Note: Descriptions are shown in the official language in which they were submitted.
CA 02306754 2000-04-19
- WO 99IZ1877 PCT/CA98/01002
1
HISTOGRANIN PEPTIDE AND THEIR ANALGESIC USE
FIELD OF THE INVENTION
This invention relates to novel peptides and
pseudopeptides useful as analgesics.
BACKGROUND OF THE INVENTION
Chronic pain may have multiple causes including
inflammation, peripheral nerve injury, cancer, AIDS, and
diabetes. Treatment of chronic pain has included the
administration of analgesics.
Analgesic compounds are agents which alleviate pain
without causing a loss of consciousness; they may also reduce
inflammation. Known analgesics have not been particularly
effective in the treatment of chronic pain. For instance,
aspirin derivatives and non-steroidal anti-inflammatory agents
have limited efficacy and have a number of side-effects
including interference with blood coagulation and the
exacerbation of peptic ulcers; morphine and opioid analgesics
have shown some beneficial effects, but cause side-effects such
as marked tolerance, and addiction and withdrawal syndromes;
and the known N-methyl-D-aspartate (NMDA) receptor antagonists
are effective in certain animal models, but produce behavioural
side-effects including motor impairment, learning impairment,
and ataxia.
U.S. Patent No. 5,656,267 (August 12, 1997) describes
a method of alleviating chronic pain involving the
transplantation of cells into a region of the central nervous
system of patients suffering from chronic pain. However, this
method is not practical.
SUMMARY OF THE INVENTION
In one aspect the invention provides novel linear
peptides and pseudopeptides, having analgesic properties, of
Formula I:
SU8ST1TUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/OIOOZ
2
Formula I
~5 O ~2 ~7 ~ ~4
R ~N~CH CAN CH~C~N~CH c~N CH~C~RIo
R R O R Rg IO
1 6 3
wherein
R1 represents hydrogen, alkyl, alkenyl, alkynyl, -(CH2)n-NH2,
-(CH2)n-NH-C(=NH)NH2, or
(CHI )n
N~NH
wherein "n" is an integer from 0 to 10;
R2 represents -(CH2)nCONH2, wherein "n" represents an integer
from 0 to 10;
R3 represents hydrogen, alkyl, alkenyl, alkynyl, the radical
of formula:
R11
(CH2)n
R12
R13
or the radical of formula:
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
3
(CH2)n R 11
R12
r
N R13
H
wherein
~~n° represents an integer from 0 to 10; and
R11~ R12 and R13 may be the same or different and
represent hydrogen, alkyl, alkenyl, alkynyl, -I,
-F, -Hr, -C1, or -OH; and
R4 represents -(CH2)nNH2, -(CH2)nNHC(=NH)NH2, or
(CH2)n
N~NH
wherein "n" represents an integer from 0 to 10;
R5 and R9 may be the same or different and represent hydrogen,
alkyl, alkenyl, alkynyl, alkylcarbonyl, aminocarbonyl,
alkylaminocarbonyl, dialkylaminocarbonyl, dialkylamino, or
-(CH2)naryl, wherein "n" is an integer from 1 to 10;
R6, R~, and Ra may be the same or different and represent
hydrogen, alkyl, alkenyl, or alkynyl;
R10 represents hydroxy, alkoxy, alkenyloxy, alkynyloxy, amino,
alkylamino, dialkylamino, alkylaryl, arylalkoxy, aryloxy,
alkoxyaryl, A1, A1-A2, A1-A2-A3, A1-A2-A3-A4, or A1-A2-A3_
A4-A5.
wherein
Al represents threonine or serine;
A2 represents leucine, glycine, alanine, valine, or
isoleucine;
A3 represents tyrosine, phenylalanine, or tryptophan;
A4 represents glycine, alanine, leucine, isoleucine, or
valine; and
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
- - WO 99/21877 PCT/CA98/01002
4
A5 represents phenylalanine, tyrosine, or tryptophan;
pseudopeptide analogues thereof wherein one or more of the
carbonyl groups of the peptide linkage is replaced by -C(=S)-
or by -CH2-, and/or wherein one or more of the amide bonds,
-C(0)-NH-, is replaced by the retro-verso form, -NH-C(O)-,
thereof; and
pharmaceutically acceptable salts and esters thereof.
In another aspect, the invention provides novel
cyclic peptides and cyclic pseudopeptides, having analgesic
properties, of Formula II:
R5 R1 CH-C/ ~R6
~N
Formula II XN CH,.R~
O-C
C=O
CH
~R
R4 ~N~..C-CH N 7
Rs IO ~R
3
wherein
R1, R2, R3, R4, R5, R6, R~, and Ra, are as defined above; and
X represents an amino acid or peptide fragment represented
by A1, A1-A2~ A1-A2-A3, A1-A2-A3-A4~ or A1-A2-A3-A4-A5,
wherein
A1, A2, A3, A4, and A5 are as defined above; or
a divalent group of formula:
IS O I2 (7 O I4
C CH
-f-NCH CAN CH~C~N~CH ~N~ ~C~
I I II I I II
R1 R6 O R3 Rg O
wherein
"n" represents an integer from 0 to 10; and
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PC'f/CA98/01002
R1, R2, R3, R4, R5, R6, R~, and Rg are as defined above;
pseudopeptide analogues thereof wherein one or more of the
carbonyl groups of the peptide linkage is replaced by -C(=S)-
or by -CH2-, and/or wherein one or more of the amide bonds,
5 -C(O)-NH-, is replaced by the retro-verso form, -NH-C(0)-,
thereof; and
pharmaceutically acceptable salts and esters thereof.
In another aspect, the invention provides a
pharmaceutical composition for the treatment of pain,
especially chronic pain, comprising a peptide or pseudopeptide
of the invention in admixture with a suitable pharmaceutically
acceptable diluent or carrier.
In a further aspect, the invention provides use of a
peptide or pseudopeptide of the invention for the treatment of
pain, especially chronic pain.
In another aspect, the invention provides use of a
peptide or pseudopeptide of the invention for the manufacture
of a medicament for the treatment of pain, especially chronic
pain.
The invention also provides a commercial package
which contains the peptide or pseudopeptide of the invention
together with instructions for the use thereof, for the
treatment of pain.
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
Normally, the peptides of this invention will be
based upon L-amino acids, and amino acids should be understood
to be L-amino acids, unless otherwise indicated or unless the
context requires otherwise. However, in certain instances, it
may be advantageous to utilize the D-form of the acids.
