WO2007023479A2

WO2007023479A2 - Treatment of central nervous system injury

Info

Publication number: WO2007023479A2
Application number: PCT/IE2006/000091
Authority: WO
Inventors: Jochen Prehn; Dairin Kieran; Matt Greenway; Orla Hardiman
Original assignee: Royal College Of Surgeons In Ireland
Priority date: 2005-08-23
Filing date: 2006-08-23
Publication date: 2007-03-01
Also published as: WO2007023479A3

Abstract

A method of enhancing the recovery of central nervous system function in an individual afflicted with central nervous system (CNS) injury such as that caused by ischemia or trauma is provided. The method comprises treating the individual with angiogenin, or a neuroprotective fragment or variant thereof. The CNS injury may be caused by stroke. Also described is the use of angiogenin, or a neuroprotective fragment or variant thereof, in the manufacture of a medicament for enhancing the recovery of central nervous system (CNS) function in an individual afflicted with CNS injury.

Description

TREATMENT OF CENTRAL NERVOUS SYSTEM INJURY

Technical Field

The invention relates to a method of enhancing the recovery of central nervous system function in an individual afflicted with central nervous system injury, such as that mediated by ischemia or trauma.

Background of the Invention

Stroke is a common cerebrovascular event that is responsible for over about half of all neurological hospital admissions to adult wards. It occurs when a blood vessel supplying a part of the central nervous system is obstructed by, for example, a thrombus or an embolus resulting in the brain tissue being deprived of blood and oxygen. If the obstruction is prolonged, severe, or both, the neurons in the affected area will die leading to necrosis at the affected locus. Patients who suffer a stroke, or any other form of neurovascular event or cerebral trauma, usually recover partially but in most cases will remain either severely or partially debilitated. The degree of debilitation depends on a number of factors including the severity of the stroke, and the age of the individual when the stroke occurs. Common symptoms of stroke and cerebral trauma are loss of motor coordination function, sensory perception, and speech. Currently, the only reliable treatment for stroke or similar injuries to the nervous system is physical therapy. It is an object of the invention to overcome at least some of the above problems .

Brief Description of the Invention

Accordingly, the invention relates to a method of enhancing the recovery of central nervous system function in an individual afflicted with central nervous system injury such as that caused by ischemia or trauma, which method comprises treating the individual with angiogenin, or a neuroprotective fragment or variant thereof.

In a further aspect, the invention relates to a method of treating neurological complications associated with diabetes in a diabetic individual, which method comprises the step of treating the individual with angiogenin, or a neuroprotective fragment or variant thereof. Examples of such complications would be well known to a clinician skilled in the field of diabetes, and would include, for example, diabetic neuropathy and diabetic retinopathy. Typically, the treatment of such complication would involve local administration at the site of the neuropathy.

In a further aspect, the invention relates to the use of angiogenin, or a neuroprotective fragment or variant thereof, as a neuroprotective agent.

In a further aspect, the invention relates to the use of angiogenin, or a neurotrophic fragment or variant thereof, as a neurotrophic agent. DEFINITIONS

In this specification, the term "central nervous system injury" refers to injury or damage caused by neurovascular events (especially cerebrovascular events) such as stroke, injury to the CNS of a traumatic origin such as that mediated by a hemorrhagic infarction, and injury to CNS tissue caused by disease such as neurodegenerative disease. Specific examples of such CNS injuries, and their causes, will be well known to clinicians operating in this field. The results of these injuries vary depending on the size and locus of the infarct or haemorrhage; strokes involving the cerebral hemisphere or brain stem lead to hemiplegia.

In this specification, the phrase "enhancing the recovery of central nervous system function" should be taken to mean that the treatment has a protective effect on neurons that, for example, retards or prevents the degeneration of the neurons compared to untreated neurons. Typically, this would result in a clinically relevant improvement in at least one of speech, motor coordination function (i.e. posture, balance, grasp or gait) , or sensory perception (i.e vision, touch, taste, olfaction or proprioception).

