Neuroprotective agents

Field of the Invention

The present invention relates to polypeptides, in particular polypeptides isolated from scorpion venom, and their use as neuroprotective agents for treatment of stroke, cerebral ischemia, and related conditions.

Background to the Invention Stroke has a devastating impact on public health and remains the third leading cause of death. It is also the first leading cause of long-term disability in industrialized countries (Broderick 2004) and the leading medical cause of acquired adult disability. Stroke is defined by the World Health Organization, Geneva, as the clinical syndrome of rapid onset of focal (or global) cerebral deficit, lasting more than 24h or leading to death, and with no apparent cause other than being vascular.

There are two pathological types of stroke: ischemic and haemorrhage stroke. In haemorrhagic stroke, blood bursts through the walls of an artery and leaks into the brain (intracerebral haemorrhage) or onto the surface of the brain (subarachnoid haemorrhage; Perry et al 2000) . Ischemic stroke results from a transient or permanent reduction in cerebral blood flow that is restricted to the territory of a major brain artery. The reduction in flow is, in majority of the cases, due to an occlusion of a cerebral artery either by an embolus or by local thrombosis. Different brain regions exhibit variable thresholds for ischemia, with white matter being more resilient than grey matter. Additionally, certain populations of cerebral neurons are selectively vulnerable to ischemia, such as hippocampal CAl cells and cerebral neurons, as compared to dentate granule cells and brain stem neurons, respectively (Smith, 2004) . Ischemia of cerebral tissue and the ensuing cell death underlie all forms of stroke, including

focal ischemia (reduction of nervous system blood supply to focal regions of the brain e.g. middle cerebral artery), global ischemia (declining blood flow to entire cerebral hemisphere/s) and possibly, intraparenchymal hemorrhage (Karpiak et al, 1989; Smith, 2004) .

Rapid intervention after the onset of a stroke can limit neurologic damage and improve patients' recovery. Recombinant tissue plasminogen activator (rt-PA) is the only treatment currently used to treat selected patients with acute ischaemic stroke. tPA results in revascularization of the ischemic tissue thus is regarded as 'vascular' therapy (Lo 2004) . However, only about 1 - 8.5% of hospitalised patients receive treatment with rt-PA due to the short time window for administration (within 3 h of symptom onset) , risk of intracerebral haemorrhage, edema and neurotoxicity (Kaur et al 2004; Derex et al 2000) .

Hypoxia and ischemia (hypoxia plus hypoglycemia) , are significant contributors to cell death and dysfunction and are similar to neurodegenerative diseases. In ischemic stroke, the transport of oxygen and glucose is impaired by blockage of a cerebral artery which results in neurological deficits (Sudlow and Warlow 1997) . Pathologically, a stroke results in rapid cellular necrosis in the central core of the infarcted region and delayed neuronal cell death in the surrounding area (penumbra) . Neurodegeneration may continue for several days despite tissue reperfusion. Severe ischemia and mild temporary ischemia (or hypoxia only) appears to be responsible for the neuronal cell death in the central core and penumbra, respectively.

The effects of ischemia and hypoxia are primarily due to the initiation of a neurodegenerative signaling cascade involving release of glutamate and glutamate receptor activation, accumulation of free calcium ( [Ca ²⁺ ] j.) intracellularly, free

radical formation, and subsequent necrosis and apoptosis (Davalos et al 1999) . The increase of [Ca ²⁺ Ii plays a key role in cellular necrosis, which is reflected by a rapid and long- term decrease of extracellular Ca ²⁺ ( [Ca ²⁺ ] _e ) in the central core. In contrast, the [Ca ²⁺ ] _e in the penumbra decreases only transiently (Dirnagl et al 1999) . The quantity of free radicals increases significantly in the central core during both ischemia and reperfusion, whereas they increase in the penumbra only during reperfusion (Mehta et al 2002) . Thus, it is clear that the intracellular mediators of the neurodegenerative cascade for ischemia-induced necrosis and hypoxia-induced apoptosis are, at least partially, different.

During a 2 hour middle cerebral artery (MCA) occlusion, the concentration of glucose is greatly reduced in the central core of the infarct, but only mildly in the penumbra. Restoration of glucose to a normal level occurs in both areas about Ih after reperfusion (Choi 2000) . Reperfusion also restores interstitial oxygen tension (pθ ₂ ) in the central core to its preischemic value, but penumbral pθ ₂ is recovered only partially (Hankey 2003) .

Neuroprotective agents potentially offer benefit to stroke patients without the associated haemorrhagic risk of thrombolytic therapy. Such agents target the ischaemic penumbra and aim to reduce the risk of brain injury and long- term disability by disrupting the cellular, biochemical, and metabolic consequences of infarction following acute ischaemia.

To date, drug development has generally focused on agents such as antagonists of glutamate, Ca ²⁺ channel blockers, scavengers of free radicals, and/or inhibitors of apoptosis. Unfortunately, many of these compounds either lack clinical efficacy or are hampered by dose-limiting adverse side effects (Epstein et al 1987, Beard et al 1993) . Although much

progress has been made in dissecting the intricate molecular mechanisms of how neurons die after ^" cerebral ischemia, a clinically effective neuroprotective therapy for stroke remains elusive.

There is a need, therefore, to identify new compounds that inhibit the ischemic neuronal injury cascade, thereby providing an effective treatment for stroke.

According to Xeng et al 2006, scorpion venom is composed of a large repertoire of biologically active polypeptides and has been used as a Chinese traditional medicine to relieve pain and treat neurological diseases. Nucleic acids encoding scorpion toxins are disclosed in US6768002.

Summary of the Invention

In our laboratory we have identified a protein component from the venom of the scorpion Mesobuthus martensii Karsch (BmK) , which we have called Mesopen4.6 or BπιKRP4.6. We have surprisingly found that this protein is very effective in treating animal models that represent cerebral ischemia. In particular, we have found that Mesopen4.6 reduces the focal ischemia, i.e. infarct size, caused by occlusion of the left middle cerebral artery by more than 80%. This is a significant reduction in infarct size and it was unforeseen that the venom of Mesobuthus martensii Karsch would contain a protein with such a high efficacy. This level of protection is comparable to the protection afforded by MK801, which is a known N- methyl-D-aspartate (NMDA) antagonist.

