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<td align="left" width="50%"><b>United States Patent Application</b></td>
<td align="right" width="50%"><b>20040097534 </b></td>
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<td align="left" valign="top" width="50%"><b>Kind Code</b> </td>
<td align="right" width="50%"><b>A1 </b></td>
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<td align="left" width="50%"><b>Choi, Byung-Kil ; et al.</b> </td>
<td align="right" width="50%"><b>May 20, 2004 </b></td>
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<font size="+1">Composition for the protection and regeneration of
nerve cells containing berberine derivatives </font><br>
<br>
<center><b>Abstract</b></center>
<p>Disclosed is a composition for protecting nerve cells, promoting
nerve cell growth and regenerating nerve cells comprising berberine,
derivatives thereof or pharmaceutically acceptable salts thereof. The
composition has protective effects against apoptosis of neuronal stem
cells and differentiated neuronal stem cells, an effect of inducing the
regeneration of nerve cells, a regenerative effect on neurites, a
neuroregenerative effect on central nerves and peripheral nerves, a
reformation effect on neuromuscular junctions, and a protective effect
against apoptosis of nerve cells and a neuroregenerative effect in
animals suffering from dementia and brain ischemia. Therefore, the
composition can be used as a therapeutic agent for the prevention and
treatment of neurodegenerative diseases, ischemic nervous diseases or
nerve injuries, and for the improvement of learning capability.<br>
</p>
<p>&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&<br>
</p>
<p>[0150] Next, the head of the rat was fixed on a stereotaxic
apparatus to operate on the occiput, and then the tail was fixed so
that it descended downwardly at an angle of 30.degree.. After incising
the occipital bone, an electrocauterizing needle having a diameter of 1
mm or less was inserted into the alar foramina positioned at lower part
of the first cervical vertebra under the occipital bone. At this time,
this approach must be carefully done so as not to damage the muscles in
the alar foramina. Thereafter, the vertebral artery was electrically
cauterized by intermittently applying current. After the complete
electrocauterization of the vertebral artery was confirmed, suturing
was carried out using operating clips. After 24 hours, the operating
clips were removed. Finally, the common carotid arteries were occluded
using the silicone tube rings for 10 minutes to induce ischemia. If
light reflex did not disappear within 1 minute, the cervical portion
was further tightly sutured. Rats which did not show the complete
disappearance of light reflex were excluded from the experiment because
they underwent no damage to the CA1 region. After 10 minutes, the
common carotid arteries were loosened to reperfuse. For 20 minutes
after the reperfusion, loss of consciousness was observed. At this
time, only rats which showed consciousness loss period within 20.+-.5
minutes were selected for subsequent experiments. <br>
</p>
<p>&&&&&&&&&&&&&&&&&&&&&&&&&&&&&<br>
</p>
2) Experimental Results <br>
<br>
(1) Concentration of Berberine, Influence of Body Temperature and
Ischemia Inducing Time <br>
<br>
[0157] The highest concentration of berberine was set to 300 .mu.g/0.1
kg, and 600 .mu.l (1 mg/ml) of berberine was intraperitoneally injected
to white rats weighing 200 g. In order to determine an optimal ischemia
induction time, 2.about.3 rats were selected and ischemia-induced over
5, 10, 20 and 30 minutes, respectively. 1 week after reperfusion, they
were sacrificed and their hippocampal tissue sections were obtained to
observe the number of damaged nerve cells. 10 minutes after ischemia
induction, damaged pyramidal cells in the hippocampal CA1 region were
found to be reduced to 1/4 of their original numbers. The ischemia
induction time of 10 minutes was determined to be most optimal for
evaluating the effects of berberine. <br>
<br>
[0158] For statistically analyzing the effects of berberine, a sham
operated group having undergone an operation in the same manner without
ischemia induction was used. For comparing the effects of berberine, a
control group administered with physiological saline at the same dose
as berberine was used. Berberine was intraperitoneally injected into
all experimental groups. <br>
<br>
[0159] It is well known that reduction in body temperature during
ischemia induction prevents damage to nerve cells in the hippocampus
and thus exhibits neuroprotective effects. Therefore, in order to
evaluate the neuroprotective effect of berberine, after ischemia
induction and reperfusion, the body temperature of all rats was
maintained at a constant (37.+-.1.degree. C.) for 6 hours. <br>
<br>
(2) Observation of Damaged Nerve Cells <br>
<br>
[0160] When ischemia was induced by 4-VO and then reperfusion was
performed, nerve cells in the neocortex, striatum, hippocampal CA1
region and cerebellum were damaged. Among them, pyramidal nerve cells
in the hippocampal CA1 region were the most susceptible to the induced
ischemia, and started to undergo cell death 72 hours after reperfusion.