In this specification, the term "neuroprotective" when applied to angiogenin, or a fragment or variant of the protein, is intended to mean that the protein, or fragment or variant thereof, has a protective effect on neurons that, for example, retards or prevents the degeneration of the neurons compared to untreated neurons. A molecule, such as a fragment or variant of angiogenin, will be considered to be "neuroprotective" when use of the molecule in the in- vitro model of neurodegeneration described herein increases cell viability compared with a control.

The nucleic acid sequence of human angiogenin protein is provided in the NCBI database under Accession Number M11567. The sequence coding for the mature protein extends from nucleotide 1881 to 2249. The sequence of the mature protein, without the signal peptide, is provided below:

QDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICEN KNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQS IFRRP (SEQ ID NO 1)

The sequence of the full transcript, including the signal peptide, is provided in SEQ ID NO 2, and the cDNA sequence of the human gene coding for the full transcript (mature protein plus signal peptide) is provided in SEQ ID NO. 3.

Generally, the angiogenin used in the methods and products of the invention will be human angiogenin in any of its documented isoforms. However, angiogenin obtained from, or based on, other mammalian angiogenin genes is included within the scope of the invention. Preferably, the angiogenin is recombinant angiogenin, most preferably recombinant human angiogenin. Recombinant human angiogenin is commercially available from R&D Systems Inc. (Minneapolis, USA) under Catalog Number 265-AN-250.

A "fragment" of 'the angiogenin protein means a contiguous stretch of amino acid residues of at least 5 amino acids, preferably at least 6 amino acids. Typically, the "fragment" will comprise at least 10, preferably at least 20, more preferably at least 30, and ideally at least 40 contiguous amino acids. In this regard, it would be a relatively straightforward task to make fragments of the protein and assess the neuroprotective activity of such fragments using the experimental techniques described herein, such as, for example, the in-vitro model of motoneuron degeneration described below.

A "variant" of the angiogenin protein shall be taken to mean proteins having amino acid sequences which are substantially identical to wild-type angiogenin protein, typically human wild-type angiogenin. Thus, for example, the term should be taken to include proteins or polypeptides that are altered in respect of one or more amino acid residues. Preferably such alterations involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged. The variant may have conservative amino acid changes, wherein the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted. Typically, angiogenin proteins which have been altered by substitution or deletion of catalytically-important residues will be excluded from the term "variant". Details of such catalytically-important residues will be well known to those skilled in the field of angiogenin protein modeling. Generally, the variant will have at least 70% amino acid sequence homology, preferably at least 80% sequence homology, more preferably at least 90% sequence homology, and ideally at least 95%, 96%, 97%, 98% or 99% sequence homology with wild-type angiogenin, typically mature wild- type human angiogenin (excluding the signal peptide as recited above) . In this context, sequence homology comprises both sequence identity and similarity, i.e. a polypeptide sequence that shares 70% amino acid homology with wild-type human angiogenin is one in which any 70% of aligned residues are either identical to, or conservative substitutions of, the corresponding residues in wild-type human angiogenin. Specific variants included within the scope of the invention are the mutant angiogenin proteins identified in European Patent Publication Number 0 335 243, and ideally the mutant angiogenin proteins disclosed in US Patent Serial Number 4,966,849. The contents of both of these documents, and the angiogenin mutants and variants disclosed therein, are incorporated herein by reference.

The term "variant" is also intended to include chemical derivatives of angtiogenin, i.e. where one or more residues of angiogenin is chemically derivatized by reaction of a functional side group. Also included within the term variant are angiogenin molecules in which naturally occurring amino acid residues are replaced with amino acid analogues .

Proteins and polypeptides (including variants and fragments thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. The proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984) .

The therapeutic method, and therapeutic products, of the ^■invention are directed to the treatment of central nervous system function in an individual afflicted with central nervous system injury such as that caused by ischemia or trauma. In one embodiment of the invention, the therapy is directed to individuals who have damaged central nervous system tissue as a result of a stroke.

