Patent Application
20070054860
The current invention relates to the body's innate way of modulating important physiological processes and builds on insights reported in PCT International Publications WO99/59617 and WO01/72831 and PCT International Application PCT/NL02/00639, the contents of the entirety of all of which are incorporated herein by this reference.
In the aforementioned applications, small gene-regulatory peptides are described that are present naturally in pregnant women and are derived from proteolytic breakdown of placental gonadotropins such as human chorionic gonadotropin (hCG) produced during pregnancy. These peptides (in their active state often only at about 4 to 6 amino acids long) were shown to have unsurpassed immunological activity that they exert by regulating expression of genes encoding inflammatory mediators such as cytokines. Surprisingly, it was found that breakdown of hCG provides a cascade of peptides that helps maintain a pregnant woman's immunological homeostasis. These peptides are nature's own substances that balance the immune system to assure that the mother stays immunologically sound while her fetus does not get prematurely rejected during pregnancy but instead is safely carried until its time of birth.
Where it was generally thought that the smallest breakdown products of proteins have no specific biological function on their own (except to serve as antigen for the immune system), it now emerges that the body, in fact, routinely utilizes the normal process of proteolytic breakdown of the proteins it produces to generate important gene-regulatory compounds, short peptides that control the expression of the body's own genes. Apparently, the body uses a gene-control system ruled by small, broken-down products of the exact proteins that are encoded by its own genes.
It is known that, during pregnancy, the maternal system introduces a status of temporary immunomodulation which results in suppression of maternal rejection responses directed against the fetus. Paradoxically, during pregnancy, often the mother's resistance to infection is increased and she is found to be better protected against the clinical symptoms of various autoimmune diseases such as rheumatism and multiple sclerosis. The protection of the fetus thus cannot be interpreted as only a result of immune suppression. Each of the above three applications has provided insights by which the immunological balance between protection of the mother and protection of the fetus can be understood.
It was shown that certain short breakdown products of hCG (i.e., short peptides which can easily be synthesized, if needed modified, and used as a pharmaceutical composition) exert a major regulatory activity on pro- or anti-inflammatory cytokine cascades that are governed by a family of crucial transcription factors, the NF-κB family, which stands central in regulating the expression of genes that shape the body's immune response.
Most of the hCG produced during pregnancy is produced by cells of the placenta, the exact organ where cells and tissues of mother and child most intensely meet and where immunomodulation is most needed to fight off rejection. Being produced locally, the gene-regulatory peptides which are broken down from hCG in the placenta immediately balance the pro- or anti-inflammatory cytokine cascades found in the no-man's land between mother and child. Being produced by the typical placental cell, the trophoblast, the peptides traverse extracellular space, enter cells of the immune system and exert their immunomodulatory activity by modulating NF-κB-mediated expression of cytokine genes, thereby keeping the immunological responses in the placenta at bay.
It is postulated herein that the beneficial effects seen on the occurrence and severity of autoimmune disease in the pregnant woman result from an overspill of the hCG-derived peptides into the body as a whole; however, these effects must not be overestimated, as it is easily understood that the further away from the placenta, the less immunomodulatory activity aimed at preventing rejection of the fetus will be seen, if only because of a dilution of the placenta-produced peptides throughout the body as a whole. However, the immunomodulatory and gene-regulatory activity of the peptides should by no means only be thought to occur during pregnancy and in the placenta; men and women alike produce hCG, for example, in their pituitaries, and nature certainly utilizes the gene-regulatory activities of peptides in a larger whole.
Consequently, a novel therapeutic inroad is provided, using the pharmaceutical potential of gene-regulatory peptides and derivatives thereof. Indeed, evidence of specific up- or down-regulation of NF-κB-driven pro- or anti-inflammatory cytokine cascades that are each, and in concert, directing the body's immune response was found in silico in gene arrays by expression profiling studies, in vitro after treatment of immune cells and in vivo in experimental animals treated with gene-regulatory peptides. Also, considering that NF-κB is a primary effector of disease (A. S. Baldwin, J. Clin. Invest., 2001, 107:3-6), using the hCG-derived gene-regulatory peptides offers significant potential for the treatment of a variety of human and animal diseases, thereby tapping into the pharmaceutical potential of the exact substances that help balance the mother's immune system such that her pregnancy is safely maintained.
The invention in particular relates to the treatment of an ischemic event such as a stroke or myocardial infarction.
An ischemic event refers to an event in which the blood supply to a tissue is obstructed. Due to this obstruction, the endothelial tissue lining the affected blood vessels becomes “sticky” and begins to attract circulating white blood cells. The white cells bound to the endothelium eventually migrate into the affected tissue, causing significant tissue destruction. Although neither acute myocardial infarction nor stroke is directly caused by inflammation, much of the underlying pathology and the damage that occurs after an acute ischemic event are caused by acute inflammatory responses during reperfusion, the restoration of blood flow to the affected organ. Early restitution of blood flow to ischemic tissues is essential to halt the progression of cellular injury associated with decrease of oxygen supply and nutrient delivery. This fact provides the basis for the traditional view that minimizing ischemic time is the only important intervention for diminishing the extent of ischemic injury. However, it is now well recognized that reperfusion of ischemic tissues initiates a complex series of reactions that can paradoxically injure tissues. Although several mechanisms have been proposed to explain the pathogenesis of ischemia—reperfusion injury, most attention has focused on a role for reactive oxygen and nitrogen metabolites and inflammatory leukocytes. In addition to the local tissue injury, distant organs can also be affected, particularly if the intensity of the inflammatory reaction in post-ischemic tissue (e.g., intestine) is great. The remote effects of ischemia—reperfusion injury are most frequently observed in the lung and (cardio- or cerebro-)vascular system, and can result in the development of the systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), both of which account for 30-40% of the mortality in tertiary referral intensive care units (ICUs). This application, however, mostly deals with localized ischemic events.
In PCT International Publication WO 01/72831, a method and a pharmaceutical composition are provided for modulating cardiovascular or circulatory disorders, such as heart failure, brain infarctions, Alzheimer's disease, thrombosis, arteriosclerosis, pregnancy-related cardiovascular or circulatory disorders and the like. It has been found that an immunoregulator as described in the application has a very beneficial effect on animals, including humans, suffering from a cardiovascular disorder. The immunoregulator according to PCT International Publication WO 01/72831 also widens the scope of possibilities of dotter treatments. In cases where conventionally such a treatment could not be performed because of risks of an oxygen tension becoming too low, a dotter treatment in cases of myocardial infarction is feasible when combined with treatment with the immunoregulator. Accordingly, expensive and difficult bypass surgery may, in many cases, be avoided, and the application also suggested the same protective effect of the immunoregulator in other organs as well in circulatory-related disease.
