Method of treating ischemia-related neuronal damage

A method of reducing neuronal damage related to an ischemic condition in a human patient, such as stroke-induced damage, by administration of a binding/inhibitory omega-conotoxin peptide.

FIELD OF THE INVENTION 
The present invention relates to a method of reducing neuronal damage 
associated with an ischemic condition, such as stroke. 
REFERENCES 
Ahmad, S. and Miljanich, G., Br. Res. 453:247-256 (1988). Anderson, A. and 
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57:665-700. 
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(1988) pp. 490-536. 
Kirino, T., Brain Res. 239:57-69 (1982). 
McCleskey, E., Fox, A., Feldman, D., Cruz, L., Olivera, B., Tsien, R., 
Yoshikami, D., Proc. Natl. Acad. Sci. (USA) 84:4327-4331 (1987). 
Morel, N. and Meunier, F. -M., J. Neurochem. 36:1766-1773 (1981). 
Olivera, B., Gray, W., Zeikus, R., McIntosh, J., Varga, J., Rivier, J., de 
Santos, V., Cruz, L. J., Science 230: 1338-1343 (1985). 
Pulsinelli, W. and Brierley, J., Stroke 10:267-272 (1979). 
Sano, K., Enomoto, K., Maeno, T., Eur. J. Pharmacol. 141:235-241 (1987). 
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Structure, 5 suppl. 3:353-358, Nat. Biomed. Res. Found., Washington, D.C. 
Schweitzer, E., J. Neurosci. 7:2948-2956 (1987) 
Tsien, R., Lipscombe, D., Madison, D., Bley, K., Fox, A., TINS 11:431-438. 
Van Reempts, J. and Borgers, M. (1984), Acad. Anaesthesiolica Belge 35, 
Supplement, 209-218. 
Wauquier, A., Edmonds, H., Clincke, G. (1987), Neuroscience and 
Biobehavioral Reviews 11:287-306. 
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and Siesjo, B. K., eds), Elsevier Sciencer Publ., pp 13-24 (1984). 
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BACKGROUND OF THE INVENTION 
Ischemic damage to the central nervous system (CNS) 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 occlusion of a cerebral vessel, traumatic 
head injury, edema, and brain tumors. 
Both global and focal ischemic conditions have the potential for producing 
widespread neuronal damage, even if the ischemic condition is transient. 
Although some permanent neuronal injury may occur in the initial minutes 
following cessation of blood flow to the brain, most of the damage in 
global and focal ischemia occurs over hours or more typically days 
following the ischemic onset. Much of this neuronal damage is attributed 
to secondary consequences of reperfusion of the tissue, such as the 
release of vasoactive products by damaged endothelium, and the release of 
cytotoxic products (free radicals, leukotrienes, etc.) by damaged tissues. 
Several drug strategies have been proposed for treatment of stroke and 
other neuronal conditions related to ischemia, and these have been 
reviewed in recent articles (e.g., Wauquier). Anti-coagulants, such as 
heparin, have been examined, but with mixed results. Similarly, 
antivasoconstriction agents, such as flunarazine, excitatory 
neurotransmitter antagonists, such as MK-801 and AP7, and 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. 
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-antagonist vasodilator drugs. Verapamil and related compounds 
which prevent calcium entry into smooth and striated muscles appear to be 
effective only at high drug concentrations, where serious cardiotoxicity 
effects may ensue. Dihydropyridines, such as nimodipine, 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 such as hypotension which may be 
inimical to treatment. 
In summary, drugs which have been proposed to date for the treatment of 
stroke and other ischemic-related conditions of the brain are either (i) 
relatively ineffective, (ii) effective only at dosage levels where 
undesired side effects are observed, and/or (iii) produce systemic 
effects, such as hypotension, which compromise the potential effectiveness 
of the drug. 
SUMMARY OF THE INVENTION 
It is therefore a general object of the invention to provide an effective 
drug treatment for stroke and other ischemia-related brain conditions. 
It is still another object to provide novel peptide compounds for use in 
the treatment method. 
In one aspect, the invention includes a method of reducing neuronal damage 
related to an ischemic condition in a human patient by administering to 
the patient a pharmacologically effective amount of a binding/inhibitory 
omega-conotoxin (OCT) peptide. 
Treatment with the peptide is effective to significantly reduce (a) 
anatomical damage in the brain, as evidenced by reduced damage to 
hippocampal CA1 cells, and (b) loss of brain function, as evidenced by 
reduced loss of short-term memory. 
The binding/inhibitory conotoxin is characterized by one or some of the 
following properties: 
(a) specific binding to OCT binding sites associated with calcium channels 
in neuronal-cell membranes, as evidenced by competitive inhibition of OCT 
peptide binding to electric-ray electric-organ synaptosomes, or rat brain 
synaptosomes; 
(b) inhibition of voltage-gated calcium currents selectively in neuronal 
tissue, as evidenced by inhibition of voltage gated calcium currents in 
cultured mouse neuroblastoma cells, but not voltage-gated calcium currents 
in muscle cells, such as dissociated guinea pig cardiac ventricular cells; 
and 
(c) inhibition of neurotransmitter release selectively in neuronal tissue, 
as evidenced by inhibition of induced ATP release from electric ray 
electric organ synaptosomes, but not blockade of evoked neurotransmitter 
release at a mammalian neuromuscular junction of skeletal muscle. 
OCT peptides effective in the treatment method may be further characterized 
by their ability to induce shaking in mice following intracerebral 
injection. 
The binding/inhibitory OCT peptide may be a G-group peptide, such as GVIA, 
GVIIA and RVIA peptide, including binding/inhibitory analogs thereof, or 
an M-group peptide such as SVIB, MVIIA, MVIIB peptide, including 
binding/inhibitory analogs thereof. The isoelectric point of the peptide 
is preferably between about 10.5-12.0. 
The three-dimensional structures of preferred M-group peptides are further 
characterized by the following NMR-NOE based interatomic distances of 
about 4.5 angstroms (long-range) or 2.5 angstrom (short-range) between 
protons of the designated amino acid residue amide nitrogen found in OCT 
MVIIA, or as otherwise specified: 
Long-range: 
Lys (position 2) and Cys (position 16) 
Gly (position 5) and Cys (position 28) 
Lys (position 26) and Cys (position 20) 
alpha H Cys (position 28) and Gly (position 18) 
beta H Cys (position 28) and Lys (position 2) 
alpha H Lys (position 26) and Cys (position 8) 
beta H Cys (position 1) and Cys (position 16) 
beta H Cys (position 28) and Gly (position 5) 
beta H Cys (position 28) and Gly (position 18) 
Short-range: 
Ser (position 24) and Gly (position 25) 
Asp (position 14) and Cys (position 15) 
Tyr (position 13) and Asp (position 14) 
Gly (position 25) and Lys (position 26) 
The three-dimensional structures of preferred G-group peptides are 
characterized by peptide backbones having similar three-dimensional 
conformations as that of OCT MVIIA as defined by the above NMR-NOE 
constraints. 
These and other objects and features of the invention will become more 
fully apparent when the following detailed description of the invention is 
read in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION 
I. Preparation of OCT Peptides 
Omega-conotoxin (OCT) peptides are peptide toxins produced by marine snails 
of the genus Conus, and which act as calcium channel blockers (Gray). About 
300 species of cone snails in the Conus genus have been identified, and a 
variety of OCTs from several of these species have been isolated. The 
primary sequences of seven OCT peptides of the eleven which have been 
completely sequenced are shown in FIG. 1. Conventional letter initials are 
used for the amino acid residues, and X represents 4-hydroxyproline, also 
abbreviated 4Hyp. Known C-terminal amidation is indicated by a "*" in FIG. 
1. The peptides have been grouped into M- and G-group peptides, as 
described in Section IV below. 
The identifying names of the peptides are also given in the figure, and 
these names will be used herein to refer to the specific OCT peptide. For 
example, the peptide whose sequence is designated MVIIA will be referred 
to herein as OCT MVIIA peptide, or simply MVIIA peptide. The MVIIA and 
GVIA peptides also have the common names CmTx and CgTx, respectively. 
The MVIIA and GVIA peptides are also shown in FIG. 2, along with their 
three disulfide linkages, as demonstrated chemically or spectroscopically. 
All of the OCT peptides in FIG. 1 have these three linkages between the 
first and fourth, second and fifth, and third and sixth cysteine (Cys) 
residues. 
OCT peptides, including the MVIIA, MVIIB, GVIA, GVIIA, RVIA, SVIA and SVIB 
peptides shown in FIG. 1, and binding/inhibitory analogs thereof, can be 
synthesized by solid phase synthesis, according to the methods detailed in 
Examples 1 and 2. Briefly, N-alpha-protected amino acid anhydrides are 
prepared in crystallized form and used for successive amino acid addition 
to the N-terminus. At each residue addition, the growing peptide (on a 
solid support) is acid treated to remove the N-alpha-protective group, 
washed several times to remove residual acid and to promote accessibility 
of the peptide terminus to the reaction medium. The peptide is then 
reacted with an activated N-protected amino acid symmetrical anhydride, 
and the solid support is washed. At each residue-addition step, the amino 
acid addition reaction may be repeated for a total of two or three 
separate addition reactions, to increase the percent of growing peptide 
molecules which are reacted. Typically, 1-2 reaction cycles are used for 
the first twelve residue additions, and 2-3 reaction cycles for the 
remaining residues. 
After completing the growing peptide chains, the protected peptide resin is 
treated with liquid hydrofluoric acid to deblock and release the peptides 
from the support. For preparing an amidated peptide, the resin support 
used in the synthesis is selected to supply a C-terminal amine, after 
peptide cleavage from the ring. 
The three disulfide linkages in the peptides may be formed by air oxidation 
in the presence of dithiothreitol (DTT) at room temperature or at 4.degree. 
C. over an extended reaction period. Alternatively, where the correct or 
desired bridging cannot be achieved by random oxidation, a chemically 
directed process may be used in which the bridges are formed sequentially, 
one bridge at a time. The following side-chain protecting groups could be 
used for each pair of cysteine residues: 4-methylbenzyl, ethylcarbamoyl, 
and acetamidomethyl. These protecting groups constitute an orthogonal set 
in which any one kind of protecting group can be removed under conditions 
that do not affect the other two. 
The strategy here involves removing one kind of protecting group from a 
pair of cysteine residues, followed by oxidation to form the first 
disulfide bridge. A second kind of protecting group is then removed, again 
followed by oxidation to form the second bridge. A third bridge, if needed, 
is formed in like manner. An example is the synthesis of compound (FIG. 2) 
No. 163 (with a bridge linking positions 1 and 16) followed by conversion 
to compound 172 (with the second bridge linking positions 8 and 15). 
The peptide can be isolated by an initial separation by gel filtration, to 
remove peptide dimers and higher polymers, and also to remove undesired 
salts, such as guanidine hydrochloride, used in the oxidation reaction. 
