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WO1993010145A1 - Compositions pour le traitement retarde de lesions neuronales associees a l'ischemie - Google Patents

Compositions pour le traitement retarde de lesions neuronales associees a l'ischemie Download PDF

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Publication number
WO1993010145A1
WO1993010145A1 PCT/US1992/009766 US9209766W WO9310145A1 WO 1993010145 A1 WO1993010145 A1 WO 1993010145A1 US 9209766 W US9209766 W US 9209766W WO 9310145 A1 WO9310145 A1 WO 9310145A1
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Prior art keywords
snx
oct
binding
mviia
compound
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PCT/US1992/009766
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English (en)
Inventor
George P. Miljanich
Stephen S. Bowersox
James A. Fox
Karen L. Valentino
Robert S. Bitner
Donald H. Yamashiro
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Neurex Corporation
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Priority claimed from US07/789,913 external-priority patent/US5559095A/en
Application filed by Neurex Corporation filed Critical Neurex Corporation
Publication of WO1993010145A1 publication Critical patent/WO1993010145A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to pharmaceutical compositions and methods for reducing neuronal damage with an ischemic condition, such as stroke, and for methods of screening test compounds for inclusion in such compositions and methods.
  • Ischemic damage to the central nervous system may result from either global or focal ischemic conditions.
  • Global ischemia occurs under conditions in which blood flow to the entire brain ceases for a period of time, such as may result from cardiac arrest.
  • Focal ischemia occurs under conditions in which a portion of the brain is deprived of its normal blood supply, such as may result from thrombo- e bolytic occlusion of a cerebral vessel, traumatic head injury, edema, or brain tumors.
  • Focal ischemia may be of limited or prolonged duration.
  • prolonged focal ischemia as caused by lodgement of a thromboembolus in a cerebral blood vessel, reduction of blood flow to a defined, focal region may be followed by reperfusion to part of the ischemic region, via collateral circulatory pathways.
  • Ischemic cell death following focal ischemia has been reported to be complete 24 hours after the primary ischemic event (Nedergaard, 1987) .
  • Anti ⁇ coagulants such as heparin
  • antivasoconstriction agents such as flunarizine
  • excitatory neurotrans ⁇ mitter antagonists such as MK-801 and AP7
  • anti- edemic compounds have shown mixed results, with no clear benefits to outweigh a variety of side effects, including neurotoxicity or increased susceptibility to infection.
  • vasodilators Two general classes of vasodilators have been studied for possible treatment of neuronal ischemic damage. Non-specific vasodilators, including papaverine, prostacyclin, pentoxifylline, and nitroprusside failed to demonstrate any clear benefit in reducing ischemic damage.
  • a second general class of vasodilators includes a variety of calcium-anta ⁇ gonist vasodilator 'drugs. Verapamil and related compounds which prevent calcium entry into smooth and striated muscle appear to be effective only at high drug concentrations, where serious cardiotoxicity effects may ensue. Dihydropyridines, such as nimodipine, have produced mixed results — some neurological improvement may be seen, but increased cerebral edema has also been observed. Benzothiazepines, as exemplified by diltiazem, have shown moderate protective effects, but these drugs also appear to cause undesired side effects.
  • the drugs mentioned above have been administered prior to or within a few hours of the period of experimental ischemic insult.
  • treatment is generally not feasible until well after the ischemic insult.
  • the treatment paradigms have generally included treatment commencing before the ischemic event and continuing over an extended period of time, such as continuous administration of nimodipine from one hour before until 24 hours following ischemia (Jacewicz) , or repeated doses administered before as well as after the ischemic event (Dirnagl, 1990; Bielenberg, 1990) .
  • the NMDA antagonist MK-801 was administered to Mongolian gerbils 24 hours post- ischemia, and neuroprotection was observed (Gill et al . , 1988); however, the effects of this compound have subsequently been shown to be a consequence of postischemic hypothermia rather than a direct action on NMDA receptors in this animal model (Buchan and Pulsinelli, 1990) .
  • drugs which have been proposed to date for the treatment of stroke and other ischemic- related conditionsOf the brain are either (i) relatively ineffective, (ii) effective only at dosage levels where undesired side effects are observed, and/or (iii) effective only when administered prior to or shortly after the ischemic insult.
  • test compounds were administered at the time of or up to 1 hour following the experimentally induced occlusion which caused the ischemic event.
  • the applicants show that reduction of neuronal damage can be enhanced when the N-channel blocking compound is administered between ***4-24 hours following ischemia, relative to immediate post-ischemia drug administration.
  • the improvement is based on the discovery herein that compounds effective in reducing neuronal damage associated with ischemia are characterized by (a) relatively high affinity binding to the SNX-111 synaptosomal binding site, and (b) relatively low affinity to an OCT-MVIIC (SNX-230) binding site in a synaptosomal preparation. Further, in accordance with the present invention, it has been discovered that such compounds can be administered hours after ischemic insult to a mammalian subject.
  • the method includes administering to the subject, at a time 4-24 hours following the onset of the ischemic condition, a compound which selectively binds to an OCT MVIIA binding site in neuronal tissue.
  • Selectivity of binding is evidenced by a selectivity ratio of binding of the compound for the OCT MVIIA site, as compared to the affinity of the compound in binding the OCT MVIIC site, as described herein.
  • Compounds are useful in the treatment method if they have a selective binding ratio to these two sites (MVIIA/MVIIcy of at least 100, and preferably at least 500. More generally, effective compounds exhibit a selectivity ratio of binding for the MVIIA site which is at least as great as that of an omega conotoxin selected from the group consisting of MVIIA, MVIIB, GVIA, GVIIA and RVIA omega conotoxins.
  • compounds useful in the treatment method exhibit a high affinity for the MVIIA binding site.
  • Such high affinity is defined as an affinity which is at least as great as that of an omega conotoxin selected from the group consisting of MVIIA, MVIIB, GVIA, GVIIA and RVIA omega conotoxins.
  • compounds useful in the treatment method are characterized further by their ability to selectively inhibit N-type voltage-gated calcium currents in neuronal tissue.
  • compounds useful in the treatment method are characterized by their ability to selectively inhibit N-channel mediated neurotransmitter release in neuronal tissue, as evidenced by a specific activity.
  • active compounds will exhibit activities within the range of activities of MVIIA, MVIIB, GVIA, GVIIA and RVIA omega conotoxins.
  • compounds are omega conotoxin compounds; in still another embodiment, such omega conotoxin compounds are selected from the group consisting of OCT MVIIA, OCT MVIIB, OCT GVIA, OCT GVIIA, OCT RVIA, and SNX-207.
  • the invention includes a method of screening compounds for use in reducing ischemia-related neuronal damage, such as produced by stroke, in a human subject.
  • test compounds are assayed for their binding affinities to OCT-MVIIA and OCT-MWIC binding sites in neuronal tissue, to determine a selectivity ratio of binding for the MVIIA site with respect to the MVIIC site.
  • the compound is selected if its selectivity ratio of binding for the MVIIA site is at least 100, and preferably, at least 500.
  • a compound is selected, if its selectivity ratio of binding is at least as great as that of one of the omega conotoxins MVIIA, MVIIB, GVIA, GVIIA or RVIA.
  • Test compounds can be further screened for their ability to to selectively inhibit N-type voltage- gated calcium currents in neuronal tissue, again selecting those test compounds if their specific activity, in producing such inhibition of calcium gated current, is at least as great as that of one of the omega conotoxins MVIIA, MVIIB, GVIA, GVIIA or RVIA.
  • Test compounds can be further tested for their ability to selectively inhibit N-channel mediated neurotransmitter release in neuronal tissue, again selecting those test compounds if their specific activity, in producing such inhibition of neurotransmitter release, is at least as great as that of one of the omega conotoxins MVIIA, MVIIB, GVIA, GVIIA or RVIA.
  • test compound is an an omega conotoxin.
  • These neuroprotective peptides are also useful in the method, described herein, for treatment and reduction of ischemia- related neuronal damage in a mammalian subject.