In addition to the above, we were also surprised to observe that the genes that are associated with ischemic damage are down regulated when treated with Mesopen4.6. Furthermore, our in vitro studies using organotypic hippocampal cultures have shown that Mesopen4.6 provides neuroprotection under oxygen and glucose deprived (OGD) conditions.

These observations indicate that the natural venom compound, Mesopen4.6, is a valuable new therapeutic agent for the effective treatment of stroke, cerebral ischemia, and related conditions.

In various aspects the invention relates to Mesopen4.6, variants thereof, and their use in the treatment of stroke and cerebral ischemia. These and other aspects are discussed below.

In a first aspect of the invention, there is provided a polypeptide comprising:

(i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3,

(ii) an amino acid sequence sharing at least 70 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3,

(iii) a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or

(iv) an amino acid sequence sharing at least 70 percent sequence identity with the fragment defined in (iii) , wherein the amino acid sequence has at least 6 amino acids, and wherein the amino acid sequence has less than 66 amino acids.

Alternatively, the polypeptide may consist of an amino acid sequence defined in (i) , (ii) , (iii), (iv) , or it may consist essentially of an amino acid sequence defined in (i) , (ii) , (iii) , or (iv) .

An amino sequence that shares a particular sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 may share at least 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99, or 100 percent sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. The sequence identity may be shared over at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66 contiguous amino acids of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Preferably, the sequence identity is shared over the full- length sequence of SEQ ID NO:- 1 or SEQ ID NO: 3. Preferably, the polypeptide is the polypeptide of Mesopen4.6.

A fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 may have at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. The fragment may have less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57., 58, 59, 60, 61, 62, 63, 64, 65 or 66 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

An amino acid sequence sharing at least a particular sequence identity with a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 may share at least 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,

88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 percent sequence identity with the fragment. The sequence identity is shared over the full-length sequence of the fragment.

An amino acid sequence sharing at least a particular sequence identity with a fragment of the amino acid sequence of SEQ ID

NO: 1 or SEQ ID NO: 3. may have at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,. 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 amino acids. The amino acid sequence sharing at least a particular sequence identity with a fragment of the amino acid sequence of SEQ ID NO: 1 or , SEQ ID NO: 3 may have less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66 amino acids.

In a further aspect, there is provided an isolated polypeptide obtainable from Mesobuthus martensii Karsch venom, which polypeptide has an N-terminal sequence of VKDGYIVDDRNC or VKDGYIADDRNC

Preferably, the polypeptide has a molecular mass within the range of 6500-7500 Da, 6600-7500 Da, 6700-7500 Da, 6800-7500 Da, 6900-7500 Da, 7000-7500 Da, 7100-7500 Da, 7200-7500 Da, 7300-7500 Da, 7350-7450 Da, or 7380-7410 Da. More preferably, the molecular mass is within the range of 7385-7393 Da. Most preferably, the molecular mass is 7389 Da. Preferably, the mass of the polypeptide is determined by mass spectrometry, e.g. MALDI-Tof.

Preferably, the polypeptides of the invention are capable of reducing cell death following cerebral ischemia. For example, the polypeptides of the invention are capable of reducing cell death, e.g. neuronal cell death, in the brain of an animal when a polypeptide of the invention is administered to an animal that has experienced, or is experiencing, cerebral ischemia. The reduced cell death will normally be relative to an equivalent animal that has experienced, or is experiencing, cerebral ischemia, but which animal has not been administered

with the polypeptide. The animal may be a mammal, for example a human, or a rodent, such as a rat.

In a further aspect, there is provided an isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide of the invention. For example, the nucleic acid may comprise a nucleotide sequence that encodes a polypeptide comprising:

(i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3,

(ii) an amino acid sequence sharing at least 70 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3,

(iii) a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or

(iv) an amino acid sequence sharing at least 70 percent sequence identity with the fragment defined in (iii) , wherein the amino acid sequence has at least 6 amino acids, and wherein the amino acid sequence has less than 66 amino acids.

In a further aspect, there is provided a nucleic acid comprising: (i) the nucleotide sequence of SEQ ID NO: 2 or SEQ ID

NO: 4,

(ii) a nucleotide sequence that shares at least 70 percent identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4, (iii) a fragment of the nucleotide sequence of SEQ ID NO:

2 or SEQ ID NO: 4, which fragment has at least 18 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2 or SEQ

ID NO: 4,

(iv) a nucleotide sequence sharing at least 70 percent identity with the fragment defined in (iii) , wherein the

nucleotide sequence has at least 18 nucleotides and wherein the nucleotide sequence has less than 198 nucleotides,

(v) a nucleotide sequence that hybridises with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 under stringent conditions, or

(vi) a nucleotide sequence that encodes a polypeptide of the invention.

Alternatively, the nucleic acid may consist of a nucleotide sequence defined in (i) , (ii) , (iii) , (iv) , (v) , or (vi) , or it may consist essentially of a nucleotide sequence defined in (i) , (ii) , (iii) , (iv) , (v) , or (vi) . The nucleic acid may include a start codon and/or a stop codon.

A nucleotide sequence that shares a particular sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 may share at least 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ

ID NO: 4. The sequence identity may be shared over 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2 or SEQ

ID NO: 4. Preferably, the sequence identity is shared over the full-length sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

A fragment of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 may have at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,

45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,

60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,

104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,

116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,

128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,

152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,

164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,

176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,

188, 189, 190, 191, 192, 193, 194, 195, 196, or 197 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 2 or

SEQ ID NO: 4. The fragment may have less than 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,

71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,

101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,

113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,

137,. 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,

149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,

161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,

173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,

197, or 198 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

A nucleotide sequence sharing at least a particular sequence identity with a fragment of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 may share at least 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity with the fragment. The sequence identity is shared over the full-length sequence of the fragment.

A nucleotide sequence sharing at least a particular sequence identity with a fragment of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 may have at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, or 197 nucleotides. The nucleotide sequence sharing at least a particular sequence identity with a fragment of the nucleotide sequence- of SEQ ID NO: 2 or SEQ ID NO: 4 may have less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,

111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,

123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,

147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,

159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,

171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,

183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198 nucleotides.

