Patent Application
20130058939
This invention relates to a method for the treatment of neurological or neurodegenerative disorders. More particularly, this invention relates to the protection of neurons and/or the blood brain barrier which are adversely affected as result of a neurological or neurodegenerative disorder and claims priority of the European patent application 09 011 149.3 which is hereby fully incorporated in terms of disclosure.
Stroke is a leading cause of adult death and disability with approximately 6 million deaths per year, with an alarming projected incidence over the next decade due to the increase in the elderly population. Despite the significant advances that have been made in understanding the cellular and molecular pathophysiology of cerebral ischaemia, recombinant tissue-type plasminogen activator (rt-PA) remains the only approved acute treatment for ischaemic stroke. Hundreds of compounds have been tested so far in clinical trials for ischaemic stroke, but aside from rt-PA, none has proven effective.
The use of rt-PA is limited by a short therapeutic window (4.5 hours post-onset) and by promotion of both intracerebral haemorrhage and neurotoxicity. Thus, there is a critical need for safer and more effective treatments, in order to improve the global benefit of rt-PA induced thrombolysis and to provide treatment for stroke patients who are not eligible for thrombolysis (i.e. more than 80% of stroke patients).
T-PA is a serine protease with two faces which displays key roles in the intravascular space, at the interface between blood and brain and in the brain parenchyma (for review: Yepes et al., 2008). In the intravascular compartment, t-PA's main substrate is the inactive zymogen plasminogen and its main role is to promote fibrinolysis. Blood-derived t-PA can cross both the intact and the injured blood-brain barrier (BBB) (Benchenane et al. 2005a, Benchenane et al., 2005b) and thus can, together with endogenously produced t-PA, interact in the brain parenchyma with a variety of substrates, thus extending its functions above t-PA/plasmin(ogen)-driven extracellular matrix degradation.
There is a growing body of evidence indicating that the interaction between t-PA and the N-methyl-D-aspartate receptor (NMDAR), the low density lipoprotein receptor-related protein (LRP), annexin-II in glial cells and/or neurons activate cell signaling processes results in a deleterious outcome including cerebral edema, hemorrhagic transformation and cell death. Based on these multiple pathophysiological effects of t-PA, the participation of endogenous t-PA (supported by the side effects of exogenously applied rt-PA) is discussed beyond the established role in ischaemic disorders for several neurological or neurodegenerative disorders such as epilepsy, Alzheimer's disease, multiple sclerosis or meningitis.
According to the complex behaviour of t-PA several molecular strategies for inhibiting the deleterious effects of t-PA can be envisaged. However a strategy that can be transferred into the clinical situation is still missing.
Thus, it is the objective of the present invention to provide novel means for the treatment of neurological or neurodegenerative disorders, in particular of stroke.
This objective is solved by a method. using a protein or peptide for the manufacture of a medicament for the treatment of neurological or neurodegenerative disorders in a patient, in particular stroke, wherein with the protein or peptide is selected form the group consisting of:
further embodiments of the invention are subject matter of additional independent or dependent claims.
The inventors have found that proteins comprising or consisting of a kringle domain or antibodies specifically binding to the amino-terminal domain of the NR1 subunit of the NMDA receptor (anti-ATD-NR1 antibody) are active at two t-PA relevant target sites: the blood brain barrier and the NMDA receptor and as a result exhibit vasoprotective and neuroprotective activity.
Accordingly the anti-ATD-NR1 antibody (or an antigen binding fragment thereof) and the kringle domain-containing proteins or peptides (hereinafter “kringle proteins” and/or “kringle peptide”) are together referred to as “t-PA inhibitor”.
As will be shown below the results from the animal model reveal that the t-PA inhibitors of the invention are not only effective when given together with t-PA but also when administered alone. Furthermore the t-PA inhibitors exhibit beneficial effect in thrombosis disorders when given after clot formation, hence enable an acute or post-acute treatment of thrombosis.
Thus, the invention relates to the use of a protein or peptide for the manufacture of a medicament for the treatment of neurological or neurodegenerative disorders, in particular stroke, wherein the protein or peptide is selected form the group consisting of:
The inventors could identify the kringle domain as the relevant domain for the inhibition of the t-PA transport through the BBB with the final proof that a protein consisting only of a plasminogen activator-related kringle (PARK) domain is also a t-PA transport inhibitor. Hence a protein or peptide comprising or consisting of this domain can be used as a vasoprotectant and/or neuroprotectant. This is not limited to a kringle domain of desmoteplase (Desmodus rotundus plasminogen activator) DSPA but applies also to the kringle domains of other plasminogen activators since it was found that kringle domains of different plasminogen activators were able to inhibit trans-BBB transport of rt-PA.
Hence the invention relates to the use of proteins with a plasminogen activator-related kringle (PARK) domain or a desmoteplase activator-related kringle (DARK) domain for the treatment of neurological disorders. In particular these proteins can be applied as vasoprotectants and/or neuroprotectants.
The protective activity of the kringle proteins is not due to its protease activity. Hence the proteins of the invention do not exhibit the serine protease activity commonly shared by plasminogen activators.
This could be shown by the inventors in an in vitro model of BBB penetration. Herein was demonstrated that the transport of rt-PA can be blocked not only by the plasminogen activator desmoteplase but also by an inactivated so called ‘clogged’ DSPA (cDSPA). This opened the way to a new therapy approach since an inactivated DSPA will neither augment the plasminogen activating capacity of t-PA nor interfere with the plasminogen-activating and therefore clot-lysing efficacy of t-PA. Rather, it inhibit only the detrimental side effects of t-PA related thrombolytics. Furthermore, the inactivated DSPA is not capable of cleaving the NR1 subunit of the NMDA receptor and hence poses no risk of neurotoxicity on its own.
Furthermore the inventors could show that the co-incubation of rt-PA and clogged DSPA (or the isolated and purified kringle domain) had no toxic effect on the blood brain barrier.
A kringle is a triple looped polypeptide structure formed by three disulfide bonds. Kringles vary in length from about 79 to 82 amino acid groups. A high degree of sequence homology is shared among the single kringle of human urokinase (Günzler et al., Hoppe-Seyler's Z. Physiol. Chem. 363, 1155, 1982), the two kringles of human tissue plasminogen activator (Pennica, et al. Nature, 301,214,1983), the two kringles of human prothrombin (Walz et al., Proc. Nat'l. Acad. Sci. USA, 74, 1969, 1977), and the five kringles of human plasminogen (Sottrup-Jensen et al., in Progress in Chemical Fibrinolysis and Thrombolysis (eds. Davidson et al), 3, 191, 1978). The relative positions of the six cysteines involved in the intra-kringle disulfide bridges are conserved in all kringles.
The term, “plasminogen activator related kringle domain” (PARK domain) as used in this application refers to proteins or peptides with an amino acid sequence with at least 90% identity, preferable at least 95% or 100% identity to the plasminogen activator-related kringle (PARK) motif defined by the sequence: “CY-X3-G-X2-YRGTXS-X2-ES-X3-C-X2-WNS-X2-L-X4-Y-X4-PXA-X2-LGLGXHNYCRNP-X4-KPWCXVXK-X6-EXC-X2-PXC”, wherein X denotes an arbitrary amino acid (
This term in particular encompasses proteins comprising or consisting of the amino acid sequences SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. It is also understood that polymorphic forms in the kringle region of these proteins may exist in nature where one or more amino acids may be added, deleted or substituted. Similar changes in a protein may also be brought about in vitro with the advent of the present day technology of point mutation of the gene or by chemical synthesis of the gene with any desired sequence. These modified structure(s) are therefore also included in the term, kringle(s), used in this application.
Sequences SEQ ID NO:1 and SEQ ID NO:2 show the amino acid sequences of the kringle 1 and 2 domains of the recombinant t-PA (alteplase). SEQ ID NO:3 shows the amino acid sequence of the kringle domain of desmoteplase (DSPA alpha 1).
