US007892764B2

(12) United States Patent

Xiong et a1.
(54)

SYSTEM FOR SEIZURE SUPPRESSION

(75) Inventors: Zhigang Xiong, Beaverton, OR (US);

Roger P. Simon, Portland, OR (US)

(10) Patent N0.:

(45) Date of Patent:

US 7,892,764 B2
Feb. 22, 2011

Meller,R. et al. Seizure-like activity leads to the release of BAD from

14-3 -3 protein and cell death in hippocampal neurons in vitro. Cell.

Death. Dijjer 10, 539-547 (2003).

Furshpan,E.J. & Potter,D.D. Seizure-like activity and cellular dam
age in rat hippocampal neurons in cell culture. Neuron. 3, 199-207

(73) Assignee: Legacy Emanuel Hospital & Health

Center, Portland, OR (US)
(*)

Notice:

Subject to any disclaimer, the term of this

patent is extended or adjusted under 35

U.S.C. 154(b) by 68 days.

(21) Appl.No.: 11/944,332

(22) Filed:

in vitro seizure models. Brain Res. Dev. Brain Res. 128, 113-120

Prior Publication Data

US 2008/0242588 A1

Oct. 2, 2008

Related US. Application Data

(60)

Provisional application No. 60/ 860,522, ?led on Nov.

21, 2006, provisional application No. 60/959,987,

?led on Jul. 17, 2007.

Alvarez,d.I.R. et al. Distribution, subcellular localization and

ontogeny of ASICl in the mammalian central nervous system. J'.

Physiol 546, 77-87 (2003).

Stasheff,S.F., Bragdon,A.C. & Wilson,W.A. Induction of
epileptiform activity in hippocampal slices by trains of electrical
stimuli. Brain Res. 344, 296-302 (1985).

Araki,T., Simon,R.P., Taki,W., Lan,J.Q. & Henshall,D.C. Character

ization of neuronal death induced by focally evoked limbic seizures
in the C57BL/6 mouse. J'. Neurosci. Res. 69, 614-621 (2002).

Pignataro,G., Simon,R.P. & Xiong,Z.G. Prolonged activation of

(51)

Int. Cl.
G01N 33/566

(52)
(58)

US. Cl. ...................... .. 435/72; 435/721; 436/501

Field of Classi?cation Search ..................... .. None

(2006.01)

See application ?le for complete search history.

(56)

proton-gated Na+ channels. 1. Biol. Chem. 275, 25116-25121 (2000).

Avoli,M. et al. Network and pharmacological mechanisms leading to
epileptiform synchronization in the limbic system in vitro. Prog.
Neurobiol. 68, 167-207 (2002).
Wong,M. & Yamada,K.A. Developmental characteristics of
epileptiform activity in immature rat neocortex: a comparison of four

(2001).

Nov. 21, 2007

(65)

(1989).
Escoubas,P. et a1. Isolation of a tarantula toxin speci?c for a class of

References Cited
U.S. PATENT DOCUMENTS

ASICla and the time Window for neuroprotection in cerebral

ischaemia. Brain. 130, 151-158 (2007).
Chesler,M. & Chan,C.Y. Stimulus-induced extracellular pH tran
sients in the in vitro turtle cerebellum. Neuroscience. 27, 941-948

(1988).
Wemmie,J.A., Price,M.P. & Welsh,M.J. Acid-sensing ion channels:
advances, questions and therapeutic opportunities. Trends Neurosci.
29, 578-586 (2006).
Miesenbock,G., De Angelis,D.A. & Rothman,J.E. Visualizing secre
tion and synaptic transmission With pH-sensitive green ?uorescent

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proteins. Nature. 394, 192-195 (1998).

Zha,X.M., Wemmie,J.A., Green,S.H. & Welsh,M.J. Acid-sensing ion

2009/0291150 A1

11/2009 Welsh et al.

channel 1a is a postsynaptic proton receptor that affects the density of

dendritic spines. Proc. Natl. Acad. Sci. U. S. A. 103, 16556-16561

OTHER PUBLICATIONS

(2006).

Waldmann et al. A Proton-gated Cation Channel Involved in Acid

(Continued)

Sensing, Mar. 13, 1997, Nature 386:173-177.*

Ali et a1. Evidence of the Antiepileptic Potential of Amiloride With

Neuropharmacological Bene?ts in Rodent Models of Epilepsy and

Behavior, Mar. 3, 2004, Epilepsy & Behavior5:322-328.*

McNamara,J.O., Huang,Y.Z. & Leonard,A.S. Molecular signaling

Primary Examinerilohn D Ulm

(74) Attorney, Agent, or FirmiKolisch HartWell, PC.