Fragments of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and nucleic acid molecules that share sequence identity with fragments of SEQ ID NO: 2 or SEQ ID NO: 4 are useful as probes for detecting nucleic acid encoding Mesopen4.6 or variants of Mesopen4.6. Fragments of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 are also useful as primers for amplifying the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or variants of SEQ ID NO: 2 or SEQ ID NO: 4, e.g. by polymerase chain reaction (PCR) . Methods of designing probes and their use in detecting nucleic acids of interest, and methods of designing primers and their use in amplifying nucleic acid are well known in the art. See for example Sambrook et al . , "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1989. Preferably, a fragment of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 suitable for use as a primer includes at least one nucleotide within 50, 40, 30, or 20, preferably within 10 nucleotides of the 5' or 3 ' terminus of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Most preferably, the fragment includes the 5' or 3 ' terminus nucleotide.

The nucleic acid may also comprise a regulatory sequence, e.g. a promoter, that is operably linked to the nucleotide sequence that encodes the polypeptide of the invention.

In a further aspect, there is provided a vector comprising a nucleic acid of the invention. The vector may include one or more elements that facilitate expression of nucleotide sequence in a host cell. The vector may include an element that allows the vector to replicate in a host cell. The vector may include an element that allows selection of host cells that contain the vector, e.g. a marker gene. Methods of introducing nucleic acid such as vectors into cells, e.g. by transformation or transfection, are well known in the art.

In a further aspect, there is provided a host cell comprising a nucleic acid of the invention. The host cell may be a eukaryotic cell, e.g. a yeast or a CHO cell, or a prokaryotic cell, e.g. E. coli. The host cell may be a cell that is suitable for culturing, i.e. it may be a cell that is not part of a living human or animal body. The nucleic acid of the invention may be present in the host cell as part of the host cell genome. Alternatively, it may be present in the host cell as an autonomously replicating entity, e.g. a plasmid. Where the nucleic acid is present in the genome of the host cell it is preferably heterologous nucleic acid, i.e. nucleic acid that is not naturally present within the genome of the host cell. For example, the nucleic acid may be inserted into the genome by genetic engineering.

We have found that Mesopen4.6 is able to reduce the infarct volume in rats subjected to MCA occlusion by more than 80 percent, compared to negative control, i.e. rats subjected to MCA occlusion but not administered with Mesopen4.6.

Our "transient ischemia test" involved subjecting rats to left middle cerebral artery (MCA) occlusion for 60 minutes, after which period reperfusion was initiated. Mesopen4.6 was injected intravenously at a dose of 10μg/250g body weight and the rats were sacrificed 23 hours after reperfusion was initiated. When Mesopen4.6 was administered 180 minutes after

commencement of MCA occlusion we found that the infarct volume, as measured by histopathological analysis, was 88 percent less than the infarct volume in the negative control, i.e. compared to rats that had been subjected to the MCA occlusion but which did not receive Mesopen4.6.

Preferably, the polypeptides of the invention reduce cell death, e.g. compared to negative control, in the brain of an animal subjected to MCA occlusion and administered with the polypeptide. For example, the polypeptide may reduce the infarct volume, e.g. cell death, in the brain of a rat that has been subjected to MCA occlusion, 'compared to negative control, i.e. a rat that has been subjected to MCA occlusion but which has not been administered with the polypeptide. Preferably, the reduction in infarct volume is at least 40, 50, 60, 70, or 75 percent, preferably at least 80 percent. Preferably, the reduction in infarct volume is determined in accordance with the transient ischemia test described above, i.e. the polypeptide is administered 180 minutes after commencement of MCA occlusion and at a concentration of

10μg/250g body weight. Observing cell death in the brain of the animal may involve sacrificing the animal.

We also tested the ability of Mesopen4.6 to provide neuroprotection in oxygen-glucose deprived conditions (OGD) and found that Mesopen4.6 provides 30-40% percent protection compared to positive control.

The "oxygen-glucose deprived conditions test" (OGD test) is a known in vitro test that mimics in vivo cerebral ischemia. We cultured hippocampal cultures under conditions in which oxygen was eliminated using 95% N ₂ and 5%CO ₂ for 60 minutes, and in which glucose was not used in the culture medium. Addition of 0.5μg/ml of Mesopen4.6 during the period of oxygen and glucose elimination provided at least 30% protection compared to positive control, i.e. cultures that were oxygen and glucose

deprived but which did not receive Mesopen4.6. "30% protection" means that 30% fewer cells died compared to positive control.

Percent protection was measured by both lactate dehydrogenase (LDH) assay and Propidium iodide (PI) staining. LDH assay measures release of the cytoplasmic enzyme lactate dehydrogenase into culture media. The amount of lactate dehydrogenase released in the culture medium is a measure of cytotoxicity. PI stains dead cells in hippocampal slices.

Preferably, the polypeptides of the invention reduce cell death in a culture subjected to oxygen-glucose deprived conditions, e.g. compared to positive control. A positive control is a culture subjected to oxygen-glucose deprived conditions but which does not receive the polypeptide. Preferably, the polypeptide reduces cell death by at least 20, 25, 30, 35, or even by at least 40 percent compared to negative control. Preferably, the reduction in cell death in the culture is determined in accordance with the oxygen- glucose deprived conditions test described above.

In a further aspect, there is provided a method of producing a polypeptide of the invention, which method comprises the steps of:

(a) providing a host cell comprising a nucleotide sequence that encodes a polypeptide of the invention, and

(b) causing the host cell to express the polypeptide encoded by the nucleotide sequence. The method may also comprise step (c) isolating the polypeptide and/or may also comprise, prior to step (a) , introducing the nucleic acid that encodes the polypeptide of the invention into the host cell, e.g. on a vector. The step of providing the host cell may also include culturing the host cell. Methods of culturing host cells to express polypeptides and methods of purifying polypeptides are well known in the

art. The host cell may express the polypeptide under the control of a constitutive promoter. Alternatively, expression of polypeptide may require an inducing agent, e.g. if expression of the nucleotide sequence is under the control of an inducible promoter.

In a further aspect, there is provided a polypeptide of the invention for use in a therapeutic method. In a further aspect, there is provided use of a polypeptide of the invention in the manufacture of a medicament for use in a therapeutic method.

The therapeutic method may be for reducing cell death, e.g. neuronal cell death, in the brain of a patient. For example, the therapeutic method may be for preventing or ameliorating physiological damage following ischemia, e.g. cerebral ischemia in a patient.