  • Figure 1 shows primary sequences of several natural omega-conopeptides, MVIIA/SNX-111 (SEQ ID NO: 01), MVIIB/SNX-159 (SEQ ID NO: 02), GVIA/SNX-124 (SEQ ID NO: 03), GVIIA/SNX-178 (SEQ ID NO: 04) , RVIA/SNX- 182 (SEQ ID NO: 05), MVIID/SNX-238 (SEQ ID NO: 24), SVIA/SNX-157 (SEQ ID NO: 06) , TVIA/SNX-185 (SEQ ID NO: 01), MVIIB/SNX-159 (SEQ ID NO: 02), GVIA/SNX-124 (SEQ ID NO: 03), GVIIA/SNX-178 (SEQ ID NO: 04) , RVIA/SNX- 182 (SEQ ID NO: 05), MVIID/SNX-238 (SEQ ID NO: 24), SVIA/SNX-157 (SEQ ID NO:
  • Figure 2 shows several analog omega-conopeptides SNX-190 (SEQ ID NO: 09) , SNX-191 (SEQ ID NO: 10) , SNX-193 (SEQ ID NO: 11) , SNX-194 (SEQ ID NO: 12) , SNX-195 (SEQ ID NO: 13), SNX-196 (SEQ ID NO: 14), SNX-197 (SEQ ID NO: 15) , SNX-198 (SEQ ID NO: 16) , SNX-200 (SEQ ID NO: 17), SNX-201 (SEQ ID NO: 18), SNX-202 (SEQ ID NO: 19) , SNX-207 (SEQ ID NO: 20) , SNX-260 (SEQ ID NO: 23) , and SNX-236 (SEQ ID NO: 25) and their relationships to SNX-111 (SEQ ID NO: 01) , SNX-185 (SEQ ID NO: 07) or SNX-183 (SEQ
  • Figure 3B plots the percent inhibition of peak inward calcium currents in neuroblastoma cells as a function of OCT MVIIA (SNX-111) (solid triangles) and OCT GVIA (SNX-124) (solid circles) ;
  • Figure 4A shows voltage-gated calcium current traces induced by a voltage step from -70 to -20 mV in human neuroblastoma cells (IMR-32) in the absence (lower trace) and presence (upper tracing) of 150 nM SNX-lll;
  • Figures 4B and 4C show plots of absolute values of peak inward current measured every 15 seconds in IMR-32 cells, elicited by pulses from -70 to 0 or -10 mV, versus time, where addition of compounds SNX-lll (4B) or SNX-lll, SNX-183 (4C) , and cadmium to the bathing medium are indicated by hatch marks just above the ordinate;
  • Figure 5A shows the inhibition of norepinephrine release from neuronal cells as a function of OCT MVIIA (SNX-lll) concentration (solid bars are potassium stimulated and open bars are basal values) ;
  • Figure 5B shows a plot of evoked norepinephrine release from neuronal tissue as a function of concentration of OCT peptides SNX-230, SNX-lll or SNX-183;
  • Figures 6A and 6B are a binding curve showing the amount of OCT MVIIA (SNX-lll) bound to rat synaptosomal membranes, as a function of OCT MVIIA (SNX-lll) concentration (6A) , and the same data plotted as a Scatchard plot (6B) ;
  • Figures 7A and 7B show plots of displacement of [ 125 I]SNX-lll (7A) and [ 125 I]SNX-230 (7B) by various OCT peptides;
  • Figures 8A and 8B show autoradiographs of binding of [ I25 I]SNX-lll (8A) and [ 15 I]SNX-183 (8B) to neuronal proteins separated by SDS polyacrylamide gel electrophoresis in the absence or presence of competing unlabeled ligand, as indicated;
  • Figures 8C and 8D show autoradiographs of binding of [ 125 I]SNX-lll (8C) and [ 125 I]SNX-230 (8D) to neuronal proteins separated by SDS polyacrylamide gel electrophoresis in the absence or presence of competing unlabeled ligand, as indicated;
  • Figures 9A and 9B show plots of displacement of binding of [ 125 I]SNX-lll (9A) and [ 125 I]SNX-183 (9B) by unlabeled OCT peptides SNX-lll and SNX-183 to 210 kilodalton proteins as described in the protein binding assay depicted in Figures 8A and 8B;
  • Figure 10A-10B are low-power micrographs of gerbil hippocampus CA1 region in animals after ischemia, and infusion of OCT MVIIA (SNX-lll) (10A) or after ischemia and infusion of drug vehicle (10B) ;
  • Figures 11A-11D are higher power micrographs of cells in the drug-treated ischemic animals (11A, 11C, 11D) , in animals receiving vehicle alone (11B) , in animals showing complete protection by OCT against ischemic cell damage (11C) ; and in animals showing partial protection by OCT against ischemic cell damage (11D) ;
  • Figures 12A-12H show autoradiographs of coronal sections of rat brain to which is bound radiolabeled SNX-lll or SNX-183, as indicated, in the absence or presence of unlabeled peptide;
  • Figures 13A-13D show autoradiographs of sagittal sections of rat brain to which is bound radiolabeled SNX-lll or SNX-183, as indicated, in the absence or presence of unlabeled peptide;
  • Figure 14 shows amino acid sequences of active (groups I and II) and inactive (group III) OCT peptides and conserved peptide sequences within group I, Region a (SEQ ID NO: 26), Region b (SEQ ID NO: 27), Region c (SEQ ID NO: 28) , Region d (SEQ ID NO: 29) , Region e (SEQ ID NO: 28) , and Region f (SEQ ID NO: 30), and within group II, Region s (SEQ ID NO: 27), Region t (SEQ ID NO: 31), Region u (SEQ ID NO: 32) and Region v (SEQ ID NO: 27);
  • Figure 15 shows a plot of hippocampal damage (CA1 region) as a function of dose of SNX-lll administered 1 hour (triangles) or 6 hours (circles) post-ischemia;
  • Figure 16 shows a plot of hippocampal damage as a function of time post-ischemia of administration of a constant dose of SNX-lll;
  • Figure 17 shows a bar graph of hippocampal damage at various doses of SNX-159 and SNX-lll.
  • Figure 18 shows a bar graph of hippocampal damage as observed 5 days and 12 days post-ischemia.
  • Omega-conotoxin (OCT) peptides are peptide toxins produced by marine snails of the genus Conus, and which act as calcium channel blockers (Gray) .
  • OCT peptides Omega-conotoxin peptides
  • OCT peptides Omega-conotoxin peptides
  • FIG. 1 Conventional letter initials are used for the amino acid residues, and X represents 4-hydroxyproline, also abbreviated 4Hyp. All of the peptides shown in the figure are amidated at their C-termini.
  • the peptides shown in Figure 1 are identified by names which are commonly associated with either the naturally occurring peptide (single letter followed by a Roman numeral followed by a single letter) , and by a synthetic designation (SNX-plus numeral) . Either or both of these designations will be used interchangeably throughout the specification.
  • the peptide whose sequence is designated MVIIA/SNX-lll will be referred to herein as OCT MVIIA, or alternatively, SNX-lll, the latter to signify that the compound is synthetic in origin.
  • Synthetic and naturally occurring peptides having the same sequence behave substantially identically in the assays and methods of treatment of the invention.
  • OCT MVIIA SNX-lll
  • OCT GVIA SNX-124
  • All of the OCT peptides have three disulfide linkages connecting cysteine residues 1 and 4, 2 and 5, and 3 and 6, as indicated for the MVIIA peptide in Figure 2.
  • Figure 2 shows analogs of natural OCT MVIIA, OCT TVIA, and OCT SVIB peptides which have been synthesized and tested in accordance with the invention.
  • This section describes the synthesis, by solid phase methods, of several naturally occurring omega conotoxin (OCT) peptides and additional OCT peptides which are used in the present invention.
  • OCT omega conotoxin
  • OCT peptides such as those shown in Figures 1 and 2
  • OCT peptides can be synthesized by conventional solid phase methods, such as that detailed in U.S. Patent No. 5,051,403, and PCT patent application WO 91/079, both of which are incorporated herein by reference.
  • Example 1 for the synthesis of exemplary OCT MVIIC/SNX-230.
  • OCT Peptides This section describes in vitro properties of OCT peptides. Generally, these properties are described for neuroprotective OCT peptides. For purposes of comparison, properties of OCT MVIIC/SNX- 230, a peptide lacking neuroprotective activity, are also described and distinguished in this section.
  • neuroprotective compounds of the invention are neuronal-cell calcium channel antagonists, as defined by their ability to inhibit voltage-gated ionic currents in neuronal cells.
  • Voltage-gated calcium channels are present in neurons, and in cardiac, smooth, and skeletal muscle and other excitable cells, and are known to play a variety of roles in membrane excitability, muscle contraction, and cellular secretion, such as in synaptic transmission (McCleskey) .
  • voltage-gated calcium channels In neuronal cells, voltage-gated calcium channels have been classified by their electrophysiological as well as by their biochemical (binding) properties. Electrophysiologically, these channels can be classified either as Low-voltage-activated (LVA) or High-voltage-activated (HVA) .
  • HVA channels are currently known to comprise at least three groups of channels, known as L-, N- and P-type channels (Nowycky, Sher) .
  • dihydropyridines, diphenylalkyla ines and piperidines bind to the alp a j subunit of the L-type calcium channel and block a proportion of HVA calcium currents in neuronal tissue, which are termed L-type calcium currents.
  • Omega conotoxins also block a proportion of HVA calcium currents in neuronal tissue, and, in the presence of a maximally inhibitory quantity of dihydropyridine compound, effect substantially complete inhibition the remaining HVA currents in neuronal cells.
  • These calcium currents are identified as N-type calcium currents, though recently a proposal that such currents be termed "omega" has been presented (Sher) .
  • Omega conotoxins bind to a specific population of binding sites.
  • Dihydropyridines and other L-type channel blockers do not displace omega conotoxin binding, nor do omega conotoxins displace binding of ligands to L-channels.
  • omega channels are found predominantly, although not exclusively, in nervous tissue (Sher) .
  • One suitable system for testing inhibition (blockage) of N-type or omega HVA neuronal calcium channels is an isolated cell system, such as the mouse neuroblastoma cell line, strain N1E115 or the human neuroblastoma cell line IMR32.