Typically, the individual is treated with an effective amount of angiogenin, or a fragment or variant thereof. In this specification the term "amount effective" of angiogenin (or an angiogenin fragment or variant) should be taken to mean an amount which results in a clinically significant enhancement in the recovery, or retardation in the degeneration, of central nervous system function in the individual as a result of a suitable period of treatment. Generally, this would be determined by a neurologist. Suitably, the angiogenin or variant or fragment thereof, is administered at a dose of between 1 microgram and 10 miligrams per ml, preferably between 10 micrograms and 5 miligrams per ml, more preferably between 100 micrograms and 2 miligrams ^' per ml. Typically, it is given as a bolus dose. However, when continuous infusion is used, such as by intrathecal pump, the protein, or fragment or variant thereof, may be administered at a dosage rate of between 5 and 20 μg/kg/minute, preferably between 7 and 15 μg/kg/minute. In the context of the therapeutic aspects of the present invention, the term "individual in need thereof" shall be taken to mean an individual who- has damaged central nervous system tissue as a result of suffering CNS damage, such CNS damage being caused by ischemia, stroke, or any other cause of CNS damage (i.e. damage to CNS tissue caused by chemical or physical means, or by irradiation) .

In one embodiment of the invention, an individual in treated with angiogenin by direct delivery of the protein by a means selected from the group: intravenous delivery; oral delivery; intramuscular delivery; intrathecal delivery; and inhaled delivery. Methods for achieving these means of delivery will be well known to those skilled in the art of drug delivery. Specific examples are provided below:

• Delivered intrathecially by mini-osmotoc pump. (ref: Ignacio et al . , Ann. N. Y. Acad. Sci. 2005, 1053: 121- 136) .

• Intramuscular- Ang delivery directly into muscle (s) by syringe or mini osmotic pump (Azzouz et al . , Nat Med. 2005;ll (4) :429-33) .

• Intraperitoneal- for systemic administration of Ang. Directly administered to peritoneum by syringe or mini osmotic pump (Kieran et al . , Nat Med 2004; 10(4) :402).

• Subcutaneous- for systemic administration of Ang. Directly administered below the skin by syringe (Reinholz et al . , Exp Neurol. 1999; 159 (1) : 204-16) . • Intraventricular- direct administration to the ventricles in the brain, by injection or using small catheter attached to an osmotic pump. (Sathasivam et al., 2005 Neuropath App Neurobiol; 31(5): 467)

• Implant- ang can be prepared in an implant (eg small silicon implant) that will release ang. Implant can be placed at muscles or directly onto the spinal cord

(Kieran and Greensmith, 2004 Neurosci 125 (2) : 427-39) .

In an alternative embodiment, the individual may be treated with angiogenin by transfecting the individual with an angiogenin expression vector, such as, for example, a viral vector. A suitable expression vector is a lentiviral vector. Typically, the angiogenin used in the methods of the invention is recombinant angiogenin. Methods for producing recombinant angiogenin, and expression vectors which express recombinant forms of the protein, will be well known to those skilled in the art (see for example Nature, VoI 429 (2004), P413-417 and Nature Medicine VoI 6 (2000) No 4 P405-413 and Azzouz et al., Nat Med. 2005; 11 (4) : 429-33) which describe methods of delivering .recombinant VEGF to an animal model using a lentiviral expression vector. The gene therapy approach embodiment involves encoding ang and an attached marker protein (eg a fluorescent protein) in a virus, which will be administered to animals and taken up by cells whereby its incorporated into their genome and ang is expressed. Ideally, the administration involves over- expression of ang, which involves repeating ang sequence a number of times in the virus to produce a high copy number.

A further method of administering angiogenin is described in Russian Patent Application RU 02209245 C2 (published 27 July 2003) , the Examples of which describe the use of a viral delivery system for delivering plasmids encoding angiogenin into subjects in order to promote an increase in the density of vascular (capillary) networks in ischemic limbs. In particular, Example 4 describes the intramuscular delivery of plasmids encoding angiogenin to rats' rear limbs .

In one embodiment of the invention, the method includes a further step of treating the individual with VEGF, especially recombinant VEGF, especially isoform VEGFiβs, protein. Thus, the invention also provides a medicament comprising angiogenin, or a neuroprotective fragment or variant thereof, and VEGF protein, or isoforms thereof.

