Peptide compounds for traumatic brain injury

Abstract

The present invention provides compounds, compositions and methods for prophylaxis and treatment of traumatic brain injury using a P13 peptide or P13 fusion peptide which can be administered prior to or after traumatic brain injury and which can ameliorate the biophysiological indicators of brain injury and improve neurological outcomes in subjects suffering from traumatic brain injury.

Description

PEPTIDE COMPOUNDS FOR TRAUMATIC BRAIN INJURYCROSS-REFERENCE TO REEATED APPLICATIONS

[0001] This application is an international PCT application, which claims the benefit of United States provisional application serial no. 63 / 711,793, filed 25 October 2024. The entire contents of the aforementioned application(s) is / are hereby incorporated by reference as if fully set forth herein.GOVERNMENT FUNDING SUPPORT

[0002] This invention was made with government support under grant no. C0210037 awarded by MRDC-CCCRP. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates to the general field of medicine and more specifically to compounds and compositions for treatment and prophylaxis of traumatic brain injury (TBI), and methods for their use.

[0005] 2. Background of the invention

[0006] The increased use of improvised explosive devices and other explosive weapons has greatly increased the incidence of survivable TBI among military personnel during recent conflicts. Exposure to blast has been described as the major cause of TBI and associated disabilities in the recent wars in Iraq and Afghanistan. Civilians and military personnel also can obtain a TBI from numerous traumas such as contact and other sports, workplace accidents, vehicular accidents, and the like.

[0007] Although several biochemical and histopathological changes in the central nervous system after blast exposure have been documented, the potentially complex pathophysiological mechanisms triggering both short and long-term neurobehavioral abnormalities after TBI are still not well understood, which has hampered the development of effective countermeasures orpreventative measures. Rigorous pre-clinical studies have shown the potential involvement of different pathological mechanisms of TBI of various causes including increased oxidative stress, pro-inflammatory processes, defective mitochondrial functions leading to decreased neuronal energy status, cerebral edema due to blood-brain barrier (BBB) rupture, and the like.

[0008] For example, the toll-like receptor (TLR) signaling cascade is inter-connected with MAPK / JNK signaling, reactive oxygen species (ROS) production and generation of inflammatory cytokines. TLRs are a family of proteins expressed by a variety of cell types and are central to the inflammatory response in a broad array of species. TLRs recognize both microbial ligands (pathogen-associated molecular pattern molecules) and self-antigens from damage- associated molecular patterns, such as may occur during brain injury. TLR activation of immune cells initiates an inflammatory immune response characterized by production of pro-inflammatory cytokines, such as IL-6, IL-1, and TNF-a, and the downstream activation of MAPK / JNK signaling.

[0009] When TLRs are activated, ROS are produced and inflammatory cytokines and nitric oxide are released. The MAPK signaling pathway, initiated by oxidative stress and culminating in apoptosis, can also be initiated by TLR activation. The apoptosis signal-regulating kinase 1 (ASK-1) is involved in the ROS dependent activation of the MAPK apoptotic signaling pathway. ASK-1 is a common component of both the MAPK and TLR signaling pathways, and it has been hypothesized that there is cross-talk between the two pathways.SUMMARY OF THE INVENTION

[0010] Thus, there is a need in the art for methods to treat and prevent the sequelae of TBI. Targeting intra-cellular TLR signaling was found to impact multiple pathways with involvement in neurodegeneration after brain injury and produced a beneficial result in subjects suffering from TBI, whether administered before or after receiving a TBI.

[0011] The invention described herein thus provides embodiments related to treatment and prophylaxis of TBI with P13 peptide. In particular, the invention relates to a therapy for treatment and prevention of the effects of TBI. Specifically, the invention relates to a fusion peptide comprising a P13 peptide covalently linked to a cell penetration peptide sequence. The P13 peptide preferably is DIVKLTVYDCI (SEQ ID NO:3) or a conservative variant thereof. Inpreferred embodiments, the cell penetration peptide sequence is RRRRRRRRR (SEQ ID N0:2). In preferred embodiments, the fusion peptide is DIVKLTVYDCIRRRRRRRRR (SEQ ID NO:1).

[0012] In certain embodiments, the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the fusion peptide discussed herein. Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a fusion peptide as described herein.

[0013] In additional embodiments, the invention includes a method of treating traumatic brain injury in a subject in need thereof, comprising administering to the subject a fusion peptide as described herein or pharmaceutical compositions that contain a fusion peptide as described herein. The subject can receive administration of the treatment before or after a traumatic brain injury is received. Preferably, when administered before a traumatic brain injury, the subject is administered the treatment within about 1 day to about 30 days before the subject receives the injury. Preferably, when administered after a traumatic brain injury, the subject is administered treatment within about 1 minute to about 14 days after the traumatic brain injury.

