U.S. patent application number 15/137033 was filed with the patent office on 2016-10-27 for uses of kappa opioid synthetic peptide amides.
This patent application is currently assigned to Cara Therapeutics, Inc.. The applicant listed for this patent is Cara Therapeutics, Inc.. Invention is credited to Roberta Vezza ALEXANDER, Derek T. CHALMERS, Guangcheng JIANG, Robert Zhiyong LUO, Frederique MENZAGHI, Claudio D. SCHTEINGART, Robert H. SPENCER, Javier SUEIRAS-DIAZ.
Application Number | 20160310558 15/137033 |
Document ID | / |
Family ID | 44505590 |
Filed Date | 2016-10-27 |
United States Patent
Application |
20160310558 |
Kind Code |
A1 |
SCHTEINGART; Claudio D. ; et
al. |
October 27, 2016 |
USES OF KAPPA OPIOID SYNTHETIC PEPTIDE AMIDES
Abstract
The invention relates to methods of use of synthetic peptide
amides that are ligands of the kappa opioid receptor in the
treatment and prevention of kappa opioid receptor-associated
diseases and conditions; and particularly to uses of these agonists
in the prophylaxis, inhibition and treatment of pain, inflammation
and pruritis associated with a variety of diseases, disorders and
conditions. Inflammatory conditions preventable or treatable by the
methods of the invention include diseases and conditions associated
with elevated levels of a proinflammatory cytokines, such as
TNF-.alpha., IL-1.beta., IL-6, MMP-1 and MMP-3. Such diseases and
conditions include cardiovascular inflammation, neurological
inflammation, skeletal inflammation, muscular inflammation,
gastrointestinal inflammation, ocular inflammation, otic
inflammation, inflammation due to insect bites and inflammation due
to wound healing; atherosclerosis, ischemia, restenosis and
vasculitis; of asthma, Sjogren's syndrome, pulmonary inflammation,
chronic airway inflammation and chronic obstructive pulmonary
disease (COPD), allergy, psoriasis, psoriatic arthritis, eczema,
scleroderma, atopic dermatitis and systemic lupus erythematosus,
arthritis, synovitis, osteomyelitis, rheumatoid arthritis,
osteoarthritis and ankylosing spondylitis; septicemia and septic
shock, diabetes, glucose intolerance, insulin resistance and
obesity, colitis, ulcerative colitis, Crohn's disease, IBD and IBS,
and the inflammatory diseases and conditions due to tumor
proliferation, tumor metastasis or transplantation rejection.
Inventors: |
SCHTEINGART; Claudio D.;
(San Diego, CA) ; MENZAGHI; Frederique; (Rye,
NY) ; JIANG; Guangcheng; (San Diego, CA) ;
ALEXANDER; Roberta Vezza; (San Diego, CA) ;
SUEIRAS-DIAZ; Javier; (La Jolla, CA) ; SPENCER;
Robert H.; (New Hope, PA) ; CHALMERS; Derek T.;
(Riverside, CT) ; LUO; Robert Zhiyong; (New City,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cara Therapeutics, Inc. |
Shelton |
CT |
US |
|
|
Assignee: |
Cara Therapeutics, Inc.
Shelton
CT
|
Family ID: |
44505590 |
Appl. No.: |
15/137033 |
Filed: |
April 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14557576 |
Dec 2, 2014 |
9321810 |
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15137033 |
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12868790 |
Aug 26, 2010 |
8906859 |
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14557576 |
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12480059 |
Jun 8, 2009 |
8236766 |
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12868790 |
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12119311 |
May 12, 2008 |
7713937 |
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12480059 |
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11938771 |
Nov 12, 2007 |
7402564 |
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12119311 |
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11938776 |
Nov 12, 2007 |
7842662 |
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11938771 |
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60928557 |
May 10, 2007 |
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11938776 |
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60928527 |
May 10, 2007 |
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60928557 |
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60858120 |
Nov 10, 2006 |
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60928527 |
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60858121 |
Nov 10, 2006 |
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60858120 |
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60858123 |
Nov 10, 2006 |
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60858121 |
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60928551 |
May 10, 2007 |
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60858123 |
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60858109 |
Nov 10, 2006 |
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11938771 |
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60928550 |
May 10, 2007 |
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60858109 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/095 20190101;
C07K 5/101 20130101; A61P 3/10 20180101; A61P 35/00 20180101; C07K
5/1016 20130101; A61P 25/04 20180101; C07K 5/1019 20130101; A61K
38/07 20130101; A61P 17/00 20180101; A61P 25/00 20180101; A61P
19/02 20180101; A61P 3/04 20180101; C07K 7/02 20130101; A61K 38/08
20130101; A61P 19/00 20180101 |
International
Class: |
A61K 38/07 20060101
A61K038/07; A61K 38/08 20060101 A61K038/08 |
Claims
1. A method of prophylaxis or treatment of a kappa opioid
receptor-associated disease or condition in a mammal, the method
comprising administering to the mammal a composition comprising an
effective amount of a synthetic peptide amide of the formula:
##STR00063## or a stereoisomer, mixture of stereoisomers, prodrug,
pharmaceutically acceptable salt, hydrate, acid salt hydrate,
N-oxide or isomorphic crystalline form thereof, wherein each
Xaa.sub.1 is independently selected from the group consisting of
(A)(A')D-Phe, (A)(A')(.alpha.-Me)D-Phe, D-Tyr, D-Tic,
D-tert-leucine, D-neopentylglycine, D-phenylglycine,
D-homophenylalanine, .beta.-(E)D-Ala and tert-butyl-D-Gly, wherein
each (A) and each (A') are phenyl ring substituents independently
selected from the group consisting of --H, --F, --Cl, --NO.sub.2,
--CH.sub.3, --CF.sub.3, --CN, --CONH.sub.2, and wherein each (E) is
independently selected from the group consisting of tert-butyl,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, furyl, pyridyl,
thienyl, thiazolyl and benzothienyl; each Xaa.sub.2 is
independently selected from the group consisting of (A)(A')D-Phe,
(A)(A')(.alpha.-Me)D-Phe, D-1Nal, D-2Nal, D-Tyr, (E)D-Ala, and
D-Trp; each Xaa.sub.3 is independently selected from the group
consisting of D-Nle, D-Phe, (E)D-Ala, D-Leu, (.alpha.-Me)D-Leu,
D-Hle, D-Val, and D-Met; each Xaa.sub.4 is independently selected
from the group consisting of (B).sub.2D-Arg, (B).sub.2D-Nar,
(B).sub.2D-Har, .zeta.-(B)D-Dap, .epsilon.-(B)D-Lys,
.epsilon.-(B).sub.2-D-Lys, D-Amf, amidino-D-Amf,
.gamma.-(B).sub.2D-Dbu, .delta.-(B).sub.2.alpha.-(B')D-Orn,
D-2-amino-3(4-piperidyl)propionic acid,
D-2-amino-3(2-aminopyrrolidyl)propionic acid,
D-.alpha.-amino-.beta.-amidino-propionic acid,
.alpha.-amino-4-piperidineacetic acid,
cis-.alpha.,4-diaminocyclohexane acetic acid,
trans-.alpha.,4-diaminocyclohexaneacetic acid,
cis-.alpha.-amino-4-methyl-aminocyclo-hexane acetic acid,
trans-.alpha.-amino-4-methylaminocyclohexane acetic acid,
.alpha.-amino-1-amidino-4-piperidineacetic acid,
cis-.alpha.-amino-4-guanidino-cyclohexane acetic acid, and
trans-.alpha.-amino-4-guanidinocyclohexane acetic acid, wherein
each (B) is independently selected from the group consisting of --H
and C.sub.1-C.sub.4 alkyl, and (B') is --H or (.alpha.-Me), and p
is zero or 1; G is ##STR00064## wherein q, r and s are each
independently zero or 1; p and t are each independently zero or 1;
provided that at least one of q, r, s and t is 1; L is a linker
selected from the group consisting of .epsilon.-D-Lys,
.epsilon.-Lys, .delta.-D-Orn, .delta.-Orn, .gamma.-aminobutyric
acid, 8-aminooctanoic acid, 11-amino-undecanoic acid,
8-amino-3,6-dioxaoctanoic acid, 4-amino-4-carboxylic piperidine and
bis(D-Lys-Gly)Lactam; or G is ##STR00065## wherein p is 1; and the
moiety ##STR00066## is an optionally substituted 4 to 8-membered
heterocyclic ring moiety wherein Y is C or N and Z is C, N, O, S,
SO, or SO.sub.2; provided that when such ring moiety is a 6-, 7- or
8-membered ring, Y and Z are separated by at least two ring atoms;
and provided further that when such ring moiety is aromatic, then Y
is carbon; wherein W is selected from the group consisting of:
null, provided that when W is null, Y is N;
--NH--(CH.sub.2).sub.b-- with b equal to zero, 1, 2, 3, 4, 5, or 6;
and --NH--(CH.sub.2).sub.c--O-- with c equal to 2, or 3; wherein V
is C.sub.1-C.sub.6 alkyl, and e is zero or 1, wherein when e is
zero, then V is null and, R.sub.1 and R.sub.2 are directly bonded
to the same or different ring atoms; wherein (a) R.sub.1 is --H,
--OH, halo, CF.sub.3, --NH.sub.2, --COOH, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, amidino, C.sub.1-C.sub.6 alkyl-substituted
amidino, aryl, optionally substituted heterocyclyl, Pro-amide, Pro,
Gly, Ala, Val, Leu, Ile, Lys, Arg, Orn, Ser, Thr, CN, CONH.sub.2,
COR', SO.sub.2R', CONR'R'', NHCOR', OR, or SO.sub.2NR'R''; wherein
said optionally substituted heterocyclyl is optionally singly or
doubly substituted with substituents independently selected from
the group consisting of C.sub.1-C.sub.6 alkyl, --C.sub.1-C.sub.6
alkoxy, oxo, --OH, --Cl, --F, --NH.sub.2, --NO.sub.2, --CN, --COOH,
and amidino; wherein R' and R'' are each independently --H,
C.sub.1-C.sub.8 alkyl, aryl, or heterocyclyl or R' and R'' are
combined to form a 4- to 8-membered ring, which ring is optionally
substituted singly or doubly with substituents independently
selected from the group consisting of C.sub.1-C.sub.6 alkyl,
--C.sub.1-C.sub.6 alkoxy, --OH, --Cl, --F, --NH.sub.2, --NO.sub.2,
--CN, --COOH and amidino; and R.sub.2 is H, amidino, singly or
doubly C.sub.1-C.sub.6 alkyl-substituted amidino, --CN,
--CONH.sub.2, --CONR'R'', --NHCOR', --SO.sub.2NR'R'', or --COOH; or
(b) R.sub.1 and R.sub.2 taken together can form an optionally
substituted 4- to 9-membered heterocyclic monocyclic or bicyclic
ring moiety which is bonded to a single ring atom of the Y and
Z-containing ring moiety; or (c) R.sub.1 and R.sub.2 taken together
with a single ring atom of the Y and Z-containing ring moiety can
form an optionally substituted 4- to 8-membered heterocyclic ring
moiety to form a spiro structure; or (d) R.sub.1 and R.sub.2 taken
together with two or more adjacent ring atoms of the Y and
Z-containing ring moiety can form an optionally substituted 4- to
9-membered heterocyclic monocyclic or bicyclic ring moiety fused to
the Y and Z-containing ring moiety; wherein each of said optionally
substituted 4-, 5-, 6-, 7-, 8-, and 9-membered heterocyclic ring
moieties comprising R.sub.1 and R.sub.2 is optionally singly or
doubly substituted with substituents independently selected from
the group consisting of C.sub.1-C.sub.6 alkyl, --C.sub.1-C.sub.6
alkoxy, optionally substituted phenyl, oxo, --OH, --Cl, --F,
--NH.sub.2, --NO.sub.2, --CN, --COOH, and amidino; provided that
when the Y and Z-containing ring moiety is a six or seven membered
ring comprising a single ring heteroatom and wherein such
heteroatom is N, and e is zero, then R.sub.1 is not --OH, and
R.sub.1 and R.sub.2 are not both --H; provided further that when
the Y and Z-containing ring moiety is a six membered ring
comprising two ring heteroatoms, both Y and Z are N, W is null, and
--V.sub.e(R.sub.1)(R.sub.2) is attached to Z, then
--V.sub.e(R.sub.1)(R.sub.2) is selected from the group consisting
of amidino, C.sub.1-C.sub.6 alkyl-substituted amidino,
dihydroimidazole, --CH.sub.2COOH, and --H.sub.2C(O)NH.sub.2, and
lastly, provided that if the Y and Z-containing ring moiety is a
six membered ring comprising an S or O ring heteroatom, or if the Y
and Z-containing ring moiety is a six membered ring comprising two
ring heteroatoms, wherein both Y and Z are N and W is null, or if
the Y and Z-containing ring moiety is a six membered aromatic ring
comprising a single ring heteroatom, which heteroatom is N, then,
when e is zero, R.sub.1 and R.sub.2 are not both --H.