The current invention provides additional modes and means of treatment. The invention provides a method for modulating an ischemic event in a subject believed to be in need thereof comprising providing the subject with a signaling molecule comprising a short gene-regulatory peptide or functional analogue thereof, wherein the signaling molecule is administered in an amount sufficient to modulate the ischemic event. The signal molecule is preferably a short peptide, preferably of at most 30 amino acids long, or a functional analogue or derivative thereof. In a much preferred embodiment, the peptide is an oligopeptide of from about 3 to about 15 amino acids long, preferably 4 to 12, more preferably 4 to 9, most preferably 4 to 6 amino acids long, or a functional analogue or derivative thereof. Of course, such signaling molecule can be longer, for example, by extending it (N- and/or C-terminally) with more amino acids or other side groups, which can, for example, be (enzymatically) cleaved off when the molecule enters the place of final destination. In particular, a method is provided wherein the signaling molecule modulates translocation and/or activity of a gene transcription factor. It is particularly useful when the gene transcription factor comprises an NF-κB/Rel protein or an AP-1 protein. Ischemia induces increased expression of inflammatory cytokines due to activation of NF-κB and AP-1, and in a preferred embodiment, the invention provides a method wherein translocation and/or activity of the NF-κB/Rel protein is inhibited. In one embodiment, the peptide is selected from a group of peptides including LQG, AQG, LQGV (SEQ ID NO:1 of the hereby incorporated accompanying SEQUENCE LISTING), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:3), VLPALP (SEQ ID NO:4), ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ (SEQ ID NO:7), VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV (SEQ ID NO:10), VLAALP (SEQ ID NO:11), VLPALA (SEQ ID NO:12), VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID NO:15), GVLPALP (SEQ ID NO:16), LQGVLPALPQVVC (SEQ ID NO:17), LPGCPRGVNPVVS (SEQ ID NO:18), LPGC (SEQ ID NO:19), MTRV (SEQ ID NO:20), MTR, and VVC.
The invention is further explained by the use of the following illustrative examples.
Ischemia induces increased expression of inflammatory cytokines due to activation of NF-κB and AP-1. Inflammatory cytokines can be expressed by endothelium (for example, by trauma), perivascular cells and adherent or transmigrating leukocytes, inducing numerous pro-inflammatory and pro-coagulant effects. Together these effects predispose to inflammation, thrombosis and hemorrhage. Of clinical and medical interest and value, the present invention provides the opportunity to selectively control NFκB-dependent gene expression in tissues and organs in a living subject, preferably in a primate, allowing up-regulating essentially anti-inflammatory responses such as interleukin (IL) 10, and down-regulating essentially pro-inflammatory responses such as those mediated by tumor necrosis factor α (TNF-α), nitric oxide (NO), IL-5, and IL-1β.
The invention thus provides use of an NFκB-regulating peptide or derivative thereof for the production of a pharmaceutical composition for the treatment of an ischemic event, preferably in a primate, and provides a method of treatment of an ischemic event, notably in a primate. It is preferred that the treatment comprise administering to the subject a pharmaceutical composition comprising an NF-κB-down-regulating peptide or functional analogue thereof. Examples of useful NF-κB-down-regulating peptides are VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:23), VLPALP (SEQ ID NO:4), VVC, MTR and circular LQGVLPALPQVVC (SEQ ID NO: 17). More down-regulating peptides and functional analogues can be found using the methods as provided herein. Most prominent among NF-κB-down-regulating peptides are VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV (SEQ ID NO:1), and VLPALP (SEQ ID NO:4). These are also capable of reducing production of NO by a cell. It is herein also provided to use a composition that comprises at least two oligopeptides or functional analogues thereof, each capable of reducing production of NO and/or TNF-α by a cell, in particular wherein the at least two oligopeptides are selected from a group including LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2) and VLPALP (SEQ ID NO:4), for the treatment of an ischemic event and, moreover to treat ischemia—perfusion injury.
In one such instance as provided herein, such a subject has suffered from ischemic events or has undergone anoxia or infarction. A typical clinical instance is the myocardial infarction or chronic myocardial ischemia of heart tissue in various zones or areas of a living human subject or, likewise, a cerebrovascular infarct, such as a sudden massive infarct of the brain with immediate and possibly grave consequences, but also the so-called silent infarcts that go unnoticed for long times but are thought to be involved in the development of certain forms of dementias.
Typical examples also include other cardiovascular or circulatory disorders, such as heart failure, lacunar brain infarctions, Alzheimer's disease, thrombosis, arteriosclerosis, pregnancy-related cardiovascular or circulatory disorders, retinopathies (such as associated with vascular diseases like diabetes) and the like.
In response to a variety of pathophysiological and developmental signals, the NF-κB/Rel family of transcription factors is activated and forms different types of heterodimers and homodimers among themselves to regulate the expression of target genes containing κB-specific binding sites. NF-κB transcription factors are heterodimers or homodimers of a family of related proteins characterized by the Rel homology domain. They form two subfamilies, those containing activation domains (p65-RELA, RELB, and c-REL) and those lacking activation domains (p50, p52). The prototypical NF-κB is a heterodimer of p65 (RELA) and p50 (NF-κB1). Among the activated NF-κB dimers, p50-p65 heterodimers are known to be involved in enhancing the transcription of target genes and p50-p50 homodimers in transcriptional repression. However, p65-p65 homodimers are known for both transcriptional activation and repressive activity against target genes. KB DNA-binding sites with varied affinities to different NF-κB dimers have been discovered in the promoters of several eukaryotic genes and the balance between activated NF-κB homodimers and heterodimers ultimately determines the nature and level of gene expression within the cell. The term “NF-κB-regulating peptide” as used herein refers to a peptide or a modification or derivative thereof capable of modulating the activation of members of the NF-κB/Rel family of transcription factors. Activation of NF-κB can lead to enhanced transcription of target genes. Also, it can lead to transcriptional repression of target genes. NF-κB activation can be regulated at multiple levels. For example, the dynamic shuttling of the inactive NF-κB dimers between the cytoplasm and nucleus by IκB proteins and its termination by phosphorylation and proteasomal degradation, direct phosphorylation, acetylation of NF-κB factors, and dynamic reorganization of NF-κB subunits among the activated NF-κB dimers have all been identified as key regulatory steps in NF-κB activation and, consequently, in NF-κB-mediated transcription processes. Thus, an NF-κB-regulating peptide is capable of modulating the transcription of genes that are under the control of the NF-κB/Rel family of transcription factors. Modulating comprises the up-regulation or the down-regulation of transcription. In a preferred embodiment, a peptide according to the invention, or a functional derivative or analogue thereof, is used for the production of a pharmaceutical composition for the treatment of ischemic events. Examples of such events are (but not limited to) cerebral vascular accident (CVA), circulatory diseases of the brain, retinopathies (such as associated with vascular diseases like diabetes), circulatory diseases of pregnancy, thrombosis, atherosclerosis, and so on.