The partially purified peptide is further purified by preparative HPLC 
chromatography, and the purity of the peptide confirmed by amino acid 
composition analysis. Details of the preparation and purification of the 
MVIIA peptide are given in Example 1. The same methods are applied in 
Example 2 for preparation and purification of the MVIIB, GVIA, GVIIA, 
RVIA, SVIA, and SVIB peptides. 
FIG. 2 shows portions of MVIIA and GVIA peptides which were also prepared 
for peptide fragment binding studies, described in Section IV. The heavy 
lines in the figure indicate the portion of the corresponding MVIIA or 
GVIA peptide which was synthesized, and the light-line loop, the disulfide 
linking which was formed in the peptides. A small arrow above a Cys 
position indicates that the Cys residue is blocked (with an 
acetamidomethyl group) and therefore unable to participate in disulfide 
bridging. Thus for example, the peptide fragment labeled 160 extends 
between the third and sixth Cys residues corresponding to the C terminus 
of the full-length MVIIA peptide, and has a disulfide bridge between the 
third and sixth Cys, the fourth and fifth Cys residues being blocked. The 
peptide fragment labeled 162 includes a deletion between the positions 9 
and 19, and is linked between the first and sixth Cys residues, the second 
and fifth Cys residues being blocked. The A, S, and X designations in the 
figure indicate Ala, Ser, and 4Hyp substitutions, respectively, at the 
positions shown. 
The peptide fragments shown in FIG. 2 were prepared substantially as 
described in Example 3, following the method detailed in Example 1, but 
using a single-coupling protocol. All of the peptide fragments are 
amidated at the C-terminal residues. 
II. Binding/Inhibitory OCTs 
This section describes in vitro binding and inhibitory properties of OCT 
peptides which are effective in the treatment method of the invention. 
According to one aspect of the invention, the in vitro properties are used 
as indicators selecting and identifying natural and analog conotoxin 
peptides which are effective in the treatment method of the invention. 
More generally, it has been discovered that (a) conotoxin peptides can be 
characterized as binding/inhibitory, according to selected in vitro 
binding and/or inhibitory properties, and (b) binding/inhibitory peptides 
are effective in reducing neuronal damage in ischemia-related neuronal 
conditions. 
As defined herein, binding/inhibitory OCT peptides have at least one of the 
following binding/inhibitory activities: 
(a) Specific, high-affinity binding to OCT binding sites associated with 
calcium channels in neuronal-cell membranes, as evidenced by competitive 
inhibition of OCT binding to rat brain synaptosomes or to electric-ray 
electric-organ (fish) synaptosomes; 
(b) inhibition of voltage-gated calcium currents selectively in neuronal 
tissue, as evidenced by inhibition of voltage-gated calcium currents in 
cultured mouse neuroblastoma N1E-115 cells, but not in muscle cells, such 
as dissociated guinea pig cardiac ventricular cells; and 
(c) inhibition of neurotransmitter release selectively in neuronal tissue, 
as evidenced by inhibition of ATP release from fish synaptosomes, but not 
neurotransmitter release such as mammalian neuromuscular junction of 
skeletal muscle, such as mouse diaphragm neuromuscular junction. 
A. Specific, High Affinity Binding to OCT Receptors 
High-affinity binding to OCT sites in neuronal tissue can be demonstrated 
with a variety of cell types and synaptosomal cell fractions. Example 4 
describes the determination of OCT binding constants, based on competitive 
displacement of MVIIA peptide from electric-ray electric-organ synaptosomal 
membranes, also referred to herein as fish synaptosomes. 
Fish electric organ synaptosomes are prepared by the gradient 
centrifugation method detailed in Example 4. The binding constant K.sub.d 
of the MVIIA peptide for the synaptosomes is determined by a saturation 
binding method in which increasing quantities of radiolabeled peptide are 
added to the synaptosomes, and the amount of labeled material bound at 
each concentration is determined. The plot of bound peptide as a function 
of concentration is then used to calculate a B.sub.max, the concentration 
of binding sites on the synaptosomes, and K.sub.d following standard 
methods. The K.sub.d value obtained for the MVIIA peptide is about 0.4 nM, 
as seen in Table 3 of Example 4. The corresponding pK.sub.i value is 9.4. 
Competitive binding assays, to determine K.sub.i binding constants for 
other OCT peptides, are carried out by addition of test peptides, over a 
concentration range between about 10.sup.-3 and 10.sup.-13 M peptide, to 
fish synaptosomes having bound, labeled MVIIA peptide. The synaptosome 
material is then rapidly filtered, washed and assayed for bound 
radiolabel. The binding constant, K.sub.i, of the test peptide can be 
determined using computer-fit competitive binding curves, such as shown in 
FIG. 3 for GVIA peptide, with the results shown in Table 3 of Example 4. Of 
the six peptides examined (in addition to MVIIA), five show high-affinity 
binding, as judged by a pK.sub.i greater than 6. One of the peptides, 
SVIA, shows relatively low affinity binding and thus fails to meet the 
criteria of binding/inhibitory OCT peptides. 
Table 3 in Example 3 also gives approximate K.sub.i and pK.sub.i values for 
a number of OCT MVIIA and GVIA peptides fragments shown in FIG. 1. The 
significance of the fragment binding constants will be discussed below in 
Section IV. 
Similar competitive binding studies can be carried out with mammalian 
synaptosome preparations, such as the rat brain synaptosome preparation 
described in Example 5. An analysis of the competitive binding curves, 
such as shown in FIG. 4 for the MVIIA peptide, obtained in studies with 
the rat brain preparation indicates a single binding site for OCTs, with 
the corresponding IC.sub.50 values (that concentration of competing 
compound at which specific binding is inhibited by 50%) shown in Table 4 
in Example 5. 
Studies reported in Example 6 further indicate that OCT peptide binding 
activity can be assessed with respect to several cultured neuronal cells. 
The different cell types and strains which were employed are shown at the 
left in Table 5 in this example, along with the corresponding 
concentration of specific binding sites measured by the addition of 1 nM 
labeled MVIIA peptide to the cells. Two of the neuroblastoma cell strains, 
IMR-32 and SY5Y(+RA), show relatively high concentrations of OCT surface 
binding sites. 
In summary, both synaptosome preparations and cultured neuronal cells are 
suitable for testing binding affinity of test OCT peptides. One preferred 
system is electric-ray electric organ synaptosomes, where specific, 
high-affinity binding is evidenced by a pK.sub.i of 6 or greater, as 
determined by competitive displacement of MVIIA peptide. 
B. Inhibition of voltage-gated calcium currents 
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 
cell secretion, such as in synaptic transmission (McCleskey). In neuronal 
cells, voltage-gated calcium channels have been classified into L, T, and 
N channels, each with characteristic gating voltage, inactivation rate, 
and selective modulation by neurochemicals (Tsien). 
GVIA OCT 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 inhbition by 
the peptide was not observed in cardiac, smooth, and skeletal muscles. 
One suitable system for testing inhibition (blockage) of neuronal calcium 
channels is the mouse neuroblastoma cell line, strain N1E115. Membrane 
currents are conveniently measured with the whole cell configuration of 
the patch clamp method, according to the procedure detailed in Example 7. 
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. 
FIG. 5A shows a typical inward calcium current elicited by a voltage step 
from -100 mV to -20 mV in the absence of OCT. In this, and most of the 
recordings, Ba replaced Ca as the charge-carrier through the calcium 
channels in order to increase the signal (Tsien). According to the 
procedure described in Example 7, an N1E115 neuroblastoma cell was bathed 
in saline with sodium replaced by N-methyl-D-glucamine (NMDG), and 10 mM 
Ba instead of 2 mM Ca. These substitutions reduced the sodium current that 
would otherwise have contaminated the calcium-current record, and increased 
the calcium current above what it would have been with only 2 mM Ca in the 
bath. Potassium currents were blocked by TEA in the bath and Cs in the 
pipet solution. 
As seen from FIG. 5A, 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 -1196 pA. 
The cell in FIG. 5A was also exposed to 1 .mu.M nifedipine, a 
dihydropyridine, which is expected to effectively block L-type calcium 
channels in the neuroblastoma cells. The calcium current observed is thus 
expected to be predominantly an N-type calcium channel current. 
FIG. 5B shows the effect on the calcium current when an N.sub.IE 115 cell 
was exposed to a solution containing 10 nM MVIIA peptide. The measured 
current amplitude in the same cell as shown in FIG. 5A (same scale) was 
reduced to about -857 pA. With increasing concentrations of OCT peptide, 
the measured calcium current dropped to -534 pA, at 50 nM OCT peptide, and 
to -257 pA, at 200 nM OCT peptide, as seen in FIGS. 5C and D, respectively. 
The ED.sub.50 concentration, at which 50% inhibition of calcium current is 
produced, is determined from the voltage-gated current amplitudes, plotted 
as a function cf OCT peptide concentration. 
The responses of voltage-gated calcium current to increasing dosages of 
OCTs MVIIA and GVIA are shown in FIG. 6. The calculated ED.sub.50 is 12 nM 
for GVIA and 115 nM for MVIIA, indicative of high inhibitory peptide 
activity. The ED.sub.50 concentration for test OCT peptides can be readily 
determined in a like manner. Test peptides which have ED.sub.50 values of 
less than about 1 .mu.M and preferably less than about 200 -250 nM (Table 
4, Example 5) in the above neuroblastoma system are classed as having the 
requisite calcium current inhibitory activity. 
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 (McCleskey). Cell current measurements are made 
substantially as outlined above and detailed in Example 7. 
C. Selective Inhibition of Neurotransmitter Release 
A third property of binding/inhibitory OCT peptides is the ability to 
specifically inhibit neurotransmitter release in neuronal cells, but not 
at a mammalian neuromuscular junction of a skeletal muscle. This property 
may be related to (a) specific binding of active peptide to the neuronal 
cell membrane and (b) resultant inhibition of calcium currents produced by 
such binding, and thus may represent a consequence of the binding and 
current-inhibition properties of OCT peptides. 
In one standard method for measuring neurotransmitter release, ATP release 
from synaptosomes, such as fish synaptosomes is assayed. Here, the 
synaptosomes are suspended in medium containing luciferase, and 
scintillation events are counted after exocytotic release of ATP from the 
synaptosomes. In the procedure detailed in Example 8, synaptosomal release 
of ATP is evoked by injecting potassium at a high concentration into the 
medium, producing voltage-gated calcium currents which are accompanied by 
the concommitant release of ATP and acetylcholine. 
Test peptides are included in the synaptosome medium at several 
concentrations between about 1 and 1000 nM, and the reduction in ATP 
levels is measured before and after peptide addition. From a plot of ATP 
release inhibition as a function of peptide concentration, the IC.sub.50 
value, at which 50% ATP release inhibtion occurs, is determined. Table 6 
in Example 8 gives the IC.sub.50 values determined for the MVIIA and GVIA 
active peptides. 