[0014] In a preferred embodiment, the treatment is administered to the subject as soon as possible after the subject receives a traumatic brain injury and the treatment is continued to be administrated in further doses, preferably daily doses, for about 1 day to about 6 months or until symptoms of traumatic brain injury abate.

[0015] In certain embodiments, the dose administered to the subject, either before or after the injury, is about 0.1 mg / kg to about 20 mg / kg.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Certain embodiments are illustrated by way of example, and not by way of limitation in the figures of the accompanying drawings.

[0017] FIG. 1A is a photograph of the blast simulator instrument.

[0018] FIG. IB presents a representative pressure profile from the blast simulator instrument.

[0019] FIG. 2A through FIG. 2D are graphs showing pNFH levels in the CSF (FIG. 2A and FIG.2B) and plasma (FIG. 2C and FIG. 2D) at 24 hours and 1 month post-blast, with and without P13 treatment.

[0020] FIG. 3A and FIG. 3B are bar graphs showing plasma corticosterone levels at 24 hours and 1 month post-blast, with and without P13 treatment.

[0021] FIG. 4A, FIG. 4B, and FIG. 4C are western blots showing the differential expression of TBI protein biomarkers at 24 hours post-blast in frontal cortex (FIG. 4A), cerebellum (FIG. 4B), and cortex (FIG. 4C).

[0022] FIG. 5A, FIG. 5B, and FIG. 5C are western blots showing the differential expression of TBI protein biomarkers at 1 month post-blast in frontal cortex (FIG. 5A), cerebellum (FIG. 5B), and cortex (FIG. 5C).

[0023] FIG 6 A and FIG. 6B are bar graphs showing the discrimination index (DI) measured in the novel object recognition test carried out on days 2 (FIG. 6A) and 28 (FIG. 6B) post-blast, with and without Pl 3 treatment.

[0024] FIG. 7A through FIG. 7D are bar graphs showing sleep fragmentation index data on days 1, 7, 14, and 28, as indicated for sham-treated, not treated, and P13-treated ferrets.

[0025] FIG. 8A through FIG. 8H provide qRT-PCR data showing that blast exposure upregulates multiple pro-inflammatory cytokines and downregulates anti-inflammatory cytokine in the inner ear on days 1 and 28 after blast injury. Pl 3 administration provided protection against these changes in the inner ear post-blast.

[0026] FIG. 9A through FIG. 9D provide qRT-PCR data showing that blast exposure downregulates prestin (the outer hair cell specific protein) and calretinin (the calcium buffering protein) in the inner ear on day 1. P13 administration provided protection against these changes in the inner ear post-blast.

[0027] FIG. 10A through FIG. 10F provide immunohistochemical data showing that blast exposure causes loss of outer hair cells in the apical, middle and basal regions of the cochlea on days 1 and 28 post-blast. P13 administration provided protection against this outer hair cell loss post-blast.

[0028] FIG. 11A through FIG. 1 IL shows auditory brainstem response (ABR) testing. The data showed a decrease in ABR amplitudes on days 1 and 7 post-blast, where P13 administration provided protection against this auditory functional deficit.

[0029] FIG. 12A-C provides graphs showing the results of Openfield exploratory activity test on ferrets on days 1, 7, 14 and 28 after blast exposure with and without P13 treatment. Horizontal activity count is shown in FIG. 12A. Center time is shown in FIG. 12B. Margin time is shown in FIG. 12C. The results are expressed as mean±SEM. *p<0.05; **p<0.01; (n=5 to 8); NT-Not treated.

[0030] FIG. 13 provides graphs showing patern of ferret limb movements in the CatWalk system on Days 1, 14, and 28 post-blast with and without P13 treatment. The intensity of light produced in the CatWalk system by the paw from each limb is expressed as mean±SEM. *p<0.05;**p<0.01; n=6 to 10; NT-Not treated.DETAILED DESCRIPTION OF THE INVENTION

[0031] 1. Overview

[0032] The present invention relates in particular to the use of P13 peptide for treatment and prophylaxis in subjects suffering from TBI or likely to be exposed to conditions where TBI can occur. For example, military personnel, first responders, professional or student athletes, persons in dangerous professions (e.g„ mining, construction, and the like) and any person suffering from an accidental fall, vehicular accident, and the like.

[0033] 2. Definitions

[0034] Unless defined otherwise, all technical and scientific terms use herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials used are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration, and any other measurements, quantities, or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.

[0035] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements orsteps but not the inclusion of any other item, element, step, or group of items, elements, or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element, or step modified by the article.

[0036] As used herein, the term “about” means plus or minus 20 percent of the recited value so that, for example, “about 0.125” means 0.125 ± 0.025, and “about 1.0” means 1.0 ± 0.2.Notwithstanding that the numerical ranges and parameters setting for the broad scope of the invention are approximations, the numerical values set forth in specific non-limiting examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing.