Other preferred kringle domains for said peptide or protein include a kringle domain from urokinase and prothrombin.
As used herein the “DARK domain” is defined as a peptide with an amino acid sequence of at least 90% identity and preferable at least 95% or 100% identity to the following amino acid sequence motive resulting from the kringle sequence of DSPA alpha 1 as given in
CY-X3-G-X2-YRGTXS-X2-ESR-X2-C-X2-WNS-X2-LXR-X2-Y-X3-MPXAFN-LGLGXHNYCRNPNXAXKPWCXVXK-X3-F-X2-ESC-X2-PXC; wherein X denotes an arbitrary amino acid
In a further aspect of the invention the kringle protein consists of an amino acid sequence of either the DARK or the PARK domain as of
The kringle proteins of the invention preferably have a length of not more than 200 amino acids (aa), preferably not more than 150 aa, most preferably not more than 100 aa, and comprise the PARK or DARK motif as above. This has a length of 82 aa.
The kringle proteins furthermore can include a lysine binding site, which is preferably defined by the presence of the amino acids Y36, W62 and H64, according to the numbering of the 82 aa kringle domain as used herein.
The kringle proteins of the invention preferably inhibit the t-PA transport across the blood brain barrier (BBB) in mammals by at least 20%, preferably by at least 30%, most preferred by at least 40% or 50% assessable by the method described in Example A, Chapter 2 of the present invention.
Said t-PA inhibitor can bind specifically to the low-density lipoprotein (LDL) receptor-related protein (LRP). The LRP is a multifunctional endocytosis receptor that binds a variety of ligands, including t-PA, the β-amyloid precursor protein, alpha 2-macroglobulin, apolipoprotein E-enriched beta-very-low-density lipoprotein, and Pseudomonas exotoxin A, some of which are implicated in neurological diseases such as Alzheimer's disease. Due to LRP binding said protein or peptide can block the binding of t-PA or other ligands to the LRP thereby inhibiting the LRP-mediated transport over the BBB.
Furthermore, the t-PA inhibitor of the invention can bind to the NR1 subunit of the NMDA receptor. Due to this NR1 binding said t-PA inhibitor can block the binding of other substrates or ligands to the NR1 subunit thereby inhibiting its proteolytical cleavage.
In a further aspect the invention relates to the use of an isolated antibody or an antigen-binding portion thereof for the treatment of neurological disorders, in particular of stroke, which specifically binds to the N-terminal domain of the NMDA receptor subunit NR1 (anti-ATD-NR1 antibody) or a fragment thereof, in particular to an antigen comprising or consisting of SEQ ID NO:4 or 5.
In a further embodiment the antibody of the invention or a fragment thereof specifically hybridizes to the complement of the nucleic acid molecule of SEQ ID NO 6 or 7 under conditions of high stringency. This high stringency conditions can be provided by e.g. a hybridisation that is performed in 5×SSPE, 5×Denhardt's solution and 0.5% SDS overnight at 55 to 60° C.
The inventors have now found that an antibody-based therapy targeting the interaction of t-PA with the amino-terminal domain of the NR1 subunit of the NMDA receptor can strongly and durably improve neurological outcome after stroke, by limiting brain damage and inhibiting the disruption of BBB.
This beneficial efficacy of the anti-ATD NR1 antibody effect was shown in a clinically relevant model of stroke that has the ability to reproduce the observed time window for t-PA treatment in humans (Orset et al., 2007). In this model, the in situ microinjection of purified murine thrombin triggers a local clot in the middle cerebral artery resulting in reproducible clot formation and cortical brain injury and lack of surgery-associated lethality. In this thrombosis model the efficacy of t-PA as well as the time window for t-PA treatment is similar to the human clinical situation. In sum this animal model is well suited for the preclinical evaluation of stroke therapies.
The anti-ATD NR1 antibody is directed only against a small part of one of the NMDA subunits. This allows a specific intervention at the NMDA receptor that does not interfere with the physiological function of the NMDA receptor but only inhibits the t-PA-induced potentiation of the NMDA activity.
Due to the fact that the antibody is not directed against t-PA, therapeutically administered t-PA or other plasminogen activators will retain their normal thrombolytic function that is mandatory for a beneficial activity in thrombotic disorders.
The anti-ATD NR1 antibody can be used for passive immunization of patients that are in risk of a thrombotic disorder or suffer from a thrombotic disorder. In contrast to the active immunization which is achieved by pretreatment with a suitable antigen, the blocking effect at the NMDA receptor is immediately achieved. This is of high importance, since in thrombotic disorders a timely treatment is a hallmark for a successful outcome.
As t-PA and glutamate, the endogenous agonist at the NMDA receptor, exert critical functions in synaptic remodeling and plasticity underlying memory and learning processes, the risk of corresponding side effects is apparent. For the passive immunization as used herein, no alterations of cognitive functions including spatial memory, contextual and fear conditioning were evident. This supports a superior safety profile for this strategy of brain protection.
While therapeutic efficacy of the antibodies against detrimental effects of administration of t-PA would be a logical consequence of their design, they most surprisingly were also found to be effective without co-administration of t-PA: Mice treated with antibodies in the mouse model of thromboembolic stroke displayed strongly reduced brain damage (See
As a result of these findings the anti-ATD NR1 antibody is also able to inhibit the endogenously expressed t-PA.
Hence according to the invention the anti-ATD NR1 antibody can be used as a monotherapy (i.e. without t-PA co-treatment or coadministration of any other drug substance) for the treatment of thrombotic disorders, particularly stroke.
The analysis of the blood brain barrier leakage in the stroke model revealed that both the early and the late administration of the anti-ATD NR1 antibodies alone significantly reduce the extent of BBB leakage induced by stroke. In addition the anti-ADT-NR1 treatment efficiently reduced the damaging effect of rt-PA on the integrity of the BBB in this stroke model. Furthermore the kringle domain containing protein prevents t-PA-induced damage of the BBB.
Hence in a preferred embodiment of the invention the t-PA inhibitor can be used for the treatment of neurological or neurodegenerative disorders that are associated with enhanced permeability of the blood brain barrier. These disorders comprise ischaemic or thrombotic disorders such as stroke or TIA, epilepsy such as temporal lobe epilepsy (TLE), amyotrophic lateral sclerosis, multiple sclerosis, brain tumours, Parkinson disease, Alzheimer's disease, brain oedema, or CNS complications resulting from parasitic, bacterial, fungal or viral infections such as meningitis or encephalitis.
Antibodies are commonly believed to be effectively excluded from the brain by an intact blood brain barrier and gain only access to brain tissue in case of BBB injury. As a surprising finding, immunostaining and quantification of fluorescent anti-ATD-NR1 antibodies in the brain parenchyma (see
Hence according to the invention the anti-ATD NR1 antibody can be used also for disorders without an enhanced permeability of the BBB or as a preventive treatment that even before a pathophysiological enhanced permeability of the BBB therapeutically effective amounts of the antibody are present in the brain parenchyma to initially protect neuronals cells.
In the context of the invention the antibody is directed against the N-terminal region of the NR1-1a subunit encoding the amino acids 19 to 480, preferably encoded by the nucleotide sequence of SEQ ID NO:6 or 7 or encoding amino acids 19 to 371 as disclosed in SEQ ID NO:4 or 5 or a fragment thereof comprising of 8 to 30, preferably 10 to 20 contiguous amino acids wherein the antibody which is directed said fragment prevents the cleavage of the NR1 subunit by t-PA.
SEQ ID NO:6 (rat NR1-1a, Nucleotide sequence to be inserted)
SEQ ID NO:7 (human NR1-1, Nucleotide sequence to be inserted)
According to the invention the antibody prevents cleavage of the extracellular domain of the NR1 subunit by a protease, preferably by t-PA and most preferably at the amino acid residue Arg260.
