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  Pharmaceutical Patents  


Title:  Treatment of non-convulsive seizures in brain injury using G-2-methyl-prolyl glutamate
United States Patent: 
May 11, 2010

 Gluckman; Peter David (Auckland, NZ), Brimble; Margaret Anne (Auckland, NZ), Wilson; Douglas (Taupo, NZ), Tortella; Frank Casper (Columbia, MD), Williams; Anthony Joseph (Middletown, MD), Lu; Xi-Chun May (Laurel, MD), Hartings; Jed A. (Silver Spring, MD), Gryder; Divina (Potomac Falls, VA)
  Neuren Pharmaceuticals Limited (Auckland, NZ)
Appl. No.:
 April 4, 2006


Executive MBA in Pharmaceutical Management, U. Colorado


Aspects of this invention include the use of G-2MePE to treat patients with brain injury characterized by non-convulsive seizures. G-2MePE is useful in treating brain injuries caused by traumatic brain injury, stroke, hypoxia/ischemia and toxic injury.

Description of the Invention


1. Field of the Invention

This invention relates to analogs of glycyl-L-prolyl-L-glutamic acid (GPE). In particular, this invention relates to neuroprotective GPE analogs, to methods of making them, to pharmaceutical compositions containing them, and to their use in treating neurological disorders resulting from brain injury and characterized by non-convulsive brain seizures.

2. Description of Related Art

Each year approximately 1.5 million people in the U.S.A. sustain a traumatic brain injury with an estimated 1.0 million hospitalised. Of these, 225,000 are moderate to very severe and 50,000 result in death. These injuries can be caused by concussions, penetrating injury, contusions and diffuse axonal injury resulting from tearing of brain tissue. Traumatic brain injury is a difficult and often frustrating condition to treat. Blunt head trauma can result in brain hemorrhage, swelling and increased intracranial pressure. Penetrating wounds caused by projectiles can be particularly difficult because of the rapid absorption by brain tissues of a large amount of kinetic energy and the high degree of damage that can result. As a direct result of such injuries, brain cells can be damaged or die. Additionally, secondary effects can further exacerbate the loss of functional neurons. For example, cellular damage can release cytokines and other chemoattractive molecules into the brain and can cause inflammation. Inflammation itself can cause additional damage to brain tissues and cells through the release of proteases and other inflammatory mediators, which may recruit yet additional cell types and exacerbate the problems further.

Many attempts are made to reduce the severity of brain injury. Surgery can be used to remove projectiles, bone fragments or other debris from the brain. Additionally, surgery can be successful in certain cases to relieve increased intracranial pressure, which can cause additional nerve damage, either through a direct effect on pressure, or an effect related to changes in blood flow to affected portions of the brain. For example, a focal traumatic injury that causes bleeding or increased vascular permeability can produce an area of increased hydrostatic pressure. If the pressure is sufficiently high, blood flow to nearby portions of uninjured neural tissue can be reduced, compromising oxygenation of the affected tissue. Further, decreased blood flow, if severe enough, can cause starvation of brain tissues due to decreased flow of nutrients to the affected areas. As a result of these changes, in most patients with traumatic brain injury, recovery is often slow and incomplete. With prolonged periods of injury, neurological functions can be severely compromised and neural deficits may persist for many years, or even for the remainder of the patient's lifetime.

In addition to traumatic brain injury, stroke or severe hypoxia/ischemia can also result in brain injury. In many cases, patients with stroke exhibit similar signs and symptoms as patients with traumatic brain injury, including penetrating ballistic brain injury (PBBI). Further, perinatal asphyxia and coronary artery bypass graft (CABG) surgery, brain seizures and neurotoxic agents can lead to brain injury.

In many types of brain injury, neural deficits and neurological signs may be easy to evaluate. In some cases, impairment of motor function or abnormalities in electroencephalographic (EEG) signals is observed. In many animals and humans with traumatic brain injury, stroke or severe hypoxia/ischemia, a type of delayed EEG abnormality or brain seizure may evolve that is associated with overt motor convulsions and is therefore clearly identifiable by observers of such patients and thereby treatable with established anti-epileptic drug (AED) therapy.

However, in most cases of brain injury acute/early monitoring of EEG brain function is impossible or impractical. Here seizures may occur that are not associated with overt motor abnormalities. Without continuous EEG monitoring these "non-convulsive seizures" ("NCS") or "silent brain seizures" ("SBS") are not observed as a clinical feature of the brain trauma and go untreated. Nonetheless, such non-convulsive seizures can reflect severe brain injury. In one study, a subgroup of patients with severe traumatic brain injury experienced electroencephalographic signs of seizures, but had no convulsions (Vespa et al., J. Neurosurg 91:750-760 (1999), expressly incorporated herein fully by reference.