2. The method according to claim 1, wherein the kappa opioid
receptor-associated disease or condition is an inflammatory disease
or condition.
3. The method according to claim 2, wherein the inflammatory
disease or condition is selected from the group consisting of
cardiovascular inflammation, neurological inflammation, skeletal
inflammation, muscular inflammation, gastrointestinal inflammation,
ocular inflammation, otic inflammation, inflammation due to insect
bites and inflammation due to wound healing.
4. The method according to claim 3, wherein the cardiovascular
inflammation is due to atherosclerosis, ischemia, restenosis or
vasculitis.
5. The method according to claim 2, wherein the inflammatory
disease or condition is selected from the group consisting of
asthma, Sjogren's syndrome, pulmonary inflammation, chronic airway
inflammation and chronic obstructive pulmonary disease (COPD).
6. The method according to claim 2, wherein the inflammatory
disease or condition is selected from the group consisting of
allergy, psoriasis, psoriatic arthritis, eczema, scleroderma,
atopic dermatitis and systemic lupus erythematosus.
7. The method according to claim 2, wherein the inflammatory
disease or condition is selected from the group consisting of
arthritis, synovitis, osteomyelitis, rheumatoid arthritis,
osteoarthritis and ankylosing spondylitis.
8. The method according to claim 2, wherein the inflammatory
disease or condition is selected from the group consisting of
septicemia and septic shock.
9. The method according to claim 2, wherein the inflammatory
disease or condition is selected from the group consisting of
diabetes, glucose intolerance, insulin resistance and obesity.
10. The method according to claim 2, wherein the inflammatory
disease or condition is selected from the group consisting of
colitis, ulcerative colitis, Crohn's disease, IBD (inflammatory
bowel disease) and IBS (Irritable bowel syndrome).
11. The method according to claim 2, wherein the inflammatory
disease or condition is due to tumor proliferation, tumor
metastasis or transplantation rejection.
12. The method according to claim 1, wherein the mammal is a
human.
13. The method according to claim 12, wherein the disease or
condition is migraine.
14. The method according to claim 13, wherein the synthetic peptide
amide is selected from the group consisting of compounds
(1)-(105).
15. A method according to claim 1, wherein the inflammatory disease
or condition is associated with elevated levels of a
proinflammatory cytokine.
16. The method according to claim 15, wherein the proinflammatory
cytokine is selected from the group consisting of TNF-.alpha.,
IL-1.beta., IL-6, MMP-1 and MMP-3.
17. A method of prophylaxis or treatment of an inflammatory disease
or condition in a mammal, the method comprising administering to
the mammal a composition comprising an effective amount of a
synthetic peptide amide having a structure selected from the group
consisting of compounds (1)-(105).
18. The method according to claim 17, wherein the mammal is a
human.
19. The method according to claim 17, wherein the the inflammatory
disease or condition is associated with elevated levels of a
proinflammatory cytokine.
20. The method according to claim 17, wherein the proinflammatory
cytokine is selected from the group consisting of TNF-.alpha.,
IL-1.beta., IL-6, MMP-1 and MMP-3.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 12/480,059, filed Jun. 8, 2009 which is a is a
continuation-in-part of U.S. application Ser. No. 12/119,311 filed
May 12, 2008, now U.S. Pat. No. 7,713,937, which is a
continuation-in-part of U.S. application Ser. No. 11/938,771 filed
Nov. 12, 2007, now U.S. Pat. No. 7,402,564 which claims priority to
U.S. provisional applications, Ser. No. 60/858,109 filed Nov. 10,
2006, and 60/928,550 filed May 10, 2007; and is a
continuation-in-part of U.S. Ser. No. 11/938,776 filed Nov. 12,
2007 which claims priority to U.S. provisional applications, Ser.
Nos. 60/858,120; 60/858,121 and 60/858,123 filed Nov. 10, 2006, and
60/928,527, 60/928,551, and 60/928,557 filed May 10, 2007, all of
which are expressly incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The invention relates to synthetic peptide amides
incorporating D-amino acids in the peptide chain and more
particularly to such synthetic peptide amides that are kappa opioid
receptor agonists, and methods for their use as prophylactic and
therapeutic agents for the prevention, inhibition and treatment of
kappa opioid receptor associated diseases and conditions, including
pain, inflammation and pruritis.
BACKGROUND
[0003] Kappa opioid receptors have been suggested as targets for
intervention for treatment or prevention of a wide array of
diseases and conditions by administration of kappa opioid receptor
agonists. See for example, Jolivalt et al., Diabetologia,
49(11):2775-85; Epub Aug. 19, 2006), describing efficacy of
asimadoline, a kappa receptor agonist in rodent diabetic
neuropathy; and Bileviciute-Ljungar et al., Eur. J. Pharm.
494:139-46 (2004) describing the efficacy of kappa agonist U-50,488
in the rat chronic constriction injury (CCI) model of neuropathic
pain and the blocking of its effects by the opioid antagonist,
naloxone. These observations support the use of kappa opioid
receptor agonists for treatment of diabetic, viral and
chemotherapy-induced neuropathic pain. The use of kappa receptor
agonists for treatment or prevention of visceral pain including
gynecological conditions such as dysmenorrheal cramps and
endometriosis has also been reviewed. See for instance, Riviere,
Br. J. Pharmacol. 141:1331-4 (2004).
[0004] Kappa opioid receptor agonists have also been proposed for
the treatment of pain, including hyperalgesia. Hyperalgesia is
believed to be caused by changes in the milieu of the peripheral
sensory terminal occur secondary to local tissue damage. Tissue
damage (e.g., abrasions, burns) and inflammation can produce
significant increases in the excitability of polymodal nociceptors
(C fibers) and high threshold mechanoreceptors (Handwerker et al.
(1991) Proceeding of the VIth World Congress on Pain, Bond et al.,
eds., Elsevier Science Publishers BV, pp. 59-70; Schaible et al.
(1993) Pain 55:5-54). This increased excitability and exaggerated
responses of sensory afferents is believed to underlie
hyperalgesia, where the pain response is the result of an
exaggerated response to a stimulus. The importance of hyperalgesia
in the post-injury pain state has been repeatedly demonstrated and
appears to account for a major proportion of pain experienced in
the post-injury/inflammatory state. See for example, Woold et al.
(1993) Anesthesia and Analgesia 77:362-79; Dubner et al. (1994) In,
Textbook of Pain, Melzack et al., eds., Churchill-Livingstone,
London, pp. 225-242.
[0005] Kappa opioid receptors have been suggested as targets for
the prevention and treatment of cardiovascular disease. See for
example, Wu et al. "Cardioprotection of Preconditioning by
Metabolic Inhibition in the Rat Ventricular Myocyte--Involvement of
kappa Opioid Receptor" (1999) Circulation Res vol. 84: pp.
1388-1395. See also Yu et al. "Anti-Arrhythmic Effect of Kappa
Opioid Receptor Stimulation in the Perfused Rat Heart: Involvement
of a cAMP-Dependent Pathway" (1999) J Mol Cell Cardiol. vol.
31(10): pp. 1809-1819.
[0006] It has also been found that development or progression of
these diseases and conditions involving neurodegeneration or
neuronal cell death can be prevented, or at least slowed, by
treatment with kappa opioid receptor agonists. This improved
outcome is believed to be due to neuroprotection by the kappa
opioid receptor agonists. See for instance, Kaushik et al.
"Neuroprotection in Glaucoma" (2003) J. Postgraduate Medicine vol.
49 (1): pp. 90-95.
[0007] The presence of kappa opioid receptors on immune cells
(Bidlak et al., (2000) Clin. Diag. Lab. Immunol. 7(5):719-723) has
been implicated in the inhibitory action of a kappa opioid receptor
agonist, which has been shown to suppress HIV-1 expression. See
Peterson P K et al., Biochem Pharmacol. 2001, 61(19):1145-51.
[0008] Walker, Adv. Exp. Med. Biol. 521:148-60 (2003) appraised the
anti-inflammatory properties of kappa agonists for treatment of
osteoarthritis, rheumatoid arthritis, inflammatory bowel disease
and eczema. Bileviciute-Ljungar et al., Rheumatology 45:295-302
(2006) describe the reduction of pain and degeneration in Freund's
adjuvant-induced arthritis by the kappa agonist U-50,488.
[0009] Wikstrom et al., J. Am. Soc. Nephrol. 16:3742-7 (2005)
describes the use of the kappa agonist, TRK-820 for treatment of
uremic and opiate-induced pruritis, and Ko et al., J. Pharmacol.
Exp. Ther. 305:173-9 (2003) describe the efficacy of U-50,488 in
morphine-induced pruritis in the monkey.
[0010] Application of peripheral opioids including kappa agonists
for treatment of gastrointestinal diseases has also been
extensively reviewed. See for example, Lembo, Diges. Dis. 24:91-8
(2006) for a discussion of use of opioids in treatment of digestive
disorders, including irritable bowel syndrome (IBS), ileus, and
functional dyspepsia.
[0011] Ophthalmic disorders, including ocular inflammation and
glaucoma have also been shown to be addressable by kappa opioids.
See Potter et al., J. Pharmacol. Exp. Ther. 309:548-53 (2004),
describing the role of the potent kappa opioid receptor agonist,
bremazocine, in reduction of intraocular pressure and blocking of
this effect by norbinaltorphimine (norBNI), the prototypical kappa
opioid receptor antagonist; and Dortch-Carnes et al., CNS Drug Rev.
11(2):195-212 (2005). U.S. Pat. No. 6,191,126 to Gamache discloses
the use of kappa opioid agonists to treat ocular pain. Otic pain
has also been shown to be treatable by administration of kappa
opioid agonists. See U.S. Pat. No. 6,174,878 also to Gamache.
[0012] Kappa opioid agonists increase the renal excretion of water
and decrease urinary sodium excretion (i.e., produces a selective
water diuresis, also referred to as aquaresis). Many, but not all,
investigators attribute this effect to a suppression of vasopressin
secretion from the pituitary. Studies comparing centrally acting
and purportedly peripherally selective kappa opioids have led to
the conclusion that kappa opioid receptors within the blood-brain
barrier are responsible for mediating this effect. Other
investigators have proposed to treat hyponatremia with nociceptin
peptides or charged peptide conjugates that act peripherally at the
nociceptin receptor, which is related to but distinct from the
kappa opioid receptor (D. R. Kapusta, Life Sci., 60:15-21, 1997)
(U.S. Pat. No. 5,840,696). U.S. Pat Appl. 20060052284.
SUMMARY OF THE INVENTION
[0013] The invention provides a method of prophylaxis or treatment
or inhibition of a kappa opioid receptor-associated disease or
condition, including pain, inflammation and pruritis, in a mammal,
wherein the method includes administering a composition comprising
an effective amount of a synthetic peptide amide of the formula I
to the mammal. The structure of formula I is as follows.
##STR00001##
[0014] Also useful in the methods of the present invention are
stereoisomers, prodrugs, pharmaceutically acceptable salts,
hydrates, solvates, acid salt hydrates, N-oxides and isomorphic
crystalline forms of the synthetic peptide amides of formula I.
[0015] The invention also provides uses of the synthetic peptide
amides for the preparation of medicaments and pharmaceutical
compositions useful for the prevention or treatment of a kappa
opioid receptor-associated disease or condition in a mammal.
[0016] The kappa opioid receptor-associated disease or condition
preventable or treatable by the methods of the present invention
include inflammatory diseases and conditions.
[0017] The invention also provides a method of treating or
preventing hyponatremia or hypokalemia, and thereby treating or
preventing a disease or condition associated with hyponatremia or
hypokalemia, such as congestive heart failure, liver cirrhosis,
nephrotic syndrome, hypertension, or edema, and preferably where
increased vasopressin secretion is associated with said disease or
condition, wherein the method includes administering to a mammal an
aquaretically effective amount of a synthetic peptide amide of the
invention in a pharmaceutically acceptable diluent, excipient or
carrier.
[0018] In formula I,
##STR00002##
each Xaa.sub.1 is independently chosen from the following D-amino
acids: (A)(A')D-phenylalanine,
(A)(A').alpha.-methyl-D-phenylalanine, D-tyrosine,
D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,
D-neopentylglycine, D-phenylglycine, D-homophenylalanine,
.beta.-(E)D-alanine and D-tert-Leu, wherein each (A) and each (A')
are phenyl ring substituents independently chosen from --H, --F,
--Cl, --NO.sub.2, --CH.sub.3, --CF.sub.3, --CN, --CONH.sub.2, and
wherein each (E) is independently chosen from tert-butyl,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, furyl, pyridyl,
thienyl, thiazolyl and benzothienyl.
[0019] Each Xaa.sub.2 is independently chosen from (A)(A')D
phenylalanine, 3,4-dichloro-D-phenylalanine,
(A)(A')(.alpha.-Me)D-phenylalanine, D-1-naphthylalanine,
D-2-naphthylalanine, D-tyrosine, (E)D-alanine, and D-tryptophan,
wherein (A), (A') and (E) are defined above.
