WO2015035414A1 - Traitement des troubles neurologiques - Google Patents

Traitement des troubles neurologiques Download PDF

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Publication number
WO2015035414A1
WO2015035414A1 PCT/US2014/054845 US2014054845W WO2015035414A1 WO 2015035414 A1 WO2015035414 A1 WO 2015035414A1 US 2014054845 W US2014054845 W US 2014054845W WO 2015035414 A1 WO2015035414 A1 WO 2015035414A1
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subject
inhibitor
cells
clip
peptide
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PCT/US2014/054845
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English (en)
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Martha Karen Newell
Richard Tobin
Lee Shapiro
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The Texas A&M University System
Scott & White Healthcare
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia

Definitions

  • Traumatic brain injury is the leading cause of death from injuries in the United States (U.S.).
  • U.S. Traumatic brain injury
  • TBI injuries are primarily the result of firearms, automobile accidents, and sports, and in the elderly, falls.
  • TBI is an urgent clinical problem.
  • the invention in some aspects is a method of treating a subject having a neurological disorder, by selectively reducing peripherally activated immune cells from the subject in an effective amount to treat the subject.
  • the peripherally activated immune cells are reduced by administering to the subject an isolated MHC class II specific CLIP inhibitor.
  • the CLIP inhibitor in some embodiments is a synthetic peptide. In other embodiments the CLIP inhibitor is a TNP peptide. In yet other embodiments the CLIP inhibitor is an siRNA. In other embodiments the peripherally activated immune cells are reduced by administering to the subject an antibody specific for the peripherally activated immune cells.
  • the neurological disorder may be, for instance, a traumatic brain injury.
  • the neurological disorder is selected from the group consisting of post- ischemia reperfusion injuries, chronic traumatic encephalopathies, autoimmune neuronal pathologies with unclear etiology, pediatric autoimmune neuropsychiatric syndrome, Sydenham's Chorea, stroke, schizophrenia, OCD, Tourette' s syndrome, post infectious encephalopathies, and aneurysm.
  • the neurological disorder is a chronic traumatic encephalopathy. In other embodiments it is a stroke.
  • the subject may be treated with the compounds of the invention at any time following a brain injury.
  • the treatment may be immediately following an injury or perceived injury.
  • the treatment may be within 5, 10, 20, 30, 40 ,or 50 minutes following the injury.
  • the subject may be treated within 1 hour, 2, 3, 4, 5, 10, 15, 20, 24, 36, 72, or 120 hours following the traumatic brain injury.
  • the subject is treated within a week, 2 weeks, 1 month, 2 months, 6 months or 1 year of the traumatic brain injury.
  • the subject is treated during an infection with a bacterial agent.
  • the subject has a bacterial infection and a CLIP inhibitor is administered to the subject systemically.
  • the subject may be, for instance, a military personnel or an athlete.
  • the method further involves using a head injury monitor to detect the presence of a head injury.
  • the head injury monitor may be selected from the group consisting of a CheckLightTM device or a X-PatchTM.
  • a device is provided according to other aspects of the invention.
  • the device has a housing; a needle connected to the housing; a source of a CLIP inhibitor; and optionally a spring for causing the needle to inject the CLIP inhibitor; wherein the source of the a CLIP inhibitor is within the housing or within a container, operably connected to the housing.
  • the devise further includes a locking mechanism for locking the spring such that the spring is not capable of releasing until the locking mechanism is disengaged.
  • the invention is a kit including a device described herein, a predetermined dose of CLIP inhibitor, and instructions for delivering the CLIP inhibitor to the subject using the device.
  • a method of treating a subject having a seizure disorder involves administering to a subject having a seizure disorder a CLIP inhibitor.
  • the CLIP inhibitor is an isolated MHC class II specific CLIP inhibitor.
  • the subject is treated within 8 hours of a seizure, within 2 hours of a seizure, within 30 minutes of a seizure, or within 10 minutes of a seizure.
  • the subject is administered at least 2 doses of CLIP inhibitor in some embodiments. In other embodiments the subject is administered at least 3 doses of CLIP inhibitor. In yet other embodiments the CLIP inhibitor is administered on a regular basis to the subject. For instance the CLIP inhibitor may be administered to the subject daily, every other day, or weekly.
  • the subject is further treated with neurotherapy.
  • a fatty acid oxidation inhibitor is administered to the subject.
  • the CLIP inhibitor is synthetic. In other embodiments the
  • CLIP inhibitor is a TNP peptide, an siRNA, or an MHC class II CLIP inhibitor.
  • the CLIP inhibitor comprises a peptide having the sequence:
  • X1RX 2 X 3 X 4 X5LX 6 7 (SEQ ID NO: 3), wherein each X is an amino acid, wherein R is Arginine, L is Leucine and wherein at least one of X2 and X3 is Methionine, wherein the peptide is not N- MRMATPLLM-C (SEQ ID NO: 1), and wherein the peptide is a CLIP displacer.
  • the peptide in some embodiments has any one or more of the following variables: Xi is Phenylalanine; X 2 is Isoleucine; X 3 is Methionine; X 4 is Alanine; X5 is Valine; X 6 is Alanine; and/or X 7 is Serine.
  • the peptide in some embodiments includes 1-5 amino acids at the N and/or C terminus.
  • the peptide may have 1-5 amino acid at the C terminus of
  • X1RX 2 X 3 X 4 X5LX 6 X 7 (SEQ ID NO: 3) and/or the peptide may have 1-5 amino acid at the N terminus of X1RX 2 X 3 X 4 X5LX 6 X 7 (SEQ ID NO: 3).
  • the peptide in other embodiments comprises FRIM X 4 VLXeS (SEQ ID NO: 6), wherein X 4 and X are any amino acid.
  • X 4 and X6 are Alanine.
  • the peptide comprises FRIMAVLAS (SEQ ID NO: 2),
  • IRIMATLAI SEQ ID NO: 4
  • FRIMAVLAI SEQ ID NO: 75
  • IRIMAVLAS SEQ ID NO: 76
  • the peptide in some embodiments has 9-20 amino acids.
  • the CLIP inhibitor comprises a peptide selected based on the subject's HLA-DR allele.
  • the methods further involve administering to the subject an MHC binding agent and/or an autophagy inhibitor.
  • the autophagy inhibitor in some embodiments is a 4-aminoquinoline or a pharmaceutically acceptable salt or prodrug thereof.
  • the autophagy inhibitor may be, for instance, chloroquine, 2-hydroxychloroquine, amodiaquine, passethylchloroquine, quinoline phosphate, or chloroquine phosphate or mixtures thereof.
  • compositions of an autophagy inhibitor, a CLIP inhibitor and/or an amino acid oxidation inhibitor are provided according to other aspects of the invention.
  • the composition further includes a carrier.
  • kits are provided according to other aspects of the invention.
  • the kit includes one or more containers housing an autophagy inhibitor, a CLIP inhibitor and/or an amino acid oxidation inhibitor and instructions for administering to a subject the autophagy inhibitor, the CLIP inhibitor and/or the amino acid oxidation inhibitor.
  • the invention is any of the compositions or combinations of compositions described herein for use in the treatment of a neurological disorder or in the manufacture of a medicament for the treatment of neurological disorder.
  • FIG. 1 A schematic of a cellular response to Traumatic Brain Injury (TBI) is presented. TBI sensitizes neurons by inducing death receptor expression. TBI also induces peripheral immune cell activation. A secondary inflammatory insult amplifies recruitment of activated peripheral cells to the brain.
  • Figure 2. A diagram depicting the effect of streptococcus pyogenes on blood- brain barrier, 24 hours post-infection is shown.
  • FIGS. 3A-G Activation of peripheral immune cells following fluid percussion injury can be reversed by targeted peptide therapy.
  • 3A the frequency of B cells in the spleens of mice is shown following FPI and sham, with or without TPP treatment (SEQ ID NO: 2) at 30 minutes after FPI. Note that TPP treatment significantly reduces the number of B cells in the spleen following FPI.
  • 3B the frequency of T cells in the spleen is significantly increased following FPI and this effect is reversed with TPP treatment given 30 minutes after FPI.
  • the number of ⁇ T cells in the spleen of mice is significantly increased following FPI and this effect is reversed by TPP treatment at 30 minutes following FPI.
  • the frequency of T regulatory cells (Tregs) is significantly increased in the spleens of mice following FPI. This effect is reversed by treatment with TPP at 30 minutes after FPI.
  • the frequency of CLIP positive B cells is decreased in the spleens of mice following FPI and treatment with peptide.
  • the frequency of live T cells is increased in the spleens of mice following FPI and that number is significantly decrease following treatment with peptide.