In one embodiment of the therapy of the invention, the angiogenin protein (or fragment or variant thereof) is linked to a coupling partner, e.g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule. Techniques for coupling the peptides of the invention to both peptidyl and non-peptidyl coupling partners are well known in the art.

Proteins and polypeptides (including reverse peptides, analogues and fragments thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. The proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), and M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984)).

In one embodiment of the invention, an individual is treated with angiogenin by any means known in the art, such as intravenously, orally, intramuscularly, intravascularly, intraperinoneally, subcutaneously, or transdermally. Methods for achieving these means of delivery will be well known to those skilled in the art of drug delivery. Direct administration into the CNS is also possible by means of delivery into a ventricle, for example .

In an alternative embodiment, the individual may be treated with angiogenin by transfecting the individual with an angiogenin expression vector, such as, for example, a viral vector. A suitable expression vector is a lentiviral vector. Typically, the angiogenin used in the methods of the invention is recombinant angiogenin, especially recombinant human angiogenin. Recombinant human angiogenin is commercially available from R&D Systems Inc under catalogue number 265-AN-250 (see link http : //www. rndsystems . com/product results . aspx?c=78 ) Methods for producing recombinant angiogenin, and expression vectors which express recombinant forms of the protein, will be well known to those skilled in the art (see for example Nature, VoI 429 (2004), P413-417 and Nature Medicine VoI 6 (2000) No 4 P405-413 which describe methods of delivering recombinant VEGF to an animal model using a lentiviral expression vector) . In one embodiment of the invention, the method includes a further step of treating the individual with VEGF, especially isoform VEGFi_6S protein.

The invention also relates to the use of angiogenin, or a neuroprotective fragment or variant thereof, in the manufacture of a medicament for the treatment or prevention of CNS injury, typically that caused by ischemia or trauma. Typically, the CNS injury is that caused by stroke.

Brief Description of the Figures

The invention will be more clearly understood from the following experiments that demonstrate the neuroprotective effect of angiogenin on an in-vitro model of motor neuron disease, and stroke, and an in-vivo model of nerve injury, in which:

Figure 1. MTT assay. An MTT assay was used to determine the neuroprotective effect of angiogenin treatment in an in- vitro model of motoneuron degeneration. Cell viability in treated cultures was expressed as a percentage of cell viability in untreated sister cultures (controls). As can be seen exposure to AMPA results in a significant decrease in cell viability, however co-treatment or pre-treatment with angiogenin (100ng/ml) significantly increases cell viability. Exposure to angiogenin or BSA alone has no significant effect on cell viability, (values = mean, error bars = S. E.M., N=24) . Figure 2. LDH release and PI uptake in CGN' s after 24h following a 10 min exposure to glutamate.

Figure 3. PI staining in CGN' s 24h following a 10 min exposure to glutamate.

Figure 4. Western blot for phosphorylated Akt . The activation of the PI3K/Akt pathway was examined by western blot for phosphorylated Akt. As can be seen there is a strong increase in phospho-Akt expression in AMPA exposed cultures co-treated or pre-treated with angiogenin. This suggests that the neuroprotective effect of angiogenin involves signalling through the PI3K/Akt signalling pathway.

Figure 5. The effect of sciatic nerve axotomy on motoneuron survival in the sciatic motor pool was assessed 2 days after injury, when the number of NeuN-stained motoneurons in the ventral horn of the operated and control sides of each spinal cord were counted. Motoneuron survival in the operated sciatic motor pool was expressed as a percentage of the number of motoneurons in the contralateral sciatic motor pool

Detailed Description of the Invention

EXPERIMENTAL

Methods

1. a) In-vitro model of motoneuron degeneration Primary motoneuron cultures at 7 days in-vitro were exposed to 5OuM AMPA (Tocris Cookson) in the culture medium for 24hrs, as an in-vitro model of motoneuron degeneration (as previously described by Kieran and Greensmith, Neuroscience 2004, Vol.125: 427-439) .To examine the possible neuroprotective effect of angiogenin we examined the effect of i) pre-treatment and ii) co-treatment with angiogenin (R&D systems) in the culture medium at concentrations of 25ng/ml, 50ng/ml, lOOng/ml, 200ng/ml and 500ng/ml. As controls, sister cultures were used that were either unexposed, or exposed to angiogenin (at same concentrations as detailed above) , or exposed to BSA (bovine serum albumin, Sigma) .