Accordingly, both forms are within the scope of this invention.
The preferred forms for the amino acids comprised
within Formulae I and II are the levo (L) forms for amino acids
with R1 and R3 side-chains and dextro (D) forms for amino acids
with R2 and R4 side-chains.
As alkyl groups, we mention ones with up to l0
carbons, preferably up to 4 carbons, which can be straight or
branched and can have from 0 to 4 carbon-carbon double and/or
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
- - WO 99/21877 PCT/CA98/01002
6
triple bonds. Examples include methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl, and 2-ethyl-hexyl. As
alkoxy groups we mention groups with up to l0 carbon atoms,
preferably up to 4 carbon atoms. Examples of alkoxy groups
include methoxy, ethoxy, propoxy, and tert-butoxy. As aryl
groups, we mention 5- and 6-membered single-ring aromatic
radicals which include from zero to four heteroatoms selected
from nitrogen, oxygen, and sulfur, and the corresponding benzo-
fused groups. Examples include phenyl, thienyl, furanyl,
pyridinyl, imidazolyl, pyrimidyl, isoxazolyl, thiazolyl,
triazolyl, tetrazolyl, pyrrolyl, naphthyl, indolyl, and
quinolyl.
Preferred peptides and pseudopeptides according to
Formula I have the following Formula III:
R9-Q1-Q2-Q3-Q4-R10
wherein
Q1 represents glycine, alanine, valine, leucine, isoleucine,
lysine, histidine, or arginine;
Q2 represents asparagine or glutamine;
Q3 represents glycine, alanine, valine, leucine, isoleucine,
phenylalanine, tryptophan, or tyrosine;
Q4 represents lysine, arginine, or histidine;
R9 represents hydrogen; and
R10 represents hydroxy;
pseudopeptide analogues thereof wherein one or more of the
carbonyl groups of the peptide linkage is replaced by -C(=S)-
or by -CH2-, and/or wherein one or more of the amide bonds,
-C(O)-NH-, is replaced by the retro-verso form, -NH-C(0)-,
thereof; and
pharmaceutically acceptable salts and esters thereof.
More preferred peptides and pseudopeptides are
represented by Formula III
wherein
Q1 represents glycine or arginine;
Q2 represents L-glutamine or D-glutamine;
Q3 represents glycine, alanine, or tyrosine;
Q4 represents L-arginine or D-arginine;
SUBSTITUTE SHEET (RULE 26~
CA 02306754 2000-04-19
- WO 99/21877 PCT/CA98/01002
7
Rg represents hydrogen; and
R10 represents hydroxy;
pseudopeptide analogues thereof wherein one or more of the
carbonyl groups of the peptide linkage is replaced by -C(=S)-
or by -CH2-, and/or wherein one or more of the amide bonds,
-C(0)-NH-, is replaced by the retro-verso form, -NH-C(O)-,
thereof; and
pharmaceutically acceptable salts and esters thereof.
Preferred cyclic peptides and pseudopeptides have the
following Formula IV:
~Q2
Formula IV Q1 Q3
~Q4~
wherein
Q1 represents glycine, alanine, valine, leucine, isoleucine,
lysine, histidine, or arginine;
Q2 represents asparagine or glutamine;
Q3 represents glycine, alanine, valine, leucine, isoleucine,
phenylalamine, tryptophan, or tyrosine; and
Q4 represents lysine, arginine, or histidine;
pseudopeptide analogues thereof wherein one or more of the
carbonyl groups of the peptide linkage is replaced by -C(=S)-
or by -CH2-, and/or wherein one or more of the amide bonds,
-C(O)-NH-, is replaced by the retro-verso form, -NH-C(O)-,
thereof; and
pharmaceutically acceptable salts and esters thereof.
More preferred peptides and pseudopeptides are
represented by Formula IV
wherein
Q1 represents glycine or arginine;
Q2 represents L-glutamine or D-glutamine;
Q3 represents glycine, alanine, or tyrosine;
Q4 represents L-arginine or D-arginine;
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
- - WO 99/21877 PCT/CA98/01002
8
pseudopeptide analogues thereof wherein one or more of the
carbonyl groups of the peptide linkage is replaced by -C(=S)-
or by -CH2-, and/or wherein one or more of the amide bonds,
-C(O)-NH-, is replaced by the retro-verso form, -NH-C(O)-,
thereof; and
pharmaceutically acceptable salts and esters thereof.
Specific compounds of the invention include the
following:
Peptide H-Arg-Gln-Gly-Arg-OH (SEQ ID NO:1)
1
Peptide H-Gly-Gln-Gly-Arg-OH (SEQ ID N0:2)
2
Peptide H-Gly-Gln-Ala-Arg-OH (SEQ ID N0:3)
3
Peptide H-Arg-Gln-Ala-Arg-OH (SEQ ID N0:4)
4
Peptide cyclic(-Gly-Gln-Ala-Arg-) (SEQ ID N0:5)
5
Peptide cyclic(-Gly-Gln-Ala-Arg-Gly-Gln-Ala-Arg-)
5B
(SEQ ID NO:10)
Peptide cyclic(-Arg-Gln-Ala-Arg-) (SEQ ID N0:6)
6
Peptide cyclic(-Arg-Gln-Ala-Arg-Arg-Gln-Ala-Arg-)
6B
(SEQ ID NO:11)
Peptide cyclic(-Gly-Gln-Tyr-Arg-) (SEQ ID N0:7)
7
Peptide cyclic(-Gly-Gln-Tyr-D-Arg-)
8
Peptide cyclic(-Gly-D-Gln-Tyr-D-Arg-)
9
Peptide H-Gly-Gln-Tyr-Arg-OH (SEQ ID NO: B)
10
Peptide H-Gly-Gln-Tyr-D-Arg-OH
11
Peptide H-Gly-D-Gln-Tyr-D-Arg-OH
12
Peptide H-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-OH
13
(SEQ ID N0:9)
Especially preferred are peptides 8 and 9.
The linear peptides can be synthesized by solid-phase
procedures known in the art (see for example: Merrifield, J.
Am. Chem. Soc. 85, 2149, 1963 and Prasad et al., Can. J.
Physiol,
Pharmacol.
73, 209,
1995, the
disclosures
of which
are
incorporated
by reference).