[0020] Each Xaa.sub.3 is independently chosen from D-norleucine,
D-phenylalanine, (E)-D-alanine, D-leucine, (.alpha.-Me)D-leucine,
D-isoleucine, D-allo-isoleucine, D-homoleucine, D-valine, and
D-methionine.
[0021] Each Xaa.sub.4 is independently chosen from
(B).sub.2D-arginine, (B).sub.2D-norarginine,
(B).sub.2D-homoarginine, .zeta.-(B)D-homolysine,
D-2,3-diaminopropionic acid, .epsilon.-(B)D-lysine,
.epsilon.-(B).sub.2-D-lysine, D-aminomethylphenylalanine,
amidino-D-aminomethyl-phenylalanine,
.gamma.-(B).sub.2D-.alpha.,.gamma.-diaminobutyric acid,
.delta.-(B).sub.2.alpha.-(B')D-ornithine,
D-2-amino-3(4-piperidyl)-propionic acid,
D-2-amino-3(2-aminopyrrolidyl)propionic acid,
D-.alpha.-amino-.beta.-amidino-propionic acid,
.alpha.-amino-4-piperidineacetic acid,
cis-.alpha.,4-diaminocyclo-hexane acetic acid,
trans-.alpha.,4-diaminocyclohexaneacetic acid,
cis-.alpha.-amino-4-methyl-aminocyclo-hexane acetic acid,
trans-.alpha.-amino-4-methylaminocyclohexane acetic acid,
.alpha.-amino-1-amidino-4-piperidineacetic acid,
cis-.alpha.-amino-4-guanidino-cyclohexane acetic acid, and
trans-.alpha.-amino-4-guanidinocyclohexane acetic acid, wherein
each (B) is independently chosen from --H and C.sub.1-C.sub.4
alkyl, and (B') is --H or (.alpha.-Me); and p is zero or 1, such
that when p is 1 G is bonded to Xaa.sub.4 and when p is zero, then
G is directly bonded to Xaa.sub.3.
[0022] The moiety G is selected from one of the following
moieties:
[0023] Either (i) G is
##STR00003##
wherein p, q, r, s and t are each independently zero or 1, provided
that at least one of s and t is 1, such that when t is 1 L is
bonded to Xaa.sub.4 and when t is zero, then L is directly bonded
to Xaa.sub.3. The moiety L is a linker chosen from .epsilon.-D-Lys,
.epsilon.-Lys, .delta.-D-Orn, .delta.-Orn, .gamma.-aminobutyric
acid, 8-aminooctanoic acid, 11-amino-undecanoic acid,
8-amino-3,6-dioxaoctanoic acid, 4-amino-4-carboxylic piperidine and
bis(D-Lys-Gly)Lactam.
[0024] Or (ii) G is
##STR00004##
and p is 1; The linking moiety, W can be any of the following three
alternatives: (a) null, provided that when W is null, Y is nitrogen
and is bonded to the C-terminus of Xaa.sub.4 to form an amide; (b)
--NH--(CH.sub.2).sub.b-- with b equal to 0, 1, 2, 3, 4, 5, or 6; or
(c) --NH--(CH.sub.2).sub.c--O-- with c equal to 2, or 3, provided
that Y is carbon. In each of the foregoing alternatives, (b) and
(c) the nitrogen atom of W is bonded to the C-terminus of Xaa.sub.4
to form an amide; and the moiety
##STR00005##
is an optionally substituted 4-, 5-, 6-, 7-, or 8-membered
heterocyclic ring moiety wherein Y is a carbon or a nitrogen atom
and Z is carbon, nitrogen, oxygen, sulfur, sulfoxide, or sulfonyl;
provided that when such ring moiety is a six, seven or
eight-membered ring, Y and Z are separated by at least two ring
atoms, and provided further that when such ring moiety is aromatic,
then Y is a carbon atom.
[0025] The moiety V in the substituent of the Y--Z-containing ring
in formula I is a C.sub.1-C.sub.6 alkyl linker when present. The
operator, e is zero or 1, such that when e is zero, then V is null,
and R.sub.1 and R.sub.2 are directly bonded to the same or
different ring atoms. The moiety V represents C.sub.1-C.sub.6
alkyl, and the operator, e is either zero or 1, wherein when e is
zero, then V is null and, R.sub.1 and R.sub.2 are directly bonded
to the same or different ring atoms. The groups R.sub.1 and R.sub.2
can be any one of (a), (b), (c) or (d) as follows:
[0026] (a) R.sub.1 is H, OH, halo, CF.sub.3, --NH.sub.2, --COOH,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, amidino,
C.sub.1-C.sub.6 alkyl-substituted amidino, aryl, optionally
substituted heterocyclyl, Pro-amide, Pro, Gly, Ala, Val, Leu, Ile,
Lys, Arg, Orn, Ser, Thr, CN, CONH.sub.2, COR', SO.sub.2R',
CONR'R'', NHCOR', OR, or SO.sub.2NR'R''; wherein said optionally
substituted heterocyclyl is optionally singly or doubly substituted
with substituents independently selected from the group consisting
of C.sub.1-C.sub.6 alkyl, --C.sub.1-C.sub.6 alkoxy, oxo, --OH,
--Cl, --F, --NH.sub.2, --NO.sub.2, --CN, --COOH, and amidino;
wherein R' and R'' are each independently H, C.sub.1-C.sub.8 alkyl,
aryl, heterocyclyl or R' and R'' are combined to form a 4-, 5-, 6-,
7-, or 8-membered ring, which ring is optionally substituted singly
or doubly with substituents independently selected from the group
consisting of C.sub.1-C.sub.6 alkyl, --C.sub.1-C.sub.6 alkoxy,
--OH, --Cl, --F, --NH.sub.2, --NO.sub.2, --CN, and --COOH, amidino;
and R.sub.2 is H, amidino, singly or doubly C.sub.1-C.sub.6
alkyl-substituted amidino, --CN, --CONH.sub.2, --CONR'R'',
--NHCOR', --SO.sub.2NR'R'', or --COOH; or
[0027] (b) R.sub.1 and R.sub.2 taken together can form an
optionally substituted 4-, 5-, 6-, 7-, 8- or 9-membered
heterocyclic monocyclic or bicyclic ring moiety which is bonded to
a single ring atom of the Y and Z-containing ring moiety; or
[0028] (c) R.sub.1 and R.sub.2 taken together with a single ring
atom of the Y and Z-containing ring moiety can form an optionally
substituted 4-, 5-, 6-, 7-, or 8-membered heterocyclic ring moiety
to form a spiro structure; or
[0029] (d) R.sub.1 and R.sub.2 taken together with two or more
adjacent ring atoms of the Y and Z-containing ring moiety can form
an optionally substituted 4-, 5-, 6-, 7-, 8- or 9-membered
hetero-cyclic monocyclic or bicyclic ring moiety fused to the Y and
Z-containing ring moiety.
[0030] Each of the aforementioned optionally substituted 4-, 5-,
6-, 7-, 8- or 9-membered heterocyclic ring moieties that include
R.sub.1 and R.sub.2 is optionally singly or doubly substituted with
substituents independently chosen from C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, optionally substituted phenyl (as defined
above), oxo, --OH, --Cl, --F, --NH.sub.2, --NO.sub.2, --CN, --COOH,
and amidino.
[0031] In the first of four alternative embodiments, the moiety
R.sub.1 in formula I can be any of the following groups: --H, --OH,
halo, --CF.sub.3, --NH.sub.2, --COOH, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, amidino, C.sub.1-C.sub.6 alkyl-substituted
amidino, aryl, optionally substituted heterocyclyl, Pro-amide, Pro,
Gly, Ala, Val, Leu, Ile, Lys, Arg, Orn, Ser, Thr, CN, CONH.sub.2,
COW, SO.sub.2R', CONR'R'', NHCOR', OR', or SO.sub.2NR'R''; wherein
the optionally substituted heterocyclyl is optionally singly or
doubly substituted with substituents independently chosen from
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, oxo, --OH, --Cl,
--F, --NH.sub.2, --NO.sub.2, --CN, --COOH, and amidino. The
moieties R and R'' are each independently H, C.sub.1-C.sub.8 alkyl,
aryl, or heterocyclyl. Alternatively, R and R'' can be combined to
form a 4-, 5-, 6-, 7-, or 8-membered ring, which ring is optionally
substituted singly or doubly with substituents independently chosen
from C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, --OH, --Cl,
--F, --NH.sub.2, --NO.sub.2, --CN, --COOH and amidino. The moiety
R.sub.2 can be any of --H, amidino, singly or doubly
C.sub.1-C.sub.6 alkyl-substituted amidino, --CN, --CONH.sub.2,
--CONR'R'', --NHCOR', --SO.sub.2NR'R'', or --COOH.
[0032] In a second alternative embodiment, the moieties R.sub.1 and
R.sub.2 taken together can form an optionally substituted 4-, 5-,
6-, 7-, 8- or 9-membered heterocyclic monocyclic or bicyclic ring
moiety which is bonded to a single ring atom of the Y and
Z-containing ring moiety.
[0033] In a third alternative embodiment, the moieties R.sub.1 and
R.sub.2 taken together with a single ring atom of the Y and
Z-containing ring moiety can form an optionally substituted 4-, 5-,
6-, 7- or 8-membered heterocyclic ring moiety to form a spiro
structure.
[0034] In a fourth alternative embodiment, the moieties R.sub.1 and
R.sub.2 taken together with two or more adjacent ring atoms of the
Y and Z-containing ring moiety can form an optionally substituted
4-, 5-, 6-, 7-, 8- or 9-membered heterocyclic monocyclic or
bicyclic ring moiety fused to the Y and Z-containing ring
moiety.
[0035] In formula I M the above second, third and fourth
alternative embodiments, each of the optionally substituted 4-, 5-,
6-, 7-, 8- and 9-membered heterocyclic ring moieties comprising
R.sub.1 and R.sub.2 can be singly or doubly substituted with
substituents independently chosen from C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, optionally substituted phenyl, oxo, --OH,
--Cl, --F, --NH.sub.2, --NO.sub.2, --CN, --COOH and amidino.
[0036] Alternatively, (iii) G is
##STR00006##
wherein J is a 5-, 6-, or 7-membered heterocyclic ring moiety
comprising 1, 2, or 3 heteroatoms in the ring wherein R.sub.3 and
R.sub.4 are each independently selected from H, C.sub.1-C.sub.3
alkyl, halo, --OH, --CF.sub.3, --NH.sub.2, --COOH and amidino; and
R.sub.5 and R.sub.6 are each independently selected from H,
C.sub.1-C.sub.3 alkyl, oxo, halo, --OH, --CF.sub.3, --NH.sub.2,
--COOH and amidino.
[0037] The moiety W is chosen from the following two options:
--NH--(CH.sub.2).sub.b-- with b equal to zero, 1, 2, 3, 4, 5, or 6;
and --NH--(CH.sub.2).sub.c--O-- with c equal to 2 or 3.
[0038] The above formula I definitions are subject to the following
three provisos:
[0039] (1) That when the Y and Z-containing ring moiety is a six or
seven membered ring having a single ring heteroatom and such
heteroatom is N, and e is zero, then R.sub.1 is not OH, and R.sub.1
and R.sub.2 are not both H;
[0040] (2) That when the Y and Z-containing ring moiety is a six
membered ring comprising two ring heteroatoms, both Y and Z are
nitrogen atoms, W is null, and the moiety
--V.sub.e(R.sub.1)(R.sub.2) is attached to Z, then
--V.sub.e(R.sub.1)(R.sub.2) is chosen from amidino, C.sub.1-C.sub.6
alkyl-substituted amidino, dihydroimidazole, --CH.sub.2COOH, and
--CH.sub.2C(O)NH.sub.2; and
[0041] (3) That if the Y and Z-containing ring moiety is a six
membered ring comprising a sulfur or an oxygen ring heteroatom, or
if the Y and Z-containing ring moiety is a non-aromatic six
membered ring that includes two ring heteroatoms, wherein both Y
and Z are nitrogen atoms and W is null, or if the Y and
Z-containing ring moiety is a six-membered aromatic ring that
includes a single ring heteroatom, which heteroatom is a nitrogen
atom, then, when e is zero, R.sub.1 and R.sub.2 are not both
hydrogen.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIG. 1: Concentration detected in rat plasma and brain after
administration of 3 mg/kg compound (2) over a 5 minute infusion
through a jugular vein catheter. Concentration of compound (2) in
ng/ml: open circles: plasma, solid circles: brain.
[0043] FIG. 2: Plasma concentrations of compound (6) after
subcutaneous administration of a single bolus of 1 mg/kg of the
compound to ICR mice. Plasma was sampled at 5, 10, 15, 20, 30 60,
90 120, and 180 minutes post-injection.
[0044] FIG. 3: Plasma concentrations of compound (3) after
intravenous administration of a single bolus of 0.56 mg/kg of the
compound to cynomolgus monkeys. Plasma was sampled at 2, 5, 10, 15,
30, 60, 120, and 240 minutes post injection.