In one embodiment of the invention, the anti-ATD NR1 antibody is a monoclonal antibody, preferably a humanized antibody, a single domain antibody or VHH antibody (so called nanobody), a chimeric antibody or a deimmunized antibody.
According to a further embodiment of the invention, an antigen-binding portion of the antibody can be used, preferably a scFv molecule or a Fab fragment. The antigen-binding portion can be humanized or possess the human sequence.
Further experiments in the mouse model of thrombo-embolic stroke revealed another surprising aspect of the therapeutic relevance of the anti-ATD NR1 antibody. Late t-PA thrombolysis, despite restoring cerebral blood flow comparably to early reperfusion, was associated with increased rather than reduced brain damage. However in mice treated with anti-ATD NR1 antibodies in addition, brain protection was recovered to a similar extent as observed in early thrombolysed animals.
Based on these findings the invention relates to a method for the treatment of neurological and neurodegenerative disorders, particularly stroke, whereby the patient is treated with a therapeutically effective amount of a thrombolytic drug and an effective amount of a t-PA inhibitor, in particular an anti-ATD NR1 antibody. The invention further relates to a composite comprising a thrombolytic drug and a t-PA inhibitor, particularly an antibody directed against the N-terminal domain of the NMDA receptor subunit NR1.
Accordingly, the anti-ATD NR1 antibody can be used as an adjunct to a thrombolytic therapy, in particular for late t-PA-induced thrombolysis.
The inventors could also demonstrate that a single intravenous injection of the anti-ATD NR1 antibody was highly protective even after clot formation. Early delivery (20 minutes post clot formation) of the anti-ATD NR1 antibody in the thromboembolic stroke model conferred a significant brain protection (44% of protection compared to control, see
Accordingly, in a further aspect of the invention the anti-ATD NR1 antibody can be used for the acute or post-acute treatment of a neurological or neurodegenerative disorder, in particularly stroke. This acute or post-acute treatment can be performed as a monotherapy or in combination with further drugs, preferably an anticoagulant, an antiplatelet drug, or a thrombolytic drug, more preferably with t-PA or a variant thereof or DSPA alpha 1.
Since the deleterious effects of the endogenous t-PA also counteract the beneficial effect of an exogenously applied thrombolytic drug, the combined use of the t-PA inhibitors should even improve the outcome of a thrombolytic therapy for thrombotic drugs that are not associated with neurotoxic and/or vasculotoxic side effects, such as e.g. DSPA alpha 1.
Hence the t-PA inhibitor can be given in combination with a thrombolytic drug that does not adversely interact with the BBB or the NMDA receptor or does not possess a deleterious effect on the vasculature or on the neural cells. For such a combination DSPA alpha 1 is preferred.
According to a further embodiment of the invention the thrombolytic drug and the t-PA inhibitor are given in equimolar concentrations.
The composite according to the invention can comprise either a single preparation including a therapeutically effective amount of at least both components (the thrombolytic drug and the t-PA inhibitor) or represents two separate preparations, each containing at least a therapeutically effective amount of one of the components, the thrombolytic drug or the t-PA inhibitor. When two separate preparations are used, they can be administered to the patient either simultaneously or subsequently. Accordingly, both components can either be administered as a combinatorial preparation or concomitantly as separate preparations. Where applicable and suitable, the preparations can be administered as a single bolus or as an infusion, or a combination thereof. Also, multiple consecutive infusions can be applied.
The t-PA inhibitor and the thrombolytic drug, particularly a plasminogen activator, are given preferably non-orally as a parenteral application e.g. by intravenous or subcutaneous application. An intravenous or subcutaneous bolus application is possible. Thus, according to the invention a pharmaceutical composition is provided, which is suitable for parenteral administration.
In a further embodiment of the invention an oral administration can be performed for a nanobody specifically binding to the ATD of the NR1 subunit.
According to the invention the therapy using a thrombolytic drug, in particular t-PA or variants thereof or DSPA alpha 1 in combination with said t-PA inhibitor comprises one or more of the following characteristics:
Accordingly the invention allows a thrombolytic stroke therapy beyond a 3 hours, preferably beyond a 4.5 hours therapeutic time window, even more preferred beyond 9 or even 12 hours from the onset of stroke.
Thus in one aspect of the invention the t-PA inhibitor is used to reduce, prevent or delay one or more adverse side effects that occur as a result of thrombolytic treatment. These side effects include bleedings such as intracranial or intracerebral bleedings.
In a further aspect of the invention the application of the t-PA inhibitor allows an increased dose of a thrombolytic drug above the levels given normally in the respective indication, preferably above the doses that are recommended by the manufacturer of the thrombolytic drug.
The invention allows for the use of various thrombolytic drugs. Preferably the thrombolytic drug is a plasminogen activator, more preferably recombinant t-PA (rt-PA, e.g. Alteplase) or variants of t-PA such as Pamiteplase, Lanoteplase, Reteplase, Tenecteplase or Monteplase), urokinase or DSPA variants such as DSPA alpha 1, DSPA alpha 2, DSPA beta or DSPA gamma.
As outlined above the kringle proteins of the invention can be constituted by an inactivated plasminogen activator, preferably selected from the group consisting of DSPA alpha 2, DSPA beta or DSPA gamma, urokinase, Alteplase, or variants of t-PA such as Reteplase, Pamiteplase, Lanoteplase, Reteplase, Tenecteplase or Monteplase.
The variants of DSPA (Desmodus rotundus plasminogen activator) are subject of the US patents U.S. Pat. No. 5,830,849 and U.S. Pat. No. 6,008,019, which are fully incorporated herein by reference.
Particularly the inactivated DSPA alpha 1 can be used, wherein it is preferably linked to a suicide substrate, more preferably to D-phenyl-prolyl-arginine chloromethyl ketone (PPACK).
According to this aspect of the invention said kringle protein or peptide includes one or more domains, regions or catalytic sites found in plasminogen activators or related proteins, such as e.g. the finger domain (F) the epidermal growth factor domain (EGF) or the lysine binding site. As a source for this sequence(s) recombinant t-PA (rt-PA, e.g. Alteplase) or variants of t-PA such as Pamiteplase, Lanoteplase, Reteplase, Tenecteplase or Monteplase), urokinase or DSPA variants such as DSPA alpha 1, DSPA alpha 2, DSPA beta or DSPA gamma are preferred. In a further embodiment domains, regions or catalytic sites from different plasminogen activators proteins are combined with each other.
In one preferred aspect of the invention said kringle protein or peptide consists only of the kringle domain, or a fragment thereof, wherein the fragment comprises at least 8, preferably at least 15 to 30 contiguous amino acids of a kringle domain, wherein said peptide inhibits the t-PA transport across the BBB by at least 20% as assessable according to example A, chapter 2 below.
In a specific aspect of the invention said kringle peptide consists of the kringle domain from, DSPA alpha 2, DSPA beta or DSPA gamma, urokinase, Alteplase, or variants of t-PA such as Reteplase, Pamiteplase, Lanoteplase, Reteplase, Tenecteplase or Monteplase and preferably of the kringle domain from DSPA alpha 1.
Thus the invention further relates to a protein or peptide that comprises
In another aspect of the invention an isolated antibody or fragment thereof is provided, wherein the antibody or the fragment binds to the N-terminal domain of the NMDA receptor subunit NR1 (anti-ATD-NR1 antibody) encoded by a amino acid sequence selected from SEQ ID NO: 4 or 5 or by a nucleic acid sequence selected from SEQ ID NO: 6 or 7, or alternatively binds to a protein encoded by a nucleic acid molecule that specifically hybridizes to the complement of the nucleic acid molecule of SEQ ID NO: 6 or 7 under conditions of high stringency. The high stringency can be achieved by e.g. a hybridisation that is performed in 5×SSPE, 5×Denhardt's solution and 0.5% SDS overnight at 55 to 60° C.
According to the invention said t-PA inhibitor can be used as a neuroprotectant.
