WO2012068630A1 - Methods and compositions comprising antagonists of type 1 interferon-mediated signalling for reducing a neuroinflammatory response in the central nervous system following a stroke - Google Patents
Methods and compositions comprising antagonists of type 1 interferon-mediated signalling for reducing a neuroinflammatory response in the central nervous system following a stroke Download PDFInfo
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- WO2012068630A1 WO2012068630A1 PCT/AU2011/001522 AU2011001522W WO2012068630A1 WO 2012068630 A1 WO2012068630 A1 WO 2012068630A1 AU 2011001522 W AU2011001522 W AU 2011001522W WO 2012068630 A1 WO2012068630 A1 WO 2012068630A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Definitions
- the present disclosure relates generally to the amelioration of symptoms associated with a neuropathology inflammatory response component of a stroke event within the central nervous system (CNS) in a subject.
- Agents, medicaments and pharmaceutical compositions useful in the management of stroke are also enabled herein as are diagnostic assays.
- the immune system and the central nervous system (CNS) have long been considered separate systems with limited interaction.
- CNS central nervous system
- recent findings have shown support for a potential relationship between the two systems. This occurrence has been most evident in situations of neural injury, specifically ischemia-reperfuston pathologies (Zierath ei al, Ne rocrit Care 72 ⁇ :274-284, 2010).
- the widely accepted view was that the CNS is immune-privileged as the blood brain barrier (BBB) prevents communication with peripheral immune cells (such as macrophages and lymphocytes).
- BBB blood brain barrier
- Inflammation is a complex biological response initiated to remove a foreign pathogen from a given host. It is characterized by initial pathogen recognition, subsequent tissue oedema and multiple infiltrating cell types. Most notably, macrophages are recruited to ingest and remove foreign matter. The pathogens responsible can be venoms, parasites, allergens and DNA/RNA fragments released from necrotic cells. Initially, this immune response is protective, removing the pathogen, but an exaggerated response can promote detrimental overcompensation. This results from excess differentiation resulting in hematopoietic cell infiltration and release of chemokines and cytokines.
- CNS ischemia-reperfusion injuries show characteristic macrophage and leukocyte recruitment resulting in production of various cytokines; however, the response is primarily initiated by activated resident microglia (Banati et al.. GUa 7(1): ⁇ 11-118, 1993; Giulian and Vaca., Stroke 24(12 Si ⁇ Rp/J:184-1 0, 1993).
- mRNA and chaperone proteins released from necrotic cells act as ligands for Toll-like receptors (TLRs) [Kariko el al. J Biol Chem 27Of/3;:l 2542-l 2550, 2004; Ohashi et al.
- Activated microglia secrete pro-inflammatory cytokines including TNFcc and interleukins (Block et al, Neuronport 11(5) 963-967, 2000; Lambertsen et al. J Cereb Blood Flow Metab 25(1): 119-145, 2005) alongside chemo-attractants (Shohami et al. JNeruochem 53:1541-1546, 1989).
- the CCL2/3 chemokines lead to the recruitment of macrophages (Cowell et al. Stroke 33(3).795-Z01, 2002), further exacerbating the inflammatory response to the initial ischemia.
- a characteristic cytokine 'storm' develops, which contributes to neurodcgcneration.
- the Type 1 IFN receptor is composed of IFNARl and IFNAR2 subunits.
- IFNAR IFNa receptor
- STAT Signal Transducers and Activators of Transcription
- STATi and STAT2 dimerize and form a complex with an intracellular protein, Intetferon Regulatory Factor 9 (1RF9) [Leung et al, Mai Cell Biol 5:1312-1317, 1995], This complex translocates to the nucleus, acting as a transcription factor, and up-rcgulates the expression of anti-viral and anti-proliferative proteins, including pro-inflammatory cytokines (Lloyd et al., J Newainflammalion 5:28, 2008; Wei et al., J Neuroinflammation 6: 19, 2009).
- Intetferon Regulatory Factor 9 1RF9
- Minozac small molecule inhibitor
- Minozac prevented the chronic activation of microglia, which was linked to neurodegeneration in the long term (Somera-Molina ei al., Epilepsia 45:1785-1800, 2007).
- Glyprornate is derived from Insulin-like Growth Factor (IGF-1 ) and is believed to induce repair of injured brain tissue.
- IGF-1 Insulin-like Growth Factor
- NNZ-2566 particularly suppressed expression of IL- ⁇ (which is thought to play a significant role in secondary neuronal apoptosis), thus conferring a level of neuroprotection to the tissue.
- neuroinflammation a inflammatory response
- the present disclosure teaches a method for ameliorating symptoms of a neuropathological inflammatory response in the CNS and in particular the brain following a stroke event.
- the method enabled herein is predicated in part on antagonizing Type 1 interferon (TFN)- mediated signalling.
- TNF Type 1 interferon
- an aspect enabled herein is a method for reducing a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IFN)-mcdiated signalling, for a lime and under conditions sufficient to prevent or attenuate neuroinflammation.
- reducing a neuroinflammatory response includes “ameliorating the symptoms of a neuroinflammatory response”.
- Another aspect enabled herein is a method for ameliorating the symptoms of a neuroinflammatory response within the central nervous system (CNS) of a subject following a neurological event or condition, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IFN)-mediated signalling, for a time and under conditions sufficient to prevent o attenuate newomflammation, wherein the neurological event or condition is a stroke event.
- IFN Type 1 interferon
- the present disclosure teaches a method for reducing a neuroinflammatory response within the ecmral nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an antagonist of interferon alpha receptor I (IFNARl)-mediated signalling for a time and under conditions sufficient to prevent or ameliorate the symptoms of neuroinflammation.
- IFNARl interferon alpha receptor I
- Neuroprotective formulations comprising the Type 1 IFN-mediatcd signalling antagonist are also contemplated herein, as are diagnostic assays to screen for the level of Type J IFN-mcdiated signalling prior to or following a stroke in the CNS. Aspects disclosed herein are also applicable for the development of stroke protocols for patients who present with symptoms of such a condition.
- the Type 1 IFN-mediatcd signalling antagonist may target a Type 1 IF or its receptor or a component thereof.
- a Type 1 IFN includes IFNa, ⁇ and IFNto.
- the antagonist targets the IFNa receptor subunil (IFNARl) and/or the ability for IFNARl to dimerize with I NAR2.
- IFNARl IFNa receptor subunil
- Combination therapy targeting a Type I IFN and IFNAR and optionally other components of the neuroinflamraatory cascade are also taught herein.
- the Type 1 IFN-mediated signalling antagonist may be used po$t-or pre- neurological event. Hence, it may be a therapeutic or prophylactic.
- the subject includes a higher order mammal including a human.
- a human subject may be of any age and includes a fetus.
- Figure 2 is a graphical representation showing M17 cells subjected to 3 hours OGD and time-course reperfusion (0, 0.5, 2 and 24 hours), RT-PCR was then performed.
- IFNcc mRNA levels unlike ⁇ , were significantly elevated at 2 hours reperfusion (15.1+2.3 fold change) compared to control.
- Figures 3A through C are graphical representations showing Ml 7 cells subjected to 3 hours OGD and time-course reperfusion (0, 0.5, 2 and 24 hours), RT-PCR was then performed.
- Interlcukin-6 (IL-6) mRNA levels were significantly increased at 0.5 hour (266.9 ⁇ 10.9 fold change) and 2 hours (194.6 ⁇ 39.4 fold change) reperfusion compared to control.
- FIG. 4 is a graphical representation showing collated RT-PCR data for each cytokine mRNA levels post-3 hours OGD and time-course reperfusion (0, 0.5, 2 and 24 hours).
- OGD begins at -4 hours and subsequent reperfusion occurs from 0 hour.
- IFNo m NA levels increases at 2 hours reperfusion before returning to basal levels.
- ⁇ ⁇ and IL- ⁇ mR A levels only elevate at 24 hours reperfusion.
- TNFa mRNA levels show immediate increase at 0 and 0.5 hours reperfusion before returning to control levels.
- IL-6 mRNA levels are elevated at 0.5 and 2 hours reperfusion, and then return to basal levels.
- FIG. 5 is a graphical representation showing Ml 7 cells transfected with IFNARl-GFP and IFNAR2-GFP ovorexpression plasmids and subjected to 3 hours OGD and 24 hours reperfusion.
- M 17-IFNAR1 cells demonstrated significant decrease in survival (21.6 ⁇ 5.3%) compared to wild-type (44,9 ⁇ 2.1%).
- Figure 6 is a representation of the IFNARl, AttBl and AttB2 sequences used in Gateway (Registered Trademark) cloning.
- Figure 7 is a representation of the IFNAR2, AttBl and AttB2 sequences used in Gateway (Registered Trademark) cloning.
- Figure 8 is a representation of SYBRGreen nucleotide sequences.
- FIGS 9A through C are photographic representations of fluorescent immunohistochemistry showing IFNARl localization on primary cultured mouse neurons.
- Panel A Map-2 Panel B NeuN Panel C-Mcrge.
- Map2 is a neuronal marker indicating microtubule associated proteins.
- Figure 10 is a graphical and photographic representation of IFNAR " ' " (knock out; KO) mice (C57BL6) show reduced infarct size in a stroke model.
- the model comprises 2 hours of mid-cerebral artery occlusion followed by 24 hours of reperfusion.
- IFNARl " ' " mice show a reduced infarct size.
- Figure 11 is a graphical representation showing that IFNARl mAb reduces infarct size in a middle cerebral artery occlusion (MCAO) stroke model. An amount of 0.5mg mAb was given iv 1 hour prior to stroke. The infarct volume was assessed 24 hours post- injury. The monoclonal Ab (mAb) is MAR- 1.
- SEQ ID NO Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO).
- the SEQ ID NOs correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO:l), ⁇ 400>2 (SEQ ID NO:2), etc.
- a summary of the sequence identifiers is provided in Table 1.
- a sequence listing is provided after the claims.
- a neuroinflarnmatory condition includes a single neuroinflaramatory condition, as well as two or more neuroinflarnmatory conditions
- an antagonist includes a single antagonist, as well as two or more antagonists
- reference to “the disclosure” includes a single and multiple aspects described in the disclosure; and so forth. All aspects disclosed, describe and/or claimed herein arc encompassed by term “invention”. Such aspects are enabled across the width of the present invention.
- Type 1 interferons are a super-family of pleiotropic cytokines that induce pro-inflammatory gene transcription via the classical JAK/STAT pathway.
- the Type 1 IFNs include IFNa, ⁇ and IFNco.
- Certain events or conditions within the central nervous system (CNS) have the capacity to induce a ncuroinflammatory response having pathological consequences.
- CNS central nervous system
- Taught herein is the use of an antagonist of Type J IFN- mediated signalling within the CNS to attenuate an adverse neuropathological inflammatory response following a stroke event. The attenuation of a neuroinflarnmatory response leads to a reduction in secondary neuronal damage including neuronal necrosis and apoptosis.
- Reference herein to a "type 1 IFN-mediated signalling antagonist” means an antagonist of the activity of a Type 1 1FN, an inhibitor of Type 1 IFN gene expression or translation, an antagonist of a Type 1 IFN receptor activity, function or dimerization or a subunit thereof such as IFNARl , an inhibitor of Type 2 IFN receptor gene expression and an agent which blocks Type 1 IFN:receptor interaction or any agent which blocks, inhibits or otherwise reduces Type 1 IFN-mediated inflammatory signalling.
- An aspect enabled herein is a method for reducing a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IFN)-mcdiated signalling, for a time and under conditions sufficient to prevent Or attenuate neuroinflammation.
- CNS central nervous system
- a "stroke event” includes any ischemic condition in a blood vessel in the CNS including the brain as well as any blood fluid leakage such as following rupture of a blood vessel.
- the stroke event may be classified as minor-mild, mild-moderate, moderate, moderate-severe and severe-serious and can result from minor to significant impairment to a subject.
- Another aspect of the present disclosure contemplates a method for ameliorating symptoms of a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type I interferon (IFN)-mediated signalling, for a time and under conditions sufficient to prevent or attenuate neuroinflammation.
- CNS central nervous system
- IFN Type I interferon
- An aspect enabled herein is a method for reducing a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IFN)-mediated signalling, for a time and under conditions sufficient to prevent or attenuate neuroinflammation.
- a related embodiment provides a method for ameliorating the symptoms associated with a stroke event, the method comprising administering to the subject an effective amount of an antagonist of T pe 1 interferon (IFN)-mediated signalling, for a time and under conditions sufficient to reduce or attenuate a neuroinflammatory response within the CNS.
- the present disclosure teaches antagonists of Type 1 IFN-mediated signalling in the form of a compound, agent, chemical agent, pharmacologically agent, medicament, active and drug.
- the terms "compound”, “ agent”, “chemical agent”, “pharmacologically active agent”, “medicament”, “active” and “drug” are used interchangeably herein to refer to the Type 1 IFN-mediated signalling antagonist which Induces a desired pharmacological and/or physiological effect.
- the desired effect includes reducing Type I IFN-mediated signalling via the IFN or a receptor subunit such as IFNARl .
- the desired physiological effect includes attenuation of a neuropathological inflammatory response including the potential for such an inflammatory response to reach a neuropathological level.
- the terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, mimetics functional equivalents and the like.
- pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, mimetics functional equivalents and the like.
- Reference to the antagonist in these terms includes combinations of two or more actives.
- a "combination” also includes multi-part such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation. Examples include two separate agents wherein one agent down- regulates the activity or level of the Type 1 IFN and the other down-regulates the activity or level of a Type 1 IFN receptor subunit, and in particular IFNARl . Agents when in combination with an IFN or IFNAR antagonist may also target a down-stream component of the Type 1 IFN-mediated signalling cascade.
- the antagonists enabled herein provided systcmically cross the blood brain barrier (BBB). This is possible due to the chemical nature of the agent or due to a permcalization factor such as a co-factor fused to or formulated with the antagonist. Strategies for accessing the CNS are disclosed in Misra et al, JPharm Sci £252-273 , 2003.
- the "antagonist” includes a protein, polypeptide or peptide, small chemical molecule, antibody or derivative thereof including an immunoglobulin new antigen receptor (IgNAR) or a genetic molecule.
- IgNAR immunoglobulin new antigen receptor
- a "genetic molecule” is generally one which down-regulates expression of a gene encoding a Type 1 IFN or a Type 1 IFN receptor or a subunit thereof.
- the targeted subunit is IFNAR1.
- the genetic molecule includes an antisense molecule directed to all or part of a gene or mRNA encoding the Type 1 IFN or a subunit portion of its receptor such as IFNARl .
- the antisense is from about 5 nucleotides in length to 15 to 80 nucleotides in length or from about 5 nucleotides in length to full length of the mRNA transcript or 5' or 3' regions thereof.
- Other genetic molecules include sense molecules such as single- or double-stranded RNAs, RNAi and siRNA molecules, short and long RNA duplexes, ribozymes, DNAzymes, and any DNA or RNA or synthetic DNA or RNA agent which interferes with expression of the expression of the Type 1 IFN gene or of the gene encoding a receptor subunit such as IFNARl .
- the genetic molecule may be naked or expressed by a viral or other vector or introduced as part of a formulation.
- a number of viruses may be used as nucleic acid transfer vectors or as the basis for preparing nucleic acid transfer vectors to introduce a genetic agent to the CNS, including papovaviruses (e.g.
- adeno-associated virus uzyczka, Curr Top Microbiol Immunol 58:97-129, 1992; Ohi et al, Gene 5P:279-282, 1990; Russell and Hirata, Nat Genetics 75:323-328, 1998), herpesviruses including HSV and EBV (Margolskec, Curr Top Microbiol Immunol 158:67-95, 1992; Johnson et al, J Virol 06:2952-2965, 1992; Fink et al, Hum Gene Ther 3: 1-19, 1992; Ureakefield and Geller, Mol Neurobiol /:339-371, 1987; Freese et al, Biochem Pharmaco.
- Non-viral nucleic acid transfer methods include chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor- mediated DNA transfer.
- Viral-mediated nucleic acid transfer can be combined with direct in vivo nucleic acid transfer using liposome delivery, allowing one to direct the viral vectors to particular cells.
- the retroviral vector producer cell line can be injected into particular tissue. Injection of producer cells would then provide a continuous source of vector particles.
- igNAR immunoglobulin new antigen receptor
- IgNAR response is antigen-driven in the shark, and both immune and naive molecular libraries of IgNAR variable domains have been consuueted and successfully screened for antigen-specific binding reagents (Greenberg el al, 1995 supra; Nuttall et al, 2001 supra).
- IgNAR's are bivalent, but target antigen through a single immunoglobulin variable domain ( ⁇ 14kDa) displaying two complementarity determining region (CDR) loops attached to varying numbers of constant domains (Nuttall et al, Eur J Biochem 270:3543-3554, 2003; Roux et al, Proc Natl Acad Sci USA 95:11804-11809, 1998).
- Ig antibodies have a variable heavy (VH) + variable light (V,.) domain format ( ⁇ 26kDa) and bind antigen through up to six CDRs (Chothia et al.. Nature 342:877-883, 1989; Padlan, Mol Immunol 37:169-217, 1994).
- VH variable heavy
- V,. variable light domain format
- ⁇ s thermodynamic and chemical stability of IgNAR variable domains ( ⁇ s)
- the small V NA R size enables this unusual antibody domain access to cryptic antigenic epitopes through unusually long and variable CDR3 loops (Greenber et al., 1995 supra; Ewert et al., Biochemistry 4/:3628- 2636.
- IgNAR domains have been identified that recognize a variety of target antigens including: the apical membrane protein I (AMA-1) of P.
- the present disclosure teaches analogs and derivatives of a Type 1 IFN or an IFNAR such as IFNARl such as which include a modified side chain or which incorporate an unnatural amino acid and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or its analogs.
