CN1267149C - Composition and methods to improve neural outcome - Google Patents

Abstract

The tripeptide glycine-proline-glutamine (GPE) may be administered before or usually after injury, to reduce damage to the central nervous system. GPE appears useful for neuronal rescue particularly but not exclusively within the hippocampus. Advantages of GPE include: a) that it crosses the blood-brain barrier, so is effective by injected peripheral administration; b) it is unlikely to challenge the immune system; c) it is cheap; and d) its therapeutic ratio is high. GPE may be also be infused into the CSF. It may be administered prior to parturition or elective brain or cardiac surgery. Transdermal routes may be useful for chronic neural disorders. The CNS of mammals (including foetal mammals) after trauma including hypoxic/ischaemic experimental insults showed reduced damage under GPE protection as measured by histological assesment of cell damage or death and regional shrinkage.

Description

Compositions and methods for improving central output

invention technical field

The present invention relates to methods and therapeutic compositions for the treatment or prevention of cellular damage in the mammalian central nervous system (CNS) - as well as the protection of the peripheral nervous system, and more particularly to the enhancement of specific naturally occurring or induced 2 in the central nervous system. - or 3-peptide concentration for the method of treating damage or disease affecting or prone to affect CNS (or PNS) cells.

Background of the invention

The central nervous system is unique among mammalian organs in which differentiated neurons are virtually non-regenerating. Long-lasting loss of function may be the result of severe damage to the brain. Especially sad for children is the brain damage caused by dystocia and inadequate oxygen supply. There is therefore a need for a method for protecting central nervous system cells, including glial cells, from death following injury.

Following damage to the central nervous system (CNS) of humans or other mammals by asphyxiation, trauma, poisoning, infection, degeneration, metabolism, ischemia, or hypoxia results in varying degrees of damage to several different cell types. For example, an injury generally thought to affect the periventricular oligodendrocytes, periventricular leukoplakia is the result of hypoxic ischemia that damages the developing prebrain (Bejar et al., Am. J. Obstet .Gynecol., 159:357-363 (1988); Sinha et al., Arch.Dis.Child., 65:1017-1020 (1990); Young et al., Ann.Neurol., 12:445-448 ( 1982)). Injury to the CNS by trauma, asphyxiation, ischemia, poisoning, or infection is often and can cause sensory, motor, or cognitive deficits. Glial cells, which are non-central cells in the CNS, are required for normal CNS function. Infarction is a major component of some hypoxic ischemia-induced injuries and loss of glial cells is an essential component of infarction. Various "delayed injury processes" have emerged, in which "self-destructive" neural effects are sometimes evident after injury; attempts to control this effect seem to be able to mitigate the consequences of this delayed injury process.

CNS diseases can also cause the loss of specific cell populations. For example, multiple types of necrosis are associated with loss of myelin and oligodendrocytes, and similarly, Parkinson's disease is associated with loss of dopaminergic neurons. Some situations in which CNS injury or disease causes significant loss of neurons and/or other cell types include: perinatal asphyxia associated with fetal distress such as cord closure after separation or perinatal asphyxia associated with intrauterine growth retardation ; perinatal asphyxia associated with failure of adequate resuscitation or breathing; drowning associated with imminent death, near death in hospital bed, inhalation of carbon monoxide, ammonia or other gas poisoning, cardiac arrest, collapse, coma, meningitis, anemia and conditions Severe CNS injury associated with epilepsy; cerebral ischemia associated with coronary bypass surgery; cerebral hypoxia or ischemia associated with stroke, hypotension, and hypertension; brain trauma.

There are many other situations where CNS injury or disease causes damage to CNS cells. Treatment of injuries is desirable in these situations. It is also desirable to prevent or reduce the amount of CNS damage that results from the induction of cerebral asphyxia in the case of, for example, cardiac bypass surgery.

We have previously demonstrated (New Zealand patent application №239211-"IGF-1 to improveneural outcome, IGF-1 to improve central output", the content of which is incorporated herein by reference) as insulin-like growth factor 1 (IGF-1) The so-called non-involved effect of the growth factor is related to the prevention of brain cell death after asphyxia or brain injury caused by ischemia (Gluckman et al Biochem Biophys Res Commun 182:593--599 1992). Since insulin is also neuroprotective (Voll et al Neurology 41:423-428 (1991)) and both insulin and IGF-1 are able to bind to IGF-1 receptors, this brain-salvage mode of IGF-1 action can often be speculated It is mediated by the IGF-1 receptor (Guan et al J. Cereb. Blood Flow Metab. 13: 609-616 (1993)).

It is known that protein cleavage in nerve tissue can modify IGF-1 to des 1-3NIGF-1, that is to say, IGF-1 loses the three amino acids at the amino terminal of the molecule, so after cleavage it also produces a three-amino acid Peptide gly-pro-glu, which is an N-terminal tripeptide. This tripeptide is also known as GPE. Since des 1-3N IGF-1 was also able to bind to the IGF-1 receptor, but GPE was not, it was not considered significant for the neuronal rescue effect of IGF-1 by GPE.