An ischemic event refers to an event in which the blood supply to a tissue is obstructed, such as stroke or myocardial infarction. Due to this obstruction, the endothelial tissue lining the affected blood vessels becomes “sticky” and begins to attract circulating white blood cells. The white cells bound to the endothelium eventually migrate into the brain or cardiac tissue, causing significant tissue destruction. Although neither acute myocardial infarction nor stroke is directly caused by inflammation, much of the underlying pathology and the damage that occurs after an acute ischemic event are caused by acute inflammatory responses during reperfusion, the restoration of blood flow to the affected organ. Thus, a method is provided herein for treating ischemic events, including cerebrovascular disease and ischemic heart failure, comprising administering to a subject in need of such a treatment a peptide according to the invention. In particular, a method is provided to control the acute inflammatory response during reperfusion of the affected body part by administering a peptide, or a modification thereof, capable of modulating expression of a gene encoding a pro-inflammatory cytokine. TNF-α is a pro-inflammatory and multifunctional cytokine that has been implicated in diverse pathological processes such as cancer, infection, and autoimmune inflammation. TNF-α has been recently detected in various cardiac-related illnesses including congestive heart failure, myocarditis, dilated and septic cardiomyopathy, and ischemic heart diseases. TNF mRNA and TNF-α protein were detected in explanted hearts from humans with dilated cardiomyopathy and ischemic heart disease, but TNF-α was not detected in nonfailing myocardium. Although the complete portfolio of signaling pathways that are common to both tumor necrosis factor receptor 1 (TNFR1) and tumor necrosis factor receptor 2 (TNFR2) is not known, it is of interest to note that a recently described zinc finger protein, termed tumor necrosis factor receptor associated factor 2 (TRAF2), has been shown to be involved with both TNFR1- and TNFR2-mediated signaling. Consequently, TRAF2-mediated signaling has been shown to activate NF-κB, with a resultant increase in the expression of the antioxidant protein manganese superoxide dismutase (MSOD). Previous studies suggested that the cytoprotective effects of TNF in the setting of myocardial ischemia were mediated through TNF-induced up-regulation of MSOD. It was suggested that pro-inflammatory cytokines such as TNF may play an important role in the timing of cardiac stress response, both by providing early anti-apoptotic cytoprotective signals that are responsible for delimiting cardiac injury and also by providing delayed signals that facilitate tissue repair and remodeling once myocardial damage has supervened. Given the observation that some peptides according to the invention are capable of up-regulating at least one gene in a cell, the invention now provides a method to increase the expression of gene products such as MSOD and other cytoprotective NF-κB-regulated genes. In particular, the invention provides a method for treating an ischemic-reperfusion injury comprising administering to a subject in need of such treatment a signaling molecule comprising a peptide or functional analogue thereof, the molecule capable of increasing production of IL-10 by a cell. Increased IL-10 production is, for example, achieved by treating the subject systemically or treating the subject's infarcted area locally with peptides AQGV (SEQ ID NO:2), LQGV (SEQ ID NO:1) or VLPALP (SEQ ID NO:4), or a functional analogue thereof similarly capable of modulating translocation and/or activity of a gene transcription factor present in a cell in the ischemic or infracted area. These peptides have the added advantage that TNF-α production by the cell is reduced. When taking ischemic heart failure as an example, an NF-κB-down-regulating peptide according to the invention can, for example, be introduced locally to the infracted area directly as a synthesized compound to living cells and tissues via a range of different delivery means. These include the following:
A. Intracoronary delivery is accomplished using catheter-based deliveries of synthesized peptide (or derivative) suspended in a suitable buffer (such as saline) which can be injected locally (i.e., by injecting into the myocardium through the vessel wall) in the coronary artery using a suitable local delivery catheter such as a 10 mm InfusaSleeve catheter (Local Med, Palo Alto, Calif.) loaded over a 3.0 mm×20 mm angioplasty balloon, delivered over a 0.014 inch angioplasty guide wire. Delivery is typically accomplished by first inflating the angioplasty balloon to 30 psi, and then delivering the protein through the local delivery catheter at 80 psi over 30 seconds (this can be modified to suit the delivery catheter).
B. Intracoronary bolus infusion of a peptide (or derivative) synthesized previously can be accomplished by a manual injection of the substance through an Ultrafuse-X dual lumen catheter (SciMed, Minneapolis, Minn.) or another suitable device into proximal orifices of coronary arteries over 10 minutes.
C. Pericardial delivery of a synthesized peptide (or derivative) is typically accomplished by installation of the peptide-containing solution into the pericardial sac. The pericardium is accessed via a right atrial puncture, transthoracic puncture or a direct surgical approach. Once the access is established, the peptide material is infused into the pericardial cavity and the catheter is withdrawn. Alternatively, the delivery is accomplished via the aid of slow-release polymers such as heparin-alginate or ethylene vinyl acetate (EVAc). In both cases, once the peptide (or derivative) is integrated into the polymer, the desired amount of peptide/polymer is inserted under the epicardial fat or secured to the myocardial surface using, for example, sutures. In addition, the peptide/polymer composition can be positioned along the adventitial surface of coronary vessels.
D. Intramyocardial delivery of synthesized peptide (or derivative) can be accomplished either under direct vision following thoracotomy or using a thoracoscope or via a catheter. In either case, the peptide-containing solution is injected using a syringe or other suitable device directly into the myocardium.
Up to 2 cc of volume can be injected into any given spot and multiple locations (up to 30 injections) can be done in each patient. Catheter-based injections are carried out under fluoroscopic, ultrasound or Biosense NOGA guidance. In all cases, after catheter introduction into the left ventricle, the desired area of the myocardium is injected using a catheter that allows for controlled local delivery of the material. Of course, similar techniques are applied to administer the peptide locally to other infarcted areas, such as seen with cerebrovascular incidents.