The therapeutic method may be treatment of any type of brain injury or trauma that leads to cell .death, e.g. neuronal injury. For example, the therapeutic method may be treatment of a patient suffering from, or at risk of suffering from, stroke, ischemic stroke, hemorrhage stroke, cerebral ischemia, e.g. ischemia of cerebral tissues, focal ischemia, transient focal ischemia, global ischemia, intraparenchymal haemorrhage, infarction, e.g. infarction of the brain, acute ischemia, or hypoxia, e.g. cerebral hypoxia.

In particular, the therapeutic method may be treatment of a patient suffering from, or at risk of suffering from, cerebral ischemia or stroke. For example, the therapeutic method may be for preventing (e.g. it may be prophylactic), mitigating, alleviating, reversing, reducing, controlling, and/or curing any of the above conditions in a patient. The therapeutic method may be for assisting recovery from any of the above

conditions and/or for down regulating genes in a patient that are associated with ischemic damage.

The therapeutic method may be treatment of neurological damage in a patient, such as treatment to reduce neuronal cell death in the brain, caused by, following, resulting from, and/or as a consequence of, a reduction in blood flow to brain tissue, e.g. cerebral ischemia. The polypeptides of the invention may be useful for treating a patient who is at risk of, experiencing, or has experienced, neuronal cell death, resulting from a reduced blood supply and/or flow to brain tissue, e.g. compared to the normal blood supply to the brain tissue.

The polypeptides may also be useful for treating a patient at risk of any of the conditions listed above, e.g. a person who is at risk of cerebral ischemia and/or stroke. A person may be at risk due to hereditary pre-disposition, advanced age, hypertension (high blood pressure) , previous stroke or TIA (transient ischaemic attack) , diabetes mellitus, high cholesterol, cigarette smoking, atrial fibrillation, and migraine with aura.

A polypeptide of the invention may be administered whilst the patient is experiencing a reduced blood supply to brain tissue, e.g. ischemia, or it may be administered after the blood supply has returned to normal. We have shown that administration of Mesopen4.6 to rats 120 minutes after initiation of reperfusion following 60 minutes of MCA occlusion provided significant reduction in infarct volume.

For example, it may be administered 1, 2, 3, 5, 8, 15, 24, 40 hours after the blood supply has returned to normal, for example 1, 2, 3, 4, 5, 6, 7, 10, or even 14 days after blood supply has returned to normal. In particular, our results indicate that administering mesopen4.6 to humans more than 24

hours after blood supply has returned to normal will be beneficial.

The reduction in blood supply and/or flow, e.g. cerebral ischemia, may be caused by an interruption in the blood supply to the brain tissue. Such an interruption may be caused by an occlusion, such as a thrombosis or embolism, or by a haemorrhage.

The polypeptides of the invention may be provided as a composition e.g. as a pharmaceutical composition. The composition may comprise a polypeptide of the invention and a pharmaceutically acceptable excipient, diluent and/or carrier.

In a further aspect, there is provided a method for treating a patient suffering from, or at risk of, any of the conditions mentioned above, which method comprises administering a polypeptide of the invention to the patient. In particular, the method is for treating cerebral ischemia and/or stroke in a patient.

In a further aspect, there is provided a kit for treating a patient suffering from, or at risk of, any of the conditions mentioned above, which kit comprises: (i) a container comprising a composition, e.g. a pharmaceutical composition, which composition comprises a polypeptide of the invention, and optionally

(ii) instructions for administering the pharmaceutical composition to the patient. In particular, the kit is for treating cerebral ischemia and/or stroke in a patient.

The patient to be treated may be any animal or human. The patient is preferably a mammal, more preferably a human. The patient may be male or female.

Variants of Mesopen4.6

Variant Mesopen4.6 molecules, are polypeptides that do not include the exact amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and nucleic acid molecules that do not include the exact nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The variant Mesopen4.6 molecules, of the present invention are preferably capable of reducing cell death following cerebral ischemia. For example, the variant molecules of the invention are capable of reducing cell death, e.g. neuronal cell death, in the brain of an animal when administered to an animal that has experienced, or is experiencing, cerebral ischemia. The reduced cell death will normally be relative to an equivalent animal that has experienced, or is experiencing, cerebral ischemia, but which animal has not been administered with the variant molecule.

For example, a variant according to the invention preferably has a similar, or at least the same, ability to reduce infarct volume e.g. as measured in the transient ischemia test as Mesopen4.6, or a similar, or at least the same, ability to reduce cell death e.g. as measured in the OGD test as Mesopen4.6. A "similar ability" means that the ability of the variant to reduce infarct volume in the transient ischemia test is at least 70, 75, 80, 85, 90, or 95 percent of the ability of Mesopen4.6 to reduce infarct volume, or that the ability of the variant to reduce cell death in the OGD test is at least 90 percent of the ability of Mesopen4.6 to reduce cell death.

Where the activity of a variant is described, this refers to the activity of the polypeptide encoded by the nucleic acid molecule .

Variants of Mesopen4.6 molecules of the present invention may be:

(i) novel, naturally occurring, molecules, for example obtainable from Mesobuthus martensii Karsch or any other species of scorpion. Also included are natural biological variants (e.g. allelic variants or variants from scorpions that are geographically separated and therefore unable to exchange genes) of Mesopen4.6. In addition to Mesobuthus martensii Karsch, preferred sources of naturally occurring variants of Mesopen4.6 are other species of the Buthidae family, for example Tityus serrulatus.

(ii) artificial Mesopen4.6 molecule derivatives, which can be prepared by the skilled person in the light of the present disclosure. Such derivatives may be prepared, for instance, by site directed or random mutagenesis, or by direct synthesis. Preferably, a variant nucleic acid, for example, is generated either directly or indirectly, e.g. via one or more amplification or replication steps from an original nucleic acid having all or part of the sequence shown herein.

Particularly included are truncated variants which include only a distinctive part or fragment, however produced, corresponding to a portion of the sequences described herein, for example functional parts of the polypeptide that are still capable of reducing cell death following cerebral ischemia.

Also included are molecules which have been extended at their termini with non-naturally contiguous sequences, i.e. polypeptides of the invention may also comprise additional amino acids, additional domains, or may be conjugated to additional domains or other molecules. Additional amino acids, domains, or molecules conjugated to the polypeptide may provide an additional function, for example in assisting purification of the polypeptide. Examples of additional domains that may assist in purification of the polypeptide are 6-histidine tag, and glutathione S-transferase tag.