[0037] As used herein, the term “P13” refers to NH2-DIVKLTVYDCI-COOH (SEQ ID NO:3), an A52R viral protein sequence, or to the fusion peptide with an arginine or other cell penetrating peptide (e.g., NH2-DIVKLTVYDCIRRRRRRRRR-COOH (SEQ ID NO:1).

[0038] As used herein, the term “cell penetrating peptide” refers to a sequence of amino acids that allow cells to take in molecules, such as other peptides, nucleic acids, and the like. As used herein, the term “traumatic brain injury (TBI)” is an injury to the brain that occurs when an external force such as a blow or blast hits the head or when an object pierces the skull. TBIs range from mild to severe, and can cause temporary or permanent impairment of cognitive, physical, or psychosocial function.

[0039] As used herein, the term “subject” can also be referred to as a "host" or a "patient," and can refer to any animal, including mammals such as humans, laboratory animals (e.g. rodents, ferrets, rabbits, and the like), companion animals (e.g. felines, canines, and the like), farm animals (e.g., equines, bovines, porcines, ovines, caprines, and the like, and sport animals. A suitable subject for the invention preferably is a human that is suspected of having, has been diagnosed as having, or is at risk of receiving a traumatic brain injury (TBI). A “subject in need” is any subject that has, may have or may receive a TBI.

[0040] As used herein, the term "administer" and all its cognates refers to introducing an agent to a subject, and can be performed using any of the various methods or delivery systems for administering agents, pharmaceutical compositions, and the like which are known to those skilled in the art.

[0041] As used herein, the term “treatment” and all its cognates refer to obtaining a desired pharmacologic and / or physiologic effect in a subject. "Treatment," includes: (a) preventing the condition or disease or symptom thereof from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease or symptom thereof, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease or symptom thereof, such as. for example, causing regression or improvement of the condition or disease or symptom thereof.

[0042] As used herein, the terms “prophylaxis” and “prevention” and all their cognates refer to treatment modalities provided to or administered to a subject prior to exposure to a traumatic brain injury which reduce the effects of traumatic brain injury, i.e., prevents, partially prevents, lessens the severity of the symptoms of, reduces the number of symptoms of. or otherwise ameliorates the biochemical effects and neurological sequelae (long-term and short-term effects) of a later traumatic brain injury.

[0043] 3. Summary of Results

[0044] The temporal profile of expression of TLRs in different brain regions was defined in ferrets after blast exposure. Blast exposure upregulated multiple TLRs in the ferret brain regions supporting our hypothesis that P13 and similar agents that can inhibit multiple TLRs will be beneficial for treatments after blast exposure.

[0045] P13 administered as an intraperitoneal injection was found to significantly increase in ferret brain, CSF, and plasma within 2 hours after injection, and was detectable even after 24 hours after injection.

[0046] The efficacy of P13 for protection against TBI was evaluated using biochemical, molecular, and histopathological outcome measures. Biochemical analysis carried out in the brain regions, CSF, and plasma revealed that Pl 3 treatment can inhibit the neuropathological changes occurring in the brain post-blast. Compared to 10 mg / kg dose, the neuropathological improvements were more prominent in the animals receiving a 2 mg / kg dose of P13. The data generated indicated that P13 peptide can protect against TBI.

[0047] The efficacy of P13 for protection against TBI was evaluated using neurobehavioral outcome measures. Neurobehavioral functional outcome measures conducted in ferrets showed that blast exposure (1) induces anxious and depressive-like behaviors; (2) affects short-termmemory; (3) disrupts the movements of limbs; and (4) disturbs the circadian rhythm or sleep pattern. These functional deficits after blast exposure were ameliorated with Pl 3 treatment. Thus, P13 treatment can inhibit the neuropathological changes occurring in the brain post-blast and thereby protect the neurobehavioral functions.

[0048] P13 can provide protection against blast-induced injuries to ferret eyes and ears, and perhaps other organs.

[0049] 4. Embodiments of the Invention

[0050] A. Introduction

[0051] Modulation of TLR signaling pathways in the brain has been observed in different forms of traumatic brain injury and inhibitors of TLR signaling have shown significant beneficial effects. Using an open- skull weight drop model of TBI in rats, Zhang et.al studied the differential expression of TLR2 and TLR4 along with their most common adaptor molecule, myeloid differentiation factor 88 (MyD88), by immunohistochemistry. The expression of all three proteins were upregulated around the lesion area (in microglia / macrophages) as well as subcortical white matter (astrocytes). Increased expression of TLR4 and MyD88 proteins was observed within 24 hours and showed steady increase up to 4 days, whereas the significant increase in TLR2 positive cells observed after 2 days.