[0045] FIG. 4: Dose-response curves for compound (3) in ICR mice in
the acetic acid-induced writhing assay (solid circles) and in the
locomotion assay (solid squares).
[0046] FIG. 5: Dose response of compound (2)-mediated suppression
of acetic acid-induced writhing in mice when delivered by the
intravenous route.
[0047] FIG. 6: Dose response curves for compound (54) in IRC mice
in the acetic acid-induced writhing assay (open circles) and the
mean (closed circles) and error bars; and in the locomotion assay
(open squares) and the mean (closed squares) and error bars.
[0048] FIG. 7: Plasma concentrations after intravenous
administration of a single bolus of compound (52) to cynomolgus
monkeys. Plasma was sampled at 2, 5, 10, 15, 30, 60, 120, and 240
minutes post injection.
[0049] FIG. 8: Effects of compound (2) on mechanical
hypersensitivity induced by L5/L6 spinal nerve ligation in rats.
Open circles--vehicle alone; Solid circles--compound (2) at 0.1
mg/kg; open squares--compound (2) at 0.3 mg/kg; solid
squares--compound (2) at 1.0 mg/kg. ** denotes p<0.01; ***
denotes p<0.001 vs. Vehicle (2-way ANOVA, Bonferroni).
[0050] FIG. 9: Effect of compound (2) on pancreatitis-induced
abdominal hypersensitivity in rats. Dibutyltin dichloride or
vehicle alone was administered intravenously and hypersensitivity
assessed by abdominal probing with a von Frey filament at 30 minute
intervals. Hypersensitivity is expressed as number of withdrawals
from ten probings. Open circles--vehicle alone; solid
circles--compound (2) at 0.1 mg/kg; open squares--compound (2) at
0.3 mg/kg; solid squares--compound (2) at 1.0 mg/kg. ** denotes
p<0.01; *** denotes p<0.001 vs. Vehicle (2-way ANOVA,
Bonferroni).
[0051] FIG. 10: Blocking of the effect of compound (2) on
pancreatitis-induced abdominal hypersensitivity by nor-BNI and
naloxone methiodide (NM) in rats. Open column--vehicle alone, solid
column--compound (2) at 1 mg/kg with NM or norBNI as indicated. ***
denotes p<0.001 vs. Vehicle+Vehicle (2-way ANOVA,
Bonferroni).
[0052] FIG. 11: Inhibition of proliferation of human synoviocytes
after incubation with Compound (2) in vitro.
DETAILED DESCRIPTION
[0053] As used throughout this specification, the term "synthetic
peptide amide" means a compound of the invention conforming to
formula I, or a stereoisomer, mixture of stereoisomers, prodrug,
pharmaceutically acceptable salt, hydrate, solvate, acid salt
hydrate, N-oxide or isomorphic crystalline form thereof. The
designations Xaa.sub.1, Xaa.sub.2, Xaa.sub.3, and Xaa.sub.4
represent D-amino acids in the synthetic peptide amides of the
invention. Stereoisomers of the synthetic peptide amides of the
invention conforming to formula I are limited to those compounds
having amino acids in the D-configuration where so specified in
Formula I. Stereoisomers of the synthetic peptide amides of the
invention include compounds having either a D- or L-configuration
at chiral centers other than the alpha carbons of the four amino
acids at Xaa.sub.1, Xaa.sub.2, Xaa.sub.3, and Xaa.sub.4. The term
`mixtures of stereoisomers` refer to mixtures of such stereoisomers
of the invention. As used herein `racemates` refers to mixtures of
stereoisomers having equal proportions of compounds with D- and
L-configuration at one or more of the chiral centers other than the
alpha carbons of Xaa.sub.1, Xaa.sub.2, Xaa.sub.3, and Xaa.sub.4
without varying the chirality of the alpha carbons of Xaa.sub.1,
Xaa.sub.2, Xaa.sub.3, and Xaa.sub.4.
[0054] The nomenclature used to define peptides herein is specified
by Schroder & Lubke, The Peptides, Academic Press, 1965,
wherein, in accordance with conventional representation, the
N-terminus appears to the left and the C-terminus to the right.
Where an amino acid residue has isomeric forms, both the L-isomer
form and the D-isomer form of the amino acid are intended to be
covered unless otherwise indicated Amino acids are commonly
identified herein by the standard three-letter code. The D-isomer
of an amino acid is specified by the prefix "D-" as in "D-Phe"
which represents D-phenylalanine, the D-isomer of phenylalanine.
Similarly, the L-isomer is specified by the prefix "L-" as in
"L-Phe." Peptides are represented herein according to the usual
convention as amino acid sequences from left to right: N-terminus
to C-terminus, unless otherwise specified.
[0055] As used herein, D-Arg represents D-arginine, D-Har
represents D-homoarginine, which has a side chain one methylene
group longer than D-Arg, and D-Nar represents D-norarginine, which
has a side chain one methylene group shorter than D-Arg. Similarly,
D-Leu means D-leucine, D-Nle means D-norleucine, and D-Hle
represents D-homoleucine. D-Ala means D-alanine, D-Tyr means
D-tyrosine, D-Trp means D-tryptophan, and D-Tic means
D-1,2,3,4-tetrahydroisoquinoline-3carboxylic acid. D-Val means
D-valine and D-Met means D-methionine. D-Pro means D-proline,
Pro-amide means the D- or L-form of proline amide. D-Pro amide
represents D-proline with an amide formed at its carboxy moiety
wherein the amide nitrogen may be alkyl substituted, as in
--NR.sub.aR.sub.b, wherein R.sub.a and R.sub.b are each
independently a C.sub.1-C.sub.6 alkyl group, or one of R.sub.a and
R.sub.b is --H. Gly means glycine, D-Ile means D-isoleucine, D-Ser
means D-serine, and D-Thr means D-threonine. (E)D-Ala means the
D-isomer of alanine which is substituted by the substituent (E) on
the .beta.-carbon. Examples of such substituent (E) groups include
tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
furyl, pyridyl, thienyl, thiazolyl and benzothienyl. Thus,
cyclopentyl-D-Ala means the D-isomer of alanine which is
substituted by cyclopentyl on the .beta.-carbon. Similarly,
D-Ala(2-thienyl) and (2-thienyl)D-Ala are interchangeable and both
mean the D-isomer of alanine substituted at the .beta.-carbon with
thienyl that is attached at the 2-ring position.
[0056] As used herein, D-Nal means the D-isomer of alanine
substituted by naphthyl on the .beta.-carbon. D-2Nal means naphthyl
substituted D-alanine wherein the attachment to naphthalene is at
the 2-position on the ring structure and D-1Nal means
naphthyl-substituted D-alanine wherein the attachment to
naphthalene is at the 1-position on the ring structure. By
(A)(A')D-Phe is meant D-phenylalanine substituted on the phenyl
ring with one or two substituents independently chosen from halo,
nitro, methyl, halomethyl (such as, for example, trifluoromethyl),
perhalomethyl, cyano and carboxamide. By D-(4-F)Phe is meant
D-phenylalanine which is fluoro-substituted in the 4-position of
the phenyl ring. By D-(2-F)Phe is meant D-phenylalanine which is
fluoro-substituted in the 2-position of the phenyl ring. By
D-(4-Cl)Phe is meant D-phenylalanine which is chloro substituted in
the 4-phenyl ring position. By (.alpha.-Me)D-Phe is meant
D-phenylalanine which is methyl substituted at the alpha carbon. By
(.alpha.-Me)D-Leu is meant D-leucine which is methyl substituted at
the alpha carbon.
[0057] The designations (B).sub.2D-Arg, (B).sub.2D-Nar, and
(B).sub.2D-Har represent D-arginine, D-norarginine and
D-homoarginine, respectively, each having two substituent (B)
groups on the side chain. D-Lys means D-lysine and D-Hlys means
D-homolysine. .zeta.-(B)D-Hlys .epsilon.-(B)D-Lys, and
.epsilon.-(B).sub.2-D-Lys represent D-homolysine and D-lysine each
having the side chain amino group substituted with one or two
substituent (B) groups, as indicated. D-Orn means D-ornithine and
.delta.-(B).alpha.-(B')D-Orn means D-ornithine substituted with
(B') at the alpha carbon and substituted with (B) at the side chain
.delta.-amino group.
[0058] D-Dap means D-2,3-diaminopropionic acid. D-Dbu represents
the D-isomer of alpha, gamma-diamino butyric acid and
(B).sub.2D-Dbu represents alpha, gamma-diamino butyric acid which
is substituted with two substituent (B) groups at the gamma amino
group. Unless otherwise stated, each of the (B) groups of such
doubly substituted residues are independently chosen from H-- and
C.sub.1-C.sub.4-alkyl. As used herein, D-Amf means
D-(NH.sub.2CH.sub.2-)Phe, i.e., the D-isomer of phenylalanine
substituted with aminomethyl on its phenyl ring and D-4Amf
represents the particular D-Amf in which the aminomethyl is
attached at the 4-position of the ring. D-Gmf means D-Amf(amidino)
which represents D-Phe wherein the phenyl ring is substituted with
--CH.sub.2NHC(NH)NH.sub.2. Amd represents amidino, --C(NH)NH.sub.2,
and the designations (Amd)D-Amf and D-Amf(Amd) are also
interchangeably used for D-Gmf. The designations Ily and Ior are
respectively used to mean isopropyl Lys and isopropyl Orn, wherein
the side chain amino group is alkylated with an isopropyl
group.
[0059] Alkyl means an alkane radical which can be a straight,
branched, and cyclic alkyl group such as, but not limited to,
methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, t-butyl,
sec-butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, cyclohexylethyl.
C.sub.1 to C.sub.8 alkyl refers to alkyl groups having between one
and eight carbon atoms. Similarly, C.sub.1-C.sub.6 alkyl refers to
alkyl groups having between one and six carbon atoms. Likewise,
C.sub.1-C.sub.4 alkyl refers to alkyl groups having between one and
four carbon atoms. By lower alkyl is meant C.sub.1-C.sub.6 alkyl.
Me, Et, Pr, Ipr, Bu, and Pn are interchangeably used to represent
the common alkyl groups: methyl, ethyl, propyl, isopropyl, butyl,
and pentyl, respectively. Although the linkage for an alkyl group
is typically at one end of an alkyl chain, the linkage may be
elsewhere in the chain, e.g. 3-pentyl which may also be referred to
as ethylpropyl, or 1-ethylprop-1-yl. Alkyl-substituted, such as
C.sub.1 to C.sub.6 alkyl-substituted amidino, indicates that the
relevant moiety is substituted with one or more alkyl groups.
[0060] Where a specified moiety is null, the moiety is absent and
if such moiety is indicated to be attached to two other moieties,
such two other moieties are connected by one covalent bond. Where a
connecting moiety is shown herein as attached to a ring at any
position on the ring, and attached to two other moieties, such as
R.sub.1 and R.sub.2, in the case where the connecting moiety is
specified to be null, then the R.sub.1 and R.sub.2 moieties can
each be independently attached to any position on the ring.
[0061] The terms "heterocycle", "heterocyclic ring" and
"heterocyclyl" are used interchangeably herein and refer to a ring
or ring moiety having at least one non-carbon ring atom, also
called a heteroatom, which can be a nitrogen atom, a sulfur atom,
or an oxygen atom. Where a ring is specified as having a certain
number of members, the number defines the number of ring atoms
without reference to any substituents or hydrogen atoms bonded to
the ring atoms. Heterocycles, heterocyclic rings and heterocyclyl
moieties can include multiple heteroatoms independently selected
from nitrogen, sulfur, or oxygen atom in the ring. Rings can be
substituted at any available position. For example, but without
limitation, 6- and 7-membered rings are often substituted in the
4-ring position and 5-membered rings are commonly substituted in
the 3-position, wherein the ring is attached to the peptide amide
chain at the 1-ring position.
[0062] The term "saturated" means an absence of double or triple
bonds and the use of the term in connection with rings describes
rings having no double or triple bonds within the ring, but does
not preclude double or triple bonds from being present in
substituents attached to the ring. The term "non-aromatic" in the
context of a particular ring refers to an absence of aromaticity in
that ring, but does not preclude the presence of double bonds
within the ring, including double bonds which are part of an
aromatic ring fused to the ring in question. Nor is a ring atom of
a saturated heterocyclic ring moiety precluded from being
double-bonded to a non-ring atom, such as for instance a ring
sulfur atom being double-bonded to an oxygen atom substituent. As
used herein, heterocycles, heterocyclic rings and heterocyclyl
moieties also include saturated, partially unsaturated and
heteroaromatic rings and fused bicyclic ring structures unless
otherwise specified. A heterocycle, heterocyclic ring or
heterocyclyl moiety can be fused to a second ring, which can be a
saturated, partially unsaturated, or aromatic ring, which ring can
be a heterocycle or a carbocycle. Where indicated, two substituents
can be optionally taken together to form an additional ring. Rings
may be substituted at any available position. A heterocycle,
heterocyclic ring and heterocyclyl moiety can, where indicted, be
optionally substituted at one or more ring positions with one or
more independently selected substituents, such as for instance,
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.6
alkoxy, halo C.sub.1-C.sub.6 alkyl, optionally substituted phenyl,
aryl, heterocyclyl, oxo, --OH, --F, --NH.sub.2, --NO.sub.2, --CN,
--COOH and amidino. Suitable optional substituents of the phenyl
substituent include for instance, but without limitation, one or
more groups selected from C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
alkoxy, halo C.sub.1-C.sub.3 alkyl, oxo, --OH, --F, --NH.sub.2,
--NO.sub.2, --CN, --COOH and amidino.