Polypeptides may be fusion proteins, fused to a peptide or

other protein, such as a label, which may be, for instance, bioactive, radioactive, enzymatic or fluorescent.

Variant polypeptides may comprise at least one modification compared to SEQ ID NO: 1 or SEQ ID NO: 3, e.g. addition, substitution, inversion and/or deletion of one or more amino acids .

Purely as examples, conservative replacements which may be found in such polymorphisms may be between amino acids within the following groups : alanine, serine, threonine; glutamic acid and aspartic acid; arginine and leucine; asparagine and glutamine; isoleucine, leucine and valine; phenylalanine, tyrosine and tryptophan.

Production of variants of Mesopen4.6 Variants of Mesopen4.6 may be produced by modifying SEQ ID NO: 1,2,3, or 4.

The polypeptide of the invention may be a fragment of SEQ ID NO: 1 or SEQ ID NO: 3. Such fragments may be provided in isolated form, i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region with one or two non-naturally contiguous sequences fused to it. Additionally, several fragments may be comprised within a single larger polypeptide.

Changes to nucleic acid sequences may be desirable for a number of reasons. For instance, they may introduce or remove restriction endonuclease sites or alter codon usage.

Alternatively changes to a sequence may produce a derivative by way of one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.

Such changes may modify sites which are required for post translation modification, such as cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for glycosylation, lipoylation etc. Leader or other targeting sequences (e.g. membrance or golgi locating sequences) may be added to the expressed polypeptide to determine its location following expression.

Other desirable mutations may be random or site directed mutagenesis in order to alter the activity, e.g. specificity, or stability of the encoded polypeptide. Changes may be by way of conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.

Also included are variants having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three

dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.

The variants of the invention may also be created by chemical modification of Mesopen4.6. Methods for chemical modification of polypeptides are well known in the art.

Polypeptides of the invention may be obtained by expression of a nucleic acid that encodes the polypeptide using a suitable vector and host organism. Examples of suitable vectors and hosts are well known in the art (see e.g. Sambrook, J. et al . (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York) .

Polypeptides, and particularly fragments, of the invention may also be created using chemical synthesis by any suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings. In conventional solution phase peptide synthesis, the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence . Many such methods are now commonplace to those skilled in the art.

The variants of the invention may also be created by modification, using molecular biological techniques, of the nucleic acid that encodes Mesopen4.β, or a nucleic acid that encodes a further variant or homologue of the invention. Molecular biological techniques are well known in the art.

Thus, the invention provides a method of producing and/or identifying a variant of Mesopen4.6, which method comprises the steps of:

(i) providing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3,

(ii) modifying the amino acid sequence of the polypeptide, or a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and

(iii) measuring the ability of the variant to reduce cell death following cerebral ischemia.

Step (iii) may involve measuring the ability of the variant to reduce infarct volume in rats, e.g. in the transient ischemia test, compared to Mesopen4.6, and/or measuring the ability of the variant to reduce cell death, e.g. in the OGD test, compared to Mesopen4.6. Preferably, the variant has a similar, or at least the same, ability to reduce infarct volume or cell death as Mesopen4.6.

Methods of purifying polypeptides from heterogenous mixtures are well known in the art (e.g. selective precipitation, proteolysis, ultrafiltration with known molecular weight cut- off filters, ion-exchange chromatography, gel filtration, etc.) . Typical protocols are set out in "Protein Purification - principles and practice" Pub. Springer-Verlag, New York Inc (1982), and by Harris & Angal (1989) "Protein purification methods - a practical approach" Pub. O.U.P. UK, or references therein. Further methods which are known to be suitable for protein purification are disclosed in "Methods in Enzymology VoI 182 - Guide to Protein Purification" Ed. M P Deutscher, Pub. Academic Press Inc..

Antibodies to Mesopen4.6, or variants of Mesopen4.6, may also be used to screen for Mesopen4.6, which methods are well known to those skilled in the art.

The Mesopen4.6 amino acid or nucleotide sequence, i.e. SEQ ID NO: 1 or 3, or SEQ ID NO: 2 or 4 may be used in a data-base (e.g. of ESTs, or STSs) search to find sequences that share a specified level of sequence identity, such as those which may become available in due course, and expression products of which can be tested for activity as described herein.

Alternatively, variants may be provided by standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells. Probing may optionally be done by means of so-called "nucleic acid chips", see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review.

Libraries of nucleic acid molecules may be created which may be screened for nucleic acids that encode a polypeptide of the invention. Such libraries may be created using the nucleic acid molecule that encodes Mesopen4.6. The Mesopen4.6 nucleic acid molecule sequence may be mutated, for example using random PCR mutagenesis, or gene shuffling techniques. The resulting mutated nucleic acid molecules may be ligated into a vector. Libraries of polypeptides may be created, by expressing the nucleic acid molecules in a suitable host.

Polypeptides and nucleic acids

In this specification, a polypeptide of the invention may be a a polypeptide, protein, or peptide. The polypeptide may have been post-translationally modified.

A nucleic acid of the invention may be any nucleic acid (DNA or RNA) having a nucleotide sequence specified above or a nucleotide sequence that is complementary to any of the nucleotide sequences specified above. The nucleic acid of the invention may be an RNA transcript.

The polypeptides of the invention may be isolated polypeptides. The term "isolated polypeptide" refers to a polypeptide that has undergone some degree of isolation, e.g. by purification. For example, the concentration of the polypeptide, e.g. Mesopen4.6, relative to the other components of the Mesobuthus martensii Karsch venom may be greater than the concentration of the polypeptide relative to the other components in the naturally occurring venom. Preferably, the polypeptide is substantially free of the other components of the Mesobuthus martensii Karsch venom, for example 50, 60, 70, 80, 90, 95, or even 99 percent free. For example, the polypeptide may be purified from the venom or crude extract by a gel filtration chromatography step followed by a reverse phase high pressure liquid chromatography (RP-HPLC) step.

The nucleic acids of the invention may be isolated nucleic acids. The term "isolated nucleic acid" refers to a nucleic acid that has undergone some degree of isolation, e.g. the nucleic acid is not present in a cell in which it naturally occurs .