Our previous studies have demonstrated that the brain increases its production of IGF-1 after brain injury due to ischemia-hypoxia, and additionally the brain also increases two specific binding proteins, IGF-binding protein-2 (IGFBP-2) Synthesis of IGF-binding protein-3 (IGFBP-3) (Gluckman et al Biochem Biophys Res Commun 182:593--599 1992) and Klemp et al Brain Res 18:55-61 (1992). It is hypothesized that nor-IGF-1 is absorbed into the injured area to achieve the concentrations necessary to rescue neurons. For this reason, IGF-1 is expected to reach a given site far from the site of injury more efficiently than des1-3N IGF-1, which does not bind well to the protein. Indeed for this case, des 1-3N IGF-1 had no significant activity as a neuronal rescue factor at doses equivalent to those at which IGF-1 showed neuronal rescue. Therefore, it is suggested in the prior art that IGF-1 has neuron-rescuing activity on the IGF-1 receptor.

So far, no method has been given in the prior art to cleave the tripeptide GPE itself to prevent or treat CNS injury or disease resulting from CNS injury in vivo.

purpose of invention

It is an object of the present invention to provide methods and/or medicaments (therapeutic compositions) for the treatment or prevention of CNS damage which at least to some extent satisfy a need in a simple and effective manner or are at least available to the public useful options.

Summary of the invention

Therefore, in a broad sense, the present invention includes a method for treating nerve damage in a mammal (or a patient), comprising the steps of: increasing the concentration of the tripeptide GPE (peptide gly-pro-glu of three amino acids) in the CNS of the mammal and/or the concentration of GPE analogs. In particular, effective in increasing the concentration of the tripeptide GPE in the CNS of mammals.

Preferred GPE analogs are peptides selected from the group consisting of gly-pro-glu (GPE), gly-pro and pro-glu.

In one aspect the invention relates to the treatment of injuries to the central nervous system (CNS), which is considered to include cell bodies (including neurons and support cells such as glial cells or nerve sheath cells, etc.) in order to include as much as possible the site of GPE action. ) localized parts of the nervous system. The treatment of the peripheral nerves as well as the treatment of the brain, spinal cord etc. are therefore part of the present invention.

More specifically, the invention encompasses methods for treating neuronal damage in at least the hippocampus.

(The term "treating" as used herein refers to at least attempting to achieve a reduction in the severity of CNS injury by reducing neuronal loss and loss of glia and other cells following CNS injury. It includes reducing such damage to minimal.)

(The term "injury" as used herein includes asphyxiation, ischemia, stroke, poisoning, infection, trauma, hemorrhage, and surgical injury to the CNS.)

Effectively, GPE and/or its analogs are administered directly to the patient. Alternatively, administering to the patient a drug that increases the active concentration of GPE or a naturally occurring GPE analog in the patient's CNS. For example, increasing available IGP-1 can lead to increased GPE concentrations.

Effectively, the drug is administered before injury and/or up to 100 hours after CNS injury and more preferably during 0.5-8 hours after CNS injury.

Another alternative is to administer the drug prior to the selected step if it is believed that the selected step may cause damage to the CNS, so that the level of GPE can be increased between the steps.

The first form, preferably, administers GPE selected from the following group and/or its Analogs: gly-pro-glu, gly-pro and pro-glu.

In another preferred form, GPE and/or its analogues selected from the group consisting of gly-pro-glu, gly-pro-glu, gly- pro and pro-glu.

In another preferred form of the invention, GPE and/or its analogs selected from the group consisting of gly- pro-glu, gly-pro and pro-glu. Using the peripheral route, we plan to use intravenous, oral, rectal, nasal, subcutaneous, inhalational, peritoneal or intramuscular routes. Preferably, the GPE itself is administered by means of lateral ventricle injection or with a surgically inserted shunt.

Preferably, the dosing is administered according to the type of injury or the time elapsed after the CNS injury.

Preferably, the dosage range of administration is 0.1 μg-10 mg of GPE (or said analogue or compound increasing its concentration) per 100 gm of body weight.

More preferably, the dosage range of administration is 1 mg of GPE per 100 gm of body weight.

Alternatively, artificial CSF can be infused into the lateral ventricle or other infusion site suitable for CSF assessment at a dose rate of approximately 10 μg/kg.

GPE (or said analogs or compounds that increase its concentration) can be used alone or in combination with other drugs or growth factors capable of ameliorating the loss of CNS cells such as glial cells and neurons.

By "preventing" is meant a reduction in the severity of a CNS injury sustained following a CNS injury and thus includes inhibiting symptoms of CNS injury.

In another aspect, the present invention provides the use of GPF and/or its analogues in the preparation of medicaments for treating CNS injuries.

Alternatively, the present invention comprises the use of a compound which, when administered to a patient, increases the active concentration of GPE and/or naturally occurring GPE analogs in the patient's CNS for the preparation of a medicament for treating CNS damage .