Cyclic peptides can be synthesized using the Kaiser's
oxime-resin
procedure
known in
the art
(see for
example:
Osapay et al., Tetrahedron Letters, 31, 6121-6124, and Nishino
et al, J. Chem. Soc. Kin. Traps. 1, 939-946, 1986, the
disclosures
of which
are incorporated
by reference).
SU6STITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
9
The CO-NH bond of the peptides can be replaced by CO-
N-alkyl by procedures known in the art (see for example:
Tachibana et al., Design and synthesis of metabolically stable
analogues of dynorphin-A and their analgesic characterisitics
in "Biowarning systems in the brain" Ed. H. Takagi, Y. oomoro,
M. Ito, and H. Otsuka. University of Tokyo Press, Tokyo, 1988,
the disclosure of which is herein incorporated by reference).
The amide bond or bonds, -C(O)-NH-, of the peptide
linkage may be replaced by retro-verso forms, -NH-C(O)-,
thereof. The synthesis is performed as for linear peptides
except that retro-verso forms of the peptides are introduced in
place of the normal peptide linkages. For example, the Gly-Gln
bound in Gly.E-Gln-~Ala-~Arg can be obtained by introducing:
O
H I1
Boc-N-CH-H-C-CH-C-OH
H O i HZ
H2
C=O
I
H2 N
in solid-phase synthesis. The product is a mixture of 4
racemers that are separated and isolated by HPLC.
To prepare pseudopeptides wherein one or more of the
carbonyl groups of the peptide linkage is replaced by -C(=S)-
or by -CH2- pseudopeptide bonds ~(CS-NH) or ~(CH2-NH) can be
introduced into the peptides described, as given, for example,
by Michelot et al (Solid-phase synthesis of endothiopeptides
using 3-(N-Boc-aminothioacyl)-1,3-thiazolidine-2-thiones: new
efficient thioacylating reagents in "Innovation and
perspectives in solid-phase synthesis, biological and
biomedical applications" Ed. R. Epton, Mayflower Worldwide
Inc., Birmingham, 1996) or as described by Sasaki and Coy
(Peptides 8, 119-121, 1987), respectively, the disclosures of
both of which are incorporated by reference
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
The peptides and pseudopeptides of the invention may
be formed into acid addition, base addition, metallic, and
zwitter-ionic salts. Such salts are within the scope of this
invention. For administration the salts must of course be
5 pharmaceutically acceptable, but other salts may be of value as
intermediates in synthesis or in purification. Representative-
acid addition salts include the hydrobromide, hydrochloride,
sulfate, bisulfate, phosphate, nitrate, acetate, valerate,
oleate, palmitate, stearate, laurate, benzoate, lactate,
10 phosphate, tosylate, citrate, maleate, fumarate, succinate,
tartrate, naphthylate, benzene-sulphonate, mesylate,
glucoheptonate, lactobionate, and laurylsulphonate salts and
the like; representative organic amines useful for the
formation of base addition salts and quaternary ammonium salts
include aliphatic and cyclic amides, for example, ethylamine,
diethylamine, ethylenediamine, ethanolamine, diethanolamine,
piperazine, guanidinium and the like; representative metal
salts include the lithium, sodium, potassium, calcium,
magnesium, silver and aluminum salts, and the like. All these
salts can be prepared by methods well known to those skilled in
the art.
Preferred esters are those that will undergo
hydrolysis in vitro to the free acid, for example esters of
choline, cholesterol, and salicylic acid. Hence the esters can
be regarded as pro-drugs of the compounds of the invention.
Referring to X of Formula II, when n represents zero,
X is a direct covalent bond.
The invention extends to pro-drugs and to metabolites
of the compounds of formula I and II.
Pharmaceutically-acceptable compositions which
comprise a therapeutically-effective amount of one or more of
the compounds of Formula I or II, as described hereinabove,
together with one or more pharmaceutically-acceptable carriers
and/or diluents are included in this invention.
Compositions of the present invention may
conveniently be presented in unit dosage form and may be
prepared by any methods well known in the art of pharmacy. The
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99121877 PCTlCA98101002
11
amount of active ingredient (compounds of Formula I and II)
which can be combined with a carrier material to produce a
single dosage form will vary depending upon numerous factors
such as the host being treated and the particular mode of
administration. The amount of active ingredient to be combined
with a carrier material to produce a single dosage form will be
that amount of the compound which produces a therapeutic
effect. Generally, the amount of the compound of the invention
will be in the range from about 1 percent to about ninety-nine
percent of the composition, preferably about 5 percent to about
70 percent, most preferably from about 10 to about 30 percent
although the amount of the dose and the mode of administration
will be determined by the doctor or other medical professional.
An appropriate dose may be 5-50 mg per person per
day, although the appropriate dose will, of course, be
determined by the clinician.
Suitable methods of administration of the compounds
of this invention, and the compositions formed therewith
include oral, nasal, topical (including buccal and sublingual),
rectal, vaginal and/or parenteral administration, including
injectable.
Examples
1) Synthesis of linear peptides
Synthesis of linear peptides was performed as
previously described (Prasad et al., Can. J. Physiol.
Pharmacol. 73:209-214, 1995, herein incorporated by reference)
by the use of pre-formed symmetrical anhydrides of Boc-amino
acids with the solid-phase method (Merrifield, J. Am. Chem.
Soc. 85:2149-2154, 1963, herein incorporated by reference) and
chloromethylated copolystyrene-divinylbenzene 1% crosslinkage
(0.75 mequiv. C1/g) as the resin.
Peptide 3:
H-Gly-Gln-Ala-Arg-OH
The C-terminal amino acid {Boc(Tos)-Arg) was esterified to the
resin according to the procedure of Gisin {Helv. Chim. Acta,
56:1476-1482, 1973, herein incorporated by reference) with a
final yield of 0.3 mmol/g resin. Thereafter, Boc-Ala, Boc-Gln
SU9STITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
12
and Boc-Gly were attached consecutively according to the
following coupling cycle: i) one wash with CH2C12, ii) one
prewash with 40% TFA in CH2C12, iii) 20 min deprotection with
40% TFA in CH2C12, iv) three washes (one with CH2C12, one with
50% dioxane in CH2C12 and one with CH2C12, v) prewash with 5%
DEA, vi) 5 min neutralization with 5% DEA, vii) three washes
with CH2C12, viii) one hour coupling with pre-formed
symmetrical anhydrides of Boc-amino acids (six equiv. as
compared with the resin substitution of 0.3 mmol/g), ix) three
washes with CH2C12, x) two washes with isopropanol.