  • the percentage of T regulatory cells (Tregs) of CD4+ T cells in the spleens of mice remains relatively stable.
  • FIGS 4A-F Intravital microscopy showing normal (4A) and blood brain barrier permeability induced by intranasal streptococcus pyogenes (4B) or Pam-3-Cys administration (4C).
  • This figure depicts still images of videos captured in vivo, showing cerebral blood flow through the post-capillary venules within pia matter of cerebral cortex of mice. Note that both intranasal strep and Pam-3-Cys (a TLR agonist) causes leakage of the fluorescent-conjugated dextran and can be seen in perivascular space, indicating blood brain barrier permeability.
  • 4D and 4E leukocytes were selectively labeled and recorded with intravital microscopy.
  • 4D a control brain is shown indicating normal flow of these cells with minimal adhesion.
  • the leukocytes show significant adhesion and slowing.
  • FIGS 5A-C Fas expression is reversed by treatment with TPP or B cell depletion in a CLIP KO mouse.
  • mice that experienced FPI, followed 7 days later by a secondary infection with Pam-3-Cys (i.p.) show extensive Fas-labeling in the peri- lesion area.
  • TPP treatment mostly ameliorates Fas expression when it is administered 30 minutes after the FPI.
  • Fas expression is minimal in a CLIP KO mouse at 3 days after FPI.
  • FIGS 6A-B Fluorojade labeling is ameliorated by treatment with TPP.
  • TBI results in extensive Fluorojade labeling at 3 days after FPI, indicative of neuronal degeneration.
  • TPP (i.p.) treatment at 30 mins after FPI reverses most of the neuronal damage observed 3 days after FPI.
  • FIGS 7A-F Figures 7A-F.
  • GFAP-labeling in the peri-lesion area at 7 days after FPI is noticeably less than at 3 days post-FPI, indicating a healing response.
  • PAM-3-Cys administration at 7 days after FPI exacerbates the astrocyte response and prevents healing.
  • administration of TPP at 30 mins after FPI prevents the PAM-3-Cys re- induction of astrocyte activation.
  • a similar result is seen for microglial cells, where 10 days after FPI, only moderate levels of microglial activation is observed (7D).
  • FIG. 8 CD74 deficient mice are retractile to increases in proinflmmatory cytokine in the brain following TBI. Multiplex analysis of cytokine expression in the brain comparing the cytokine response to FPI in wild type C57BL/6 mice to CD74 Def mice. Note the significantly lower levels of T Fa, IF ⁇ , RANTES, and IL-13. For these experiments we used 5 C57BL/6 and 6 CD74 Def . * indicates P ⁇ 0.05.
  • FIGS 9A-E CAP treatment after FPI reduces neurodegeneration and lesion size.
  • (9A-C) Fluoro-Jade C labeling at 3 days after FPI.
  • vehicle treated mice (9A) significantly more degenerating neurons are observed compared to mice treated with CAP after FPI (9B, 9C).
  • Analysis of the size of the lesion at 3 days after FPI revealed that CAP treatment or FPI in CD74 deficient mice had a significantly smaller lesion in the anterior/posterior plane, compared to sham mice (9D).
  • Quantitative analysis of the number of Fluor- Jade C-labeled cells showed significantly less (P ⁇ .01 for both) labeled-cells in the CAP treated and FPI in CD74 deficient mice (9E).
  • Figure 11 A graph depicting the average change in weight relative to 100% in C57BL/6 mice treated with Theiler' s murine encephalomyelitis virus (TMEV) to induce seizures. Mice were either treated on day 1 (CP1), day 1 and day 2 (CP2), or dayl, day 2 and day 3 (CP3) following viral infection or with vehicle as a control (V). 5 mice were tested per group.
  • TMEV Theiler' s murine encephalomyelitis virus
  • Figure 12 A graph depicting the Rancine score for each of the group of mice according to the protocol described for Figure 11.
  • Figure 13 Panels showing the results of flow cytometric analysis are presented in Figure 13.
  • the spleen cells were stained with CDl 1 and CD45.
  • the cells were isolated from a sham infected (received no virus) mouse.
  • the top panel shows the results of the analysis of CD45 (x-axis) versus CDl lb (Y axis) and the bottom panel is ungated.
  • Figure 14 Panels showing the results of flow cytometric analysis are presented in Figure 14.
  • the spleen cells were stained with CDl 1 and CD45.
  • the cells were isolated from infected TMEV infected mice.
  • the top panel shows the results of the analysis of CD45 (x-axis) versus CDl lb (Y axis) and the bottom panel is ungated.
  • Figure 15 Panels showing the results of flow cytometric analysis are presented in Figure 15.
  • the CNS cells were stained with CDl 1 and CD45.
  • the cells were isolated from a sham infected (received no virus) mouse.
  • the top panel shows the results of the analysis of CD45 (x-axis) versus CDl lb (Y axis) and the bottom panel is ungated.
  • Figure 16 Panels showing the results of flow cytometric analysis are presented in Figure 16.
  • the CNS cells were stained with CDl 1 and CD45.
  • the cells were isolated from TMEV infected mice.
  • the top panel shows the results of the analysis of CD45 (x- axis) versus CDl lb (Y axis) and the bottom panel is ungated.
  • Figures 17A-B Pictures of stained cells from mouse brain.
  • Figure 17A shows quiescent resident microglia in hippocampus from sham infected mice.
  • Figure 17B shows activated microglia and macrophage in hippocampus of TMEV-infected mice.
  • Figures 18A-H Selective expansion of splenic immune cell subsets in response to FPL
  • (18A) The number of cells in the spleens (cellularity) of C57BL/6 mice 24 hours following sham surgery or FPL
  • FIGS 19A-E Differential expression of MHCII and CLIP on B and Gamma
  • Delta T cells in response to FPI The mean fluorescence intensity (MFI) of CLIP on the surface of B cells in the spleens of C57BL/6 mice 24 hours following sham surgery or FPL
  • MFI mean fluorescence intensity
  • 19B The MFI of MHCII on the surface of B cells in the spleens of C57BL/6 mice 24 hours following sham surgery or FPI.
  • (19C) The MFI of CLIP on the surface of ⁇ T cells in the spleens of C57BL/6 mice 24 hours following sham surgery or FPI.
  • FIGS. 21A-D FPI- induced alterations in peripheral lymphcytes require CD74.
  • the invention in aspects, involves new methods for treating neurological disorders. It has been discovered that peripheral immune cells play a key role in neurological disorders, such as traumatic brain injury, stroke and others, and that these disorders can be prevented, treated and/or reversed using the therapeutic strategies described herein.
  • a peripheral immune response includes both an innate, acute response to infection or insult as well as a specific response involving the antigen specific arm of the adaptive, specific immune response that includes B and T lymphocytes.
  • PAMPs or DAMPS pathogen or danger associated molecular patterns
  • TLR Toll-Like Receptors
  • this expansion includes subsets known as T regulatory (Tregs) cells and ⁇ T cells, and appears to foster innate immunity and acute inflammation. Thereafter, there is a slower transition to the more specific activation of antigen specific cells, including antibody producing B cells and/or antigen- specific T effector (Teff) cells.
  • T regulatory T regulatory
  • Teff antigen-specific T effector
  • the Treg populations appear to dampen autoreactive T cells and improve the clinical outcome.
  • the Tregs delay recovery and Teff, presumably autoreactive and specific for brain antigens, improve the outcome, such that
  • Tregs are beneficial.
  • at least two different populations of ⁇ T cells distinguished by their respective ⁇ T cell receptors, can be either destructive or beneficial subsequent to infection or injury, in the periphery as well as in the CNS.
  • TLR activation integrates innate and adaptive immune responses by influencing the nature and the strategic group of cytokines produced as a result of infection or injury, including the pro-inflammatory cytokines IL-1, IL-6, and TNFa, or the anti- inflammatory IL-10, IL-4, and TGFP cytokines.
  • the timing and the nature of the cytokines released during an immune response to a great extent will determine the outcome from traumatic brain injury.
  • Chemokines were originally identified by their chemo-attracting functions, but it is now known that they also are involved with additional immune-modulatory activities. They direct leukocyte migration by forming a chemical gradient and by inducing integrin expression on the cell surface, thereafter immobilizing the CCL5 cell to a CCR5 target cell.
  • CCL5 also known as RANTES, is predominantly a T cell chemoattractant for CCR5-bearing cells (the CCL5/CCR5 Axis).
  • CCL5 is expressed primarily on T cells, particularly "Thl" cells, in inflammatory diseases, and CCR5 has been shown to be expressed on B cells, Tregs, and ⁇ T cells during infection, making these interactions potentially relevant in brain trauma pathogenesis.