The present invention also includes a medicament suitable for the treatment of CNS injury sustained after CNS injury comprising human dosage form of GPE and/or its analogs provided in a pharmaceutically acceptable carrier or diluent.

In a related aspect, GPE-containing medicaments may be provided together with suitable pharmaceutically acceptable excipients.

In another related aspect, a GPE-containing medicament may be provided in a mammalian dosage form.

In another related aspect, the medicament for treating CNS injury may also comprise a human dosage form of a compound or composition which, when administered to a patient after suffering a CNS injury, increases GPE and/or its natural Active concentration of GPE analog present.

Alternatively, the drug that stimulates GPE levels can be provided in a mammalian dosage form.

The invention further provides a method for treating a patient suffering from a chronic degenerative form of the nervous system comprising administering GPE and/or an analog thereof for a prolonged period of time.

Preferably, GPE and/or its analogs (with suitable pharmaceutically acceptable carriers etc. if necessary) are administered to such patient.

Alternatively, GPE and/or its analogs can be provided with charged molecules and facilitate their uptake by electrophoretic manipulation.

Alternatively, the invention further provides a substance (GPE or said analogue or compound increasing its concentration) as a prophylactic application, so that during the indicated event (such as a specific procedure such as open heart surgery) Minimize CNS damage.

While the invention has been broadly defined above, those skilled in the art will recognize that it is not limited thereto and includes the described embodiments.

Brief description of the drawings

The invention can be better understood with reference to the following examples and accompanying drawings, in which:

Figure 1 : Cortical infarction after treatment with 50 μg of IGF-1 vehicle alone or the NMDA antagonist MK801 (1 mg) or IGF-1 plus MK801 after 2 hours of hypoxia. Similar to previous studies, cortical infarcts were reduced in the IGF-1-treated group, while MK801 had a smaller effect.

Figure 2: Example showing the effect of treatment with 1 μg IGF-1 after 2 hours of fetal sheep ischemia. The names below the horizontal axis are standard abbreviations for the various parts of the brain. This dose is neuroprotective but unlike MK801, does not suppress seizures.

Figure 3: Shows cortical infarction and hippocampal damage following treatment with 3 μg GPE or vehicle following hypoxia for 2 hours. [Hippocampal damage decreased after treatment with 3 μg GPE. ^* p<0.05]

Figure 4: Shows the results of the same experiment; where the left two columns show the area of surviving cortical tissue preserved after treatment (columns are considered stereologically) as a ratio of the right and left (injured) cases of the brain , while the two columns labeled CA-1 show the proportion of viable neurons remaining after injury (right and left comparison).

Figure 5: shows the dose-response effect of GPE on hippocampal (CA1-2 area) neuronal output following peripheral (peritoneal) administration of GPE. The vertical axis shows the ratio of R/L; the ratio between the unligated and ligated sides of the brain.

Figure 6: is a photomicrograph showing GPE binding to the injured side of the hippocampus.

Technical details of the invention

We already know that improving protein cleavage in nerve tissue can modify insulin-like growth factor (IGF-1) to des 1-3N IGF-1, which means that IGF-1 loses the three amino acids at the amino terminal end of the molecule and produces An N-terminal tripeptide gly-pro-glu (GPE) consisting of three amino acids was identified. Since des 1-3N, IGF-1 is also able to bind to the IGF-1 receptor, but GPE is not, it is not considered significant for the neuronal rescue effect of IGF-1 by GPE. Surprisingly, GPE works.

Our previous studies have demonstrated that the brain increases its production of IGF-1 after brain injury due to ischemia-hypoxia and that the brain also increases two specific binding proteins, IGF-binding protein-2 (IGFBP-2) and Synthesis of IGF-binding protein-3 (IGFBP-3) (Gluckman et al Biochem Biophys Res Commun 182:593--599 1992) and Klemp et al Brain Res 18:55-61 (1992). It is hypothesized that IGF-1 is absorbed into the injured area to achieve the concentrations necessary to rescue neurons. For this reason, IGF-1 is expected to reach a given site far from the site of injury more efficiently than des 1-3N IGF-1, which does not bind well to the protein. Indeed for this case, des 1-3N IGF-1 had no significant activity as a neuronal rescue factor at doses equivalent to those at which IGF-1 showed neuronal rescue. Therefore, it is suggested in the prior art that IGF-1 has neuron-rescuing activity on the IGF-1 receptor.

So far, no method has been given in the prior art to cleave the tripeptide GPE itself to prevent or treat CNS injury or disease resulting from CNS injury in vivo.

Surprisingly, we found that GPE itself underlies the phenomenon of neuronal rescue. (See Example 3). This suggests to us that treatment of nerve injured or diseased patients with IGF-1 is a less soundly fundamental topic, since a tripeptide is easily prepared and it is a more mobile and less immunologically aggressive compound , so it is expected to be more efficient.