In a further embodiment, the invention provides a method for modulating a cerebral ischemic event in a subject comprising providing the subject with a signaling molecule comprising a gene-regulatory peptide or functional analogue thereof in combination therapy with thrombolysis. Two major strategies can be used to reduce the neuronal damage following cerebral ischemia: restoration of cerebral blood perfusion through usage of thrombolytics and inhibition of the apoptotic and inflammatory cascades which result from ischemia through usage of a peptide or functional analogue according to the invention. Combining both treatment strategies provides additional benefits to those achieved by using the individual strategies alone. For instance, restoration of blood flow improves perfusion of the ischemic brain tissue with peptide compositions and enhances their protective effects. Thrombolysis and/or prevention of thrombi are, for example, achieved by intravenous injection of heparin, in a bolus of 5,000 IU, followed by infusion of 15,000 units/hour to induce an APTT-ratio of 2.0. Alternatively, intramuscular injections of low-molecular-weight heparin, such as fragmin of 200 IU/kg/day in two daily doses, are given. Intra-arterial thrombolysis is preferably applied within three hours of onset of ischemic stroke. In short, selective intra-arterial digital subtraction angiography is performed on a biplane, high-resolution angiography system (for example, a Toshiba CAS 500) with a matrix of 1024×1024 pixels. A 5.5.F-JB2 catheter (Valavanis) is inserted in the femoral artery and guided to the cerebral arteries for diagnostic four-vessel angiography. A microcatheter, mostly a Fast Tracker 18 (Target Therapeutics) through the 5.5-F JB2 catheter, is navigated into the cerebral arteria corresponding with the ischemic brain area. A microcatheter is navigated into the occluded cerebral artery. Urokinase (Urokinase HS Medac) in a mean dose usually ranging from 20,000 to 1,250,000 IU is infused directly into or near the proximal end of the occluding thrombus over 60 to 90 minutes. For mechanical disruption and removal of the thrombotic material, additional usage of a very flexible hydrophilic guide wire catheter with a J-shaped tip to avoid perforation of the vessel wall (for example, a Silver Speed MTI 0.008 or 0.010 inch) may be necessary. In addition to agents for thrombolysis and/or prevention of thrombosis, whether applied intravenously, intramuscularly or intra-arterially, treatment with a peptide composition is preferably started at the same time. The invention also provides a method for modulating an ischemic event in a subject comprising providing the subject with a signaling molecule comprising a gene-regulatory peptide or functional analogue thereof for the prevention of cerebral ischemia in patients with defined at-risk periods. Some conditions are frequently followed by cerebral ischemia, in which a peptide or functional analogue thereof is valuable to prevent infarction, illustrated in two specific examples. (1) Cardiac or aortic surgery is frequently complicated with severe hypotensive periods and/or thrombo-embolic events which may result in cerebral or myelum ischemia and infarction. The peptide composition according to the invention can be given in all or a specific subgroup of these patients, before, during and/or after surgery to prevent cerebral ischemia. (2) The final outcome in patients with aneurysmatic subarachnoid hemorrhagia (SAH) is largely determined by the development of cerebral ischemia in the subsequent three weeks. SAH is a life-threatening intracerebral bleeding, usually due to a rupture of an aneurysm of the cerebral arteries in the circulus Willisi. SAH affects 10.5 per 100,000 persons per year of which one-third will die. Up to one-third of the patients will develop cerebral ischemia in the three weeks after SAH, which determines the final outcome and for which all patients with SAH will be admitted to intensive care units. The pathophysiology of cerebral ischemia after SAH is not precisely known, but a specific role is claimed for the presence of subarachnoid blood and/or intracerebral inflammation and vasospasms. Treatment to prevent cerebral ischemia, including triple H-therapy (hypervolemia, hemodilution, hypertension), vasodilators, and endovascular approaches to symptomatic vasospasms, thus far are insufficient in many patients. An NF-κB-down-regulating peptide should be given in this three weeks following SAH, alone or in combination with other forms of preventive treatments, during which these patients are at risk to develop cerebral ischemia and can be monitored at the intensive care unit. In these two and other conditions in which there is a limited period with a significant increase to develop cerebral ischemia, an NF-κB-down-regulating peptide can be used to prevent (further) cerebral ischemia and improve final clinical outcome.
The invention furthermore provides a method to monitor and titrate therapeutic effects of a treatment with a peptide according to the invention in patients with cerebral ischemia. This is foremost achieved by clinical evaluation according to predefined neurological deficit-, disability- and handicap scales, such as the Oxford-handicap scale. CT, CT-angiography, MRI, MR-angiography, and SPECT-scan can be done. Also, cytokines, soluble cytokine-receptors, and chemokines are determined in follow-up plasma and cerebrospinal fluid (CSF) samples. Follow-up CSF samples can be obtained by permanent monitoring via ventricular catheters. Intracerebral HPLC sensors provide for determining parenchyma oxygen, pH and small metabolites including lactate, pyruvate and glucose. This device is already in use in combination with intracranial pressure bolds to monitor the cerebral parenchyma of patients with contusio cerebri.
Preferred routes of administration of a peptide or functional analogue thereof according to the invention in patients with cerebral ischemia are: Intravenously in 0.9% saline solutions according to protocol; intrathecally. In short, the peptide composition may be given after a lumbar puncture with an 18 G needle or after subsequent insertion of an extralumbal catheter with the tip in the intrathecal space. This way of drug administration cannot be used in patients with large infarctions and danger of replacement of brain tissue or herniation, but is a useful way in treating patients with an SAH. Intrathecal drug administration is an established route of drug administration in patients with leukemia and multiple sclerosis. In patients with SAH, extralumar drains are already frequently used to prevent or treat hydrocephalus, a common complication in SAH. Intra-arterial: A similar protocol is used as in intra-arterial thrombolysis. In short, selective intra-arterial digital substraction angiography is performed on a biplane, high-resolution angiography system (for example, a Toshiba CAS 500) with a matrix of 1024×1024 pixels. A 5.5.F-JB2 catheter (Valavanis) is inserted in the femoral artery and guided to the cerebral arteries for diagnostic four-vessel angiography. A microcatheter, mostly a Fast Tracker 18 (Target Therapeutics) through the 5.5-F JB2 catheter, is navigated into the cerebral arteria corresponding with the ischemic brain area. Perfusion of this area with a peptide is achieved according to this protocol. This route of administration is of special interest in the case of combination therapy with intra-arterial thrombolysis. In that case, the same devices and protocols are used in which the microcatheter is navigated into the occluded cerebral artery. Urokinase (Urokinase HS Medac) in a mean dose usually ranging from 20,000 to 1,250,000 IU is infused directly into or near the proximal end of the occluding thrombus over 60 to 90 minutes. For mechanical disruption and removal of the thrombotic material, additional usage of a very flexible hydrophilic guide wire catheter with a J-shaped tip to avoid perforation of the vessel wall (for example, a Silver Speed MTI 0.008 or 0.010 inch) may be necessary. Furthermore, a peptide or functional analogue may be applied locally after craniotomy. A range of suitable pharmaceutical carriers and vehicles is known conventionally to those skilled in the art. Thus, for parenteral or systemic administration, the peptide compound will typically be dissolved or suspended in sterile water or saline. Typically, systemic administration involves intravenous administration, for example, per infusionem. Especially when the subject is at risk to experience iatrogenic reperfusion injury occurring after the ischemic event, for example, due to treatment with an anticoagulant or a thrombolytic agent, systemic administration per infusionem is advantageous, as the risk of bleeding is increased in such patients, necessitating the reduction of invasive measures such as the use of catheters or other puncturing techniques.