- This term also does not exclude modification of the glycosylation. acctylation and phosphorylation patterns. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid or polypeptides with substituted linkages. Such polypeptides may need to be able to enter the cell and/or cross the BBB or promote cerebral fluid half-life survival.
- Mimetics of the Type 1 IFN or IFNAR such as IFNARl are another useful group of agents to test for neuroprotective ability.
- the term is intended to refer to a substance which has some chemical similarity to the molecule it mimics and which acts as an antagonist.
- a peptide mimetic of a Type I IFN or IFNA 1 may be a peptide- containing molecule that mimics elements of protein secondary structure (Johnson ei al., Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzuto et al (Eds), Chapman and Hall, New York, 1993).
- peptide mimetics The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions such as with a receptor or ligand.
- a peptide mimetic therefore, is designed to permit molecular interactions similar to the natural molecule but block signalling.
- a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
- the template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
- the mimetic is peptide-based
- further stability can be achieved by cyclizing the peptide, increasing its rigidity.
- the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
- Non-human animals are contemplated such as in veterinary applications as well as for use in animal models.
- Reference to a mammal includes a mouse, rat, hamster, guinea pig, rabbit, pig, sheep, horse, goat, cow, camel and non-human primate (such as orangutan, gorilla, marmoset and a macaque).
- the subject is a human.
- Reference to a human includes a fetus, in uiero as well as a human of any age.
- the present disclosure teaches a method for reducing a neuroinflammatory response within the central nervous system (CNS) of a human subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IF )-mediated signalling, for a time and under conditions sufficient to prevent, reduce or attenuate neuroinflammation.
- IF interferon alpha receptor 1
- the present disclosure enables a method for reducing a neuroinflammatory response within the CNS of a subject following a stroke event, the method comprising administering to the subject an antagonist of interferon alpha receptor 1 (IFNA 1 )-mediated signalling for a time and under conditions sufficient to prevent or 5 ameliorate the symptoms of neuroinflammation.
- IFNA 1 interferon alpha receptor 1
- the present disclosure further describes a method for ameliorating the symptoms of a neuroinflammatory response within the CNS of a human subject following a stroke event the method comprising administering to the human subject an antagonist of interferon 1 alpha receptor 1 (IFNAR l)-mediated signalling for a time and under conditions sufficient to prevent or ameliorate the symptoms of neuroinflammation.
- IFNAR l interferon 1 alpha receptor 1
- an agent as used herein mean a sufficient amount of an agent (i.e. a Type 1 IFN-mediaied signalling
- I S antagonist to provide the desired therapeutic or physiological effect or outcome as indicated above.
- Undesirable effects e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount”.
- the exact amount required will vary from subject to subject, depending on the species, age and0 general condition of the subject, mode of administration and the like. T us, it may not be possible to specify an exact "effective amount”. However, an appropriate "effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation. In general, the amount is effective to attenuate a neuroinflammatory response within the CNS or to prevent or reduce the severity of a5 neuroinflammatory response developing or continuing.
- the prevention of a neuroinflammatory response developing is also useful in at risk subjects such as those with a genetic disposition or family history of strokes.
- the present disclosure teaches reducing, preventing or attenuating neuroinflammation anywhere in the CNS, including in the brain.
- the antagonist may be used as a therapeutic to treat a condition or as a preventative (i.e.0 prophylactically) to reduce the risk of neuroinflammation in anticipation of a stroke.
- the agent in the form of an antagonist may also be administered with a pharmaceutically acceptable carrier, excipient or diluent.
- a pharmaceutically acceptable carrier excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
- Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
- agent in the form of an antagonist may also be a pharmacologically acceptable form or derivative of a compound.
- pharmacologically acceptable means a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.
- treating and “treatment” as used herein refer to reduction in severity and/or f equency of symptoms of the condition being treated, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms of the condition and/or their underlying cause and improvement or remediation or amelioration of damage following a stroke event leading to or having the potential to lead a ncuroinflammatory response in the CNS.
- treatment may involve ameliorating symptoms of a neuroinflammatory response. Such symptoms may arise from neuronal necrosis, apoptosis, senescence or arrest, demyelination and/or axonal or neuronal degeneration. Amelioration of a downstream physiological, psychological or mental condition is also a useful indicator of treatment.
- Treating" a subject may involve prevention or reduction in extent of development of a condition or other adverse physiological or psychological event in a susceptible individual associated with a neuroinflammatory response as well as treatment of a clinically symptomatic individual by ameliorating the symptoms of the neuroinflammatory response.
- a "subject” as used herein refers to an animal, such as a mammal including a human who can benefit from the pharmaceutical agents and formulations and methods of the present disclosure.
- a subject regardless of whether a human or non-human animal may be referred to as an individual, patient, animal, host or recipient.
- the compounds and methods enabled herein have particular applications in human medicine.
- the neuroprotective agent enabled herein is an agent which down-rcgulatcs the extent or activity of Type 1 IFN-mediated signalling.
- Such an agent is referred to herein as a "Type 1 IFN signalling antagonist”.
- This includes an antagonist of a Type 1 IFN or a portion of the Type 1 IFN receptor and in particular 1FNAR.
- Reference to an "IFNAR" includes the IFNA receptor or a subunit thereof such as IFNARl.
- the present disclosure further teaches combinations of neuroprotective agents or neuroprotective formulations comprising a Type 1 IFN signalling antagonist and another neuroprotective agent such as leukemia inhibitory factor (LIF) or ciliary neurotrophic factor (CNTF) or a homolog, derivative, analog or mimetic thereof.
- a Type 1 IFN signalling antagonist such as leukemia inhibitory factor (LIF) or ciliary neurotrophic factor (CNTF) or a homolog, derivative, analog or mimetic thereof.
- LIF leukemia inhibitory factor
- CNTF ciliary neurotrophic factor
- the present disclosure teaches a method for the treatment or prophylaxis of a stroke event in a subject, the method comprising administering to the subject an effective amount of a Type 1 IFN signalling antagonist for a time and under conditions sufficient to ameliorate adverse neurological inflammation or prevent or reduce its progression, [0074J
- a method is provided for the treatment or prophylaxis of a stroke event in a subject or at least delaying onset of symptoms thereof, the method comprising administering to the subject an effective amount of a Type 1 IFN alpha receptor (IFNARl) antagonist for a time and under conditions sufficient to ameliorate adverse neurological inflammation or prevent or reduce its progression in the CNS.
- IFNARl Type 1 IFN alpha receptor
- the neuroprotective agent includes a Type 1 IFN antagonist or an IFNAR antagonist such as IFNARl antagonist.
- ITie antagonist may act at the level of protein activity or function or gene expression including transcription, translation or processing.
- the present disclosure teaches a method for ameliorating the symptoms of a ncuroinflammatory response following a stroke event in a subject or delaying development of symptoms thereof, the method comprising administering to the subject an effective amount of a neuroprotective formulation comprising a Type 1 IFN antagonist for a time and under conditions sufficient to reduce neurological inflammation or prevent or reduce its progression.
- Still another aspect taught herein to a method for ameliorating the symptoms of a neuroinflammatory response following a stroke event or delaying development of symptoms thereof comprising administering to the subject an effective amount of a neuroprotective formulation comprising an IFNAR1 antagonist for a time and under conditions sufficient to reduce neurological inflammation or prevent or reduce its progression.
- the amount or time sufficient to treat the neurodegenerative disease or condition may be the amount or time required to ameliorate one or more symptoms of the stroke event.
- a symptom includes a psychological or mental symptom.
- the antagonist may be provided with a pharmaceutically acceptable carrier, excipicnt or diluent. ' I " he antagonist itself is considered to be pharmacologically acceptable.
- the Type 1 IFN-mcdiatcd signalling antagonist may also be provided in combination with another neuroprotective agent such as LIF and or CNTF or their homologs, derivatives, analogs or mimctics.
- the present disclosure enables a method for the treatment or prophylaxis of a neuropathological event, disease or condition in a subject selected irom a stroke event, the method comprising administering to the subject an effective amount of a neuroprotective formulation comprising a Type 1 IFN-mediated signalling antagonist and . onc or both of LIF and CNTF or a homolog, derivative, analog or mimetic thereof for a time and under conditions sufficient to reduce neurological inflammation or prevent or reduce its progression.
- a neuroprotective formulation comprising a Type 1 IFN-mediated signalling antagonist and . onc or both of LIF and CNTF or a homolog, derivative, analog or mimetic thereof for a time and under conditions sufficient to reduce neurological inflammation or prevent or reduce its progression.
- compositions taught herein may be administered in a number of ways depending upon whether local or systemic treatment as desired including with means for the agent to cross the BBB.
- Administration includes intravenous, mtra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, e.g., intrathecal or intraventricular, administration; or oral administration; or via a spinal tap.
- Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
- the formulation needs to enable the agent to cross the BBB.
- the agent itself may need to be modified.
- the formulation may enable retrograde transport.
- the agents may also be specifically targeted to the brain or other parts of the CNS.
- compositions described herein may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s).
- compositions described herein may be formulated into any of many possible dosage forms such as, but not limited to, injectable formulations, and tablets, capsules, gel capsules and liquids.
- compositions herein include, hut are not limited to, solutions, emulsions, foams and liposome-containing formulations.
- the pharmaceutical compositions and formulations herein described may comprise one or more penetration enhancers, carriers, excipicnts or other active or inactive ingredients.
- Emulsions are typically heterogeneous systems of ne liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment taught herein.
- Liposome means a vesicle composed of amphophilic lipids arranged in a spherical bilayer or bi layers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed Scorn a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitivc or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
- Liposomes also include "stericaily stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
- stericaily stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is dcrivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
- PEG polyethylene glycol
- various penetration enhancers may be employed to effect the efficient delivery of nucleic acids.
- penetration enhancers also enhance the permeability of lipophilic drugs.
- Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants.
- formulations are routinely designed according to their intended use, i.e. route of administration. [0090] The formulation of therapeutic compositions and their subsequent administration (dosing) are within the skill of those in the art.
- Dosing is dependent n severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
- Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
- Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on ECjos found to be effective in vitro and in vivo animal models. In general, dosage is from 0.01 to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years.
- oligonucleotide is administered in maintenance doses, ranging from 0.01 ⁇ g to 100 g per kg of body weight, once or more daily, to once every 20 years.
- the antagonists are provided in amounts of 1 , 10, 100 or 1000 ⁇ g ml or amounts in between.
- the present disclosure teaches a neuroprotective formulation comprising a Type 1 IFN-mcdiated signalling antagonist and one or more pharmaceutically acceptable carriers and or diluents.
- the neuroprotective formulation is provided to patients or at risk subjects of stroke.
- the present disclosure teaches a neuroprotective formulation comprising a Type 1 IFN-mediated signalling antagonist and one or both of LIF and/or CNTF and one or more pharmaceutically acceptable carriers and/or diluents.
- the neuroprotective formulation is provided to patients or at risk subjects of stroke.
- the level of Type 1 IFN-mediated signalling may be determined such as via the level of a Type 1 IFN, its corresponding mR A levels, activity of IFNAR1 or its corresponding mRNA levels or via a down-stream effector such as T Fa, IL-6 or a chemokine or other pro-inflammatory effector molecule.
- a medical protocol is enabled herein to treat a subject which has or may experience a neuropathological event, disease or condition selected from a stroke event, the protocol including:
- Inflammatory parameters may be detected by any means including HPLC, TLC, ELISA, RIA, immuno-fluorescent assay, Southern analysis,
- Another aspect of the present disclosure enables a medical protocol for treating acute neuronal injury in the form of a stroke event in a subject, the protocol comprising administering to the subject, within from 1 to 120 minutes of the injury, a neuroprotective formulation comprising the antagonist of Type 1 interferon-mediated signalling.
- a neuroprotective formulation comprising the antagonist of Type 1 interferon-mediated signalling.
- 1 to 120 include 1, 2, 3, 4, 5, 6, 7, 8, 10. 1 1. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25. 26, 27, 28, 29. 30, 31, 32, 33. 34. 35, 36. 37, 38, 39, 40, 41 , 42, 43, 44, 45. 46, 47, 48, 49, 50.
- mice (eight-week-old male mice 23 ⁇ 3 g) used were of a C57BI ⁇ -6 strain with -FNARl ⁇ (knockout or KO) or IFNARl ⁇ (wild-type or wt) genotypes (Hwang et a Proc Natl Acad Sci USA 92(24) ⁇ 1284-11288, 1995).
- Pregnant female mice were killed by cervical dislocation when embryos were 14 days old.
- the embryos were extracted from the female uterus and their heads removed into a petridish of Solution 1 (30ml Krebs stock (3625g NaCl, 2g KCl, 0.7g NaH 2 P0 4 .H 2 0, I3g d-Glucose, O.OSg Phenol red, 20.7g HEPES (acid form) in 500ml 3 ⁇ 40, pH7.4), 270ml H 2 0, 0.9g bovine serum albumin (BSA), 2.4ml 3.4% w/v MgS0 4 , pH 7.4).
- BSA bovine serum albumin
- the brains were removed, cerebral cortices were isolated and the meninges removed.
- the cortices were placed into a fresh petri dish of solution 1 and minced to a fine paste.
- the paste was transferred to a 50ml Falcon tube containing solution 2 (15ml sol 1, 1.5ml lOx trypsin (25mg trypsin in 10ml)) using a pasture pipette.
- the tube was incubated for 15-20 minutes at 37*0 with occasional mixing by inversion.
- 12.5ml of Solution 4 (8.5ml Sol 1, 4ml Sol 3) was added to the digested tissue.
- the tissue was then centrifuged (lOOOrpm, 45 seconds), supernatant discarded and pellet resuspended in solution 3 (7.5ml Sol 1, 0.75ml lOx DNase soybean trypsin inhibitor (SBT1) stock (8mg DNase, 52mg SBT1 in 10ml), 75 ⁇ . 3.4% w v MgSOi) by passing through a pasture pipette for 2 minutes, the solution was then transferred to a fresh 50ml Falcon tube. Remaining tissue was washed from the old tube with 2 pasture pipettes of solution 5 (6.25ml Sol 1, 50 ⁇ , MgS0 4 stock, 7.5 ⁇ 1.2% w/v C Cy.
- SBT1 DNase soybean trypsin inhibitor
- neuronal culture medium Ncurobasal media containing 10ml B27 supplement (Gibco), 1.25ml 200mM 1-glutamine and 0.5ml Oentaroycin (Oibco) in 500ml
- ARAC 5-chlorocytosine arabinoside
- Ml 7 cells (ATCC [Registered Trademark] number: CRL-2267 [Trademark]) were cultured in T75 flasks with culture medium (OptiMEM (Gibco), 5% v/v FBS, 0.5% w/v Penicillin-Streptomycin (Gibco)) at 37°C until 90% confluent. Cells were then plated at the densities listed in Table 3. For the Alzheimer's disease studies, M17 cells were housed in an incubator at 37°C and 5% v/v CO, in OPTI- EN (Invitrogen) in 10% v/ FBS and 1% w/v penicillin/streptomycin.
- OptiMEM Gibco
- 5% v/v FBS 0.5% w/v Penicillin-Streptomycin
- a 14 day pregnant mouse was killed by cervical dislocation and uterine horns were removed and placed in a 10cm dish containing working solution l(For 300ml: 30ml Krebs lOx stock, 270ml MUliQ, 0.9g BSA, 2.4ml 3.82% w/v MgSO*).
- Fetuses were removed from placental sacks, decapitated and heads were placed in a new dish containing solution 1. Under a dissection microscope skull and meninges were removed and cortical tissue was placed in a dish and chopped finely with a scalpel. Flamed Pasteur pipettes were then used to separate tissue into tubes using 20ml solution 1 with 10% w/v trypsin added.
- cell pellet was resuspended in 10ml of Dulbecco's Modified Eagle Medium (Invitrogen) with 10% v/v FBS. The suspension was placed in a T-75 flask and allowed to incubate at 37°C for 1 hour to allow fibroblast adhesion. Cell media containing only neurons was then removed from the flask, cell density determined, and plated into appropriate poly-L-lysine coated plates and kept in an incubator (37 e C and 5% v/v CX1 ⁇ 2). 4 hours following plating Neurons are given a full media change to DMEM with 10% FBS and 0.5% w/v penicilin/streptomycin.
- Dulbecco's Modified Eagle Medium Invitrogen
- Oxygen Glucose Deprivation OGD
- IFNaffFNfi cytokine treatment 6cm dishes of M17 cells were treated with human (hu) lFNcc (huIFNa) and human IFNp (huIF p) at 1, 10, 100 or lOOOU/ml for a time course of 5, 10 or 30 minutes whilst incubated at 37°C. Post-treatment harvested for Western blot analysis. SDS PAGE gel protein separation and western blotting
- PVDF Polyvinylidene fluoride
- Membranes were blocked with 5% w/v skim milk powder in TBS-T (lOmM Tris, 15mM NaCI, 0.01% v v Tween-20) for 1 hour.
- Phospho-STAT-1, Phospho-STAT-3 (Cell Signalling) and 0-tubulin (Millipore) antibodies were diluted 1:1000 in 5% w/v BSA in TBS-T and incubated on membranes at 4 D C overnight.
- TR oi (Registered Trademark) mRNA extraction
- RNA pellet was air-dried and redissolved in
- RNAasc free water was then analyzed by the nanodrop 1000 spectrophotometer (Thermo-scientific).
- Tumor Necrosis Factor a (TNFa, HsG0174l28_m I), Interleukin- ip (IL-lp ⁇ Hs00174097_ml), Interleukin-6 (IL-6, Hs00985639_ml), Interferon-al (IFNal, Hs00256882_sl), Interferon- ⁇ (IFNpl, HsOl 077958_sl) and 18s ribosomal RNA (18s rRNA, 4352930E).