Sara (patent EP 0366638 A2) suggests that GPE can be used as a neuromodulator to alter the activity of neuronal cells. Because it contains a glutamic acid and a glycine, she suggested that GPE appears to act as a partial agonist or antagonist at NMDA-like receptors. A common NMDA receptor antagonist is MK801. We therefore compared the effects of IGF-1 and MK801 following a given injury and also looked for any auxiliary effects.

Experiment 1 of this specification is a non-limiting example showing that the use of NMDA receptor antagonists does not mimic or increase the effects of IGF-1 in rat patients with hypoxic-ischemic injury. This study shows that IGF-1 does not function by regulating neural activity. In contrast to IGF-1, both GPE and MK801 have the same effect on the release of gonadotropins from hypothalamic tissue (Bourgignon et al Growth Regulation (in press)), implying that IGF-1 has a role as a GPE prohormone, with a role for GPE regulation NMDA-mediated neuroactivity, ie, hormone release, plays a role, so there is no reason in the prior art to suggest that GPE is a neuronal rescue factor. Thus there is no suggestion in the prior art that IGF-1 has the ability to act as a prohormone to form GPE which in turn prevents neuronal death. It is also suggested in the prior art that IGF-1 acts through the IGF-1 receptor.

A non-limiting example of Experiment 2 performed on fetal sheep, showing that IGF-1, which induces neuronal rescue in a fetal sheep ischemia model, fails to suppress cortical EEG activity while MK801 does (Tan et al Ann Neurol 32 : 677-682 (1992)).

Experiment 3 is a non-limiting example showing that GPE has potency as a neuronal rescue factor despite prior art implications that IGF-1 acts as a neuronal rescue factor via the IGF-1 receptor but fails to modulate neuronal activity Like IGF-1. GPE was administered shortly after hypoxic-ischemic damage but before the onset of DNA degradation to specific areas that had previously been shown to show neuronal death in control animals. The reduction in hippocampal neuronal damage and cortical infarcts reflected less neuronal and glial damage due to asphyxia. The mechanism of the protection against cell death caused by GPE is not known, but apparently does not act through modulation of neuronal activity.

Experiment 4 is a non-limiting example performed on 21 day old rats, showing a significant potent effect of GPE on neuronal output after intraperitoneal administration when administered 2 hours after an injury involving hypoxia.

Sara has demonstrated that GPE can modulate neuronal activity, and since factors such as NMDA have a role in the treatment of neuronal damage, suggests that no evidence for its use in the treatment of neurological diseases can be provided. But there is nothing in the prior art to our claim that GPE can be used to prevent neuronal diseases by preventing neuronal and glial cell death. The type of clinical application that our invention is aimed at is generally different from Sara's method.

Our recent studies tend to support the following findings: In the hippocampus itself; the CA1-2 region where the effects of GPE are most volatile. So our data on GPE etc. may firstly be most relevant for diseases primarily involving the hippocampus, and secondly involve other neuronal populations once the principles of operation are better understood.

Description of the preferred embodiment

The present invention relates to methods of manipulating neuronal injury. In one aspect, the invention relates to methods of treating CNS injury after the injury has occurred. For example, the patient has suffered from perinatal asphyxia or stroke-related asphyxia or cerebral ischemia or other non-limiting examples of CNS injury described earlier herein. For these conditions, reduction or removal of CNS damage is desirable.

CNS damage is measured clinically, for example, in terms of the degree to which neurocognitive deficits persist, and/or the degree to which they tend toward seizure disorders. (In our trials we have used histological techniques).

It has been suggested that the concentration of GPE and/or its analogues in the CNS and brain of patients should be increased especially for the treatment of CNS damage. Thus, GPE and/or analogs thereof can be administered directly to a patient. We speak specifically of the term "GPE" referring to gly-pro-glu or gly-pro or pro-glu. An analog of GPE refers to a compound having similar biological activity to GPE. These compounds can be produced by humans or other animals. GPE and analogs can be purified from natural sources or produced by synthetic techniques. Synthetic GPE is commercially available.

Alternatively, compounds may be used which, when administered to a patient, increase the active concentration of GPE and/or naturally occurring analogs of GPE in the patient's CNS. "Active concentration" refers to the biological concentration of GPE and/or analogs thereof in the CNS of a patient that is effective in treating CNS damage. For example, increasing the active concentration of IGF-1 can increase the formation of GPE.

GPE, its analogs, and compounds that increase the concentration of its activity can be administered centrally or systemically. Desirably, the composition is administered directly to the CNS of the patient. Thus, the compositions are administered directly to the brain or cerebrospinal fluid using techniques including lateral ventricle drilling, or bregma, lumbar or lymphatic space puncture, and the like.

The compounds may be administered in combination, if desired. Additionally they may be administered with other agents or growth factors such as transforming growth factor beta (TGF-beta).

Previous studies have shown that IGF-1 expression follows a specific time course and occurs in specific body regions following nerve injury. Therefore, the composition should be administered according to the degree of CNS injury and the time elapsed after the injury in order to produce the most satisfactory effect. The composition can be administered directly to the area of the body where CNS damage is most severe.