An additional aspect of the invention provides a pharmaceutical composition selected from the group consisting of one or more of said t-PA inhibitors together with or without a thrombolytic drug, wherein said pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or excipients.
In a further aspect of the invention a method of treating a neurological or neurodegenerative disorder, in particular stroke is provided, comprising administering a therapeutically effective amount of said t-PA inhibitor, alone or in combination with therapeutically effective amount of a thrombotic drug, preferably a plasminogen activator, more preferably t-PA.
The term “neurological disorder” as used herein is defined as disease, disorder or condition which directly or indirectly affects the normal functioning or anatomy of a subject's nervous system.
In the context of the invention the term “neurodegenerative disorder” is defined as disease, in which cells of the central or peripheral nervous system are lost.
Examples for neurological and/or neurodegenerative disorders are spinal cord injury, intracranial or intravertebral lesions including, but not limited to, contusion, penetration, shear, compression or laceration lesions of the spinal cord or whiplash shaken infant syndrome.
In the context of the present invention the neurological disorders also include ischaemic events, or ischaemia or ischaemic disorders which can be defined as any local or regional state of hypoxia in cells or tissues which are usually due to an inadequate blood supply (circulation), e.g. caused by a blockage or obstruction of a blood vessel in this area.
The hypoxia can cause acute injury as in hypoxia and/or ischemia including, but not limited to, cerebrovascular insufficiency, cerebral ischemia or cerebral infarction (including cerebral ischemia or infarctions originating from embolic occlusion and thrombosis), retinal ischemia (diabetic or otherwise), glaucoma, retinal degeneration, multiple sclerosis, ischemic optic neuropathy, reperfusion following acute cerebral ischemia, perinatal hypoxic-ischemic injury, or intracranial hemorrhage of any type (including, but not limited to, epidural, subdural, subarachnoid or intracerebral hemorrhage).
Accordingly the term “ischaemic disorder” encompasses thrombotic disorders which include conditions associated with or resulting from thrombosis or a tendency towards thrombosis. These conditions include conditions associated with arterial or venous thrombosis that can be treated with a thrombolytic drug.
The term “t-PA” as used herein includes native t-PA and recombinant t-PA, as well as modified forms of rt-PA that retain the enzymatic or fibrinolytic activities of native t-PA. The enzymatic activity of t-PA can be measured by assessing the ability of the molecule to convert plasminogen to plasmin. The fibrinolytic activity of t-PA may be determined by any in vitro clot lysis activity known in the art. Recombinant t-PA has been described extensively in the prior art and is known to the person of skill. rt-PA is commercially available as alteplase (Activase® or Actilyse®). Modified forms of rt-PA (“modified rt-PA”) have been characterized and are known to those skilled in the art. Modified rt-PAs include, but are not limited to, variants having deleted or substituted amino acids or domains, variants conjugated to or fused with other molecules, and variants having chemical modifications, such as modified glycosylation. Several preferred modified rt-PAs have been described in PCT Publication No. WO93/24635; EP 352,119; EP382174.
In the context of the invention the term “suicide substrate” is defined as a compound that resembles a normal substrate closely enough that it undergoes an enzymatic reaction to a product that inhibits the enzyme. As a substrate analogue it often binds irreversibly to an amino acid of the enzyme, usually the catalytic amino acid, thereby blocking the active site of the enzyme to other molecules and effectively and mostly irreversibly inhibits the enzyme.
The term “stroke” as used herein is used for any event of cerebral ischaemia.
The term “treating” or “treatment” refers to any medical measure for preventing, reducing, mitigating or curing physiological disorders within a mammal, in particular a human, in the need thereof.
A “therapeutically effect amount” is defined as the amount of active ingredient that will reduce the symptoms associated with a neurological or neurodegenerative disease, such as stroke. “Therapeutically effective” also refers to any improvement in disorder severity or the frequency of incidences compared to no treatment. The term “treatment” encompasses either curing or healing as well as mitigation, remission or prevention.
The term “N-terminal domain of the NMDA receptor subunit NR1” (anti-ATD NR1) as used in the context of the present is defined as the region of the NR1 subunit of the NMDA receptor that interacts with t-PA. This encompasses NMDA receptors of different species like mouse, rat, pig, bovine, cat, dog, or monkey. Preferably the human NMDA receptor is used. Different isoforms of the NR1 subunits are also included in this definition, preferably the isoforms generated by alternative splicing and including the isoforms NR1-1a, NR1-1b, NR1-2a, NR1-2b, NR1-3a, NR1-3b, NR1-4a, NR1-4b. Preferably the region of the NR1-1a subunit encoding the amino acids 19 to 480, more preferably encoding the amino acids 19 to 371 (see SEQ ID NO:4 or SEQ ID NO:5) is used.
The term “antibody” encompasses the various forms of antibodies including but not being limited to whole antibodies, human antibodies, humanized antibodies and genetically engineered antibodies like monoclonal antibodies, chimeric antibodies or recombinant antibodies as well as fragments of such antibodies as long as the characteristic properties according to the invention are retained.
The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of a single amino acid composition. Accordingly, the term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g. a transgenic mouse, having a genome comprising a human heavy chain transgene and a light human chain transgene fused to an immortalized cell. Preferably said type of anti-ATD NR1 antibody is a monoclonal antibody.
The term “chimeric antibody” refers to a monoclonal antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are especially preferred. Such murine/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding murine immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of “chimeric antibodies” encompassed by the present invention are those in which the class or subclass has been modified or changed from that of the original antibody. Such “chimeric” antibodies are also referred to as “class-switched antibodies.” Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat. No. 5,204,244.
The term “humanized antibody” refers to antibodies in which the framework or “complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the “humanized antibody.” See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S., et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric and bifunctional antibodies.
The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374). Based on such technology, human antibodies against a great variety of targets can be produced. Examples of human antibodies are for example described in Kellermann, S. A., et al., Curr Opin Biotechnol. 13 (2002) 593-597.
The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences in a rearranged form.
As used herein, the term “neuroprotectant” means an agent that is capable of providing neuroprotection, i.e., protecting a neural entity, such as a neuron, at a site of injury, for example, an ischaemic injury or traumatic injury.
In the context of the invention the term “acute treatment” refers to a short-term medical treatment, usually in a hospital, for patients having an acute illness or injury or recovering from surgery. The term “post-acute treatment” refers to a mid-term medical treatment, usually in a hospital, for patients having an acute illness or injury or recovering from surgery.
Materials and Methods
The ability of some proteins comprising the PARK domain (DSPA alpha 1, cDSPA, Reteplase, DSPA alpha 2, DSPA beta, and kringle 2 (K2) of alteplase) to inhibit alteplase trans-BBB transport was demonstrated using the materials and methods outlined below.
DSPA alpha 1, cDSPA, Reteplase, DSPA alpha 2, and DSPA beta were provided by PAION Deutschland GmbH. Reteplase was purchased in a Pharmacy and provided by PAION Deutschland GmbH. kringle 2 (from alteplase) is a compound developed and provided by UMR CNRS 61185/INSERM-Avenir, Caen France.
To provide an in vitro system for studying brain capillary functions, a co-culture has been developed that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and glial cells on the other (
Bovine brain endothelial cells were isolated from brain capillaries, grown in DMEM supplemented with 10% (v/v) heat-inactivated calf serum and 10% (v/v) horse serum (Hyclone Laboratories, Logan, Utah, USA), 2 mM glutamine, 50 μg/mL gentamicin and basic fibroblast growth factor (1 ng/mL, added every other day). Sub-clones of endothelial cells frozen at passage 3 were recultured on a 60-mm-diameter gelatin-coated Petri dish and grown to confluency.
Primary glial cultures were isolated from newborn rat cerebral cortex. After removing the meninges, the brain tissue was gently forced through a nylon sieve. DMEM supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM glutamine, and 50 μg/mL of gentamycin was used for the dissociation of cerebral tissue and development of glial cells. The glial cells were plated at a concentration of 1.25×105 cells ml−1 on plastic in six-well plates and incubated at 37° C. with 5% CO2. The medium was changed twice a week. Three weeks after seeding, glial cultures were confluent and composed of astrocytes (˜60%), oligodendrocytes and microglial cells.