- RT-PCR was performed in a 384-well plate with 4 ⁇ diluted cDNA, 0.5 ⁇ DEPC dH 2 0, 0.5 ⁇ Taqman primer, 5 ⁇ . Taqman Fast Universal PCR master mix (2x) per well. Each sample was repeated in triplicate and RT-PCR performed using the 7900ht Fast Real-Time PCR system (Applied Biosystems) under the following conditions: 50°C for 2 minutes, 94.5°C for 10 minutes, (97°C for 30 seconds, 59.7°C for 1 minute)x 40 repeats. Data were collated and fold change calculated using the AACt method.
- RNA samples were diluted 1 :10 in RNAse free H 2 0.
- the following Taqman primers were obtained from Applied Biosciences: TNFa (ID: Mm00443258_ml), ⁇ (ID: m00439552_sl), IL- ⁇ ⁇ (ID: Mm0l336l 89_ml) and 18S rRNA (ID: 4352930E), Taqman primers were incubated along with sample in a 384 well plate (MicroAmp, Singapore) in the following ratios: 0.5 ⁇ 1 primer, 0.5 ⁇ H 2 0, 5 ⁇ 1 2x Taqman Mix (Invitrogen) and 4 ⁇ 1 diluted cDNA.
- SybrGreen primers were obtained from Genc orks (sequences are listed in Figure 8): Allalpha forward, Allalpha Reverse 1, Allalpha Reverse 2, OADPH forward and GADPH reverse. SybrGreen primers were incubated in the following ratios: 2 ⁇ 1 diluted cDNA, 5ul Fast SybrGreen Master Mix, ⁇ Forward GADPH primer, ⁇ Reverse GADPH primer and ⁇ 3 ⁇ 40. The plate was incubated according to the following conditions and read using the 7900ht spectrophotometer (Applied Biosciences): 95X for 20 minutes, 95°C for 3 minutes (40 repeats), 60°C for 30 minutes, 95°C for 15 minutes, 60°C for 15 minutes and 95°C for 15 minutes. Ct values were obtained for each sample, and relative transcript levels for each gene were calculated using the SSCT method. MTT eell viability assay
- RNA to cDNA Conversion of RNA to cDNA involved the use of a High Capacity RNA to cDNA kit (Applied Biosystems) as per manufacturers instructions. Briefly this involved loading 1 ug of RNA with 1 ⁇ 2X reaction mix with 1 ⁇ RT enzyme mix and DEPC H 2 0 to 2 ⁇ 1. Tube was then heated at 35°C for 30 minutes, and 95°C for 5 minutes (termination step) and chilled on ice.
- Relative expression levels of R.NA were measured using the prepared cDNA in accordance with the TaqMan (Registered Trademark) [Applied Biosystems] primer preparation. This involves adding in triplicate 4 ⁇ 1 cDNA with 5 ⁇ 1 2X TaqMan Mix, 0.5 ⁇ DECP H2O, and 0.5 ⁇ of primer of raRNA in question. PCR plate was then read on a 7900ht Fast Real-Time PCR System. Fold increase was determined by using the delta-delta method.
- cDNA encoding mouse interferon receptor a 1 (mIFNARl) and mouse interferon receptor a 2 (IFNAR2) were custom synthesized and inserted into vector puc57 by GeneScript (U.S.A.).
- Subclone forward primers containing AttBl motif and subclone reverse primers containing AttB2 motif were designed and synthesized (Geneworks) for both mIFNARl and mtFNAR2 genes to use with the Gateway (Registered Trademark) cloning system (Invitrogen). These primers were ligated to the mIFNARl and mIFNAR2 genes via PCR using GoTaq (Registered Trademark) polymerase (Promega). Nucleotide sequences of IFNAR1 & 2 and AttBl and AttB2 sequences are shown in Figures 6 and 7.
- the gel containing AttBi-mIFNARl/2-AttB2 was rcdissolved and DNA was retained on a silicon filter whilst impurities were discarded. Post-washing with ethanol buffer the purified DNA was then dissolved in elurion buffer (l0mM Tris-HCL, pH8.5).
- the Invitrogen Gateway (Registered Trademark) cloning system was used.
- the purified AttB-mlFNARl and AttB-mIFNAR2 PCR products were translocated into vector pDONR20l (Invitrogen) using the BP Clonase (Trademark) Q enzyme kit (Invitrogen), according to manufacturers guidelines.
- a ⁇ 0 ⁇ xh reaction comprised of 0.6uL pDONR20l (289ng ⁇ L), 1.8 ⁇ AttB-mlFNARl PCR product (33n / ⁇ L) or 1.5 ⁇ .
- AttB-mlFNAR2 PCR product 37ng L
- 2 ⁇ BP Clonase Trademark
- I en2yme mix TE buffer to ⁇ .
- the reaction was incubated for 24 hours at 25°C and subsequently terminated upon addition of Proteinase K ( ⁇ ⁇ ,
- the BP reaction product was transformed into DHSa competent E. coli cells (Invitrogen) by adding 1 1 exert BP reaction product to each DI I5a aliquot (50 ⁇ . ⁇ «). Cells were then incubated on ice for 30 minutes and heat-shocked by incubation at 42°C for 30 seconds. 250 ⁇ / ⁇ of S.O.C.
- PCR was performed using the conditions above to confirm successful mIFNARl 2 insertion into pDONR20l and the subsequent DI-I5a colony.
- the PCR products were then electrophoresized on a 1% w/v agarose gel and 1.77kb (for mIFNA l) or 1.54kb (for mIFNAR2) bands were visualized using the IQ-350.
- the mIFNARl 2 genes were removed from the pDONR201-raIFNARl/2 plasmid and translocated into a pcDNA6.2 cEM-GFP destination vector using the LR Clonase (Trademark) II kit (Invitrogen) as per manufacturers protocol.
- a lOpL reaction consisted of ] ⁇ pDONR- mIFNARl (I 22.6ng/ L) or ⁇ ⁇ pDONR-mIFNAR2 (156.9ng L) entry clones, 2 ⁇ L ⁇ pcDNA6.2/cEM-GFP (75ng/pL) destination vector, 2 ⁇ , LR Clonase (Tradeamrk), TE buffer to l0 L.
- reaction samples were then transformed into DH5a competent E. coli cells as per above.
- a colony was selected from an ampicillin (50mg/rnl) agar plate and grown in ⁇ LB media of which 5 ⁇ . was lysed by heating (99°C, 5 minutes).
- PCR was then performed under the conditions described above and samples run on a 1% w/v agarose gel. Imaging the gel using the IQ-350 showed a 1.77kb mIFNARl specific or a 1.54kb mJFNAR2 specific band for successful LR reaction and transformation samples. Positive colonies were then selected and shaken in 250ml ampicillin (50 ⁇ 1) selective LB media for 16 hours (225rpm, 37°C).
- Bacterial plasmid DNA was isolated using EndoFree (Registered Trademark) plasmid Maxi Kit (QIAGEN) following manufacturers protocol. Briefly, bacteria were lysed, excess protein precipitated and DNA retained in the filter of a gravity flow column. After subsequent washing with an ethanol buffer DNA was eluted from the gravity flow column. The DNA was then precipitated using isopropanol, washed with endotoxin-free ethanol and resuspended in supplied TE buffer. DNA yields of pcDNA6.2/cEM-GFP- mIFNARl 2 plasmids were then determined using the nanodrop 1000.
- mice were transcardially perfused at various time points after injury (or sham surgery) with 0.1% v/v heparinized Phosphate-Buffered Saline (Pfizer), followed by 4% v v paraformaldehyde (Scharlab S.L.), and their brains dissected. Brains were cut by the Histology facility, University of Melbourne. Brain sections were cut into ⁇ coronal sections starting at the rostral end, paraffin-embedded and mounted onto glass slides. Every 10* slide was stained with Haematoxylin and Eosin (H&E).
- H&E Haematoxylin and Eosin
- Infarct volume analysis was conducted on slides, which had been stained with H&E. Analysis was performed using Image J. Area of infarct was calculated by measuring around regions appearing less intensely stained for viable cells. The average area of infarct per brain was then multiplied by the number of sections per slide by the thickness of each section (1 ⁇ ) and the number of slides in the sequence to get the volume of infarct. Immunohistochemistry
- Paraffin-embedded sections were put in a 60°C oven for 20 minutes and taken 5 through a series of Histolenc (Lornb Scientific) incubations, followed by 100, 95 and 70% cthanol. Sections were incubated in Phosphate-Buffered Saline for a further 5 minutes. Sections were blocked firstly for endogenous peroxidase activity with a Peroxidase blocking solution (DA O). followed by a protein-blocking buffer (5% v/v whole Goat serum [Invitrogen] and Triton X-100 solution [Sigma] in lx Phosphate Buffered Saline)
- DA O Peroxidase blocking solution
- Fluorescent secondary antibodies Alexa Fluor 594 anti-mouse and rabbit, Alexa Fluor 488 anti-mouse, rabbit and rat
- Biotinylated secondary antibody Anti-Mouse IgG, horse biotinylated
- Biotinylated primary antibodies were visualized using the Vector Vecstain ABC kit0 (Vector Laboratories) using Oiamtnobenzidene (DAB [DAKO]) as a substrate.
- Wild-type mice were intra-venously injected with IgG Isotype control (25mg kg) or MARl antibody (25mg kg)prior to and subsequently to subjecting the mice to a stroke5 event.
- Mice were initially anacstheti-3 ⁇ 4d with approximately 3% v/v Isoflurane in a 1: 1 mixture of medical grade air and oxygen. Anesthesia was maintained throughout scanning with 0.25-1.5% v/v Isoflurane through a nosecone placed over the animal's snout and respiration was continuously monitored throughout the experiment with a pressure sensitive probe positioned over the animal's diaphragm.
- Anaesthetized animals were laid0 supinely on a purpose built animal holder and their head fixed into position with ear and bite bars.
- a surface receive coil was placed over the animals head and the cradle was inserted into a transmit coil fixed inside a BGA12S gradient set for imaging with a 4.7 Tesla Broker Biospcc 47/30 scanner.
- the scanning protocol consisted of a 3-plane localizer sequence followed by multi-slice axial, coronal and sagittal scout images to ascertain the orientation and position of the brain.
- the effective echo times (TEeff) were: 45 ms, 60 ms and 80 ms.
- MTS Reagent (Promega) was prepared according to manufacturer's instructions. Briefly, this involved 42mg of MTS Reagent powder dissolved in 1 ml Dulbecco's PBS solution, pH to 6.0-6.5 and filter sterilized. For viability assays, 20 parts MTS solution was combined with 1 part phenazine methosulfate (PMS, Sigma) and 1/5 final well volume was added to cell wells in triplicate (i.e. 80 ⁇ MTS PMS in 400uJ well). MTS well preparation was then allowed to incubate for 2 hours at 37*C and absorbance was measured at 490nm. A Students T-Tcst was used to test statistical significance using Graphpad Prism 5 Software.
- the 12% w v resolving gel (8ml 30% w/v Acrylamide, 5ml 1.5M Tris, 200 ⁇ 10% w/v SDS, 6.68ml mQHjO, 200 ⁇ 1 10% APS, 20 ⁇ 1 TEMED) was prepared days in advance with the 5% w v stacking gel (1.67ml 30% w/v Acrylamide, 2.5ml 0.5M Tris, ⁇ ! 10% w/v SDS, 5.67ml mQH 2 0, ⁇ 10% w/v APS, 1 ⁇ TEMED) poured the day the gel was run.
- IFN is known to induce STAT phosphorylation governed by the JAK-STAT pathway.
- Ml 7 cells were treated with increasing concentrations of IFNa and IFNp (1, 10, 100 and lOOOU/ml) for 15 minutes and harvested for Western blot analysis.
- IFNa and IFNp (1, 10, 100 and lOOOU/ml) for 15 minutes and harvested for Western blot analysis.
- lOOOU/ml of IFNa induced the most prominent phosphorylation of STAT-1 in contrast to IFNp treatment which induced similar levels of STAT-1 phosphorylation across all concentrations.
- IFNa preferentially induces P-STA T-l not PSTA T-3
- IFNp also stimulated STAT-3 phosphorylation at all time-points but the more robust responses came earlier than that of P-STAT-1 induction (5 and 10 minutes treatment). Interestingly, IFNa treatment was causative of only minimal STAT-3 phosphorylation at 10 minutes compared to the robust P-STAT-I induction (IFNa, 10 minutes).
- IFNamRNA levels are elevated earlier in OGD reperfusion than IFNfimRNA levels
- IFNa mRNA levels were significantly increased at 2 hours reperrusion (15.1 ⁇ 2.3 fold change) before returning to basal levels by 24 hours.
- TNF-a and IL-10 were also analyzed via RT-PCR to confirm the inflammatory state created by OGD treatment.
- IL-6 mRNA levels were significantly increased at 0.5 and 2 hours reperfusion (266.9 ⁇ 10.9 and l£>4.6 ⁇ 39.4 fold change, respectively) before returning to basal levels by 24 hours reperfusion.
- TNF-a mRNA levels were significantly elevated at an earlier time-course of 0 and 0.5 hours reperfusion (10.0 ⁇ 3.1 and 10.1 ⁇ 2.8 fold change respectively) and returned to control levels by 2 hours reperfusion.
- Figure 4 shows a summary of collated RT-PCR data.
- Ml 7 cells were subjected to 3 hours OGD and 24 hours reperfusion. MTT assay was then used to assess cell viability.
- M17-1FNAR1 cells showed decreased viability (21.6 ⁇ 5.3%) in comparison to wild-type (44. ⁇ 2.1%).
- IFNAR1 is both membrane-bound and intra-nuclear in Wl neurons
- IFNAR1 and IFNA 2 over-expression in terms of cell viability IFNAR1 cellular trafficking was briefly investigated. Isolated CBL57/6 WT neurons were used for immunohistochemistry probed with mAb-IF ARl. Fluorescence microscopy shows membrane-bound, but more interestingly, intra-nuclear localization of the lWARl subunit.
- Magnetic Resonance Imaging (0142) Both procedures represent traditional methods used to assess neuronal damage following stoke.
- Magnetic Resonance Imaging (MRI) is a particularly powerful approach used to n l ze brain damage, as it allows an in vivo assessment of the progression of oedema and injury, done in real time. MRI scans were done on mice, 2 and then 24 hours after a stroke event. T2 images show the diffusion of water inside tissue, and are thus ideal for visualization of tissue oedema. The development of the infarct and penumbra (surrounding area of damage) is shown, with the si2e of the infarct and penumbra increasing drastically after 24 hours.
- Diffusion Weighted Imaging allowed an assessment of the movement of water molecules within injured and uninjured tissue.
- An ADC map of two sequential slices is taken of an injured brain, showing both infarct and penumbra size, in voxels.
- a T2 map is generated by T2-weighted Imaging of the same brain, showing again infarct and penumbra size. The two types of sequence give different values for infarct and penumbra size.
- IFNARl ⁇ mice underwent mid cerebral artery occlusion (MCAO) surgery.
- Western blot and immmiohistochemistry showed alterations in the Stat-1 and 3 phosphorylation profiles in the IFNARl ⁇ " .
- Neuroprotection conferred by the absence of IFN signalling was confirmed in IFNAR-deficient primary cultures that were protected from cell death when exposed to oxygen glucose deprivation (0GD).
- IFNARl localization (0146J Figures 9A through C show by fluorescent irnmunohistochemistr that IFNAR1 is localized on primary cultured mouse neurons.
- IFNAR 1 ' ' (KO) CS7 BL6 mice exhibited reduced infarct si2e in this stroke model.
- the infarct size was assessed 24 hours post injury in wild-type (IFNAR +/* ) and IFNAR " ' " C57 BL6 mice.
- Figure 11 shows that a mAb specific for IFNARl (IFNARl mAb) reduces infarct size when 0.5mg mAb is given iv 1 hour prior to the stroke.
- the infarct volume is assessed at 24 hours post-injury. This further shows the utility of an IFNARl antagonist (e.g.
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Abstract
The present disclosure relates to the amelioration of symptoms associated with a neuropathological inflammatory response component of a stroke event within the central nervous system (CNS) in a subject. Pharmaceutical formulations comprising antagonists of Type 1 interferon-mediated signalling or antagonists of interferon alpha receptor 1 mediated signalling; and methods and medical protocols for reducing neuroinflammation following a stroke are disclosed.
Description
METHODS AND COMPOSITIONS COMPRISING ANTAGONISTS OF TYPE 1 INTERFERON-MEDIATED SIGNALLING FOR REDUCING A NEUROINFLAMMATORY RESPONSE IN THE CENTRAL NERVOUS
SYSTEM FOLLOWING A STROKE
FILING DATA fOOOl) This application is associated with Australian Patent Application No. 2010905239 filed 26 November 2010 and Australian Patent Application No. 201 1902499, filed 24 June 201 1, the entire contents of both of which, are incorporated herein by reference.
FIELD
[0002] The present disclosure relates generally to the amelioration of symptoms associated with a neuropathology inflammatory response component of a stroke event within the central nervous system (CNS) in a subject. Agents, medicaments and pharmaceutical compositions useful in the management of stroke are also enabled herein as are diagnostic assays.
BACKGROUND
(0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
(0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
[0005] The immune system and the central nervous system (CNS) have long been considered separate systems with limited interaction. However, recent findings have shown support for a potential relationship between the two systems. This occurrence has been most evident in situations of neural injury, specifically ischemia-reperfuston pathologies (Zierath ei al, Ne rocrit Care 72^:274-284, 2010). The widely accepted view was that the CNS is immune-privileged as the blood brain barrier (BBB) prevents communication
with peripheral immune cells (such as macrophages and lymphocytes). This view is now being rc-shapcd with findings of CNS and immune system communication in neural injury without the disruption of the BBB (Kaur and Ling, Curr Neurovasc Res 5(7,1:71-81, 2008). The mechanism of this CNS neuroinflammation is unknown, thus posing a new approach to investigate neuropathologies demonstrating distinct neuroinflammation.