1. b) In-vitro model of stroke

Cerebellar granular neurons were treated with glutamate and glycine for 10 mins and the neurons were allowed to recover for 24 hours. The Angiogenin treated neurons were pre- incubated with 100ng/ml of the compound for lhr prior to treatment and the incubated with 100ng/ml of the compound for the 24 h after the initial treatment.

2. Cell viability assays

In this study to examine the neuroprotective effect of angiogenin in a) an in-vitro. model of motoneuron degeneration and b) an in-vitro model of stroke an MTT cell viability assay and LDH release assays, respectively, were performed.

MTT assay: MTT (Sigma) dissolved in PBS (5mg/ml) and diluted 1:10 in culture medium was added to cultures and incubated for 4 hours at 37C. After incubation, the media was replaced with isopropanol containing 0.04M HCl. Emission values were then read on a micro-ELISA plate reader at 570nm._. Cell viability in treated cultures was expressed as a percentage of cell viability in untreated sister cultures (100%) and results were compared for significance using a Mann Whitney U-test.

LDH assay: The LDH assay was performed according to manufacturers instructions (Roche, catalog no. 1644793).

3. Western blotting

To examine the signalling pathway involved in mediating the neuroprotective effect, of angiogenin, the activation of the PI3K/Akt pathway was examined using western blotting. In particular, we examined the activation of Akt, as demonstrated by its phosphorylation. Primary motoneuron cultures exposed to AMPA, AMPA and angiogenin, or angiogenin alone (as described above) were lysed and protein extracted. Protein ^• concentration in each experimental condition was determined using a Micro BCA Assay (Pierce) . Samples were run on a 10% SDS gel and transferred onto nitrocellulaose membranes, and were probed with antibodies to Akt and phospho-Akt (both Cell Signalling) . Blots were visualised using the ECL system. Equal protein loading was confirmed by re-probing blots with antibodies to alpha-tubulin (Sigma) .

4. Nerve injury using sterile surgical procedure, the sciatic nerve in the left hindlimb of 3-day old wild-type C57 mouse pups was axotomised as previously described (Kieran and Greensmith, 2004) . Briefly, animals were anaesthetized and using sterile surgical procedure the sciatic nerve was exposed in one hindlimb. The sciatic nerve was then cut and the incision scutured. Animals were allowed to recover for 2days after injury before being sacrificed.

Angiogenin treatment

We administered 2 ^g of Angiogenin (R&D Systems) intraperitoneally 1 day before the surgery and then daily until 2 days after surgery.

Motoneuron survival

Animals were terminally anaesthetized and perfused transcardially with cold saline followed by 4% paraformaldehyde. The lumbar region of the spinal cord was removed and 20-^m transverse sections were cut and immunostained with a neuron specific marker (NeuN) . NeuN- stained motoneurons located within the sciatic motor pool in the ventral horn on the operated side of the spinal cord as well as the contralateral control side were counted, as previously described (Kieran et al, 2004).

Results

1. The neuroprotective effect of angiogenin in an in-vitro model of motoneuron degeneration and stroke. a) Using an MTT cell viability assay the effect of increasing concentrations of angiogenin was examined in an in-vitro model of motoneuron degeneration where primary motoneuron cultures were exposed to 5OuM AMPA for 24hrs. It was found that the optimum concentration of angiogenin to demonstrate a neuroprotective effect was lOOng/ml. Exposure of primary motoneuron cultures to 5OuM AMPA for 24hrs is a well described in-vitro model of motoneuron degeneration (Kieran and Greensmith, 2004) . As can be seen in Figure 1, exposure to AMPA results in a significant decrease in cell viability to 58.5% (+/-3.3 S. E. M., n=24, p=<0.05). However, co-treatment .or pre-treatment with lOOng/ml angiogenin significantly increased cell viability to 82.5 (+/- 3.9 S. E.M., n=24) and 92.3% (+/- 4.8 S. E. M., n=24), respectively (p=<0.05). Treatment of primary motoneuron cultures with either angiogenin or BSA had no significant effect on cell viability.