In order to observe delayed neuronal death in the hippocampal CA1
region, 1 week after reperfusion, the time when almost all nerve cells
were damaged, white rats were sacrificed and tissue sections from the
hippocampus were observed under an optical microscope. In a sham
operated group having undergone no ischemia, normal hippocampal nerve
cells were observed in the stratum pyramidale (490 .mu.m long)(see,A
and B of FIG. 21). <br>
<br>
[0161] C and D of FIG. 21 as control groups show apoptosis. When cells
are induced to undergo apoptosis by an external or an internal
stimulus, they shrink to lose their original shapes. This shrinkage
breaks the junctions with other adjacent cells so that the interaction
between cells is disrupted. When the shrinkage proceeds to some extent,
the cell membranes form apoptotic bodies like a bulla. In the
hippocampal CA1 region of the control group administered with
physiological saline (D of FIG. 21), it was observed that nerve cells
underwent apoptotic morphological changes after ischemia induction. In
addition, it was observed that tissues was relaxed and separated from
adjacent cells, unlike B of FIG. 21. From these observations, it was
confirmed that the cell bodies of nerve cells lost their original
pyramidal shape and were condensed, thereby appearing to be single
cells. Furthermore, it was confirmed that subsequent nuclear chromatin
condensation and nuclear envelope collapse led to apoptosis of nerve
cells. On the contrary, nerve cells in the hippocampal CA1 region
administered with berberine were similar to normal cells in terms of
their morphology (see, E and F of FIG. 21). At this time, because
necrotic nerve cells around the CA1 region were very difficult to
distinguish from microglias, only viable pyramidal nerve cells in the
CA1 region were counted. In F of FIG. 21, separated cells were observed
above and below the hippocampal region and cell bodies were condensed.
This demonstrates that the damage to nerve cells was great enough to
induce apoptosis. Nevertheless, it was observed that a great number of
nerve cells were protected from apoptosis and their original pyramidal
morphology was maintained. This suggests that berberine has a
protective effect against damages to nerve cells in the hippocampal CA1
region induced by 4-VO. Although it was not confirmed what stage during
apoptosis influences nerve cell survival, it was certain that berberine
has a significant protective effect against apoptosis of nerve cells
(see, E and F of FIG. 21). <br>
<br>
(3) Protective Effect of Berberine Against Damage to Nerve Cells <br>
<br>
[0162] In order to examine the neuroprotective effect of berberine
after ischemia induction, berberine was intraperitoneally injected 0
and 90 minutes after ischemia induction. <br>
<br>
[0163] In the sham groups, the density of viable cells was measured to
be 308.+-.6.6 cells/mm.sup.2 (at 37.degree. C.). In the control groups
administered with physiological saline, the density of viable cells was
measured to be 28.+-.3.8 cells/mm.sup.2 (at 37.degree. C.). There was
cell loss in these two groups. On the other hand, in the experimental
groups administered with berberine, the density of viable cells was
measured to be 257.+-.9.6 cell/mm.sup.2. In conclusion, berberine was
determined to have a significant neuroprotective effect (p<0.05). <br>
<br>
[0164] As described above, the composition according to the present
invention regenerates axons and dendrites of nerve cells, thereby
having a protective effect against nerve cell injuries, a positive
effect on nerve cell growth and a regenerative effect on nerve cells.