The test OCT peptide may also be assayed for non-inhibition of 
neuromuscular transmission at a mammalian neuromuscular junction of 
skeletal muscle. Various model systems for measuring the effect of test 
compounds on neurotransmitter release at a neuromuscular junction have 
been described (Sano; Anderson). In both an isolated mouse diaphragm 
system described by Sano, and a mouse triangularis sterni system described 
by Anderson, OCT MVIIA was reported to show little or no inhibitory efects. 
D. Mouse Shaker Test 
Peptides which are can be classed as binding/inhibitory peptides in the in 
vitro binding and inhibition activity tests described above can be further 
tested for activity in an in vivo mouse shaker model system. This test 
system is modeled on an earlier reported finding that 
intracerebroventricular (ICV) administration of MVIIA peptide in mice 
produces whole-body tremors (Olivera). 
In the shaker test, unanesthetized animals are given a selected dose of the 
test peptide by intracerebral administration. Animals which show 
spontaneous tremors within about 30 minutes after peptide administration 
are scored as positives. Table 7 in Example 9 shows the shaker results 
obtained on tests with increasing doses of OCT's MVIIA, GVIA, and SVIA. 
The middle column shows the number of animals which were tested at each 
dose, and the right column, the percent number of "shakers", i.e., animals 
showing shaking behavior. The MVIIA and GVIA OCT peptides, which are both 
active as judged by the binding and inhibition criteria described above, 
give a dose-dependent shaker response, with both peptides showing a high 
percentage of shakers at 0.3 .mu.g dose. By contrast, the SVIA peptide, 
which is inactive by the above binding activity criterion, gives only a 
low percentage of shakers, even at a dose of 10 .mu.g. 
The shaker model data above demonstrate that the mouse shaker model may 
provide a reliable in vivo test of OCT peptide activity, i.e., the model 
provides a useful predictor of OCT activity in the treatment method of the 
invention. The shaker model may also be useful in determining effective 
treatment dose ranges of a test OCT peptide. As will be seen in Section 
III below, dose ranges effective for stroke treatment are roughly 
comparable to the doses at which a low-to-moderate percentage of shakers 
are observed. The shaker model system can also be used to test toxicity 
effects at projected treatment doses. 
III. Treatment Method 
The present invention provides a treatment method for reducing 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 flood, 
such as due to cerebral hemorrhaging, or localized thrombotic or embolic 
events, or head trauma. 
The ischemic condition to be treated is generally associated with stroke, 
defined as the sudden diminution or loss of neurological function caused 
by an obstruction or rupture of blood vessels in the brain. In stroke, as 
well as in other types of cerebral ischemic conditions, the peptide 
treatment 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 vulnerability in lesions, 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. As will be seen below, the treatment method of the 
invention is effective in reducing or preventing both anatomical and 
functional secondary damage related to ischemia. 
The OCT peptide administered in the treatment method is a 
binding/inhibitory OCT peptide as defined in Section II above, including 
the MVIIA, MVIIB, GVIA, SVIB, GVIIA, and RVIA OCT peptides and 
binding/inhibitory analogs of natural OCT peptides having amino acid 
substitutions selected according to the constraints discussed in Section 
IV. 
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 0.1-10 mg/ml. 
The dose administered will be determined by route of administration. One 
suitable route is intracerebroventricular (ICV), at a dose level of about 
0.1-50 .mu.g peptide/kg body weight, depending on the binding and 
inhibitory values of the peptide. A pharmaceutically effective dose, i.e., 
a dose effective to produce significant reduction in anatomical and/or 
functional damage, can be estimated, as noted above, from the dose 
response seen in the mouse shaker model. The dose level can also be 
estimated, for new OCT peptides, by comparison with established effective 
doses for known peptides, corrected for observed differences in in vitro 
binding and inhibitory activities. 
As reported below, and according to an important feature of the invention, 
it has been found that there is little or no loss of protective effect of 
the peptide when administered well after the ischemic effect, e.g., one 
hour following the period of transient occlusion. The 
delayed-administration protective effect indicates that the peptide is 
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. Thus, the delayed administration may be 
effective to reduce secondary cerebral damage when administered several 
hours, or even a day or more, following the onset of ischemia. 
The treatment method has been demonstrated in two animal systems which are 
widely employed as model systems for global ischemia and secondary stroke 
damage. The first system is the gerbil model of global ischemia produced 
by transient occlusion of carotid arteries of the neck. For clinical 
comparisons, 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. 
Although some differences in particular sequelae have been noted between 
species, gerbils exhibit the same kind of selective regional damage to 
ischemia as is found in other mammals, including humans. In particular, 
the characteristic secondary damage observed in the hippocampal CA1 region 
is similar to that seen in other mammals, including humans (Kirino; 
Yamaguchi). Neurons in this area, and especially pyramidal neurons, 
exhibit a delayed neuronal death over a period of up to 4 days after 
ischemic injury. 
The second model 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. In addition, the 
occlusion procedure allows the animals to be easily monitored, maintained 
and analysed. 
A. Reduction in Anatomical Damage 
Ischemia in the gerbil model system was induced in anesthetized animals by 
occluding the two carotid arteries for eight minutes, as detailed in 
Example 10. OCT peptide was administered ICV during the occlusion period, 
or one hour following occlusion. Four days after occlusion and peptide 
treatment, the animals were examined histologically for anatomical damage 
in the hippocampal CA1 region, as detailed in Example 10. 
FIGS. 7A and 7B are low-power micrographs of gerbil hippocampus CA1 region 
in animals after ischemia, and infusion of MVIIA OCT (7A) or drug vehicle 
(7B). The arrows in the figures indicate the approximate borders of the 
CA1 region. At higher power, cells in the drug-treated ischemic animals 
appear normal (FIG. 8A), whereas damage is apparent in the ischemic 
animals receiving vehicle alone (FIG. 8B). Another example of complete 
drug protection is seen in FIG. 8C, and an example of partial protection 
is seen in FIG. 8D, where there are a small number of damaged cells. 
Anatomical sections, such as those seen in FIGS. 7 and 8, were scored 
according to the criteria set out in Example 10. The extent of anatomical 
damage in ischemic animals treated with MVIIA or GVIA OCT or receiving 
vehicle alone (control), based on the above scoring system, is given in 
Table 8 in Example 10. The peptide was administered by ICV infusion during 
the eight minutes of ischemia, at a total dose indicated in Table 8 in 
Example 10. As seen, the extent of damage in the higher-dose MVIIA OCT 
treated animal was only 25% of that in untreated animals. The GVIA peptide 
also produced more than a 50% reduction in damage, and the lower dose was 
near maximal effectiveness. 
Ischemia in the rat model system was induced by first surgically closing 
the vertebral arteries, and after surgical recovery, transiently blocking 
the carotid arteries (thus completely blocking blood flow to the brain) 
for a period of 15 minutes. During occlusion, animals were given 0.3 .mu.g 
OCT MVIIA peptide ICV. Four days after occlusion, the animals were examined 
histologically, as above, to determine the extent of damage in the 
hippocampal CA1 region, as above. The mean scores are given in Table 10 in 
Example 11. As seen, the extent of damage in the treated animals was only 
about 1/3 that in untreated animals. 
B. Functional Activity Protection: Hyperactivity 
One common consequence of cerebral ischemia in animals is hyperactivity, 
which can be seen as pacing (exploratory) behavior within a few hours of 
occlusion, and can be observed up to several days later. Hyperactivity in 
ischemic gerbils was monitored as described in Example 12. Briefly, 
gerbils were tested individually for 60 min, with cumulative activity 
counts recorded every 15 min for statistical analysis. Baseline activity 
was measured before surgery to ensure comparability of the different 
treatment groups on this measure, and activity measurements were made at 1 
and 3 days after occlusion. 
The results of the tests are plotted in FIG. 9. The downward slope in each 
test curve is due to the decrease in activity over the four 15 minutes 
intervals of the test (1-4 for baseline, 5-8 at day 1, and 9-12 at day 
three), as the animal becomes more familar with the test environment. 
Occlusion alone (open triangles) produced a significant rise in activity 
level over baseline levels 1 day after occlusion, and an elevated activity 
level was observed over a three-day period, indicating permanent behavioral 
damage. Non-occluded control animals receiving ICV administration of 
vehicle (open circles) remained at baseline activity levels through the 
test period. OCT peptide itself, in the absence of ischemia (solid 
circles) reduces activity, and this effect persists slightly even at three 
days. Occluded animals which had been treated with OCT MVIIA (solid 
triangles) showed lower-than baseline values at 1 day, apparently 
reflecting the reduced activity produced by the peptide alone. At three 
days, treated animals showed near-normal levels of activity, indicating 
that the OCT peptide treatment provided protection against 
ischemia-induced hyperactivity. 
B. Functional Activity Protection: Spontaneous Alternation 
One test which has been widely applied as a measure of short-term memory in 
experimental animals is the Y maze, in which animals are placed in the base 
of the stem of a Y "maze," and allowed to enter either of the two Y arms. 
When the animal enters an arm, a door is shut behind it. After 5 sec, the 
gerbil is returned to its home cage for an intertrial interval (ITI) of 2 
to 12 min. At the end of that interval the animal is run in the maze again 
in the same way. Most normal animals will alternate, that is, will enter 
the arm that was not entered on the previous trial. The test is scored by 
a "Y" for alternation and an "N" for repeat selection of the same Y arm. 
In the test procedure, ischemia in gerbils was induced as above, with 
simultaneous ICV administration of vehicle (control) or 0.1 or 0.3 .mu.g 
OCT MVIIA or GVIA peptide (results from all drug treatments were combined, 
as described in Example 12). Three days after occlusion, the animals were 
tested in the Y maze. Results of the spontaneous alternation tests are 
summarized in Table 11 of the example for animals for which there was 
anatomical protection from doses of at least 0.1 .mu.g of either compound. 
As seen from the data in the table, the normal Y/N ratio for control 
animals (no occlusion, ICV administration of vehicle) was about 2:1. 
Ischemic injury produced a drop in this ratio to less than 1, indicating 
substantially random behavior in the Y test. The loss of short-term memory 
seen in ischemic animals was completely prevented by peptide treatment, 
with Y/N ratios of about 2:1 being obtained. Peptide alone in the absence 
of ischemic injury appeared to enhance the Y/N ratio, and this enhancement 
may contribute to the improved performance of treated, ischemic animals. 
In summary, ischemic animals in which OCT peptide treatment was shown to 
significantly reduce anatomical damage, also showed statistically improved 
functional activity, as evidenced by peptide protection against 
ischemia-induced hyperactivity and loss of short-term memory. 