Improvement in neurological diseases is limited due to the restricted regeneration capacity of neurons, especially in the central nervous system (CNS). For this reason, and for the high susceptibility of neurons to ischemia and inflammation, treatment strategies in neurology, more than in other medical disciplines, focus on an immediate prevention of (further) neural damage. Ischemia and inflammation of neural tissue are mediated by similar pathogenic pathways leading to and mediated by release and activation of transcription factors, such as NF-κB, and cytokines, such as TNF-α. In addition, in many neurological diseases, both ischemic and inflammatory processes contribute to (further) tissue damage. More than other diseases, neurological disorders will therefore profit from immune-mediating agents, such as by treatment with an β-hCG oligopeptide derivate such as an NF-κB-down-regulating peptide according to the invention, that have an immediate and pleiotropic effect and inhibit these common pathways in both ischemic and inflammatory processes. The invention also provides a method for treating cerebral infarction with an NF-κB-down-regulating peptide according to the invention.
Cerebral infarction is a common and disabling neurological disease which results from an acute onset, insufficient arterial blood supply and ischemia of the associated territorial brain. The causes of the acute insufficient perfusion are (1) thrombo-embolic events related to atherosclerosis of large cerebral arteria and/or cardiac diseases leading to cortical infarctions, (2) hypotension leading to so-called “watershed infarctions,” and (3) small vessel diseases related to hypertension and atherosclerosis leading to lacunar infarctions. Each type of infarction may induce distinct patterns of neurological deficits related to the function of the damaged brain area. All these types of infarctions, especially multiple lacunar infarctions, may contribute to the development of vascular (or post-stroke) dementia.
Neurological deficits in stroke are potentially reversible, provided the duration of ischemia is short, such as in “transient ischemic attacks” (TIA's). Partial spontaneous improvement in ischemic strokes most likely results from reversible dysfunction of the penumbra area, where ischemia does not evolve into infarction. The invention also provides treatment of ischemic stroke patients with thrombolytic agents combined with treatment with an NF-κB-down-regulating peptide according to the invention, preferably within three hours after onset of neurological symptoms when cerebral ischemia in potential is a treatable state.
Permanent neurological deficit in stroke patients is due to apoptotic cell death of infarcted brain tissue caused by long-lasting ischemic periods and subsequent activation of apoptotic pathways during the reperfusion phase. Ischemia induces depolarization and release of excitatory amino acids such as glutamate leading to Ca2+ and water influx, which successively leads to cerebral edema and Ca2+-mediated inflammatory and degenerative processes. Ischemia induces increased expression of TNF and activation of NF-κB. TNF can be expressed by endothelium (for example, by trauma), perivascular cells and adherent or transmigrating leukocytes, inducing numerous pro-inflammatory and procoagulant effects. Together these effects predispose to local inflammation, thrombosis and hemorrhage. As such, they can contribute to stroke initiation, progression of brain damage and development of tolerance to ischemia. In addition, TNF may contribute to repair and recovery after stroke as an important mediator and modulator of inflammation. β-hCG oligopeptide derivates are known to inhibit TNF expression and NF-κB activation and successive inflammatory and apoptotic pathways. These characteristics should enable single β-hCG oligopeptide derivates or cocktails of derivates to prevent the further brain ischemia and infarction and the occurrence of complications, including cerebral edema and secondary hemorrhages, which may contribute to improvement of clinical outcomes in stroke patients. Ischemia and infarction secondary to cerebral contusion and to epi-, subarachnoid- and subdural hemorrhages play a significant role in final brain damage and clinical outcome in patients with these disorders. Local TNF expression and NF-κB activation due to ischemia in these diseases will predispose to local inflammation, thrombosis and hemorrhage, similar to ischemic stroke patients. Therefore, administration of single NF-κB-down-regulating peptides or mixtures thereof contributes to improvement of final outcomes also in these diseases. In patients with contusio cerebri and intracranial pressure treatment, it is advantageous to combine treatment with the peptides or functional analogues thereof with osmotic agents like mannitol to reduce intracranial pressure and stimulate cerebral perfusion, i.e., by administering intravenous infusions of mannitol 20% in 0.9% saline solutions of 200 ml, or another hypertonic solution, 1 to 6 times a day. NF-κB-regulating peptides can be given in the same infusion, the peptide (or analogue) concentration preferably being from about 1 to about 1000 mg/L, but the peptide can also been given in a bolus injection. Doses of 1 to 5 mg/kg bodyweight, for example, every eight hours in a bolus injection or per infusionem until the patient stabilizes, are recommended. For example, in cases where large infarcted areas are expected or diagnosed, it is preferred to monitor cytokine profiles, such as TNF-α or IL-10 levels, in the plasma (or cerebrospinal fluid) of the treated patient, and to stop treatment when these levels are normal. In patients with contusio cerebri, intracranial pressure and intraparenchymal oxygen and metabolites can be monitored using intracranial sensors. In a preferred embodiment, the invention provides a method of treating a subject suffering from an ischemic event with a method and signaling molecule according to the invention concomitantly, or at least timely, with a thrombolytic agent, such as (recombinant) tissue plasminogen activator, or truncated forms thereof having tissue plasminogen activity, or streptokinase, or urokinase. In the case of a cerebrovascular incident, such treatment can, for example, take the form of intravenous infusions of recombinant tissue plasminogen activator (rt-PA) at a dose of 0.9 mg/kg (maximum of 90 mg) in 0.9% saline solutions, whereby it is preferred that 10% of the rt-PA dose is given within one to two minutes and the remaining dose of rt-PA in 60 minutes. In the case of an acute myocardial infarction, such treatment can, for example, take the form of intravenous infusions of rt-PA at a dose of 15 mg as an intravenous bolus, followed by 50 mg in the next 30 minutes followed by 35 mg in the next 60 minutes. For the sake of treating the resulting perfusion injury that occurs due to the lysis of the thrombus and the subsequent perfusion of the ischemic area, it is herein provided to also provide the patient with a bolus injection of NF-κB-down-regulating peptide such as AQGV (SEQ ID NO:2), LQGV (SEQ ID NO:1) or VLPALP (SEQ ID NO:4) at 2 mg/kg and continue the infusion with an NF-κB-down-regulating peptide such as AQGV (SEQ ID NO:2), LQGV (SEQ ID NO:1) or VLPALP (SEQ ID NO:4) or a functional analogue thereof at a dose of 1 mg/kg bodyweight for every eight hours. Dosages may be increased or decreased, for example, depending on the outcome of monitoring the cytokine profile in the plasma of the patient. In one embodiment of the present invention, a signal molecule is administered in an effective concentration to an animal or human systemically, e.g., by intravenous, intramuscular or intraperitoneal administration. Another way of administration comprises perfusion of organs or tissue, be it in vivo or ex vivo, with a perfusion fluid comprising a signal molecule according to the invention. Topical administration, e.g., in ointments or sprays, may also apply, e.g., in or around infarcted areas in brain or heart, etc. The administration may be done as a single dose, as a discontinuous sequence of various doses, or continuously for a period of time sufficient to permit substantial modulation of gene expression. In the case of a continuous administration, the duration of the administration may vary depending upon a number of factors which would readily be appreciated by those skilled in the art.