[0063] D-Phe and substituted D-Phe are examples of a suitable amino
acid for residue Xaa.sub.1 in Formula I. The phenyl ring can be
substituted at any of the 2-, 3- and/or 4-positions. Particular
examples of permitted substitutions include, for instance, chlorine
or fluorine at the 2- or 4-positions. Also the alpha-carbon atom
may be methylated. Other equivalent residues which represent
conservative changes to D-Phe can also be used. These include
D-Ala(cyclopentyl), D-Ala(thienyl), D-Tyr and D-Tic. The residue at
the second position, Xaa.sub.2 can also be D-Phe or substituted
D-Phe with such substitutions including a substituent on the
4-position carbon of the phenyl ring, or on both the 3- and
4-positions. Alternatively, Xaa.sub.2 can be D-Trp, D-Tyr or
D-alanine substituted by naphthyl. The third position residue,
Xaa.sub.3 can be any non-polar amino acid residue, such as for
instance, D-Nle, D-Leu, (.alpha.-Me)D-Leu, D-Hle, D-Met or D-Val.
However, D-Ala(cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl)
or D-Phe can also be used as Xaa.sub.3. The fourth position residue
Xaa.sub.4 can be any positively charged amino acid residue, such as
for instance, D-Arg and D-Har, which can be optionally substituted
with lower alkyl groups, such as one or two ethyl groups.
Alternatively, D-Nar and any other equivalent residues can be used,
such as, for instance, D-Lys or D-Orn (either of which can be
.omega.-amino group alkylated, for example by methyl or isopropyl
groups, or methylated at the .alpha.-carbon group). Moreover,
D-Dbu, D-4-Amf (which can be optionally substituted with amidino),
and D-Hlys are also suitable amino acids at this position.
[0064] Compounds of the invention contain one or more chiral
centers, each of which has two possible three-dimensional spatial
arrangements (configurations) of the four substituents around the
central carbon atom. These are known as "stereoisomers", and more
specifically as "enantiomers" (all chiral centers inverted) or
"diastereoisomers" (two or more chiral centers, at least one chiral
center remaining the same). In a specific embodiment of the
invention, the amino acids which make up the tetrapeptide backbone,
Xaa.sub.1Xaa.sub.2Xaa.sub.3Xaa.sub.4 are specified to be D-amino
acids i.e., the opposite configuration to those generally found in
mammals. Reference to stereoisomers of the synthetic peptide amides
of the invention concerns chiral centers other than the alpha
carbons of the D-amino acids which make up Xaa.sub.1-Xaa.sub.4.
Thus, stereoisomers of synthetic peptide amides that are
embodiments of the invention wherein each of Xaa.sub.1-Xaa.sub.4
are specified to be D-amino acids, do not include L-amino acids or
racemic mixtures of the amino acids at these positions. Similarly,
reference to racemates herein concerns a center other than the
alpha carbons of the D-amino acids which make up
Xaa.sub.1-Xaa.sub.4. Chiral centers in the synthetic peptide amides
of the invention for which a stereoisomer may take either the R or
S configuration include chiral centers in the moiety attached to
the carboxy-terminus of Xaa.sub.4, and also chiral centers in any
amino acid side chain substituents of Xaa.sub.1-Xaa.sub.4.
[0065] The synthetic peptide amides of the invention described
herein (also interchangeably referred to as synthetic peptide amide
compounds, compounds of the invention, compound (number), or simply
"the compounds") can be used or prepared in alternate forms. For
example, many amino-containing compounds can be used or prepared as
an acid salt. Often such salts improve isolation and handling
properties of the compound. For example, depending on the reagents,
reaction conditions and the like, compounds such as the synthetic
peptide amides described herein can be used or prepared, for
example, as the hydrochloride or tosylate salts. Isomorphic
crystalline forms, all chiral and racemic forms, N-oxide, hydrates,
solvates, and acid salt hydrates, are also contemplated to be
within the scope of the present invention.
[0066] Certain acidic or basic synthetic peptide amides of the
present invention may exist as zwitterions. All forms of these
synthetic peptide amide compounds, including free acid, free base
and zwitterions, are contemplated to be within the scope of the
present invention. It is well known in the art that compounds
containing both amino and carboxyl groups often exist in
equilibrium with their zwitterionic forms. Thus, for any compound
described herein that contains, for example, both amino and
carboxyl groups, it will also be understood to include the
corresponding zwitterion.
[0067] As used herein, the chemical designation
"tetrapeptide-[.omega.(4-amino-piperidine-4-carboxylic acid)]" is
used to indicate the aminoacyl moiety of the synthetic peptide
amides of the invention derived from 4-aminopiperidine-4-carboxylic
acid, wherein the nitrogen atom of the piperidine ring is bound to
the C-terminal carbonyl-carbon of the tetrapeptide fragment, unless
otherwise indicated.
[0068] In one embodiment, the invention provides a synthetic
peptide amide wherein each Xaa.sub.1 is D-Phe, each Xaa.sub.2 is
D-Phe, each Xaa.sub.3 is D-Leu and each Xaa.sub.4 is D-Lys. In
another embodiment, each Xaa.sub.1 is D-Ala(2-thienyl), each
Xaa.sub.2 is D-Phe, each Xaa.sub.3 is D-Nle, and each Xaa.sub.4 is
D-Arg.
[0069] In another embodiment, G is
##STR00007##
and the dipeptide Xaa.sub.3-Xaa.sub.4 is chosen from D-Leu-D-Orn
and D-Nle-D-Arg. In another embodiment Xaa.sub.1-Xaa.sub.2 is
D-Phe-D-Phe. In another embodiment Xaa.sub.1 is D-(4-F)Phe, and
Xaa.sub.2 is D-(4-Cl)Phe.
[0070] In another embodiment each Xaa.sub.1 is D-Phe or
D-Ala(2-thienyl) and each Xaa.sub.2 is D-(4-Cl)Phe. In another
embodiment, each Xaa.sub.3 is D-Leu or D-Nle.
[0071] In another embodiment G is
##STR00008##
and Xaa.sub.1 is chosen from D-Phe, D-(4-F)Phe, D-(2-F)Phe,
cyclopentyl D-Ala, 2-thienyl D-Ala, Xaa.sub.2 is chosen from D-Phe,
D-(4-F)Phe, D-(4-Cl)Phe, D-1Nal, D-2Nal, and D-Trp, and
Xaa.sub.3-Xaa.sub.4 is chosen from D-Nle-D-Arg, D-Leu-D-Lys and
D-Leu-D-Orn.
[0072] In one embodiment Xaa.sub.1 is (A)(A')D-Phe, and in one
aspect, each Xaa.sub.1 is D-Phe. In another embodiment each
Xaa.sub.2 is D-Phe. In another embodiment, each Xaa.sub.3 is chosen
from D-Nle, and D-Leu. In another embodiment each Xaa.sub.4 is
chosen from .delta.(B).sub.2D-Orn, D-Lys and D-Arg. In one aspect,
each Xaa.sub.4 is .delta.(B).sub.2D-Orn and each (B) chosen from
--H, methyl and isopropyl. In another aspect each Xaa.sub.4 is
(B).sub.2D-Orn, wherein one (B) is H, and the other (B) selected
from the group consisting of methyl and isopropyl. In another
particular aspect each Xaa.sub.4 is D-Orn.
[0073] In another embodiment each Xaa.sub.4 is chosen from
.epsilon.(B).sub.2D-Lys, (B).sub.2D-Arg, and
.delta.-(B).sub.2D-Orn. In another particular aspect each Xaa.sub.4
is chosen from D-Arg, (Et).sub.2D-Arg, and .delta.-(B)D-Orn, and
(B) is H, Me, iPr, or Bu.
[0074] In another embodiment G is
##STR00009##
and W is null.
[0075] In another embodiment G is
##STR00010##
and W is --N--(CH.sub.2).sub.b with b equal to 0, 1, 2, 3, or 4. In
one aspect b is zero and Y is a carbon atom. In another aspect b is
1 or 2 and Y is a nitrogen atom. In another embodiment W is
--N--(CH.sub.2).sub.c--O--. In one particular aspect c is 1 or 2.
In another aspect the Y and Z-containing ring moiety is a four or
five membered ring and Y is a nitrogen atom. In another embodiment
the Y and Z-containing ring moiety is a four or five membered ring
and Y is a carbon atom.
[0076] In another embodiment the Y and Z-containing ring moiety is
a six or seven membered ring, Y is nitrogen and Z is a carbon atom.
In another alternative, the Y and Z-containing ring moiety is a six
membered ring. In one aspect the Y and Z-containing ring moiety is
a seven membered ring. In still another aspect the Y and
Z-containing ring moiety is a six or seven membered ring and both Y
and Z are nitrogen atoms.
[0077] In another embodiment e is zero and R.sub.1 and R.sub.2 are
bonded directly to the same ring atom. In one aspect e is zero,
R.sub.2 is --H and R.sub.1 is bonded directly to a carbon ring atom
adjacent to Z. In another aspect R.sub.1 is H, amidino,
C.sub.1-C.sub.3 alkyl substituted amidino, C.sub.1-C.sub.3 alkyl,
dihydroimidazole, D-Pro, D-Pro amide, or --CONH.sub.2 and wherein e
is zero and R.sub.2 is --H. In another aspect R.sub.1 is --H,
amidino, or methyl amidino. In one aspect the Y and Z-containing
ring moiety is a five membered ring, e is zero and R.sub.1 is
--COOH.
[0078] In another embodiment G is
##STR00011##
and Xaa.sub.1 is D-Phe, Xaa.sub.2 is D-Phe, Xaa.sub.3 is D-Leu,
Xaa.sub.4 is .epsilon.(B).sub.2D-Lys, or .delta.-(B).sub.2D-Orn,
wherein (B) is --H, methyl, or isopropyl; further wherein W is
null, the Y and Z-containing ring moiety is a six or seven membered
ring, Y is a nitrogen atom, e is zero, R.sub.1 is --NH.sub.2,
amidino, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 alkyl-substituted
amidino, dihydroimidazole, D-Pro, or D-Pro amide, and R.sub.2 is H
or --COOH.
[0079] In another embodiment, G is chosen from the following
groups:
##STR00012## ##STR00013## ##STR00014## ##STR00015##
[0080] In certain embodiments of the synthetic peptide amides of
the invention, there are two independent occurrences of the
residues Xaa.sub.1, Xaa.sub.2, Xaa.sub.3 and Xaa.sub.4. For
instance, in embodiments having the formula:
##STR00016##
wherein G is:
##STR00017##
and one or more of q, r, and s is 1 or both p and t are 1, then
there are two occurrences of Xaa.sub.1, Xaa.sub.2, Xaa.sub.3 and
Xaa.sub.4 respectively. In such embodiments, each instance of each
of the residues Xaa.sub.1, Xaa.sub.2, Xaa.sub.3 and Xaa.sub.4 can
be identical.
[0081] Alternatively, and in other embodiments, each instance of
one or more of the pairs of residues Xaa.sub.1, Xaa.sub.2,
Xaa.sub.3 or Xaa.sub.4 can be different. For example, one instance
of Xaa.sub.1 can be D-phenylalanine, while the second instance of
Xaa.sub.1 in the same molecule can be a different Xaa.sub.1
residue, such as D-(4-F)phenylalanine. Similarly, one instance of
Xaa.sub.2 can be D-phenylalanine, while the second instance of
Xaa.sub.2 in the same molecule can be D-Ala(2-thienyl). Likewise,
one instance of Xaa.sub.3 can be D-norleucine, while the second
instance of Xaa.sub.3 in the same molecule can be D-leucine. In the
same manner, one instance of Xaa.sub.4 can be D-ornithine, while
the second instance of Xaa.sub.4 in the same molecule can be
D-arginine, and so on.
[0082] In one embodiment, the invention provides a synthetic
peptide amide wherein Xaa.sub.1 is D-Ala(2-thienyl). In another
embodiment Xaa.sub.1 is D-(4-F)phenylalanine and Xaa.sub.2 is
D-(4-Cl)phenylalanine. In another embodiment each Xaa.sub.1 is
D-phenylalanine or D-Ala(2-thienyl) and each Xaa.sub.2 is
D-(4-Cl)phenylalanine. In another embodiment Xaa.sub.1-Xaa.sub.2 is
D-phenylalanine-D-phenylalanine.