  • Membrane currents are conveniently measured with the whole cell configuration of the patch clamp method, according to the procedure detailed in Example 2. Briefly, a voltage clamp protocol was performed in which the cell potential was stepped from the holding potential of about -100 mV to test potentials that ranged from -60 mV -to +20 mV, and the cell was held at the holding potential for 5 seconds between pulses.
  • Figure 3A shows a typical inward calcium current in an N1E115 neuroblastoma cell elicited by a voltage step from -80 mV to -20 mV in the absence of OCT, as detailed in Example 2.
  • barium (Ba) replaced calcium (Ca) as the charge-carrier through the calcium channels in order to increase the signal (McCleskey) .
  • curve a the calcium current activates quickly (within about 20 ms) and inactivates with a time constant of 30 to 40 ms.
  • the calcium current is measured by the amplitude of the peak inward current elicited by the depolarization peak, and has a measured value of about -1200 pA.
  • the cell in Figure 3A (curve a) was also exposed to lM nifedipine, a dihydropyridine, which is expected to effectively block L-type calcium channels in the neuroblastoma cells, and no effect on the measured calcium current was observed. The calcium current observed is thus not dihydropyridine-sensitive.
  • the responses of voltage-gated calcium current to increasing concentrations of OCTs MVIIA (SNX-lll) and GVIA (SNX-124) are shown in Figure 3B.
  • the ED 50 concentration, at which 50% inhibition of calcium current is produced, is determined from the voltage- gated current amplitudes, plotted as a function of OCT peptide concentration.
  • the calculated ED 50 is about 10 nM for GVIA and 100 nM for MVIIA, indicative of high inhibitory peptide activity.
  • the ED 50 concentration for these and OCT peptides SVIA (SNX- 157) and SVIB (SNX-183) are given in Table l below.
  • the two compounds with relatively low IC 50 values (below 1 ⁇ K) are both active as neuroprotective agents, as will be'seen in Section III below, whereas the OCT SVIA and SVIB peptides with IC 50 values above this threshold are not.
  • the compounds of the invention are classified as antagonists of voltage-gated calcium channels by their ability to inhibit voltage-gated calcium channel currents characterized as above with an ED J0 value of less than about 1 ⁇ l ⁇ in the assay detailed in Example 2.
  • Figures 4B and 4C show cumulative data from many consecutive currents, elicited at 15 second intervals as described above, in IMR-32 cells. In these plots, peak inward current recorded from each stimulus is recorded sequentially as a single data point.
  • Figure 4B addition of SNX- lll to the bathing medium resulted in decreased peak inward currents; restoration of substantially normal calcium currents was achieved after washing of the compound from the cell chamber, shown on the right side of the figure.
  • Figure 4C shows the effects of 150 nM SNX-lll and SNX-183 added sequentially to a single cell preparation. Both compounds resulted in attenuation of peak inward current; though recovery following SNX-183 exposure was not observed. Addition of cadmium (Cd) to the medium resulted in blockade of all remaining voltage-gated calcium currents in this preparation.
  • Cd cadmium
  • Test peptides which are inhibitory for neuronal cell calcium currents can be further tested in non- neuronal cells, to confirm that the peptide activity in blocking calcium currents is specific to neuronal cells.
  • a variety of muscle cell types which are refractory to calcium-current inhibition by OCTs such as vertebrate embryo heart and skeletal muscle cells, are suitable. Cell current measurements are made substantially as outlined above and detailed in Example 2.
  • OCT MVIIA for example, has been reported to block voltage-gated calcium channels in a variety of neuronal cells, including dorsal root ganglion (DRG) neurons (McCleskey) . This blockage or inhibition of calcium channel currents has been reported to be neuron-specific, since calcium current inhibition by the peptide was not observed in cardiac, smooth, and skeletal muscles.
  • DRG dorsal root ganglion
  • OCT MVIIC a non-neuroprotective OCT peptide also inhibits certain calcium currents.
  • these currents like the OCT peptides described above, OCT MVIIC (SNX-230) has no effect on dehydropyridine-sensitive currents.
  • This compound exhibits a distinctive electrophysiological profile which includes blockade of a significant fraction of dehydropyridine-resistant, OCT GVIA-resistant (non-L, non-N type) current in hippocampal CA1 neuronal cells, as well as inhibition of high threshold calcium channel current in cerebella Purkinje neurons; as described in co-owned U.S. Patent Application 916,478 and subsequently published (Hillyard, et al . ) .
  • OCT MVIIC additionally blocks calcium uptake in rat brain synaptosomes at low (2.5 ⁇ M) concentration. Unlike MVIIA, it also shows high potency in inhibition of phrenic nerve-mediated muscle concentrations in an isolated mouse diaphragm preparation.
  • a second requisite property of neuroprotective compounds is the ability to specifically inhibit depolarization-evoked and calcium-dependent norepinephrine release in brain (CNS) neuronal cells, but not inhibit neurotransmitter release at a mammalian neuromuscular junction of a skeletal muscle.
  • Inhibition of norepinephrine release in neuronal cells can be assayed in mammalian brain hippocampal slices by standard methods, such as detailed in Example 3.
  • Figure 5A shows effects of increasing concentrations of OCT MVIIA peptide on norepinephrine release from rat brain hippocampal slices which were first bathed in normal wash solution (open bars) , then stimulation medium (solid bars) .
  • the compound produces a strong concentration-dependent inhibition of norepinephrine release in the presence, but not in the absence of stimulation medium. From the concentration-dependent inhibition data, the compound concentration effective to produce 50% inhibition of norepinephrine release (IC 50 ) is calculated.
  • Figure 5B shows a comparison of the effects of the three OCT peptides, MVIIA/SNX-lll, SVIB/SNX-183, and MVIIC/SNX-230, on the release of norepinephrine evoked by potassium depolarization in vitro, as detailed in Example 3.
  • the IC 50 values given in Table 2 for a variety of OCT peptides which have been examined by this method represent the average IC 50 values calculated from thin (200 ⁇ ) and thick (400 ⁇ ) hippocampal slices.
  • the OCT peptides SNX-195 and SNX-201 are OCT MVIIA with amino acid substitutions or modifications at key residue sites ( Figure 2) , as will be discussed in Section IV below.
  • the higher IC 50 values measured for these modified peptides is reflected in substantial reduction or loss of neuroprotective activity.
  • SVIA (SNX-157) and SVIB (SNX-183) are representative of OCT compounds which show no neuroprotective activity, and this is reflected by high IC 50 values for norepinephrine release.
  • the SNX-202 peptide is a modification of SVIB peptide in which the Ser-Arg-Leu-Met residues at positions 9-12 in OCT MVIIA (SNX-lll) are substituted for the Arg- Lys-Thr-Ser residues at the same positions in OCT SVIB (SNX-183) .
  • pronounced neuroprotective activity is associated with an ability to inhibit norepinephrine release with an IC 50 value which is within the range of IC 50 values measured for active OCT peptides MVIIA (SNX-lll) , GVIA (SNX-124) , and TVIA (SNX-185) ; i.e., less than the largest of the IC 50 values measured for these active OCT peptides.
  • IC 50 value which is within the range of IC 50 values measured for active OCT peptides MVIIA (SNX-lll) , GVIA (SNX-124) , and TVIA (SNX-185) ; i.e., less than the largest of the IC 50 values measured for these active OCT peptides.
  • inhibition of NE release is not sufficient in itself to predict neuroprotective activity.
  • Another property of neuroprotective compounds, in accordance with the invention, is high-affinity binding to an OCT MVIIA (SNX-lll) binding site in neuronal cells.
  • the binding affinity can be characterized either by the binding constant of the compound for the MVIIA (SNX-lll) binding site, or by the ratio of binding constants measured for binding to neuronal-cell MVIIA-selective binding site (designated site 1) and SVIB/MVIIC- selective binding site (designated site 2) .
  • MVIIA binding site in neuronal tissue can be demonstrated with a variety of cell types and synaptosomal cell fractions.
  • One preferred neuronal membrane is a mammalian brain synaptosomal preparation, such as the rat brain synaptosome preparation described in Example 4.
  • the binding constant of a compound for the MVIIA binding site is typically determined by competitive displacement of radiolabeled OCT MVIIA (SNX-lll) from the synaptosomal preparation, as follows.
  • the binding constant K d of the MVIIA (SNX-lll) peptide for the synaptosomal membranes is determined by a saturation binding method detailed in Example 5A.
  • the plot of bound peptide as a function of concentration is then used to calculate a B ⁇ , the concentration of binding sites on the synaptosomes, and K d following standard methods.
  • the K d value is the calculated concentration of peptide needed to half saturate the synaptosomal specific binding sites.
  • Figure 6A shows the specific binding of radiolabeled OCT MVIIA (SNX-lll) to rat brain synaptosomes, plotted as a function of OCT peptide concentration
  • Figure 6B the same data in Scatchard plot form. From the slope of the
  • test compound for the MVIIA binding site, the test compound is added, at increasing concentrations to the synaptosome preparation having bound, radiolabeled OCT MVIIA.
  • the synaptosomal material is then rapidly filtered, washed and assayed for bound radiolabel, as detailed in Example 5B.