The administration dose of the active molecule may be varied over a fairly broad range. The concentrations of an active molecule which can be administered would be limited by efficacy at the lower end and the solubility of the compound at the upper end. The optimal dose or doses for a particular patient should and can be determined by the physician or medical specialist involved, taking into consideration well-known relevant factors such as the condition, weight and age of the patient, etc.
Ten male C57BL/six mice (23 to 26 g), five controls and five test animals, were used as a model for ischemic stroke by middle cerebral artery occlusion/reperfusion. These mice are initially anesthetized with metofane and maintained with i.p. ketamine (60 mg/ml) and xylazine (5 mg/ml). Atropine methyl nitrate (0.18 mg/kg i.p.) is given to prevent airway obstruction. Animals are allowed to breathe spontaneously. A modified intravascular middle cerebral artery (MCA) occlusion technique is used to induce stroke. A nonsiliconized, uncoated 6-0, 8-mm-long prolene suture with a rounded tip (diameter 0.20 mm) is advanced into the internal carotid artery to occlude the MCA for one hour, followed by 24 hours of reperfusion.
Cerebral blood perfusion (CBF) is monitored by laser Doppler flowmetry (Transonic Systems). Laser Doppler flowmetry probes (0.8 mm in diameter) are positioned on the cortical surface 2 mm posterior to the bregma, both 3 and 6 mm to each side of midline. The procedure is considered to be successful if a >85% drop in CBF was observed immediately after placement of the suture.
Survival and neurological deficits are monitored and scored as follows: no neurological deficit (0), failure to extend forepaw fully (1), turning to left (2), circling to left (3), unable to walk spontaneously (4), stroke-related death (5).
Arterial blood gases (pH, PaO2, PaCO2) are measured before and during MCA occlusion with an ABL 30 Acid-Base Analyzer (Radiometer).
Reducing cerebral ischemia by gene-regulatory peptides in murine model.
Each of the five test mice receives a 1:1 mixture of LQGV (SEQ ID NO:1) and VLPALP (SEQ ID NO:4) at 5 mg/kg in a volume of 0.5 ml 0.9% saline which is given intravenously at ten minutes, when blood flow is at a minimum, since these conditions would give a reasonable test of the bioactivity of these peptides during ischemia, because the time course of pathophysiological changes in the present murine model is different from that of human strokes, and the occlusion is experimentally removed after one hour. Each of the control mice receives 0.5 ml 0.9% saline i.v.
Administration of peptides after two to three hours in this murine model would actually be during the reperfusion phase, which may not be fully relevant to the human clinical situation where treatment would be desired within one hour after the stroke, because complete reopening of major occluded blood vessels in humans who experience ischemic stroke might not typically happen spontaneously one hour after the onset of ischemic stroke.
Early results control mice score: 3, 5, 4, 2, 4; test mice score: 3, 1, 3, 2, 1
Possible mechanisms by which peptides reduce cerebral ischemia in a murine model. Not wishing to be bound by theory, the thrombomodulin-protein C (TM-PC) pathway is known to function on endothelium and to counterbalance coagulation. In addition, the TM-PC pathway provides protective signaling that counteracts apoptosis in response to oxygen deprivation. Activated protein C (APC) is a systemic anticoagulant and anti-inflammatory factor which has been demonstrated to protect the brain from ischemic injury. Cytoprotection of brain endothelium by APC in vitro required endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), as did in vivo neuroprotective activity in the murine stroke model. It reduces organ damage in animal models of sepsis, ischemic injury and stroke. This invention shows that the above-used gene-regulatory peptides reduce inflammatory mediators, activation of transcription factors, including NF-κB, and directly interfere with the TM-PC pathway. The neuroprotection induced by these peptides is mediated by one or combinations of these effects. Peptides in this way act as a direct cell survival factor and reduce secondary ischemia by their anticoagulant and anti-inflammatory effects.
The peptides as mentioned in this document, such as LQG, AQG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:3), VLPALP (SEQ ID NO:4), ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ (SEQ ID NO:7), VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV (SEQ ID NO:10), VLAALP (SEQ ID NO:11), VLPALA (SEQ ID NO:12), VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID NO:15), GVLPALP (SEQ ID NO:16), VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:24), RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT (SEQ ID NO:25), SKAPPPSLPSPSRLPGPS (SEQ ID NO:26), LQGVLPALPQVVC (SEQ ID NO:17), SIRLPGCPRGVNPVVS (SEQ ID NO:27), LPGCPRGVNPVVS (SEQ ID NO:18), LPGC (SEQ ID NO:19), MTRV (SEQ ID NO:20), MTR, and VVC, were prepared by solid-phase synthesis using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based methodology with 2-chlorotrityl chloride resin as the solid support. The side chain of glutamine was protected with a trityl function. The peptides were synthesized manually. Each coupling consisted of the following steps: (i) removal of the α-amino Fmoc-protection by piperidine in dimethylformamide (DMF), (ii) coupling of the Fmoc amino acid (3 eq) with diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) in DMF/N-methylformamide (NMP) and (iii) capping of the remaining amino functions with acetic anhydride/diisopropylethylamine (DIEA) in DMF/NMP. Upon completion of the synthesis, the peptide resin was treated with a mixture of trifluoroacetic acid (TFA)/H2O/triisopropylsilane (TIS) 95:2.5:2.5. After 30 minutes, TIS was added until decolorization. The solution was evaporated in vacuo and the peptide precipitated with diethyl ether. The crude peptides were dissolved in water (50-100 mg/ml) and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). HPLC conditions were: column: Vydac TP21810C18 (10×250 mm); elution system: gradient system of 0.1% TFA in water v/v (A) and 0.1% TFA in acetonitrile (ACN) v/v (B); flow rate 6 ml/minute; absorbance was detected from 190-370 nm. There were different gradient systems used. For example, for peptides LQG and LQGV (SEQ ID NO:1): ten minutes 100% A followed by linear gradient 0-10% B in 50 minutes. For example, for peptides VLPALP (SEQ ID NO:4) and VLPALPQ (SEQ ID NO:13): five minutes 5% B followed by linear gradient 1% B/minute. The collected fractions were concentrated to about 5 ml by rotation film evaporation under reduced pressure at 40° C. The remaining TFA was exchanged against acetate by eluting two times over a column with anion exchange resin (Merck II) in acetate form. The eluant was concentrated and lyophilized in 28 hours. Peptides were prepared for use later by dissolving them in PBS.