In addition, the composition according to the present invention can be
used as a therapeutic agent for the prevention and treatment of
neurodegenerative diseases or nerve injuries, in particular, dementia,
Parkinson's disease, Alzheimer's disease, epilepsy, palsy, ischemic
brain diseases, trauma to the spinal cord and peripheral nerve
injuries. <br>
<br>
[0165] Although the preferred embodiments of the present invention have
been disclosed for illustrative purposes, those skilled in the art will
appreciate that various modifications, additions and substitutions are
possible, without departing from the scope and spirit of the invention
as disclosed in the accompanying claims. <br>
<br>
<center><b>* * * * *</b></center>
Aside from its carrier capacity DMSO was chosen for its ability to
inhibit damage from a light freeze<br>
<br>
There are a number of complementary chemistries besides from the ones
cited readily available.<br>
<br>
<br>
<br>
Eugen Leitl wrote:<br>
<blockquote cite="mid20041215104301.GT9221@leitl.org" type="cite">
<pre wrap="">On Wed, Dec 15, 2004 at 03:01:49AM -0600, Extropian Agroforestry Ventures Inc. wrote:
</pre>
<blockquote type="cite">
<pre wrap="">The concept goes thusly:
The liferaft =
-sleeping /body bag like sack made so that no 2 arms or legs touch each
other or body
-zipper + ziplock seal
- control/RFID biomonitor keypad with on outside
-stage 1- evacuate liner to ensure good skin contact with sack
-person put inside without outer clothes , shoes etc
-put cooling hood or cap over all of head less face, face cover ziplock
cover after body cooled off
-start in 2 parts; activation of emergency cooling packs layer of sack
to quick cool body; hood cooling cycle
</pre>
</blockquote>
<pre wrap=""><!---->
Useless. This gives you no advatage over an ice bath.
</pre>
<blockquote type="cite">
<pre wrap="">co2 based for higher cooling rate
</pre>
</blockquote>
<pre wrap=""><!---->
More than useless. You can't go below 0 C, or you'll get freezing injury.
</pre>
<blockquote type="cite">
<pre wrap="">-infusion of adjuvants may include:
Caffeinol as neuroprotectant; berberine in DMSO solution as
neuroprotectant; cannabidiol in DMSO solution as neuroprotectant
-optional defib cycle to pump neuroprotectants into cooling body uniformly
</pre>
</blockquote>
<pre wrap=""><!---->
You have to maintain the circulation. Best do achieve this is life support.
</pre>
<blockquote type="cite">
<pre wrap="">The adjuvants are designed to allow the brain to survive a longer
cool-off time than the usual 3-5 minutes.
</pre>
</blockquote>
<pre wrap=""><!---->
Sorry, but your science is garbage. I'm being delibertely harsh here, because
otherwise you won't get the message.
</pre>
<blockquote type="cite">
<pre wrap="">as well as allow for easier re-start of body by hospital medical team
-once body temp is near 32F optional external hookups to maintain cooled
body during extended transport
Once the working prototype is designed and tested , the actual mfg
costs may be quite reasonable
</pre>
</blockquote>
<pre wrap=""><!---->
If you have to live in the sticks, you have to rely on people. No machinery
is going to help.
</pre>
<blockquote type="cite">
<pre wrap="">Morris Johnson
Hara Ra wrote:
</pre>
<blockquote type="cite">
<pre wrap="">For Eugen:
Thanks for taking my point and clarifying it. If you read my sig
file, I don't think I am an "Armchair cryonicist". Please correct me
if you think so, and explain why.
</pre>
</blockquote>
</blockquote>
<pre wrap=""><!---->
Of course I wasn't commenting on what you wrote, but on periodical resurgence
of well-meaning-but-clueless armchair cryonicists.
</pre>
<blockquote type="cite">
<blockquote type="cite">
<pre wrap="">PS We currently do no longer use the Thumper, which is an obscene
bastard of equipment fully capable of major injury to both patient and
rescue team. Respiratory support is no longer in the protocol, because
</pre>
</blockquote>
</blockquote>
<pre wrap=""><!---->
Oh yeah, if the cup breaks off you'll get a massive metal rod puncturing the
ribcage.
</pre>
<blockquote type="cite">
<blockquote type="cite">
<pre wrap="">basically all patients are over the 4 minute limit, and restoring O2
is a bad idea. We use an Ambi product, a suction cup with handles, to
</pre>
</blockquote>
</blockquote>
<pre wrap=""><!---->
Not if you add neuroprotectants via IV push, and maintain artificial
circulation.
</pre>
<blockquote type="cite">
<blockquote type="cite">
<pre wrap="">maintain circulation for the 3-5 minutes it takes to circulate the
medicines (a proprietary cocktail of anticoaguants, clot busters and
other stuff)
</pre>
</blockquote>
</blockquote>
<pre wrap=""><!---->
</pre>
<pre wrap="">
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</pre>
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