(57)

ABSTRACT

mechanisms underlying epileptogenesis. Sci. STKE. 2006, re12

(2006).
Pitkanen,A. & Halonen,T. Prevention of epilepsy. Trends Pharmacol.
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Meldrum,B.S. & RogaWski,M.A. Molecular targets for antiepileptic

drug development. Neurother. 4, 18-61 (2007).

Siesjo,B.K., Katsura,K. & Kristian,T. Acidosis-related damage. Adv.
Neurol. 71, 209-233 (1996).

Systems, including methods and compositions, for seizure

suppression, such as inhibition of epileptic seizures. In some
embodiments, the methods may provide a screen for anti
seizure drugs. One or more compositions may be selected
based on an ability to affect a response of biological cells to a

Simon RP. Status epilepticus mechanisms and management .

change in extracellular pH and/or to affect an activity of at

Wasterlain, C.G & Treiman D.M. (eds), pp. 149 (The MIT

least one acid sensing ion channel (ASIC). Based on the one
or more compositions selected, at least one drug candidate

Press,2006).
Chesler,M. & Kaila,K. Modulation of pH by neuronal activity.
Trends. Neurosci 15, 396-402 (1992).

Siesjo,B.K. & Wieloch,T. Epileptic brain damage: pathophysiology

and neurochemical. Adv. Neurol. 44:813-47., 813-847 (1986).
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perpetrator. Cell 118, 665-666 (2004).

Benveniste,M. & Dingledine,R. Limiting stroke-induced damage by

targeting an acid channel. N. Engl. J'. Med. 352, 85-86 (2005).
Xiong,Z.G. et al. Neuroprotection in ischemia: blocking calcium

permeable Acid-sensing ion channels. Cell 118, 687-698 (2004).

may be assayed for inhibition of seizure-like electrical activ

ity and/or seizures. In some embodiments, the methods and
compositions may, respectively, administer and provide an
effective amount of PcTXl, a peptide derivative of PcTXl
amiloride, an amiloride derivative, or a combination thereof
to a subject prone to seizures and/ or having a seizure, in order

to suppress seizure activity.

17 Claims, 23 Drawing Sheets

US 7,892,764 B2
Page 2
OTHER PUBLICATIONS

Hesselager,M., Timmermann,D.B. & Ahring,P.K. pH Dependency

Urbanics,R., Leniger-Follert,E. & Lubbers,D.W. Time course of

changes of extracellular H+ and K+ activities during and after direct
electrical stimulation of the brain cortex. P?ugers Arch. 378, 47-53

and desensitization kinetics of heterologously expressed combina

tions of acid-sensing ion channel subunits. J'. Biol. Chem. 279, 11006

Wemmie,J.A. et al. The acid-activated ion channel ASIC contributes

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to synaptic plasticity, learning, and memory. Neuron. 34, 463-477

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acid-induced increase of intracellular Ca2+, and acidosis-mediated
neuronal injury by intracellular pH. 1. Biol. Chem. 281, 29369-29378

(2002).
Na+ channel on ischemia-sensing neurons. Nat. Neurosci 4, 869-870

(2006).

(2001).

DeVries,S.H. Exocytosed protons feedback to suppress the Ca2+

Immke,D.C. & McCleskey,E.W. Lactate enhances the acid-sensing

(2001).

Steen,K.H., Reeh,P.W., Anton,F. & HandWerker,H.O. Protons selec

tively induce lasting excitation and sensitization to mechanical
stimulation of nociceptors in rat skin, in vitro. J'. Neurosci. 12, 86-95

Krishtal,O.A., Osipchuk,Y.V., Shelest,T.N. & Smirnoff,S.V. Rapid

(1992).

current in mammalian cone photoreceptors. Neuron. 32, 1107-1117

extracellular pH transients related to synaptic transmission in rat

hippocampal slices. Brain Res. 436, 352-356 (1987).

Chesler,M. Regulation and modulation of pH in the brain. Physiol
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Seizure Termination by Acidosis Depends on ASIC 1 a by Ziemann

et el., vol. 11 No. 7, Jul. 2008, Nature Neuroscience, pp, 816-822.