[0006) Inflammation is a complex biological response initiated to remove a foreign pathogen from a given host. It is characterized by initial pathogen recognition, subsequent tissue oedema and multiple infiltrating cell types. Most notably, macrophages are recruited to ingest and remove foreign matter. The pathogens responsible can be venoms, parasites, allergens and DNA/RNA fragments released from necrotic cells. Initially, this immune response is protective, removing the pathogen, but an exaggerated response can promote detrimental overcompensation. This results from excess differentiation resulting in hematopoietic cell infiltration and release of chemokines and cytokines. These cells adhere to endothelium through intracellular adhesion molecule- 1 OCAM-l) and infiltrate the inflamed tissue (Caprio et al, Ore Res Jft∑Y7/ :1359-l367, 2008). Although this is considered a peripheral event, this is also relevant to many neuropathologies including AlJtheimer's disease (Akiyama et al, leurobmol Aging 2/(^:383-421, 2000; Rogers et al. Gtia 40(2):26Q-269, 2002) which exhibit neuroinflammation within the CNS.
[0007] CNS ischemia-reperfusion injuries show characteristic macrophage and leukocyte recruitment resulting in production of various cytokines; however, the response is primarily initiated by activated resident microglia (Banati et al.. GUa 7(1):\ 11-118, 1993; Giulian and Vaca., Stroke 24(12 Si<Rp/J:184-1 0, 1993). Amongst others, mRNA and chaperone proteins released from necrotic cells, act as ligands for Toll-like receptors (TLRs) [Kariko el al. J Biol Chem 27Of/3;:l 2542-l 2550, 2004; Ohashi et al. J Immunol l64(2):55%-56\, 2000]. Activated microglia secrete pro-inflammatory cytokines including TNFcc and interleukins (Block et al, Neuronport 11(5) 963-967, 2000; Lambertsen et al. J Cereb Blood Flow Metab 25(1): 119-145, 2005) alongside chemo-attractants (Shohami et al. JNeruochem 53:1541-1546, 1989). The CCL2/3 chemokines lead to the recruitment of macrophages (Cowell et al. Stroke 33(3).795-Z01, 2002), further exacerbating the inflammatory response to the initial ischemia. Upon macrophage infiltration, a
characteristic cytokine 'storm' develops, which contributes to neurodcgcneration.
(00081 The inflammatory component of a brain injury contributes to widespread cellular damage in the brain (Grcve et al., Mt Sinai J Med 75:97-104, 2009). Studies of gene expression changes between control and traumatized brain samples revealed that genes playing a role in inflammation were up-regulated at an early stage after head injury (Crack et al., J Neural Transm 116Λ-Μ, 2009). The up-regulation of these genes suggests they play a major role in the secondary injury processes after ischemia-reperfusion injury. (0009J The Type 1 interferons (IFNa, IFNp and ΙΡΝω) play a role in the production of pro-inflammatory cytokines and chemokines (Schindler et al.t J Biol Chem 282:20059- 20063, 2007). Studies have shown an up-regulation in Type 1 interferon gene expression as a result of microglial activation (Field et al.. Brain Behav Immuti 2 996-1007, 2010). Interferons signal through interferon receptors, located widely on cells, including microglia and other immune cells (de Weerd et al., J Biol Chem 252:20053-20057, 2007).
(0010] The Type 1 IFN receptor, the IFNa receptor (IFNAR), is composed of IFNARl and IFNAR2 subunits. {0011 ] Upon binding of Type 1 IFN proteins, the IFNARl and DFNAR2 subunits dimeri2e, activating the receptor-bound kinases JAK1 and Tyk2 (Schindler et a!., 2007 supra). Activation of these proteins recruits Signal Transducers and Activators of Transcription (STAT) proteins, and consequently, STATs arc phosphorylated and activated by JAK1 and Tyk2. STATi and STAT2 dimerize and form a complex with an intracellular protein, Intetferon Regulatory Factor 9 (1RF9) [Leung et al, Mai Cell Biol 5:1312-1317, 1995], This complex translocates to the nucleus, acting as a transcription factor, and up-rcgulates the expression of anti-viral and anti-proliferative proteins, including pro-inflammatory cytokines (Lloyd et al., J Newainflammalion 5:28, 2008; Wei et al., J Neuroinflammation 6: 19, 2009).
(0012] The effects of certain treatments in reducing the neuroinflammatory cascade have been studied. For instance, one study investigated the effects of a small molecule inhibitor,
Minozac, on the induction of pro-inflammatory cytokines after a traumatic brain injury (TBI) [Lloyd et al., 2008 supra]. The exact mechanism of action of Minozac is not known, but it is believed to reduce the overproduction of pro-inflammatory cytokines in microglia post-injury, which was seen in this study. In addition to this, in an animal model of epilepsy, administration of Minozac prevented the chronic activation of microglia, which was linked to neurodegeneration in the long term (Somera-Molina ei al., Epilepsia 45:1785-1800, 2007).
[0013] Another study looked at the synthetic tripeptide analog (Glyprornate) [NNZ-2566] which was found to attenuate the increased expression in pro-inflammatory cytokine levels and attenuate neuropathological events following injury in a rat model of TBI (Wei el al., 2009 supra). Glyprornate is derived from Insulin-like Growth Factor (IGF-1 ) and is believed to induce repair of injured brain tissue. In this study, NNZ-2566 particularly suppressed expression of IL-Ιβ (which is thought to play a significant role in secondary neuronal apoptosis), thus conferring a level of neuroprotection to the tissue.
[0014) These studies illustrate the relevance of blocking certain aspects of the inflammatory response after head injury. However, whilst studies of drug therapies which targeted inflammation in TBI animal models have shown some benefit, when these therapies were put through clinical trials, they were not successful ( enon, Crii Care Med 37:5129-5135, 2009). This illustrates the complexity of this pathology.
[0015] There is, therefore, a need to design a therapy which can effectively attenuate secondary neuronal damage resulting from a neuroinflammatory response following stroke.
SUMMARY
[0016] It is determined herein that stroke leads to inflammation and to neuronal necrosis and apoptosis within the central nervous system (CNS). Such an inflammatory response is referred to herein as "neuroinflammation" which has pathological consequences. The present disclosure teaches a method for ameliorating symptoms of a neuropathological inflammatory response in the CNS and in particular the brain following a stroke event. The method enabled herein is predicated in part on antagonizing Type 1 interferon (TFN)- mediated signalling. The reduction or attenuation of a neuroinflammatory response reduces secondary neuronal damage after stroke.
[0017] Accordingly, an aspect enabled herein is a method for reducing a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IFN)-mcdiated signalling, for a lime and under conditions sufficient to prevent or attenuate neuroinflammation. By "reducing a neuroinflammatory response" includes "ameliorating the symptoms of a neuroinflammatory response". [0018] Another aspect enabled herein is a method for ameliorating the symptoms of a neuroinflammatory response within the central nervous system (CNS) of a subject following a neurological event or condition, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IFN)-mediated signalling, for a time and under conditions sufficient to prevent o attenuate newomflammation, wherein the neurological event or condition is a stroke event.
(0019] In an embodiment, the present disclosure teaches a method for reducing a neuroinflammatory response within the ecmral nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an antagonist of interferon alpha receptor I (IFNARl)-mediated signalling for a time and under conditions sufficient to prevent or ameliorate the symptoms of neuroinflammation.
[0020] Neuroprotective formulations comprising the Type 1 IFN-mediatcd signalling antagonist are also contemplated herein, as are diagnostic assays to screen for the level of Type J IFN-mcdiated signalling prior to or following a stroke in the CNS. Aspects disclosed herein are also applicable for the development of stroke protocols for patients who present with symptoms of such a condition.
[0021] The Type 1 IFN-mediatcd signalling antagonist may target a Type 1 IF or its receptor or a component thereof. A Type 1 IFN includes IFNa, ΙΡΝβ and IFNto. In an embodiment, the antagonist targets the IFNa receptor subunil (IFNARl) and/or the ability for IFNARl to dimerize with I NAR2. Combination therapy targeting a Type I IFN and IFNAR and optionally other components of the neuroinflamraatory cascade are also taught herein. The Type 1 IFN-mediated signalling antagonist may be used po$t-or pre- neurological event. Hence, it may be a therapeutic or prophylactic. [0022] The subject includes a higher order mammal including a human. A human subject may be of any age and includes a fetus.
[0023] Abbreviations used in the subject specification are defined in Table 2.
TABLE 1
Summary of sequence identifiers
SEQUENCE I» NO: DESCRIPTION
1 Nucleotide sequence of the IFNARl gene
2 Nucleotide sequence of the IFNA l AttB ] subclone
3 Nucleotide sequence of the IFNARl AttB2 subclone
4 Nucleotide sequence of the IFNAR2 ene
5 Nucleotide sequence of the IFNAR2 AttB 1 subolone
6 Nucleotide sequence of the IFNAR2 AttB2 subclone
7 Nucleotide sequence of the GADPH forward primer
8 Nucleotide sequence of the GADPH reverse primer
9 Nucleotide sequence of the Allalpha forward primer
10 Nucleotide sequence of the Allalpha reverse primer (1)
11 Nucleotide sequence of the Allalpha reverse primer (2)
TABLE 2
Abbreviations
Abbreviation Definition
18srRNA 18s ribosomal RNA
ADC Apparent Diffusion Coefficient
ANOVA Analysis of Variance
APS Ammonium Persulphate
BBB Blood Brain Barrier
BSA Bovine Serum Albumin
CCI Controlled Cortical Impact
CNS Central Nervous System
DMEM Dulbccco's Modified Eagle Medium
DMSO Dimethyl Sulfoxide
DWI Diffusion Weighted Imaging
ECL Enhanced Chcmilumi ncsence
EDTA Ethylenediaminetetraacetic acid
FBS Fetal Bovine Serum
H&E Hematoxylin &. Eosin
HEPES 4- 2-hydroxyethyl)-l-piperazineethane sulphonic acid hulFN human Type- 1 Interferon
IFN Interferon
IFNA Interferon Alpha Receptor
IFNAR1 - IFNAR 1 genetic knock-out
ΪΡΉΑΚΙ* IFNAR2 genetic knock-out
1L Interleukin
II-- Ι β lnierleukin-i p
IL-6 Interleukin-6
IRF Interferon Regulatory Factor
JAK Janus Kinase
JAK-1 Janus Associated Kinase 1
Abbreviation defi ition
KO Knock out (-1-)
LB Luria Broth
J 7 human neuroblastoma Ml 7 cell line
Map2 Neuronal marker indicating microtubule associated proteins AR1 Monoclonal antibody against IFNAR1
MCAO Middle Cerebral Artery Occlusion
MRI Magnetic Resonance Imaging
MTT 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
NeuN Neuronal nuclei
NMR Nuclear Magnetic Resonance
OGD Oxygen and Glucose Deprivation
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
P-STAT Phosphorylated-STAT
qRT-PCR Quantitative Real Time Polymerase Chain Reaction
RT-PCR Real-Time Polymerase Chain Reaction
SDS Sodium Dodecyl Sulphate
SEM Standard Error of the Means
STAT Signal Transducers and Activators of Transcription
TBI Traumatic Brain Injury
TBS-T Tris Buffered Salinc-Twccn 20
TE Tris-EDTA
TEMED N, N, N", N'-tetramethylethylenediamine
TNF Tumor Necrosis Factor
TNFa Tumor Necrosis Factor Alpha
Tris Tris(hydroxymethyl)aminomethane
TYK Tyrosine Kinase
ΊΎΚ-2 Tyrosine Kinase 2
BRIEF DESCRIPTION OF THE FIGURES
[0024] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.
[0025] Figure 1 is a graphical representation showing isolated wild-type and ΙΡ ΑΚ.." " C57B/6 neurons subjected to 4 hours oxygen and glucose deprivation (OGD) and 24 hours reperfusion. A subsequent MTT assay showed increased cell viability of IFNARI'1'" neurons at 91.0+4.9% compared to wild-type with 49.8+0.8% (n=6\ P<0.05, Student's t- test).
[0026] Figure 2 is a graphical representation showing M17 cells subjected to 3 hours OGD and time-course reperfusion (0, 0.5, 2 and 24 hours), RT-PCR was then performed. IFNcc mRNA levels, unlike ΙΡΝβ, were significantly elevated at 2 hours reperfusion (15.1+2.3 fold change) compared to control. IFN^ mRNA levels were only increased at 24 hours reperfusion (2.4+0.1 fold change) compared to control. (n=3, *P.05, One-way ANOVA, Dunnett's Post-hoc test.) [0027] Figures 3A through C are graphical representations showing Ml 7 cells subjected to 3 hours OGD and time-course reperfusion (0, 0.5, 2 and 24 hours), RT-PCR was then performed. A) Interlcukin-6 (IL-6) mRNA levels were significantly increased at 0.5 hour (266.9±10.9 fold change) and 2 hours (194.6±39.4 fold change) reperfusion compared to control. B) Tumor Necrosis Factor a (TNF-a) mRNA levels were elevated at 0 hour (10.0±3.1 fold change) and 0.5 hour (10.1±2.8 fold change) as to control. C) lnterleukin-ΐβ (1L-1|5) mRNA levels showed a significant increase at 24 hours reperfusion (4.6±1.2 fold change) compared to control. (n-3,*P<0.05, Dunnett's Post-hoc test.)
[0028] Figure 4 is a graphical representation showing collated RT-PCR data for each cytokine mRNA levels post-3 hours OGD and time-course reperfusion (0, 0.5, 2 and 24 hours). OGD begins at -4 hours and subsequent reperfusion occurs from 0 hour. IFNo
m NA levels increases at 2 hours reperfusion before returning to basal levels. Ι Νβ and IL-Ι mR A levels only elevate at 24 hours reperfusion. TNFa mRNA levels show immediate increase at 0 and 0.5 hours reperfusion before returning to control levels. IL-6 mRNA levels are elevated at 0.5 and 2 hours reperfusion, and then return to basal levels.
[0029] Figure 5 is a graphical representation showing Ml 7 cells transfected with IFNARl-GFP and IFNAR2-GFP ovorexpression plasmids and subjected to 3 hours OGD and 24 hours reperfusion. Curiously, M 17-IFNAR1 cells demonstrated significant decrease in survival (21.6±5.3%) compared to wild-type (44,9±2.1%). Furthermore, M17-IFNAR2 cells showed no difference in survival (39.8±5.7%) compared to wild-type. (n=4,* PO.05, One-way ANOVA, Dunnett's Post-hoc test)
[0030] Figure 6 is a representation of the IFNARl, AttBl and AttB2 sequences used in Gateway (Registered Trademark) cloning.
[0031] Figure 7 is a representation of the IFNAR2, AttBl and AttB2 sequences used in Gateway (Registered Trademark) cloning.
[0032] Figure 8 is a representation of SYBRGreen nucleotide sequences.
[0033] Figures 9A through C are photographic representations of fluorescent immunohistochemistry showing IFNARl localization on primary cultured mouse neurons. Panel A Map-2, Panel B NeuN Panel C-Mcrge. Note, Map2 is a neuronal marker indicating microtubule associated proteins.
[0034] Figure 10 is a graphical and photographic representation of IFNAR"'" (knock out; KO) mice (C57BL6) show reduced infarct size in a stroke model. The model comprises 2 hours of mid-cerebral artery occlusion followed by 24 hours of reperfusion. IFNARl"'" mice show a reduced infarct size.
[0035] Figure 11 is a graphical representation showing that IFNARl mAb reduces infarct size in a middle cerebral artery occlusion (MCAO) stroke model. An amount of 0.5mg
mAb was given iv 1 hour prior to stroke. The infarct volume was assessed 24 hours post- injury. The monoclonal Ab (mAb) is MAR- 1.
DETAILED DESCRIPTION [0036] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method step or group of elements or integers or method steps.
[0037) Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:l), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.
[0038] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a neuroinflarnmatory condition" includes a single neuroinflaramatory condition, as well as two or more neuroinflarnmatory conditions; reference to "an antagonist" includes a single antagonist, as well as two or more antagonists; reference to "the disclosure" includes a single and multiple aspects described in the disclosure; and so forth. All aspects disclosed, describe and/or claimed herein arc encompassed by term "invention". Such aspects are enabled across the width of the present invention. (0039] Type 1 interferons (IFNs) are a super-family of pleiotropic cytokines that induce pro-inflammatory gene transcription via the classical JAK/STAT pathway. The Type 1 IFNs include IFNa, ΙΡΝβ and IFNco. Certain events or conditions within the central nervous system (CNS) have the capacity to induce a ncuroinflammatory response having pathological consequences. Taught herein is the use of an antagonist of Type J IFN- mediated signalling within the CNS to attenuate an adverse neuropathological inflammatory response following a stroke event. The attenuation of a neuroinflarnmatory response leads to a reduction in secondary neuronal damage including neuronal necrosis
and apoptosis.
[0040] Reference herein to a "type 1 IFN-mediated signalling antagonist" means an antagonist of the activity of a Type 1 1FN, an inhibitor of Type 1 IFN gene expression or translation, an antagonist of a Type 1 IFN receptor activity, function or dimerization or a subunit thereof such as IFNARl , an inhibitor of Type 2 IFN receptor gene expression and an agent which blocks Type 1 IFN:receptor interaction or any agent which blocks, inhibits or otherwise reduces Type 1 IFN-mediated inflammatory signalling. [0041) An aspect enabled herein is a method for reducing a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IFN)-mcdiated signalling, for a time and under conditions sufficient to prevent Or attenuate neuroinflammation.