  • targeting peripheral immune activation provides a rationale therapeutic approach for treating post-ischemia reperfusion injuries, chronic traumatic encephalopathies, autoimmune neuronal pathologies with unclear etiology, pediatric autoimmune neuropsychiatric syndrome, Sydenham's Chorea, stroke, schizophrenia, OCD, Tourette' s syndrome, post infectious encephalopathies, and aneurysm.
  • the invention involves several important discoveries. For instance, it was found that targeting the elimination of selective, peripherally activated immune cell populations is useful for preventing neuronal cell damage.
  • a method of treating a subject having a neurological disorder by selectively reducing peripherally activated immune cells from the subject in an effective amount to treat the subject is provided.
  • Several methods can be used in order to reduce the peripherally activated immune cells in the subject.
  • the subject can be treated with a CLIP inhibitor, as described in more detail below.
  • the cells can be targeted using antibodies that are specific for cell surface markers.
  • Peripheral immune cell activation is an early event after TBI and that several systemic components contribute to detrimental effects mediating CNS pathology.
  • the TBI sensitizes glia and neurons, leaving them susceptible to secondary insults.
  • Such secondary insults can include common infectious or inflammatory stimuli that typically show minimal to no neuropathogenic response despite their ability to cause blood brain barrier permeability (Fig. 5).
  • traumatic brain injury induces activation of B and some T cells, including ⁇ T-cells that can enter the brain via compromised blood brain barrier.
  • Peripheral pro-inflammatory cells express death- inducing ligands and chemokine receptors that guide them to target the neurons that express these death receptors.
  • the expression of these cell surface markers can be used to selectively eliminate peripherally activated immune cells in order to prevent or reverse the neuronal damage.
  • CLIP inhibitors i.e. a death-inducing peptide
  • therapeutic use of selective immune cell depletion using highly specific therapeutic antibodies is an important component of the invention.
  • CLIP inhibitors i.e. a death-inducing peptide
  • CLIP inhibitors can target pro-inflammatory, MHCII-expressing immune cells by causing MHCII-mediated death of pro-inflammatory antigen presenting cells.
  • MHCII-mediated cell death has been described as a part of T cell recognition resulting in both T cell activation and the death of antigen presenting cells.
  • Our preliminary data suggest that traumatic brain injury activates pro-inflammatory peripheral immune cells.
  • Such immune cells are implicated in detrimental immune responses in the CNS, culminating in permanent loss of neurons following traumatic brain injury.
  • These peptides or depleting antibodies can be used to eliminate the expanded subsets of peripherally activated immune cells as novel therapies for TBI and other neuropathologies.
  • CLIP inhibitors ameliorate Theiler's virus-induced seizures and thus are useful for treating epilepsy.
  • Epilepsy is a disease effecting over two million people in the United States.(Hirtz D, Thurman DJ, Gwinn-Hardy K, Mohamed M, Chaudhuri AR, Zalutsky R. How common are the "common” neurological disorders?. Neurology, 2007, 68:326-337). It is a neurological condition characterized by the occurrence of repeated seizures. (Epocrates Premium. "Overview of Seizure Disorder” Updated: 2013-05-23.
  • the mouse model provides a platform for identifying potential therapeutics for the treatment of epilepsy. It is demonstrated herein that synthetic CLIP peptides can completely amelioarate seizure activity in a TMEV model of virus-induced epilepsy.
  • a CLIP inhibitor as used herein is any molecule that reduces the association of a CLIP molecule with MHC, for instance, by binding to the MHC and blocking the CLIP-MHC interaction or inhibiting the expression of CLIP.
  • the CLIP inhibitor may function by displacing CLIP from the surface of a CLIP molecule expressing cell.
  • a CLIP molecule expressing cell is a cell that has MHC class I or II on the surface and includes a CLIP molecule within that MHC.
  • Such cells include, for example, epithelial cells, endothelial cells, and cells of the vascular endothelium.
  • the CLIP molecule refers to intact CD74 (also referred to as invariant chain) or intact CLIP, as well as the naturally occurring proteolytic fragments thereof.
  • Intact CD74 or intact CLIP refer to peptides having the sequence of the native CD74 or native CLIP respectively.
  • the CLIP molecule is one of the naturally occurring proteolytic fragments of CD74 or CLIP in some embodiments.
  • the CLIP molecule may be, for example, at least 90% homologous to the native CD74 or CLIP molecules.
  • the CLIP molecule may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the native CD74 or CLIP molecules
  • An example of native CLIP molecule is MRMATPLLM (SEQ ID NO: 1), and in three-letter abbreviation as: Met Arg Met Ala Thr Pro Leu Leu Met (SEQ ID NO: 1).
  • An example of native CD74 molecule is MHRRRSRSCR EDQKPVMDDQ RDLISNNEQL
  • CLIP inhibitors include peptides and small molecules that can replace CLIP.
  • the CLIP inhibitor is a peptide.
  • a number of peptides useful for displacing CLIP molecules are described in U.S. Patent Application Nos.: 12/508,543 (publication number US-2010-0166782-A1); 12/739459 (publication number US -2011- 0118175) and 12/508,532 (publication number US-2010-0166789-A1) each of which is herein specifically incorporated by reference. For instance a number of these peptides are "thymus nuclear protein (TNP)" peptides.
  • TNP thymus nuclear protein
  • CLIP inhibitors include for instance but are not limited to competitive CLIP fragments, MHC class II binding peptides and peptide mimetics.
  • the CLIP inhibitor includes peptides and peptide mimetics that bind to MHC class II and displace CLIP.
  • an isolated peptide comprising X1RX 2 X3X 4 X5LX6X7 (SEQ ID NO: 3), wherein each X is an amino acid, wherein R is Arginine, L is Leucine and wherein at least one of X 2 and X 3 is Methionine, wherein the peptide is not N- MRMATPLLM-C (SEQ ID NO: 1), and wherein the peptide is a CLIP displacer is provided according to the invention.
  • X refers to any amino acid, naturally occurring or modified.
  • the Xs referred to the in formula X 1 RX 2 X 3 X 4 X5LX 6 7 (SEQ ID NO: 8) have the following values:
  • X ! is Ala, Phe, Met, Leu, He, Val, Pro, or Trp
  • X 2 is Ala, Phe, Met, Leu, lie, Val, Pro, or Trp
  • X 3 is Ala, Phe, Met, Leu, lie, Val, Pro, or Trp.
  • X 5 is Ala, Phe, Met, Leu, lie, Val, Pro, or Trp
  • X 7 is Ala, Cys, Thr, Ser, Gly, Asn, Gin, Tyr.
  • the peptide preferably is FRIM X 4 VLX 6 S (SEQ ID NO: 6), such that X 4 and X 6 are any amino acid and may be Ala.
  • FRIMAVLAS SEQ ID NO: 5
  • Other preferred peptides of the invention include: IRIMATLAI (SEQ ID NO: 4), FRIMAVLAI (SEQ ID NO: 75), and IRDVIAVLAS (SEQ ID NO: 76).
  • the minimal peptide length for binding HLA-DR is 9 amino acids. However, there can be overhanging amino acids on either side of the open binding groove. For some well-studied peptides, it is known that additional overhanging amino acids on both the N and C termini can augment binding. Thus the peptide may be 9 amino acids in length or it may be longer. For instance, the peptide may have additional amino acids at the N and/or C terminus. The amino acids at either terminus may be anywhere between 1 and 100 amino acids. In some embodiments the peptide includes 1-50, 1-20, 1-15, 1- 10, 1-5 or any integer range there between.
  • N- FRIMAVLAS-C SEQ ID NO: 7
  • N-X 1 RX 2 X3X 4 X5LX6X7-C ' SEQ ID NO: 9
  • the -C and -N refer to the terminus of the peptide and thus the peptide is only 9 amino acids in length.
  • the 9 amino acid peptide may be linked to other non-peptide moieties at either the -C or -N terminus or internally.
  • Other peptides useful as CLIP inhibitors, including some TNP peptides and synthetic peptides are shown in Table 1.
  • the peptides may be mixed with cystatin A and/or histones and in other instances the composition is free of cystatin A or histones.
  • Histone encompasses all histone proteins including HI, H2A, H2B, H3, H4 and H5.
  • the peptide may be cyclic or non-cyclic. Cyclic peptides in some instances have improved stability properties. Those of skill in the art know how to produce cyclic peptides.
  • the peptides may also be linked to other molecules.
  • the peptide and molecule may be linked directly to one another (e.g., via a peptide bond); linked via a linker molecule, which may or may not be a peptide; or linked indirectly to one another by linkage to a common carrier molecule, for instance.