Non-convlusive seizures are not only symptomatic, but also can contribute to poor patient outcome. Thus, it is desirable to identify useful treatments for NCS. Although gabapentin and ethosuximide have been reported to reduce experimental NCS, many conventional antiepileptic agents are ineffective (Williams et al., J. Pharmacol. Exp. Therap. 311:220-227 (2004), expressly incorporated herein fully by reference). Furthermore, efforts to treat NCS in human TBI with standard AED therapies have proven ineffective thereby identifying a critical care need in the art for improved methods of treating NCS.

It had been previously believed that mature nervous tissue is incapable of regeneration or recovery after severe injuries. Thus, few attempts have been made to treat brain damage to restore neural function. Fortunately, this misapprehension is being reversed, due in large part to recent studies on neural regeneration. For example, insulin-like growth factor 1 (IGF-1) has been shown to promote neural survival in animals with brain injuries. The N-terminal tripeptide of IGF-1, glycyl-prolyl-glutamate (Gly-Pro-Glu; GPE or Glypromate.TM.) has similar neuroprotective effects. In fact, GPE has been used both in vitro and in vivo to treat neurodegeneration. However GPE is rapidly hydrolyzed by enzymes in plasma and in tissues thereby contributing to a relatively short half-life in vivo. Therefore, there is a great need for new types of therapies that can be used to treat neural damage associated with brain injuries resulting from stroke, various traumatic brain insults, coronary artery by-pass graft, hypoxic-ischemic episodes, etc.

EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-Pro-Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0 366 638 discloses that GPE is effective as a neuromodulator and is able to affect the electrical properties of neurons.

WO95/172904 discloses that GPE has neuroprotective properties and that administration of GPE can reduce damage to the central nervous system (CNS) by the prevention or inhibition of neuronal and glial cell death.

WO 98/14202 discloses that administration of GPE can increase the effective amount of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and nitric oxide synthase (NOS) in the central nervous system (CNS).

WO99/65509 discloses that increasing the effective amount of GPE in the CNS, such as by administration of GPE, can increase the effective amount of tyrosine hydroxylase (TH) in the CNS in order to increase TH-mediated dopamine production in the treatment of diseases such as Parkinson's disease.

WO02/16408 discloses GPE analogs capable of inducing a physiological effect equivalent to GPE within a patient. The applications of the GPE analogs include the treatment of acute brain injury and neurodegenerative diseases, including but not limited to, injury or disease in the CNS.

The disclosures of these and other documents referred to in this application (including in the Figures (see Original Patent)) are expressly incorporated herein by reference as if each one was individually incorporated by reference.


To address the above and other problems in the art, we have recently discovered that an analog of GPE, namely glycyl-2-methylprolyl-glutamate (G-2MePE) has neuroprotective properties. We have unexpectedly found that G-2MePE can be used to protect neural tissue in animals with traumatic brain injuries, including penetrating brain injuries, stroke, hypoxia/ischemia and toxic injury. Furthermore, G-2MePE can be effective in reducing the incidence and severity of non-convulsive brain seizures, and can be effective in restoring motor coordination in animals with traumatic brain injury.


Description of Embodiments

Non-convulsive seizures (NCS) can be present in patients having brain injuries caused by a variety of insults. In fact, some patients present with a history of brain injury but with few signs or symptoms. Thus, many of those patients are untreated or undertreated. Because NCS can lead to worsening in outcome, including more significant brain pathology and later neurological impairment, it can be important to intervene in treatment early after an insult that may lead to brain injury. Thus, a sub-group of patients with brain injury can be identified by the presence of non-convulsive brain seizures, even in the absence of motor or other frank neurological abnormalities. By diagnosing NCS at an early stage, patients can be treated and worsening of outcome be minimized, if not improved. Thus, in certain embodiments of this invention, patients with NCS can be treated effectively with G-2MePE, decreasing the overall severity of the SBS contribution to brain damage by 1) decreasing the incidence of NCS, and/or 2) increasing the time before NCS signs appear. This strategy can be useful in treating patients with stroke, hypoxia/ischemia, toxic and/or traumatic brain injury, including penetrating ballistic brain injury (PBBI).

Studies of stroke can be carried out using a well-characterized animal system in which a middle cerebral artery is occluded (MCAo). In animals with MCAo, a portion of the brain is deprived of oxygen, resulting in an infarct, similar to that seen in non-hemorrhagic stroke in human patients. Using this system, we have unexpectedly found that G-2MePE can decrease the incidence of NCS and can prolong the time period before which NCS signs appear. Similarly, hypoxia/ischemia can be studied in animal systems by temporarily or permanently occluding a carotid artery. We have previously demonstrated that G-2MePE can reduce the severity of neural degeneration in animals subjected to hypoxia/ischemia (U.S. application Ser. No. 10/155,864; U.S. application Ser. No. 11/314,424), both applications herein expressly incorporated fully by reference.