For example the composition may be administered about 0.5-100 hours after injury and only one treatment is necessary. Another option is to repeat the treatment to the patient.

A suitable dosage range when administered directly to the central system is, for example, about 0.1-1000 [mu]g of GPE (and/or analogues or compounds increasing its concentration) per 100 gm of body weight.

Said treatment is performed before (and after) the injury - eg, before selected surgery. Concerning selected surgical examples include neurological surgery, where retraction of the lobes of the brain leads to brain tumor glands, or cardiac surgery, such as valve replacement, where small emboli inevitably lead to detectable brain function in 75% of cases repair.

The invention also relates to medicaments for treating CNS injuries. The medicament may contain GPE or an analog thereof or a compound that increases the concentration of GPE in the CNS such as IGF-1. The compound may be provided with those pharmaceutically acceptable carriers or diluents well known in the art, as required. GPE or its analogs or compounds having increased concentrations thereof can be prepared using peptide synthesis techniques. Alternatively, the compound is isolated from a natural source.

Compounds with little or no immunological effect may be administered chronically provided other side effects prove insignificant. We suggest, for example, that patients with chronic CNS disorders such as Parkinson's disease, multiple necrosis, Alzheimer's disease, etc. can be chronically administered oral doses of pharmaceutical compounds (GPE itself) that can induce higher concentrations of GPE in the brain. In this case, GPE should be able to enter the circulation due to its properties as a lozenge placed under the tongue by direct absorption from the buccal mucosa. We have shown that GPE administered via the peritoneal route (young rats) is effective, so at least not limited to injection into the CSF. It is difficult to establish the therapeutic effect of GPE for this disease unless clinical trials are performed.

The following experimental data supports the invention. Found in the following research:

1) The use of NMDA receptor antagonists cannot mimic or increase the neuronal rescue effect of IGF-1.

2) Unlike NMDA receptor antagonists, neuron salvage therapy with IGF-1 does not suppress seizures. Thus, neuronal salvage therapy with IGF-1 is not primarily mediated through NMDA receptors.

3) Changes in the content of the N-terminal tripeptide of IGF-1 called GPE in the CNS can alter the CNS injury due to CNS injury.

The present invention is further described by the following examples. These examples are for illustration only and are not intended to limit the invention in any way. All patents and references described throughout this specification are incorporated by reference. The described research was sponsored by the Animal Ethical Committee of the University of Auckland.

Experiment 1

The purpose of this study was to compare the effects of administration of IGF-1 and the NMDA receptor antagonist MK801 following CNS injury in order to clarify the site of action of IGF-1. The assay involved treating rats with vehicle, IGF-1, MK801 or IGF-1 plus MK801 2 hours after CNS injury. These rats had a hypoxic-ischemic injury of one cerebral hemisphere induced in a standard manner. One carotid hemisphere was ligated and the animals were subjected to hypoxia for a predetermined period of 2 hours. The degree of hypoxia, length, room temperature and humidity were selected to normalize the degree of injury. The animals were sacrificed five days later for histological analysis using a dye specific for necrotic neurons (acid-fuchsin). Cell death in these experiments was usually limited to the side of the arterial ligation and mainly in the hippocampus, axial gyrus and outer cortex of the ligated hemisphere.

Adult Wistar rats (68 280-320 g) were prepared under 3% halothane/O ₂ anesthesia. The right carotid artery was ligated. A guide cannula was placed in the dura 8.2 mm anterior to bregma and 1.4 mm from the right midline. Rats were allowed to recover from anesthesia for 1 h, and then placed in an incubator with a humidity of 85 ± 5% and a temperature of 34 ± 0.5 °C 1 h before hypoxia. The oxygen concentration was reduced and maintained at 6±0.5% _O2 , and the rats were hypoxic for 10 minutes. Rats were kept in the incubator for 2 hours after the end of hypoxia and then treated with IGF-1 (n=17), MK801 (n=17), MK801+IGF-1 (n=17) or vehicle alone deal with. 50 mg of IGF-1 or vehicle alone (0.1% BSA in 0.15M PBS (pH 7.3)) was given by intracerebroventricular (IVC) infusion. Rats were simultaneously treated with 1 mg MK801/0.5 ml or saline alone subcutaneously. Intraventricular injections of 50 μg of IGF-1 or vehicle alone were injected into the right lateral ventricle at a rate of 1 μl/min in the presence of 1.5%-2% halothane anesthesia. Rats were infused simultaneously in each treatment group. Rats were allowed to eat freely during the experiment and were euthanized with an overdose of sodium pentobarbitol 120 hours after hypoxia. Briefly, brains were in situ perfused with FAM (formaldehyde, acetic acid, methanol 1:1:8) and then embedded in paraffin. Sections were stained with Thionin and acid fuchsin. The presence of cortical infarcts, defined as areas of tissue death or parenchymal total necrosis resulting from the death of glial cells and neurons, was determined by means of light microscopy by an assessor blinded to the experimental group.

