appreciated by those skilled in the art.

  The peptide can be administered in the form of a fusion protein or transport protein conjugate. Typically, to form a fusion protein, this peptide is linked to another peptide by a bond other than a Cys-Cys bond. An amide bond from the C-terminus of one peptide to the N-terminus of another peptide is an example of a bond in a fusion protein. The second peptide to which the δPKC agonist / antagonist peptide is attached can be virtually any peptide selected for therapeutic or transport purposes. For example, it may be desirable to link a ψεRACK peptide to a cytokine or other peptide that elicits a biological response.

  Where the peptide is part of a conjugate, the peptide is typically linked to a carrier peptide (eg, a Tat-derived transport polypeptide (Vives et al., 1997), polyarginine (Mitchell et al., 2000) by Cys-Cys coupling. Rolhbad et al., 2000), or Antennapedia peptides). See U.S. Pat. No. 5,804,604. In another general embodiment, the peptide can be introduced into the entire cell, tissue, or organ using a carrier or capsule material (eg, liposomes in liposome-mediated delivery).

  It is also understood that ψεRACK as well as any compound having similar activity can be used in the treatment methods described herein. Other compounds (eg, peptidomimetics, chemicals, or other peptides) can be identified, for example, by the screening methods disclosed in US Pat. No. 6,165,977, and this column 14, line 45- The part of column 15 line 54 is incorporated herein by reference. Briefly, as an example for identifying compounds that are effective in protecting cells or tissues from ischemia, δPKC introduces a solution containing δPKC into a multiwell plate and binds δPKC to plastic. To be fixed in the wells of a multi-well plate. The wells can be pre-coated with a substrate that enhances δPKC binding and / or a substrate that reduces the level of non-specific binding.

  The plate is then incubated with a blocking solution (eg containing bovine serum albumin) and then washed several times. In the reporter labeled (eg, radiolabeled by fluorescent labeling) peptide ψεRACK (SEQ ID NO: 2) and test well, a solution containing the test compound is added, as opposed to the control well. Different wells can contain different test compounds or different concentrations of the same test compound. Each test compound at each concentration is typically tested in duplicate, and each assay is typically negative (well without test compound) control as well as positive ("test compound" unlabeled) Wells that are peptides) control. The free peptide is then washed away and the degree of binding in this well is assessed.

  A test compound is identified as an active test compound if the binding of the peptide is reduced (ie, the effect on the degree of binding is above a threshold level). More specifically, a compound can be considered to have binding activity if the decrease in binding differs several fold between the control sample and the experimental sample. Typically, a binding difference between the test sample and the control sample that is 2 or 4 times the threshold is determined.

  Detection methods useful in such assays include antibody-based methods, direct detection of reporter moieties (eg, fluorescent labels) introduced into peptides, and the like.

  Various test compounds can be screened, including other peptides, macromolecules, small molecules, chemical and / or biological mixtures, fungal extracts, bacterial extracts or algae extracts. These compounds can occur biologically or synthetically.

(IV. Usefulness and route of administration)
The present invention has application, for example, in the treatment of surviving cardiac paralysis patients and in the treatment of persons who die immediately due to heart disease after hospital admission. Delivery of an εPKC selective peptide agonist (ψεRACK) is useful both acutely and chronically in the management of these patients.

  Acutely, in patients who are transported to hospital with an impending infarction, medical treatment has traditionally been directed to eliminating the cause of coronary artery occlusion, either by thrombolysis or catheter angioplasty . However, reperfusion of damaged sites can be one of the major mechanisms of cardiomyocyte damage. To induce cardioprotection, administration of a ψεRACK peptide PKC agonist, delivered to the occlusion site by a catheter or injected intravenously, immediately before or simultaneously with thrombolysis or angiogenesis, is contemplated by the present invention. Is done.

  Chronically, in patients with angina, the current medical approach is to stop angina symptoms without replacing the protective effects of angina preconditioning. An AεPKC selective agonist (eg, ψεRACK) replaces the preconditioning effect induced by angina in patients with angina and faster myocardium during future onset of more severe ischemia Provide salvage.

  A further application of the present invention is in clinical situations where the timing of ischemia is controlled by a physician. In such cases, pharmacological enhancement of the preconditioning response may provide significant benefits for the patient undergoing treatment. Specifically, each year, in the United States alone, 600,000 adults and 12,000 children undergo thoracotomy using cardiopulmonary bypass, during which time the heart takes a few minutes to an hour or so Subject to controlled ischemia for a period spanning over. Despite advances in cardioprotection, myocardial failure during the immediate postoperative period remains the leading cause of morbidity and mortality in these patients. The exact timing of ischemic injury is known prior to the patient and allows administration of ψεRACK prior to ischemia. Administration of ψεRACK reduces myocardial damage by inducing a preconditioning response for hours or days prior to surgery.

  Similar benefits can be seen in the field of heart transplantation, where there are about 2500 cases each year in the United States. Prolonged graft ischemia is one of the factors limiting long-range donor organ acquisition for such heart transplants. Administration of ψεRACK peptide at the time of organ acquisition can prolong the time between organ collection and organ transplantation and can reduce the risk of post-operative myocardial failure.

  Of course, it is understood that the ψεRACK peptide can be used in the treatment of various ischemic and hypoxic conditions in addition to cardiac ischemia. For example, ψεRACK can be administered to a wide variety of cells and tissues before, during, or after an ischemic or hypoxic event. Examples include, but are not intended to be limited to, the central nervous system, where the kidney, vascular endothelium, liver, eye, and tissue damage to the brain and other central nervous system tissue can occur due to seizures .