IV. Binding/Inhibitory OCT Peptides 
As noted above, the correlation between specific binding and inhibitory 
properties of OCT peptides and efficacy in treating ischemic allows for 
the selection and identification of OCT peptides based on the in vitro 
properties discussed in Section II. The binding/inhibitory OCT peptides, 
i.e., those which have the requisite in vitro binding and/or inhibitory 
properties, may be either natural OCT peptides or analogs thereof with 
amino acid substitutions which are compatible with the requisite binding 
and inhibitory activities of the peptide. This section describes 
constraints for amino acid substitutions in binding/inhibitory OCT peptide 
analogs. 
A. Primary Structure Constraints 
Based on a sequence homology analysis of seven of the peptides whose full 
sequences are known (FIG. 1), the active natural OCT peptides were grouped 
into two distinct groups, each with internal homologies distinct to that 
group, as can be appreciated from FIG. 1. The two groups are designated 
"M," including the MVIIA, MVIIB, and SVIB peptides, and "G," including the 
GVIA, GVIIA, RVIA, and SVIA peptides. 
The two groups of OCT peptides are arranged in FIG. 1 with their six Cys 
residues aligned, which places these residues at positions 1, 8, 15, 16, 
20, and 28. To make this alignment, a gap was introduced at the 18 
position of the G-group peptides, and at the 23 and 27 positions of the 
M-group peptides. In the analysis below, these gaps retain the assigned 
number shown in FIG. 1, even though they represent amino acid deletions in 
the respective groups of active OCT peptides. 
The sequence variation in the two peptide groups, based on primary 
structure considerations alone, was analysed by adopting the following 
constraints: 
1. The peptides in both groups include the Cys residues at position 1, 8, 
15, 16, 20, and 28. Other Cys residues may be substituted at the positions 
indicated below only if they are selectively protected during oxidation of 
the peptide to form the three disulfide linkages. 
2. The peptides in both groups include three disulfide linkages connecting 
the Cys residues at positions 1 and 16, 8 and 20, and 15, and 30. As 
described above, the disulfide bridges are preferably formed by air 
oxidation of the full sequence peptide in the presence of DTT without 
Cys-residue protection, but may also be formed by selective deprotection 
of each pair of Cys residues. 
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 excludes 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. 
3. The SVIA peptide sequence is used as a negative predictor, based on its 
low binding and inhibitory properties, as discussed above. This was done 
by including amino acid variations which are consistent among the other 
group-G peptides, but inconsistent with the SVIA sequence, and conversely, 
by excluding variations found in the SVIA peptide which are not found in 
the other group-G peptides. 
4. Within each of the two peptide groups, the residue positions which are 
invariant are most likely to be retained. Thus, for the G group peptides, 
the position 2 Lys, position 4 4Hyp, position 5 Gly, position 19 Ser, 
position 23 Tyr, and the position 26 Lys are retained, so that the G-group 
peptides have a total of 12 invariant residues (plus a deletion at position 
18). Similarly, among the M-group peptides, the position 2 Lys, position 4 
Lys, position 5 Gly, position 13 Tyr, position 14 Asp, position 18 Gly, 
position 19 Ser, position 22 Arg, position 25 Gly, and position 26 Lys are 
retained, for a total of 16 retained positions (plus deletions at positions 
23 and 27). It is possible, of course, that invariant residues may be 
substituted by closely related amino acids, such as an Arg to Lys 
substitution, or a Tyr to Phe substitution. 
5. At each position within the two groups which exhibit variance, i.e., 
represented by two or more different amino acid variations, the variants 
are analysed for membership in a common amino acid class. The classes 
which were selected are six standard 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 (Schwartz). The six classes are Class I: Cys; Class II: 
Ser, Thr, Pro, 4Hyp, Ala, and Gly, representing small aliphatic side 
chains and OH-group side chains; Class III: Asn, Asp, Glu, and Gln, 
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: Ile, Val, and Leu, representing branched aliphatic side 
chains, and Met; and Class VI: Phe, Tyr, and Trp, representing aromatic 
side chains. In addition, each group may include related amino acid 
analogs, such as ornithine, homoarginine, N-methyl lysine, dimethyl 
lysine, or trimethyl lysine in class IV, and halogenated tyrosine in Group 
VI. Further, the classes may include both L- and D-stereoisomers, although 
L-amino acids are preferred for substitutions. 
6. If the variants in natural peptides can be placed in a single class, the 
most probable amino acid substitution that can occur at that position 
involves a member of the identified class. In the G-group peptides, these 
single-class variations are found at position 3 (class II), position 11 
(class II), and position IV (class III). In the M-group peptides, the 
single-class variations are found at position 24 (class II). 
7. If the variants in natural peptides do not fall within a single class, 
the variants are examined for other common properties which may be shared 
by other members of two or more of the above classes. In particular, if 
the natural variants are in two or more of the groups II, III IV, all of 
which are relatively polar amino acids, substitution is permitted among 
all members of the three classes. Similarly, if the natural variants are 
in groups V and VI, both of which are relatively nonpolar, substitution 
may occur within all members of theses two groups. In the G-group 
peptides, these groupings are present at position 9 (polar), position 17 
(polar), position 23 (polar), and position 29 (polar). In the M-group 
peptides, these groupings are present at position 6 (polar), and position 
21 (polar). 
9. If variants are found in both polar and nonpolar groups, the position is 
considered open to substutition. In the G-group peptides, these open 
positions are 10, 12, 13, and 21. In the M-group peptides, the 
open-substitution positions are 3, 11, and 12. The substitutions 
preferably include L-amino acids. 
10. Positions 25, 27, 29, or 30 of the G-group peptides may include a 
deletion. Positions 21, 23, or 24 of the M-group peptides may include a 
deletion. 
11. The C-terminal amino acid residue of the peptide is preferably 
amidated. 
B. Binding and Charge Constraints 
The necessary binding regions or domains of OCT peptides have been examined 
by binding studies on the OCT peptide fragments shown in FIG. 2. The 
binding constants for the several peptides, shown in Table 3 in Example 4, 
were determined from competitive displacement of MVIIA peptide in 
electricorgan synaptosomes, as previously described. 
The peptide fragments whose pK.sub.i 's are greater than 6 are those 
labeled 170, 162, 173, and 174. All of these peptides have the first 9 
N-terminal amino acids of M-group OCT peptides, indicating that this 
region, which contains three Lys groups, is critical for binding 
activities in fish electric organ synaptosomes. Similarly, peptide 
fragments that did not include the first 9 N-terminals generally showed 
low binding activities in fish synaptosomes. Additional binding studies 
carried out in support of the invention indicate that substitutions of 
acidic amino acids (Glu, Asp) at any of the first 10 N-terminal amino acid 
residues severely reduces binding activity. Therefore, another constraint 
which is imposed on the amino acid variations given above is that Asp and 
Glu substitutions are forbidden at the 1-10 N-terminal positions. 
The net charge of OCT peptides has been plotted as a function of pH for 
several of the natural peptides, where the C-terminal amino acid is 
amidated. The plots, which are shown in FIGS. 10A-10B were constructed 
using standard pK's of the amino acids. As seen from the figures, all of 
the sequences examined which are active OCT peptides (all OCT peptides 
except SVIA) have isoelectric points between about 10.5 and 12. A further 
constraint, therefor, which may be imposed on the selection of amino acid 
variations in active peptides is the requirement for an isoelectric point 
between about 10.5 and 12. The M-group peptides (FIGS. 10E-10G) may be 
further constrained by the requirement for an isoelectric point between 11 
and 12. 
C. Secondary Structure Constraints 
Several of the natural OCT peptide sequences were examined for secondary 
structure properties, using computer analysis programs available from 
Intelligenetics (Palo Alto, Calif.). The secondary structure prediction 
program known as NOVOTNY (Novotny) calculates the relative probability for 
a residue to adopt a helical or extended (.beta.-sheet configuration), and 
the likelihood that the residue is part of a .beta.-turn. The tendency to 
form helical or sheet states is determined using the method of Chou and 
Fasman (Chou), and this is then compared to the charge profile of the 
sequence (this is simply a map of the location of the charged residues and 
their associated charge). The probability values are averaged over groups 
of consecutive residues in a moving average to reduce random statistical 
fluctuations. 
Secondary structure predictions can be made from these calculations. For 
example, regions having high .beta.-turn potential, and charged residues, 
associated with low helix, sheet, and hydrophobicity probabilities are 
likely to be in a .beta.-turn configuration. This is a non-quantitative 
method: it utility lies in comparing the relative tendencies of residues 
to hold different configurations, thus serving as a guide with which to 
judge possible secondary structures. 
The analyses carried out with six of the OCT peptide sequences using the 
NOVOTNY program are shown in FIGS. 11A-11G. The alpha-helix and beta-sheet 
plots were considered only as features related to the folding of the 
peptide, prior to forming disulfide bridges, rather than as structures 
present in the mature peptide. 
The plots in FIG. 11 were examined for particularly common secondary 
structure features. The most prominent of these are: 
(a) a relatively low probability of alpha-helix and beta-sheet formation in 
the first 10 amino acid positions, and a correspondingly high likelihood of 
beta turn in the same region; 
(b) a relatively low hydrophobicity over the first 8-10 amino acid 
positions, and an hydrophobicity peak in the region approximately between 
positions 12-15; and 
(c) for the G-group peptides, a second hydrophobicity peak in the region 
approximately between positions 19-25. 
None of the above features are unique to the active peptides--i.e., present 
in the GVIA, GVIIA, RVIA, MVIIA, SVIB, and MVIIB, but not the SVIA, 
peptide--and therefore provide useful, but not sufficient constraints for 
evaluating possible amino acid substitutions which are consistent with 
binding/inhibitory activity in OCT peptide. 
D. Tertiary Structure Constraints 
The MVIIA peptide, which is exemplary of M-group OCT peptides, was studied 
by nuclear magnetic resonance (NMR) spectroscopy, to define distance 
relationships between selected proton sites in the peptide. The NMR method 
known as Two-Dimensional Nuclear Overhauser Effect (2D-NOE, or NOESY), was 
used. The NOE NMR phenomenon is based on the perturbation of the intrinsic 
magnetic fields of hydrogen nuclei by the presence of nearby hydrogen 
nuclei. The perturbation effect is transmitted through the space between 
the nuclei, and is inversely proportional to the sixth power of the 
distance between nuclei (Kessler). By measuring the NOE of the hydrogen 
nuclei in the sample, and correlating this measurement with the amino 
acids to which the hydrogen nuclei are attached, distance relationships 
between pairs of hydrogen nuclei, typically associated with the amide 
nitrogen proton, can be determined. The maximum proton/proton spacing 
which can be detected is about 5 angstroms. 