[0052] In other TBI models, studies have shown that the mRNA and protein levels of TLR-4 are upregulated at 24 hours after brain injury and are maximally increased at 7 days, followed by a decline. Known neuroprotective molecules such as resveratrol, curcumin, docosahexanoic acid etc. appear to elicit their effects at least partially through inhibition of TLR-4. Resatorvid (TAK-242), a selective inhibitor of TLR-4 has elicited significant improvements after TBI; treatment with resatorvid initiated at 4 hours post-injury and sustained over 5 days decreased brain damage and improved neurobehavioral functions after TBI.

[0053] Intravenous injections of brain-specific microRNA (MIR- 124) encapsulated in exosomes increased hippocampal neurogenesis by inhibiting TLR-4. Hyperbaric oxygen therapy was found to alleviate brain injury through inhibition of TLR4 signaling pathway. Silencing TLR-4 gene expression in the brain using shRNA inhibited the release of several pro-inflammatory cytokines in the brain, decreased astrocyte proliferation, alleviated hippocampal neuronal damage, decreased brain edema and improved functional outcome measures. In a controlledcortical impact model of TBT in mice in two independent laboratories, TLR4 knock out mice showed decreased infarct volume and significantly improved neurobehavioral outcome measures, revealing across multiple measures the key role of TLR-4 in the pathogenesis of TBI. Knocking out the TLR-2 gene also significantly decreased secondary brain injury severity after weight-drop impact in mice, suggesting that inhibiting multiple TLRs may have added benefit.

[0054] In support of the above proposed pathological mechanisms, treatment with therapeutic agents having antioxidant and or anti-inflammatory properties have shown beneficial effects after blast-induced neurotrauma. Treatment with a-lipoic acid, an oxidative stress inhibitor, improved neuropathological and neurobehavioral outcomes after blast exposure. In a rat model of TBI, a combinatorial antioxidant treatment was found to significantly decrease the number of both hyperphosphorylated and oligomeric Tau-positive hilar mossy cells and somatic accumulation of endogenous Tau protein in oligodendrocytes in the hippocampus. In addition, the non-steroidal anti-inflammatory drug, minocycline, treatment prevented development of the deleterious effects of TBI in a rat model using cylindrical shock tube. Minocycline treated rats showed biomarker levels and neurobehavioral functions similar to un-injured sham controls. Treatment with antioxidants or anti-inflammatory drugs have shown significant protection in other models of TBI including controlled cortical impact (CCI), fluid percussion injury (FPI), head impact acceleration-induced injury etc. suggesting a common mechanism between different forms of brain injuries.

[0055] Oxidative stress, neuroinflammation, mitochondrial dysfunction, cerebral edema etc. have been reported in TBI. Thus, we postulated that therapeutic agents which can counteract multiple pathological mechanisms of injury would be more effective in treating blast injury. Drugs having both anti-oxidant and anti-inflammatory properties have shown substantial improvements in different animal models of TBI. Melatonin, the endogenous molecule having both antioxidant and anti-inflammatory properties has shown significant protection against TBI. N-acetyl cysteine (NAC), another endogenous molecule having both antioxidant and antiinflammatory properties, has shown promising effects in the treatment of TBI. A combination of NAC with 2,4-disulfonyl a-phenyl tertiary butyl nitrone (HPN-07), both having antioxidant and anti-inflammatory properties, protected blast-induced hippocampal neurodegeneration in rats. In a rat model of TBI using the cylindrical shock tube, post-blast treatment with N-acetylcysteine amide (NACA, which can penetrate the BBB and deliver NAC to the brain) decreasedintracranial pressure (TCP). Pre-treatment with NACA completely prevented the elevation in ICP after blast exposure, suggesting the role of oxidative stress and / or neuroinflammation in the development of ICP after blast exposure. Thus. NAC has shown promising efficacy against blast exposure in military personal suggesting that molecules having both anti-oxidant and antiinflammatory properties will be suitable candidates for treatment.

[0056] B. Traumatic Brain Injury

[0057] Traumatic brain injury (TBI) is an injury to the brain caused by an external force (or a combination of both movement and sudden impact) and can range from a mild concussion to severe TBI. TBI can result in physical, cognitive, social, cognitive, and behavioral symptoms. Outcome after TBI can include permanent disability or death. TBI causes an initial injury to the brain, but also results in a cascade of events that can result in even further injury, such as inflammation and swelling.

[0058] Symptoms which occur after TBI include loss of consciousness, headache, nausea, vomiting, lack of motor coordination, dizziness, poor balance, blurred vision, tinnitus, dilated pupils, confusion, weakness, numbness, cognitive changes, alexithymia, poor executive function, and more. Diagnosis currently is usually based on magnetic resonance imaging (MRI), but often is not used in the acute phase of injury because MRI produces relatively poor imaging not useful for detection of bleeds and fractures, and the inability to access and stabilize or treat the injured patient during the lengthy MRI procedure.