* cited by examiner

US. Patent

Feb. 22, 2011

Sheet 1 0123

US 7,892,764 B2

Fig. 1
[

20

r22

SELECTA SUBJECT PRONE TO SEIZURES AND/OR

HAVING A SEIZURE

r24

ADMINISTER AN INHIBITOR OF AN ACID SENSING

ION CHANNEL (ASIC) TO THE SUBJECT IN
ORDER TO SUPPRESS SEIZURE ACTIVITY

Fig. 2
30

r32

TEST COMPOSITIONS USING AN ASIC ASSAY

r34

SELECT ONE OR MORE COMPOSITIONS

HAVING AN EFFECT IN THE ASIC ASSAY

/36

OBTAIN AT LEAST ONE DRUG CANDIDATE BASED

ON THE ONE OR MORE COMPOSITIONS SELECTED

I
ASSAY THE DRUG CANDIDATE FOR AN ABILITY
TO SUPPRESS SEIZURE-LIKE ELECTRICAL
ACTIVITY AND/OR SEIZURES

r38

US. Patent

Feb. 22, 2011

Sheet 2 0f 23

US 7,892,764 B2

Fig. 5A

Kyneurenic acid withdraw

30 pM Amiloride
_60

Kyneurenic acid withdraw

20 sec
_20 _

Wash

Kyneurenic acid withdraw

Control
2 sec

US. Patent

Feb. 22, 2011

Sheet 3 0f 23

US 7,892,764 B2

Fig. 50

bcAor>mutE?oavm

321
4

O0

d*T=

4321

O0U
1n*a
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T
O

mn

m
_l

Am

.n

0 u. M

mT

1T

n
9
P 0TX

m I. m

.w

Fig. 5D

A52.N060I.V"

43251

Con Amiloride 30 |.|M Wash

76543210

Con

PcTX1 100nglml Wash

US. Patent

Feb. 22, 2011

Sheet 4 0f 23

US 7,892,764 B2

Fig. 4
mV

Control

Kyneuric acid withdraw

3 sec

mV

PcTX1

Kyneuric acid withdraw

40

Wash

Kyneuric acid withdraw

US. Patent

Feb. 22, 2011

Sheet 5 0f 23

hcomwo'nwro

NmmrmNrQ

|m0.V5 30ms

20ms

Mg*-fre
Fig.
5Aa

MM1Amil0orde

US 7,892,764 B2

Wash

Mg2*-fre
Fig.
515a

n2Pcg0T/Xm1l

Wash

US. Patent

.5m6

Feb. 22, 2011

2:.5

|_|

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Sheet 7 0f 23

US 7,892,764 B2

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2we

0.0

vmwoaEz co /Vi

US. Patent

Feb. 22, 2011

Sheet 11 0123

US 7,892,764 B2

PcTX1

C O n t rM

I
I

m
m
w
o

US. Patent

Feb. 22, 2011

400

(aEofTDucyrstpievGoc-nt)4y

300

200

100

Sheet 12 0123

US 7,892,764 B2

US. Patent

Feb. 22, 2011

Sheet 13 0f 23

US 7,892,764 B2

_2:o.3=5u4

3au.t5o-E0d
we 0._, 0.0 0.0 #0 N0 0.0 m._, 0._, 0.0 0.0 10 N0 0.0

AauanbaH pazueuJJoN uogezyqodaq pazueuuoN

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>8 00

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ON. 8.

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r

US. Patent

Feb. 22, 2011

Sheet 15 0f 23

US 7,892,764 B2

gcm5E3QE.w

|a.

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ow
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007W.

o W.
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cm.m.

W
cm.1

US. Patent

Feb. 22, 2011

Sheet 16 0123

US 7,892,764 B2

mm

*0cBm6520mo5iwm0n EmmdEw0zmE.n

n_I2.:ow-n

8
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US. Patent

Feb. 22, 2011

Sheet 17 0123

US 7,892,764 B2

Fig. 105
Duration of acid pulse: 0.3 sec
Time in between:
0.5 sec
6.5

0.4

50

6.5

6.5

0.5 sec

6.5

US. Patent

Feb. 22, 2011

Sheet 18 0f 23

US 7,892,764 B2

Fig. 11
Benzam il
control

1 |JM

10 M

30 pM

100 pM

ARemlpativude

WASH

HP = 60 mV
pH = 6.0

1-0
Benzam il (pNl)

160