Culture plate inserts (Millicell PC 3 μm pore size) were coated on the upper side with rat-tail collagen prepared according to the method of Bornstein (1958).
For the analysis of sucrose permeation and compounds tested at 0.3 μM, filters with a surface of 4.2 cm2 were used. For compounds tested at 10 nM, filters with a surface of 3 cm2 were used.
Coated filters were placed in six-well dishes containing glial cells for co-culture. The co-culture medium was the same as that for brain capillary endothelial cells. Confluent endothelial cells were trypsinized and plated on the upper side of the filters at a concentration of 4-10 cells/ml. Under these conditions, endothelial cells formed a confluent monolayer within 12 days. Experiments were performed 5 days after confluence.
Sucrose was used as a paracellular marker allowing the evaluation of possible effects of the test compounds on the integrity of the BBB. This small hydrophilic molecule shows a low cerebral penetration and its endothelial permeability coefficient is a measure of the endothelial cell monolayer integrity.
The day of the experiments, Ringer-HEPES was added to the lower compartment (abluminal side) of a six-well plate (2.5 ml per well). One filter containing a confluent monolayer of endothelial cells was transferred to the first well of the plate.
1.5 ml Ringer-HEPES containing the compound in co-incubation with [14C]-sucrose (addition of 0.5 μCi of [14C]-sucrose per filter) was placed in the upper compartment (luminal side). At defined times after addition of the tested compound, the insert was transferred to another well of the six-well plate to minimize the possible reflux of substances from the lower to the upper compartment (refer to
During the 60-min experiment, the clearance volume increased linearly with time. The average cleared volumes were plotted versus time, and the slope was estimated by linear regression analysis to yield the mean and the associated Standard Error. The slope of the clearance curves for the coculture is denoted PSt, where PS is the permeability x surface area product (in microliters per minute). The slope of the clearance curve for the filter only covered with collagen is denoted PSf.
The PS value for the endothelial monolayer (PSe) was calculated from:
The PSe values were divided by the surface area of the filter (4.2 cm2 for Millicell PC and CM and 4.7 cm2 for Transwell inserts) to obtain the endothelial permeability coefficient (Pe, in centimeter per minute).
Stability of sucrose permeability was tested with each study compound in triplicate monolayers to confirm the absence of toxic effects on the BBB.
Translocation of rt-PA was determined as described under 4.1 for sucrose.
In these and all other experiments, the integrity of the BBB was monitored using [14C]-sucrose as a paracellular marker, allowing the evaluation of possible effects on the integrity of the BBB.
Data evaluation: The passage of alteplase was evaluated by means of a plasminogen-based zymography assay. It was quantified using an image quantitative system. Moreover, triplicates of luminal and abluminal solutions were assayed for alteplase by means of a spectrozyme assay.
The sequential stages of the study are described in
Filter Study
In this step, the ability of alteplase to cross the filter was tested to exclude the possibility of transport restriction by the filter. Alteplase was applied at the non-toxic concentration determined in the integrity test.
Transport Studies
DSPA-related molecules, reteplase and kringle 2 (alteplase) were studied at equimolar concentrations for their abilities to influence alteplase permeation, evaluated by means of a plasminogen-based zymography assay and quantification by spectrozyme assay.
Endothelial permeability coefficients obtained with sucrose alone and with alteplase alone or alteplase in combination with a DSPA-related molecule, reteplase or K2 (alteplase) are summarized in the following table 1.
The endothelial permeability coefficients obtained for sucrose showed that alteplase in coincubation with DSPA-related molecules does not exert toxic effects on the in vitro BBB at the respective concentrations of 0.3 μM or 10 nM.
The transport of alteplase (0.3 μM) through collagen-coated filter was investigated over a period of 120 minutes.
After the experimental period, the concentration of alteplase was determined in luminal and abluminal samples by means of spectrozyme assays (table 2).
§ PERCENTAGE OF ABLUMINAL ALTEPLASE ALONE
The results show that alteplase permeation is not restricted by the empty filter, since alteplase showed the potential to cross the filter to an extent of 7.88%. This value was registered as the maximum percentage of diffusional passage through the empty filter and used in the endothelial transport studies as a reference (the so called “versus filter”).
Translocation of alteplase alone and in coincubation with DSPA-related molecules (DSPAα1, cDSPA, DSPAα2), Reteplase and K2 (alteplase) was studied over a 120 min period. Equimolar concentrations (0.3 μM) were used.
After the experimental period, the luminal and abluminal concentrations of alteplase were determined by means of a plasminogen containing zymography assay (
As shown in
§ PERCENTAGE OF ABLUMINAL ALTEPLASE ALONE
In a further series, spectrozyme assays were performed in both the luminal and abluminal compartment to quantify the passage of alteplase (0.3 μM) during coincubation with DSPA-related molecules and K2 (alteplase) (all at 0.3 μM) (table 4).
§ PERCENTAGE OF PASSAGE VERSUS FILTER FOR ALTEPLASE ALONE.
Because of the protease activity of reteplase the spectrozyme assay could not be used, as it was impossible to distinguish between activities related to alteplase and reteplase.
As illustrated in
The inhibitory effect of a protein comprising a PARK domain was also shown in an animal model as outlined below:
Human recombinant alteplase (rt-PA, alteplase) was from Boehringer Ingelheim (France), NMDA from Tocris (U.K.). Desmoteplase was provided by PAION Deutschland GmbH (Germany).
Animals
Male Sprague Dawley rats (270 to 330 g) were housed in a temperature-controlled room on a 12-hour light/12-hour dark cycle, with food and water ad libitum. Experiments were performed in accordance with French (act no. 87 to 848; Ministere de l'Agriculture et de la Forêt) and European Communities Council (Directives of Nov. 24, 1986, 86/609/EEC) guidelines for the care and use of laboratory animals.
Striatal Excitotoxic Lesions
Rats were anesthetized with isoflurane (5%, maintenance 2% in oxygen/N2O (1:3) at 0.8 l/min). Body temperature was maintained at 37±0.5° C. Injection pipette (internal diameter 0.32 mm and calibrated at 15 mm/μl; Hecht Assistent, Germany) was stereotaxically implanted in the right striatum (3.5 lateral and 5.5 ventral to the bregma). NMDA (50 nmol) was injected in a volume of 1 μl and the pipette was removed 3 minutes later. In the first set of experiments, excitotoxic treatment was complemented after 15 minutes by intravenous injection of alteplase (1 mg/kg), desmoteplase (1 mg/kg), alteplase plus desmoteplase (1 mg/kg each), alteplase vehicle (L-Arg 35 mg/kg, phosphoric acid 10 mg/kg and polysorbate 80, 0.2%) or desmoteplase vehicle (glycine 4 mg/kg and mannitol 10.64 mg/kg). In the second set of experiments, NMDA was coinjected into the striatum with alteplase (3 μg), desmoteplase (3 μg), alteplase plus DSPA (3 μg each) or the corresponding vehicles, all in a volume of 1 μl.
Histological Analysis
After 24 hours, rats were euthanized and the whole brain was removed and frozen in isopentane. For volume analysis, one coronal section (20 μm) out of every 20 was stained with thionine and analyzed, covering the entire lesion. Regions of interest were determined using a stereotaxic atlas for the rat (Paxinos and Watson). An image-analysis system (BIOCOM RAG 200, Paris, France) was used to measure the lesion given by the non stained area.
This analysis showed that Desmoteplase antagonized rt-PA mediated neurotoxic effects when both were co-injected intravenously but not when co-administered into the striatum (
These results show the ability of various proteins comprising a PARK domain to inhibit alteplase trans-BBB transport.