  • linker molecules may optionally be used to link the peptide to another molecule.
  • Linkers may be peptides, which consist of one to multiple amino acids, or non-peptide molecules.
  • Examples of peptide linker molecules useful in the invention include glycine-rich peptide linkers (see, e.g., US 5,908,626), wherein more than half of the amino acid residues are glycine.
  • glycine-rich peptide linkers consist of about 20 or fewer amino acids.
  • the peptide for instance, may be linked to a PEG or TEG molecule.
  • a PEG or TEG molecule is referred to as a PEGylated or TEGylated peptide.
  • the CLIP inhibitor is an inhibitory nucleic acid such as a small interfering nucleic acid molecule such as antisense, RNAi, or siRNA
  • CD74 CLIP molecule
  • Small interfering nucleic acid include, for example: microRNA
  • siRNA small interfering RNA
  • dsRNA double-stranded RNA
  • shRNA short hairpin RNA
  • siNA useful in the invention can be unmodified o chemically-modified.
  • An siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. Such methods are well known in the art. Exemplary single stranded regions of siRNA for CLIP are shown below. The invention contemplates others as well.
  • AGAACAAAAAAAAAAAAAA (SEQ ID NO: 82)
  • one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target RNA or a portion thereof
  • the second strand of the double- stranded siNA molecule comprises a nucleotide sequence identical to the nucleotide sequence or a portion thereof of the targeted RNA.
  • one of the strands of the double- stranded siNA molecule comprises a nucleotide sequence that is substantially complementary to a nucleotide sequence of a target RNA or a portion thereof, and the second strand of the double- stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the target RNA.
  • each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
  • an siNA is an shRNA, shRNA-mir, or microRNA molecule encoded by and expressed from a genomically integrated transgene or a plasmid-based expression vector.
  • a molecule capable of inhibiting mRNA expression, or microRNA activity is a transgene or plasmid-based expression vector that encodes a small-interfering nucleic acid.
  • Such transgenes and expression vectors can employ either polymerase II or polymerase III promoters to drive expression of these shRNAs and result in functional siRNAs in cells. The former polymerase permits the use of classic protein expression strategies, including inducible and tissue- specific expression systems.
  • transgenes and expression vectors are controlled by tissue specific promoters.
  • transgenes and expression vectors are controlled by inducible promoters, such as tetracycline inducible expression systems.
  • inhibitor molecules that can be used include ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, antibodies, and aptamers and modified form(s) thereof directed to sequences in gene(s), RNA transcripts, or proteins.
  • Antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine Br. J.
  • neoplastic reversion was obtained using a ribozyme targeted to an H-Ras mutation in bladder carcinoma cells (Feng et al., Cancer Res. 55(10):2024-8, 1995).
  • Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994;
  • Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al., Nucleic Acids Symp Ser. (29): 121-2, 1993).
  • the invention involves methods for treating a subject.
  • a subject shall mean a human or vertebrate mammal including but not limited to a dog, cat, horse, goat and primate, e.g., monkey.
  • the invention can also be used to treat diseases or conditions in non-human subjects.
  • the subject is a human.
  • the subject has a neurological disorder.
  • a neurological disorder refers to a disorder of the central nervous system.
  • Neurological disorders include but are not limited to traumatic brain injury, post-ischemia reperfusion injuries, chronic traumatic encephalopathies, autoimmune neuronal pathologies with unclear etiology, pediatric autoimmune neuropsychiatric syndrome, Sydenham's Chorea, stroke, schizophrenia, OCD, Tourette's syndrome, post infectious encephalopathies, and aneurysm.
  • the neurological disorder may be caused by a number of different sources. For instance, traumatic brain injury may be associated with military accidents, sports injuries, car accidents, etc. Schizophrenia may be caused, at least in part, by genetic factors. While the cause of the neurological disorder varies, a common mechanism is useful for treating the disorders because it involves targeting the peripheral immune cells, which are common to all of these disorders.
  • treat, treated, or treating when used with respect to a disorder refers to a prophylactic treatment which increases the resistance of a subject to development of the disease or, in other words, decreases the likelihood that the subject will develop the disease as well as a treatment after the subject has developed the disease in order to fight the disease, prevent the disease from becoming worse, or slow the progression of the disease compared to in the absence of the therapy.
  • the dosages of known therapies may be reduced in some instances, to avoid side effects.
  • the CLIP inhibitor can be administered in combination with other therapeutic agents and such administration may be simultaneous or sequential.
  • the other therapeutic agents When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time.
  • the administration of the other therapeutic agent and the CLIP inhibitor can also be temporally separated, meaning that the therapeutic agents are administered at a different time, either before or after, the administration of the CLIP inhibitor. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.
  • the active agents of the invention are administered to the subject in an effective amount for treating disorders such as neurological disorders.
  • An "effective amount", for instance, is an amount necessary or sufficient to realize a desired biologic effect.
  • An effective amount for treating neurological disorders may be an amount sufficient to reduce neurological deficits and/or to reduce blood brain barrier permeability and/or to reduce circulating peripheral cells.
  • an effective amount is that amount of a compound of the invention alone or in combination with another medicament, which when combined or co-administered or administered alone, results in a therapeutic response to the disease, either in the prevention or the treatment of the disease.
  • the biological effect may be the amelioration and or absolute elimination of symptoms resulting from the disease. In another embodiment, the biological effect is the complete abrogation of the disease, as evidenced for example, by the absence of a symptom of the disease.
  • the cells are exposed to a fatty acid metabolism inhibitor.
  • Metabolic disruption of fatty acids can be achieved using inhibitors of fatty acid metabolism.
  • a "fatty acid metabolism inhibitor,” as used herein, is a compound able to inhibit (e.g., prevent, or at least decrease or inhibit the activity by an order of magnitude or more) a reaction within the fatty acid metabolism pathway, such as an enzyme-catalyzed reaction within the pathway.
  • the inhibitor may inhibit the enzyme, e.g., by binding to the enzyme or otherwise interfering with operation of the enzyme (for example, by blocking an active site or a docking site, altering the configuration of the enzyme, competing with an enzyme substrate for the active site of an enzyme, etc.), and/or by reacting with a coenzyme, cofactor, etc. necessary for the enzyme to react with a substrate.
  • the fatty acid metabolism pathway is the pathway by which fatty acids are metabolized within a cell for energy (e.g., through the synthesis of ATP and the breakdown of fatty acids into simpler structures, such as CO 2 , acyl groups, etc.) or to produce a carbohydrate source.
  • inhibitors of fatty acid metabolism include inhibitors of fatty acid oxidation, fatty acid transporter inhibitors, reductase inhibitors, and isomerase inhibitors within the fatty acid metabolism pathway.
  • the fatty acid metabolism inhibitor in some embodiments is an inhibitor of fatty acid oxidation, a fatty acid transporter inhibitor, a reductase inhibitor, or an isomerase inhibitor within the fatty acid metabolism pathway.
  • the reductase is 2,4-dienoyl-CoA reductase.
  • the isomerase is 2,4-dienoyl-CoA isomerase.
  • the inhibitor of fatty acid metabolism is an inhibitor of fatty acid oxidation and is any one or more of the following: oxirane carboxylic acid compound, such as etomoxir (2-(6-(4-chlorophenoxy)-hexyl)-oxirane-2- carboxylic acid ethyl ester), 2-(4-(3-chlorophenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(4-(3-trifluoromethylphenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(5(4-chlorophenoxy)-pentyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(3,4- dichlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(4-fluorophenoxy)-
  • the fatty acid metabolism pathway includes several enzymatic reactions, which use various enzymes such as reductases or isomerases. Specific examples of enzymes within the fatty acid metabolism pathway include 2,4-dienoyl-CoA reductase, 2,4- dienoyl-CoA isomerase, butyryl dehydrogenase, etc, as further discussed below.
  • the fatty acid metabolism inhibitor is an inhibitor able to inhibit a beta- oxidation reaction in the fatty acid metabolism pathway.
  • the inhibitor is an inhibitor for a fatty acid transporter (e.g., a transporter that transports fatty acids into the cell, or from the cytoplasm into the mitochondria for metabolism).
  • the inhibitor may react or otherwise inhibit key steps within the fatty acid metabolism pathway.
  • the inhibitor may be an inhibitor of fatty acids as a source of energy in the mitochondria.
  • the inhibitor may inhibit the breakdown of intermediates such as butyryl CoA, glutaryl CoA, or isovaleryl CoA.
  • 2,4-dienoyl-CoA reductase is an enzyme within the fatty acid metabolism pathway that catalyzes reduction reactions involved in the metabolism of polyunsaturated fatty acids.