The peptide was cleaved from the resin and
deprotected with liquid HF at 0°C in the presence of anizole
(10%, v/v). HF was removed in vacuo, and the residue was
washed with ether before extraction of the peptide with 25%
acetic acid followed by lyophilization. The peptide was then
purified by passage through SephadexTM G-10 resin (2 x 25 cm
column) and HPLC on a Bio-SilTM C18 column (Waters, Milford,
MA). The product was eluted from the HLPC column with a
gradient of acetonitrile (0-30% in 0.1% TFA), detected by W at
24o nm and lyophilized to yield 20-25% of the pure compound
(based on the starting Boc-amino acid resin). The purity of
the peptide was verified by thin layer chromatography on silica
gel plates (layer thickness 0.25 mm) BDH Chemicals Associate of
E. Merck, Darmstadt, Germany)(one spot, Rf:0.19;
nBuOH:EtOH;HOAc:H20, 1:1:1:1). Amino acid analysis of an acid
digest gave: Glu, 0.99; Arg, 1.02; Gly, 0.96 and Ala, 1.03.
The title compound was obtained as a white powder.
The molecular formula is C16H30N806. Mol Wt/ MS-ESI: 430.23
(M+H: 431). Percentage purity based on HPLC was 95%-98%.
Soluble in water and DMSO.
The following linear peptides were synthesized based
on the method given above for peptide 3:
Peptide 1:
H-Arg-Gln-Gly-Arg-OH
The title compound was obtained as a white powder. The
molecular formula is C19H3~N1106~ Mol Wt/ MS-ESI: 515.29
(M+H: 516). Percentage purity based on HPLC was 95%-98%. The
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
13
compound was soluble in water and DMSO.
Peptide 2:
H-Gly-Gln-Gly-Arg-OH
The title compound was obtained as a white powder. The
molecular formula is C15H28N806~ Mol Wt/ MS-ESI: 416.21 (M+H:
417). Percentage purity based on HPLC was 95%-98%. Soluble iri
water and DMSO.
Peptide 4:
H-Arg-Gln-Ala-Arg-OH
The title compound was obtained as a white powder. The
molecular formula is C2pH3gN1106. Mol Wt/ MS-ESI: 529.31
(M+H: 530). Percentage purity based on HPLC was 95%-98%.
Soluble in water and DMSO.
Peptide 10:
H-Gly-Gln-Tyr-Arg-OH
Peptide 11:
H-Gly-Gln-Tyr-D-Arg-OH
Peptide 12:
H-Gly-D-Gln-Tyr-D-Arg-OH
Peptide 13:
H-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-OH
2) Synthesis of cyclic peptides
Peptide 8:
Gl~ln-Tyr-D-~Arg
Boc-Gly-Oxime-Resin was prepared by mixing oxime-resin
(Novabiochem, 1.5 g O.S7 meq/g) with Boc-Gly (1.3 g, 9 eq) in
the presence of N'N-dicyclohexylcarbodiimide (DCC) (9.9 ml of
DCC 8%, 4.5 eq), 4-dimethylaminopyridine (DMAP) (0.3g, 3 eq),
1-hydroxybenzotriazole hydrate (HOBt) (0.4g, 3 eq) in
dichloromethane (DCM) at room temperature for 12 hours. The
resin was subjected to two washes with DCM, one wash with 2-
propanol and one wash with DCM. The free oxime groups were
capped by acetylation with acetic anhydride (0.4 ml, 5 eq) for
30 minutes. The peptide chain was then assembled according to
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
14
the following coupling steps: (i) one wash with 25%
trifluoroacetic acid in DCM (TFA-DCM); (ii) deprotection with
25% TFA-DCM (30 min); (iii) two washes with DCM; (iv) one wash
with 2-propanol; (v) three washes with DCM; (vi) one wash with
dimethylformamide (DMF); (viii) coupling of Boc-D-Arg(Tos)-OH
(1.1 g, 3 eq) in presence of benzotriazole-yl-oxy-tris-
pyrrolidino-phosphonium hexafluorophosphate (PyBOP), (1.3 g,
3 eq), HOBt (0.13 g, 1 eq) and diisopropylethylamine (DIEA)
(0.95 ml, 6.5 eq) in DMF (45 min.). In cycles 2 and 3, step
viii is performed with Boc-Tyr(2,6-di-C1-Bzl)-OH (1.1 g, 3 eq)
and Boc-D-Gln (0.6 g, 3 eq), respectively; (ix) three washes
with DMF; (x) two washes with DCM. Solvent volumes were 15m1/g
resin. Coupling efficiency was checked at each coupling cycle
by the Kaiser test (Kaiser et al., Anal. Biochem., 34, 595-598,
1970, the disclosure of which is incorporated by reference).
The peptide was cleaved from the resin by intrachain aminolysis
in the presence of AcOH (0.097 ml, 2 eq) and DIEA (0.293 ml,
2 eq) in 30 ml DMF at room temperature for 24 hours. The
product was obtained from solution phase by filtration.
Protecting groups were removed with anhydrous hydrogen fluoride
(HF) at 0°C fox 30 minutes. The crude product was purified by
SephadexTM G-10, then RP-HPLC (Hondapak C1g column, l0 ~,m x
125A, 25 x 100 mm), with a gradient of 0-50% water-
acetonitrile, 0.1%TFA over 65 minutes. The final yield was
22mg (5%) based on starting resin. The purity and identity of
the synthetic peptide was assessed by analytical HPLC on
Bondapak C18 column, 10~m x 125A, 3.9 x 300 mm with a gradient
of 0-50% water-acetonitrile, 0.1% TFA over 50 minutes, k':2.7,
molecular mass by FAB-MS: 505 (calcd: 504.24), amino acid
analysis: D-Arg (1.1), Gln (1.0), Gly(1.0), Tyr (1.0). The
following cyclic peptides were synthesized based on the method
given above for peptide 8.
Peptide 5:
G1' y-Gln-A1a-Arg
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
The title compound was obtained as a white powder. The
molecular formula is C16H28N805~ Mol Wt/ MS-ESI: 824.44 (M+H:
413). Percentage purity based on HPLC was 95%-98%. Soluble in
water and DMSO. K'(HPLC): 2.25. Rf (B:A:W:P/15:3:10:12) -
5 0.75.
Peptide 5H:
Gly-Gln-Ala-Arg-Gly-Gln-Ala-Arg
The title compound was obtained as a white powder. The
molecular formula is C32H56N16010~ Mol Wt/ MS-ESI: 824.44
(M+H: 825). Percentage purity based on HPLC was 98%.