The following pairs of amino acids given below represent proximate amino 
acid pairs in which the amide hydrogens (or otherwise as indicated) in the 
paired residues give detectable long-range (about 4.5 angstrom) or 
shortrange (about 2.5 angstrom) distance couplings: 
Long-range: 
Lys (position 2) and Cys (position 16) 
Gly (position 5) and Cys (position 28) 
Lys (position 26) and Cys (position 20) 
alpha H Cys (position 28) and Gly (position 18) 
beta H Cys (position 28) and Lys (position 2) 
alpha H Lys (position 26) and Cys (position 8) 
beta H Cys (position 1) and Cys (position 16) 
beta H Cys (position 28) and Gly (position 5) 
beta H Cys (position 28) and Gly (position 18) 
Short-range: 
Ser (position 24) and Gly (position 25) 
Asp (position 14) and Cys (position 15) 
Tyr (position 13) and Asp (position 14) 
Gly (position 25) and Lys (position 26) 
These distance constraints can be used to generate a minimum-energy 3-D 
configuration of the protein using one of a variety of commercially 
available molecular dynamics (MD) programs, such as the computer modelling 
program available from by Biosym.TM. (San Diego, Calif.). 3-D models of the 
MVIIA have been generated using the Biosym.TM. program. Amino acid 
substitutions can be tested in this modelling system by allowing the 
system to reach a new energy minimum, with the substituted amino acid 
residue(s), and determining whether the above distance constraints are 
preserved. 
The above subsections A-D provide criteria for selecting amino acid 
substitutions in natural binding/inhibitory OCT peptides. Assuming a 
proposed amino acid substitution meets the criteria set forth in these 
subsections, the peptide can be synthesized and tested for in vitro 
binding or inhibitory activity, as described in Section II, to confirm 
that the peptide has the desired binding/inhibitory properties. 
The following examples are intended to illustrate methods for preparing OCT 
peptides, test methods for determining in vitro and in vivo binding and 
inhibitory activities, and exemplary treatment results. The examples are 
in no way intended to limit the scope of the invention. 
EXAMPLE 1 
Preparation of MVIIA OCT Peptide 
Abbreviations used in this example are BOC, tertiary butoxycarbonyl; DCM, 
dichloromethane; TFA, trifluoroacetic acid; IPM, N-isopropylmorpholine; 
BOC-AA-OH, BOC amino acid; DIEA, diisopropylethylamine; 2-ClZ, 
2-Chlorobenzyloxycarbonyl; tosyl, p-toluenesulfonyl; DMF, 
N,N-dimethylformamide; TFE, trifluoroethanol; SA, symmetrical anhydride of 
BOC-AA-OH; DCCI, N,N-dicyclohexylcarbodiimide; DCM, dichloromethane; E, 
ethyl ether; P, petroleum ether; 2-BrZ, 2-bromobenyloxycarbonyl. 
Commercially available benzhydrylamine-resin hydrochloride, Lot No. B30101, 
was obtained from Beckman Instruments Inc., Palo Alto, Calif. With this 
resin, cleavage of a peptide formed on the resin, under the conditions 
described below, produces a peptide which is amidated at its carboxy end. 
A. Preparing Protected Amino Acid Anhydrides 
Each BOC-AA-OH (2.4 mmol) was dissolved in 5 ml DCM and cooled to 0.degree. 
C. The volume of DCM used for BOC-Leu-OH (dried in vacuo) was 12 ml, and 
the BOC-Leu-OH solution was not cooled. 2 ml 0.6M DCCI in DCM was added 
and the mixture stirred at 0.degree. C. for 15 min. For BOC-Leu-OH, the 
mixture was also cooled after this addition. Precipitation of 
N,N,-dicyclohexylurea was completed by storage at -20.degree. C. for 1.5 
hour, after which the precipitate was filtered and washed with ethyl ether 
(5 ml). The filtrate was evaporated to remove solvents and the product was 
crystallized in the solvent system given in Table 1. Residual amounts of 
DCM can affect the exact conditions for crystallization. Recrystallization 
was performed by dissolving in DCM, evaporating most of the solvent, and 
recrystallizing from the appropriate solvent. 
TABLE 1 
______________________________________ 
Amino Acid Solvent 
______________________________________ 
Ala DCM 
Asp (Benzyl) E:P 
Cys (4-MeBenzyl) DCM 
Gly E:P 
Leu P 
Lys (2-ClZ) E:P 
Met E:P 
Ser (Benzyl) E:P 
Thr (Benzyl) E:P 
Tyr (2-BrZ) DCM 
______________________________________ 
B. Coupling Procedure 
Synthesis of MVIIA peptide was performed in a Beckman Model 990 Peptide 
Synthesizer by a solid-phase method based on the following primary 
structure: 
HCysLysGlyLysGlyAlaLysCysSerArgLeuMetTyrAspCysCysThrGlySer 
CysArgSerGlyLysCysNH.sub.2. 
A double coupling protocol was used for the incorporation of residues 
Cys-28 through Tyr-13, and a triple coupling protocol, for amino acids 
Met-12 through Cys-1. Symmetrical anhydrides were used in crystalline form 
according to published methods described in (Yamashiro). Crystalline 
symmetrical anhydrides (1.0 mmole) were each dissolved in 6 ml DCM and 
stored in the amino acid reservoirs at 4.degree. C. Side-chain protecting 
groups used were: Cys, 4-MeBenzyl; Lys, 2-ClZ; Ser, Benzyl; Arg, Tosyl; 
Thr, Benzyl; Asp, Benzyl; Tyr, 2-BrZ. 
Unless specified, volumes were 8 ml, except for step 2 below, which was 10 
ml, and all reactions were carried out at room temperature. After 
incorporation of the Asp-14 residue, the volume of step 2 was increased to 
15 ml while all other volumes were raised to 10 ml after incorporation of 
the Arg-10 residue. The double coupling protocol consisted of steps 1-16 
listed in Table 2 below. 
Amino acids Met-12 through Cys-1 were added by a triple coupling protocol 
which included, in addition to steps 1-16, steps 17-19 in Table 2. 
TABLE 2 
______________________________________ 
Step Reagent 
______________________________________ 
1 DCM wash (3 times) 
2 67% TFA/DCM (20 min.) 
3 DCM wash (2 times) 
4 25% dioxane/DCM wash (2 times) 
5 5% DIEA/DCM wash 
6 DCM wash 
7 5% DIEA/DCM wash 
8 DCM wash (5 times) 
9 1.0 mmol SA in DCM (5 min) 
10 0.5 mmol IPM in 3 ml TFE plus 1 ml DCM (5 min) 
11 0.5 mmol IPM in 5 ml DCM (5 min) 
12 DMF wash (3 times) 
13 1.0 mmol SA in DMF (5 min) 
14 0.5 mmol IPM in 5 ml DCM (5 min) 
15 0.5 mmol IPM in 4 ml DMF (5 min) 
16 DCM wash 
17 DCM wash (2 times) 
18 1.0 mmol SA in DCM (5 min) 
19 0.5 mmol IPM in 4 ml DMF (5 min) 
______________________________________ 
Crystalline symmetrical anhydrides (1 mmole) were each dissolved in 6 ml 
DCM or DMF and stored in the amino acid reservoirs at 4.degree. C. 
Side-chain protecting groups used were: Cys, 4-MeBzl; Lys, 2-ClZ; Ser, 
Bzl; Arg, tosyl; Thr, Bzl; Asp, Bzl; Tyr, 2-BrZ. 
For BOC-Arg(tosyl)-OH, the following mixture was prepared: 1.87 
BOC-Arg(tosyl)-OH, 0.57 g 1-hydroxybenzotriazole, 15 ml DMF, stirred to 
dissolve, cooled to 4.degree. C., added 0.52 ml diisopropylcarbodiimide, 
and split in half for steps 9 and 13. For this coupling, the protocol was 
modified as follows: step 8 was 3 times DCM wash and 2 times DMF wash; 
step 9 was for 10 min; step 11 was for 10 min; step 13 was for 10 min; 
step 14 was 0.4 mmol IPM in 4 ml DMF for 10 min; step 15 was for 10 min; 
step 16 was 1 times DMF wash and 1 time DCM wash. Reaction mixtures in 
steps 9, 10, 13, 14 and 18 were not drained. 
The mixture for a third coupling for incorporating the Arg-10 residue 
consisted of 1.00 g BOC-Arg(tosyl)-OH, 1 ml DMF, 5 ml DCM, stirred to 
dissolve, and cooled to 4.degree. C. to which is then added 1.67 ml 0.6M 
DCCI in DCM. 
After the last amino acid had been incorporated, the protected peptide 
resin was subjected to steps 1-4 to remove the N-terminal BOC group, 
collected on a filter with use of ethanol, and dried in vacuum to yield 
2.61 g. 
C. Deblocking and cleavage in liquid HF 
A mixture of protected peptide resin (1.32 g), 2-mercaptopyridine (0.50 g), 
p-cresol (2.6 g), and liquid hydrogen fluoride (HF) (25 ml) was stirred at 
0.degree. C. for 80 min. The liquid HF was evaporated with a rapid stream 
of nitrogen gas, first below 0.degree. C., then at 24.degree. C. The 
mixture was stirred in ethyl acetate (25 ml) until a finely divided solid 
was obtained. The solid was filtered, washed with ethyl acetate, and air 
dried to yield 1.09 g. This solid was stirred in 50% aqueous acetic acid 
(10 ml) to dissolve the peptide material, filtered, and washed with 20 ml 
water. The filtrate was freeze-dried to yield 450 mg of fluffy powder. 
D. Formation of disulfide bridges 
A sample (300 mg) of the fluffy powder was dissolved in 30 ml of 0.05M 
ammonium bicarbonate, 10 mM dithiothreitol (DTT), and 2M guanidine 
hydrochloride. The solution, which had a pH of 6.7, was allowed to stand 
at 24.degree. C. for 2 hr, then diluted with 120 ml of water and stirred 
for 20 hr at 24.degree. C. DTT (25 mg) was added and the solution allowed 
to stand at 24.degree. C. for 80 min. The mixture was then stirred at 
4.degree. C. for 3 days. 
E. Isolation of MVIIA OCT 
The solution from Part D was acidified with glacial acetic acid (2 ml), 
evaporated in vacuo to a low volume, and fractionated by gel filtration on 
Sephadex G-25 in a 2.5.times.48 cm column, using 1N acetic acid, to remove 
peptide polymeric species (exclusion volume), and salts (slowest moving 
peak). Fractions (5 ml) were collected, with peptide absorbance monitored 
at 280 nm. Fractions corresponding to the monomer peptide were pooled and 
freeze-dried to give 127 mg of fluffy powder. A sample of the monomeric 
material (34 mg) was purified by preparative HPLC on a Vydac 218TP1022 
column with a gradient of 10-20% acetonitrile in 0.1% trifluoroacetic acid 
over 50 min at 8 ml/min, with detection at 226 nm and collection of 4 ml 
fractions. Fractions corresponding to the major peak were pooled, 
evaporated in vacuo to remove acetonitrile, and freeze-dried to yield 7.7 
mg. Analytical HPLC on a Vydac 218TP104 column with the same solvent and 
gradient over 10 min followed by 10 min of isocratic elution at the 20% 
composition (1.5 ml/min) gave a single peak identical in behavior to an 
authentic sample of OCT MVIIA. Amino acid analysis of a 24-hr 
HCl-hydrolysate gave: Asp, 0.93; Thr, 1.05; Ser, 2.85; half-cystine, 5.2; 
Gly, 4.08; Ala, 1.07; Met 0.94; Leu, 1.02; Tyr,0.85; Lys, 3.98; Arg, 2.09. 