  • Certain fatty acids are substrates for 2,4-dienoyl-CoA reductases located within the mitochondria.
  • fatty acids may be transported into the mitochondria through uncoupling proteins.
  • the uncoupling protein may, in certain instances, increase the mitochondrial metabolism to increase the availability of fatty acids within the mitochondria and/or increase the throughput of beta- oxidation within the mitochondria.
  • the enzyme 2,4-dienoyl-CoA isomerase is an enzyme within the fatty acid metabolism pathway that catalyzes isomerization of certain fatty acids.
  • ROI reactive oxygen intermediates
  • fatty acid metabolism inhibitors compounds useful for inhibiting fatty acid metabolism are also useful for altering cellular production of reactive oxygen; compounds described in reference to fatty acid metabolism inhibition should also be understood herein to be able to alter reactive oxygen production within a cell.
  • fatty acid metabolism inhibitors By altering the ability of a cell to metabolize a fatty acid, the ability of the cell to produce reactive oxygen may also be affected, since one pathway for a cell to produce reactive oxygen intermediates is through the metabolism of fatty acids.
  • the production of reactive oxygen can be affected by exposing a cell to, or removing a cell from, a fatty acid metabolism inhibitor.
  • the inhibitor of fatty acid metabolism may be an inhibitory nucleic acid.
  • the inhibitory nucleic acid may be, for instance, specific for an enzyme selected from the group consisting of 2,4-dienoyl-CoA reductase, 2,4-dienoyl-CoA isomerase, and butyryl dehydrogenase.
  • the inhibitor of fatty acid metabolism is oxamate.
  • the oxamate may be, for instance an alkyl oxamate such as, ethyl oxamate or sodium oxamate.
  • the inhibitor of fatty acid metabolism is a compound having the following structure:
  • the fatty acid inhibitor is an oxamate including, for example, each of the following:
  • the method involves the use of a fatty acid metabolism inhibitor that is an oxirane carboxylic acid compound capable of inhibiting fatty acid metabolism, or a pharmacologically acceptable salt thereof in some embodiments.
  • a fatty acid metabolism inhibitor that is an oxirane carboxylic acid compound capable of inhibiting fatty acid metabolism, or a pharmacologically acceptable salt thereof in some embodiments.
  • the subject may not have an indication otherwise indicated for treatment with the compound.
  • the oxirane carboxylic acid compound has the formula:
  • R5 represents a hydrogen atom, a halogen atom, a 1-4C alkyl group, a 1-4C alkoxy group, a nitro group or a trifluoromethyl group
  • R6 has one of the meanings of R5
  • R7 represents a hydrogen atom or a 1-4C alkyl group
  • Y represents the grouping— O— (CH 2 ) m —
  • m is 0 or a whole number from 1 to 4
  • n is a whole number from 2 to 8 wherein the sum of m and n is a whole number from 2 to 8.
  • R 6 is a hydrogen atom
  • m is 0, and n is 6.
  • R7 is an ethyl group.
  • the oxirane carboxylic acid compound is etomoxir in some embodiments.
  • etomoxir i.e., 2-(6-(4-chlorophenoxy)- hexyl)-oxirane-2-carboxylic acid ethyl ester.
  • examples of other oxirane carboxylic acid compounds useful in the invention are 2-(4-(3-chlorophenoxy)-butyl)-oxirane-2- carboxylic acid ethyl ester, 2-(4-(3-trifluoromethylphenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(5(4-chlorophenoxy)-pentyl)-oxirane-2-carboxylic acid ethyl ester, 2- (6-(3,4-dichlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(4- fluorophenoxy)-hexyl)-oxirane-2-carboxylic acid eth
  • oxirane carboxylic acid compounds including etomoxir
  • Horst Wolf and Klaus Eistetter in United States Patent 4,946,866 for the prevention and treatment of illnesses associated with increased cholesterol and/or triglyceride concentration
  • Horst Wolf in United States Patent 5,739,159 for treating heart insufficiency.
  • the preparation of oxirane carboxylic acid compounds, and their use for blood glucose lowering effects as an ant diabetic agent, is described in Jew et al United States Patent 6,013,666.
  • Etomoxir has been described as an inhibitor of mitochondrial carnitine palmitoyl transferase-I by Mannaerts, G. P., L. J. Debeer, J.
  • fatty acid metabolism inhibitors include fatty acid transporter inhibitors, beta-oxidation process inhibitors, reductase inhibitors, and/or isomerase inhibitors within the fatty acid metabolism pathway.
  • Specific examples of other fatty acid metabolism inhibitors include, but are not limited to, cerulenin, 5- (tetradecyloxy)-2-furoic acid, oxfenicine, methyl palmoxirate, metoprolol, amiodarone, perhexiline, aminocamitine, hydrazonopropionic acid, 4-bromocrotonic acid, trimetazidine, ranolazine, hypoglycin, dichloroacetate, methylene cyclopropyl acetic acid, and beta-hydroxy butyrate.
  • the inhibitor may be a non- hydrolyzable analog of carnitine.
  • the fatty acid metabolism inhibitor has the structure:
  • each of R15 and R1 ⁇ 2 independently comprises organic moiety.
  • either or both of R15 and R1 ⁇ 2 may independently be an alkyl, such as a straight- chain alkyl, for instance, methyl, ethyl, propyl, etc.
  • R i6 may have at least 5 carbon atoms, at least 10 carbon atoms, or at least 15 or more carbon atoms.
  • R 1 ⁇ 2 may be a tetradecyl moiety.
  • R 16 may include an aromatic moiety, for example, a benzene ring.
  • Ri6 may have the structure:
  • Ri 7 may be a an alkyl, such as a straight-chain alkyl.
  • Ar may be a benzene ring or a derivative thereof, i.e., having the structure:
  • each of R 18 , R 19 , R 2 o, R21, and R 2 2 is hydrogen, a halogen, an alkyl, an alkoxy, etc.
  • the fatty acid metabolism inhibitor has the structure:
  • each of R23, R24, R25, R26, R27 28 and R29 independently comprises hydrogen, a halogen, or an organic moiety, such as an alkyl, an alkoxy, etc.
  • R 2 3 and R 2 4 together may define an organic moiety, such as a cyclic group.
  • the fatty acid metabolism inhibitor may have the structure:
  • R30 comprises an organic moiety, such as an alkyl, an alkoxy, an aromatic moiety, an amide, etc.
  • An example, of R30 is:
  • Ar 2 comprises an aromatic moiety, such as a benzene ring or a benzene derivative, as previously described.
  • the cells may be exposed to an agent that inhibits the synthesis or production of one or more enzymes within the fatty acid metabolism pathway. Exposure of the cells to the agent thus inhibits fatty acid metabolism within the cell.
  • an inhibitory oligonucleotide such as a RNAi or antisense oligonucleotide may be used that selectively binds to regions encoding enzymes present within the fatty acid metabolism pathway, such as 2,4-dienoyl-CoA reductase or 2,4-dienoyl-CoA isomerase.
  • agents that inhibit enzymes of the fatty acid metabolism pathway include enzymes of the fatty acid metabolism pathway expression inhibitors.
  • a enzymes of the fatty acid metabolism pathway expression inhibitor as used herein is molecule that knocks down expression of an enzyme of the fatty acid metabolism pathway.
  • the invention also features the use of small nucleic acid molecules, referred to as short interfering nucleic acid (siNA).
  • siNA short interfering nucleic acid
  • the diverse array of suppression strategies that can be employed includes the use of DNA and/or RNA aptamers that can be selected to target, for example, a protein of interest such as enzymes of the fatty acid metabolism pathway.
  • 2,4-dienoyl-CoA reductase has been described in for instance Koivuranta et al Biochemical Journal 1994, 304, p. 787. It is also disclosed in NCBI gene ID 1666 (DECR1) as well as NCBI genbank Accession number U78302. The sequence of 2,4- dienoyl-CoA isomerase is disclosed in NCBI gene ID 1891 (ECH1).
  • Fatty acid oxidation inhibitors also include dichloroacetate (DCA) compounds.
  • a DCA compound as used herein is a compound that is a structural and functional analog of DCA, as well as DCA.
  • DCA is a small molecule mimetic and antagonist of pyruvate, a key metabolic intermediate derived from glucose metabolism in the cytosol. When pyruvate levels are low, thereby signaling conditions of starvation, the cell will initiate fatty acid oxidation. The process begins with the activation of the enzyme pyruvate dehydrogenase kinase that phosphorylates pyruvate dehydrogenase so that the metabolism of glucose-derived carbons is halted in favor of an alternate carbon source, fat.