[0059] The present invention provides a new way to treat and prevent the sequelae of TBI by administering Pl 3 to the subject, prior to or after the injury.

[0060] C. Compounds

[0061] Certain embodiments of the invention relate to the preferred compound, peptide Pl 3, which is useful in therapy for TBI. This peptide is a potent inhibitor of TLR signaling and is a 20 amino acid peptide, consisting of 11 active amino acids from the A52R viral protein from poxvirus, preferably attached to a 9-arginine cell penetrating peptide, which allows intracellular delivery of the active peptide sequence without loss of potency. The 9-arginine cell-penetrating motif internalizes cargo from the cell surface into the cytoplasm, facilitating transport of the cargo across intact tissues. The peptide is synthesized commercially by BioSynthesis™ andstabilized as a trifluoroacetate salt. The molecular weight of Pl 3 is 2686.24 Daltons; isoelectric point: 12.41; solubility: 10 mg / mL in phosphate buffered saline (PBS) or water.

[0062] The sequence of P13 with the arginine cell penetrating peptide is NH2-DIVKLTVYDCIRRRRRRRRR-COOH (SEQ ID NO:1), a fusion of an A52R viral protein sequence with an arginine cell penetrating protein.

[0063] The peptide DIVKLTVYDCI (SEQ ID NO:3) can be joined to any suitable cellpenetrating motif as determined by the skilled person, however the RRRRRRRRR (SEQ ID NO: 2) sequence is preferred.

[0064] Conservative variants of SEQ ID NO:3 also can be used as a fusion with any suitable cell penetrating peptide according to the invention. Such conservative variants include peptides according to SEQ ID NO:3 which have one deletion, substitution, or addition. Thus, herein, the term “Pl 3” refers to the P13 sequence provided above and conservative variants thereof.

[0065] P13 blocks TLR signaling upstream of MAPK / JNK signaling and is a potent modulator of TLR- induced inflammatory cytokines. P13 targets multiple intracellular TLR signaling pathways including TLR4 and TLR2, with varying degrees of activity ranging from >95% inhibition of TLR9 induced cytokine secretion to <40% of TLR2 induced cytokines. These characteristics allow P13 to modulate the inflammatory immune response without completely inhibiting all Toll pathways.

[0066] In vitro preliminary studies demonstrated that P13 can inhibit the secretion of cytokines in primary human monocyte-derived dendritic cells induced by a combination of P. Aeruginoa (3.3 x 106 / mL) and H. Influenza (3.3 x 104 / mL). Using the RAW264.7 macrophage cell line, the studies showed that P13 can inhibit cellular apoptosis induced by H2O2. and therefore indicated that P13 is a powerful anti-oxidant and anti-inflammatory agent.

[0067] The peptide was developed originally to treat noise-induced hearing loss and ear infections. Here, studies were designed to test the efficacy of P13 for blast-induced auditory dysfunctions and to test its efficacy against TBI using a simulated blast exposure. P13 was effective in preventing all the tested pathological changes in the ferrets exposed to blast.

[0068] D. Compositions

[0069] In a preferred embodiment, the therapeutic agents of some embodiments are administered as a pharmaceutical composition that includes a pharmaceutically acceptable carrier or vehicle.A suitable carrier depends on the route of administration contemplated for the pharmaceutical composition. Routes of administration are determined by the person of skill according to convenience, the health and condition of the subject to be treated, and the location and stage of the condition to be treated.

[0070] Such routes can be any route which the practitioner deems to be most effective or convenient using considerations such as the patient, the patient’s general condition, and the specific condition to be treated. For example, routes of administration can include, but are not limited to: local or parenteral, including: oral, intravenous, intraarterial, intrathecal, injection into the cerebrospinal fluid, intraperitoneal, nasal, local injection, and the like. The administration can be given by transfusion or infusion, and can be administered by an implant, an implanted pump, or an external pump, or any device known in the art.

[0071] Therefore, the forms which the pharmaceutical composition can take will include, but are not limited to: tablets, capsules, granules, oral solutions, powders for dilution, sterile solutions or other liquids for injection or infusion, inserts and implants, suspensions, emulsions, lipid vesicles, and the like.

[0072] Treatment regimens include a single administration or a course of administrations lasting two or more days, including a week, two weeks, several weeks, a month, two months, several months, a year, or more, including administration for the remainder of the subject’s life. The regimen can include multiple doses per day, one dose per day or per week, for example, or a long infusion administration lasting for an hour, multiple hours, a full day, or longer.