F. Radio-Iodination of MVIIA 
MVIIA peptide was iodinated by reaction with Iodogen.TM. in the presence of 
NaI according to Cruz et al., with minor modification. 2 m Ci of 
carrier-free Na.sup.125 I, 75 ul 0.5M phosphate buffer pH 7.4 and 20 ul of 
1 ug/ul peptide were added to a polypropylene test tube coated with 10 ug 
Iodogen.TM.. The tube was agitated for 8 minutes, and the solution was 
chromatographed by HPLC through a 10.times.0.46 cm C-8 reverse phase 
column with a pore size of 300 .ANG. (Brownlee Labs, Santa Clara, Calif.). 
The sample material was eluted with a gradient from 0.1% trifluoroacetic 
acid to 60% acetonitrile in 0.1% trifluoroacetic acid. The major peak of 
active radio-iodinated peptide was resolved at about 2 minutes greater 
retention time than the underivatized peptide. 
The fractions containing this peak were collected and later diluted for use 
in binding experiments. MVIIA, iodinated under the conditions as above 
except with non-radioactive NaI, was tested for the ability to inhibit 
depolarization-dependent ATP release from synaptosomes, using the method 
described in (Ahmad). The iodinated MVIIA peptide was found to be as 
potent in this regard as the underivatized peptide. 
EXAMPLE 2 
Preparation of GVIA, SVIA, GVIIA, SVIB, and RVIA OCT Peptides 
Syntheses of GVIA, SVIA, MVIIB, GVIIA, and RVIA OCTs were based on the 
following sequences, which are shown in FIG. 1: 
GVIA: 
HCysLysSer4HypGlySerSerCysSer4HypThrSerTyrAsnCysCysArgSerCysAsn4HypTyrLysA 
rgCysTyr-NH.sub.2. 
SVIA: H-CysArgSerSerGlySer4HypHisCysGlyValThrSerIleCysCysGly 
ArgCysTyrArgGlyLysCysThr-NH.sub.2. 
MVIIB: H-CysLysGlyLysGlyAlaSerCysHisArgThrSerTyrAspCysCysThr 
GlySerCysAsnArgGlyLysCys-NH.sub.2. 
GVIIA: H-CysLysSer4HypGlyThr4HypCysSerArgGlyMetArgAsp 
CysCysThrSerCysLeuLeuTyrSerAsnLysCysArgArgTyr-NH.sub.2. 
RVIA: H-CysLysPro4HypGlySer4HypCysArgValSerSerTyrAsnCys 
CysSerSerCysLysSerTyrAsnLysLysCysGly-NH.sub.2. 
SVIB: 
H-CysLysLeuLysGlyGlnSerCysArgLysThrSerTyrAspCysCysSerGlySerCys-GlyArgSerGl 
yLysCys-NH.sub.2. 
Synthesis of each OCT was performed according to the solid-phase method 
described in Example 1, except that a single coupling protocol involving 
steps 1-12 in Example 1, Part C was used for coupling the first 10 
C-terminal amino acids residues, and a double coupling method involving 
steps 1-16 in Example 1, Part C was used for coupling the remaining 
N-terminal residues.oosefi Releasing the peptide from the solid support, 
removing the blocking groups, and joining the disulfide bridges were 
carried out substantially as above. The peptide was separated from salts 
and polymeric peptide species by gel filtration on Sephadex G-25, and 
purified on preparative HPLC as described in Example 1. For binding 
studies, each peptide can be radioiodinated essentially as above. 
EXAMPLE 3 
Preparation of OCT Peptide Fragments 
The following peptide sequences, representing the aligned portions of the 
MVIIA OCT peptide, as shown in FIG. 2, were synthesized, where boldface A 
is Ala replacing Cys, and X is 4Hyp: 
##STR1## 
The following peptide sequences, representing the aligned portions of the 
GVIA OCT peptide, were synthesized: 
##STR2## 
Synthesis of each OCT peptide fragment was performed according to the 
solid-phase method described in Example 1, except that a single coupling 
protocol involving steps 1-12 in Example 1, Part C was used for coupling 
all of the residues in the each fragment. 
Releasing the peptide fragment from the solid support, removing the 
blocking groups, and joining the disulfide bridges were carried out 
substantially as above. The peptide fragments were purified on preparative 
HPLC as described in Example 1, Part E. 
EXAMPLE 4 
Competitive Peptide Binding to Fish Synaptosomes 
A. Preparation of Fish Synaptosomes 
Electric organ synaptosomes were prepared by dissection from marine 
electric rays (Ommata dyscopyge) that had been stunned with 0.25 g/liter 
tricaine HCl and cooled to 4.degree. C. immediately prior to dissection. 
All subsequent manipulations were carried out at 0.degree.-4.degree. C. 
whenever possible. Organs were diced and homogenized for 4 15-second 
periods in a Waring blender with an equal weight of plain synaptosome 
buffer (PSB) consisting of 280 mM NaCl, 3 mM KCl, 1.8 mM MgCl.sub.2, 300 
mM urea, 100 mM sucrose, and 5.5 mM glucose in 20 mM HEPES containing 5 mM 
Mg-EGTA at pH 7.2. 
The homogenate was loaded into a 60 ml syringe, force filtered through a 
40-mesh stainless-steel screen and centrifuged at 30,000.times.g for 15 
min. The supernatant was discarded and the pellet taken up in 20 ml PSB. 
The resuspended pellet was further disrupted with 5 strokes of a 
loose-fitting Teflon pestle in a glass homogenizer and then centrifuged at 
400 rpm. The resulting suspension was centrifuged once more at 
30,000.times.g for 15 min. The supernatant was discarded at the pellet 
resuspended with the Teflon-glass homogenizer by 10 passes of the pestle 
at 400 rpm. This homogenate was layered onto six 32 ml 3-20% Ficoll 
gradiants in PSB and centrifuged at 76,6000.times.g for 1 hour in a 
swinging bucket rotor. The synaptosome band (the first band below the 
buffer-gradient interface) of each gradient was aspirated off and diluted 
at more than 1:1 with PSBG25. The diluted synaptosome suspension was 
pelleted at 30,000.times.g for 15 min and resuspended in 1 ml PSB. 
B. Competitive Binding Assay 
The OCT peptides and peptide fragments whose synthesis is described in 
Examples 1-3 were tested for binding to elasmobranch electric organ 
synaptosomes (also referred to herein as fish synaptosomes), using a 
competitive binding assay based on displacement of radiolabeled OCT MVIIA 
by the test peptide or peptide fragment. Total binding was measured using 
.sup.125 I-MVIIA OCT alone, and non-specific binding was determined by 
measuring .sup.125 I-MVIIA OCT bound in the presence of 1 uM unlabeled 
MVIIA. The difference between these values constituted .sup.125 I-MVIIA 
OCT specifically bound to OCT binding sites. 
The binding constant (K.sub.d) for MVIIA OCT to fish synaptosomes was 
determined by a saturation binding method in which increasing quantities 
of the OCT were added to aliquots of a synaptosome preparation. The amount 
of labeled peptide specifically bound at each concentration was used to 
determine B.sub.max, the concentration of specific binding sites on the 
synaptosomes, and K.sub.d, following standard binding analysis methods 
(Bennett). 
In the competitive binding assays, binding was initiated by adding a 
quantity of synaptosomal membranes (5-10 .mu.g protein) suspended in 400 
ul binding medium to a series of tubes containing both 50-275 pM 
125I-MVIIA and test substances ranging in concentration from approximately 
10.sup.-3 M to 10.sup.-13 M. An additional tube containing 1 uM unlabeled 
MVIIA OCT was included to measure non-specific MVIIA binding. Binding 
medium consisted of 0.1 mM EDTA, 0.1 mM EGTA, 1 mM PMSF, 2 uM leupeptin, 1 
uM pepstatin, and 125 mM NaCl in 20 mM HEPES (pH 7.4) containing 0.1% 
gelatin. Final assay volume was 500 ul. Assay tubes were incubated for 100 
minutes at 4.degree. C. on a rotating mixer. 
Separation of bound .sup.125 I-MVIIA OCT from unbound peptide was achieved 
by rapid vacuum filtration of 150 ul aliquots of binding mixture from each 
tube through Whatman GF/C glass-fiber filters presoaked in an aqueous 
solution containing 0.6% polyethylenimine. Tissue-bound .sup.125 I-MVIIA 
OCT retained on the filters was washed three times with 3.5 ml of ice-cold 
wash buffer consisting of 20 mM HEPES (pH 7.4), 125 mM NaCl, and 0.1% 
gelatin. Filters were dried and their radioactivity measured in an LKB 
gamma counter at 75% counting efficiency. 
The binding constant K.sub.i for each test substance was calculated using 
non-linear, least-squares regression analysis of competitive binding data 
from 2 assays performed in duplicate on separate occasions, and employing 
the K.sub.d value determined for the MVIIA OCT, according to reported 
K.sub.i analysis procedures (Bennett). Approximate K.sub.i values and 
corresponding pKi values for the test peptides are shown in the table. 