(0042] A "stroke event" includes any ischemic condition in a blood vessel in the CNS including the brain as well as any blood fluid leakage such as following rupture of a blood vessel. The stroke event may be classified as minor-mild, mild-moderate, moderate, moderate-severe and severe-serious and can result from minor to significant impairment to a subject.
[0043] Another aspect of the present disclosure contemplates a method for ameliorating symptoms of a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type I interferon (IFN)-mediated signalling, for a time and under conditions sufficient to prevent or attenuate neuroinflammation.
[0044] An aspect enabled herein is a method for reducing a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IFN)-mediated signalling, for a time and under conditions sufficient to prevent or attenuate neuroinflammation.
(0045] A related embodiment provides a method for ameliorating the symptoms associated with a stroke event, the method comprising administering to the subject an effective amount of an antagonist of T pe 1 interferon (IFN)-mediated signalling, for a time and under conditions sufficient to reduce or attenuate a neuroinflammatory response within the CNS.
(0046) The present disclosure teaches antagonists of Type 1 IFN-mediated signalling in the form of a compound, agent, chemical agent, pharmacologically agent, medicament, active and drug. The terms "compound", " agent", "chemical agent", "pharmacologically active agent", "medicament", "active" and "drug" are used interchangeably herein to refer to the Type 1 IFN-mediated signalling antagonist which Induces a desired pharmacological and/or physiological effect. The desired effect includes reducing Type I IFN-mediated signalling via the IFN or a receptor subunit such as IFNARl . The desired physiological effect includes attenuation of a neuropathological inflammatory response including the potential for such an inflammatory response to reach a neuropathological level. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, mimetics functional equivalents and the like. When the terms "compound", " agent", "chemical agent" "pharmacologically active agent", "medicament", "active", "drug" and "antagonist" are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salt, ester, amide, prodrug, metabolite and analogs thereof.
(0047] Reference to the antagonist in these terms includes combinations of two or more actives. A "combination" also includes multi-part such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation. Examples include two separate agents wherein one agent down- regulates the activity or level of the Type 1 IFN and the other down-regulates the activity or level of a Type 1 IFN receptor subunit, and in particular IFNARl . Agents when in combination with an IFN or IFNAR antagonist may also target a down-stream component
of the Type 1 IFN-mediated signalling cascade.
[0048] The antagonists enabled herein provided systcmically cross the blood brain barrier (BBB). This is possible due to the chemical nature of the agent or due to a permcalization factor such as a co-factor fused to or formulated with the antagonist. Strategies for accessing the CNS are disclosed in Misra et al, JPharm Sci £252-273 , 2003.
(0049J The "antagonist" includes a protein, polypeptide or peptide, small chemical molecule, antibody or derivative thereof including an immunoglobulin new antigen receptor (IgNAR) or a genetic molecule.
(0050] A "genetic molecule" is generally one which down-regulates expression of a gene encoding a Type 1 IFN or a Type 1 IFN receptor or a subunit thereof. In an embodiment, the targeted subunit is IFNAR1. The genetic molecule includes an antisense molecule directed to all or part of a gene or mRNA encoding the Type 1 IFN or a subunit portion of its receptor such as IFNARl . In an embodiment, the antisense is from about 5 nucleotides in length to 15 to 80 nucleotides in length or from about 5 nucleotides in length to full length of the mRNA transcript or 5' or 3' regions thereof. Other genetic molecules include sense molecules such as single- or double-stranded RNAs, RNAi and siRNA molecules, short and long RNA duplexes, ribozymes, DNAzymes, and any DNA or RNA or synthetic DNA or RNA agent which interferes with expression of the expression of the Type 1 IFN gene or of the gene encoding a receptor subunit such as IFNARl . The genetic molecule may be naked or expressed by a viral or other vector or introduced as part of a formulation. [0051] A number of viruses may be used as nucleic acid transfer vectors or as the basis for preparing nucleic acid transfer vectors to introduce a genetic agent to the CNS, including papovaviruses (e.g. SV40, adzak et al, J Gen Virol 75:1533-1536, 1992), adenovirus (Berkner, Curr Top Microbiol Immunol J 58:39-66, 1992; Berkner et al, BioTechniques (5:616-629, 1 88; Gorziglia and Kapikian, ./ Virol (56:4407-4412, 1 92; Quantin et al, Proc Nail Acad Set USA 59:2581 -2584, 1992: Rosenleld et al Cell (55:143-155, 1992; Wilkinson et al. Nucleic Acids Res 20:233-2239, 1992; Stratford-Perricaudet et al, Hum Gene Ther 7:241-256, 1990; Schneidei- et al, Nat Genetics 75:180-183, 1998), vaccinia
virus (Moss, Curr Top Microbiol Immunol 158: 5-38, 1992; Moss, Proc Nail Acad Sci USA 93:11341-11348, 1996). adeno-associated virus ( uzyczka, Curr Top Microbiol Immunol 58:97-129, 1992; Ohi et al, Gene 5P:279-282, 1990; Russell and Hirata, Nat Genetics 75:323-328, 1998), herpesviruses including HSV and EBV (Margolskec, Curr Top Microbiol Immunol 158:67-95, 1992; Johnson et al, J Virol 06:2952-2965, 1992; Fink et al, Hum Gene Ther 3: 1-19, 1992; Ureakefield and Geller, Mol Neurobiol /:339-371, 1987; Freese et al, Biochem Pharmaco. 40:2189-2199, 1990; Fink et al, Ann Rev Neurosci 9:265-287, 1996), lenti viruses (Naldini et al, Science 272:263-267, 1 96), Sindbis and Semliki Forest virus (Berglund et al, Biotechnology 11:9 6-920, 1993) and retroviruses of avian (Bandyopadhyay and Temin, Mol Cell Biol 4:749-754. 1984; Petropoulos et al, J Virol 66:3391-3397, 1992), murine (Miller, Curr Top Microbiol Immunol 158:1-24, 1992; Miller et al, Mol Cell Biol J :431 -437, 1985; Sorge et al. Mol Cell Biol 4: 1730-1737, 1984; Mann and Baltimore, J Virol 54:401-407, 1 85; Miller et al, J Virol 62:4337-4345, 1988) and human (Shiroada et al, J Clin Invest 88: 1043-1047, 1991; Helseth et al, J Virol 64:2416-2420, 1990; Page et al, J Virol 64:5270-5276, 1990; Buchschacher and Pangantban, J Virol 66:2731-2739, 1982) origin.
(00521 Non-viral nucleic acid transfer methods include chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor- mediated DNA transfer. Viral-mediated nucleic acid transfer can be combined with direct in vivo nucleic acid transfer using liposome delivery, allowing one to direct the viral vectors to particular cells. Alternatively, the retroviral vector producer cell line can be injected into particular tissue. Injection of producer cells would then provide a continuous source of vector particles.
(0053] An igNAR (immunoglobulin new antigen receptor) is an antibody isotype found in cartilaginous marine animals (sharks and rays), which has evolved over hundreds of millions of years to be stably expressed in the potent urea environment of the blood stream (Greenberg et al '., Nature 374:168-173. 1995; Nuttall et al, Mol Immunol 3<S:313-326, 2001). The IgNAR response is antigen-driven in the shark, and both immune and naive molecular libraries of IgNAR variable domains have been consuueted and successfully
screened for antigen-specific binding reagents (Greenberg el al, 1995 supra; Nuttall et al, 2001 supra). IgNAR's are bivalent, but target antigen through a single immunoglobulin variable domain (~14kDa) displaying two complementarity determining region (CDR) loops attached to varying numbers of constant domains (Nuttall et al, Eur J Biochem 270:3543-3554, 2003; Roux et al, Proc Natl Acad Sci USA 95:11804-11809, 1998). In contrast, traditional immunoglobulin (Ig) antibodies have a variable heavy (VH) + variable light (V,.) domain format (~26kDa) and bind antigen through up to six CDRs (Chothia et al.. Nature 342:877-883, 1989; Padlan, Mol Immunol 37:169-217, 1994). The small size, and thermodynamic and chemical stability of IgNAR variable domains (\ s), offer distinct advantages over conventional antibodies. Furthermore, the small VNAR size enables this unusual antibody domain access to cryptic antigenic epitopes through unusually long and variable CDR3 loops (Greenber et al., 1995 supra; Ewert et al., Biochemistry 4/:3628- 2636. 2002; Nuttall et al.. Proteins 55: 187- 1 7, 2004; Stanfield et al. Science 305:1770- 1773, 2004; Strcltsov el al, Proc Natl Acad Sci USA J 07: 12444- 12449, 2004; Streltsov et al, Protein Sci 7 :2901-2909, 2005). IgNAR domains have been identified that recognize a variety of target antigens including: the apical membrane protein I (AMA-1) of P. falciparum (Nuttall et al., 2004 supra); the Kgp protease from Porphyromonas gingtvalis (Nuttall et al., FEBS Lett J/d:80-86, 2002); cholera toxin (Goldman et al, Anal Chem 7#:8245-8255, 2006); the Tom70 mitochondrial membrane spanning protein (Nuttall et al., 2003 supra), and lyso2yme (Streltsov et al, 2004 supra).
(0054] The present disclosure teaches analogs and derivatives of a Type 1 IFN or an IFNAR such as IFNARl such as which include a modified side chain or which incorporate an unnatural amino acid and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or its analogs. This term also does not exclude modification of the glycosylation. acctylation and phosphorylation patterns. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid or polypeptides with substituted linkages. Such polypeptides may need to be able to enter the cell and/or cross the BBB or promote cerebral fluid half-life survival.
|0055) Mimetics of the Type 1 IFN or IFNAR such as IFNARl are another useful group of
agents to test for neuroprotective ability. The term is intended to refer to a substance which has some chemical similarity to the molecule it mimics and which acts as an antagonist. A peptide mimetic of a Type I IFN or IFNA 1 , for example, may be a peptide- containing molecule that mimics elements of protein secondary structure (Johnson ei al., Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzuto et al (Eds), Chapman and Hall, New York, 1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions such as with a receptor or ligand. A peptide mimetic, therefore, is designed to permit molecular interactions similar to the natural molecule but block signalling.
(0056) The designing of mimetics to a pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g. peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
[0057] There arc several steps commonly taken in the design of a mimetic from a compound having a given target property. First, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. Alanine scans of peptides, for example, are commonly used to refine such peptide motifs. These parts or residues constituting the active region of the compound are known as its "pharmacophore".
(0058) Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.
[0059] In a variant of this approach, the three-dimensional structure of a receptor and ligand are modeled. This can be especially useful where the receptor and/or Hgand change conformation on binding, allowing the model to take account of this in the design of the mimetic. Modeling can be used to generate agents which interact with the linear sequence or a three-dimensional configuration.
[0060] A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
(0061 J Hence, the present disclosure teaches antagonists of Type 1 IFN-rnediated signalling for use in mammals including higher order mammals' such as humans, Such antagonists facilitate an attenuation of a neuroinflammatory response following a su-oke event. Non-human animals are contemplated such as in veterinary applications as well as for use in animal models. Reference to a mammal includes a mouse, rat, hamster, guinea pig, rabbit, pig, sheep, horse, goat, cow, camel and non-human primate (such as orangutan, gorilla, marmoset and a macaque). In an embodiment, the subject is a human. Reference to a human includes a fetus, in uiero as well as a human of any age.
[0062] The present disclosure teaches a method for reducing a neuroinflammatory response within the central nervous system (CNS) of a human subject following a stroke event, the method comprising administering to the subject an effective amount of an antagonist of Type 1 interferon (IF )-mediated signalling, for a time and under conditions sufficient to prevent, reduce or attenuate neuroinflammation.
10063] In an embodiment, the present disclosure enables a method for reducing a neuroinflammatory response within the CNS of a subject following a stroke event, the method comprising administering to the subject an antagonist of interferon alpha receptor 1 (IFNA 1 )-mediated signalling for a time and under conditions sufficient to prevent or 5 ameliorate the symptoms of neuroinflammation.
(0064) The present disclosure further describes a method for ameliorating the symptoms of a neuroinflammatory response within the CNS of a human subject following a stroke event the method comprising administering to the human subject an antagonist of interferon 1 alpha receptor 1 (IFNAR l)-mediated signalling for a time and under conditions sufficient to prevent or ameliorate the symptoms of neuroinflammation.
[0065] The terms "effective amount" and "therapeutically effective amount" of an agent as used herein mean a sufficient amount of an agent (i.e. a Type 1 IFN-mediaied signalling
I S antagonist) to provide the desired therapeutic or physiological effect or outcome as indicated above. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount". The exact amount required will vary from subject to subject, depending on the species, age and0 general condition of the subject, mode of administration and the like. T us, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation. In general, the amount is effective to attenuate a neuroinflammatory response within the CNS or to prevent or reduce the severity of a5 neuroinflammatory response developing or continuing. The prevention of a neuroinflammatory response developing is also useful in at risk subjects such as those with a genetic disposition or family history of strokes. The present disclosure teaches reducing, preventing or attenuating neuroinflammation anywhere in the CNS, including in the brain. The antagonist may be used as a therapeutic to treat a condition or as a preventative (i.e.0 prophylactically) to reduce the risk of neuroinflammation in anticipation of a stroke.
[0066] The agent in the form of an antagonist may also be administered with a
pharmaceutically acceptable carrier, excipient or diluent. By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
[00671 The agent in the form of an antagonist may also be a pharmacologically acceptable form or derivative of a compound. By "pharmacologically acceptable" means a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.
(0068] The terms "treating" and "treatment" as used herein refer to reduction in severity and/or f equency of symptoms of the condition being treated, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms of the condition and/or their underlying cause and improvement or remediation or amelioration of damage following a stroke event leading to or having the potential to lead a ncuroinflammatory response in the CNS. In general terms, treatment may involve ameliorating symptoms of a neuroinflammatory response. Such symptoms may arise from neuronal necrosis, apoptosis, senescence or arrest, demyelination and/or axonal or neuronal degeneration. Amelioration of a downstream physiological, psychological or mental condition is also a useful indicator of treatment.
[0069) " Treating" a subject, therefore, may involve prevention or reduction in extent of development of a condition or other adverse physiological or psychological event in a susceptible individual associated with a neuroinflammatory response as well as treatment of a clinically symptomatic individual by ameliorating the symptoms of the neuroinflammatory response. (0070| As indicated above, a "subject" as used herein refers to an animal, such as a mammal including a human who can benefit from the pharmaceutical agents and formulations and methods of the present disclosure. A subject regardless of whether a
human or non-human animal may be referred to as an individual, patient, animal, host or recipient. The compounds and methods enabled herein have particular applications in human medicine. [0071J Taught herein is the use of neuroprotective agents to reduce a neuroinflammatory response or risk of such a response developing or the consequences of degenerative inflammatory processes such as immunodegenerative processes or conditions which induce cell cycle arrest, necrosis and/or apoptosis following a stroke event. [0072] The neuroprotective agent enabled herein is an agent which down-rcgulatcs the extent or activity of Type 1 IFN-mediated signalling. Such an agent is referred to herein as a "Type 1 IFN signalling antagonist". This includes an antagonist of a Type 1 IFN or a portion of the Type 1 IFN receptor and in particular 1FNAR. Reference to an "IFNAR" includes the IFNA receptor or a subunit thereof such as IFNARl. The present disclosure further teaches combinations of neuroprotective agents or neuroprotective formulations comprising a Type 1 IFN signalling antagonist and another neuroprotective agent such as leukemia inhibitory factor (LIF) or ciliary neurotrophic factor (CNTF) or a homolog, derivative, analog or mimetic thereof. [0073] Hence, the present disclosure teaches a method for the treatment or prophylaxis of a stroke event in a subject, the method comprising administering to the subject an effective amount of a Type 1 IFN signalling antagonist for a time and under conditions sufficient to ameliorate adverse neurological inflammation or prevent or reduce its progression, [0074J In an embodiment, a method is provided for the treatment or prophylaxis of a stroke event in a subject or at least delaying onset of symptoms thereof, the method comprising administering to the subject an effective amount of a Type 1 IFN alpha receptor (IFNARl) antagonist for a time and under conditions sufficient to ameliorate adverse neurological inflammation or prevent or reduce its progression in the CNS.
[0075J As indicated above, the neuroprotective agent includes a Type 1 IFN antagonist or an IFNAR antagonist such as IFNARl antagonist. ITie antagonist may act at the level of
protein activity or function or gene expression including transcription, translation or processing.
(0076] In another aspect, the present disclosure teaches a method for ameliorating the symptoms of a ncuroinflammatory response following a stroke event in a subject or delaying development of symptoms thereof, the method comprising administering to the subject an effective amount of a neuroprotective formulation comprising a Type 1 IFN antagonist for a time and under conditions sufficient to reduce neurological inflammation or prevent or reduce its progression.
[0077] Still another aspect taught herein to a method for ameliorating the symptoms of a neuroinflammatory response following a stroke event or delaying development of symptoms thereof, the method comprising administering to the subject an effective amount of a neuroprotective formulation comprising an IFNAR1 antagonist for a time and under conditions sufficient to reduce neurological inflammation or prevent or reduce its progression.
[0078] As indicated above, the amount or time sufficient to treat the neurodegenerative disease or condition may be the amount or time required to ameliorate one or more symptoms of the stroke event. A symptom includes a psychological or mental symptom. Furthermore, the antagonist may be provided with a pharmaceutically acceptable carrier, excipicnt or diluent. 'I"he antagonist itself is considered to be pharmacologically acceptable. [0079] The Type 1 IFN-mcdiatcd signalling antagonist may also be provided in combination with another neuroprotective agent such as LIF and or CNTF or their homologs, derivatives, analogs or mimctics.