  • a glycolytic inhibitor may also be used in the methods of the invention.
  • Glycolytic inhibitors are inhibitors of gluconeogenesis.
  • Preferred glycolytic inhibitors are 2-deoxyglucose compounds, defined herein as homologs, analogs, and/or derivatives of 2-deoxy-D-glucose.
  • Glycolytic inhibitors particularly useful herein can have the formula:
  • Rc >; Rio, Rn, Ri 2 , and Ri 3 are herein; wherein X represents an O or S atom; R 9 represents a hydrogen atom or a halogen atom; Ri 0 represents a hydroxyl group, a halogen atom, a thiol group, or CO-Re; Rn, R1 2 , and Ri 3 each represent a hydroxyl group, a halogen atom, or CO- Ri 4 , Rn represents an alkyl group of from 1 to 20 carbon atoms, and at least two of Rn, R 12 , and Ri 3 are hydroxyl groups.
  • the 2-deoxyglucose compound is 2-deoxy-D-glucose.
  • the cells are exposed to an autophagy inhibitor.
  • An autophagy modulator as used herein, is a lysosomotropic agent, meaning that it accumulates preferentially in the lysosomes of cells in the body and blocks pathways involved in breakdown of cellular components.
  • An autophagy inhibitor as used herein, is any compound which blocks the collection or metabolism of lipids in the lysosome. The inhibitor is effective for killing cells by inhibiting autophagy in cells that depend on autophagy to survive. While no one knows exactly the mechanism by which autophagy inhibitors function, it may well be through the inhibition of the acidic hydrolases (enzymes in the lysosomes) that are necessary to break down proteins, lipids, etc. for processing and removal by increasing the pH to decrease the necessary acidity for the enzymes to work.
  • the autophagy inhibitor is selected from the group consisting of: chloroquine compounds, 3-methyladenine, bafilomycin Al, 5-amino-4- imidazole carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1, analogues of cAMP, and drugs which elevate cAMP levels, adenosine, N6-mercaptopurine riboside, wortmannin, and vinblastine.
  • AICAR 5-amino-4- imidazole carboxamide riboside
  • the autophagy inhibitor is preferably a chloroquine compound.
  • Chloroquine is a synthetically manufactured drug containing a quinoline nucleus (The Merck Index, p. 2220, 1996).
  • the chloroquine compounds useful according to the invention include chloroquine analogs and derivatives. A number of chloroquine analogs and derivatives are well known.
  • suitable compounds include but are not limited to chloroquine, chloroquine phosphate, hydroxychloroquine, chloroquine diphosphate, chloroquine sulphate, hydroxychloroquine sulphate, quinacrine, primaquine, mefloquine, halofantrine, lumefantrine and tafenoquine or enantiomers, derivatives, analogs, metabolites, pharmaceutically acceptable salts, and mixtures thereof.
  • Chloroquine and hydroxychloroquine are generally racemic mixtures of (-)- and (+)-enantiomers.
  • the (-)-enantiomers are also known as (R)-enantiomers (physical rotation) and 1 -enantiomers (optical rotation).
  • the (+)-enantiomers are also known as (S)-enantiomers (physical rotation) and r-enantiomers (optical rotation).
  • the (-)-enantiomer of chloroquine is used.
  • hydroxychloroquine can be prepared by procedures known to the art.
  • the compounds of the invention may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism, and/or optical isomerism.
  • the invention covers any tautomeric, conformational isomeric, optical isomeric and/or geometric isomeric forms of the compounds described herein, as well as mixtures of these various different forms.
  • the autophagy inhibitor useful in the invention is a 4- aminoquinoline.
  • 4-aminoquinolines include compounds having the following structure:
  • each instance of the dotted line independently represents a single bond or a double bond which can be in the cis or trans configuration
  • Ri is 1 or 2 hydrogens, alkyl, cycloalkyl, aryl, substituted alkyl, substituted cycloalkyl or substituted aryl.
  • the 4-aminoquinoline has the following structure:
  • each instance of the dotted line independently represents a single bond or a double bond which can be in the cis or trans configuration
  • R 2 and R 3 is independently a hydroxalkyl, an alkyl, alkyloxy, alkylcarboxy, alkylene or alkenylene having from one to six carbon atoms.
  • 4-aminoquinolines useful according to the invention include but are not limited to chloroquine, 2-hydroxychloroquine, amodiaquine,
  • the effective amount of a compound of the invention in the treatment of a disease described herein may vary depending upon the specific compound used, the mode of delivery of the compound, and whether it is used alone or in combination.
  • the effective amount for any particular application can also vary depending on such factors as the disease being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular molecule of the invention without necessitating undue experimentation.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.
  • Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED5 0 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5 0 /ED5 0 .
  • Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans.
  • the dosage of such agents lies preferably within a range of circulating concentrations that include the ED5 0 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5 0 (i.e., the concentration of the test compound that achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • IC5 0 i.e., the concentration of the test compound that achieves a half- maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • Subject doses of the compounds described herein typically range from about 0.1 ⁇ to 10,000 mg, more typically from about 1 ⁇ g/day to 8000 mg, and most typically from about 10 ⁇ g to 100 g. Stated in terms of subject body weight, typical dosages range from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1
  • milligram/kg/body weight about 50 milligram/kg/body weight, about 100
  • milligram/kg/body weight about 200 milligram/kg/body weight, about 350
  • milligram/kg/body weight about 500 milligram/kg/body weight, to about 1000 mg kg/body weight or more per administration, and any range derivable therein.
  • a derivable range from the numbers listed herein a range of about 5 mg kg/body weight to about 100 mg/kg/body weight, about 5 microgram kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
  • the absolute amount will depend upon a variety of factors including the concurrent treatment, the number of doses and the individual patient parameters including age, physical condition, size and weight. These are factors well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
  • DCA Dichloroacetate
  • Chloroquine typically is administered in a dosage of 300mg-600mg to adults for the treatment of malarial infection.
  • DCA can be used, for example, in dosages of 1- 25 mg/kg of body weight per day, 1- 15 mg/kg of body weight per day, or 5- 10 mg/kg of body weight per day.
  • a sub-therapeutic dosage of either or both of the molecules may be used.
  • a “subtherapeutic dose” as used herein refers to a dosage which is less than that dosage which would produce a therapeutic result in the subject if administered in the absence of the other agent.
  • compositions of the present invention comprise an effective amount of one or more agents, dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • animal e.g. , human
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • the compounds are generally suitable for administration to humans. This term requires that a compound or composition be nontoxic and sufficiently pure so that no further manipulation of the compound or composition is needed prior to administration to humans.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. , antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and
  • the agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in creams, in lipid compositions (e.g. , liposomes), or by other method or
  • the composition may comprise various antioxidants to retard oxidation of one or more components.
  • the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the agent may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g. , those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g. , glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g. , triglycerides, vegetable oils, liposomes) and combinations thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods.
  • the compounds of the invention may be administered directly to a tissue.
  • Direct tissue administration may be achieved by direct injection.
  • the compounds may be administered once, or alternatively they may be administered in a plurality of administrations. If administered multiple times, the compounds may be administered via different routes. For example, the first (or the first few) administrations may be made directly into the affected tissue while later administrations may be systemic.
  • the compounds of the invention may be formulated in a device for individual delivery.
  • the device may be similar to an epipen.
  • the device may have a housing connected to a needle and a spring-loaded mechanism for a single delivery of a predetermined amount of active agent.
  • the advantage of these devices is that they can be used by an individual who has experienced a brain injury, very close in time to the injury. For instance, military personnel who are at risk of head injuries, or athletes competing in sports that are associated with the risk of head injury can maintain a device of the invention.
  • compositions of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • a pharmaceutical composition comprises the compound of the invention and a pharmaceutically-acceptable carrier.
  • Pharmaceutically- acceptable carriers for peptides, monoclonal antibodies, and antibody fragments are well- known to those of ordinary skill in the art.
  • a pharmaceutically- acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art. Exemplary pharmaceutically acceptable carriers for peptides in particular are described in U.S. Patent No. 5,211,657. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically- acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically- acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • the compounds of the invention may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections, and usual ways for oral, parenteral or surgical administration.
  • the invention also embraces pharmaceutical compositions which are formulated for local administration, such as by implants.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active agent.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids, such as a syrup, an elixir or an emulsion.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymefhylcellulose, and/or
  • polyvinylpyrrolidone PVP
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
  • the compounds when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g. , by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer' s, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer' s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
  • the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient.
  • exemplary bioerodible implants that are useful in accordance with this method are described in PCT International Application No. PCT/US/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15, 1994).
  • PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing a biological macromolecule. The polymeric matrix may be used to achieve sustained release of the agent in a subject.
  • the agent described herein may be encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agent is stored in the core of a polymeric shell).
  • Other forms of the polymeric matrix for containing the agent include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted.
  • the size of the polymeric matrix device further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular, pulmonary, or other surface.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the agents of the invention to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
  • the agents of the invention may be delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,
  • non-biodegradable polymers examples include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
  • biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compound, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075, 109.
  • Delivery systems also include non- polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • hydrogel release systems silastic systems
  • peptide based systems such as wax, but are not limited to: (a) erosional systems in which the platelet reducing agent is contained in a form within a matrix such as those described in U.S.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • Therapeutic formulations of the compounds i.e., peptides, small molecules, nucleic acids or antibodies may be prepared for storage by mixing a compounds having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
  • benzalkonium chloride benzethonium chloride
  • phenol butyl or benzyl alcohol
  • alkyl parabens such as methyl or propyl paraben
  • catechol resorcinol
  • cyclohexanol 3- pentanol
  • m-cresol low molecular weight (less than about 10 residues)
  • polypeptides proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine
  • the compounds may be administered directly to a cell or a subject, such as a human subject alone or with a suitable carrier. Additionally, a peptide may be delivered to a cell in vitro or in vivo by delivering a nucleic acid that expresses the peptide to a cell.
  • Various techniques may be employed for introducing nucleic acid molecules of the invention into cells, depending on whether the nucleic acid molecules are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid molecule- calcium phosphate precipitates, transfection of nucleic acid molecules associated with DEAE, transfection or infection with the foregoing viruses including the nucleic acid molecule of interest, lipo some-mediated transfection, and the like.
  • a vehicle used for delivering a nucleic acid molecule of the invention into a cell can have a targeting molecule attached thereto.
  • a targeting molecule e.g., a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid molecule delivery vehicle.
  • monoclonal antibodies are especially preferred.
  • proteins that bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.
  • proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life, and the like.
  • Polymeric delivery systems also have been used successfully to deliver nucleic acid molecules into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acid molecules.
  • the peptide of the invention may also be expressed directly in mammalian cells using a mammalian expression vector.
  • a mammalian expression vector can be delivered to the cell or subject and the peptide expressed within the cell or subject.
  • the recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art.
  • tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid- specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters.
  • Developmentally-regulated promoters are also encompassed, for example the murine hox promoters and the a-fetoprotein promoter.
  • a "vector" may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids, phagemids and virus genomes.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.
  • the invention also includes articles, which refers to any one or collection of components.
  • the articles are kits.
  • the articles include pharmaceutical or diagnostic grade compounds of the invention in one or more containers.
  • the article may include instructions or labels promoting or describing the use of the compounds of the invention.
  • promoted includes all methods of doing business including methods of education, hospital and other clinical instruction, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with compositions of the invention in connection with treatment of neurological disorders.
  • Instructions can define a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the invention. Instructions also can include any oral or electronic instructions provided in any manner.
  • kits may include one or more containers housing the components of the invention and instructions for use.
  • kits may include one or more agents described herein, along with instructions describing the intended therapeutic application and the proper administration of these agents.
  • agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents.
  • the kit may be designed to facilitate use of the methods described herein by physicians and can take many forms.
  • Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder).
  • compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
  • a suitable solvent or other species for example, water or a cell culture medium
  • "instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the invention. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc.
  • the written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflects approval by the agency of manufacture, use or sale for human administration.
  • the kit may contain any one or more of the components described herein in one or more containers.
  • the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject.
  • the kit may include a container housing agents described herein.
  • the agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely.
  • the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.
  • Example 1 Peripheral Immune cells are activated by brain injury, and that activation can be reversed using CLIP inhibitors.
  • FPI activates B and T cells in the spleen that can be reversed by administering TPP.
  • Neuroinflammation after TBI is well documented. The contributions of peripheral inflammation have not been documented, despite the fact that infiltration of a large number of systemic factors occurs following FPL In order to determine the role for these cells, we performed preliminary studies to examine several components of peripheral inflammation. Our results show that at 24 hrs after TBI, a significant increase in activated B (Fig. 3A) and T (Fig. 3B) cells are observed in the spleen and that administration of the small peptide TPP (SEQ ID NO: 5) ameliorates this effect (Fig. 3).
  • TPP treatment reverses the peripheral activation of immune cells and neuronal FAS expression
  • this treatment would also be neuroprotective.
  • Fig. 6 Fluorojade-labeled cells following TPP treatment
  • TPP did not prevent the activation of astrocytes of microglial cells following the initial TBI (not shown), but did prevent neuronal degeneration (Fig. 6). This finding provides additional support for the idea that peripheral immune cells contribute to neurodegeneration following TBI and blocking this response is neuroprotective.
  • FIG.3 FPI results in Fas-expression on neurons (Fig. 5); 3) Preventing peripheral immune cell activation after FPI using a peptide (TPP) that targets pro-inflammatory cells, reverses peripheral B and T cell activation.
  • TPP peptide
  • Such treatment is also neuroprotective (Fig. 6) and reduces neuronal Fas expression (Fig. 5); 4)TBI followed by administration of PAM-3-Cys (which represents a product of Borrelia Burgdorferi and other gram- negative bacterium) at a time when substantial healing has occurred in the brain, exacerbates Fas expression (Fig. 5) and glial activation (Fig.
  • Example 2 Administration of varying doses of peptide show a dose dependent response in the alleviation of TMEV-induced epilepsy
  • FLOW CYTOMETRY TMEV infected mice and sham infected mice were terminated on day 3 post infection. Flow cytometry was used to examine the effects of Theiler's virus infection on cell populations in the CNS and spleen. Lymphocytes were extracted from the tissues using a percoll gradient. 4 2X10 5 cells from each tissue were labeled with specific antibodies labeled with fluorescent dyes for 20mins at 4°C. 4 Cells were washed 2 times with 500 ⁇ 1 and re- suspended in 250 ⁇ 1 in PBS +2% FBS buffer and tested by flow cytometer.
  • a flow cytometer is a device that detects and counts individual cells passing in a stream through a laser. 5 Cells in a mixed population are tagged with specific antibodies labeled with fluorescent dyes. As a cell passes through a laser light is scattered and any dye molecule is excited. Characteristics of cells are measured by scattered light: forward scatter (cell size) and side scatter (cell complexity).
  • mice treated with Theiler' s murine encephalomyelitis virus (TMEV) to induce seizures is shown as Figure 11.
  • Mice were either treated on day 1 (CP1), day 1 and day 2
  • mice were tested per group.
  • FIG. 13 includes panels showing the results of flow cytometric analysis.
  • the spleen cells were stained with CDl 1 and CD45.
  • the cells were isolated from a sham infected (received no virus) mouse.
  • the top panel shows the results of the analysis of CD45 (x-axis) versus CDl lb (Y axis) and the bottom panel is ungated.
  • Panels showing the results of flow cytometric analysis on spleen cells stained with CDl 1 and CD45 that were isolated from infected TMEV infected mice are presented in Figure 14.
  • the top panel shows the results of the analysis of CD45 (x-axis) versus CD 1 lb (Y axis) and the bottom panel is ungated.
  • cytometric analysis of CNS cells stained with CD11 and CD45 isolated from TMEV infected mice are presented in Figure 16.
  • the top panel shows the results of the analysis of CD45 (x-axis) versus CD1 lb (Y axis) and the bottom panel is ungated.
  • Figures 17A and 17B are pictures of stained cells from mouse brain.
  • Figure 17A shows quiescent resident microglia in hippocampus from sham infected mice.
  • Figure 7B shows activated microglia and macrophage in hippocampus of TMEV-infected mice.
  • CAP competitive antagonist peptide
  • Fluid percussion injury (FPI) - FPI was induced as previously described. 29 Briefly, mice were anesthetized with isoflurane, prepped, cleaned and shaved, then put into a stereotactic instrument (Stoelting, Inc. Illinois, USA). A 2 mm craniotomy was made over the left parietal cortex making sure to keep the dura intact. The female end of a lure lock syringe was cemented over the craniotomy and attached to the fluid percussion apparatus. A 12-16 ms FPI was delivered at a pressure of -1.4-1.6 atm. Sham mice received identical treatment except the pressure pulse was never delivered. After injury or sham, suture was used to close the scalp over the wound and mice were returned to their home cage resting on a heating pad. Mice were monitored to ensure that they resumed walking, feeding, drinking and grooming behavior.