TABLE 3 
______________________________________ 
Compound Ki pKi 
______________________________________ 
MVIIA 0.40 nM 9.4 
MVIIB 22 nM 7.66 
GVIA 14 nM 7.85 
SVIA &gt;&gt;1 mM &lt;3 
RVIA 324 nM 6.45 
GVIIA 3.1 nM 8.51 
SVIB 3.0 nM 8.52 
MVIIA 170 280 nM 6.55 
MVIIA 171 4.6 .mu.M 5.33 
MVIIA 161 &gt;&gt;1 mM &lt;3 
MVIIA 160 120 .mu.M 3.92 
MVIIA 162 170 nM 6.77 
MVIIA 174 530 nM 6.28 
MVIIA 172 20.9 .mu.M 4.68 
MVIIA 173 720 nM 6.14 
MVIIA 179 &gt;&gt;1 mM &lt;3 
MVIIA 163 217 .mu.M 3.66 
MVIIA 175 3.6 .mu.M 5.44 
MVIIA 176 1.8 .mu.M 5.74 
GVIA 177 &gt;&gt;1 mM &lt;3 
GVIA 180 123 .mu.M 3.91 
______________________________________ 
EXAMPLE 5 
Competitive Peptide Binding to Mammalian-Brain Synaptosomes 
A. Preparing Rat Brain Membranes 
Rodents were sacrificed by cervical dislocation and their brains quickly 
removed, weighed, then transferred to approximately 10 ml ice-cold 0.32M 
sucrose, and homogenized two brains at a time with a motor-driven Teflon 
glass homogenizer. Homogenates were transferred to two 40 ml centrifuge 
tubes and spun at 950.times.g for 10 minutes at 4.degree. C. Supernatants 
were transferred to two 40 ml tubes and recentrifuged at 8,500.times.g for 
15 minutes. The resulting supernatants were discarded and the pellets were 
resuspended in 10 ml ice cold 0.32M sucrose by vortexing. Suspensions were 
combined and recentrifuged at 8,500.times.g for 15 minutes. After removing 
and discarding the supernatant, the pellet was resuspended in 20 ml 
ice-cold 0.32 M sucrose plus protease inhibitors, and the mixture was 
layered in 5 ml aliquots over four stepped sucrose density gradients in 35 
ml ultracentrifuge tubes. Centrifugation was at 150,000.times.g for 60 
minutes at 4.degree. C. in a swinging bucket rotor. 
After centrifugation, top layers were aspirated off; the 1.0M sucrose layer 
and the material at the 1.0M-1.2M sucrose interface were collected, and 
centrifuged at 30,000.times.g for 15 minutes. Pellets were resuspended in 
3 volumes of ice cold water, (Polytron setting 6, 10 seconds) then 
recentrifuged at 40,000.times.g for 15 minutes. Supernatants were 
discarded and pellets were resuspended in 5 ml ice-cold distilled water. 
Aliquots of this suspension were frozen at -120.degree. C. until used. 
The competitive binding assay was performed substantially as described in 
Example 4B, for MVIIA OCT. Computer-fit competitive binding curves for 2 
assays performed in duplicate on separate occasions are shown in FIG. 3. 
Assuming a single binding site, a four-parameter logistic function fitted 
to the data yielded the IC.sub.50 values (OCT peptide concentration at 
which half the labeled NVIIA OCT is displaced by the test peptide) shown 
below in Table 4. 
TABLE 4 
______________________________________ 
OCT peptide IC.sub.50 [nM] 
______________________________________ 
MVIIA 0.019 
MVIIB 0.49 
GVIA 0.48 
SVIB 5.7 
GVIIA 22.9 
RVIA 229. 
SVIA &gt;&gt;1 mM 
______________________________________ 
EXAMPLE 6 
Binding of MVIIA to Cultured Neuronal Cells 
A. Preparing Cultured Neuronal Cells 
A variety of human and rat cultured cells were examined for their ability 
to bind MVIIA OCT, to identify cell types which are rich in OCT receptor 
sites. 
The human and rat cell types indicated at the right in Table 5 were grown 
in a culture medium containing 10% fetal bovine serum. Cells were frozen 
in medium containing 10% DMSO, stored in liquid nitrogen until use, and 
rapidly thawed and diluted 10 fold in phosphate buffered saline (20 mM 
sodium phosphate, 150 mM NaCl, pH 7.4). All procedures were carried out at 
4.degree. C. The cells were then centrifuged at 1300.times.g for 10 min. 
The supernatant was decanted, and the cells were resuspended in 3 volumes 
(relative pellet volume) distilled water. The cells were broken and 
homogenized using a Polytron mixer (10 seconds), then centrifuged at 
24,500.times.g for 10 min. The resulting membrane pellet was resuspended 
in 3 volumes distilled water, and used in the binding assay. Protein 
concentrations were estimated using the BioRad protein assay reagent. 
For binding, cells were suspended in a mixture containing 20 mM HEPES 
buffer pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, (PMSF), 2 uM leupeptin, 1 uM 
pepstatin, 125 mM NaCl, 0.1% gelatin. Radiolabeled MVIIA OCT was added at 
a concentration of 1 nM. Nonspecific binding was estimated by inclusion of 
3 uM unlabeled MVIIA in replicate tubes. The results are shown in Table 5 
below. 
TABLE 5 
______________________________________ 
(fmol/mg. prot.) 
Cell Type 
______________________________________ 
Kelly 44.7 Human neuroblastoma 
NG-108 45.6 Rat glioma x mouse 
neuroblastoma 
IMR-32 369.1 Human neuroblastoma 
SKNSH 27.1 Human neuroblastoma 
SY5Y(-RA).sup.a 
46.8 Human neuroblastoma 
SY5Y(+RA).sup.a 
135.4 Human neuroblastoma 
PC-12(-NGF).sup.b 
56.6 Rat pheochromocytoma 
PC-12(-NGF).sup.b 
79.1 Rat pheochromocytoma 
293 &lt;4.6 Human kidney 
______________________________________ 
EXAMPLE 7 
Inhibition of Ionic Currents 
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 have been examined. 
A. Current Measurement Methods 
Most measurements were obtained using a bath saline that allowed 
examination of the calcium currents in the absence of other ionic 
currents. These solutions contained 80 mM NMDG (as a sodium replacement), 
30 mM TEACl (to block potassium currents), 10 mM BaCl.sub.2 (as a 
charge-carrier through the calcium channels), and 10 mM HEPES at pH 7.3. 
Some solutions also contained 2 mM quinidine (to block potassium currents) 
and 3 .mu.M tetrodotoxin (to block sodium currents). Normal bath saline was 
(mM): 140 NaCl, 10 glucose, 3 KCl, 2 CaCl.sub.2, 1 MgCl.sub.2, 10 mM HEPES 
pH 7.3. Intracellular solutions contained 150 mM CsCl, 0.5 mM CaCl.sub.2, 
5 mM EGTA, 5 mM MgCl.sub.2, 2 mM K.sub.2 ATP 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, Claremont, 
Calif. 91711), coated with Sylgard (Dow Corning, Midland, Mich. 48640) and 
fire-polished before use. Bubble numbers were typically 5 to 6, with pipet 
resistances typically 2-5 MOhms. Corning 8161, Kimble, and other glasses 
were also used without noticeable effect on the calcium currents observed. 
Recordings were carried out at room temperature with an Axopatch 1-C 
amplifier (Axon Instruments, Foster City, Calif. 94404) and analyzed with 
pCLAMP software (Axon Instruments). Data were filtered at 1000 Hz for a 
typical sampling rate of 0.1 kHz; in all cases data was filtered at a 
frequency at most 1/5 of the sampling rate to avoid aliasing. Data were 
collected on-line by the software. Analysis was performed on-screen with 
print-out via a Hewlett-Packard LaserJet Printer (Hewlett-Packard, Palo 
Alto, Calif. 94306). 
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 holding potentials usually covered the same 
range of test potentials. 
B. Current Inhibition Measurement 
FIG. 5A shows a calcium current trace from an N1E115 mouse neuroblastoma 
cell. The figure is read from left to right, with downward deflections of 
the trace indicating positive current flowing into the cell. Currents were 
elicited by a voltage step from -100 mV to -20 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 FIGS. 5B-5C show decreasing calcium currents, with 
increasing OCT peptide concentrations of 10 nM (5B), 50 nM (5C), and 200 
nM (5D). The response of voltage-gated calcium current to increasing 
dosages of OCTs MVIIA and GVIA are shown in FIG. 6. The calculated ED50 is 
12 nM for GVIA and 115 nM for MVIIA. These values indicate extremely high 
specificity of the pep ides for their site of action. 
EXAMPLE 8 
Inhibition of ATP Release from Fish Synaptosomes 
Electric-ray, electric-organ (fish) synaptosomes were prepared 
substantially as described in Example 4. The diluted synaptosome 
suspension from the final centrifugation step was pelleted at 
30,400.times.g for 15 min and resuspended in 1 ml of PBS (with the 
inclusion, for some experiments, of 1% BSA to enhance stability of the 
synaptasomes). This final synaptosome preparation was stored at 0.degree. 
C. and used for ATP release experiments within 30 hours. Storage for 
longer periods resulted in the almost complete loss of 
depolarization-dependent ATP release activity. 
Luminometry was performed by the method of Morel et al. (Morel), as 
modified by Schweitzer. Into a 5 ml polypropylene test tube were mixed 465 
.mu.l PSB, 5 .mu.l of 5 .mu.g/ml luciferin in PSB, 20 .mu.l firefly 
lantern extract (1 Sigma FLE-50 bottle reconstituted in 1 ml PSB and spin- 
dialyzed through 3 ml of Sephadex G-25 pre-equilibrated in PSB), 5 .mu.l 
100 mM CaCl.sub.2, and 5 .mu.l synaptosome suspension (5-7 mg/ml protein, 
excluding BSA). The tube was placed in the chamber of a custom-built 
luminometer and the light output produced by extracellular ATP was 
continuously monitored by a chart recording of the voltage generated by 
the photomultiplier tube. Exocytotic release of ATP was evoked by 
injecting 0.5 ml of high K+ buffer (PSB with equimolar replacement of Na+ 
by K+) into the reaction mixture in the luminometer. 
ATP release was quantitated by comparing the peak heights of unknowns with 
the heights of peaks generated by ATP standards that were injected into 
each reaction mixture at the end of each trial. Over the range 
investigated, light output was linear with respect to the amount of ATP 
injected. 
The results obtained are given in Table 6 below for neurotransmitter (ATP) 
release from synaptosomes prepared from electric organ of Ommata 
discopyge. 
TABLE 6 
______________________________________ 
Compound 
IC.sub.50 
______________________________________ 
MVIIA 30 nM 
GVIA 200 nM 
______________________________________ 
EXAMPLE 9 
Mouse Shaking Test for OCTs 
Male Swiss-Webster mice were given intracerebral (IC) injections of MVIIA, 
GVIA, or SVIA, or its vehicle, at doses between 0.01-10.0 ug/animal. 
During the ICV injection the animal was unanesthesized, using a method 
previously described (Haley and McCormick, 1956). Animals were observed 
for the presence or absence of shaking behavior 15 minutes following 
injection. The percentage of animals exhibiting this behavior is shown in 
Table 7. 
TABLE 7 
______________________________________ 
OCT dose n percent "shakers" 
______________________________________ 
vehicle 0.0 100 4.0 
MVIIA .01 30 23.3 
.03 60 31.7 
.1 60 60.0 
.3 50 78.0 
1.0 10 100 
GVIA .01 10 20.0 
.03 30 60.0 
.1 30 63.0 
.3 10 90.0 
SVIA .01 10 0.0 
.03 10 10.0 
.1 20 0 
.3 20 0 
1.0 20 10 
3.0 30 13.3 
10.0 10 10 
______________________________________ 
EXAMPLE 10 
Reduction in Anatomical Damage: 1 
A. Global Ischemia 
Global ischemic damage was examined in the gerbil model, according to 
standard procedures (Kirino). Male mongolian gerbils (Meriones 
unguiculatus, Tumblebrook Farm, West Brookfield, Mass.) weighing 50-80 g 
were anesthetized in a small chamber with 4% halothane carried by 70% 
nitrous oxide (0.44 L/min) and 30% oxygen (0.19 L/min). They were then 
maintained throughout surgery with 2% halothane by placing their noses 
through a hole in a rubber dam on a gas delivery tube. Using aseptic 
techniques, 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. 