In this specification the term "operably linked" may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide sequence under

the influence or control of the regulatory sequence. Thus a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide sequence which forms part or all of the selected nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.

Sequence identity Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in SEQ ID NO: 1 or SEQ ID NO: 3 after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences.

Where the aligned sequences are of different length, sequence identity of the shorter comparison sequence may be determined over the entire length of the longer given sequence or, where the comparison sequence is longer than the given sequence, sequence identity of the comparison sequence may be determined over the entire length of the shorter given sequence.

For example, where a given sequence comprises 100 amino acids and the candidate sequence comprises 10 amino acids, the candidate sequence can only have a maximum identity of 10% to the entire length of the given sequence. This is further illustrated in the following example:

(A)

Given seq: XXXXXXXXXXXXXXX (15 amino acids) Comparison seq: XXXXXYYYYYYY (12 amino acids)

The given sequence may, for example, be that encoding SEQ ID NO: 1 or SEQ ID NO: 3.

% sequence identity = the number of identically matching amino acid residues after alignment divided by the total number of amino acid residues in the longer given sequence, i.e. (5 divided by 15) x 100 = 33.3%

Where the comparison sequence is longer than the given sequence, sequence identity may be determined over the entire length of the given sequence. For example:

(B)

Given seq: XXXXXXXXXX (10 amino acids) Comparison seq: XXXXXYYYYYYZZYZZZZZZ (20 amino acids)

Again, the given sequence may, for example, be that encoding SEQ ID NO: 1 or SEQ ID NO: 3.

% sequence identity = number of identical amino acids after alignment divided by total number of amino acid residues in the given sequence, i.e. (5 divided by 10) x 100 = 50%.

Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW, T-coffee or Megalign (DNASTAR) software. When using such software, the default parameters, e.g. for gap and exention penalty, are preferably used. Preferably, sequences are aligned using ClustalW 1.82 software, and using the default parameters, e.g. DNA Gap Open Penalty = 15.0, DNA Gap Extension Penalty = 6.66, DNA Matrix = Identity, Protein Gap Open Penalty = 10.0, Protein Gap Extension Penalty = 0.2, Protein matrix = Gonnet, Protein/DNA ENDGAP = -1, and Protein/DNA GAPDIST = 4.

Identity of nucleic acid sequences may be determined in a similar manner involving aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and calculating sequence identity over the entire length of the respective sequences. Where the aligned sequences are of different length, sequence identity may be determined as described above and illustrated in examples (A) and (B) .

Hybridisation stringency

In accordance with the present invention, nucleic acid sequences may be identified by using hybridization and washing conditions of appropriate stringency.

Complementary nucleic acid sequences will hybridise to one another through Watson-Crick binding interactions. Sequences which are not 100% complementary may also hybridise but the strength of the hybridisation usually decreases with the decrease in complementarity. The strength of hybridisation can therefore be used to distinguish the degree of complementarity of sequences capable of binding to each other.

The "stringency" of a hybridization reaction can be readily determined by a person skilled in the art.

The stringency of a given reaction may depend upon factors such as probe length, washing temperature, and salt concentration. Higher temperatures are generally required for proper annealing of long probes, while shorter probes may be annealed at lower temperatures. The higher the degree of desired complementarity between the probe and hybridisable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so.

For example, hybridizations may be performed, according to the method of Sambrook et al., ("Molecular Cloning, A Laboratory- Manual, Cold Spring Harbor Laboratory Press, 1989) using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes .

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules is to calculate the melting temperature T _m (Sambrook et al., 1989) :

T _m = 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/n

where n is the number of bases in the oligonucleotide.

As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T _1n is 57°C. The T _m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in sequence complementarity .

Accordingly, nucleotide sequences can be categorised by an ability to hybridise to a target sequence under different hybridisation and washing stringency conditions which can be selected by using the above equation. The T _n , may be used to provide an indicator of the strength of the hybridisation.

The concept of distinguishing sequences based on the stringency of the conditions is well understood by the person skilled in the art and may be readily applied.

Sequences exhibiting 95-100% sequence complementarity are considered to hybridise under very high stringency conditions, sequences exhibiting 85-95% complementarity are considered to hybridise under high stringency conditions, sequences exhibiting 70-85% complementarity are considered to hybridise under intermediate stringency conditions, i.e. "stringent conditions", sequences exhibiting 60-70% complementarity are considered to hybridise under low stringency conditions and sequences exhibiting 50-60% complementarity are considered to hybridise under very low stringency conditions. Hybridisations performed at 42 ⁰ C or higher may be considered to be under high stringency conditions.

Pharmaceutical compositions

Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intraturαoural, oral and nasal. The medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body. Injectable formulations may comprise the selected compound in a sterile or isotonic medium. The polypeptides of the invention are preferably administered intravenously, or by intraperitoneal or intraventricular injection.

Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time- course of administration, will depend on the nature and severity of the disease being treated. Prescription of

treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington' s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins. We have found that a dosage of 10μg/250g (0.04μg/g) of mesopen4.6 was effective in rats. A suitable administration regime may be formulated in light of this finding.

Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

-The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be

apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Brief Description of the Figures Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1 : Figure l(a) shows gel filtration chromatography of Mesobuthus martensii Karsch (BmK) venom on Sephadex G50 column. BmK crude venom (lOOmg) was subjected to gel filtration chromatography. Figure 1 (b) shows fractionation of Peak (II) fraction on RP- HPLC. Peak 2 of the gel filtration elution (protein) profile was subjected to RP-HPLC chromatography. Figure l(c) shows purification of BmKRP4 on RP-HPLC. BmKRP4 was further subjected to RP-HPLC, using a similar buffer composition to Figure l(a) . Figure l(d) shows purification of BmKRP4.6 (Mesopen4.6) . Purification was carried out on a Jupiter C4 reverse phase column. Figure l(e) shows mass spectrometry analysis of Mesopen4.6. Mesopen4.6 was subjected to mass analysis (7.3kDa) followed by N-terminal protein sequencing. The N-terminal sequence was determined to be VKDGYIADDRNC.