10 Soluble in water and DMSO.
Peptide 6:
Arg-Gln-Al~Arg
The title compound was obtained as a white powder. The
molecular formula is C2pH37N1105~ Mol Wt/ MS-ESI: 511.58
(M+H: 512). Percentage purity based on HPLC was 95%-98%.
15 Soluble in water and DMSO. K'(HPLC): 2.17. Rf
(B:A:W:P/15:3;10:12) - 0.79.
Peptide 6B:
Arg-Gln-A1a-Arg-Arg-Gln-Ala-Arg
The title compound was obtained as a white powder. The
molecular formula is C40H7N22O10. Mol Wt/ MS-ESI: 1023.12.
Percentage purity based on HPLC was 95%. Soluble in water and
DMSO. K'(HPLC): 2.23. Rf (B:A:W:P/15:3:10:12) - 0.85.
Peptide 7:
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA9$/01002
16
~Gly-GIn-Tyr-
The title compound was obtained as a white powder. The
molecular formula is C22H32N806~ Mol Wt/ MS-ESI: 504.24 (M+F~:
505). Percentage purity based on HPLC was 95%-98%. Soluble in
water and DMSO. K'(HPLC): 2.7. Rf (B:A:W:P/15:3:10:12) -
0.49.
Peptide 9:
Gly-D-Gln-Tyr-D-
The title compound was obtained as a white powder. The
molecular formula is C22H32N806. Mol Wt/ MS-ESI: 504.24 (M+H:
505). Soluble in water and DMSO. K'(HPLC): 2.7. Rf
(B:A:W:P/15:3:10:12) - 0.47.
3) Administration of the peptides
Mice (male 20-25 g, Swiss Webster) were obtained from
Charles River (Canadian Breeding Farm, St. Constant, Quebec).
They were housed five per cage in a room with controlled
temperature (22 ~ 2°C), humidity and artificial light (06.30-
19h). The animals had free access to food and water and were
used after a minimum of 4 days of acclimatisation to housing
conditions. Experiments were carried out between 10:00 a.m.
and 4:00 p.m. in an air-regulated and soundproof laboratory
(23 ~ 1°C, 40 % humidity), in which mice were habituated at
least 3o min before each experiment. The experiments were
authorized by the animal care committee of the University of
Ottawa in accordance withe the guidelines of the Canadian
Council on Animal Care.
The i.c.v. administrations of the peptides were
performed as described by Shukla et al. (Shukla et al., Brain
Res. 591, 176, 1992 the disclosure of which is incorporated by
reference). Peptides were dissolved in double-distilled
sterile water (vehicle) and 10 ~Cl of the peptide solution or
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
- - WO 99/Z1877 PCTICA98/01002
17
vehicle was delivered gradually within approximately 3 sec.
The mice exhibited normal behaviour within 1 min after
injection. The administration site was confirmed by injecting
Indian ink in preliminary experiments.
4) Antinociceptive assay
Mouse Writhing Test: Antinociceptive activity of the
peptides was evaluated using the acetic acid-induced writhing
test according to a modification (Shukla et al. Brain Research
591, 176, 1992 the disclosure of which is incorporated by
reference) of the method of Hayashi and Takemori (Eur.J.
Pharmacol. 16 63, 1971 the disclosure of which is incorporated
by reference). Male Swiss Webster [(SW)f BR] mice were
injected intraperitoneally (i.p.) with 1.0% acetic acid
(lOml/kg) 5 min after i.c.v. injection of 0 (saline), 0.5, 1,
10, 25, 50, 75, or loo nmol of the peptides. The number of
writhes displayed by each mouse was counted for a period of 10
min after the injection of the acetic acid solution. An
abdominal stretch is characterized by the contraction of the
abdominal muscles, the arching of the back ventrally such that
the abdomen touches the bedding surface and the extension of
one or both hind limbs. Mice were used once and then killed
immediately. Groups of l0 mice were used for each dose.
The analgesic activity of the peptides was assessed
in terms of either 1) the number of mice out of ten in which a
given dose of a peptide is considered to be active, expressed
as a percentage, or 2) the percent analgesia displayed by a
test group of 1o mice.
In the first case (method #1), the compound is said
to be active at a given dose if, after its administration, the
number of writhes elicited by a mouse injected with acetic acid
is equal to, or less than, one half the median number of
writhes recorded for the saline-treated control group of mice
that day, as described by Taber (Adv. Biochem.
Psycholpharmacol. 8:191, 1974, the disclosure of which is
incorporated by reference). The ED50 value (the dose of the
peptide that produced analgesia in 50% of the animals) with 95%
confidence limits (95% CL) and potency ratios with 95% CL were
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
- WO 99/21877 PCT/CA98/01002
18
measured by the method of Lichfield and Wilcox on (J.
Pharmacol. Exp. Ther. 96 99, 1949, the disclosure of which is
incorporated by reference) using procedure 47 of the computer
program of Tallarida and Murray (in "Manual of pharmacological
calculations with computer programs", 2nd ed., Springer, New
York, 1987, the disclosure of which is incorporated by
ref erence ) .
In the second case (method #2), the percentage of
analgesia is calculated for each dose by the formula:[(mean
l0 number of writhes in control group - mean number of writhes for
the test group)/(mean number of writhes in control group) x
100]. The doses producing 50% analgesia (AD50) with 95%
confidence limits (95% CL) and potency ratios with 95% CL are
measured by the method of Lichfield and Wilcoxon (J. Pharmacol.
Exp. Ther 96, 99, 1949 the disclosure in which is incorporated
by reference) using procedure 47 of the computer program of
Tallarida and Murray (in "Manual of pharmacological
calculations with computer programs". 2nd ed., Springer, New
York, 1987, the disclosure in which is incorporated by
reference).
In order to determine the length of action of the
peptides of the invention, the acetic acid solution was
administered at different times after the administration of the
peptide, as indicated. For verifying the blockade of the
analgesic effect of the peptides with receptor antagonists,
naloxone (lnmol), MK-801 (0.3 nmol) or CPP (0.1 nmol) were
administered i.c.v. in an aliquot of l0 ~,1, alone or in
combination with the peptides of the invention (50 nmol).