[0083] In one embodiment each Xaa.sub.3 is chosen from D-norleucine
and D-leucine. In another embodiment each Xaa.sub.2 is
D-phenylalanine, each Xaa.sub.3 is D-norleucine, and each Xaa.sub.4
is D-arginine. In another embodiment each Xaa.sub.3 can be
D-leucine or D-norleucine.
[0084] In another embodiment Xaa.sub.4 is chosen from
.delta.(B).sub.2D-ornithine and D-arginine. Alternatively, each
Xaa.sub.4 is .delta.(B).sub.2D-ornithine and each (B) is chosen
from --H, methyl and isopropyl. In still another embodiment, each
Xaa.sub.4 is (B).sub.2D-ornithine, wherein one (B) is --H, and the
other (B) chosen from methyl and isopropyl. In one aspect, each
Xaa.sub.4 is (B).sub.2D-arginine, or .delta.-(B).sub.2D-ornithine.
In another embodiment each Xaa.sub.4 can be a residue chosen from
D-arginine, (Et).sub.2D-arginine, and .delta.-(B)D-ornithine, and
wherein (B) is --H, methyl, isopropyl, or butyl. In one embodiment
the dipeptide Xaa.sub.3-Xaa.sub.4 is chosen from
D-leucine-D-ornithine and D-norleucine-D-arginine.
[0085] In one particular embodiment the synthetic peptide amide of
the invention has the formula
##STR00018##
wherein G is:
##STR00019##
and b is zero and Y is a carbon atom. In another embodiment, b is 1
or 2 and Y is a nitrogen atom. In a particular aspect of the
invention, b is 2.
[0086] In another embodiment G is
##STR00020##
and the Y- and Z-containing moiety is
[.omega.(4-aminopiperidine-4-carboxylic acid)]-OH.
[0087] In one particular embodiment Xaa.sub.1 is chosen from D-Phe,
D-(4-F)Phe, D-(2-F)Phe, cyclopentyl D-Ala, 2-thienyl D-Ala,
Xaa.sub.2 is chosen from D-(4-F)Phe, D-(4-Cl)Phe, D-1Nal, D-2Nal,
and D-Trp, and Xaa.sub.3-Xaa.sub.4 is chosen from D-Nle-D-Arg and
D-Leu-D-Orn.
[0088] In another embodiment W is an N-alkoxyl linker of the
formula: --N--(CH.sub.2).sub.2--O--. In an alternative embodiment W
is null and Xaa.sub.1Xaa.sub.2Xaa.sub.3Xaa.sub.4 is directly bonded
to Y. In a second alternative embodiment, W is
--NH--(CH.sub.2).sub.2--.
[0089] In another particular embodiment, the Y and Z-containing
ring moiety is a four or five membered ring and Y is a nitrogen
atom. Alternatively, the Y- and Z-containing ring moiety can be a
four or five membered ring wherein Y is a carbon atom. In a
different embodiment, the Y and Z-containing ring moiety is a 6- or
7-membered ring, Y is a nitrogen atom and Z is a carbon atom. In
one aspect of this embodiment, the Y and Z-containing ring moiety
is a 6-membered ring. Alternatively, the Y and Z-containing ring
moiety can be a seven membered ring. In one aspect of this
embodiment, the Y and Z-containing ring moiety is a 6- or
7-membered ring and both Y and Z are nitrogen atoms.
[0090] In another particular embodiment the Y- and Z-containing
ring moiety is a six or seven membered ring, or an eight-membered
ring, Y is a carbon atom, and Z is a nitrogen atom. In one aspect,
Y is a nitrogen atom and Z is a carbon atom. In an alternative
embodiment Y and Z are each nitrogen atoms.
[0091] In another particular embodiment the Y- and Z-containing
ring moiety is an optionally substituted 4-, 5-, 6-, 7-, or
8-membered heterocyclic ring moiety wherein Y is a carbon or a
nitrogen atom and Z is carbon, nitrogen, oxygen, sulfur, sulfoxide,
or sulfonyl; and the 4-, 5-, 6-, 7-, or 8-membered heterocyclic
ring moiety is optionally singly or doubly substituted with
substituents independently chosen from C.sub.1-C.sub.6 alkyl,
--C.sub.1-C.sub.6 alkoxy, oxo, --OH, --Cl, --F, --NH.sub.2,
--NO.sub.2, --CN, --COOH, and amidino. In one aspect when the Y-
and Z-containing ring moiety is a six, seven or eight-membered
ring, then Y and Z are separated by at least two ring atoms. In
another aspect, when the Y- and Z-containing ring moiety is
non-aromatic and Z is a carbon or a nitrogen atom, then such ring
moiety includes at least one sulfur or oxygen ring heteroatom. In a
particular aspect, when the Y- and Z-containing ring moiety is
aromatic, then Y is a carbon atom.
[0092] In one embodiment of the synthetic peptide amide of the
invention, R.sub.1 is --H, --OH, --NH.sub.2, --COOH,
C.sub.1-C.sub.3 alkyl, amidino, C.sub.1-C.sub.3 alkyl-substituted
amidino, dihydroimidazole, D-Pro, D-Pro amide, or --CONH.sub.2. In
another particular embodiment R.sub.2 is --H, --COOH, or
C.sub.1-C.sub.3 alkyl. In one aspect, only one of R.sub.1 and
R.sub.2 is a hydrogen atom. In a particular embodiment R.sub.1 is
--H, D-Pro, D-Pro amide, or --NH.sub.2 and R.sub.2 is H or --COOH.
In one aspect of this embodiment, R.sub.1 is --NH.sub.2 and .sub.2
is --COOH.
[0093] In one embodiment, the operator, e is zero and R.sub.1 and
R.sub.2 are bonded directly to the same ring atom. In a particular
embodiment, e is zero, R.sub.2 is --H and R.sub.1 is bonded
directly to a carbon ring atom adjacent to Z. In another particular
embodiment, R.sub.1 is --H, amidino, C.sub.1-C.sub.3 alkyl
substituted amidino, C.sub.1-C.sub.3 alkyl, dihydroimidazole,
D-Pro, D-Pro amide, or --CONH.sub.2 and e is zero and R.sub.2 is
--H.
[0094] In one embodiment of the synthetic peptide amide of the
invention, Xaa.sub.1 is D-Phe, Xaa.sub.2 is D-Phe, Xaa.sub.3 is
D-Leu, Xaa.sub.4 is .delta.-(B).sub.2D-Orn, wherein (B) is --H,
methyl, or isopropyl; such that wherein W is null, the Y and
Z-containing ring moiety is a six or seven membered ring, Y is an N
atom, e is zero, R.sub.1 is NH.sub.2, amidino, C.sub.1-C.sub.3
alkyl, C.sub.1-C.sub.3 alkyl-substituted amidino, dihydroimidazole,
D-Pro, or D-Pro amide, and R.sub.2 is H or COOH.
[0095] In one embodiment of the synthetic peptide amide of the
invention: Xaa.sub.1 is chosen from (A) D-Phe, (.alpha.-Me)D-Phe,
D-Tyr, D-Tic, (tert-butyl)D-Gly, and .beta.-(E)D-Ala, wherein (A)
is chosen from --H, --F, --Cl, --NO.sub.2, and --CH.sub.3, and (E)
is chosen from tert-butyl, cyclopentyl and thienyl; Xaa.sub.2 is
chosen from (A)(A')D-Phe, D-1Nal, D-2Nal, D-Tyr, and D-Trp, wherein
(A') is H or Cl; Xaa.sub.3 is chosen from D-Nle, D-Phe,
(cyclopentyl) D-Ala, D-Leu, (.alpha.-Me)D-Leu, D-Hle, D-Val, and
D-Met; and Xaa.sub.4 is chosen from D-Arg, (ethyl).sub.2, D-Arg,
D-Nar, D-Har, (ethyl).sub.2D-Har, .epsilon.-(isopropyl)D-Lys,
D-Lys, D-Amf, amidino-D-Amf, .beta.-amidino-D-Dap, D-Dbu, D-Orn,
.alpha.-(methyl)D-Orn and .delta.-(isopropyl)D-Orn.
[0096] In another embodiment of the synthetic peptide amide of the
invention: Xaa.sub.1Xaa.sub.2 is D-Phe-D-Phe, Xaa.sub.3 is D-Leu or
D-Nle and Xaa.sub.4 is chosen from (B).sub.2D-Arg, D-Lys,
(B).sub.2D-Nar, (B).sub.2D-Har, .zeta.-(B)D-Dap, amidino-D-Dap,
.epsilon.-(B)D-Lys, .epsilon.-(B).sub.2-D-Lys, D-Amf,
amidino-D-Amf, .gamma.-(B).sub.2D-Dbu and
.delta.-(B).sub.2.alpha.-(B')D-Orn.
[0097] In another embodiment of the synthetic peptide amide of the
invention: Xaa.sub.4 is chosen from D-Lys, (B).sub.2D-Har,
.epsilon.(B)-D-Lys, .delta.(B).sub.2-.alpha.(B')D-Orn and
.epsilon.(B).sub.2-D-Lys.
[0098] In another embodiment of the synthetic peptide amide of the
invention: G is
##STR00021##
[0099] In another embodiment of the synthetic peptide amide of the
invention: the integers p, q, r, s and t are each 1.
[0100] In another embodiment of the synthetic peptide amide of the
invention: Xaa.sub.1 is chosen from (A) D-Phe, (.alpha.-Me)D-Phe,
D-Tyr, D-Tic, (tert-butyl)D-Gly, and .beta.-(E)D-Ala, wherein A is
selected from the group consisting of --H, --F, --Cl, --NO.sub.2,
and --CH.sub.3, and (E) is selected from the group consisting of
tert-butyl, cyclopentyl and thienyl; Xaa.sub.2 is selected from the
group consisting of (A)(A')D-Phe, D-1Nal, D-2Nal, D-Tyr, and D-Trp,
wherein (A') is H or Cl; Xaa.sub.3 is selected from the group
consisting of D-Nle, D-Phe, (cyclopentyl)D-Ala, D-Leu,
(.alpha.-Me)D-Leu, D-Hle, D-Val, and D-Met; and Xaa.sub.4 is
selected from the group consisting of D-Arg, (ethyl).sub.2D-Arg,
D-Nar, D-Har, (ethyl).sub.2D-Har, .epsilon.-(isopropyl)D-Lys,
D-Lys, D-Amf, amidino-D-Amf, 3-amidino-D-Dap, D-Dbu, D-Orn,
.alpha.-(methyl)D-Orn and .delta.-(isopropyl)D-Orn.
[0101] In another embodiment of the synthetic peptide amide of the
invention: Xaa.sub.1 is D-Phe; Xaa.sub.2 is D-Phe; Xaa.sub.3 is
D-Leu and Xaa.sub.4 is chosen from D-Nar, D-Orn, and
(isopropyl)D-Orn.
[0102] In another embodiment of the synthetic peptide amide of the
invention: L is a linker chosen from .epsilon.-D-Lys,
.epsilon.-Lys, .delta.-D-Orn, .delta.-Orn, 4-amino-4-carboxylic
piperidine and bis(D-Lys-Gly)Lactam.
[0103] In another embodiment of the synthetic peptide amide, J is a
five-membered heterocyclic ring moiety. In an alternative
embodiment, J is a five-membered heterocyclic ring moiety that
includes two heteroatoms, which two heteroatoms are both N.
[0104] In another embodiment of the synthetic peptide amide, G
is
##STR00022##
[0105] In a particular aspect of this embodiment, W is null, and Y
is nitrogen. In another particular aspect of this embodiment, the Y
and Z-containing ring moiety is a five-membered saturated ring.
[0106] In another embodiment of the synthetic peptide amide, G is
an optionally substituted proline radical.
[0107] In another embodiment of the synthetic peptide amide, the Y
and Z-containing ring moiety is a six-membered saturated ring. In a
particular aspect of this embodiment, the Y and Z-containing ring
moiety comprises a single heteroatom and e is zero, and R.sub.1 and
R.sub.2 taken together or with one or two ring atoms of the Y and
Z-containing ring moiety comprise an optionally substituted
monocyclic or bicyclic 4-, 5, 6-, 7, 8- or 9-membered heterocyclic
ring moiety. In a particular aspect of this embodiment, R.sub.1 and
R.sub.2 taken together with one ring atom of the Y and Z-containing
ring moiety comprises a five-membered heterocyclic ring moiety
having only heteroatoms chosen from N and O, which heterocyclic
ring moiety with the Y and Z-containing ring moiety forms a spiro
structure.
[0108] In another embodiment of the synthetic peptide amide, the Y
and Z-containing ring moiety includes two heteroatoms. In a
particular aspect of this embodiment, the two heteroatoms of the Y
and Z-containing ring moiety are both nitrogen. In another
particular aspect of this embodiment, the integer e is zero,
R.sub.2 is hydrogen and the Y- and Z-containing ring moiety is
3-substituted with R.sub.1. In still another particular aspect of
this embodiment, the two heteroatoms of the Y- and Z-containing
ring moiety are nitrogen and oxygen. In one particular aspect the
Y- and Z-containing ring moiety is 3-substituted with R.sub.1, the
integer e is zero and R.sub.2 is hydrogen. In another particular
aspect the two heteroatoms of the Y- and Z-containing ring moiety
are nitrogen and sulfur. In still another particular aspect the Y-
and Z-containing ring moiety is 3-substituted with R.sub.1, e is
zero and R.sub.2 is H.