[0080] Hence, the present disclosure enables a method for the treatment or prophylaxis of a neuropathological event, disease or condition in a subject selected irom a stroke event, the method comprising administering to the subject an effective amount of a neuroprotective formulation comprising a Type 1 IFN-mediated signalling antagonist and .
onc or both of LIF and CNTF or a homolog, derivative, analog or mimetic thereof for a time and under conditions sufficient to reduce neurological inflammation or prevent or reduce its progression. [0081| Also enabled herein are pharmaceutical compositions and formulations which include one or more of the neuroprotective agents hereinbefore described. The pharmaceutical compositions taught herein may be administered in a number of ways depending upon whether local or systemic treatment as desired including with means for the agent to cross the BBB. Administration includes intravenous, mtra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, e.g., intrathecal or intraventricular, administration; or oral administration; or via a spinal tap. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Clearly, the formulation needs to enable the agent to cross the BBB. Hence, the agent itself may need to be modified. Alternatively, the formulation may enable retrograde transport. The agents may also be specifically targeted to the brain or other parts of the CNS.
[0082} The pharmaceutical formulations described herein may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s).
[0083] The compositions described herein may be formulated into any of many possible dosage forms such as, but not limited to, injectable formulations, and tablets, capsules, gel capsules and liquids.
[0084] Pharmaceutical compositions herein include, hut are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations herein described may comprise one or more penetration enhancers, carriers, excipicnts or other active or inactive ingredients.
(0085) Emulsions are typically heterogeneous systems of ne liquid dispersed in another in
the form of droplets usually exceeding 0.1 μιη in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment taught herein.
[0086 J Formulations include liposomal formulations. The term "liposome" means a vesicle composed of amphophilic lipids arranged in a spherical bilayer or bi layers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed Scorn a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitivc or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells. (0087] Liposomes also include "stericaily stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of stericaily stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is dcrivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
(0088) In an embodiment, various penetration enhancers may be employed to effect the efficient delivery of nucleic acids. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants. (0089) One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.
[0090] The formulation of therapeutic compositions and their subsequent administration (dosing) are within the skill of those in the art. Dosing is dependent n severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on ECjos found to be effective in vitro and in vivo animal models. In general, dosage is from 0.01 to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years. Alternatively, the antagonists are provided in amounts of 1 , 10, 100 or 1000 μg ml or amounts in between. (0091) The present disclosure teaches a neuroprotective formulation comprising a Type 1 IFN-mcdiated signalling antagonist and one or more pharmaceutically acceptable carriers and or diluents. The neuroprotective formulation is provided to patients or at risk subjects of stroke. [0092] In another embodiment, the present disclosure teaches a neuroprotective formulation comprising a Type 1 IFN-mediated signalling antagonist and one or both of LIF and/or CNTF and one or more pharmaceutically acceptable carriers and/or diluents. In this aspect, the neuroprotective formulation is provided to patients or at risk subjects of stroke.
[0093] Diagnostic assays to assess the presence of a neuroinflammatory response following a neuropathological event, disease or condition selected from a stroke event are
enabled herein. For example, following ordering a stroke or infection, the level of Type 1 IFN-mediated signalling may be determined such as via the level of a Type 1 IFN, its corresponding mR A levels, activity of IFNAR1 or its corresponding mRNA levels or via a down-stream effector such as T Fa, IL-6 or a chemokine or other pro-inflammatory effector molecule.
[0094] Hence, a medical protocol is enabled herein to treat a subject which has or may experience a neuropathological event, disease or condition selected from a stroke event, the protocol including:
(i) assessing physical trauma to the brain or other parts of the CNS;
(ii) determining parameters of inflammation;
(Hi) administering an antagonist of Type 1 IFN-mediated signalling; and/or (iv) monitoring patients including subjecting the patient to behavoral modification procedures. Inflammatory parameters may be detected by any means including HPLC, TLC, ELISA, RIA, immuno-fluorescent assay, Southern analysis,
Western blot. Northern analysis, gel electrophoresis, nucleic acid expression and the like.
(00951 Another aspect of the present disclosure enables a medical protocol for treating acute neuronal injury in the form of a stroke event in a subject, the protocol comprising administering to the subject, within from 1 to 120 minutes of the injury, a neuroprotective formulation comprising the antagonist of Type 1 interferon-mediated signalling. By "1 to 120" include 1, 2, 3, 4, 5, 6, 7, 8, 10. 1 1. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25. 26, 27, 28, 29. 30, 31, 32, 33. 34. 35, 36. 37, 38, 39, 40, 41 , 42, 43, 44, 45. 46, 47, 48, 49, 50. 51, 52, 53, 54, 55, 56, 57, 58, S9, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 0, 91, 92, 93, 94, 95, 96, 97, 98, 99. 100, 101, 102, 103, 104, 105, 106. 107, 108, 109, 110, U I, 112, 113, 114, 115, 11 , 117, 118, 119 or 120 minutes. In an embodiment, the time is 30 minutes.
(0096) Still another aspect taught herein the use of a Type I IFN-mediated signalling antagonist in the manufacture of a medicament in the treatment or prophylaxis of neuroinflammation associated with a neurological condition or event selected from a stroke event.
EXAMPLES
(0097) Aspects taught herein are further described by the following non-limiting Examples. In these Examples, materials and methods as outline below are employed.
Animals
[0098J Mice (eight-week-old male mice 23±3 g) used were of a C57BI^-6 strain with -FNARl^ (knockout or KO) or IFNARl^ (wild-type or wt) genotypes (Hwang et a Proc Natl Acad Sci USA 92(24)\\ 1284-11288, 1995).
Tissue Culture
Mixed hippocampal and cortical neurons
[0099) Pregnant female mice were killed by cervical dislocation when embryos were 14 days old. The embryos were extracted from the female uterus and their heads removed into a petridish of Solution 1 (30ml Krebs stock (3625g NaCl, 2g KCl, 0.7g NaH2P04.H20, I3g d-Glucose, O.OSg Phenol red, 20.7g HEPES (acid form) in 500ml ¾0, pH7.4), 270ml H20, 0.9g bovine serum albumin (BSA), 2.4ml 3.4% w/v MgS04, pH 7.4). The brains were removed, cerebral cortices were isolated and the meninges removed. The cortices were placed into a fresh petri dish of solution 1 and minced to a fine paste. The paste was transferred to a 50ml Falcon tube containing solution 2 (15ml sol 1, 1.5ml lOx trypsin (25mg trypsin in 10ml)) using a pasture pipette. The tube was incubated for 15-20 minutes at 37*0 with occasional mixing by inversion. 12.5ml of Solution 4 (8.5ml Sol 1, 4ml Sol 3) was added to the digested tissue. The tissue was then centrifuged (lOOOrpm, 45 seconds), supernatant discarded and pellet resuspended in solution 3 (7.5ml Sol 1, 0.75ml lOx DNase soybean trypsin inhibitor (SBT1) stock (8mg DNase, 52mg SBT1 in 10ml), 75μΙ. 3.4% w v MgSOi) by passing through a pasture pipette for 2 minutes, the solution was then transferred to a fresh 50ml Falcon tube. Remaining tissue was washed from the old tube with 2 pasture pipettes of solution 5 (6.25ml Sol 1, 50μΙ, MgS04 stock, 7.5μί 1.2% w/v C Cy. Upon centrifugation (lOOOrpm, 5 minutes) and supernatant removal, the resulting pellet was resuspended in 1 Oml plating medium ( eurobasal media (Gibco) containing 10% v/v fetal bovine scrum (FBS, Gibco)). Cells were then counted and diluted to achieve a final concentration of 11.2 x 10* cells/6cm dish. All tissue culture dishes were coated with poly-
l-lysine before neurons were plated. Following 5 hours in plating medium, all medium was exchanged for neuronal culture medium (Ncurobasal media containing 10ml B27 supplement (Gibco), 1.25ml 200mM 1-glutamine and 0.5ml Oentaroycin (Oibco) in 500ml), thereafter half medium changes were performed every 2-3 days with a final concentration of 20μ 5-chlorocytosine arabinoside (ARAC) added to prevent proliferation of murine embryonic fibroblasts.
Human neuroblastoma Ml 7 cells (Ml 7)
[0100) Ml 7 cells (ATCC [Registered Trademark] number: CRL-2267 [Trademark]) were cultured in T75 flasks with culture medium (OptiMEM (Gibco), 5% v/v FBS, 0.5% w/v Penicillin-Streptomycin (Gibco)) at 37°C until 90% confluent. Cells were then plated at the densities listed in Table 3. For the Alzheimer's disease studies, M17 cells were housed in an incubator at 37°C and 5% v/v CO, in OPTI- EN (Invitrogen) in 10% v/ FBS and 1% w/v penicillin/streptomycin.
TABLE 3
Densities
[01011 Plated cultures were then incubated for 24 hours at 37¾C. Following incubation cells were serum-starved to halt the cell cycle with culture medium (no FBS) for 24 hours at 37°C, cells were then prepared for treatment.
Isolation of Murine Cortical Neurons
(0102] A 14 day pregnant mouse was killed by cervical dislocation and uterine horns were removed and placed in a 10cm dish containing working solution l(For 300ml: 30ml Krebs lOx stock, 270ml MUliQ, 0.9g BSA, 2.4ml 3.82% w/v MgSO*). Fetuses were removed from placental sacks, decapitated and heads were placed in a new dish containing solution 1. Under a dissection microscope skull and meninges were removed and cortical tissue was placed in a dish and chopped finely with a scalpel. Flamed Pasteur pipettes were then used to separate tissue into tubes using 20ml solution 1 with 10% w/v trypsin added. Cells were then incubated for 15 minutes at 37°C with occasional shaking. 25ml of solution 1 with 3% w/v added 10X DNase (Sigma D-502S)/SBTI (Sigma T-9003) was then added and gently inverted until DNA strings appeared. Cell suspension was then spun for 1 minute (lOOOrpm, RT) and supernatant removed. Cell pellet was then resuspended in 8ml of solution 1 with 10% w v DNase/STBI by 60 pasteur pipette passages and spun for 5 minutes at lOOOrpm at RT. Following discarding of supernatant, cell pellet was resuspended in 10ml of Dulbecco's Modified Eagle Medium (Invitrogen) with 10% v/v FBS. The suspension was placed in a T-75 flask and allowed to incubate at 37°C for 1 hour to allow fibroblast adhesion. Cell media containing only neurons was then removed from the flask, cell density determined, and plated into appropriate poly-L-lysine coated plates and kept in an incubator (37eC and 5% v/v CX½). 4 hours following plating Neurons are given a full media change to DMEM with 10% FBS and 0.5% w/v penicilin/streptomycin.
Tissue culture treatments
Oxygen Glucose Deprivation (OGD)
[0103] To engender OGD, culture media was replaced with no glucose and FBS DMEM (0.5 standard culture media volume) and cells were introduced into an hypoxic chamber (1% v/v 0¾ 5% v v CO& 94% v/v N2 and 60% humidity) for 3 hours (Ml 7 cells) or 4 hours (WT or IFNARl '" neurons). Cells were then removed from the chamber and reperfused with DMEM (high glucose, 0.5 standard culture media volume) for 0, 0.5, 2 or 24 hours. After this reperfusion event, cells were immediately utilized for MTT assay, RT-PCR or Western blot analysis.
IFNaffFNfi cytokine treatment
[0104] 6cm dishes of M17 cells were treated with human (hu) lFNcc (huIFNa) and human IFNp (huIF p) at 1, 10, 100 or lOOOU/ml for a time course of 5, 10 or 30 minutes whilst incubated at 37°C. Post-treatment harvested for Western blot analysis. SDS PAGE gel protein separation and western blotting
[0105] Cells (6cm dish) were scraped in I ml PBS/plate and centrifuged at SOOOx g for 5 minutes. The resultant cell pellet was resuspended in ΙΟΟμί of lysis buffer (50m Tris HC1, ISOmM NaCl, 1% v/v Triton x-100, 1% v v SDS, 1 tablet PhosphoSTOP (Registered Trademark) and protease inhibitor (Roche), pH 7.4) and lysed via sonication. 60μg of protein was added to 1:1 v/v reducing buffer (2.5ml 0.5M Tris, 2ml Glycerol, 4ml 10% w/v SDS, 1ml (3-mecaptoethanoI, bromophenol blue, in 10ml) and heated to 99°C for 5 minutes. 10% w/v acrylamide SDS PAGE (17.85ml dH20, l l.25ml I .5M Tris, 450μ[. 10% w v SDS, 15ml 30% w/v Acrylamide/BIS (Bio-rad), 15μΙ, N,N,N',N'tetramethylethylenediamine (TE ED, Bio-rad), 450μΙ, 10% w/v ammonium persulphate (APS), per 5 gels) in wiirung buffer (0. l%w/v SDS, 1.44% v/v glycine, 0.3% w/v Tris, pH 8.4) was used to separate the denatured protein. Samples were loaded on a stacking gel (3m! dH20, 1.25ml 0.5M Tris, ΙΟΟμί 10% w/v SDS, 650μΙ Acrylamide BIS, 7.5μί TEMED, ΙΟΟμί APS) and run at 80-120V for 1.5 hours. Polyvinylidene fluoride (PVDF) membrane was prcsoaked in 100% v/v methanol for 5 minutes and washed in transfer buffer (20% v/v methanol in running buffer). Separated proteins were transferred from PAGE gel to PVDF membranes utilizing a semi-dry transfer unit (60mA/gel for 75 minutes). Membranes were blocked with 5% w/v skim milk powder in TBS-T (lOmM Tris, 15mM NaCI, 0.01% v v Tween-20) for 1 hour. Phospho-STAT-1, Phospho-STAT-3 (Cell Signalling) and 0-tubulin (Millipore) antibodies were diluted 1:1000 in 5% w/v BSA in TBS-T and incubated on membranes at 4DC overnight. Membranes were then washed 3 times for 5 minutes with TBS-T and incubated with goat anti-rabbit or goat anti-mouse (for (3-tubulin) horseradish peroxidase (HRP) conjugated secondary antibodies, diluted 1 : 1000 in 5%w/v BSA in TBS-T, for 60 minutes at 25°C. HRP product was detected using an ECL advance western blotting detection kit (Amersham) After 4 washes of 5 minutes in TBS-T the ECL solution was applied to the membrane and visualized using the IQ350 imaging machine (GG Healthcare).
Kcal-Time Polymerase Chain Reaction (RT-PCR)
TR oi (Registered Trademark) mRNA extraction
[0106] Cells were plated at 2xl0 cells 6cm dish and incubated overnight in OptiMEM (5% w/v FBS, 0.5% w v Penicillin-Streptomycin). Post 3 hours OGD and 0, 0.5, 2 or 24 hours rcperfusion media was removed and cells were washed in lml/6cm dish PBS. Cells were lysed in lml/6cm dish TRIzol (Registered Trademark) [Invitrogen], transferred to a 1.7ml microcentrifuge tube and incubated for 10 minutes at 25°C. 0.2ml/sample chloroform was added and after vigorous shaking was incubated at 25°C for 3 minutes. Samples were oentrifuged (12,000x g, 15 minutes, 4°C) and resultant aqueous layer containing RNA was removed to fresh 1.7ml microcentrifuge tube. RNA was precipitated by adding 0.5ml/sample isopropanol and incubating at 25°C for 10 minutes. Following centrirugation (12,000x g, 10 minutes, 4°C), the supernatant was removed and resultant pellet was washed with 1 ml/sample 75% v/v EtOH in DEPC treated d O and centrifuged again (7 ,500* g, 5 minutes, 4°C). The RNA pellet was air-dried and redissolved in
RNAasc free water. Sample RNA concentration was then analyzed by the nanodrop 1000 spectrophotometer (Thermo-scientific).
Reverse transcription of RNA to cDNA
(0107] RNA was reversed transcribed into cDNA using Superscript (Registered Trademark) III kit (Invitrogen) according to manufacturer guidelines. Briefly, ^g of sample RNA was converted to cDNA using reverse transcriptase under the following conditions: 25°C for 10 minutes, 50°C for 30 minutes then 85° for 5 minutes. Residual RNA was digested by the addition of RNase H to ensure purification of cDNA which was 1 : 10 in d¾0 for use in RT-PCR.
RT-PCR using Taqman (Registered Trademark) probes
|0108| Taqman probes (all reagents from Applied Biosystems) were used as RT-PCR primers, these included: Tumor Necrosis Factor a (TNFa, HsG0174l28_m I), Interleukin- ip (IL-lp\ Hs00174097_ml), Interleukin-6 (IL-6, Hs00985639_ml), Interferon-al (IFNal, Hs00256882_sl), Interferon-β (IFNpl, HsOl 077958_sl) and 18s ribosomal RNA (18s rRNA, 4352930E). RT-PCR was performed in a 384-well plate with 4μί diluted cDNA, 0.5μί DEPC dH20, 0.5μί Taqman primer, 5μΙ. Taqman Fast Universal PCR master
mix (2x) per well. Each sample was repeated in triplicate and RT-PCR performed using the 7900ht Fast Real-Time PCR system (Applied Biosystems) under the following conditions: 50°C for 2 minutes, 94.5°C for 10 minutes, (97°C for 30 seconds, 59.7°C for 1 minute)x 40 repeats. Data were collated and fold change calculated using the AACt method.
Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
(0109] Brains were dissected into ipsilateral and contralateral hemispheres 2, 4 and 24 hours after injury, or sham operation. Hemispheres were homogenized in 2 ml Trizol (Invitrogen), and placed at room temperature for 10 minutes. 0.2ml Chloroform (Chem Supply) per 1ml Trizol was added to the samples, and samples were centrifuged at 12000g for 15 minutes at 4°C to separate samples into phases. The colorless, aqueous phase of each sample, which contained RNA, was transferred into a new 1.7ml microcentriiuge tube. RNA was precipitated by adding 0.5ml Propan-2-oI (Chem Supply) per 1ml Trizol, and samples were again centrifuged at 12000g for 10 minutes at 4°C. The supernatant from the tubes was discarded, and the RNA pellet was washed with 75% Ethanol (Chem Supply) in Diethyl pyrocarbonatc (DEPC)-treated water (Sigma), vortexed and centrifuged at 7500g for 5 minutes at 4°C. The RNA pellet was air-dried and redissolved in RNAse- free H20 (Invitrogen). Concentration of the RNA samples was assessed using the NanoDrop 1000 Spectrophotometer (ThermoScientific).
(0110] 1 g of RNA was transcribed per cDNA reaction using the Superscript (Registered Trade Mark) III kit (Invitrogen) according to these conditions: 10 minute incubation at 30°C, 30 minute incubation at 50°C, 5 minute incubation at 85°C, and finally, a 20 minutes incubation at 37°C with I Ι £ coll RNAse H (from the same kit).
(0111] Samples were diluted 1 :10 in RNAse free H20. The following Taqman primers were obtained from Applied Biosciences: TNFa (ID: Mm00443258_ml), ΙΡΝβ (ID: m00439552_sl), IL-Ι β (ID: Mm0l336l 89_ml) and 18S rRNA (ID: 4352930E), Taqman primers were incubated along with sample in a 384 well plate (MicroAmp, Singapore) in the following ratios: 0.5μ1 primer, 0.5μΙ H20, 5μ1 2x Taqman Mix (Invitrogen) and 4μ1 diluted cDNA. SybrGreen primers were obtained from Genc orks (sequences are listed in Figure 8): Allalpha forward, Allalpha Reverse 1, Allalpha Reverse
2, OADPH forward and GADPH reverse. SybrGreen primers were incubated in the following ratios: 2μ1 diluted cDNA, 5ul Fast SybrGreen Master Mix, Ιμΐ Forward GADPH primer, Ιμΐ Reverse GADPH primer and Ιμΐ ¾0. The plate was incubated according to the following conditions and read using the 7900ht spectrophotometer (Applied Biosciences): 95X for 20 minutes, 95°C for 3 minutes (40 repeats), 60°C for 30 minutes, 95°C for 15 minutes, 60°C for 15 minutes and 95°C for 15 minutes. Ct values were obtained for each sample, and relative transcript levels for each gene were calculated using the SSCT method. MTT eell viability assay
(0112] M 17 cells in 24-well plates were subjected to OGD and subsequent reperfusion (24 hours). Post-reperfusion 50μ1_Λ«!ΐΙ MTT reagent (2mg/ml, Sigma) was added to media and incubated for 1 hour at 37°C. Culture medium was then removed and cells were solubilteed with 200μΙ<Λνβ11 of dimethyl sulfoxide (DMSO) of which ΙΟΟμΙ. was transferred to a clean 96- well plate and absorbance read at 595nM. cDNA Preparation
{0113) Conversion of RNA to cDNA involved the use of a High Capacity RNA to cDNA kit (Applied Biosystems) as per manufacturers instructions. Briefly this involved loading 1 ug of RNA with 1 Ομΐ 2X reaction mix with 1 μΐ RT enzyme mix and DEPC H20 to 2θμ1. Tube was then heated at 35°C for 30 minutes, and 95°C for 5 minutes (termination step) and chilled on ice.
RTPCR
[011 ) Relative expression levels of R.NA were measured using the prepared cDNA in accordance with the TaqMan (Registered Trademark) [Applied Biosystems] primer preparation. This involves adding in triplicate 4μ1 cDNA with 5μ1 2X TaqMan Mix, 0.5μΙ DECP H2O, and 0.5μΙ of primer of raRNA in question. PCR plate was then read on a 7900ht Fast Real-Time PCR System. Fold increase was determined by using the delta-delta method.
Gateway (Registered Trademark) cloning system
Ligatio of subclone AttBl and subclone Attbl primers to mIFNARl/mlFNAR2 genes [0115] cDNA encoding mouse interferon receptor a 1 (mIFNARl) and mouse interferon receptor a 2 (IFNAR2) were custom synthesized and inserted into vector puc57 by GeneScript (U.S.A.). Subclone forward primers containing AttBl motif and subclone reverse primers containing AttB2 motif were designed and synthesized (Geneworks) for both mIFNARl and mtFNAR2 genes to use with the Gateway (Registered Trademark) cloning system (Invitrogen). These primers were ligated to the mIFNARl and mIFNAR2 genes via PCR using GoTaq (Registered Trademark) polymerase (Promega). Nucleotide sequences of IFNAR1 & 2 and AttBl and AttB2 sequences are shown in Figures 6 and 7.
Polymerase chain reaction conditions
[0116] Briefly a 20μί sample reaction consisted of μL· 5x GoTaq buffer, Ιμϊ, MgC12 (25m ), 0.4μΙ. dNTPs (2mM), 0.2μΙ. Subclone AttBl primer (20μΜ), 0.2μΙ. Subclone AttB2 primer (20μΜ), 0.1 μί GoTaq polymerase (5Ό/μΙ). DEPC H20 (to 20μί). The ligation reaction was performed under the conditions given in Table 4.
TABLE 4
Ligation conditions
Step Temperature Duration
Initial Denaturation 95°C 2 minutes
Denaturation 95°C 40 seconds
Annealing 60°C 40 seconds
Extension 72°C 1 10 seconds
Final Extension 72°C 5 minutes
Purificatlon of AttSl-mIFNARl 2 -Att82 PCR product
[0117] Samples were then electrophoresized on a 1% w v agarose gel (lg agarose, 2μΙ_ Ethidium Bromide per 100ml TAB buffer (242g Tris Base, 57.1ml glacial acetic acid, 100ml 0.5M Ethylenediaminetetra-acetic acid (EDTA) per Liter)) and a 1.77kb (mlFNARl) or 1.54kb (mIF AR2) was selectively cut from the entire gel. The gel bound AttBl -mJF ARl 2-AttB2 ligation product was then purified from the PCR reaction using the QIAquick (Registered Trademark) PCR Purification Kit (QIAGEN) according to manufacturer's protocol. Briefly, the gel containing AttBi-mIFNARl/2-AttB2 was rcdissolved and DNA was retained on a silicon filter whilst impurities were discarded. Post-washing with ethanol buffer the purified DNA was then dissolved in elurion buffer (l0mM Tris-HCL, pH8.5).
The BP reaction
[0118] The Invitrogen Gateway (Registered Trademark) cloning system was used. The purified AttB-mlFNARl and AttB-mIFNAR2 PCR products were translocated into vector pDONR20l (Invitrogen) using the BP Clonase (Trademark) Q enzyme kit (Invitrogen), according to manufacturers guidelines. A \0\xh reaction comprised of 0.6uL pDONR20l (289ng^L), 1.8μί AttB-mlFNARl PCR product (33n /μL) or 1.5μΙ. AttB-mlFNAR2 PCR product (37ng L), 2μΙ BP Clonase (Trademark) (I en2yme mix, TE buffer to ΙΟμί. The reaction was incubated for 24 hours at 25°C and subsequently terminated upon addition of Proteinase K (Ι ί,
DHSa bacterial transformation
(0119) The BP reaction product was transformed into DHSa competent E. coli cells (Invitrogen) by adding 1 1„ BP reaction product to each DI I5a aliquot (50μΙ.Λυο«). Cells were then incubated on ice for 30 minutes and heat-shocked by incubation at 42°C for 30 seconds. 250μί/ηιοβ of S.O.C. medium (Invitrogen) was added and cells were shaken (37°C, 225rpm, 1 hour) before 20μ1, was spread evenly on a kanamycin (50μg ml) selective agar plate (1% w/v Tryptone, 1% w v NaCI, 0.5% w/v Yeast extract, 0.75% w/v agar) and incubated for 16 hours at 37°C. A single bacterial colony was picked for colonies PCR to verify transformation success. The selected colony was grown in ΙΟμί Luria Broth (LB, 1% w/v Tryptone, 1% w/v NaCI, 0.5% w v Yeast lixlraci) of which 5μΙ_ was lysed at
99°C for 5 minutes. PCR was performed using the conditions above to confirm successful mIFNARl 2 insertion into pDONR20l and the subsequent DI-I5a colony. The PCR products were then electrophoresized on a 1% w/v agarose gel and 1.77kb (for mIFNA l) or 1.54kb (for mIFNAR2) bands were visualized using the IQ-350.
Isolation of pDONR20J-miFNARl/2 plasmid
[0120] Once the selected colony was deemed positive for pDONR20l-mlFNARl 2 insertion, the remaining 5μΙ. of culture (not used in PCR) was supplemented with a further 5ml kanamycin (50 g/ml) selective LB media and shaken for 16 hours (225rpm, 37°C). Plasmid DNA was then isolated from the bacterial culture using QIAprep (Registered Trademark) Spin Miniprep kit (QIAOE ) as per manufacturer's protocol. Briefly, bacteria were lysed, excess protein precipitated and DNA retained in the filter of a spin column. After subsequent washing with an ethanol buffer, DNA was eluted from the filter column giving isolated pDONR201 -mIFNARl/2 plasmid. DNA yields were then measured using the nanodrop 1000.
The LR reaction
[01211 The mIFNARl 2 genes were removed from the pDONR201-raIFNARl/2 plasmid and translocated into a pcDNA6.2 cEM-GFP destination vector using the LR Clonase (Trademark) II kit (Invitrogen) as per manufacturers protocol. A lOpL reaction consisted of ] μΙ pDONR- mIFNARl (I 22.6ng/ L) or Ι μΙ pDONR-mIFNAR2 (156.9ng L) entry clones, 2μL· pcDNA6.2/cEM-GFP (75ng/pL) destination vector, 2μΙ, LR Clonase (Tradeamrk), TE buffer to l0 L. The reaction was incubated for 24 hours at 25°C and subsequently terminated upon addition of Proteinase K (Ι ί, 2μg/μL). Reaction samples were then transformed into DH5a competent E. coli cells as per above. A colony was selected from an ampicillin (50mg/rnl) agar plate and grown in ΙΟμί LB media of which 5μΙ. was lysed by heating (99°C, 5 minutes). PCR was then performed under the conditions described above and samples run on a 1% w/v agarose gel. Imaging the gel using the IQ-350 showed a 1.77kb mIFNARl specific or a 1.54kb mJFNAR2 specific band for successful LR reaction and transformation samples. Positive colonies were then selected and shaken in 250ml ampicillin (50μ^Γη1) selective LB media for 16 hours (225rpm, 37°C).
„ isolation of final pcDNA6,2 EM-GFP-ndFNARl/2 plasmids
(0122) Bacterial plasmid DNA was isolated using EndoFree (Registered Trademark) plasmid Maxi Kit (QIAGEN) following manufacturers protocol. Briefly, bacteria were lysed, excess protein precipitated and DNA retained in the filter of a gravity flow column. After subsequent washing with an ethanol buffer DNA was eluted from the gravity flow column. The DNA was then precipitated using isopropanol, washed with endotoxin-free ethanol and resuspended in supplied TE buffer. DNA yields of pcDNA6.2/cEM-GFP- mIFNARl 2 plasmids were then determined using the nanodrop 1000.
Statistical analysis
[0123) All numerical data are stated as mean ± SEM and analyzed using GraphPad Prism 5.0. RT-PCR and MTT assay data were analyzed using Student's Most or one-way ANOVA and subsequent Dunnett's Post-hoc test where applicable. For all statistical tests P<0.05 was considered significant. For qRT-PCR data, a one way Analysis of Variance (ANOVA) was also performed followed by Bonferroni's post-hoc analysis, with a value of P<0.05 considered statistically significant. Infarct volume values were analyzed using an unpaired Student's t-tcst, with a value of P<0.05 considered statistically significant. Preparation of serial sections for staining
[0124] Mice were transcardially perfused at various time points after injury (or sham surgery) with 0.1% v/v heparinized Phosphate-Buffered Saline (Pfizer), followed by 4% v v paraformaldehyde (Scharlab S.L.), and their brains dissected. Brains were cut by the Histology facility, University of Melbourne. Brain sections were cut into ΙΟμπι coronal sections starting at the rostral end, paraffin-embedded and mounted onto glass slides. Every 10* slide was stained with Haematoxylin and Eosin (H&E).
Infarct volume analysis
[0125] Infarct volume analysis was conducted on slides, which had been stained with H&E. Analysis was performed using Image J. Area of infarct was calculated by measuring around regions appearing less intensely stained for viable cells. The average area of infarct per brain was then multiplied by the number of sections per slide by the thickness of each
section (1 Ομιη) and the number of slides in the sequence to get the volume of infarct. Immunohistochemistry
(0126) Paraffin-embedded sections were put in a 60°C oven for 20 minutes and taken 5 through a series of Histolenc (Lornb Scientific) incubations, followed by 100, 95 and 70% cthanol. Sections were incubated in Phosphate-Buffered Saline for a further 5 minutes. Sections were blocked firstly for endogenous peroxidase activity with a Peroxidase blocking solution (DA O). followed by a protein-blocking buffer (5% v/v whole Goat serum [Invitrogen] and Triton X-100 solution [Sigma] in lx Phosphate Buffered Saline)
10 for another hour. Where sections were used for immunofluorescence, only protein- blocking buffer was used. The primary antibody was diluted in an appropriate dilution buffer (1% w/v SA [Iiovogcn] in 1 x Phosphate Buffered Saline), and slides were incubated with the antibody overnight at 4'C. Primary antibodies used were: NeuN (Milliporc) and Mac-1 (Monash University), [Flentjar et al, Exp Neurol ]77(]):9-2Q,
15 2002]). Sections were washed in PBS, and incubated in an appropriate secondary antibody.
Fluorescent secondary antibodies (Alexa Fluor 594 anti-mouse and rabbit, Alexa Fluor 488 anti-mouse, rabbit and rat) were obtained from Invitrogen and the biotinylated secondary antibody (Anti-Mouse IgG, horse biotinylated) was obtained from Vector Laboratories. Biotinylated primary antibodies were visualized using the Vector Vecstain ABC kit0 (Vector Laboratories) using Oiamtnobenzidene (DAB [DAKO]) as a substrate.
Magnetic Resonance Imaging
[0127) Wild-type mice were intra-venously injected with IgG Isotype control (25mg kg) or MARl antibody (25mg kg)prior to and subsequently to subjecting the mice to a stroke5 event. Mice were initially anacstheti-¾d with approximately 3% v/v Isoflurane in a 1: 1 mixture of medical grade air and oxygen. Anesthesia was maintained throughout scanning with 0.25-1.5% v/v Isoflurane through a nosecone placed over the animal's snout and respiration was continuously monitored throughout the experiment with a pressure sensitive probe positioned over the animal's diaphragm. Anaesthetized animals were laid0 supinely on a purpose built animal holder and their head fixed into position with ear and bite bars. A surface receive coil was placed over the animals head and the cradle was inserted into a transmit coil fixed inside a BGA12S gradient set for imaging with a 4.7
Tesla Broker Biospcc 47/30 scanner. The scanning protocol consisted of a 3-plane localizer sequence followed by multi-slice axial, coronal and sagittal scout images to ascertain the orientation and position of the brain. To calculate a T2 map, three T2- weighted images were acquired using a rapid acquisition, relaxation enhanced (RARE) sequence with the following imaging parameters: recovery time (TR) = 3000 ms, RARE factor = 8, field of view (FOV) - 12.8 x 12.8 mm2, matrix size = 128 x 128, in-plane resolution = 100 x 100 μιη2, number of slices = 15, slice thickness = 0.6 mm, averages (NEX) = 16 and total scan time of 12 minutes. The effective echo times (TEeff) were: 45 ms, 60 ms and 80 ms. Diffusion weighted images (DWI) were also acquired with the following parameters: TR =3000 ms, TE = 60 ms, FOV = 12.8 x 12.8 mm2, matrix size - 128 x 128, in-plane resolution -100 x 100 urn2, number of slices = 6, slice thickness * 0.6 mm, number of repetitions ( R) -4 and total scan time of 12 minutes 48 seconds per repetition. A single bO and diffusion image where acquired with the following diffusion parameters: = 7 ms, 0 - 14 ms and b-value■ 1500 s/tnm Images were analyzed for infarct volume using Matlab R2010b (Mathworks).
MTS Viability Assay
(0128] CellTiter 96 (Registered Trademark) AQueous MTS Reagent (Promega) was prepared according to manufacturer's instructions. Briefly, this involved 42mg of MTS Reagent powder dissolved in 1 ml Dulbecco's PBS solution, pH to 6.0-6.5 and filter sterilized. For viability assays, 20 parts MTS solution was combined with 1 part phenazine methosulfate (PMS, Sigma) and 1/5 final well volume was added to cell wells in triplicate (i.e. 80μΙ MTS PMS in 400uJ well). MTS well preparation was then allowed to incubate for 2 hours at 37*C and absorbance was measured at 490nm. A Students T-Tcst was used to test statistical significance using Graphpad Prism 5 Software.
Western Blot Analysis
[0129] Protein was extracted by removal of media and scrapping cells into a tube using PBS. Cells were spun to a pellet at 5000g (5mins, 4°C). Supernatant was removed and cells were re-suspended in 150 ul Lysis Buffer (lOuM Tris-HCl. 2% w v SDS, Protease inhibitor, PMSI) and machinated using a probe sonicator.