Naloxone is an opioid antagonist. MK-801 ((+)-5-methyl-10,11-
dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate) and CPP
((~)3-(2-carboxypiperazine-4-yl)-propyl-1-propionic acid) are
non-competitive and competitive NMDA receptor antagonists,
respectively. These two latter compounds were obtained from
Tocris Neuramin, Essex, England. The experiments for
assessment of the peripheral antinociceptive activity of the
peptides were performed by i.p. administration of 5 ~.mol/kg of
the tested compounds 10 min prior to the injection of the
SU8ST1TUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
19
acetic acid solution. Data were analyzed by the Wilcoxon's
paired non-parametric test. The criterion for statistical
significance was P < 0.05.
Mouse tail flick assay: Antinociception is also
determined using the radiant heat tail-flick technique (D'Amour
and Smith, J. Pharmacol. Exp. Ther. 72, 74, 1941, the
disclosure of which is herein incorporated by reference).
Briefly, the latency to withdraw the tail from a focused light
stimulus is determined using a photocell. The light intensity
is set to give a control reading of about 3 sec. Baseline
latencies are determined before experimental treatment as the
mean of two trials, and a maximal latency of 12 s is used to
minimize tissue damage. Post-treatment latencies are determined
5 min after i.c.v. injection. The antinociceptive effect is
expressed as the percentage of the maximum possible effect, as
calculated by the formula: %MPE=t(post-injection latency-
baseline latency)/(cutoff latency-baseline latency)] x100. The
use of %MPEs takes into account differences in baseline
latencies so that these differences do not bias the
quantification of antinociception. Group %MPE means are
compared using one-way ANOVAs and P s 0.05 is considered
significant .
Antinociceptive efficacy of tested peptides in the
mouse writhing test and the mouse tail-flick assay:
Intracerebroventricular administration of certain peptides of
the invention and related peptides in mice, induced dose- and
structure-dependent analgesic activities as assessed by their
ability to inhibit writhing in response to intraperioneal
(i.p.) injection of acetic acid (1%) as given below. Certain
peptides also produced potent antinociceptive effects in the
mouse tail-flick assay as compared with morphine.
Besides testing certain peptides of the invention, a
certain number of related peptides were tested for the purpose
of comparison. Histogranin (HILT) is an adrenal ~edullary
peptide possessing N-methyl-D-Aspartate (NMDA) receptor
antagonist activity and has analgesics properties. HN(7-15) is
a fragment of histogranin (Rogers et al. (1993) J. Pharmacol.
SUBSTITUTE SHEET (RULE 26)
L.v rY YV IY.,.oo~n rmn- + ~ ~ T-ZZZ r P.05~1p'~~J 5S3~J'~
'"'-- -- CA 02306754 2000-04-19
- Exp. Th2r. 267, 350-356, herein incorpox~ai=ed by reference).
The other pEp'ci.des used for purposas of compar~.scr. are rel ated
peptides. The sequences of Lhese peptides are as given below:
HN:
5 H-Met-Asn-Tyr-Ala-Leu-Lys-Gly-G1n-Gly-Axg-Thr-Leu-ryr-uly-Phe-
OH
(SerflBN:
H-Ser-Asn-TSrr-Ala-Leu-Lys-G1y-Gln-Gly--Arg-Thr-Leu-Tyr-G1y-Phe-
Oi~
~,0 AN (7-7.5)
H-Gly-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-DH
HN (1-10
H-Met-Asn-Tyr-Ala-Leu-Lye-Gly-Gln-Gly-Arg-OH
s~ ~asmoo~
1S H-Val-~lal-Tyr-Ala-Leu-Lys-Arg-Glr__-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-
QH (S~Q ID N0:12)
H4 (89-102 tOGP)
H-Tyr-Ala-Leu-Lys-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-Gly-Gly-
OH
20 Comparisons were also performed with morphine, an
Qpio~.d analgesic.
The results are shown in Tablas ? and 2 and FigurES 1
to 3, S, and 6.
N»A x~ceptor-mediated analgesia activity: T_n arder
to verify which receptor was invol'~ed in :he antinociceptive
activity of 4Serl]HN in the writhing test, the peptide was co-
administered with the apioid antagnoist naloxone, or the
competitive or non competitive NMDA antagonists, CPE and MK-
301, respecti~cre~.y. The results are gi~ren in F:.gure 4.
S) Measur~meat of prostc~glam,din E2 (PGE2y rslea~e ~xom Rat
l~lvaal.ar ~da.~roghages
~Iaie histar rats weighing 250 to 300 g were purchased
from Harlan Sprague Dawlay Ir.c. tIndianapolis). These animals
A
_ ~--~ +4;3 89 239~J9~46p : # ~;
vav-cu-ov l4:Laam trom- T-ZZi P.U6/iC~ =-5Q3
CA 02306754 2000-04-19
20a
Mal a Trt'istar rats weighing ~~0 to 300 g were purcha3ed
from Harlan Sprague Dawley Inc. (Tndianapolis). ~hESe animals
wExe derived from a pathog°n-free coinny, s~:; p~a~d behind fi-_tar
baxriers, and housed in a riorizort al laminar flow isolator
Johns Scientific :Inc. , '~oronto) . 8rc~r_croalveolax calls were
obtained by brorchoalveolar lavage as i~nown in the art.
A~I~iDED ~E~'
CA 02306754 2000-04-19
WO 99/21$77 PCT/CA98/01002
21
Briefly, after the animals were killed, the abdominal aorta was
severed and the trachea cannulated. A total volume of 48 ml of
PBS (pH 7.4) in 8-ml aliquots was infused in each animal, 93%
(45 ml) of which was recovered. The bronchoalveolar cells were
obtained by centrifugation at 2o0g at 4°C for 5 minutes and
resuspended in RPMI-1640 medium containing 0.5% dialysed FHS
(Wisent Inc., St-Bruno, Quebec) and o.8% Hepes, which will
henceforth be referred to as tissue culture media.
Differential cellular analysis, made from cytocentrifuged
smears (4 x 104 cells) stained with Wright-Giemsa, indicated
that the bronchoalveolar cells represent a pure population of
alveolar macrophage (AM, 99%). Alveolar macrophages (0.2 x
106) were incubated in 0.2 ml tissue culture media for 20 h at
37°C in a humidified 95%air-5%C02 atmosphere alone or with
lipopolysaccharide (LPS)(l~.g/ml)(Sigma chemical Co., St-Louis,
MO) in the presence and absence of one of [SerIJHN, Peptide 5,
Peptide 6, Peptide 7, and Peptide 8 at various concentrations
(10-9M-10-7M). The culture supernatants were collected,
centrifuged and frozen at -80°C.