Figure 2 :

Figure 2 shows histopathological analysis of brain sections. Figure 2 (a) (i-iii) shows photographs showing serial coronal sections, for rostral to caudal slices stained with TTC. Surviving tissue stains red whilst dead cells remain white. The brain sections were obtained from rats treated with

10μg/250g body weight of BmK (Figure 2 (a) (i)), BmKRP4 (Figure 2 (a) (ii)), and BmkRP4.6 (Figure 2(a) (iii)) intravenously at 0, 30min, 60min, 120min and 180mins, post-occlusion; and MK801, injected intraperitoneally in three doses: 2.5mg/kg, 30min prior to MCAo, followed by 1.25mg/kg, 6hr and 14hr post- occlusion. Figure 2 (b) shows the percentage of neuroprotection

which was calculated by measuring infarct volumes. Infarct volumes were quantitated using Scion Image software (Scion Corporation, USA) .

Figure 3 :

Figure 3 shows neuroprotection conferred by Mesopen4.6 in OGD culture. Figure 3 (a) shows organotypic hippocampal culture subjected to OGD for 60mins and treated with Mesopen4.6 during the insult. Figure 3 (a) (i) shows neuroprotection conferred by 62μg MK801, Figure 3 (a) (ii) shows neuroprotection conferred by 3μg and 6μg BmKII, Figure 3 (a) (iii) shows neuroprotection conferred by 0.5μg and lμg BmKRP4, Figure 3 (a) (iv) shows neuroprotection conferred by 0.5μg and lμg BmKRP4.6. Figure 3 (a) shows that Mesopen4.6 protected the hippocampal tissues from the ischemic induced cell death. Figure 3(b) shows the percent protection provided by Mesopen4.6 (BmKII, BmKRP4 and BmKRP4.6) in a cytotoxicity assay (LDH assay), showing that Mesopen4.6 treatment reduced the cell death experienced by the hippocampal tissues.

Figure 4 :

Figure 4 shows oligonucleotide microarray global gene expression analysis. Figure 4 (a) shows a hierarchical clustering (average linkage clustering) tree of the differentially regulated genes (tree selection; gene tree and sample tree; distance metric selection, Euclidean selection) . Figure 4 (b) shows the number of gene changes upon MCAo and Mesopen4.6 treatment, Figure 4 (c) shows classes of molecular functions of the regulated genes.

Figure 5 :

Figure 5 (a) shows an amino acid sequence alignment of Mesopen4.6 and its isoform ("pCR4topl0rp4.6" corresponds to Mesopen4.6, "pCR4topl9rp4.6" corresponds to Mesopen4.6 isoform) . Figure 5(b) shows the amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 2) of Mesopen4.β.

Figure 5(c) shows the amino acid sequence (SEQ ID NO: 3) and nucleotide sequence (SEQ ID NO: 4) of an isoform of Mesopen4.6.

Table 1:

Table 1 (presented as Table 1 (a) and Table 1 (b) ) shows the list of genes whose expression was changed by MK801 and BmKRP4.6 treatment when compared to saline-treated MCAo controls. Fold changes greater or equal to 2.0 and detection p value <0.01, were deemed significant. The gene numbers in Table 1 (a) correspond to the gene numbers in Table 1 (b) .

Detailed Description of the Invention

The vast majority of experimental stroke research has been aimed at preventing or ameliorating morphological and physiological damage after an ischemic insult. A common paradigm in experimental stroke research involves histological examination of the post-stroke damage in animal models that did or did not receive a pharmacological treatment. These comparisons are generally made within days of the ischemic insult. The experimental models (rodent) of cerebral ischemia that are largely being used in the laboratory are the models for focal (which includes MCAo) and global ischemia. The focal ischemic model represents the clinical consequences of ischemic stroke, while the global ischemic model represents the consequences of cardiac arrest. Middle cerebral artery- occlusion (MCAo) in rodents is considered as a convenient, reproducible and reliable model that represents the largely occurring cerebral ischemia in humans. MCAo has been routinely used to delineate the pathophysiology of stroke as well to identify new interventions in stroke therapies (Mergenthaler et al 2004) . The two most commonly used methods for quantifying the amount of histological damage following MCAo are calculating the volume of infarcted tissue via image analysis or counting the number of surviving neurons with norma1 morphology.

Specific details of the best mode contemplated by the inventors for carrying out the invention are set forth below, by way of example. In particular, the purification and characterization of the protein Mesopen4.6 as well as the neuroprotective effects of the protein in both in vivo (MCAo rat model) as well as in vitro (organotypic hippocampal and primary tissue culture) conditions is discussed. It will be apparent to one skilled in the art that the present invention may be practiced without . limitation to these specific details.

Results

Isolation, purification and characterization of BmKRP4.6 (Mesopen4.6) Mesobuthus martensii Karsch venom was fractionated on a gel filtration chromatography (Figure Ia) . The resulting protein profile showed 2 peaks of which peak 2 (BmKII), consisting of low molecular weight proteins/peptides showed potency to reduce infarct volume in MCAo rats. BmKII was fractionated on a RP-HPLC (Figure Ib) . The fraction showing reduction on the infarct size, BmKRP4 was subjected to RP-HPLC (Figure Ic) . The partially purified proteins were further resolved on RP-HPLC to yield a homogenously pure fraction BmKRP4.6 (Figure Id) . Mass spectrometry (MALDI-Tof) and N-terminal protein sequencing analysis (Figure Ie) showed that the mass of

BmKRP4.6 is 7389Da and the N-terminal amino acid sequence to be VKDA(g)Y(f )I (p) A(t) K (d) P (n) H (r)NX (a) , where letters in brackets represent minor components or residues that were detected during sequencing. For example, at cycle 4 apart from alanine (A), glycine (g) residue also shows up. However, subsequent sequencing of the nucleotide sequence showed that the correct N-terminal amino acid sequence of this molecule is VKDGYIADDRNC. This molecule has been named as Mesopen4.6. The nucleotide sequence and deduced amino acid sequence of Mesopen4.6 and an isoform of Mesopen4.6 are shown in Figure 5.

Intravenous administration of Mesoρen4.6 confers protection during transient Ischemia.

Transient cerebral focal ischemia was induced by the intraluminal insertion of a nylon suture to occlude the left middle cerebral artery (MCA) . The presence of focal occlusion was confirmed using the Laser Doppler that measures the cerebral blood flow in the region of middle cerebral artery. The ischemic period lasted 60min, after which the suture was removed and reperfusion initiated. Animals were sacrificed 23hr later. Mesopen4.6 from Mesobuthus martensii Karsch venom, was injected intravenously, at a dose of 10μg/250g body weight, immediately- (Omin) , 30min, 60min, 120min and 180min after MCAo. The concentration of Mesopen 4.6 used did not induce any toxicity since the LD ₅₀ of the crude venom is 7mg/kg body weight. Histopathological analysis was carried out to assess the extent of infarction in the brains of rats (Figure 2a i-iii) .