[0073] Dosage amounts per administration include any amount determined by the practitioner, and will depend on the size of the subject to be treated, the state of the health of the subject, the route of administration, the condition to be treated or prevented, and the like. A therapeutically effective dose can be administered in a single dose or in a series of doses over a period of time. In general, it is contemplated that for the majority of subjects, a dose in the range of about 0.01 mg / kg to about 100 mg / kg is suitable, preferably about 0.1 mg / kg to about 20 mg / kg, more preferably about 0.1 mg / kg to about 10 mg / kg, and most preferably about 0.2 mg / kg to about 5 mg / kg are useful. This dose can be administered weekly, daily, or multiple times per day. A dose of about 0.1 mg, 0.2 mg, 0.25 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 250 mg, 500 mg, or 1000 mg can be administered. A preferred dose is in the range of about 0.25 mg / kg up to about 2 mg / kg.

[0074] E. Prophylaxis and Treatment Methods

[0075] When a subject has received a TBI, methods according to embodiments of the invention provide a treatment which ameliorates the short- and long-term effects of TBI. A subject who has received a TBI can be administered a pharmaceutical composition containing the P13 fusion peptides described herein as soon as possible after the injury and preferably within one hour or several hours of the injury. The dose preferably is about 0.1 mg / kg to about 20 mg / kg or any suitable dose as determined by the clinical practitioner. The subject preferably also is put on a regimen of P13 peptide administration at least daily, which will continue for a week, a month, six months, a year, or longer, or will continue until the symptoms of TBI abate. A preferred treatment for a subject who receives a traumatic brain injury involves a first dose by injection as soon as possible after the injury and further daily doses of the pharmaceutical composition for about 1 day to about 6 month to a year. Preferably, the treatment is administered within about 1 minute after the injury up to about 14 days after the injury. Most preferably, the treatment should be administered from about 1 hour to about 7 days after the injury or about 2 hours to about 1 day after the injury or about 2 hours to about 16 hours after the injury.When a subject is or will be in an environment where TBI is likely or could occur, methods according to embodiments of the invention provide prophylaxis which reduces the short- and long-term effects of TBI. In such cases, a subject can be placed on a regular regimen of P13 administration as long as the danger of TBI is likely. For example, military personnel or professional athletes in certain sports can be administered a dosage of a Pl 3 pharmaceutical composition prior to engaging in the dangerous activity or can be placed on a regular regimen of treatment with P13. Such prophylactic treatments can include daily or weekly doses. Preferably, the prophylactic treatment is administered at a time within about 1 days to about 30 days before the subject receives a traumatic brain injury. More preferably, the treatment is administered daily or weekly with at least one treatment administered prior to the injury; most preferably the treatment is administered several times prior to the injury, for example 2 days, three days, four days, five days, six days, one week, or longer prior to the injury, and optionally continuing after the injury as well.

[0076] 5. Examples

[0077] This invention is not limited to the particular processes, compounds, compositions, or methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, systems, compounds, compositions and materials are now described.

[0078] Example 1: General Methods and Materials.

[0079] A. Materials

[0080] The P13 peptide used for this study was synthesized in-house.

[0081] B. Ferret TB I Model

[0082] The ferret model is used here to study TBI because ferrets have genetically conserved proteins that function in the cellular activities similar to that of humans. In addition, ferrets have gyrencephalic (folds of the brain) brains, which can result in higher degree of translation of the studies to humans. The ferrets are exposed to a blast injury as described below and examined with or without treatment with Pl 3.

[0083] C. Blast TBI Simulation

[0084] To produce traumatic brain injury in the experimental animals, ferrets were subjected to single blast overpressure (about 20 psi) waves using an advanced blast simulator (20 psi peak pressure, impulse: 29.87 psi*msec). The blast simulator used was an advanced blast simulator. See FIG. 1A, which shows a photograph of the instrument, and FIG. IB, which shows blast pressure profiles generated from the advanced blast simulator that closely resemble the exposures experienced by military and civilian personnel from an explosion or detonation or weapon systems exposures. This simulated blast injury is approximately equivalent to a mild to moderate brain injury in a human patient.

[0085] Example 2: Differential Expressions of TLRsin Ferret Brain after TBI.

[0086] In a preliminary study, male ferrets were exposed to two tightly coupled 20 psi blasts within 2 minutes under isoflurane anesthesia using an advanced blast simulator (ABS). The results obtained indicated that multiple TLRs were significantly upregulated in brain regionspost-blast suggesting that molecules such as Pl 3 which can inhibit multiple TLRs will be more effective that those molecules which can inhibit only a single TLR.

[0087] Example 3: Pharmacokinetic Study of P13 in Ferrets.

[0088] In a pilot pharmacokinetic study, ferrets were given an intraperitoneal injection of P13 at a dose of 10 mg / kg. The concentrations of P13 in the plasma, cerebrospinal fluid (CSF) and brain were determined using mass spectrometry. Results indicated that the maximum levels of P13 were detected at 2 hours, and the peptide was detectable in the plasma, CSF and brain even after 24 hours post-injection, although the concentrations were less at later time points after 6 hours.

[0089] Example 4: P13 Treatment for Protection against Effects of TBI.