The results are shown in Figure 1, showing the R (ligated carotid) and L sides of the brain, where column A is vehicle, column B is 50 μg IGF-1, column C is 1 mg MK801, and column D is 50 μg IGF -1+1 mg MK801. (p( ^* )=0.031)

Similar to our own previous studies, cortical infarcts were reduced (33%) after IGF-1 treatment compared with controls (65%) (Guan et al J Cereb BloodFlow metab 13:609-616 (1993)); The incidence was 50% after MK801 treatment. 41% after IGF-1 combined with MK801. Thus, administration of NMDA receptor antagonists does not mimic or augment the effects of IGF-1 in rats subjected to hypoxic-ischemic injury.

Experiment 2

The aim of this study was to compare the effects of IGF-1 treatment (Fig. 2) and administration of the NMDA receptor antagonist MK801 after cerebral ischemic injury in fetal sheep on postischemic epilepsy and The effects of neuronal damage were compared. (Tan et al Ann Neurol 32:677-682 (1992)).

The method was the same as the earlier study (Tan et al Ann Neurol 32:677-682 (1992)). Briefly, late-gestation fetal sheep were gradually recorded with instruments for EEG, scruff activity, and blood pressure before returning to the uterus. Cortical EEG activity, scruff activity and blood pressure were recorded throughout the experiment and fetal sheep were subjected to 30 min ischemia. Treatment was performed 2 hours later by infusion of 1 μg IGF-1 (n=6) or vehicle (artificial CSF) (n=6) into the lateral ventricle. Five days later the brains were fixed and evaluated for neuronal damage as previously described (Tan et al Ann Neurol 32:677-682 (1992)).

Figure 2 shows the grading of neuronal damage (indicated by abbreviations on the horizontal axis), expressed as a percentage of the untreated side, for a number of regions of the brain. Excipient in all cases is the left hand column and the effect of 1 μg IGF-1 is the right hand column.

The results showed that unlike NMDA antagonist-treated sheep, when charge activity was significantly inhibited (Tan et al Ann Neurol 32:677-682 (1992)), IGF-1 rescued neurons (Figure 2) but did not inhibit Post-ischemic epileptic activity in fetal sheep. The study also suggests that the neuroprotective effects of IGF-1 do not primarily arise through NMDA receptors or alter the electrical charge of the brain.

Experiment 3

The aim of this study was to compare the effect of GPE treatment with vehicle treatment 2 hours after ischemic-hypoxic brain injury.

The effect of the selected dose of 3 μg GPE was equivalent to that shown previously by 50 μg IGF-1 (Guan et al J Cereb Blood Flow metab 13:609-616 (1993)) which has been shown to be neuroprotective. Unilateral hypoxic-ischemic injury was induced in adult 300±10 g male Wistar rats. The rat carotid artery was ligated unilaterally under mild halothane anesthesia. After 1 hour of recovery they were placed in an incubator at 34°C, 85±5% humidity, 1 hour before injury. They were inhaled asphyxiated ( _FiO2 6.0%) for 10 minutes and kept in the incubator for 1 hour after asphyxiation. A single stereotaxically controlled lateral ventricle was injected 2 hours after termination of inhalation injury with 3 μg of GPE (n=15) or with phosphate buffered saline alone (n=15). Animals were then maintained for 120 hours, anesthetized and brains fixed in situ for histological evaluation.

Utilize thionin/acid fuchsin staining technique to distinguish living and dead neurons (C.Williams, A.Gunn, C.Mallard, P.Gluckman Ped Res, (1990).A.Brown, J.Brierley, J.Neurol Sci, 16:59-84 (1971)). The results using the scoring technique are shown in Figure 2. Having demonstrated neuronal damage on the non-ligated side, GPE treatment reduced the appearance of hippocampal damage in the ligated hippocampus compared to vehicle-treated controls (p<0.05, as determined by Fisher&apos;s exact test). Similar to our previous study with IGF-1, cortical Infarct reduction was 27%.

Figure 3 shows - Cortical infarction (Panels A and B) and hippocampal injury (Panels C and D) after treatment with vehicle (Panels A and C) or 3 μg GPE (Panels B and D) 2 hours after hypoxia. [Hippocampal damage decreased after treatment with 3 μg GPE. Asterisks indicate probability p<0.05. ]

Figure 4 shows a more critical evaluation of the later stages of the same experiment. For this figure, columns A and B show the ratio of area loss between the left and right sides of the cerebral cortex after treatment with control vehicle or 3 μg GPE (from which area can be inferred using known principles of stereology output volume). The volume can be measured using computer-aided image analysis techniques. Columns C and D relate to the hippocampus and represent the proportion of live neurons remaining after the experiment; compare right and left numbers again. Asterisks indicate a probability of 0.04. Neuronal numbers were counted with the aid of a microscope after staining. The number of damaged cells was significantly reduced after GPE administration. Thus, a single injection of GPE was associated with significantly improved output of histological evaluation following asphyxial injury in adult rats.