  • the binding constant (K d ) of the test compound is determined from computer-fit competitive binding curves, such as shown in Figure 7A for MVIIA (SNX-lll) peptide, to determine first the IC 50 value of the compound, i.e., the concentration which gives 50% displacement of labeled MVIIA peptide, then calculating K ( from the K d value of OCT MVIIA and the IC 50 value of the compound, as detailed in Example 5.
  • IC 50 values for a number of OCT peptides for inhibition of OCT MVIIA binding are shown in Table 3. The compounds are arranged in order of increasing IC 50 values.
  • Compounds with known neuroprotective activity such as SNX-207, OCT MVIIA (SNX-lll), GVIA (SNX-124) , and TVIA (SNX-185) , have IC 50 values between about 15 and 300 pM, in the assay shown, and corresponding K ; values between about 1 and lOOpM.
  • OCT peptides such as OCT SVIA (SNX-157) and SVIB (SNX- 183) , which are not neuroprotective have substantially greater IC 50 and K ; values for displacement of OCT MVIIA binding.
  • OCT peptide compounds which were tested gave IC 50 and K ; values lower than or within the ranges of those of OCT peptides MVIIA (SNX-lll) , GVIA (SNX-124), and/or TVIA (SNX-185) , and these compounds should thus be considered candidates as neuroprotective compounds.
  • some of these compounds such as SNX-201, SNX-195, and SNX-202 have IC 50 values for inhibition of norepinephrine release which are outside the range of neuroprotective com- pounds (Table 2) , and thus these compounds do not meet all of the criteria for neuroprotective compounds.
  • IC 50 and K values for compound binding to this site are calculated, as above, by determining the K d of radioiodinated OCT SVIB (SNX-183) or of radiolabeled OCT MVIIC (SNX-230) for binding to a synaptosome preparation, then using competitive displacement of the labeled compound by the test compound, to determine the IC J0 and K ; values of the test compound.
  • SVIB/SNX-183 this one from C. striatus , displayed substantially lower affinity to both sites with a slight preference for site 1 (Table 4) .
  • neuroprotective compounds OCT MVIIA (SNX-lll) , GVIA (SNX-124) , SNX207 and TVIA (SNX-185) exhibited highest relative displacement potencies at the MVIIA site.
  • Table 4 also shows ratios of relative potencies of binding for each compound at the MVIIA (site 1) and MVIIC (site 2) binding sites. These ratios accentuate the difference in binding properties between neuroprotective compounds, and those which show no neuroprotective activity within the range of concentrations tested.
  • neuroprotective compounds in accordance with the invention are characterized by a high binding affinity for the MVIIA binding site, OCT site 1, on neuronal membranes.
  • the binding affinity for this site may be characterized in one of two ways. In the first approach, the binding affinity of the compound for the MVIIA site, as estimated by IC 50 at the site, is compared directly with those of SNX-lll, SNX-207, SNX-124, or SNX-185.
  • a neuroprotective compound is one whose binding affinity is at least as high as and preferably within the range of binding affinities measured for the OCT's MVIIA (SNX-lll) , GVIA (SNX- 124), and TVIA (SNX-185) , i.e., the binding constant is no greater than the highest binding constant among these four OCT peptides.
  • the binding affinity for the MVIIA site can be characterized by the ratio of binding constants or relative potencies for the MVIIA-selective and SVIB/MVIIC-selective (site 2) sites, as just described.
  • a neuroprotective compound is one whose binding ratio is within the range of such binding ratios measured for the OCT's SNX-207, MVIIA (SNX-lll), GVIA (SNX-124) , and TVIA (SNX-185) , i.e., the binding ratio is no lower than the smallest ratio among these four OCT peptides.
  • the hybrid peptide SNX-201 is identical to SNX-lll except for residues 9-12 (-S-R-L-M-) which have been replaced by the corresponding residues (-R-K-T-S-) from SNX-183.
  • SNX-202 contains residues 9-12 from SNX- 111 in place of the corresponding residues in the sequence of SNX-183.
  • Conopeptide receptor polypeptides in rat brain or hippocampal synaptic membranes were chemically cross-linked to radio-iodinated SNX-lll, SNX-183, or SNX-230 with a water-soluble carbodiimide, as detailed in Example 6.
  • the radiolabeled peptides were separated by SDS-PAGE and visualized by autoradiography.
  • [ 125 I]-SNX-lll, [ 125 I]-SNX-183, and [ 125 I]-SNX-230 were chemically crosslinked to synaptosomal membrane preparations and then subjected to SDS-PAGE followed by autoradiography.
  • [ I25 I]-SNX-lll essentially only one polypeptide band of M r 210-220 kDa was specifically labeled.
  • SNX-lll inhibited the incorporation of radioactive SNX-lll into this band with an IC J0 of 0.03 nM, in good agreement with the IC 50 for site 1 determined by binding assays (Fig. 8A) .
  • SNX-230 binds specifically to the 210-220 band plus three additional protein bands which migrate as 170, 150 and 140 kDa ( Figure 8C and Figure 8D) .
  • [ 125 I]SNX-230 was incorporated at a concentration (0.1 nM) at which site 2, but not site 1 should be labeled, according to the equilibrium binding data shown above.
  • Analysis of the inhibition of incorporation of [ 125 I ⁇ -SNX-183 into the 210-220 kDa band by SNX-lll provides good evidence for the presence of two distinct polypeptides of approximate M r 210 kDa corresponding to site 1 and site 2 (Fig. 9B) .
  • SNX-lll displaces [ 125 I]-SNX-183 from the 210 kDa polypeptide in a biphasic manner with IC 50 values of 0.006 nM and 65 nM.
  • SNX-lll effectively competes for binding to site 1; the binding of [ I25 I]-SNX-183 to site 2 is competed out only at much higher concentrations of SNX-lll because of the poor affinity of SNX-lll for site 2.
  • [ 125 I]-SNX-lll and [ 125 I]-SNX-183 revealed overlapping but differential distributions of binding sites. Both ligands labeled the cortex, CA1, dentate gyrus, and caudate-putamen. In these regions, binding of [ 125 I]-SNX-183 was unaffected by concentrations of SNX-lll that completely displace [ 125 I]-SNX-lll labeling, suggesting both a co-localization of sites 1 and 2 and a preponderance of [ 125 I]-SNX-183 labelled site 2 receptors in these regions.
  • Site 1 and site 2 binding was also characterized by comparison of binding of [ 125 I]-SNX-lll and [ 125 I]- SNX-230 in sagittal sections of rat brain as shown in Figure 13(A-D) .
  • [ I25 I]-SNX-lll ( Figure 13A) and [ 125 I]-SNX-230 ( Figure 13B) bind differentially to various brain structures.
  • Figures 13C and 13D show non-specific binding of [ I25 I]-SNX-lll and [ 125 I]-SNX- 230, respectively, carried out in the presence of 25 mM uniodinated peptide.
  • the present invention provides a method and composition of the invention effective to reduce neuronal damage related to an ischemic condition in a human patient.
  • the ischemic conditions may be due to an interruption in cerebral circulation, such as caused by cardiac failure, or other condition leading to global loss of blood supply to the brain, or to localized interruptions in blood flow, such as due to cerebral hemorrhage, or localized thrombotic or embolic events, or head trauma.
  • the ischemic condition which is to be treated using the method and composition is generally associ ⁇ ated with stroke, defined as the sudden diminution or loss of neurological function caused by an obstruction or rupture of blood vessels in the brain, or by complete cessation of blood flow to brain, as in cardiac failure.
  • the treatment method is aimed at preventing or reducing secondary brain damage resulting from the original ischemic event.
  • the secondary damage typically includes cerebral cell destruction, or lesions, in the area surrounding the ischemic injury, in the case of focal ischemia, and also in areas of selective vulner- ability, such as the hippocampus or basal ganglia, in the case of global ischemia.
  • the secondary damage may often be manifested by functional impairment, such as loss of short-term or long-term memory.
  • the treatment method of the invention is effective in reducing or preventing both anatomical and functional secondary damage related to ischemia.
  • compositions include a neuronal- cell calcium channel antagonist compound having activities for selectively blocking norepinephrine release in mammalian neuronal cells, and for binding to neuronal-membrane omega-conotoxin MVIIA binding site (OCT site 1) , which are within the ranges of such activities for OCT peptides SNX-207, MVIIA (SNX- 111) , GVIA (SNX-124) , or TVIA (SNX-185) .
  • OCT site 1 neuronal- cell calcium channel antagonist compound having activities for selectively blocking norepinephrine release in mammalian neuronal cells, and for binding to neuronal-membrane omega-conotoxin MVIIA binding site (OCT site 1) , which are within the ranges of such activities for OCT peptides SNX-207, MVIIA (SNX- 111) , GVIA (SNX-124) , or TVIA (SNX-185) .
  • the binding activities may be expressed either as binding constants for the MVIIA site on neuronal membranes, or as a ratio of the binding constants for the MVIIA and MVIIC/SVIB binding sites (sites 1 and 2, respectively) , as discussed in Section II above.
  • the compound is carried in a suitable pharmaceutical carrier, such as a sterile injectable solution.