Alteplase alone and in co-incubation with DSPAα1, cDSPA, DSPAα2, Reteplase and K2 (alteplase), all at a concentration of 0.3 μM, had no toxic effects on BBB integrity. As analyzed by means of the spectrozyme assay, DSPAα1 and DSPα2 were able to reduce alteplase transport across the BBB to approximately 67 and 62%, respectively, confirming that DSPA-related molecules can be used to block alteplase blood-to-brain translocation. Similar results (reduction to about 76%) were obtained for clogged DSPA, a DSPA variant that does not exert proteolytic activity. Inhibition was confirmed in zymography quantitation of alteplase.
In addition, isolated kringle 2 was able to reduce alteplase transport by about 50% (spectroscopy data, confirmed by zymography).
Also Reteplase was able to inhibit alteplase translocation as demonstrated in alteplase zymography analysis. As Reteplase possesses protease activity, its effect could not be quantified by means of the spectrozyme assay.
The molecules tested in this study are able to inhibit rt-PA trans-BBB transport, most likely by competing for LRP-mediated transport. According to the spectrozyme data, they can be ranked as follows: K2 (t-PA)>DSPAα2>DSPAα1>cDSPA.
Production of Recombinant ATD/Leucine-Isoleucine-Valine-Binding Protein (LIVBP)-like Domain:
The region of the NR1-1a subunit encoding amino acids 19-371 corresponding to the amino terminal domain of NR1, identified as the domain of interaction with t-PA, was amplified from the full-length rat NR1-1a cDNA, as previously described (Fernandez-Monreal et al., 2004). The recombinant protein, designed rATD-NR1, was purified from inclusion bodies of isopropyl 1-thio-β-D-galactopyranoside-induced bacterial cultures (Escherichia coli, M15 strain) on a nickel affinity matrix as described by the manufacturer (Qiagen, France).
Active Immunisation:
Briefly, as previously described with slight modifications (Benchenane et al., 2007), mice were immunised by intraperitoneal injection of immunogenic mixtures: Complete Freund's adjuvant (Sigma Aldrich, France used for the first injection) and Incomplete Freund's adjuvant (Sigma Aldrich, France; later injections, once a week during 3 weeks) containing the rATD-NR1 (30 μg); the same mixture without the rATD-NR1 was used as a control serum.
Production and Purification of Polyclonal Antibodies:
Sera were collected 2 weeks after the last inoculation from mice immunised with rATD-NR1 or control adjuvant as described for active immunisation. Polyclonal antibodies (anti-ATD-NR1 antibodies or control Igs) were then purified from the serum hydroxyapatite columns (Proteogenix, France).
Immunoblotting:
rATD-NR1 (20 μg) or protein extracts as mentioned in the corresponding figures were loaded and separated by 10% SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked with Tris-buffered saline (10 mM Tris and 200 mM NaCl, pH 7.4) containing 0.05% Tween-20, 5% dry milk. Blots were exposed overnight either with a mouse anti-ATD-NR1 primary antibody (1:2000; 4° C.), mouse control Igs primary antibody (1:2000; 4° C.) or a control goat histidine protein primary antibody (1:1000; 4° C., Qiagen, France). After mouse or goat peroxydase-conjugated secondary antibodies (1:5000; Sigma Aldrich, France), proteins were visualized with an enhanced chemiluminescence ECL-Plus detection system (Perkin Elmer-NEN, France).
Thromboembolic Focal Cerebral Ischaemia:
As previously described (Orset et al., 2007), male Swiss mice (28-30 g, CERJ, Paris, France) were deeply anesthetised (using isoflurane 5% with 70%/30% mixture of NO2/O2) and placed in a stereotaxic device; the skin between the orbit and ear was incised and the temporal muscle was retracted. A small craniotomy was performed, the dura was excised and the middle cerebral artery (MCA) exposed. The pipette was introduced into the lumen of the MCA and 1 μL of purified murine alpha-thrombin (0.75 Ul, Gentaur, Belgium) was pneumatically injected (by applying positive pressure with a syringe connected to the pipette via a catheter) to induce the in situ clot formation. The pipette was removed 10 min after the injection of the alpha-thrombin at which time the clot is stabilized. The muscle and soft tissue was replaced and the incision sutured. Cerebral blood velocity was monitored by laser Doppler flowmetry (Oxford Optronix, United Kingdom) and the temperature was controlled over the whole surgical procedure. To induce thrombolysis, rt-PA (10 mg/kg; Actilyse®, Boehringer Ingelheim, Germany) was intravenously injected (tail vein, 10% bolus, 90% perfusion during 40 minutes) 20 minutes or 4 hours after the injection of alpha-thrombin. The control group received the same volume of saline under identical conditions.
Passive Immunisation:
20 minutes or 4 hours after clot formation, purified polyclonal antibodies raised against the rATD-NR1 were intravenously injected in the tail vein by a bolus of 0.2 mg of anti-ATD-NR1 or control immunoglobulins (200 μl).
Histological Analysis of Brain Lesions:
After 24 hours, mice were killed by anaesthetic overdose and the brains were removed and frozen in isopentane. Cryostat-cut coronal brain sections (20 μm) were stained with thionine and analysed by using an image analyser (BIOCOM RAG 200, Paris, France). For volume analysis, one section of 20 μm out of 10 was stained and analysed (covering the whole lesion). Regions of interest (non-stained areas corresponding to infarcted tissues) were determined through the use of a stereotaxic atlas for the mouse (Franklin and Paxinos, 1997). The infarct volume was determined as the sum of the unstained areas of the sections multiplied by their thickness according to the equation: Vischemic lesion=P (Areas of ischaemic lesion) distance between sections. This method has been validated previously, demonstrating an excellent reproducibility of the volumetric assessment of the ischaemic lesion (Osborne et al., 1987).
MRI analyses:
were performed at 24 hours, 72 hours, 7 days, 15 days after thromboembolic ischaemia performed in mice intravenously injected with saline or the α-ATD-NR1 antibody 4 hours after clot formation. MRI experiments were carried out on a Pharmascan 7 T/12 cm system with Paravision 4.0 software (Bruker, Ettlingen, Germany) using a 72 mm inner diameter birdcage for radio frequency transmission and a 25 mm diameter surface coil for reception. During the MRI experiments, anesthesia was maintained using isoflurane (70%/30% mixture of NO2/O2). Mice were monitored for changes in their respiratory rate in order to adjust the anesthetic concentration. T2-weighted images were acquired using a RARE sequence: TE/TR 411 3000 ms with 4 averages (Matrix 256×256, FOV 23.7×20 mm). Diffusion-weighted images were acquired with a 6 directions EPI-DTI sequence: TE/TR 34/3750 ms using two b values (0 and 800 s/mm2) with 2 experiments for each direction (Matrix 128×128, FOV 20.5×19.2 mm). Apparent diffusion coefficients (ADCs) in mm2/s were calculated by pixel-by-pixel curve fitting using a monoexponential model. Angiographies were acquired with TE/TR 12/7 ms with 2 averages (Matrix 256×256, FOV 20×20 mm).
Evan's blue extravasation: Evan's blue (200 μl, 2%, Sigma-Aldrich) was intravenously injected in ischaemic mice 3 hours before euthanasia. The mice were perfused at their mean arterial pressure with heparinised saline prior to harvesting their brain. The cortices were dissected, homogenised in phosphate buffered saline with sodium dodecyl sulphate 1% and centrifuged (10000 g, 15 minutes). Evan's blue was quantified in supernatants from the absorbance at 620 nm and divided by the wet weight of each hemisphere. Each observation was repeated three times.