  As shown by the studies described herein, the peptide can be administered before, during, or after ischemic or hypoxic events. When delivered prior to ischemic injury, this peptide effectively reduces the extent of cell damage. Preferably, the peptide is perfused through or through the tissue prior to ischemia for about 1-180 minutes, more preferably for about 1-120 minutes, and most preferably for about 1-60 minutes. Is done. In one embodiment, perfusion is performed within about 3 hours, more preferably within about 120 minutes, and most preferably within about 60 minutes (pause of peptide perfusion and ischemic or low Separated from the oxygen phenomenon). The peptide can be delivered during a period following an ischemic injury of 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, or longer.

  If the polypeptide is delivered following an ischemic event, in a preferred embodiment, the period of no more than about 2 hours, more preferably no more than 1 hour, and even more preferably no more than 30 minutes Separated from the start of administration of the peptide. The peptide can be delivered during a period following an ischemic injury of 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, or longer.

  The peptide can also be administered during an ischemic event. Specifically, during a time of controlled ischemia (eg, during surgery), a healthcare professional may begin administering ψεRACK immediately before or simultaneously with the onset of an ischemic event.

(IV. Examples)
The following examples further illustrate the invention described herein and are in no way intended to limit the scope of the invention.

(Method)
(1. Peptide preparation)
ΨεRACK (HDAPIGYD, SEQ ID NO: 2) was synthesized and purified (> 95%) with Stanford Protein and Nucleic Acid Facility. Similarly, a scrambled ψεRACK peptide (PDYHDAGI, SEQ ID NO: 4) was prepared. In some studies, this peptide was used as a Drosophila antennapedia homeodomain-derived peptide (C-RQIKIWFQNRRMKWKK, SEQ ID NO: 3; Theodore, L. et al., 1995; Jhonson, JA et al., 1996a) using a carrier peptide. Through the N-terminal Cys-Cys bond or by cross-linking with a Tat protein-derived peptide (C-YGRKKRRQRRR, SEQ ID NO: 6) via the N-terminal Cys-Cys bond.

Example 1
(Ex vivo administration of ψεRACK before ischemia)
Mice or rats i. p. Anesthetized with avertin, their hearts were rapidly removed and the hearts were intubated through the aorta for reperfusion as described in the art (Colbert et al., 1997). Care was taken to perfuse these hearts within 90 seconds of removal. Left ventricular pressure and real time derivative (dP / dt) were monitored via a catheter placed at the apex of the ventricle. Hemodynamic parameters were recorded every 20 minutes throughout the procedure. Oxygenated Krebs-Henseleit solution (NaCl 120; KCl 5.8; NaHCO ₃ 25; NaH ₂ O ₄ 1.2; MgSO ₄ 1.2; CaCl ₂ 1.0; and glucose 10 (unit is nmol / L) The heart was perfused with a pH of 7.4, 37 ° C.).

  After an equilibration period of 10-20 minutes, the heart was treated with ψεRACK peptide (SEQ ID NO: 2) or scrambled ψεRACK peptide (SEQ ID NO: 4) for 20 minutes. Perfusion was maintained with constant flow (10 mL / min) using a Krebs-Henseleit solution containing 0.5 μM of the appropriate peptide. The Langendorff method used used retrograde flow from the ventricle to the aorta and coronary artery, bypassing the pulmonary artery.

  Flow was interrupted for 45 minutes to induce global ischemia. After the ischemic event, the heart was reperfused with Krebs-Henseleit solution for 30-160 minutes. During reperfusion, ischemia-induced cell damage was determined by measuring the activity of creatine phosphokinase (CPK) (absorption at 520 nm) in the perfusate using the Sigma kit. The results are shown in FIGS.

(Example 2)
(Ex vivo administration of ψεPKC after ischemia)
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 heart was then perfused with 0.5 μM ψεRACK peptide for 20 minutes and then reperfused for 40 minutes without the peptide. As a control, some hearts were left untreated after ischemia. During the 60 minute period after ischemia, cell damage induced by ischemia was monitored by monitoring creatine phosphokinase (CPK) activity (absorption at 520 nm) in the perfusate collected during reperfusion. Were determined. The results are shown in FIGS.

(Example 3)
(In vivo administration of ψεPKC before ischemia)
Young adult sows (35-40 kg in weight) were anesthetized and a catheter was introduced into the heart via the carotid artery. Using conventional procedural cardiology techniques, a wire was placed through the catheter to the left anterior descending artery (LAD). This wire was routed to deliver the balloon to the occlusion site. The balloon was then inflated to block blood flow for 30 minutes. During the first 10 minutes of ischemic injury, either a control scrambled ψεRACK peptide (SEQ ID NO: 4, n = 5) or a biologically active ψεRACK peptide (SEQ ID NO: 2, n = 5) was transferred to the balloon. Delivered directly downstream of the occlusion by diffusion through. About 20 μg of peptide (about 400 ng / kg body weight) was administered.

  After 30 minutes of occlusion, the balloon was removed and the pig was left to recover from surgery. After 5 days, the pig was euthanized and the heart was collected. After cardiac removal, the LAD was occluded. With the occlusion fixed, Evans Blue dye was injected (which stains all sites with no risk of infarction in blue but leaves all sites without access to the blood in red). The heart was then cut into sections and stained with tetrazolium red dye, which stains all living sites red and occluded dead tissue white. Each heart had multiple tissue sections with unique sites marked with ischemic risk sites and infarct sites. This was used to determine the percentage of infarct to the area at risk for each section and the entire heart. The results are shown in FIGS.

  Although the invention has been described with reference to particular embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.

  These and other objects and features of the invention will be more fully appreciated when the detailed description of the invention is read in conjunction with the accompanying drawings.

Claims (1)

The invention described herein.

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