Penetrating ballistic brain injury (PBBI) has been more difficult to study due to the lack of suitable animal systems. However, with the recent development of an animal model system for generating and evaluating PBBI (Williams et al., Journal of Neurotrauma 22(2):314-332 (2005), expressly incorporated herein fully by reference, it is now possible to evaluate prospective therapies. Although PBBI has certain unique features, many of the signs and symptoms of PBBI are similar to those of other types of brain injuries including impairment if cerebral blood flow (cerebral ischemia) as may be encountered in clinical ischemic insults including stroke. For example, in an animal model of stroke (the system described in U.S. patent application Ser. No. 11/314,424), we found that motor deficits associated with loss of brain tissue caused by occlusion of the middle cerebral artery (MCAO) can be reduced by G-2MePE. Herein, we unexpectedly found that in other animals without motor seizure activity, G-2MePE can delay and in many cases prevent the appearance of NCS in animals with MCAo.

Animal Model System for Studying PBBI

With the development of an animal model for studying PBBI it is now possible to carry out careful studies of PBBI under controlled conditions. Briefly, the animal system was designed to mimic effects in human beings who suffer from PBBI, for example caused by a penetrating round (bullet). When a penetrating round enters the brain, the increased drag forces and yaw angle cause the round to tumble. During such unstable flight, an estimated 83% of the round's available energy is dissipated into the tissue, forming a large temporary cavity in the shape of an ellipsoid. This temporary cavity, which is estimated to be 10-20 times larger than the size of the permanent cavity caused by the missile track, compresses the surrounding tissue and is considered to be a major source of damage from a ballistic wound.

The penetrating ballistic brain injury model described in Williams et al (2005), which is that used in this application was designed to model two aspects of a high-energy bullet wound to the head: (1) the permanent injury tract created by the path of the bullet itself, and (2) the large temporary cavity generated by energy dissipation from the penetrating missile. The injury is produced by insertion of a specially designed probe into the brain of an anesthetized rat at the desired location (permanent injury tract) and rapid inflation of an attached balloon to mimic the temporary cavity induced by a penetrating bullet. Parameters for the size and shape of the temporary cavity were calculated based on the cavitation produced in the human brain by a NATO 7.62 mm round. The size of the probe and the volume of the expanded balloon were scaled to the rat brain by a ratio of 762.5:1. Due to the linear relationship between the bullet's impact velocity and the diameter of the temporary cavity, different injury severities can be modelled by expanding the balloon to different volumes.

As a result of such injuries, hemispheric swelling and intracranial pressure increases, reactive astrocytes increase in number, microglia and leukocyte infiltration is observed. Neurological tests and behavioural tests (e.g., balance beam test) demonstrate sensory-motor deficits. Additionally, severe, electroencephalographic disturbances occur, including the presence of cortical spreading depression, slow-waves and brain seizure activity. Thus, the rat system used herein is a reproducible system that produces quantifiable measures of outcome of PBBI and is scalable to the severity of injury. Thus, results obtained using this animal system closely mimic human PBBI, and studies using this system are therefore predictive of outcomes in humans. Additionally, this animal system can provide highly predictive results of studies on agents that protect neurons from damage caused by PBBI, making positive results obtained with G-2MePE highly predictive of effects that are expected in treating humans with G-2MePE.

Non-Convulsive Seizures

Brain injury can, in many cases, cause a type of seizure without motor effects. Such seizures, non-convulsive seizures (NCS), also known as silent brain seizures (SBS) have been observed in humans with severe traumatic brain injury (Vespa et al., J. Neurosurg 91:750-760 (1999). In these patients, many have abnormal electroencephalographic (EEG) activity that can be monitored using continuous EEG measurements. Such measurements show both epileptic types of activity, including a sudden onset of repetitive spike-and-wave discharges that can increase in amplitude and evolve over time. One type of EEG pattern is pseudoperiodic lateralized epileptiform discharges ("PLEDs"). Other subjects showed non-epileptiform discharges, including a symmetrical disorganized slowing in the 5 to 7 Hz range or asymmetrical disorganized delta waves. Importantly, although classical therapies for epileptiform EEG abnormalities (e.g., phenyloin, midazolam, lorazepam, phenobarbital and the like) may be effective in reducing delayed post-traumatic epileptiform abnormalities, those agents may not be effective by themselves in treating patients suffering from SBS.

Similarly, acute seizures can be observed after intracerebral hemorrhage (Vespa et al. Neurology 60:1441-1446 (2003). In a population of patients with ischemic stroke or intraparenchymal hemorrhage, EEG recordings showed electrographic seizures. Additionally, seizures after intercerebral hemorrhage may be of the non-convulsive type (NCS or SBS).