Behavioural Analyses:
Mice were subjected to a global neurological function test 24 hours post cerebral ischaemia This behavioral test was performed in a chamber (67×53×55 cm, BIOSEB®, Chaville, France) formed by black methacrylate walls and featuring a Plexiglas front door. The floor of the chamber consisted of 22 stainless steel bars (3 mm in diameter, spaced 1.1 cm apart, center-to-center), which were wired to a shock generator with scrambler for the delivery of unconditioned foot shock stimuli. The signal generated by mouse movement was recorded over a 5 min period and analysed by means of a high sensitivity Weight Transducer system. The analog signal was transmitted to the Freezing software module through the load cell unit for recording and off-line analysis for mobility as indicated by periods of activity/immobility (Freezing). An additional interface associated with corresponding hardware allowed controlling the intensity of the shock from the Freezing software.
Cell Culture:
Primary cultures of cortical neurons were prepared from foetal mice (E15-E16). Dissociated cortical cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum, 5% horse serum and 2 mmol/L glutamine and plated in 24 well dishes previously coated with poly-D-lysine and laminin. After 3 days, the cells were exposed to 10 μmol/L of Ara-C to inhibit glial proliferation. Cultures were used after 12 days in vitro.
Excitotoxicity:
Slowly or rapidly triggered excitotoxicity respectively, was induced at 37° C. by exposing neuronal cultures to 10 μmol/L NMDA for 24 hours and 50 μmol/L NMDA for 1 hour in DMEM supplemented with glycine (10 μmol/L). The exposure to NMDA was performed alone, or in combination with rt-PA (20 μg/mL) and/or α-ATD-NR1 or control Igs (0.01 mg/ml). In both cases, neuronal death was assessed after 24 hours using phase-contrast microscopy and quantified by measurement of lactate dehydrogenase (LDH) release from damaged cells into the bathing medium.
Calcium Videomicroscopy Analysis:
Primary cultures of cortical neurons were loaded with HEPES-buffered saline solution containing 5 μmol/L of fura-2/AM plus 0.1% pluronic F-127 (Molecular Probes, Leiden, the Netherlands) (30 minutes, at 37° C.) and incubated for an additional 30 minutes period in a HEPES-buffered saline solution. Experiments were performed at 22° C. on the stage of a Nikon Eclipse inverted microscope equipped with a 75 W Xenon lamp and a Nikon x40, 1.3 numerical aperture epifluorescence oil immersion objective. Fura-2 (excitation: 340, 380 nm, emission: 510 nm) ratio images were acquired with a CCD camera (Princeton Instrument, Trenton, N.J.), and digitized (256×512) using Metafluor 4.11 software (Universal Imaging Corporation, Cherter, Pa.).
Induction of Apoptosis:
Serum deprivation (SD) was induced by the exposure of neuronal cultures (DIV7) to a serum-free DMEM supplemented with 10 microM of glycine and characterized as previously described (Koh et al. 1995; Nicole et al. 2001a; Liot et al. 2004). Controls were maintained in serum-containing fresh medium. MK-801 (1 microM) was added to prevent secondary NMDA receptor activation. Before fixation in 4% paraformaldehyde, cells were stained with 0.4% trypan blue for 15 min. SD was performed alone, or in combination with rt-PA (20 μg/mL) and/or anti-ATD-NR1 antibodies (0.01 mg/ml). Neuronal cell injury was quantified by counting trypan blue positive cells in three random fields per well. The percentage of neuronal death was determined as the number of trypan blue positive neurones after SD compared with the total number of neurones. The mean values of trypan blue positive neurones in sham washed control conditions were subtracted from experimental values. Densitometric analysis data (Image J software) of corresponding immunoblots were normalized to mean values obtained for control conditions.
Gelatin Zymography Analysis for MMP-2 and -9:
Gelatinase assay was performed to evaluate MMP-9 and MMP-2 levels in ischaemic mice cerebral sample. Briefly, tissues lysates (25 μg of proteins) were loaded onto 10% SDS-polyacrylamide gels copolymerized with 1 mg/mL gelatin (Sigma Aldrich, France) for electrophoresis. After electrophoresis, gels were washed in 2.5% Triton X-100 and then incubated for 36 hours at 37° C. with a developing Tris buffer before staining with Coomassie blue. Gels were then destained and MMP-2 and -9 band intensity was quantified.
Focal ischaemia was induced in mice by in situ injection of plasma purified murine thrombin into the MCA (Orset et al., 2007). Immediately after thrombin injection, clot formation was evidenced by a dramatic reduction (mean reduction of 80%) of the cerebral blood velocity (CBV) as measured by laser Doppler flowmetry (
Previous active immunization of mice with the recombinant amino-terminal domain of the NR1 subunit of the NMDAR (rATD-NR1) as an antigen altered neither clot formation nor rt-PA-induced reperfusion (
The results given in example 1 provided proof of concept for the idea that antibodies against the amino-terminal domain of the NR1 subunit of the NMDA receptor are beneficial in acute ischemic stroke. However, as active immunization is not feasible as a means to treat an acute disorder, the inventors developed a strategy of passive immunization (antibody-based immunotherapy), based on purified serum immunoglobulin from rATD-NR1-vaccinated mice. The inventors first controlled by immunoblotting that purified polyclonal anti-ATD-NR1 antibodies can recognize the immunogenic peptide. The anti-ATD-NR1 antibodies could independently recognize two forms of rATD-NR1, coupled with either a histidine-tag (
The next step was to validate the ability of these antibodies to prevent the aggravating effect of rt-PA on NMDA toxicity (Nicole et al., 2001). In primary cultures of murine cortical neurons exposed to either a high concentration of NMDA (50 μM) for a short period (1 hour) or a moderate concentration of NMDA (10 μM) for a long period (24 hours), the co-application of the anti-ATD-NR1 antibodies completely prevented the promotion of neuronal loss induced by rt-PA (0.3 μM) (
The therapeutic value of the anti-ATD-NR1 antibodies (passive immunisation) was then investigated in vivo. First, excitotoxic lesions were induced in mice by administrating NMDA (10 nmol) into the striatum together with a single intravenous injection of control or purified antibodies. In control animals, NMDA led to an excitotoxic lesion of 17±2 mm3, while in anti-ATD-NR1 antibodies-treated mice, the lesion (9±1 mm3) was reduced in size by 47.06% (n=8 in each group, p<0.01;
Twenty four hours post-occlusion, stroke-induced cognitive impairment was evaluated in a contextual freezing task (n=10), which is considered to allow a global cognitive evaluation of anxiety, memory and learning processes. Compared to sham animals, ischaemic mice displayed an increase in freezing (+214%, p<0.025), an effect which was dramatically reduced (−170%; p<0.05) by the intravenous administration of the α-ATD-NR1 antibodies (
Since ischaemic brain injuries as well as t-PA's beneficial or deleterious effects are highly related to the blood-brain barrier (BBB), Evan's blue (EB) extravasation and matrix metalloproteinases activation (MMP-2 and MMP-9) were evaluated in the conditions described above. As expected, rt-PA was found to be able to induce the activity of MMP-9 and enhance extravasation of Evan's Blue into brain parenchyma. The later after clot formation rt-PA was administered, the stronger were the effects on MMP-9 activity (
Of highest clinical relevance are long-term follow-ups of the consequences of acute treatment. Thus, an MRI-based longitudinal follow-up of animals treated 4 hours post clot onset with a single injection of anti-ATD-NR1 antibodies was undertaken over a 3 month period. T2 MRI analyses clearly confirmed the patterns of thionine staining observed earlier, evidencing a brain protective effect of the antibodies which was already visible 24 hours after inducing ischaemia and maintained for at least 15 days post-surgery (X % of reduction when compared to control animals, n=3, p<x;
(A, C) Ischaemic lesions were performed 11 days after the last inoculation of the recombinant rATD-NR1, by an in situ injection of thrombin (0.75 U.I) into the MCA in Swiss mice (n=10 per group). After 20 minutes (A) or 4 hours (C), rt-PA (10 mg/kg) or saline was injected over 40 minutes (10% Bolus, 90% infusion). Brains were harvested 24 hours later, and cerebral lesions were measured by analysing thionine-stained cryostat sections (see representative section for each group). Results are expressed as mean±SD. *: p<0.01
(B, D) The normalized cerebral blood velocity measured by laser Doppler showed that immunisation against the ATD of the NMDA receptor NR1 subunit does not affect the ability of early (20 min) or late (4 hours) rt-PA administration to induce reperfusion (n=10 per group, results were normalised to baseline values and expressed as mean±SD, *: p<0.001).