[0130] Protein levels were detected using Western Blot Analysis. The 12% w v resolving gel (8ml 30% w/v Acrylamide, 5ml 1.5M Tris, 200μΙ 10% w/v SDS, 6.68ml mQHjO, 200μ1 10% APS, 20μ1 TEMED) was prepared days in advance with the 5% w v stacking gel (1.67ml 30% w/v Acrylamide, 2.5ml 0.5M Tris, ΙΟΟμ! 10% w/v SDS, 5.67ml mQH20, ΙΟΟμΙ 10% w/v APS, 1 Ομΐ TEMED) poured the day the gel was run. 50 g of protein was taken with an equal volume of 2XTris-glycine sample buffer with 5% v v b- mercaptoethanol, boiled for 5 minutes at 95 degrees and loaded into the well. A ladder consisting of 2μ1 SeeBJue (Registered Trademark) Marker 2ul MagicMark (Trademark) [Invitrogen] and 6μ1 PBS was used. Gels were run at a voltage of 80mV until protein passed the stacking gel, when voltage was kept constant at 120mV. Protein was transferred from gel to membrane using a semi-dry apparatus at a constant current of 60mA per gel for 1 hour. Membranes were then blocked in 5% w/v milk powder (In TBST) for 1 hour. Primary antibodies (1/1000) were incubated at 4°C overnight on a roller. Membranes were rinsed three times with TBST and secondary antibodies (1/1000) were then incubated at RT in 5% Milk for 1 hour. Membrane were developed using ECL (Amersham) and imaged on an IQ350.
Immunohistocbemistry
[0131] Cells plated on glass cover slips were harvested for immunohistochemistry by removing cell media, rinsing three times with ice-cold PBS and fixing with 4% v/v paraformaldehyde for 15 minutes at RT. Cells were then permeabili2ed using 0.2% v/v Triton X-100 in PBS at RT for 20 minutes and rinsed three times in PBS. CAS-Block (Trademark) [Invitrogen] was then used to block at RT for 1 hour. Cells were then incubated overnight with primary antibody (1/100) in CAS-Block (Trademark). Following primary antibody binding, cells were rinsed in PBS three times and incubated 2 hours with the secondary antibody in CAS-Block (Trademark). Cells were then rinsed three times in PBS and mounted onto slides using Vcctashield (Registered Trademark) mounting media with DAPI.
EXAMPLE l
IfNART^ neurons demonstrate increased viability in response to OGD
[0132] Previous data had demonstrated that Middle Cerebral Artery Occlusion (MCAO) surgery performed on IFNAR1"'" and IFNAR2 " mice showed a decreased infarct size of IFNAR1''" (24.9*7. ! mm3), but not IFNAR2 '' (52.3 4.9mm3), mice compared to wild- type (65.1±4.8mm3) (n-6-8, PO.05, One-way ANOVA, Dunneu's Post-hoc test, Figure 1)'. To support these findinp primary cultures of IFNAR1V" and wild-type neurons were subjected to 4 hours OGD with 24 hours reperfusion and an MTT assay was performed. IFNARl^" neurons showed a significant increase in cell viability (9l.0±4.9%) compared to wild-type (49.8±0.8%) in response to OGD (n=6, *P<0.05, students t-test, Figure 1).
EXAMPLE 2
IFN-dependent STAT phosphorylation
[0133) IFN is known to induce STAT phosphorylation governed by the JAK-STAT pathway. To identify the optimal concentration of IFN required to induce STAT phosphorylation, Ml 7 cells were treated with increasing concentrations of IFNa and IFNp (1, 10, 100 and lOOOU/ml) for 15 minutes and harvested for Western blot analysis. lOOOU/ml of IFNa induced the most prominent phosphorylation of STAT-1 in contrast to IFNp treatment which induced similar levels of STAT-1 phosphorylation across all concentrations.
EXAMPLE 3
IFNa preferentially induces P-STA T-l not PSTA T-3
[0134] To investigate the STAT phosphorylation profile induced from IFN-dependant signalling, M17 cells were treated with lOOOU/ml IFNa or ΙΡΝβ (concentration selected from previous concentration-response Western blots) for a time-course of 5, 10 or 30 minutes. Western blot analysis of STAT-1 and STAT-3 phosphorylation was then conducted. IFNa treatment induced P-STAT-I across all time-points with 10 minutes treatment showing the most prominent phosphorylation. IFNp treatment also induced P- STAT-1 across all time-points with noticeable phosphorylation at 10 and 30 minutes treatment. IFNp also stimulated STAT-3 phosphorylation at all time-points but the more robust responses came earlier than that of P-STAT-1 induction (5 and 10 minutes treatment). Interestingly, IFNa treatment was causative of only minimal STAT-3 phosphorylation at 10 minutes compared to the robust P-STAT-I induction (IFNa, 10 minutes).
EXAMPLE 4
M17 cells subjected to OGD show Induction ofPSTAT-1 but not PSTAT-3 [0I3SJ STATs initiate pro-inflammatory gene transcription leading to increased cytokine release. In the OGD environment, cells are exposed to an inflammation state which contributes to the persistent cell death. To analyze if STATs play a role in the signalling events responsible for OGD-dependant cell death, cells were exposed to 3 hours OGD and a time-course reperfusion period (0, 2 and 24 hours). OGD treatment induces P-STAT-1 across all reperfusion time-points (0, 2 and 24 hours) but is most prominent at 2 hours reperfusion. Interestingly, OGD treatment did not cause STAT-3 phosphorylation across any reperfusion lime-pomis.
EXAMPLE 5
IFNamRNA levels are elevated earlier in OGD reperfusion than IFNfimRNA levels
(0136| Given the STAT-1 phosphorylation in the OGD response, the time-course of IFN mRNA transcript production was investigated. Ml 7 cells were subjected to 3 hours OGD with a time-course reperfusion (0, 0.5, 2 and 24 hours) and cellular RNA was isolated. RNA was reverse transcribed to give cDNA and RT-PCR was performed. IFNa mRNA levels were significantly increased at 2 hours reperrusion (15.1±2.3 fold change) before returning to basal levels by 24 hours. In contrast, IFNp mRNA levels were only increased alter 24 hours reperrusion (2.4*0.1 Ibid change) (iv=3, *P<0,05, One-way ANOVA, Dunnctt's Post-hoc test, Figure 2).
EXAMPLE 6
IL-6, TNF-a and ϋΛβ cytokine mRNA levels shew elevation post-OGD
J0I37] Apart from IFNa and ΙΡΝβ mRNA common pro-inflammatory cytokines IL-6, TNF-a and IL-10 were also analyzed via RT-PCR to confirm the inflammatory state created by OGD treatment. IL-6 mRNA levels were significantly increased at 0.5 and 2 hours reperfusion (266.9±10.9 and l£>4.6±39.4 fold change, respectively) before returning to basal levels by 24 hours reperfusion. TNF-a mRNA levels were significantly elevated at an earlier time-course of 0 and 0.5 hours reperfusion (10.0±3.1 and 10.1±2.8 fold change respectively) and returned to control levels by 2 hours reperfusion. IL-10 mRNA levels showed a later onset significant increase at 24 hours reperfusion (4.6*1.2 fold change). (n=3, *P.<0.05, One-way ANOVA, Dunnett's Post-hoc test, Figure 3). Figure 4 shows a summary of collated RT-PCR data.
EXAMPLE 7
Verification ofmIFNARl/2gene insert within pcDNA6.2cEM-GFP plasmid
(0138] In order to investigate the effects of IFNAR1 and IFNAR2 subunit over-expression during OGD and subsequent reperfusion, the genes were inserted into the pcDNA6.2/cEM- GFP plasmid for subsequent cellular transfection. To verify both the IFNARI and IFNAR2 gene inserts in the pJasmid, PGR using a forward plasmid-specific T7 and reverse gene- specific attB2 primers was used to amplify the gene product A 1.77kb genomic band specific for the IFNARI gene insert was identified. A 1.54kb genomic band specific for the IFNAR2 gene insert was also observed.
EXAMPLE 8
Confirmation of miFNARl/2-GFP plasmid cellular transfection [0139] To evaluate the efficiency of M17 cell-line transfection with pcDNA6.2/cEM- GFP-M1FNARI/ IFNAR2 constructs, fluorescence microscopy was used to detect GFP expression in the transient transfected Ml 7 cell-line. Results show >50% transfection efficiency, with IFNA l-GFP located in the cytoplasm and bound to the cell membrane of Ml 7 cells.
EXAMPLE 9
Over-expression of IFNARI in OOD treated Ml 7 ceils is detrimental
(0140) In order to evaluate the in vitro effect of IFNARI and IFNAR2 over-expression, Ml 7 cells were subjected to 3 hours OGD and 24 hours reperfusion. MTT assay was then used to assess cell viability. Interestingly, M17-1FNAR1 cells showed decreased viability (21.6±5.3%) in comparison to wild-type (44. ±2.1%). Furthermore. M17-IFNAR2 cells showed no difference in cell viability (39.8*5.7%) when compared to wild-type (n=4, *P<0.05, One-way ANOVA, Dunnett's Post-hoc test, Figure 5).
EXAMPLE 10
IFNAR1 is both membrane-bound and intra-nuclear in Wl neurons
[0141] Considering the difference in IFNAR1 and IFNA 2 over-expression in terms of cell viability IFNAR1 cellular trafficking was briefly investigated. Isolated CBL57/6 WT neurons were used for immunohistochemistry probed with mAb-IF ARl. Fluorescence microscopy shows membrane-bound, but more interestingly, intra-nuclear localization of the lWARl subunit.
EXAMPLE 11
H&E Staining and NeuN immunohistochemistry and
Magnetic Resonance Imaging (0142) Both procedures represent traditional methods used to assess neuronal damage following stoke. Magnetic Resonance Imaging (MRI) is a particularly powerful approach used to n l ze brain damage, as it allows an in vivo assessment of the progression of oedema and injury, done in real time. MRI scans were done on mice, 2 and then 24 hours after a stroke event. T2 images show the diffusion of water inside tissue, and are thus ideal for visualization of tissue oedema. The development of the infarct and penumbra (surrounding area of damage) is shown, with the si2e of the infarct and penumbra increasing drastically after 24 hours. Another imaging sequence used, Diffusion Weighted Imaging (DWI) allowed an assessment of the movement of water molecules within injured and uninjured tissue. This generated an Apparent Diffusion Coefficient (ADC) map, and using this sequence, brains were again measured for infarct volume. An ADC map of two sequential slices is taken of an injured brain, showing both infarct and penumbra size, in voxels. A T2 map is generated by T2-weighted Imaging of the same brain, showing again infarct and penumbra size. The two types of sequence give different values for infarct and penumbra size. To determine whether this difference is also observed through a more conventional way of measuring infarct volume (H&E sections measured through ImageJ), the infarct sizes, in voxels, of the three wild-type brains, which had been previously imaged at 24 h, are converted into mm3 (one voxel in this scanning sequence is equivalent to
100χ100χ600μηι3). A comparison of all the methods is used to obtain infarct volumes (T2- weighted imaging, DWI and H&E infarct analysis). The mean infarct volumes obtained by DWI (1.50mm3 * 0.22) and H&E analysis (2.56mm3 ± 0.13) are not statistically different (P>0.05, n=3), and both differ to the infarct volume obtained using T2-weighted imaging (5.65mm3 ± 0.48, P<0.05, n=3).
EXAMPLE 12
Role of Type- J interferon signalling in stroke outcomes (0143) To examine the role of Type- 1 interferon signalling in stroke, IFNARl ~ mice underwent mid cerebral artery occlusion (MCAO) surgery. The mice demonstrated a decreased infarct size (24.9dt7.lmra3 n**8) compared to wild-type controls (65.1±4.8mm3 n=*8). Western blot and immmiohistochemistry showed alterations in the Stat-1 and 3 phosphorylation profiles in the IFNARl^". Neuroprotection conferred by the absence of IFN signalling was confirmed in IFNAR-deficient primary cultures that were protected from cell death when exposed to oxygen glucose deprivation (0GD).
(0144] Co-culture experiments using IFNAR1"'" glia and WT neurons and WT glia and IFNARl*7' neurons were carried out in the OGD model. IFNARl "*" neurons in the presence of WT glia no longer displayed a neuroprotective phenotype suggesting the glia are a major driver of the neuroinflammatory response. In an attempt to block IFNAR signalling in vivo a blocking monoclonal antibody targeting the IFNARl receptor (IFNARl mAb) was injected into WT mice via the tail vein (0,5mg) 30 minutes prior to MCAO. This resulted in a 60% decrease in infarct size when compared to the IgG control.
[0145] Collectively these results indicate signalling through the IFNARl subunit is deleterious in stroke. Furthermore, data indicate That therapeutic agents targeting the IFNARl subunit are beneficial in reducing the severity of a ncuro-inflammatory event following stroke and in doing so limit infarct size.
EXAMPLE 13
IFNARl localization (0146J Figures 9A through C show by fluorescent irnmunohistochemistr that IFNAR1 is localized on primary cultured mouse neurons.
EXAMPLE 14
Effects on IFNAR1 mAb in a stroke model
{0147] IFNAR1'' (KO) CS7 BL6 mice exhibited reduced infarct si2e in this stroke model. The infarct size was assessed 24 hours post injury in wild-type (IFNAR+/*) and IFNAR"'" C57 BL6 mice. The results ait shown in figure 10. Figure 11 shows that a mAb specific for IFNARl (IFNARl mAb) reduces infarct size when 0.5mg mAb is given iv 1 hour prior to the stroke. The infarct volume is assessed at 24 hours post-injury. This further shows the utility of an IFNARl antagonist (e.g. on IFNARl mAb) to reduce an infarct prophylactically. (0148] Those skilled in the art will appreciate that aspects described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications to these aspects. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.
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Claims
1. A method for reducing a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, said method comprising administering to said subject an effective amount of an antagonist of Type 1 interferon-mediated signalling for a time and under conditions sufficient to prevent or attenuate neuroinflammation.
2. The method of Claim 1 wherein the neuroinflammatory response is in the brain.
3. The method of Claims 1 or 2 wherein the antagonist targets a Type 1 interferon.
4. The method of Claim 3 wherein the Type 1 interferon is IFNa or IFNp.
5. The method of Claims 1 to 2 wherein the antagonist targets the Type I interferon receptor or a component thereof.
6. The method of Claim 5 wherein the receptor component is Type 1 interferon alpha receptor 1 (IFNA I).
7. The method of Claim 1 wherein the subject is a human.
8. The method of Claim 1 or 3 or 6 wherein the antagonist is selected from a protein, small molecule, antibody including an immunoglobulin new antigen receptor (IgNAR.) specific, for a Type 1 interferon or IFNARI, a genetic molecule which down-regulates expression of a Type 1 interferon or IFNARI , a modified Type 1 interferon and a soluble IFNARI .
9. A method for reducing a neuroinflammatory response within the central nervous system (CNS) of a subject following a stroke event, said method comprising administering to said subject an antagonist of interferon alpha receptor 1 (IFNARI )-mediated signalling for a time and under conditions sufficient to prevent or ameliorate the symptoms of neuroinflammation.
10. The method of Claim 9 wherein the ncuroinflammatory response is in the brain.
11. The method of Claim 9 wherein the subject is a human.
1 2. The method of Claim 9 wherein the antagonist is selected from a protein, small molecule, antibody including an immunoglobulin new antigen receptor (IgNAR) specific for IFNARl, a genetic molecule which down-regulates 1J';NAR1, a modified form of a Type 1 interferon and a soluble IFNARl.
13. A neuroprotective formulation comprising an antagonist of Type 1 interferon- mediated signalling and one or more pharmaceutically acceptable carriers, diluents and or excipients.
14. The neuroprotective formulation of Claim 13 wherein the antagonist is selected from a protein, small molecule, antibody including an immunoglobulin new antigen receptor (IgNAR) specific, for a Type 1 interferon or IFNARl, a genetic molecule which down-regulates expression of a Type 1 interferon or IFNARl, a modified Type 1 interferon and a soluble IFNARl .
15. A medical protocol for treating acute neuronal injury following a stroke in a subject, said protocol comprising administering to said subject, from 1 to 120 minutes of the injury, a neuroprotective formulation of Claim 13 r 14.
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AU2010905239 | 2010-11-26 | ||
AU2010905239A AU2010905239A0 (en) | 2010-11-26 | Method of treatment and prophylaxis | |
AU2011902499A AU2011902499A0 (en) | 2011-06-24 | Method of treatment and prophylaxis | |
AU2011902499 | 2011-06-24 |
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PCT/AU2011/001533 WO2012068636A1 (en) | 2010-11-26 | 2011-11-25 | Methods and compositions comprising antagonists of type 1 interferon-mediated signalling for reducing a neuroinflammatory response in the central nervous system |
PCT/AU2011/001522 WO2012068630A1 (en) | 2010-11-26 | 2011-11-25 | Methods and compositions comprising antagonists of type 1 interferon-mediated signalling for reducing a neuroinflammatory response in the central nervous system following a stroke |
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US10947295B2 (en) | 2017-08-22 | 2021-03-16 | Sanabio, Llc | Heterodimers of soluble interferon receptors and uses thereof |
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WO2018136625A2 (en) * | 2017-01-20 | 2018-07-26 | Children's Medical Center Corporation | Compositions and methods for treating diseases characterized by reactive microglia mediated synapse loss |
WO2020154656A1 (en) | 2019-01-25 | 2020-07-30 | Brown University | Compositions and methods for treating, preventing or reversing age-associated inflammation and disorders |
AU2022293556A1 (en) | 2021-06-18 | 2024-01-18 | Ionis Pharmaceuticals, Inc. | Compounds and methods for reducing ifnar1 expression |
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WO2016009430A1 (en) * | 2014-07-14 | 2016-01-21 | Yeda Research And Development Co. Ltd | Reduction of aging-induced type i interferon signaling at the brain's choroid plexus or within the cns for treatment of disease or injury of the cns |
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