[0109] In another embodiment of the synthetic peptide amide, G
is
##STR00023##
W is null, and Y is nitrogen and the Y and Z-containing ring moiety
is a seven-membered saturated ring comprising two heteroatoms. In a
particular aspect of this embodiment, Y and Z are both nitrogen
atoms and the moiety V.sub.eR.sub.1R.sub.2 is bonded to Z. In an
alternative aspect of this embodiment, Y is nitrogen and the second
heteroatom of the Y and Z-containing ring moiety is chosen from S
and O. In another alternative aspect of this embodiment, W is
--NH.sub.2--(CH.sub.2).sub.b-- and b is zero, 1, 2, or 3. In a
particular aspect the Y- and Z-containing ring moiety is a
five-membered saturated ring such as for instance, an optionally
substituted proline radical. Alternatively, the Y- and Z-containing
ring moiety can be a six-membered saturated ring, wherein for
example, Y can be carbon and Z can be nitrogen; alternatively, Y
and Z can both be nitrogen atoms.
[0110] In another embodiment of the synthetic peptide amide, G is
chosen from substituted piperidinyl, piperidinyl forming a spiro
structure with an optionally substituted heterocycle, piperidinyl
fused with an optionally substituted heterocycle, substituted
piperazinyl, 4-sulfonamidyl piperazinyl, 3-substituted piperazinyl,
substituted homopiperazinyl, optionally substituted
homomorpholinyl, optionally substituted homothiomorpholinyl,
3-substituted morpholinyl, 3-substituted thiomorpholinyl, 4-4 dioxo
thiomorpholinyl, and optionally substituted proline, and W is
null;
or G is
##STR00024##
[0111] wherein the moiety
##STR00025##
is chosen from substituted pyrazinyl, substituted pyridinyl,
substituted piperazinyl, optionally substituted pyrimidinyl,
substituted "reverse" piperidinyl (i.e. not bonded to W through the
ring nitrogen), optionally substituted heterocyclic bicycle,
optionally substituted proline, optionally substituted thiazolyl,
optionally substituted dioxolanyl, and optionally substituted
tetrahydropyranyl, and W is --NH.sub.2--(CH.sub.2).sub.b-- and b is
zero, 1, 2, or 3.
[0112] In another embodiment of the synthetic peptide amide, G is
chosen from substituted piperidinyl, piperidinyl forming a spiro
structure with an optionally substituted heterocycle, and
piperidinyl fused with an optionally substituted heterocycle. In a
particular aspect of this embodiment, G is chosen from substituted
piperazinyl, 4-sulfonamidyl piperazinyl, 3-substituted piperazinyl,
and substituted homopiperazinyl. In another alternative aspect, G
is chosen from optionally substituted homomorpholinyl, optionally
substituted homothiomorpholinyl, 3-substituted morpholinyl,
3-substituted thiomorpholinyl, and 4-4 dioxothiomorpholinyl. In
still another alternative aspect, G is an optionally substituted
proline.
[0113] In another embodiment of the synthetic peptide amide, W is
--NH.sub.2--(CH.sub.2).sub.b--, b is zero, 1, 2, or 3 and the
moiety
##STR00026##
is chosen from optionally substituted thiazolyl, optionally
substituted dioxolanyl, and optionally substituted
tetrahydropyranyl. Alternatively, W is
--NH.sub.2--(CH.sub.2).sub.b--, b is zero, 1, 2, or 3 and the
moiety
##STR00027##
is chosen from substituted pyrazinyl, substituted pyridinyl,
optionally substituted pyrimidinyl, and optionally substituted
heterocyclic bicycle. In another alternative, W is
--NH.sub.2--(CH.sub.2).sub.b--, b is zero, 1, 2, or 3 and the
moiety
##STR00028##
is chosen from substituted piperazinyl and 4-substituted
piperidinyl.
[0114] In another embodiment of the synthetic peptide amide, W is
--NH.sub.2--(CH.sub.2).sub.b--, b is zero, 1, 2, or 3 and the
moiety
##STR00029##
is an optionally substituted proline moiety.
[0115] In another embodiment of the synthetic peptide amide, e is
zero and R.sub.1 and R.sub.2 are bonded directly to the same ring
atom.
[0116] In another embodiment of the synthetic peptide amide,
R.sub.1 is chosen from H, OH, NH.sub.2, COOH, CH.sub.2COOH,
C.sub.1-C.sub.3 alkyl, amidino, C.sub.1-C.sub.3 alkyl-substituted
amidino, dihydroimidazole, D-Pro, D-Pro amide and CONH.sub.2; and
R.sub.2 is H, COOH, or C.sub.1-C.sub.3 alkyl.
[0117] The synthetic peptide amides of the invention are useful as
kappa opioid receptor ligands and exhibit biological activity in
vitro and in vivo.
[0118] A variety of assays may be employed to test whether the
synthetic peptide amides of the invention exhibit high affinity and
selectivity for the kappa opioid receptor, long duration of in vivo
bioactivity, and lack of CNS side effects. Receptor assays are well
known in the art and kappa opioid receptors from several species
have been cloned, as have mu and delta opioid receptors. Kappa
opioid receptors as well as mu and delta opioid receptors are
classical, seven transmembrane-spanning, G protein-coupled
receptors. Although these cloned receptors readily allow a
particular candidate compound, e.g., a peptide or peptide
derivative, to be screened, natural sources of mammalian opioid
receptors are also useful for screening, as is well known in the
art (Dooley C T et al. Selective ligands for the mu, delta, and
kappa opioid receptors identified from a single mixture based
tetrapeptide positional scanning combinatorial library. J. Biol.
Chem. 273:18848-56, 1998). Thus, screening against both kappa and
mu opioid receptors, whether of recombinant or natural origin, may
be carried out in order to determine the selectivity of the
synthetic peptide amides of the invention for the kappa over the mu
opioid receptor.
[0119] In a particular embodiment, the synthetic peptide amides of
the invention are selective kappa opioid receptor agonists. The
potency of the synthetic peptide amides of the invention as
agonists for a particular receptor can be measured as a
concentration at which half maximal effect is achieved expressed as
an EC.sub.50 value. Potency of the synthetic peptide amides of the
invention as kappa opioid agonists, expressed as the percent of
maximal observable effect, can be determined by a variety of
methods well known in the art. See for example, Endoh T et al.,
1999, Potent Antinociceptive Effects of TRK-820, a Novel
.kappa.-Opioid Receptor Agonist, Life Sci. 65 (16) 1685-94; and
Kumar V et al., Synthesis and Evaluation of Novel Peripherally
Restricted .kappa.-Opioid Receptor Agonists, 2005 Bioorg Med Chem
Letts 15: 1091-1095.
[0120] Examples of such assay techniques for determination of
EC.sub.50 values are provided below. Many standard assay methods
for characterization of opioid ligands are well known to those of
skill in the art. See, for example, Waldhoer et al., (2004) Ann.
Rev. Biochem. 73:953-990, and Satoh & Minami (1995) Pharmac.
Ther. 68(3):343-364 and references cited therein.
[0121] In certain particular embodiments, the synthetic peptide
amides of the invention are kappa opioid receptor agonists with an
EC.sub.50 of less than about 500 nM. In other embodiments, the
synthetic peptide amides have an EC.sub.50 of less than about 100
nM as kappa opioid receptor agonists. In still other embodiments,
the synthetic peptide amides have an EC.sub.50 of less than about
10 nM as kappa opioid receptor agonists. In particular embodiments
the synthetic peptide amides of the invention have an EC.sub.50 of
less than about 1.0 nM, or less than about 0.1 nM, or even less
than about 0.01 nM as kappa opioid receptor agonists. The compounds
of the foregoing embodiment can have an EC.sub.50 that is at least
10 times greater for a mu and a delta opioid receptor than for a
kappa opioid receptor, preferably at least 100 times greater, and
most preferably at least 1000 times greater, such as for instance,
an EC.sub.50 of less than about 1 nM for a kappa opioid receptor,
and EC.sub.50 values of greater than about 1000 nM for a mu opioid
receptor and a delta opioid receptor.
[0122] In particular embodiments, the synthetic peptide amides of
the invention are highly selective for kappa over mu opioid
receptors. In certain embodiments the synthetic peptide amides of
the invention have EC.sub.50 values for the mu opioid receptor that
are at least about a hundred times higher than the corresponding
EC.sub.50 values for the kappa opioid receptor. In particular
embodiments, the synthetic peptide amides of the invention have
EC.sub.50 values for the mu opioid receptor that are at least about
a thousand times higher than the corresponding EC.sub.50 values for
the kappa opioid receptor. Alternatively, the selectivity of the
synthetic peptide amides of the invention can be expressed as a
higher EC.sub.50 for a mu opioid receptor than for a kappa opioid
receptor. Thus, in particular embodiments, the synthetic peptide
amides of the invention have EC.sub.50 values of greater than about
10 .mu.M for the mu opioid receptor and EC.sub.50 values of less
than about 10 nM, and in other embodiments less than about 1.0 nM,
or even less than about 0.01 nM for the kappa opioid receptor. In
another embodiment, the particular synthetic peptide amide can have
an EC.sub.50 of less than about 1 nM for a kappa opioid receptor
and an EC.sub.50 of greater than about 1000 nM for a mu opioid
receptor, or for a delta opioid receptor.
[0123] Another property of the synthetic peptide amides of the
invention is their characteristic property of low inhibition of the
cytochrome P.sub.450 isozymes. The cytochrome P.sub.450 isozymes
constitute a large superfamily of haem-thiolate proteins
responsible for metabolic oxidative inactivation of many
therapeutics and other bioactive compounds. Usually, they act as
terminal oxidases in multicomponent electron transfer chains, also
referred to as cytochrome P.sub.450-containing monooxygenase
systems.
[0124] Over fifty different cytochrome P.sub.450 isozymes have been
identified and have been classified into families grouped by
genetic relatedness as assessed by nucleic acid sequence homology.
Most abundant among the cytochrome P.sub.450 isozymes in human
cells are the 1A2 and 3A4 isozymes, although isozymes 2B6, 2C9,
2C19, 2D6, and 2E1 also contribute significantly to oxidative
inactivation of administered therapeutics. While inhibition of the
cytochrome P.sub.450 isozymes may be useful in prolonging the time
after in vivo administration during which an effective
concentration of the synthetic peptide amides of the invention is
maintained, it also prolongs the persistence of any co-administered
therapeutic compound that is subject to oxidation by cytochrome
P.sub.450. This increase in persistence may cause the
co-administered therapeutic to persist beyond the period that is
optimal for therapy, or may cause the in vivo concentration to
exceed the desired levels or safely tolerated levels. Such
increases in persistence and/or increases in concentration are
difficult to accurately quantify and are preferably avoided.
Therapeutics that show little or no inhibition of the activity of
the cytochrome P.sub.450 isozymes do not have this potential
problem and can be more safely co-administered with other
therapeutics without risk of affecting the rate of inactivation of
the co-administered therapeutic compound by the cytochrome
P.sub.450 isozymes.
[0125] Particular embodiments of the synthetic peptide amides of
the invention show low inhibition of the cytochrome P.sub.450
isozymes at therapeutic concentrations of the synthetic peptide
amides, while others show essentially no inhibition of the
cytochrome P.sub.450 isozymes at therapeutic concentrations. In
some embodiments, the synthetic peptide amides at a concentration
of 10 .mu.M show less than about 50% inhibition of cytochrome
P.sub.450 isozymes CYP1A2, CYP2C9, CYP2C19 or CYP2D6. In particular
embodiments, the synthetic peptide amides at a concentration of 10
.mu.M show less than about 20% inhibition of any of these
cytochrome P.sub.450 isozymes. In very particular embodiments, the
synthetic peptide amides at a concentration of 10 .mu.M show less
than about 10% inhibition of any of these cytochrome P.sub.450
isozymes.
[0126] In another embodiment, the synthetic peptide amides of the
invention at an effective concentration exhibit no more than about
50% inhibition of any of P.sub.450 CYP1A2, CYP2C9, CYP2C19 or CYP
2D6 by the synthetic peptide amide at a concentration of 10 .mu.M
after 60 minutes incubation with human liver microsomes.
[0127] The synthetic peptide amides of the invention when
administered to a mammal or a human patient at a therapeutically
effective concentration exhibit low or essentially no penetration
across the blood-brain barrier. Kappa opioid receptors (hereinafter
interchangeably referred to as kappa receptors) are distributed in
peripheral tissues, including the skin and somatic tissues, as well
as the viscera in humans and other mammals. Kappa receptors are
also found in the brain. Activation of the kappa receptors in
peripheral tissues causes suppression of pain and inflammatory
responses, while activation of the kappa receptors in the brain
causes sedative effects and may also lead to severe dysphoria and
hallucinations. In certain embodiments, the synthetic peptide
amides of the invention when administered at therapeutically
effective concentrations exhibit little or essentially no
penetration across the blood-brain barrier and therefore minimize
or even completely obviate the sedative, hallucinogenic effects of
many other kappa agonists that show some penetration across the
blood-brain barrier.