[0090] For the P13 treatment efficacy study using biochemical outcome measures, we have used two doses (2mg / kg and lOmg / kg) of P13 to determine a dose response. Male ferrets were exposed to blast as described above. P13 was given at 30 minutes post-blast intraperitoneally and the injections were repeated every day until euthanasia. Brain regions (frontal cortex, hind cortex, midbrain, hippocampus, cerebellum and brainstem), plasma and CSF samples were collected at 24 hours and 1 month post-blast. CSF samples were used for measuring the levels of phosphorylated neurofllament heavy chain (pNFH) protein. Plasma samples were used for the measurements of pNFH and corticosterone levels.

[0091] Example 5: Expression of TBI Biomarkers.

[0092] Neuronal axonal degeneration occurs after blast exposure, resulting in the release of pNFH into the CSF and plasma. pNFH levels in the CSF and plasma at 24 hours and 1 month post-blast, with and without P13 treatment, are shown in FIG. 2. The results shown in FIG. 2 indicate that prior P13 treatment significantly protected against this neuropathology at 24 hours in CSF and in plasma. Values are given as ±SEM; *-p<0.05; **p<0.01; n = 4; ns = not significant.

[0093] The levels of the stress response molecule, corticosterone, increased significantly in the plasma post-blast at 24 hours. P13 treatment reduced the release of this stress hormone after blast exposure. See FIG. 3, which shows the plasma corticosterone levels at 24 hours and 1month post-blast, with and without Pl 3 treatment. Values are mean +SEM; *p<0.05; n = 4; ns = not significant.

[0094] Example 6: Western Blot Analysis.

[0095] Brain samples were used for determining the differential expressions of known biomarkers of TBI using Western blot analysis in the brain regions. Antibodies specific to pNFH, neurofilament light chain (NFL present in degenerating neurons), phosphorylated Tau (phosphorylated at serine 396 or threonine 231), glial fibrillary acidic protein (GFAP) and actin (for loading control) were used.

[0096] Western blotting revealed that blast exposure increases the phosphorylation Tau protein which can lead to neurodegeneration, but treatment with P13 reduced this tau phosphorylation post-blast. See FIG. 4, which shows the differential expression of TBI protein biomarkers at 24 hours in different brain regions post-blast, with and without P13 treatment, and FIG. 5, which shows the differential expression of TBI protein biomarkers at 1 month post-blast in different brain regions, with and without P13 treatment. The immunoreactivity of the antibody which selectively binds to NFL present in degenerating neurons indicated accelerated neurodegeneration post-blast and the P 13 treatment can protect against this accelerated neurodegeneration .

[0097] The data discussed in Examples 4-6 show that expression of pNFH in different brain regions decreased after blast exposure, supporting its increased levels in the CSF (FIG. 2 and FIG. 3), but P13 treatment inhibited this axonal degeneration process post-blast (FIG. 4 and FIG.5). The levels of pNFH in some brain regions after P13 treatment were higher than even sham controls, suggesting the inhibition of normal axonal degeneration occurring under physiological conditions. The levels of GFAP in different brain regions post-blast revealed that blast-induced astrogliosis can be inhibited by treatment with P13. The western blot analyses indicated that P13 at 2 mg / kg dose showed more efficacy compared to 10 mg / kg dose warranting future studies with doses lower than 2 mg / kg. See FIG. 4 and FIG 5.

[0098] Example 7: Protection Against Neurobehavioral Outcomes of TBL

[0099] The efficacy of a 2 mg / kg dose of P13 (which provided maximum protection in the biochemical outcome measures) for protection against blast-induced TBI was tested usingneurobehavioral functional outcome measures. Male ferrets were exposed to blast as described above (two 19 psi blasts in two minutes) and treated with P13 (2 mg / kg) by injection at 30 minutes post-blast with continued treatment every day until euthanasia. Neurobehavioral functional assessments were done at different intervals post-blast in animals with and without P13 treatment (day 1, day 7, day 14, and day 28). Neurobehavioral functions in ferrets were assessed using the openfield locomotion test (for anxious and depressive-like behaviors), the novel object recognition test (NOR, for short-term memory function), gait analysis using the CatWalk XT system, and actigraphy measurements for sleep pattern.

[0100] The NOR test for short-term memory function indicated that blast exposure significantly disrupted the short-term memory function in the ferrets and that Pl 3 treatment improved the memory function. See FIG. 6, which presents data on the discrimination index measured in the novel object recognition test carried out on days 2 (FIG. 6A) and 28 (FIG. 6B) post-blast, with and without P13 treatment. *p<0.05; NT = not treated; DI = discrimination index.

[0101] The sleep fragmentation index measured using the actigraph device revealed that blast exposure disrupted the circadian rhythm or normal sleep pattern on day 14 post-blast and that this disruption is inhibited by P13 treatment. See FIG. 7, which shows the sleep fragmentation index (SFI) measured using actigraphy on the days indicated. *p<0.05; NT = not treated.