  • One exemplary class of neuronal cell calcium channel antagonists is that class of OCT peptides having the requisite inhibitory and binding activities.
  • the peptide is formulated for parenteral administration in a suitable inert carrier, such as a sterile physiological saline solution.
  • the concentration of peptide in the carrier solution is typically between about 0.1-20 mg/ l.
  • the dose administered will be determined by route of administration.
  • One suitable route is intracerebroventricular (ICV) , at a dose level of about 0.1 to 20 ⁇ g peptide/kg body weight, depending on the binding and inhibitory values of the peptide.
  • the peptide compound may alternatively be administered intravenously (IV) as a bolus or as a continuous infusion as demonstrated below. It may be desirable for IV administration to pretreat the subject with antihistamines specific for HI and H2 histamine receptors, to reduce possible blood pressure lowering after peptide administration.
  • the delayed-administration protective event indicates that these compounds are effective in blocking the events leading from ischemic injury to secondary cerebral injury, since these events may occur over a period of many hours or even days after injury.
  • the delayed administration may be effective to reduce secondary cerebral damage over a several hour period, or even a day or more, following the onset of ischemia.
  • the effectiveness of the composition in reducing neuronal damage related to ischemic injury has been examined in three animal systems which are widely employed as model systems for global ischemia and secondary stroke damage.
  • the first system is the gerbil two vessel occlusion model of global ischemia produced by transient occlusion of carotid arteries of the neck.
  • the ischemia produced in this model has been likened to that produced by cardiac arrest, since all blood flow to the brain is stopped for a fixed period, typically 5- 10 minutes.
  • gerbils exhibit the same kind of selective regional damage from ischemia as is found in other mammals, including humans.
  • the characteristic secondary damage observed in the hippocampal CA1 region is similar to that seen in other mammals, including humans (Kirino; Yamaguchi) .
  • the second animal model utilized in experiments carried out in support of the present invention is the rat four-vessel occlusion model.
  • the experimental procedure for producing temporary occlusion produces an ischemia that mimics conditions in the human brain following cardiac arrest, including the following similarities: the ischemic event is temporary, typically 5-30 minutes; it occurs in an unanesthetized state; in most rats, the ischemic event is not accompanied by generalized seizures, and animals that have seizures can be excluded from the study.
  • the occlusion procedure allows the animals to be easily monitored, maintained and analyzed (Pulsinelli) .
  • the third animal model is the rat cerebral artery occlusion model of focal ischemia.
  • the left middle cerebral artery is permanently occluded by electrocoagulation.
  • Twenty-four hours after the occlusion the animals are anesthetized and areas of damage are examined by magnetic resonance imaging.
  • Neuroprotective activity of OCT peptide MVIIA was demonstrated in this model of ischemia by the applicants, published in PCT application WO 91/079.
  • Figures 10A and 10B are low-power micrographs of gerbil hippocampus CA1 region in animals after ischemia, and infusion of MVIIA OCT (SNX-lll) (10A) or drug vehicle (10B) .
  • the arrows in the figures indicate the approximate borders of the CA1 region.
  • cells in the drug-treated ischemic animals appear normal ( Figure 11A)
  • damage is apparent in the ischemic animals receiving vehicle alone ( Figure 11B) .
  • Figure lie Another example of complete drug protection is seen in Figure lie, and an example of partial protection is seen in Figure 11D, where there is a small number of damaged cells.
  • the extent of protection from ischemic damage in animals treated with neuroprotecting OCT peptides MVIIA and OCT GIVA was reported in PCT publication WO 91/079 for peptides administered prior to or 1 hour subsequent to ischemia. Ischemia in the rat model system was induced using the rat 4-VO method described in Example 9.
  • Table 5 shows protection by OCT peptides MVIIA (SNX-lll) and SNX-207 against ischemic damage to the hippocampus. Both compounds showed significant neuroprotection at the doses indicated, when the compounds were administered intravenously 1 hour post-ischemia (Table 5) .
  • OCT MVIIA SNX-lll
  • OCT TVIA SNX-185
  • SNX-195 while not significantly different from control, did show a trend toward neuroprotection.
  • OCT SVIB SNX-183
  • SNX-202 SNX-202
  • SNX-201 SNX-201
  • OCT SVIA SNX-157
  • Test compound was administered intravenously 6, 12, or 24 hours post- occlusion to a ratsubjected to 4-VO as detailed in Example 9B.
  • Results of a study in which saline, 1, or 5 mg/kg of OCT MVIIA was given as a bolus intravenously 6 hours post-occlusion are shown in Table 7.
  • Table 7 results of a study in which saline, 1, or 5 mg/kg of OCT MVIIA was given as a bolus intravenously 6 hours post-occlusion.
  • Table 7 In contrast to administration 1 hour post- occlusion, when compound was given 6 hours post- occlusion, a significant reduction in neuronal damage was observed at the 5 mg/kg dose. Significant reductions in neuronal damage were also observed when the same total dose drug was administered intravenously as a slow infusion over a time period of up to 25 hours.
  • FIG. 15 shows a comparison of dose-response data obtained from rats subjected to 15 minutes of reversible forebrain ischemia in the four- vessel occlusion paradigm, then given varying doses of SNX-lll 1 hour (triangles) or 6 hours (circles) after reperfusion. ' In the plot shown, damage scores from 7-21 animals are expressed as percentages of saline control values.
  • the monophasic dose-response curves yield ED 50 values of 2.3 mg/kg for the animals given SNX-lll 6 hours post-occlusion and 12.5 mg/kg for animals given SNX-lll 1 hour post occlusion. This represents about a five-fold increase in potency in the compound when administered 6 hours post- occlusion.
  • Shown in the bar graph is a comparison of the effects of 3 and 10 mg/kg SNX-159 and 3.5 mg/kg SNX-lll to saline treated animals.
  • time at which neuronal damage is observed after temporary forebrain ischemia varies among neuronal populations, maximal damage is typically manifested within 72 hours after reperfusion (Pulsinelli, Kirino) .
  • Pulsinelli, Kirino reperfusion
  • hippocampal damage was compared in SNX-lll treated 'animals after 5 or 12 days of survival. Damage scores of SNX-lll treated animals were comparably reduced at both time points (Fig.
  • Group I includes active OCT peptides MVIIA (SNX-lll) and MVIIB (SNX-159) which possesses a binding constant to the MVIIA site within the range of compounds with neuroprotective activity.
  • Group II includes neuroprotective peptides GVIA (SNX-124), TVIA (SNX-185) and SNX-207.
  • a third group includes inactive peptides SVIA (SNX-157) , SVIB (SNX-183), SNX-230 and OCT peptides whose binding activities for the MVIIA site on neuronal membranes and/or activity in norepinephrine inhibition are outside the range of active compounds.
  • the peptides in both active groups include the Cys residues at position 1, 8, 15, 16, 20, and 28. Other Cys residues could be substituted at the positions indicated below only if they are selectively protected during oxidation of the peptide to form the three disulfide linkages.
  • the peptides in the active groups include three disulfide linkages connecting the Cys residues at positions 1 and 16, 8 and 20, and 15 and 28. According to the synthetic method described in PCT publication WO 91/079, the disulfide bridges are formed by air oxidation of the full sequence peptide in the presence of DTT.
  • the ability of the peptide to form the three desired disulfide linkages would therefore require that the peptide, prior to disulfide bridging, be able to adopt a conformation which allows the three selected linkages, with or without the Cys protecting-group strategy discussed above. This constraint would thus exclude amino acid variations which prevent or otherwise hinder the formation of the three selected bridges.
  • Constraints 1 and 2 preserve the basic conformation of the OCT peptides imposed by the three disulfide bridges.
  • the position 2 amino acid may be lysine or leucine
  • the position-3 amino acid may be glycine or serine
  • the position 4 amino acid, hydroxyproline or arginine if the two or more amino acids at a variant position are in a common substitution class, substitution within that class may be favorable.
  • Standard substitution classes are the six classes based on common side chain properties and highest frequency of substitution in homologous proteins in nature,- as determined, for example, by a standard Dayhoff frequency exchange matrix (Dayhoff) . These classes are Class I: Cys; Class II: Ser, Thr,
  • Class III Asn, Asp, Glu, and Gin, representing neutral and negatively charged side chains capable of forming hydrogen bonds
  • Class IV His, Arg, and Lys, representing basic polar side chains
  • Class V lie, Val, and Leu, representing branched aliphatic side chains, and Met
  • Class VI Phe, Tyr, and Trp, representing aromatic side chains.
  • each group may include related amino acid analogs, such as ornithine, homoarginine, N-methyl lysine, dimethyl lysine, or tri ethyl-lysine in class IV, and cyclohexylalanine or a halogenated tyrosine in Group VI.
  • the classes may include both L and D stereoisomers, although L-amino acids are preferred for substitutions.
  • the peptide is further screened for the requisite calcium channel antagonist activity, and the requisite activities for inhibition of norepinephrine release and binding to the MVIIA (SNX- lll) binding site of neuronal membranes, as described above.