RAW264.7 macrophages, obtained from American Type Culture Collection (Manassas, Va.), were cultured at 37° C. in 5% CO2 using DMEM containing 10% FBS and antibiotics (100 U/ml of penicillin and 100 μg/ml streptomycin). Cells (1×106/ml) were incubated with peptide (10 μg/ml) in a volume of 2 ml. After eight hours of culturing, cells were washed and prepared for nuclear extracts.
Nuclear extracts and Electrophoretic Mobility Shift Assays (EMSA) were prepared according to Schreiber et al., Methods (Schrieber et al., 1989, Nucleic Acids Research 17). Briefly, nuclear extracts from peptide-stimulated or nonstimulated macrophages were prepared by cell lysis followed by nuclear lysis. Cells were then suspended in 400 μl of buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitors), vigorously vortexed for 15 seconds, left standing at 4° C. for 15 minutes, and centrifuged at 15,000 rpm for two minutes. The pelleted nuclei were resuspended in buffer (20 mM HEPES (pH 7.9), 10% glycerol, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitors) for 30 minutes on ice, then the lysates were centrifuged at 15,000 rpm for two minutes. The supernatants containing the solubilized nuclear proteins were stored at −70° C. until used for the (EMSA).
Electrophoretic mobility shift assays were performed by incubating nuclear extracts prepared from control (RAW264.7) and peptide-treated RAW264.7 cells with a 32P-labeled double-stranded probe (5′ AGCTCAGAGGGGGACTTTCCGAGAG 3′ (SEQ ID NO:28)) synthesized to represent the NF-κB-binding sequence. Shortly, the probe was end-labeled with T4 polynucleotide kinase according to the manufacturer's instructions (Promega, Madison, Wis.). The annealed probe was incubated with nuclear extracts as follows: in EMSA, binding reaction mixtures (20 μl) contained 0.25 μg of poly(dI-dC) (Amersham Pharmacia Biotech) and 20,000 rpm of 32P-labeled DNA probe in a binding buffer consisting of 5 mM EDTA, 20% Ficoll, 5 mM DTT, 300 mM KCl and 50 mM HEPES. The binding reaction was started by the addition of cell extracts (10 μg) and was continued for 30 minutes at room temperature. The DNA-protein complex was resolved from free oligonucleotide by electrophoresis in a 6% polyacrylamide gel. The gels were dried and exposed to x-ray films.
The transcription factor NF-κB participates in the transcriptional regulation of a variety of genes. Nuclear protein extracts were prepared from LPS- and peptide-treated RAW264.7 cells or from LPS-treated RAW264.7 cells. In order to determine whether the peptide modulates the translocation of NF-κB into the nucleus, EMSA was performed on these extracts. The amount of NF-κB present in the nuclear extracts of RAW264.7 cells were treated with LPS or LPS in combination with a peptide for four hours. Here we determined that, indeed, some peptides are able to modulate the translocation of NF-κB since the amount of labeled oligonucleotide for NF-κB is reduced. In this experiment, peptides that show the modulation of translocation of NF-κB are: VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:23), VLPALP (SEQ ID NO:4), VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID NO:16), VVC, MTRV (SEQ ID NO:20), and MTR.
RAW264.7 mouse macrophages were cultured in DMEM, containing 10% or 2% FBS, penicillin, streptomycin and glutamine, at 37° C., 5% CO2. Cells were seeded in a 12-well plate (3×106 cells/ml) in a total volume of 1 ml for two hours and then stimulated with LPS (E. coli 026:B6; Difco Laboratories, Detroit, Mich., USA) and/or NMPF (1 mg/ml). After 30 minutes of incubation, plates were centrifuged and cells were collected for nuclear extracts. Nuclear extracts and EMSA were prepared according to Schreiber et al. Cells were collected in a tube and centrifuged for five minutes at 2000 rpm (rounds per minute) at 4° C. (Universal 30 RF, Hettich Zentrifuges). The pellet was washed with ice-cold Tris buffered saline (TBS pH 7.4) and resuspended in 400 μl of a hypotonic buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail (Complete™ Mini, Roche)) and left on ice for 15 minutes. Twenty-five microliters of 10% NP-40 were added and the sample was centrifuged (two minutes, 4000 rpm, 4° C.). The supernatant (cytoplasmic fraction) was collected and stored at −70° C. The pellet, which contains the nuclei, was washed with 50 μl buffer A and resuspended in 50 μl buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail and 10% glycerol). The samples were left to shake at 4° C. for at least 60 minutes. Finally, the samples were centrifuged and the supernatant (nucleic fraction) was stored at −70° C.
Bradford reagent (Sigma) was used to determine the final protein concentration in the extracts. For electrophoretic mobility shift assays, an oligonucleotide representing an NF-κB-binding sequence (5′-AGC TCA GAG GGG GAC TTT CCG AGA G-3′ (SEQ ID NO:28)) was synthesized. One hundred picomoles of sense and antisense oligo were annealed and labeled with γ-32P-dATP using T4 polynucleotide kinase according to the manufacturer's instructions (Promega, Madison, Wis.). Nuclear extract (5-7.5 μg) was incubated for 30 minutes with a 75,000 cpm probe in a binding reaction mixture (20 microliters) containing 0.5 μg poly dI-dC (Amersham Pharmacia Biotech) and binding buffer BSB (25 mM MgCl2, 5 mM CaCl2, 5 mM DTT and 20% Ficoll) at room temperature. The DNA-protein complex was resolved from free oligonucleotide by electrophoresis in a 4-6% polyacrylamide gel (150 V, two to four hours). The gel was then dried and exposed to x-ray film. The transcription factor NF-κB participates in the transcriptional regulation of a variety of genes. Nuclear protein extracts were prepared from either LPS (1 mg/ml), peptide (1 mg/ml) or LPS in combination with peptide-treated and untreated RAW264.7 cells. In order to determine whether the peptides modulate the translocation of NF-κB into the nucleus, EMSA was performed on these extracts. Peptides are able to modulate the basal as well as LPS-induced levels of NF-κB. In this experiment, peptides that show the inhibition of LPS-induced translocation of NF-κB are: VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:23), VLPALP (SEQ ID NO:4), VVC, MTR and circular LQGVLPALPQVVC (SEQ ID NO:17). Peptides that promote LPS-induced translocation of NF-κB in the experiment are: VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID NO:16) and MTRV (SEQ ID NO:20). Basal levels of NF-κB in the nucleus were decreased by VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG and LQGV (SEQ ID NO:1) while basal levels of NF-κB in the nucleus were increased by GVLPALPQ (SEQ ID NO:23), VLPALPQ, (SEQ ID NO:13) GVLPALP (SEQ ID NO:16), VVC, MTRV (SEQ ID NO:20), MTR and LQGVLPALPQVVC (SEQ ID NO:17). In other experiments, QVVC (SEQ ID NO:29) also showed the modulation of translocation of NF-κB into the nucleus (data not shown).