Quantitative receptor autoradiography was used to localize in coronal sections of the brain following the previous histological experiments of Dragunow et al. (1988, Brain Research 462, 252-257) for mapping GPE binding sites in rat brain [3H ]-GPE combination. Fresh frozen brain sections were cut on a cryostat and stored at -80°C until use. Sections were then thawed and preincubated with 50 mM Tris HCl (pH 7.4) for 10 minutes at room temperature (250 μl per section). Sections were then dried and 250 μl of [3H]-GPE supplemented in Tris HCl buffer (50 mM pH 7.4) was added to each section at room temperature at a rate of 5 x ¹⁰⁵ counts/min for 1 hour. Sections were then washed twice for 1 min each in chilled Tris HCl followed by 1 min in chilled distilled water. Sections were then dried overnight at 4°C and exposed to [3H]-sensitive film for 2 weeks, then developed to prepare autoradiograms.

The results are shown in Figure 6, which shows that the left hippocampus has bound to the radioactive material, while the corresponding side on the right has little response. Neurons were lost on this side due to pre-existing injury. The autoradiogram shows a specific binding site for GPE and tends to support our view that there is a specific role for GPE at this important core.

Experiment overview

A single dose of GPE (dissolved in 0.15 phosphate-buffered saline in these experiments) from the time of onset of CNS injury to about 8 hours after injury (including the 2-hour time point after nerve injury) has been shown to reduce or eliminate nerve injury There is a therapeutic effect in terms of the severity of the CNS injury suffered afterwards. GPE is particularly well suited to reduce neuronal loss, infarction, and loss of glia and other cells associated with CNS injury. It will thus be seen that at least in the preferred form of the invention there is provided a method and/or medicament for treating CNS damage which substantially prevents or treats CNS damage. CNS injury is associated with asphyxia, hypoxia, poisoning, infection, infarction, ischemia or trauma. It should be appreciated that the present invention is primarily applicable to humans. However, the use of the present invention is not limited thereto, and the treatment of other non-human animals, especially mammals, is also within the scope of the present invention.

The present invention thus recognizes the effect of administering a drug containing GPE and/or other compounds of similar action to a patient at or after a CNS injury, resulting in the destruction of the CNS by preventing the otherwise corollary, self-induced injury that follows the injury. Minimizing damage does not involve repairing damage that has already occurred but involves reducing the occurrence of such damage by treating it at or after the damage but before the eventual long-term damage occurs.

Example 1

Attenuation of brain damage in infants or newborn mammals born as a result of perinatal asphyxia

A suitable approach to attenuate brain damage was modeled with dose ratios established in our rat and fetal sheep models, with either GPE or An analogue thereof is injected intravenously into the infant's circulation at a preferred dosage rate ranging from 0.1 μg/kg to 10 mg/kg, and more preferably at a dosage of about 1 mg/kg. Higher loading doses can be used at the beginning of processing. Alternatively, GPE is initially administered at a higher iv dose rate of about 5 mg/kg via the maternal circulation while the placenta is extremely functional. Alternatively, 10 μg/kg in artificial CSF can be infused intravenously into the lateral ventricle as needed.

Example 2

Attenuation of human or mammalian brain damage caused by stroke

An appropriate method model to attenuate brain damage at the dose ratios we have established in rat and fetal sheep models is to use a solution in normal saline within 12 hours of the onset of neurological symptoms in the fetus until 120 hours later GPE, or an analogue thereof, is injected intravenously into the infant's circulation at a preferred dosage rate ranging from 0.1 μg/kg to 10 mg/kg and more preferably at a dosage of about 1 mg/kg. Higher loading doses can be used at the beginning of processing. Alternatively, the same dose is administered by juxtacarotid injection. Alternatively, 10 μg/kg in artificial CSF can be infused intravenously into the lateral ventricle as needed.

Example 3

Mitigation of human or mammalian brain damage caused by intracerebral hemorrhage

An appropriate approach to attenuate brain damage was modeled at the dose ratios we established in rat and fetal sheep models, using either GPE in normal saline or An analogue thereof is injected intravenously into the infant's circulation at a preferred dosage rate ranging from 0.1 μg/kg to 10 mg/kg, and more preferably at a dosage of about 1 mg/kg. Higher loading doses can be used at the beginning of processing. As an alternative, 10 μg/kg-infused into the lateral ventricle in artificial SCF is administered intravenously as needed.

Example 4

Attenuation of human or mammalian brain damage caused by head trauma

An appropriate method to attenuate brain damage was modeled with dose ratios established in our rat and fetal sheep models by administering GPE in normal saline or its equivalent within 12 hours of injury and up to 120 hours after injury. The drug is injected intravenously into the infant's circulation at a preferred dosage rate ranging from 0.1 μg/kg to 10 mg/kg and more preferably at a dosage of about 1 mg/kg. Higher loading doses can be used at the beginning of processing. Alternatively, 10 μg/kg in artificial CSF can be infused intravenously into the lateral ventricle as needed.