  • the SNX-195 compound contains a Lys to Ala substitution at the position corresponding to position 26 in the MVIIA structure shown in Figure 14. Since this substitution is at a conserved-sequence position, it is predicted that the neuroprotective activity would be lost or reduced. As discussed above, the SNX-195 peptide shows retention of MVIIA binding activity, but reduced norepinephrine release inhibitory activity, and weak neuroprotective activity compared with the unsubstituted MVIIA OCT.
  • the SNX-201 compound contains substitutions at positions 9-12 from Ser- Arg-Leu-Met to Arg-Lys-Thr-Ser, the sequence at positions 9-12 in the inactive SVIB OCT peptide.
  • the position-9 substitution is not favored since Arg is present at this position in a non-neuroprotective compound, but not in one of the neuroprotective OCT peptides.
  • the position-10 substitution is disfavored for the same reason.
  • the position-11 substitution is favored, however, since the Leu to Thr substitution occurs within the neuroprotective peptides.
  • the Met to Ser substitution at position 12 is favored for the same reason. Since the peptide modification contains two disfavored substitutions, it is predicted that the neuroprotective activity would be lost or reduced.
  • the SNX-201 peptide shows retention of MVIIA binding activity (Table 3) , but reduced norepinephrine inhibitory activity (Table 2) , and no neuroprotective activity at a concentration at which the unsubstituted MVIIA OCT/SNX-lll was found to be active ( Figure 12) .
  • the invention further includes the active OCT peptides formed according to amino acid selection rules 3 and 4 above, excluding the natural C-terminal amidated OCT peptides MVIIA (SNX-lll) , MVIIB (SNX- 159), GVIA (SNX-124) , and TVIA (SNX-185) . More specifically, with reference to conserved regions a-f and s-v illustrated in Figure 14, the peptide compounds of the invention have the form: Region a
  • peptides are intended for formulation with a suitable pharmaceutical carrier, in the composition of the invention.
  • compounds are tested for their binding affinities to OCT-MVIIA and OCT-MVIIC binding sites, as described above, by their ability to displace OCT MVIIA and OCT MVIIC, respectively, from synaptosomal preparations. Binding affinities for the two sites are determined, as described in Example 5. The binding affinities are compared to produce a selectivity ratio of binding to the MVIIA binding site using the formula:
  • the test compound is selected as a candidate for a neuro ⁇ protective agent if (i) the compound exhibits relatively high affinity binding to the MVIIA binding site (site 1) , and (ii) the selectivity ratio of binding for the MVIIA site is at least as great as such ratios of the omega conopeptides MVIIA, MVIIB, GVIA, GVIIA and RVIA.
  • relatively high affinity to the MVIIA site is approximated by the range of binding affinities of OCT peptides MVIIA, MVIIB, GVIA, GVIIA and RVIA.
  • Test compounds selected by the binding assay may be further screened for their ability to selectively inhibit N-type voltage-gated calcium currents in neuronal tissue, as detailed in co-owned U.S. Patent No. 5,051,403, incorporated herein by reference.
  • Candidate compounds are further selected if their specific activity, expressed, for example, as IC 50 , in producing such inhibition of N-type voltage-gated calcium currents, is within the range of specific activities of an omega conotoxin MVIIA, MVIIB, GVIA, GVIIA and RVIA.
  • Test compounds which are selected on the basis of the above two screens may be further selected for their ability to selectively inhibit N-channel mediated neurotransmitter release in neuronal tissue, as exemplified by evoked release of norepinephrine from central nervous tissue described above and in Example 3.
  • Candidate compounds are further selected if their specific activity, in producing such selective inhibition of N-channel mediated neurotransmitter release is within the range of spe ⁇ cific activities of specific activities of an omega conotoxin MVIIA, MVIIB, GVIA, GVIIA and RVIA.
  • compositions containing compounds of the invention may be administered in any expedient formulation and route which results in delivery to the site of action, which is likely to be at or in close proximity to the ischemic region.
  • routes of administration are intracerebral and intravenous
  • OCT MVIIC Synthesis of ⁇ -Conopeptide OCT MVIIC OCT MVIIC was synthesized on a replumbed ABI model 430A peptide synthesizer, using standard t- butyloxycarbonyl (tBOC) chemistry, as described below.
  • the synthesis was started from 0.4 mmole methyl-benzhydralamine (MBHA) resin (0.61-0.66 equivalent NH 2 /g; Advanced Chemical Technology) , single coupling the first nine amino acids (except Arg and Asn, which are always double coupled as active esters formed from 1-hydroxybenzotriazole (HOBt) with dicyclohyxyl carbodiimide (DCC) ) , and double coupling the remainder (the first couplings were in dichloromethane (DCM) , the second couplings in dimethylformamide (DMFA) solution) .
  • MBHA methyl-benzhydralamine
  • Amino acid side chain protections were Arg(Tosyl) , Asp(OBzl) , Cys(4-MeBzl) , Hyp(Bzl), Lys(chloro-benzyloxycar- bonyl) , Ser(Bzl), Thr(Bzl) , Tyr(BrZ) .
  • the coupling steps were monitored with the ninhydrin test and were repeated to achieve 99.5%+ amino acid incorporation yield in each cycle.
  • the peptide was cleaved from the resin (1 g) in liquid HF (15 ml, -10°C- 0°C) containing 10% p- Cresol. The cleavage time varied between 80-95 minutes.
  • the HF was removed with a strong stream of nitrogen, the oily residue was washed with cold AcOEt (3x20 ml, 30°C) , filtered, and the peptide was extracted by washing the residue with 1x15 ml water, 3x15 ml 50% AcOH, 1x15 ml water.
  • the combined aqueous extracts were lyophilized.
  • the solution was acidified to pH -3.5 with acetic acid, concentration under vacuum to -15-20 ml, and gel- filtered on a Sephadex G-25 column (2.5 x 60 cm) eluting with 0.5 M AcOH.
  • the pooled prepurified peptide fractions were further purified on a preparative HPLC column (Rainin Dynamax system, 4.14 x 30 cm, C-18 reversed phase packing material, 300 A pore size, 12 ⁇ m particle size) using 0.1% TFA in water/0.1% TFA in acetonitrile gradient elution solvent system (40 ml/min pumping rate) .
  • the pure fractions were pooled, and lyophilized.
  • the yield of purified peptide was usually 10-16% based on the loading capacity of the MBHA-resin. Synthesis of other OCT peptides has been described in U.S. Patent No. 5,051,403, incorporated herein by reference.
  • Ionic currents through calcium channels were examined in cells that were voltage-clamped by a single patch-clamp electrode. These whole-cell patch-clamp studies were performed mainly on N1E115 mouse neuroblastoma cells, although a variety of cell types, including human neuroblastoma cell line IMR- 32, have been examined.
  • Normal bath saline was (mM) : 140 NaCl, 10 glucose, 3 KC1, 2 CaCl 2 , 1 MgCl 2 , lOmM HEPES pH 7.3.
  • Intracellular solutions contained 150 mM CsCl, 0.5 mM CaCl 2 , 5 mM EGTA, 5 mM MgCl 2 , 2 mM KA P at pH 7.3-7.4. Bath saline and all internal solutions were filtered before use.
  • Pipets were made from Corning 7052 glass (Garner Glass Company, Clare ont, CA 91711) , coated with Sylgard (Dow Corning, Midland, MI 48640) and fire-polished before use. Bubble numbers were typically 5 to 6, with pipet resistances typically 2-5 MOh s. Corning 8161, Kimble, and other glasses were also used without noticeable effect on the calcium currents observed.
  • the typical experiment was conducted as follows: after seal formation followed by series resistance compensation and capacitative transient cancellation, a voltage clamp protocol was performed wherein the cell potential was stepped from the holding potential (typically -100 mV) to test potentials that ranged from -60 mV to +20 mV in 10 mV increments. The cell was held at the holding potential for 5 seconds between pulses. Protocols starting from other hold- ing potentials usually covered the same range of test potentials.
  • Figure 3A shows calcium current traces from an N1E-115 mouse neuroblastoma cell. The figure is read from left to right in time, with downward deflections of the trace indicating positive current flowing into the cell. Currents were elicited by a voltage step from 100 mV to -10 mV. The cell was bathed in saline with sodium replaced by NMDG and 10 mM Ba ++ instead of 2 mM Ca ++ . Potassium currents were blocked by TEA in the bath and Cs + in the pipet solution.
  • the three traces in Figure 3, labeled b-d, show decreasing calcium currents, with increasing MVIIA OCT peptide concentrations of 10 nM (b) , 50 nM (c) , and 200 nM (d) .
  • the response of voltage-gated calcium current to increasing dosages of OCTs MVIIA and GVIA are shown in Figure 3B.
  • the calculated IC J0 is approximately 10 nM for GVIA and 100 nM for MVIIA. These values indicate extremely high specificity of the peptides for their site of action.
  • Table 1 compares IC 50 values for GVIA, MVIIA, SVIB and SVIA OCTs. Whereas OCT GVIA and OCT MVIIA show 50% inhibition of the measured calcium current at nanomolar concentration range, IC 50 values for OCT SVIB and OCT SVIA were not measurable within the range of concentrations tested, and are therefore listed as having IC 50 values above the micromolar concentrations indicated. OCT SVIB and OCT SVIA are considered to be inactive in this assay.