Further modes of identification of gene-regulatory peptides by NF-κB analysis:
Cells: Cells will be cultured in appropriate culture medium at 37° C., 5% CO2. Cells will be seeded in a 12-well plate (usually 1×106 cells/ml) in a total volume of 1 ml for two hours and then stimulated with a regulatory peptide in the presence or absence of additional stimuli such as LPS. After 30 minutes of incubation, plates will be centrifuged and cells collected for cytosolic or nuclear extracts.
Nuclear Extracts: Nuclear extracts and EMSA could be prepared according to Schreiber et al., Methods (Schreiber et al., 1989, Nucleic Acids Research 17). Cells are collected in a tube and centrifuged for five minutes at 2000 rpm (rounds per minute) at 4° C. (Universal 30 RF, Hettich Zentrifuges). The pellet is washed with ice-cold Tris buffered saline (TBS pH 7.4) and resuspended in 400 μl of a hypotonic buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail (Complete™ Mini, Roche)) and left on ice for 15 minutes. Twenty-five microliters of 10% NP-40 is added and the sample is centrifuged (two minutes, 4000 rpm, 4° C.). The supernatant (cytoplasmic fraction) is collected and stored at −70° C. for analysis. The pellet, which contains the nuclei, is washed with 50 μl buffer A and resuspended in 50 μl buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail and 10% glycerol). The samples are left to shake at 4° C. for at least 60 minutes. Finally, the samples are centrifuged and the supernatant (nucleic fraction) is stored at −70° C. for analysis.
Bradford reagent (Sigma) could be used to determine the final protein concentration in the extracts.
EMSA: For electrophoretic mobility shift assays, an oligonucleotide representing an NF-κB-binding sequence such as (5′-AGC TCA GAG GGG GAC TTT CCG AGA G-3′ (SEQ ID NO:28)) is synthesized. One hundred picomoles of sense and antisense oligo are annealed and labeled with γ-32P-dATP using T4 polynucleotide kinase according to the manufacturer's instructions (Promega, Madison, Wis.). Cytosolic extract or nuclear extract (5-7.5 μg) from cells treated with regulatory peptide or from untreated cells is incubated for 30 minutes with a 75,000 cpm probe in a binding reaction mixture (20 μl) containing 0.5 μg poly dI-dC (Amersham Pharmacia Biotech) and binding buffer BSB (25 mM MgCl2, 5 mM CaCl2, 5 mM DTT and 20% Ficoll) at room temperature, or cytosolic and nuclear extract from untreated cells or from cells treated with stimuli could also be incubated with a probe in a binding reaction mixture and binding buffer. The DNA-protein complex is resolved from free oligonucleotide by electrophoresis in a 4-6% polyacrylamide gel (150 V, two to four hours). The gel is then dried and exposed to x-ray film. Peptides can be biotinylated and incubated with cells. Cells are then washed with phosphate-buffered saline and harvested in the absence or presence of certain stimulus (LPS, PHA, TPA, anti-CD3, VEGF, TSST-1, VIP or known drugs, etc.). After culturing, cells are lysed and cell lysates (whole lysate, cytosolic fraction or nuclear fraction) containing 200 micrograms of protein are incubated with 50 microliters of Neutr-Avidin-plus beads for one hour at 4° C. with constant shaking. Beads are washed five times with lysis buffer by centrifugation at 6000 rpm for one minute. Proteins are eluted by incubating the beads in 0.05 N NaOH for one minute at room temperature to hydrolyze the protein-peptide linkage and analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoprecipitation with agarose-conjugated anti-NF-κB subunit antibody or immunoprecipitated with antibody against the target to be studied. After hydrolyzing the protein-peptide linkage, the sample could be analyzed by HPLS and mass-spectrometry. Purified NF-κB subunits or cell lysate interaction with biotinylated regulatory peptide can be analyzed on biosensor technology. Peptides can be labeled with FITC and incubated with cells in the absence or presence of different stimulus. After culturing, cells can be analyzed with fluorescent microscopy, confocal microscopy, or flow cytometry (cell membrane staining and/or intracellular staining) or cell lysates are made and analyzed on HPLC and mass-spectrometry. NF-κB-transfected (reporter gene assay) cells and gene array technology can be used to determine the regulatory effects of peptides.
HPLC and mass-spectrometry analysis: Purified NF-κB subunit or cytosolic/nuclear extract is incubated in the absence or presence of (regulatory) peptide, diluted (2:1) with 8 N guanidinium chloride and 0.1% trifluoroacetic acid, injected into a reverse-phase HPLC column (Vydac C18) equilibrated with solvent A (0.1% trifluoroacetic acid), and eluted with a gradient of 0 to 100% eluant B (90% acetonitrile in solvent A). Fractions containing the NF-κB subunit are pooled and concentrated. Fractions are then dissolved in appropriate volume and could be analyzed on mass-spectrometry.
Further references: PCT International Publications WO99/59617, WO97/49721, WO01/10907, and WO01/11048, the contents of the entireties of incorporated herein by this reference.
This application is a continuation of U.S. patent application Ser. No. 10/409,642, filed Apr. 8, 2003, pending, which is a continuation-in-part of U.S. patent application Ser. No. 10/028,075, filed Dec. 21, 2001, pending. This application also claims priority from U.S. patent application Ser. No. 10/262,522, filed Sep. 30, 2002, pending. The contents of the entirety of all of which is incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10409642 | Apr 2003 | US |
| Child | 11593329 | Nov 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10028075 | Dec 2001 | US |
| Child | 10409642 | Apr 2003 | US |