b) Using an LDH assay to measure the release of LDH activity from the cytosol of damaged cells in the supernatant, the neuroprotective effect of angiogenin in an in-vitro model of stroke was determined. As can be seen from Figures 2 and 3, angiogenin was found to have a neuroprotective effect.

2. The role of Akt in angiogenin signalling

Using western blotting we examined the cell survival pathways involved in mediating the neuroprotective effect of angiogenin. In particular the activation of the PI3K/Akt cell survival pathway was examined by examining the expression of the active form of Akt (phosphorylted Akt) . As can be seen in Figure 4, the level of activated Akt (phosphorylated Akt) is increased in AMPA exposed cultures co-treated with angiogenin, compared to AMPA-only or angiogenin-only treated cultures. This increase in the activated form of Akt demonstrates that the neuroprotective effect of angiogenin in this in-vitro model of motoneuron degeneration involves the_< activation of the PI3K/Akt pathway.

3. To examine the possible neuroprotective effect of angiogenin treatment in-vivo, we used an injury-induced model motoneuron degeneration. Injury to a peripheral nerve in rodents during the first 5 postnatal days is known to result in significant motoneuron loss (Kieran and Greensmith, 2004), such as occurs in ALS. In this study the sciatic nerve was axotomised (cut) in one hindlimb of 3-day old mouse pups (Ή=8) . The effect of sciatic nerve axotomy on motoneuron survival in the sciatic motor pool was assessed 2 days after injury, when the number of NeuN- stained motoneurons in the ventral horn of the operated and control sides of each spinal cord were counted. Motoneuron survival in the operated sciatic motor pool was expressed as a percentage of the number of motoneurons in the contralateral sciatic motor pool. In untreated (control) mice sciatic nerve axotomy resulted in a significant loss of sciatic motoneurons, such that 2 days after injury only 80% (±3.1 S. E.M., n=4) motoneurons survive. In mouse pups treated with angiogenin there was no significant increase in motoneuron survival and 86% (±2.9 S. E.M., n=4) motoneurons survived (P>0.05).

The invention is not limited to the embodiment hereinbefore described which may be varied in both construction and detail without departing from the spirit of the invention.

Claims

1. A method of enhancing the recovery of central nervous system function in an individual afflicted with central nervous system (CNS) injury such as that caused by ischemia or trauma, which method comprises treating the individual with angiogenin, or a neuroprotective fragment or variant thereof.

2. A method as claimed in Claim 1 in which the individual is afflicted with CNS injury caused by ischemia or trauma.

3. A method as claimed in Claim 1 or 2 in which the CNS injury is caused by stroke.

4. A method as claimed in any preceding Claim, in which the angiogenin, or fragment or variant thereof, is delivered directly into the neuraxis by means of injection or gene therapy.

5. A method as claimed in Claim 4 in which delivery is effected by direct intrathecal injection.

6. A method as claimed in any preceding Claim, the method including a further step of treating the individual with VEGF, especially isoform VEGFies-

7. A use of angiogenin, or a neuroprotective fragment or variant thereof, in the manufacture of a medicament for enhancing the recovery of central nervous system (CNS) function in an individual afflicted with CNS injury.

8. A use as claimed in Claim 7, in which the CNS injury is that caused by ischemia or trauma.

9. A use as claimed in Claim 8 in which the CNS injury is caused by stroke.

10. A use as claimed in any of Claims 7 to 9, which employs a combination of angiogenin, or fragment or variant thereof, and isoform VEGFiβs, in the manufacture of the medicament.

11. Use of angiogenin, or a neuroprotective fragment or variant thereof, in the treatment of stroke or cerebral trauma .