  • Flow cytometry - Mice were sacrificed and spleens were removed. The tissues were dissociated from which single cell suspensions were made by passing spleens through 40 ⁇ cell strainers. Red blood cells were lysed using GEY'S buffer. 12 Splenocytes were first blocked with FC Block (BD Bioscience) and then stained with the following antibodies; CD3, CD4, CD8, CD25, MHCII (clone M5/114.15.1), B220, ⁇ -TCR (BD Bioscience), and CLIP (15G4, Santa Cruz Biotechnology). For regulatory T cell (Treg) staining we used the eBioscience mouse regulatory T cell staining Kit according to the manufacturer's directions.
  • Live cells were assessed using the Life Technologies LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit according to the manufacturer's directions.
  • Splenocytes were analyzed using a BD FACS Canto II flow cytometer and data were analyzed using FlowJo software (TreeStar Inc.). See Fig. 10 for gating strategy.
  • Fluoro-Jade C method Fluoro-Jade C staining took place as previously decribed. 29 Briefly, sections were mounted onto gelatin-coated slides and Fluoro-Jade C histology was performed according to the packaging instructions (AG325,Millipore Inc.,Billerica,MA,USA). The slides were allowed to air dry, then cleansed in xylenes, after which cover slips were applied using Vectasheild with DAPI (Vector Labs, Burlingame, CA). Sections were then visualized using an Olympus 1X80 (Olympus Inc., Center Valley, PA, USA) inverted microscope equipped to visualize FITC.
  • Olympus 1X80 Olympus Inc., Center Valley, PA, USA
  • the following Olympus Uplan objectives were used to capture the images: 10X NA0.40 /0.17 FN26.5; 20X nA0.75 ⁇ /0.17 F 26.5; 40 NA0.75 ⁇ /0.17 FN26.5. Images were captured using the FV1000 and the FluoView software (V.1.7.1.0). Images were saved as 24 Bit Tiff files and brightness and contrast were adjusted using Adobe Photoshop (V. 12.0). For quantitative analysis of Fluoro-Jade C labeling, the peri-lesion area was traced and 400 ⁇ 2 grids were randomly placed throughout the peri-lesion area. All cells which fell within the grid, or contacted the left and upper borders of the grid were included in the counts, whereas those that touched the right or lower border were excluded. The peri-lesion area consisted of +/- 1 mm anterior/posterior (AP) from the injury focus (AP +1.5 mm; medio-lateral: 1.2 mm).
  • AP anterior/posterior
  • Cytokine analysis - Tissue was isolated from the ipsilateral cortex including the lesion area and flash frozen in liquid nitrogen. Frozen tissue was homogenized following the manufacturer's instructions (Milliplex MAP kit, Millipore). Protein was estimated with a Bradford assay and similar concentrations were made such that 25 ⁇ of homogenate was added to 25 ⁇ of assay buffer. Then, 25 ⁇ of magnetic beads coated with specific antibodies
  • FPI-induced changes in components of antigen presentation by peripheral lymphocytes A hallmark of the transition between innate and adaptive (specific) immunity is successful antigen processing and presentation. Antigen processing requires proteolytic cleavage of invariant chain (CD74) into peptides known as class II invariant peptide (CLIP). CLIP functions as a "placeholder" for antigen specific binding to the peptide-binding groove of MHCII molecules. Therefore, we examined the effects of FPI on CLIP and MHCII from peripheral lymphocytes. No signs of B cell activation were observed, as indicated by no net change in the cell surface levels of MHC II (Fig. 19A). However, a significant decrease in cell surface CLIP associated with MHCII on B cells was observed (Fig. 19B). Conversely, examination of ⁇ T cells revealed no significant difference in the level of cell-surface CLIP (Fig. 19C), but a significant decrease in MHCII on ⁇ T cells (Fig. 19D).
  • CD74 which is required for processing and presentation of full-length proteins, 7 can serve as a survival factor and can function as a growth promoting receptor.
  • mice lacking CD74 exhibited no net change in spleen cellularity 24 hours following FPI (Fig. 21A).
  • wild type mice exhibit a significant expansion of lymphocytes in the spleen 24 hours after FPI (Fig. 18). Furthermore, there were no
  • CD74 Def mice have a reduced lesion size and decreased neurodegeneration after TBI supports the involvement of CD74 and CLIP in the CNS. Both CD74 and its products including CLIP, are widely accepted as central players in antigen processing and presentation, 7 and are central to the transition between innate and adaptive immunity. The neuroprotection observed in this study supports the involvement of antigen processing and presentation in the exacerbation of brain injury. While it is clear that CD74 is involved in the damage to the CNS following TBI, several different functions have also been attributed to CD74. These include: survival, signal transduction through cell surface CD74, binding to pro-inflammatory macrophage inhibitory factor (MIF), along with its well-established role in antigen processing and . present ,at .i ⁇ on.19,'22,'26,'34
  • MIF macrophage inhibitory factor
  • Tregs have been well described to dampen antigen-specific responses in the periphery, their role in the CNS is controversial. 6 ' 20 ' 21 Moalem et al has proposed that following injury to the nervous system Tregs may be detrimental, because the antigen specific T cell response that they limit may be protective. 27 ' 32 ' 33 Alternatively, some investigators have proposed protective effects of Tregs following cerebral insults. 20 ' 21 ' 23 ' 38 It is pertinent to note that two distinct subsets of Tregs have been characterized, natural Tregs and inducible Tregs. 16 ' 36 Therefore, it is possible that one population of Tregs is beneficial and the other is detrimental.
  • B cells are also well described as antigen specific, capable of producing antibodies, and as extraordinarly efficient professional antigen presenting cells. 18 The finding that after FPI, B cells have significantly reduced CLIP, but not significantly reduced MHCII, suggests that these cells have a higher capacity for antigen presentation after TBI. In support of this notion, a recent study showed that the antigen presentation function of B cells is critical for the pathology of experimental autoimmune encephalitis (EAE), more so than autoantibody production. 35
  • EAE experimental autoimmune encephalitis
  • Denzin LK Cresswell P. HLA-DM induces CLIP dissociation from MHC class II alpha beta dimers and facilitates peptide loading. Cell 1995;82: 155-165.
  • Hyder AA Wunderlich CA, Puvanachandra P, et al. Kobusingye. The impact of traumatic brain injuries: a global perspective. NeuroRehabilitation 2007;22:341-353.

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Abstract

L'invention concerne des méthodes de traitement des troubles neurologiques par ciblage de l'élimination de populations sélectives de cellules immunes activées en périphérie. Les cellules peuvent être ciblées, par exemple, à l'aide d'inhibiteurs CLIP.
PCT/US2014/054845 2013-09-09 2014-09-09 Traitement des troubles neurologiques WO2015035414A1 (fr)

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US201361875670P 2013-09-09 2013-09-09
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020097079A1 (fr) * 2018-11-05 2020-05-14 Bcell Solutions, Inc. Méthodes de traitement de lésion cérébrale traumatique
WO2021016049A1 (fr) * 2019-07-19 2021-01-28 Bcell Solutions, Inc. Modulation par l'acétylcholine de la fonction immunitaire

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997025344A1 (fr) * 1996-01-03 1997-07-17 The Australian National University Analogues des clip et maladie auto-immune
WO2009055005A2 (fr) * 2007-10-23 2009-04-30 The Regents Of The University Of Colorado Inhibiteurs compétitifs de l'expression de chaînes invariantes et/ou d'une liaison d'un clip ectopique
US20110087173A1 (en) * 2009-10-12 2011-04-14 Sibbitt Jr Wilmer L Automatic syringes
EP2633864A1 (fr) * 2008-07-25 2013-09-04 The Regents of the University of Colorado Inhibiteurs de clip et procédés de modulation de la fonction immune

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997025344A1 (fr) * 1996-01-03 1997-07-17 The Australian National University Analogues des clip et maladie auto-immune
WO2009055005A2 (fr) * 2007-10-23 2009-04-30 The Regents Of The University Of Colorado Inhibiteurs compétitifs de l'expression de chaînes invariantes et/ou d'une liaison d'un clip ectopique
EP2633864A1 (fr) * 2008-07-25 2013-09-04 The Regents of the University of Colorado Inhibiteurs de clip et procédés de modulation de la fonction immune
US20110087173A1 (en) * 2009-10-12 2011-04-14 Sibbitt Jr Wilmer L Automatic syringes

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020097079A1 (fr) * 2018-11-05 2020-05-14 Bcell Solutions, Inc. Méthodes de traitement de lésion cérébrale traumatique
WO2021016049A1 (fr) * 2019-07-19 2021-01-28 Bcell Solutions, Inc. Modulation par l'acétylcholine de la fonction immunitaire

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