The following day, PGE2 was determined in cell-tree
supernatants using a competitive enzymeimmunoassay (EIA) system
(Biotrak, Amersham Pharmacia Biotech). The assay is based on
competition between unlabelled PGE2 and a fixed quantity of
peroxidase-labelled PGE2 for a limited number of binding sites
on a PGE2 specific antibody. It was performed according to the
manufacturer's instruction. Results are expressed as percent
(%) of LPS response and represent the mean~SEM of at least 3
experiments measured in triplicate.
Inhibition of prostaglandin E2 production by
macrophages in response to LPSs The effect of certain peptides
of the invention and related peptides on the production of PGE2
by macrophages in response to LPS was tested and the results
are given in Figure 7.
BRIEF DESCRIPTION OF THE DRAWINGS
Certain results of the examples are illustrated with the
aid of the Figures.
Figure 1 gives the dose response curves of the analgesic
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99121877 PCT/CA98/01002
22
effects of the peptides of the invention compared with several
other peptides. The analgesic effect is calculated as the
percent of mice showing analgesia using method #1 explained in
the examples section.
Figure 2 gives the dose response curves of the
analgesic effects of Peptide 6, Peptide 7, and Peptide 8 as
calculated as percent analgesia using method #2 explained in
the examples section.
Figure 3 gives the time response curves of the
analgesic effects of [Serf]HN, Peptide 3, Peptide 5, Peptide 6,
Peptide 7, and Peptide 8 measured as percent analgesia using
method #2 explained in the examples section.
Figure 4 shows the effects of naloxone (1 nmol,
i.c.v.), MK-801 (0.1 nmol, i.c.v.) and CPP (0.3 nmol, i.c.v.)
on the analgesic effects of [Serf]HN (50 nmol/mouse, i.c.v.) in
the mouse writhing pain assay. *P s 0.05 as compared with
[Serf] HN alone.
Figure 5 shows the analgesic effects of peripheral
(intraperitoneal) administration (5 ~.mol/kg) of morphine,
histone H4(86-100), [Serf]HN, HN(7-15), Peptide 3, and Peptide
5 in the mouse writhing test. *P s 0.05 as compared with
control saline.
Figure 6 shows the analgesic effects of morphine (5
~.g/mouse, i.c.v.), Peptide 5 (50 ninol/mouse, i.c.v.), Peptide 6
(50 nmol/mouse, i.c.v.), Peptide 7 (10 nmol/mouse, i.c.v.) and
Peptide 8 (10 nmol/mouse, i.c.v.) in the mouse tail flick
assay. *P s 0.05 as compared with control saline.
Figure 7 shows the inhibitory effects of [Serf]HN,
Peptide 5, Peptide 6, Peptide 7, and Peptide 8 on the LPS-
induced production of prostaglandin E2 by primary cultures of
rat alveolar macrophages.
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
23
Table 1: Relative potencies of tested peptides (i.c.v.) in
producing analgesia in the mouse writhing pain assay calculated
as percent of mice showing analgesia (method #1 in the examples
section).
Peptide ED50 Potency
(nmol/mouse) b (95% CL)
a i
(95% CL) o
rat
22.3 (12.1-41.1) 1.0
[Serl]HN 17.4 (7.0-43.0) 1.3 (0.4-3.8)
H4-(86-100) 4.1 (0.9-17.9) 5.4 (0.7-40.1)
H4-(89-102)(OGP)c 40.9 (25.8-65) 0.5 (0.25-1.17)
2 11.3 (4.2-30.4) 2.0 (0.6-6.3)
3 3.9 (1.7-9.1) 5.7 (2.0-15.9)
4 4.9 (1.8-13.2) 4.5 (1.4-14.3)
5 2.9 (0.8-9.8) 7.7 (1.3-46.6)
13 7.5 (2.3-24.4) 3.0 (0.8-11.2)
a95% Confidence limit. bAs compared with HN. cOGP:
osteoblastic growth peptide. *P s 0.05 as compared with HN.
Table 2: Relative potency of tested peptides (i.c.v.) in
producing analgesia in the mouse writhing pain assay as
calculated as percent analgesia.
Peptide ADS (nmol/mouse) Potency
a b (95% CL)
i
(95% CL) o
rat
23.6 (12.4-45.0) 1.0
[Serl]HN 31.2 (18.5-52.6) 0.75 (0.23-2.43)
H4-(86-100) 22.7 (13.3-38.7) 1.04 (0.32-3.38)
H4-(89-102)(OGP)c 49.1 (33.0-73.1) 0.48 (0.17-0.91)
1 12.8 (3.6-46.1) 1.84 (0.3 -12.5)
2 25.9 (5.9-113) 0.91 (0.11-7.63)
3 3.41 (0.8-14.6) 6.92 (0.84-56.2)*
4 12.7 (3.5-46.1) 1.86 (0.27-12.8)
5 13.3 (1.6-114) 1.77 (0.11-28.1)
5B 25.3 (11.6-55.1) 0.93 (0.22-3.9)
6 25.1 (11.3-55.6) 0.94 (0.22-3.98)
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
- - WO 99/21877 PCT/CA98/01002
24
7 4.21 (1.80-9.87) 5.6 (1.25-25)*
g 4.41 (1.7-11.9) 5.35 (1.04-26.4)*
g 2.23 (1.45-3.43) 10.6 (3.6-31.0)*
13 9.7 (2.5-37.4) 2.43 (0.2-18)
a95% Confidence limit. bAs compared with HN. cOGP:
osteoblastic growth peptide. *P s 0.05 as compared with HN.
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
- WO 99/21877 PCT/CA98/01002
The following references are herein incorporated by
reference .
1. Lemaire, S., Shukla, V.K., Rogers, C., Ibrahim, I.H.,
Lapierre, C. and Dumont, M.(1993). Isolation and
5 characterization of histogranin, a natural peptide with N-
methyl-D-aspartate antagonist activity. Or. J. Pharmacol:
Molec. Pharm. Section. 245, 247-256.
2. Shukla, V.K., Lemaire, S., Dumont, M. and Merali, Z.
(1995). N-methyl-D-aspartate receptor antagonist activity
l0 and phencyclidine-like behavioral effects of the
pentadecapeptide, [Sere]histogranin. Pharmacol. Biochem.
Behav. 50, 49-54.