The types of EEG activity described for human are mimicked by similar epileptiform or NCS activity in rats (Hartings et al. Experimental Neurology 179:139-149 (2003). In rats subjected to middle cerebral artery occlusion (MCAo), EEG abnormalities include 1-3 Hz rhythmic spiking, PLEDs and intermittent rhythmic delta activity (IRDA). PLEDs were characterized by appearance of interictal spikes, sharp or slow waves recurring with a variable period of from 1 to 8 seconds. IRDA events were characterized by presence of readily identifiable, brief (e.g., <10 second) bursts of rhythmic, large-amplitude waves in the delta-theta (3 to 8 Hz) frequency range. In many cases, electrographic seizures were not associated with motor convulsant activity.

Thus, we can define a sub-population of patients with acute brain injury and the presence of NCS activity without overt motor manifestations. For example, a patient having a history of brain trauma or IRDA and PLEDs may be well suited for therapy with G-2MePE. In these patients, G-2MePE can be useful in decreasing the magnitude and number of NCS events, and thereby can decrease the likelihood of later neurological damage caused by NCS activity.

In some cases, it can be desirable to treat a subject experiencing NCS with G-2MePE and another agent to inhibit neurodegeneration. Several other neuroprotective agents are known in the art, and include Gly-Pro-Glu (GPE), insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), transforming growth factor-.beta.1, activin, growth hormone, nerve growth factor, growth hormone binding protein, IGF-binding protein-3 (IGFBP-3], basic fibroblast growth factor, acidic fibroblast growth factor, hst/Kfgk gene product, FGF-3, FGF-4, FGF-6, keratinocyte growth factor, androgen-induced growth factor, int-2, fibroblast growth factor homologous factor-1 (FHF-1), FHF-2, FHF-3 and FHF-4, keratinocyte growth factor 2, glial-activating factor, FGF-10, FGF-16, ciliary neurotrophic factor, brain derived growth factor, neurotrophin 3, neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial-cell line derived neurotrophic factor, activity-dependant neurotrophic factor, cytokine leukaemia inhibiting factor, oncostatin M, interleukin), .alpha.-, .beta., .gamma., or consensus interferon, TNF-.alpha., clomethiazole; kynurenic acid, Semax, tacrolimus, L-threo-1-phenyl-2-deca-noylamino-3-morpholino-1-propanol, adrenocorticotropin-(4-9) analog (ORG 2766), dizolcipine (MK-801), selegiline, glutamate antagonists NPS1506, GV1505260, MK-801, GV150526, AMPA antagonists 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070, LY300164, MAdCAM-1mAb, MECA-367 (ATCC accession no. HB-9478), an anti-.alpha.4.beta.1 receptor antibody and an anti-.alpha.4.beta.17 receptor antibody.

It can be appreciated that along with G-2MePE, classical anti-epileptic medications can be used if desired. Combination therapy using G-2MePE and one or more other anti-epileptic drugs (AEDs) can be of benefit in treating a non-convulsive event. Thus, in certain embodiments, one or more hydantoins, including phenyloin, fos-phenyloin, mephenyloin and ethotoin may be used along with G-2MePE. In other embodiments, one or more barbiturates, including phenobarbital, mephobarbital, primidone and its metabolite phenylethylmalonamide (PEMA) can be used along with G-2MePE. In still other embodiments, G-2MePE and one or more iminostilbenes, including carbamazepine, can be used, as well as one or more succinimides, including ethosuximide. Additionally, valproic acid and/or its salt valproate and G-2MePE can be used in combination to achieve desired therapeutic effects. In other situations, G-2MePE can be used with one or more oxazolidinediones, including trimethadione and paramethadione, one or more benzodiazepines, including clonazepam, clorazepate, lorazepam, diazepam and its metabolites N-desmethyldiazepam and oxazepam, gabapentin, lamotrigine, .gamma.-vinyl gamma amino butyric acid (.gamma.-vinyl GABA), one or more carbonic anhydrase inhibitors including acetazolamide, one or more dicarbamates including felbamate. In still further embodiments, G-2MePE can be used in combination with one or more agents including midazolam and dextromethorphan. It can be appreciated that G-2MePE can be used with one or more agents from different classes noted herein.

Claim 1 of 19 Claims

1. A method of treating an animal having a brain injury and an electroencephalographic (EEG) pattern characteristic of a non-convulsive seizure (NCS), comprising administration to an animal in need thereof a therapeutically effective amount of a neuroprotective agent which is glycyl-L-2-methylpropyl-L-glutamate (G-2MePE).

If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.


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