(A) 20 g of recombinant N-terminal domain of NR1 (ATD-NR1, amino acids 19-371) (coupled to a Histidine-tag or an Fc-tag) was separated by SDS-PAGE prior to detection with anti-ATD-NR1 polyclonal antibody, anti-histidine antibody, anti-Fc antibody and control IGs. Immunoblot is representative of three independent experiments. (B) Protein extracts from cerebral cortices of human and mice were separated by SDS-PAGE prior to detection with anti-ATD-NR1 polyclonal antibody or a commercial NR1-ct antibody which is able to recognize the NMDA receptor NR1 subunit. Immunoblots are representative of three independent experiments.
Neuronal death rate was assessed by the LDH released into the bathing medium assessed 24 h after excitotoxic exposure to NMDA. (A) Mixed cortical cultures underwent a prolonged exposure to NMDA (10 μM) alone or in the presence of rt-PA (20 μg/ml), of anti-ATD-NR1 (0.01 mg/ml), or both. rt-PA caused a significant increase in the neuronal death rate, which was antagonized by anti-ATD-NR1. (C) Mixed cortical cultures with a short exposure to NMDA (50 μM), otherwise conditions and effects as in panel A. (B, D) Controls were performed in the presence of the control IGs. For each experiments N=3; n=12 well; p<0.01.
(A, C) A single intravenous injection of anti-ATD-NR1 antibodies (0.8 mg/ml) against the rATD-NR1, targeting the potentiating effects of endogenous and exogenous t-PA after an in situ thromboembolic stroke, either with early (A: 20 min) or late (C: 4 hours) reperfusion by rt-PA (10 mg/kg) (filled bars), or without reperfusion (saline injection, empty bars) (n=8-10 mice per group, p<0.05; * p<0.01). Antibodies were injected as boli after the bolus of t-PA or saline solution. (B, D) baseline values) Cerebral blood velocity measured by laser Doppler and normalised to baseline, showed that immunisation against the ATD of the NMDA receptor NR1 subunit does not affect rt-PA-induced reperfusion, implemented early (20 min, B) or late (4 hours, D) after clot onset (n=8-10 mice per group, p<0.001).
(A) Evans blue dye extravasation 24 hours after MCAO was normalized to mean values of sham condition (mice without clot). A single injection of anti-ATD-NR1 polyclonal antibodies (0.01 mg/ml), 20 min or 4 hr post-stroke, was able to decrease stroke-induced BBB leakage, combined or not with rt-PA (10 mg/kg) (n=3 mice per group, p<0.05). This ability could be linked with the MMP-9 activity. (B) MMP-9 proteolytic activity was normalized to mean values obtained with saline injection. A single injection of anti-ATD-NR1 polyclonal antibodies, 20 min or 4 hr post stroke, was able to decrease the MMP-9 activity in the ipsilateral cortex, combined or not with rt-PA (10 mg/kg) (n=3 mice per group, p<0.05). Panel (C) shows representative pictures of MMP-2 and MMP-9 activities. MMP-9 was increased in saline and rt-PA conditions. Most interestingly, they were decreased after a single injection of anti-ATD-NR1 antibodies.
Mice were treated with saline or anti-ATD-NR1 antibody (0.8 mg/ml), 20 minutes (A) or 4 hours after stroke (B). 24 hr post ischaemia, they were placed in a fear conditioning room. Results are expressed in percentage of freezing time during a 5 min period and normalized to results obtained with naive mice (n=10 per group, * p<0.025; $ p<0.05).
Excitotoxic lesions were produced by injecting NMDA (10 nmol) into the right striatum (coordinates: 0.0 mm posterior, 2.0 mm lateral, 4.0 mm ventral to the bregma) After 30 minutes, anti-ATD-NR1 antibodies or control IGs were injected in the tail vein (0.8 mg/ml). Compared to controls, anti-ATD-NR1 antibodies afforded a significant protection of the striatum (n=8, p<0.01)
Protein extracts were isolated post mortem from the ipsilateral and contralateral hemisphere of a patient died two days after ischemic stroke, subjected to SDS-PAGE gel chromatography, blotted onto a nylon membrane and analysed in a Western blot using (A) the anti-ATD NR1 antibody or (B) the anti-human NR1 antibody.
(A) XX, (B) XX. (C) Immunohistochemical analysis directed against the anti-ATD-NR1 antibody using brain tissue from non-operated or sham animals reveal the presence of anti-ATD-NR1 antibodies in the brain parenchyma.
Tested was the influence of rt-PA (20 μg/mL) and anti-ATD-NR1 antibodies (0.01 mg/ml) on apoptosis caused by serum deprivation of neuronal cultures (DIV7). Upper panels: Neuronal death caused by serum deprivation, ability of rt-PA to antagonize this effect (p<0.01), and lack of influence of anti-ATD-NR1 antibodies on the effect of rt-PA. The percentage of neuronal death was determined by the proportion of trypan blue positive neurons (counting trypan blue positive cells in three random fields per well). Results were corrected for the respective values obtained in sham washed control neurons.
Lower panel: Cleavage of caspase 3 as an indicator of an apoptotic setting. Cleaved molecules were revealed with an antibody raised against the cleaved caspase-3. Again, the protective effect of rt-PA was not influenced by the anti-ATD-NR1 antibodies. (N=3, n=12)
A single intravenous injection of anti-ATD-NR1 antibodies is sufficient to cause dramatic neuroprotection when applied either early (20 minutes) or late (4 hours) after clot onset in a murine model of thrombo-embolic stroke. Of note, the current invention shows a time-dependent loss of the benefit of rt-PA treatment: Although early rt-PA-induced reperfusion produces benefits, as observed in the clinical use of this drug, delayed rt-PA-induced reperfusion (4 hours post clot formation) is even associated with a deleterious outcome, despite comparable levels of reperfusion. This negative outcome is healed by the co-administration of anti-ATD-NR1 antibodies, which restore the neuroprotective efficacy and, by this, extend the time window, in which rt-PA-induced thrombolysis is able to confer therapeutic benefit.
Further, the present invention of immunotherapy with anti-ATD-NR1 antibodies provides means to reduce the increase in BBB (considered a prelude to intracerebral hemorrhage), which is attendant to cerebral ischaemia and aggravated by late rt-PA-treatment under ischemic conditions. These observations reinforce the therapeutic interest in the anti-ATD-NR1 antibodies, since brain edema and bleeding are associated with a poor clinical outcome in stroke patients.
To be borne in mind, t-PA has also been reported to serve critical physiological functions in: (i) synaptic remodeling and plasticity, (ii) neuronal migration during perinatal development, (iii) control of mechanisms involved in memory and learning processes such as the Late-phase Long Term Potentiation (L-LTP) in the hippocampus, and (iv) activation of pro-BDNF into BDNF. Hence it is important to note that the antibodies of the current invention have been found innocuous where tested for a possible impact on these functions: In the absence If ischaemic lesions, no changes of cognitive functions, including spatial memory, contextual and cue fear conditioning, could be identified, suggesting a safe profile of the presented strategy of immunotherapy.
| Number | Date | Country | Kind |
|---|---|---|---|
| EP09011149.3 | Aug 2009 | EP | regional |
This application is a Continuation of International Application No. PCT/EP2010/000544, filed Jan. 29, 2010, which designated the United States and has been published as International Publication No. WO 2011/023250 and which claims the priority of European Patent Application, Serial No. 09011149.1, filed Aug. 31, 2009 pursuant to 35 U.S.C. §119(a)-(d), the content of which is hereby incorporated verbatim by reference.