3. Rogers, C., and Lemaire, S. (1993). Characterization of
[ 125I ] [ Serl] histogranin binding sites in rat brain . J .
15 Pharmacol. Exp. Ther. 267, 350-356.
4. Dumont, M., Prasad, J. and Lemaire, S. (1994). Interaction
of histogranin and related peptides with
[3H]dextromethorphan binding sites in rat brain. Neurosci.
Lett. 173, 135-138.
20 5. Prasad, J.A., and Lemaire, S. (1997). Modulation by
histogranin and related peptides of Gly potentiation of
[3H]MK-801 binding to rat brain membranes. Submitted.
6. Yamamoto, T., and Yaksh, T.L. (1992). Spinal pharmacology
of thermal hyperalgesia induced by constriction injury of
25 sciatic nerve. Excitatory amino acid antagonists. Pain 49,
121-128.
7. Ren, K., Williams, G.M., Hylden, J.L., Ruda, M.A., and
Dubner, R., (1992). The intrathecal administration of
excitatory amino acid receptor antagonists selectively
attenuated carrageenan-induced behavioral hyperalgesia in
rats. Or. J. Pharmacol. 219, 235-243.
S. Coderre, T.J. and Melzack, R. (1991). Central neural
mediators of secondary hyperalgesia following heat injury
in rats: neuropeptides and excitatory amino acids.
Neurosci. Lett. 131, 71-74.
9. Coderre, T.J. and Van Empel, I. (1994). The utility of
excitatory amino acid (EAA) antagonists as analgesic
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
26
agents: Comparison of the antinociceptive activity of
various classes of EAA antagonists in mechanical, thermal,
and chemical nociceptive tests. Pain 59, 345-359.
10. Wilcox, G.L., (1993), Spinal mediators of nociceptive
neurotransmission and hyperalgesia:relationships among
synaptic plasticity, analgesic tolerance and blood flow.
APS J. 2, 265-275.
11. Trujillo, K.A. and Akil, H., (1991), Inhibition of
morphine tolerance and dependence by the NMDA receptor
antagonist MK-801. Science 251, 85-87.
12. Marek, P., Ben-Eliyahu, S. Vaccarino, A.L., Liebeskind,
J.C., (1991), Delayed application of MK-801 attenuates the
development of morphine tolerance in rats. Brain Res. 558,
163-165.
13. Elliot, K., Minami, N., Koleskinov Y. et al., (1994), The
NMDA receptor antagonist, LY274614 and MK-801, and the
nitric oxide synthetase inhibitor, NG-nitro-L-
arginine, attenuate analgesic tolerance to the mu opioid
morphine but not to kappa opioids. Pain 56, 69-75.
14. Sagen, J., Wang, H. and Pappas, G.G., (1990), Adrenal
medullary implants in the rat spinal cord reduce
nociception in a chronic model of pain model. Pain 42, 69-
79.
15. Wang, H. and Sagen, J., (1995), Attenuation of pain-
related hyperventilation in adjuvant arthritic rats with
adrenal medullary transplants in the spinal subarachnoid
space. Pain 63, 313-320.
16. Sortwell, C.E., Pappas, G.D., and Sagen, J., (1995),
Chromaffin cell xenografts in the rat neocortex can
produce antidepressive activity in the forced swimming
test. Exp. Brain Res. 103, 59-69.
17. Wang, H. and Sagen, J., (1994), Absence of appreciable
tolerance and morphine cross-tolerance in rats with
adrenal medullary transplants in the spinal cord.
Neuropharmacology 33, 681-692.
18. Hama, A.T., Pappas, G.D., Sagen, J., (1996), Adrenal
medullary implants reduce degeneration in the spinal cord
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/O1(102
27
of rats following chronic constriction of nerve injury.
Exp. Neurology 137, 81-93.
19. Hama, A.T. and Sagen, J., (1994), Alleviation of
neuropathic pain symptoms by xenogenic chromaffin cell
grafts in the spinal subarachnoid space. Brain Res. 651,
183-193.
20. Siegan, J.B., Hama, A.T., and Sagen, J., (1996),
Histogranin attenuates chronic pain induced by peripheral
neuropathy, formalin-induced nociception and direct
application of NMDA. Soc. Neurosci. 22, 1349.
21. Siegan J.H. and Sagen, J., (1997), A natural peptide with
NMDA inhibitory activity reduces tonic pain in the
formalin model. Neuroreport 8, 1379-1381.
22. Siegan, J.B., Hama, A.T. and Sagen, J., (1997),
Suppression of neuropathic pain by naturally-derived
peptide with NMDA antagonist activity. Brain Res. 755,
331-334.
23. Ruan, H., Prasad, J. and Lemaire, S., (1997), Central and
peripheral non-opioid analgesic activity of histogranin
and related peptides. Unsubmitted manuscript.
24. Mogil, J.C., Sternberg, W.F., Balian, H., Liebeskind, J.C.
and Sudowski, B., (1996), Opioid and non-opioid swim
stress-induced analgesia. A parametric analysis in mice.
Physiol. Behav. 59, 123-133.
25. Lemaire, S., Griffiths, J., Lapierre, C., Lemaire, I.,
Merali, Z. and Ravindran, A.V., (1993), Characterization
of histogranin receptors in human peripheral blood
lymphocytes. Biochem. Biophys. Res. Commun. 194, 1323-
1329.
26. Litchfield, J.T. and F. Wilcoxon, (1949), A simplified
method of evaluating dose-effect experiments. J.
Pharmacol. Exp. Ther. 96, 99.
27. Bab I., D. Gazit, M. Chorev, A. Muhlrad, A. Shteyer,
Z. Greenberg, M. Namdar and M. Kahn, (1992), Histone H4-
related osteogenic growth peptide (OGP): a novel
circulating stimulator of osteoblastic activity. EMBO J.
11, 1867.
SUBSTITUTE SHEET (RULE 26)
CA 02306754 2000-04-19
WO 99/21877 PCT/CA98/01002
28
28. Hooke, L.P., L. He, and N.M. Lee, (1995), [Des-
Tyrl]dynorphin A-(2-17) has naloxone-insensitive
antinociceptive effect in the writhing assay. J.
Pharmacol. Exp. Ther. 273, 802.
29. E. Kaiser, Colescott, R,L., Bossingre, C.D. and Cook,
P.I., (1970), Anal. Biochem., 595.
30. U.S. Patent No. 5,169,833.
SUBSTITUTE SHEET (RULE 26)