The administration of Mesopen4.6 at all time points resulted in a significant reduction in infarct size, as revealed by TTC staining of serial brain sections. Ischemic damage was markedly attenuated in the striatum and cortex of the brain, although effects were more pronounced in striatal tissue. Administration of Mesopen4.6 at Omin post-MCAo (upon occlusion) also reduced the infarct size but to a lesser extent than its 60min, 120min and 18 Omin treatments. Quantitative assessment of neuroprotection was carried out by measuring the infarct volume of brain sections (Figure 2b) , that takes into consideration the insult size as well as extent of edema. Administration of Mesopen4.6 significantly- reduced the infarct volume at all time points but was more pronounced at the 60min & 12 Omin (81%) and 18 Omin (88%) .

MK801 confers neuroprotection Dizocilpine, or MK801, is a non-competitive antagonist of the NMDA receptor and has been shown in several animal studies to

confer neuroprotection by inhibiting glutamate-induced excitotoxicity (Buchan et al, 1992) .

MK801 was also used in our studies and its efficacy in reducing infarct volume was compared to that of Mesopen4.6. The administration of MK801 in three doses, 2.5mg/kg body 30min prior to MCAo followed by 1.25mg/kg body weight 6hr and 14hr post-occlusion, significantly reduced infarct volume as compared to the saline-treated control (Figure 2a&b; 61.1% ± 8.6%, p<0.01) . The extent of neuroprotection conferred by MK801 (84%) appeared to be slightly higher than that by Mesopen4.6 at 0 min (27%) . Nevertheless, the protection by Mesopen4.6 was observed to be higher at 180mins and somewhat similar to each other at both 60 & 120min time points. It should be noted that, the concentration of MK801 used for our study was much higher than that of the Mesopen4.6. The concentration of Mesopen4.6 used in this study was 0.04ug/g (10ug/250g) and the concentration of MK801 was 5ug/g (5mg/kg or 1.25mg/250g) of rat.

Neuroprotection in Oxygen-glucose deprived (OGD) organotypic hippocampal slice culture.

In vivo cerebral ischemia is known to be best mimicked in vitro by the oxygen-glucose deprived (OGD) conditions. Thus, we carried out experiments using the organotypic hippocampal cultures, in conditions where oxygen is eliminated by using the 95% N ₂ and 5% CO ₂ and glucose was deprived in cultures by using culture media without glucose. Addition of Mesopen4.6 during the insult period (60mins) rendered protection to the hippocampal slices that were subjected to ischemic conditions (Figure 3a) . The results showed that Mesopen4.6 at 0.5ug/ml and l.Oug/ml concentration had similar percentage of protection (30-40%) . However, the MK801 (NMDA antagonist, positive control) showed much higher protection (-80%) .

We also performed a Cytotoxicity assay. This assay measured the release of the cytoplasmic enzyme lactate dehydrogenase

(LDH) into the culture medium which was used as a measure of cytotoxicity. The amount of enzyme released was determined using a lactate dehydrogenase detection kit (Roche Molecular Biochemicals, Germany) . Briefly, lOOul of culture media from the experimental culture were added to 10 OuI of reaction mixture containing solution 1 (enzyme solution) and solution 2

(dye mixture) . The kinetics of the enzyme reactions were measured at 490nm using the microplate reader. Culture media from OGD experiment without any treatment is considered to give 100% cytotoxicity/cell death.

The cytotoxicity assay also showed that Mesopen4.6 treatment reduced the cytotoxicity due to OGD and in a manner almost similar to the cultures under normoxic conditions. In all cases, the Mesopen4.6 mediated protection has been seen in the both CAl as well as in the CA2/3 region of the hippocampal slices but more pronounced in the CA2/3 region (Figure 3b) . The NMDA antagonist, MK801, which showed protection of neuronal injury upon OGD was used as positive control.

Gene expression analysis of ischemia and Mesopen4.6-induced neuroprotection Cerebral ischemia is a powerful stimulus for the de novo expression of many gene systems and identification of genes differentially expressed in the ischemic and non-ischemic brain will provide valuable information regarding the pathophysiology and provide therapeutic targets in ischemia. Analysis of gene expression can also aid in elucidating the role of various pathways or specific genes in the mechanism of action of neuroprotective agents. Consequently, the power of high-density oligonucleotide arrays was harnessed to generate global expression profiles of non-ischemic (Sham operated) , ischemic (MCAo) and Mesopen4.6-treated brains. RNA isolated from the brains of sham-operated rats (sham-op; n=4) , rats

subjected to 60min of MCAo (MCAo; n=4) and rats subjected to βOmin of MCAo and treated with 10μg/200g body weight Mesopen4.6 at different time points post-occlusion (MCAo+Mesopen4.6; n=4) was pooled before microarray probe preparation and hybridization to minimize individual variations. Expression analysis was carried out using GeneChips from the RAE-230A Array set, with each chip representing ~15,900 genes and expressed sequence tags (ESTs) . Genes whose expression changed by 2-fold or greater in at least one pairwise comparison (between sham-op and MCAo or between MCAo and MCAo+Mesopen4.6) and detection p value<0.01, were deemed significant. Hierachical clustering of the differentially regulated genes is shown in Figure 4a. Based on the fold change value of ± 2.0 and above, a total of 309 genes have been found to be differentially . regulated by the

Mesopen4.6 (Table 1) . Among them, the number of genes showing no significant change (NC) in expression represented a large part of it than those exhibiting either decrease (D) or increase (I) levels of expression (Figure 4b) . The molecular function of the differentially regulated genes are grouped into genes that are unclassified (EST) , actin binding/cytoskeletal/structural components, calcium/ion binding, catalytic/peptidases/ inhibitory activity, transferase/carrier activity, hormone activity, nucleic acid/DNA binding, growth factor/ion binding, protein binding, receptor binding, transcription factor/signal transducer activity and transporter/channel activity (Figure 4c) . We found that genes involved in transporter/channel activity regulation were mostly downregulated with Mesopen4.6 administration.

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