[0102] The results of the openfield locomotion test are provided in FIGs 12A-C. The results revealed that blast exposure causes anxious and depressive- like behaviors in the ferrets and P13 treatment provided significant protection. The results of gait analysis using the CatWalk XT system is provided in FIG. 13. The results indicated that blast exposure disrupted the front and hind limb movements of ferrets and P13 treatment provided significant protection.

[0103] Example 8: Effects of Pl 3 Treatment on Inner Ear and Hearing Function in Rats.

[0104] For testing the efficacy of P13 applied as ear drops, rats were exposed to repeated low-level blasts (one 4psi blast per day for three consecutive days) under isoflurane anesthesia. Preexposure treatment, when administered, consisted of P13 ear drop administration (100 pg / ear in 20 pl saline under isoflurane anesthesia) at 24 hours and 1 hour before the first blast exposure and then continued once per day until euthanasia. Post-exposure treatment, when administered, consisted of ear drop administration started only at 1 hour after the third blast exposure and continued once per day until euthanasia.

[0105] Treatment efficacy was evaluated using auditory functional testing using auditory brainstem response (ABR) on days 1, 7, 14 and 28 after the third blast. A pathological evaluation (qRT-PCR analysis of inner ear and phalloidin staining of the cochlear hair cells) was performed on days 1 and 30 post-blast. See FIG. 8 through FIG. 11 for results.

[0106] FIG. 8A through FIG. 8H provide qRT-PCR data showing that blast exposure upregulates multiple pro-inflammatory cytokines and downregulates anti-inflammatory cytokine in the inner ear on days 1 and 28 after blast injury. P13 administration provided protection against these changes in the inner ear post-blast.

[0107] FIG. 9A through FIG. 9D provide qRT-PCR data showing that blast exposure downregulates prestin (the outer hair cell specific protein) and calretinin (the calcium buffering protein) in the inner ear on day 1. P13 administration provided protection against these changes in the inner ear post-blast.

[0108] FIG. 10A through FIG. 10F provide immunohistochemical data showing that blast exposure causes loss of outer hair cells in the apical, middle and basal regions of the cochlea on days 1 and 28 post-blast. P13 administration provided protection against this outer hair cell loss post-blast.

[0109] FIG. 11 A through FIG. 1 IL shows auditory brainstem response (ABR) testing. The data showed a decrease in ABR amplitudes on days 1 and 7 post-blast, where P13 administration provided protection against this auditory functional deficit.

[0111] References.

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Claims

CLAIMSWhat is claimed is:

1. A fusion peptide comprising a Pl 3 peptide covalently linked to a cell penetration peptide sequence.

2. The fusion peptide of claim 1 wherein the P13 peptide is DIVKLTVYDCI (SEQ ID N0:3).

3. The fusion peptide of claim 1, wherein the P13 peptide is a conservative variant of DIVKLTVYDCI (SEQ ID NO:3).

4. The fusion peptide of claim 1, wherein the cell penetration peptide sequence is RRRRRRRRR (SEQ ID NO:2).

5. The fusion peptide of claim 1, wherein the fusion peptide is DIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 1).

6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the fusion peptide of claim 1.

7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the fusion peptide of claim 5.

8. A method of treating traumatic brain injury in a subject in need thereof, comprising administering to the subject the fusion peptide of claim 1.

9. A method of treating traumatic brain injury in a subject in need thereof, comprising administering to the subject the fusion peptide of claim 5.

10. A method of treating traumatic brain injury in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 6.

11. A method of treating traumatic brain injury in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 7.

12. A method of treating traumatic brain injury of claim 6, wherein the pharmaceutical composition is administered before the subject receives a traumatic brain injury.

13. The method of claim 12, wherein the pharmaceutical composition is administered within about 1 day to about 30 days before the subject receives a traumatic brain injury.

14. A method of treating traumatic brain injury of claim 6, wherein the pharmaceutical composition is administered after the subject receives a traumatic brain injury.

15. The method of claim 14, wherein the pharmaceutical composition is administered within about 1 minute to about 14 days after the subject receives a traumatic brain injury.

16. The method of claim 14, wherein the pharmaceutical composition is administered as soon as possible after the subject receives a traumatic brain injury and is administered further daily doses of the pharmaceutical composition for about 1 day to about 6 months.

17. The method of claim 14, wherein the pharmaceutical composition is administered as soon as possible after the subject receives a traumatic brain injury and is administered further daily doses of the pharmaceutical composition until symptoms of traumatic brain injury abate.

18. The method of claim 12, wherein the dose administered is about 0.1 mg / kg to about 20 mg / kg.

19. The method of claim 14, wherein the dose administered is about 0.1 mg / kg to about 20 mg / kg.

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