During or after the occlusion, an intracerebroventricular (ICV) injection 
aimed at the lateral ventricle was made. To accomplish this, 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. It was advanced until 
the sleeve abutted the skull, and 5 microliters of injectate was infused 
over a period of approximately 3 sec. The skin was then sutured shut. 
Occluded animals received either drug or its vehicle. Injected, unoccluded 
controls were anesthetized, and received the ICV injection only. 
B. Histological Examination of Gerbil brains 
Animals were anesthetized with CO.sub.2. The chest cavity was opened and 
the animal was perfused through the heart with approximately 3 milliliters 
of phosphate-buffered saline (PBS; 0.10M sodium phosphate; 0.15M sodium 
chloride) containing heparin (10 Units/ml), followed by approximately 10 
ml of Zamboni's fix (15% (vol/vol) picric acid 4% (wt/vol) 
paraformaldehyde in 0.1M phosphate buffer pH 7.4. Brains 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.degree. C. The block containing the hippocampus was frozen 
with liquid Freon onto a cryostat chuck using Tissue-Tek.RTM. 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. 
Coverslips were then placed over the sections, using Permount.TM. as an 
adhesive. FIGS. 7A and 7B are low-power micrographs of gerbil hippocampus 
(CA) in animals after ischemia, after infusion of MVIIA OCT (7A) or after 
drug vehicle (7B). The arrows in the figures indicate the approximate 
borders of the CA. At higher power, cells in the drug-treated ischemic 
animals appear normal (FIG. 8A), whereas damage is apparent in the 
ischemic animals receiving vehicle alone (FIG. 8B). Another example of 
complete drug protection is seen in FIG. 8C, and an example of partial 
protection is seen in FIG. 8D, where there are a small number of damaged 
cells. 
Sections, such as those seen in FIGS. 7 and 8, were viewed and scored by an 
investigator having no knowledge of the treatment of any particular sample. 
Ischemic damage was scored in the CA-1 region of the hippocampus. Damage 
was generally seen as pink (eosinophilic) cytoplasm and shrunken, dark 
blue nuclei. Scoring was as described below: 
______________________________________ 
Score Observation 
______________________________________ 
0 No damaged cells were apparent. 
1 Less than 25% damaged cells in a CA field, or 
damage was restricted to the extreme edges of the 
CA 1 region. 
2 Approximately 50% damaged cells in a CA 1 field, or 
damage to less than half the length of CA 1. 
3 Damaged cells outnumber normal cells to a maximum 
of 75%, with damage extending throughout most of CA 
1. 
4 Complete damage to CA 1, with fewer than 25% normal 
cells surviving. 
______________________________________ 
The extent of anatomical damage in ischemic animals treated with MVIIA or 
GVIA OCT or receiving vehicle alone (control), based on the above scoring 
system, is given in Table 8 below. The peptide was administered by ICV 
infusion during the eight minutes of occlusion, at a total dose indicated 
in the table below. As seen, the extent of damage in the higher-dose MVIIA 
OCT treated animal was only 25% of that in untreated animals. The GVIA 
peptide also produced more than a 50% reduction in damage, and the lower 
dose was near maximal effectiveness. 
TABLE 8 
______________________________________ 
Treatment N Mean score (S.E.M) 
Percent Damage 
______________________________________ 
Vehicle 20 3.1 (.32) 100% 
0.02 ug MVIIA 
4 1.9 (.83) 61% 
0.1 ug MVIIA 
18 0.8 (.09)*** 25% 
0.02 ug GVIA 
3 1.3 (.33)* 42% 
0.1 .mu.g GVIA 
11 1.2 (.39)** 39% 
______________________________________ 
*p&lt; .05 compared to vehicle (Student's Ttest) 
**p&lt; .005 compared to vehicle (Student's Ttest) 
***p&lt;.0005 compared to vehicle (Student's Ttest) In a second treatment 
method, the OCT peptide was administered by ICV infusion 1 hour after the 
8-min occlusion, at the same drug dosage level as indicated above. The 
anatomical damage in the presence and absence of drug, scored as above, is 
given in Table 9 below. A comparison of the data in Table 8 indicates 
little loss of protective effect at a comparable dose (0.1 .mu.g) when the 
drug is administered 1 hour after the ischemic event (8 min of occlusion). 
TABLE 9 
______________________________________ 
Treatment N Mean score (S.E.M) 
Percent Damage 
______________________________________ 
Vehicle 15 3.0 (.31) 100% 
0.1 .mu.g MVIIA 
16 0.9 (.13)*** 30% 
0.3 .mu.g MVIIA 
3 0.7 (.17)** 23% 
______________________________________ 
**p&lt; .005 compared to vehicle (Student's Ttest) 
***p&lt;.0005 compared to vehicle (Student's Ttest) 
EXAMPLE 11 
Reduction in Anatomical Damage: 2 
A. Global Ischemia 
Global ischemic damage was examined in the rat brain model, employing the 
four-vessel occlusion method of Pulsinelli and Brierly (Pulsinelli) for 
introducing temporary global ischemia in rats. Although the two carotid 
arteries supply blood to the forebrain, their occlusion alone has only 
moderate effects on forebrain blood flow because the posterior 
communicating arteries allow blood to be shunted from the brainstem blood 
supply, which is fed by the two vertebral arteries. Therefore, in order to 
effect severe forebrain ischemia, all four vessels must be occluded. The 
procedure used allows ischemia to be produced in conscious animals, by 
closing surgically implanted clamps, and therefore avoid possible 
interactions with drug treatment. 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. Under sodium pentobarbital anesthesia (60 mg/kg) 
male Fisher 344 rats were placed in a sterotaxic holder and the first 
cervical vertebra was exposed with the aid of a dissecting microscope. The 
vertebral arteries were occluded through the alar foramina with a 
thermocautery 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 4 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 (RR) within about 1 min of occlusion. When the 
animal does not lose the RR or regains it during occlusion, the loops are 
pulled tighter to assure complete carotid occlusion. Animals that do not 
lose their RR are eliminated from the study, because this suggests that 
there is still significant cerebral blood flow. 
Neuropathological analysis (see below) of such animals confirms this 
because the damage is less than in animals that do lose their RR. Some 
animals right themselves once or twice during the occlusion, but 
immediately lose the RR again, and are not eliminated from the study. An 
animal that rights itself and remains up is eliminated. 
Immediately following reperfusion, rats were anesthetized with halothane 
(as for gerbils) and 0.3 .mu.g MVIIA OCT in 5 .mu.L saline (n=7) or saline 
alone (n=5) was injected into the lateral ventricle as for gerbils. The 
coordinates were 1.2 mm left of midline and 0.5 mm posterior to bregma. 
Rectal temperature was monitored from just before occlusion until the end 
of the day. 
Neuropathologic analysis was conducted in a manner similar to that 
described for gerbils, with the results shown in Table 10. 
TABLE 10 
______________________________________ 
Treatment N Mean score (S.E.M) 
______________________________________ 
vehicle 4 3.6 (0.38) 
MVIIA OCT (0.3 .mu.g) 
5.sup.a 
1.2 (0.36)** 
______________________________________ 
.sup.a Animals give OCT MVIIA ICV were included in the study only if they 
exhibited characteristic shaking behavior. 
**p&lt;.005 unpaired Student's t test. 
As seen from the data, treatment with MVIIA OCT reduced anatomical damage 
to about 1/3 that seen in the absence of peptide treatment. 
EXAMPLE 12 
Protection Against Loss of Functional Activity 
A. Hyperactivity 
One common sequence of cerebral ischemia is hyperactivity, which can be 
seen as pacing behavior within a few hours of occlusion and can be 
measured up to several days later. Hyperactivity was quantitated with 
Automex activity monitors (Columbia Instruments, Columbus, Ohio), which 
record perturbations of a radiofrequency field. Gerbils were tested 
individually in 17.times.27-cm plastic cages for 60 min, with cumulative 
activity counts recorded every 15 min for statistical analysis. Baseline 
activity was measured before surgery to ensure comparability of the 
different treatment groups on this measure. FIG. 9 shows the activity 
counts measured at 0, 1 and 3 days after occlusion, over a 60-minute 
period each. Occluded animals are indicated by triangles; non-occluded by 
circles; untreated, by open symbols; and administration of MVIIA by closed 
symbols. 
B. Spontaneous Alternation 
Short-term memory changes in gerbils were tested in a Y maze, in which the 
animal is placed in the base of the stem of the maze, and when the animal 
enters an arm, a door is shut behind it. After 5 sec, the gerbil is 
returned to its home cage for an intertrial interval (ITI) of 2 to 12 min. 
At the end of that interval the gerbil is run in the maze again in the same 
way. Most normal animals will alternate, that is, will enter the arm that 
was not entered on the first trial. Occasionally an animal did not enter 
an arm within about 1 min because it had had a seizure, so it was 
eliminated from that test. 
Because individual experiments include too few animals per group to allow 
meaningful statistical evaluation of the data, the results were combined 
for all experiments in which there was good evidence of protection by drug 
treatment against hippocampal damage (Example 10). Only experiments with 
positive results were combined to determine if the anatomical protection 
was associated with behavioral protection. 
Results of the spontaneous alternation tests are summarized in Table 11 for 
experiments in which there was anatomical protection from doses of at least 
0.1 .mu.g of either compound. A chi square test on the combined data was 
significant at p&lt;0.01. Combining treatment groups to examine each factor 
separately (e.g., all occluded vs. all unoccluded, regardless of drug 
treatment) indicated that each was significant by chi square at p&lt;0.05; 
that is, (a) ischemia caused worse performance and (b) the level of 
performance was largely restored in treated animals 
TABLE 11 
______________________________________ 
Gerbils Alternating (Y) or Repeating (N) 
Experminents 
3 4 5 6 Combined 
Ischemia 
Drug* Y N Y N Y N Y N Y N 
______________________________________ 
No No 9 3 2 2 3 3 6 2 20 10 
Yes -- -- 4 0 5 1 4 0 13 1 
Yes No 2 6 4 4 4 4 3 4 13 18 
Yes 4 3 5 2 7 1 7 4 23 10 
______________________________________ 
*Drug doses are from 0.1 to 0.3 .mu.g of MVIIA or GVIA. 
Although the invention has been described with respect to particular 
treatment methods and composition, it will be apparent to those skilled 
that various changes and modifications can be made without departing from 
the invention.