  • 0.1 ml stimulation buffer (0.1 % BSA in mM: NaCl, 97; KCl, 30; CaC12, 0.4; MgS0 4 , 1.2; KH,P0 4 ,
  • Synaptosomes were prepared from rat whole brain or hippocampal region of brain. Rats were sacrificed, and forebrains were removed and trans ⁇ ferred to 10 ml ice-cold 0.32 M sucrose containing the following protease inhibitors (PI) : 1 mM EGTA; 1 mM EDTA; 1 uM pepstatin; 2 uM leupeptin. Brains were homogenized using a motor-driven Teflon-glass homogenizer (approx. 8 passes at 400 rpm) . Homoge- nates from 4 brains were pooled and centrifuged at 900 xg for 10 minutes at 4 degrees.
  • PI protease inhibitors
  • the 1.0 M sucrose layer plus the interface between the 1.0 and 1.2 M sucrose layers were collected and diluted with ice cold deionized water plus PI to yield a final sucrose concentration of 0.32 M.
  • the resulting suspension was centrifuged at 20,000 xg for 15 minutes. Pellets were then resuspended in 5 ml ice-cold phosphate buffered saline plus PI.
  • the resulting rat brain synaptosomes were then aliquoted and stored in a liquid nitrogen containment system. Prior to use in binding assays, synaptosomes were thawed and diluted with 3 volumes of ice cold deionized water plus PI.
  • This suspension was homogenized using a PT 10-35 Polytron (setting 6) for two 10-second bursts.
  • the homogenate was centrifuged at 40,000 xg for 20 minutes at 4 degrees.
  • the resulting pellets were resuspended in about 5 ml of ice cold phosphate buffered saline plus PI.
  • the resulting brain synaptosomal membrane preparation was aliquoted and stored at -80°C until use. Protein concentration of the membrane preparation was determined using Bradford reagent (BioRad) , with bovine serum albumin as standard.
  • MVIIA OCT was radiolabeled with 125 I-iodine by reaction with IodogeriTM, essentially according to the method of Ahmad and Miljanich. Following the Iodogen reaction, the peptide solution was chromatographed by HPLC through a C-8 reversed phase column and eluted with a gradient from 0.1% trifluoroacetic acid in water to 0.1% trifluoroacetic acid in water/acetonitrile (40:60 vol/vol) . The major peak of radioactivity following the underivatized MVIIA OCT was collected.
  • the binding constant (K d ) for [ 125 I]-MVIIA OCT to rat brain synaptosomal membranes was determined by a saturation binding method in which increasing quantities of [ 125 I] MVIIA OCT were added to aliquots of a synaptosomal membrane preparation (10 ug membrane protein, suspended in binding buffer consisting of 20 mM HEPES, pH 7.0, 75 mM NaCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 / /M leupeptin, .035 / /g/ml aprotinin, and 0.1% bovine serum albumin (BSA), in a total volume of 0.5 ml).
  • BSA bovine serum albumin
  • Binding at each concentration of labeled compound was determined in the absence and presence of 1 nM unlabeled MVIIA OCT to determine specific binding (as described in part B, below) .
  • the amount of labeled peptide speci ⁇ fically bound at each concentration was used to determine B ⁇ , the concentration of specific binding sites on the synaptosomes, and K d , following standard binding analysis methods (Bennett) .
  • Figure 6A shows a saturation binding curve of [ 125 I]MVIIA to rat synaptosomal membranes.
  • Figure 6B shows a Scatchard transformation of the data, from which a calculated K d of about 10 pM is determined.
  • Binding of f 125 II-SNX-lll fMVIIA Rat brain synaptosomal membranes prepared as described in Example 3 were suspended in a binding buffer consisting of 20 mM HEPES, pH 7.0, 75 mM NaCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 / /M leupeptin, .035 / g/ml aprotinin, and 0.1% bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • [ I25 I]-MVIIA (SNX-lll) OCT (25-30,000 cpm, approximately 1500-2000 Ci/mmol) and test compound were aliquoted into polypropylene tubes, in the absence or presence of 1 nM MVIIA (SNX-lll) OCT to determine non-specific binding.
  • the membrane su ⁇ spension was diluted and aliquoted last into the test tubes, such that each assay tube contained 10 ⁇ g membrane protein and the total volume was 0.5 ml.
  • IC 50 values were computed from line fit curves generated by a 4-parameter logistic function. These values represent the concentration of test compound required to inhibit by 50% the total specific binding of [ 125 I]-MVIIA (SNX-lll) OCT to rat brain synaptosomal membranes, where specific binding is defined as the difference between binding of [ I2 I]-MVIIA (SNX-lll) OCT in the absence and presence of excess (1 nM) unlabelled MVIIA OCT.
  • Non-specific binding is that binding of radiolabeled compound which is measured in the presence of excess unlabeled MVIIA OCT.
  • IC 50 is the concentration of test substance required to reduce specific binding of labeled ligand by 50%
  • [L] is the concentration of [ 125 I]-MVIIA (SNX- 111) OCT used in the experiment
  • K d is the binding constant determined for binding of [ 125 I]- MVIIA (SNX-lll) OCT to rat brain synaptosomal membranes in saturation binding experiments.
  • Table 3 summarizes computed IC 50 for various OCT peptides for the MVIIA binding site of rat brain synaptosomal membranes.
  • OCTMVIIC/SVIB Binding Proteins Conopeptide receptor polypeptides in rat brain or hippocampal synaptic membranes were chemically cross linked to radio-iodinated SNX-lll, SNX-183, or SNX-230 with the water-soluble carbodiimide (l-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide) procedure. The radiolabeled peptides were separated by SDS-PAGE and visualized by autoradiography.
  • the tubes were cooled on ice and lO ⁇ l of freshly prepared 25mM EDC (in 25mM PIPES buffer, pH 6.1) and 10 l of freshly prepared N-hydroxysulfosuccinimide (NHS; in 25mM PIPES buffer, pH 6.1) was added to each sample.
  • 25mM EDC in 25mM PIPES buffer, pH 6.1
  • NHS N-hydroxysulfosuccinimide
  • binding buffer NaCl (75mM)
  • EGTA O.lnM
  • EDTA O.lnM
  • leupeptin 2 ⁇ M
  • aprotinin 0.5 unit/ml
  • bovine serum albumin (1.5%w/v
  • HEPES/NaOH 20mM, pH 7.5
  • the concentrations of radio-iodinated peptide and corresponding non-radioactive peptide for determination of specific binding were as follows: 0.1-0.15nM [ I25 I]-SNX-lll and 25nM SNX-lll; 0.3 to 0.5nM [ 125 I]-SNX-183 and lOOnM SNX-183. After the incubation period, the solution was poured off the slides and unbound peptide was then removed by serially passing the slides through four dishes (4 min. incubation each) of washing buffer (HEPES)
  • the slides were dipped quickly five times in water and dried under a stream of air at room temperature. Dried slides were exposed to XAR-2 X-ray film, and the film developed. The developed images were examined either directly over a light box, by inspec ⁇ tion of enlarged photographic prints, or by a computer assisted image analyzer. The assignment of binding to specific neuro-anatomical sites was made using an anatomical atlas of the rat brain (Paxinos) .
  • both common carotid arteries were exposed, dissected free of surrounding tissue, and occluded with microvascular clamps approximately 3 to 4 mm above the clavicle.
  • the occlusions were maintained for 8 minutes, timed while both arteries were occluded. There was generally a period of approximately 1 minute between clamping of each of the two arteries, and approximately 4 seconds between unclamping them. After the clamps were removed, the skin was sutured shut and anesthesia discontinued.
  • an intracerebroventricular (ICV) injection aimed at the lateral ventricle was made.
  • a 10 microliter Hamilton syringe with a 27 gauge needle was filled with injectate by backloading to assure the absence of air in the system.
  • a stiff plastic sleeve was slipped onto the needle so that 3.5 mm of the needle protruded past the sleeve.
  • the skull around the bregma was exposed, a distance of 1.1 mm left of the midline was measured with a compass, and a distance of 0.4 mm posterior to bregma was approximated by eye.
  • the needle tip was held perpendicular to the skull and inserted through it at that point by applying gentle pressure while twisting.
  • mice were anesthetized with C0 2 .
  • the chest cavity was opened and the animal was perfused through the heart with approximately 3 illiliters of phosphate- buffered saline (PBS; 0.10 M sodium phosphate; 0.15 M sodium chloride) containing heparin (10 Units/ml) , followed by approximately 10 ml of Za boni's fix (15% (vol/vol) picric acid, 4% (wt/vol) paraformaldehyde in 0.1 M phosphate buffer pH 7.4) or 10% phosphate buffered formalin. Brains were removed and left immersed in the same fixative for several hours.
  • PBS phosphate- buffered saline
  • Za boni's fix (15% (vol/vol) picric acid, 4% (wt/vol) paraformaldehyde in 0.1 M phosphate buffer pH 7.4
  • 10% phosphate buffered formalin were removed and left immersed in the same fixative for several hours.