Example 5

Peripheral administration of GPE is effective

The aim of this study was to compare the effect of GPE treatment with vehicle treatment 2 hours after ischemic-hypoxic brain injury. The dose range of 2-200 μg was chosen to estimate a range of systemic doses greater than that required for concentration.

At 21 days of age, 45±5 g Wistar rats were induced with unilateral ischemic-hypoxic injury. The rat carotid artery was ligated unilaterally under mild halothane anesthesia. After 1 hour of recovery they were placed in an incubator at 34°C, 85±5% humidity, 1 hour before injury. They were inhaled asphyxiated ( _FiO2 8.0%) for 1 min, then returned to room temperature (22°C) and normoxic. Each rat was injected intraperitoneally with 0.25 ml of 2, 20 or 200 [mu]g GPE (n=15) 2 hours after the termination of injury. Animals were then maintained for 120 hours, anesthetized and brains fixed in situ for histological evaluation.

Live and dead neurons were distinguished using the thionin/acid fuchsin staining technique (Guan et al JCereb Blood Flow Metab 13:609-616 (1993)). The results are shown in Figure 5, where the ratio of the percentage of live neurons in the CA1-2 area on the right to the left is the dot height. Column A is excipient, column B is 2 μg GPE, column C is 20 μg GPE, and column D is 200 μg GPE. In this figure, the P value (0.031) was calculated using the method of one-way ANOVA comparing many groups after Arcsin conversion.

GPE treatment (20 μg) reduced neuronal damage in the CA1-2 region of the hippocampus (p<0.05). A single peripheral injection of GPE after asphyxial injury in rats was therefore associated with a significant increase in the output of histological evaluation.

Selection procedure: We chose the peritoneal injection route at least in part due to the difficulty of performing other routes in such small animals. While it is possible that the peritoneal route allows GPE to better enter the circulation and thus cross the brain-blood barrier, although other routes such as intravenous, intramuscular, or subcutaneous routes are also effective, effective doses may be greater in rats.

The above experiments show that the advantages of GPE over previously favored IGF-1 therapy include that, unlike IGF-1, it can cross the brain-blood barrier and can enter the CNS from peripheral sites.

Pharmacology

Apart from the dose-response experiments on which Figure 5 is based, we have not investigated the pharmacological properties of GPE. We expect it to have a half-life in the blood similar to other peptides; we expect the liver and kidneys to absorb circulating GPE very rapidly, and we expect it to have a substantial therapeutic rate. Intravenous administration is preferred as a steady infusion due to the expected rapid absorption.

advantage

Advantages provided by the present invention, especially compared with IGF-1 etc., include:

(1) The active ingredient is easy to synthesize in vitro or by other means such as recombinant technology.

(2) The small molecule readily diffuses in vivo and between compartments (eg, blood-brain barrier, and mucosa), aiding in the choice of administration method and in reaching the site of injury.

We have shown that peritoneal administration is effective for non-CSF cases.

(3) The small molecule cannot attack the immune system. So it can be administered over a longer period of time and it can be administered prophylactically.

(4) Differences between species may be important.

While the invention has been broadly defined above, those skilled in the art will recognize that the invention is not limited thereto and includes the embodiments provided in the specification. Finally, it will be recognized that various changes and modifications are made within the scope of the claimed invention.

Claims (7)

1. A pharmaceutical composition for treating trauma-induced nerve damage, comprising an effective amount of a peptide selected from gly-pro-glu, gly-pro and pro-glu, and an effective amount of insulin-like growth factor-1 , Insulin-like growth factor-1 increases the concentration of gly-pro-glu, gly-pro or pro-glu in the nervous system of a recipient mammal. 2. Use of gly-pro-glu, gly-pro or pro-glu in the production of a pharmaceutical composition for the treatment of glial cell trauma-induced nerve damage or the treatment of trauma-induced neuronal damage, the drug combination substances suitable for administration to the nervous system of mammals. 3. Use according to claim 2, wherein said treatment is administration of the composition during the period from 12 hours before to 100 hours after the acute injury. 4. Use according to claim 3, wherein said treatment is administration of the composition 0.5-8 hours after acute injury, so that there is an increased level of cytoprotection in the nervous system, at least partly under conditions unfavorable for the survival of nerve cells the GPE. 5. Use according to claim 3, wherein said treatment is used together with a selected step that is considered to cause CNS damage, wherein an effective amount of the composition is administered prophylactically prior to the selected step, so that nerve Increased levels of GPE within the system. 6. The use according to claim 2, wherein the composition is administered in a dosage range of about 1 [mu]g to about 100 mg of the peptide per kilogram body weight of the recipient mammal. 7. A pharmaceutical composition for the treatment of trauma-induced nerve damage, said composition comprising insulin-like growth factor-1, which causes the body of a mammal to synthesize and release high levels of gly-pro-glu, gly-pro or pro-glu, said composition is suitable for administration to the nervous system of mammals.

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