  • Brains were blocked just posterior to the optic chiasm and posterior to the mammillary bodies. They were then placed in 10% (wt/vol) sucrose in PBS overnight at 4 degrees. The block containing the hippocampus was frozen with liquid Freon onto a cryostat chuck using Tissue-TekTM O.C.T. embedding medium for frozen tissue specimens (Miles Inc. , Elkhart, IA) . Sections 10 microns in thickness were cut. Series of 5 sections were collected, with each series approximately 100 microns apart, until the relevant part of the hippocampus was obtained (40-50 sections per brain) . At least 8 sections per brain were stained with hematoxylin and eosin, substantially according to reported procedures.
  • Figures 10A and 10B are low-power micrographs of gerbil hippocampus (CA1 region) in animals after ischemia, after infusion of MVIIA OCT (10A) or after drug vehicle (10B) .
  • the arrows in the figures indicate the approximate borders of the CA1'region of the hippocampus.
  • FIG 11A shows that cells in the drug-treated ischemic animals appear normal ( Figure 11A) , whereas damage is apparent in the ischemic animals receiving vehicle alone ( Figure 11B) .
  • Figure lie Another example of complete drug protection is seen in Figure lie, and an example of partial protection is seen in Figure 11D, where there are a small number of damaged cells.
  • Damaged cells outnumber normal cells to a maximum of 75%, with damage extending throughout most of CA1.
  • the procedure was modified to allow carotid occlusion without the need for reopening a skin wound in conscious animals.
  • Surgery was performed to permanently occlude both vertebral arteries and to implant an arterial clasp to allow temporary occlusion of the carotid arteries at a later time.
  • sodium pentobarbital anesthesia 60-65 mg/kg, intraperitoneally, i.p.
  • male Fisher 344 rats were placed in a stereotaxic holder and the first cervical vertebra was exposed with the aid of a dissecting microscope.
  • the ver ⁇ tebral arteries were occluded at the first cervical artery, through the alar foramina, with a ther- mocautery device and the skin closed with wound clips.
  • the animal was placed on its back and the carotid arteries were carefully dissected free of the surrounding nerves and vessels under the microscope.
  • the loose end of the Silastic loop of the clasp was passed behind the artery and put through the open side of the clasp and secured as for the other end. This was then repeated for the other carotid.
  • the clasps were tied into the skin with 3-0 suture as the skin was closed so as to externalize the ends of the loop.
  • Ischemia was produced 2 days after surgery. To occlude the carotid arteries, the animal was held by lightly pinching the skin at the back of the neck and the ends of each loop were pulled out and secured with a bulldog clamp. At the end of the 15 min. occlusion, the clamps were removed to allow reperfusion. An effective occlusion causes the animal to lose its righting response within about 1 min. of occlusion. If the animal did not lose the righting response or if it regained it during occlusion, the loops were pulled tighter to assure complete carotid occlusion. Animals that did not lose their righting response were eliminated from the study, because this suggested that there was still significant cerebral blood flow.
  • Rats receiving intracerebroventricular (ICV) compound were anesthetized using halothane i medi- ately following reperfusion, and compound contained in 5 ⁇ L saline or saline alone was injected into the lateral ventricle as for gerbils.
  • the coor ⁇ dinates of the injection were 1.2 mm left of midline and 0.5 mm posterior to bregma, at a depth of 3-4 mm.
  • Rectal temperature was monitored from just before occlusion, and for 4-6 hours post occlusion.
  • Rats were maintained normothermic (rectal temperature at about 37 degrees) for 4-6 hours following occlusion, by means of a heating apparatus. The degree of neuroprotection was assessed as in Example 8 and is shown in Tables 7 and 8.
  • Reversible tourniquets were applied to tail veins, and OCT compound was injected in a total volume of 0.25 ml, at the times and doses indicated in Tables 8-11.
  • rats were maintained normothermic (rectal temperature at about 37 degrees) for 4-6 hours following occlusion, by means of heating apparatus. The degree of neuroprotection was assessed as in Example 8.

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Abstract

L'invention se rapporte à des procédés et à des compositions pour réduire les lésions neuronales associées à un état ischémique chez un mammifère. Ce procédé consiste à administrer au sujet les compositions décrites dans la présente invention, 4 à 24 heures après le début de l'état ischémique. Les compositions de la présente invention ont le pouvoir de fixer sélectivement les sites de liaison de la conotoxine oméga, et de préférence de se lier avec une grande affinité aux sites de liaison de la conotoxine oméga MVIIA, et de bloquer sélectivement la libération des neurotransmetteurs à partir des cellules neuronales du système nerveux central du mammifère. L'invention décrit également de nouvelles structures peptidiques utilisables dans le procédé thérapeutique de la présente invention.
PCT/US1992/009766 1991-11-12 1992-11-12 Compositions pour le traitement retarde de lesions neuronales associees a l'ischemie WO1993010145A1 (fr)

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US07/789,913 US5559095A (en) 1989-11-22 1991-11-12 Delayed treatment method of reducing ischemia-related neuronal damage
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US91647892A 1992-07-17 1992-07-17
US916,478 1992-07-17

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5591821A (en) * 1993-07-16 1997-01-07 The University Of Utah Omega-conotoxin peptides
WO1997001351A1 (fr) * 1995-06-27 1997-01-16 Neurex Corporation Compositions et formulations permettant de produire une analgesie et d'inhiber la progression de troubles lies a des douleurs neuropathiques
US5670622A (en) * 1996-02-15 1997-09-23 University Of Utah Research Foundation Conotoxin peptide PIIIA
US5672682A (en) * 1996-03-18 1997-09-30 University Of Utah Research Foundation Conotoxin peptide PVIIA
US5739276A (en) * 1994-10-07 1998-04-14 University Of Utah Research Foundation Conotoxin peptides
US5830998A (en) * 1992-09-28 1998-11-03 Maccecchini; Maria-Luisa Allosteric modulators of the NMDA receptor and their use in the treatment of CNS disorders and enhancement of CNS function
US5965534A (en) * 1995-11-22 1999-10-12 Alcon Laboratories, Inc. Use of ω-conotoxin analogs for treating retinal and optic nerve head damage
US6054429A (en) * 1996-03-08 2000-04-25 Elan Pharmaceuticals, Inc. Epidural method of producing analgesia
US6268473B1 (en) 1999-01-22 2001-07-31 University Of Utah Research Foundation α-conotoxin peptides
US6855805B2 (en) 1999-01-22 2005-02-15 University Of Utah Research Foundation α-conotoxin peptides

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Publication number Priority date Publication date Assignee Title
WO2008024660A2 (fr) * 2006-08-23 2008-02-28 The University Of Montana Procédé de réduction d'un dommage aux cellules neuronales

Citations (1)

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WO1991007980A1 (fr) * 1989-11-22 1991-06-13 Neurex Corporation Compositions de traitement de dommages des neurones dus a l'ischemie

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WO1991007980A1 (fr) * 1989-11-22 1991-06-13 Neurex Corporation Compositions de traitement de dommages des neurones dus a l'ischemie

Non-Patent Citations (2)

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Title
BIOCHEMISTRY vol. 26, no. 8, 21 April 1987, EASTON, PA US pages 2086 - 2090 B.M. OLIVEIRA ET AL. 'Neuronal Calcium Channel Antagonists. Discrimination between Calcium Channel Subtypes Using omega-Conotoxin from Conus magus Venom' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA vol. 84, June 1987, WASHINGTON US pages 4327 - 4331 E.W. MCCLESKEY ET AL. 'omega-Conotoxin: Direct and persistent blockade of specific types of calcium channels in neurons but not muscle' cited in the application *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5830998A (en) * 1992-09-28 1998-11-03 Maccecchini; Maria-Luisa Allosteric modulators of the NMDA receptor and their use in the treatment of CNS disorders and enhancement of CNS function
US5591821A (en) * 1993-07-16 1997-01-07 The University Of Utah Omega-conotoxin peptides
US5739276A (en) * 1994-10-07 1998-04-14 University Of Utah Research Foundation Conotoxin peptides
WO1997001351A1 (fr) * 1995-06-27 1997-01-16 Neurex Corporation Compositions et formulations permettant de produire une analgesie et d'inhiber la progression de troubles lies a des douleurs neuropathiques
EP1336409A1 (fr) * 1995-06-27 2003-08-20 Elan Pharmaceuticals, Inc. Compositions et formulations permettant de produire une analgésie et d'inhiber la progression de troubles liés à des douleurs neuropathiques
US5965534A (en) * 1995-11-22 1999-10-12 Alcon Laboratories, Inc. Use of ω-conotoxin analogs for treating retinal and optic nerve head damage
US5670622A (en) * 1996-02-15 1997-09-23 University Of Utah Research Foundation Conotoxin peptide PIIIA
US6054429A (en) * 1996-03-08 2000-04-25 Elan Pharmaceuticals, Inc. Epidural method of producing analgesia
US5672682A (en) * 1996-03-18 1997-09-30 University Of Utah Research Foundation Conotoxin peptide PVIIA
US6268473B1 (en) 1999-01-22 2001-07-31 University Of Utah Research Foundation α-conotoxin peptides
US6855805B2 (en) 1999-01-22 2005-02-15 University Of Utah Research Foundation α-conotoxin peptides

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