[0128] One useful measure of the extent to which the synthetic
peptide amides of the invention cross the blood-brain barrier is
the ratio of the peak plasma concentration to the concentration in
brain tissue. In particular embodiments, the synthetic peptide
amides of the invention when administered at a dose of about 3
mg/kg, exhibit at least about a five fold lower peak concentration
of the synthetic peptide amide in brain than the peak concentration
in plasma.
[0129] Another useful measure of the extent to which the synthetic
peptide amides of the invention cross the blood-brain barrier is
the ratio of the dose required to achieve a sedative effect and the
dose required to achieve an analgesic effect. The analgesic and
sedative effects of kappa receptor stimulation by kappa receptor
agonists can be measured by standard assays well known to those of
skill in the art.
[0130] In particular embodiments, the synthetic peptide amides of
the invention have an ED.sub.50 for a sedative effect that is at
least about ten times the ED.sub.50 for an analgesic effect. In
particular embodiments, the synthetic peptide amides of the
invention have an ED.sub.50 for a sedative effect that is at least
about thirty times the ED.sub.50 for an analgesic effect. In still
other embodiments, the synthetic peptide amides of the invention
have an ED.sub.50 for a sedative effect that is at least about
fifty times the ED.sub.50 for an analgesic effect.
[0131] In one aspect, the synthetic peptide amide of the invention
has an ED.sub.50 for a sedative effect in a locomotion-reduction
assay in a mouse at least about ten times the ED.sub.50 of the
synthetic peptide amide for an analgesic effect in a writhing assay
in a mouse.
[0132] Another useful predictor of the extent to which the
synthetic peptide amides of the invention would be expected to
cross the blood-brain barrier is provided by the membrane
permeability values of the synthetic peptide amides into a human
cell or other mammalian cell when delivered at a therapeutically
relevant concentration. In certain embodiments, the synthetic
peptide amides of the invention at therapeutically relevant
concentrations exhibit low or essentially no ability to penetrate a
monolayer of suitably cultured human or other mammalian cells. This
permeability parameter can be expressed as an apparent
permeability, P.sub.app, representing the permeability of the
particular cell monolayer to a compound of interest. Any suitably
culturable mammalian cell monolayer can be used to determine its
permeability for a particular compound of interest, although
certain cell lines are frequently used for this purpose. For
instance, the Caco-2 cell line is a human colon adenocarcinoma that
can be used as a monolayer culture test system for determination of
membrane permeability towards compounds of the invention. In
certain embodiments, the synthetic peptide amides of the invention
have a P.sub.app of less than about 10.sup.-6 cm/sec. In certain
other embodiments, the synthetic peptide amides of the invention
have a P.sub.app of less than about 10.sup.-7 cm/sec.
[0133] In one embodiment, the synthetic peptide amide of the
invention at a dose of about 3 mg/kg in rat reaches a peak plasma
concentration and exhibits at least about a five fold lower peak
concentration in brain than such peak plasma concentration.
[0134] In another embodiment, the synthetic peptide amides of the
invention have at least about 50% of maximum efficacy at about 3
hours post administration of a dose of about 3 mg/kg of the
synthetic peptide amide in a rat.
[0135] In one embodiment the synthetic peptide amide of the
invention exhibits a long lasting duration of action in a mammal,
such as a human. In one aspect, the synthetic peptide amide has a
duration of action that is at least about 50% of maximum efficacy
at three hours post administration of 0.1 mg/kg of the synthetic
peptide amide. In another aspect the synthetic peptide amide has a
duration of action that is at least about 75% of maximum efficacy
at three hours post administration of 0.1 mg/kg of the synthetic
peptide amide. In a particular aspect the synthetic peptide amide
has a duration of action that is at least about 90% of maximum
efficacy at 3 hrs post administration of 0.1 mg/kg of the synthetic
peptide amide. In a specific aspect, the synthetic peptide amide
has a duration of action that is at least about 95% of maximum
efficacy at three hours post administration of 0.1 mg/kg of the
synthetic peptide amide.
[0136] In another embodiment, the invention provides a
pharmaceutical composition that includes a synthetic peptide amide
according to any of the above embodiments and a pharmaceutically
acceptable excipient or carrier. The invention provides methods,
compositions, or dosage forms that include synthetic peptide amides
of the invention that are selective for the kappa opioid receptor.
In particular embodiments, the synthetic peptide amides of the
invention exhibit a strong affinity for the kappa opioid receptor
and have a high potency as kappa opioid receptor agonists.
[0137] A pro-drug of a compound such as the synthetic peptide
amides of the invention include pharmaceutically acceptable
derivatives which upon administration can convert through
metabolism or other process to a biologically active form of the
compound. Pro-drugs are particularly desirable where the pro-drug
has more favorable properties than does the active compound with
respect to bioavailability, stability or suitability for a
particular formulation.
[0138] As used herein, a kappa opioid receptor-associated disease
or condition is any disease, condition or disorder that is
preventable or treatable by activation of a kappa opioid receptor.
In one aspect, the synthetic peptide amides of the invention are
kappa opioid receptor agonists that activate the kappa opioid
receptor. In some embodiments, a particular dose and route of
administration of the synthetic peptide amide of the invention can
be chosen by a clinician to completely prevent or cure the disease,
condition or disorder. In other embodiments a particular dose and
route of administration of the synthetic peptide amide of the
invention chosen by the clinician ameliorates or reduces one or
more symptoms of the disease, condition or disorder.
[0139] As used herein, "effective amount" or "sufficient amount" of
the synthetic peptide amide of the invention refers to an amount of
the compound as described herein that may be therapeutically
effective to inhibit, prevent, or treat a symptom of a particular
disease, disorder, condition, or side effect. As used herein, a
"reduced dose" of a mu opioid agonist analgesic compound refers to
a dose which when used in combination with a kappa opioid agonist,
such as a synthetic peptide amide of the invention, is lower than
would be ordinarily provided to a particular patient, for the
purpose of reducing one or more side effects of the compound. The
dose reduction can be chosen such that the decrease in the
analgesic or other therapeutic effect of the compound is an
acceptable compromise in view of the reduced side effect(s), where
the decrease in analgesic or other therapeutic effects of the mu
opioid agonist analgesic are wholly or at least partially offset by
the analgesic or other therapeutic effect of the synthetic peptide
amide of the invention. Co-administration of a mu opioid agonist
analgesic compound with a synthetic peptide amide of the invention
which acts as a kappa opioid agonist also permits incorporation of
a reduced dose of the synthetic peptide amide and/or the mu opioid
agonist analgesic compound to achieve the same therapeutic effect
as a higher dose of the synthetic peptide amide or the mu opioid
agonist analgesic compound if administered alone.
[0140] As used herein, "pharmaceutically acceptable" refers to
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for contact
with the tissues of human beings and animals without severe
toxicity, irritation, allergic response, or other complications,
commensurate with a benefit-to-risk ratio that is reasonable for
the medical condition being treated.
[0141] As used herein, "dosage unit" refers to a physically
discrete unit suited as unitary dosages for a particular individual
or condition to be treated. Each unit may contain a predetermined
quantity of active synthetic peptide amide compound(s) calculated
to produce the desired therapeutic effect(s), optionally in
association with a pharmaceutical carrier. The specification for
the dosage unit forms may be dictated by (a) the unique
characteristics of the active compound or compounds, and the
particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such active compound
or compounds. The dosage unit is often expressed as weight of
compound per unit body weight, for instance, in milligrams of
compound per kilogram of body weight of the subject or patient
(mg/kg). Alternatively, the dosage can be expressed as the amount
of the compound per unit body weight per unit time, (mg/kg/day) in
a particular dosage regimen. In a further alternative, the dosage
can be expressed as the amount of compound per unit body surface
area (mg/m.sup.2) or per unit body surface area per unit time
(mg/m.sup.2/day). For topical formulations, the dosage can be
expressed in a manner that is conventional for that formulation,
e.g., a one-half inch ribbon of ointment applied to the eye, where
the concentration of compound in the formulation is expressed as a
percentage of the formulation.
[0142] As used herein, a "pharmaceutically acceptable salt" refers
to a derivative of a compound wherein the parent compound is
modified by making an acid or a base salt thereof. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as carboxylic acids
and the like. The pharmaceutically acceptable salts include the
conventional non-toxic salts or the quaternary ammonium salts of
the parent compound formed, for example, from non-toxic inorganic
or organic acids. For instance, such conventional non-toxic salts
include those derived from inorganic acids such as hydrochloric,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric acids and the
like; and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isethionic acids, and the like. These physiologically
acceptable salts are prepared by methods known in the art, e.g., by
dissolving the free amine bases with an excess of the acid in
aqueous alcohol, or neutralizing a free carboxylic acid with an
alkali metal base such as a hydroxide, or with an amine. Thus, a
pharmaceutically acceptable salt of a synthetic peptide amide can
be formed from any such peptide amide having either acidic, basic
or both functional groups. For example, a peptide amide having a
carboxylic acid group, may in the presence of a pharmaceutically
suitable base, form a carboxylate anion paired with a cation such
as a sodium or potassium cation. Similarly, a peptide amide having
an amine functional group may, in the presence of a
pharmaceutically suitable acid such as HCl, form a salt.
[0143] An example of a pharmaceutically acceptable solvate of a
synthetic peptide amide is a combination of a peptide amide with
solvent molecules which yields a complex of such solvent molecules
in association with the peptide amide. Combinations of a drug and
propylene glycol (1,2-propanediol) have been used to form
pharmaceutical drug solvates. See for example U.S. Pat. No.
3,970,651. Other suitable solvates are hydrates of drug compounds.
Such hydrates include hydrates which either have comparable
activity or hydrates which are converted back to the active
compound following administration. A pharmaceutically acceptable
N-oxide of a synthetic peptide amide is such a compound that
contains an amine group wherein the nitrogen of the amine is bonded
to an oxygen atom.
[0144] A pharmaceutically acceptable crystalline, isomorphic
crystalline or amorphous form of a synthetic peptide amide of the
invention can be any crystalline or non-crystalline form of a
pharmaceutically acceptable acidic, basic, zwitterionic, salt,
hydrate or any other suitably stable, physiologically compatible
form of the synthetic peptide amide according to the invention.
[0145] The synthetic peptide amides of the invention can be
incorporated into pharmaceutical compositions. The compositions can
include an effective amount of the synthetic peptide amide in a
pharmaceutically acceptable diluent, excipient or carrier.
Conventional excipients, carriers and/or diluents for use in
pharmaceutical compositions are generally inert and make up the
bulk of the preparation.
[0146] In a particular embodiment, the synthetic peptide amide is a
kappa opioid receptor agonist. In another embodiment, the synthetic
peptide amide is a selective kappa opioid receptor agonist. The
target site can be a kappa receptor in the patient or subject in
need of such treatment or prophylaxis. Certain synthetic peptide
amide kappa opioid receptor agonists of the invention are
peripherally acting and show little or no CNS effects at
therapeutically effective doses.
[0147] The pharmaceutical excipient or carrier can be any
compatible, non-toxic substance suitable as a vehicle for delivery
the synthetic peptide amide of the invention. Suitable excipients
or carriers include, but are not limited to, sterile water
(preferably pyrogen-free), saline, phosphate-buffered saline (PBS),
water/ethanol, water/glycerol, water/sorbitol, water/polyethylene
glycol, propylene glycol, cetylstearyl alcohol,
carboxymethylcellulose, corn starch, lactose, glucose,
microcrystalline cellulose, magnesium stearate,
polyvinylpyrrolidone (PVP), citric acid, tartaric acid, oils, fatty
substances, waxes or suitable mixtures of any of the foregoing.
[0148] The pharmaceutical composition according to the invention
can be formulated as a liquid, semisolid or solid dosage form. For
example the pharmaceutical preparation can be in the form of a
solution for injection, drops, syrup, spray, suspension, tablet,
patch, capsule, dressing, suppository, ointment, cream, lotion,
gel, emulsion, aerosol or in a particulate form, such as pellets or
granules, optionally pressed into tablets or lozenges, packaged in
capsules or suspended in a liquid. The tablets can contain binders,
lubricants, diluents, coloring agents, flavoring agents, wetting
agents and may be enteric-coated to survive the acid environment of
the stomach and dissolve in the more alkaline conditions of the
intestinal lumen. Alternatively, the tablets can be sugar-coated or
film coated with a water-soluble film. Pharmaceutically acceptable
adjuvants, buffering agents, dispersing agents, and the like, may
also be incorporated into the pharmaceutical compositions.
[0149] Binders include for instance, starch, mucilage, gelatin and
sucrose. Lubricants include talc, lycopodium, magnesium and calcium
stearate/stearic acid. Diluents include lactose, sucrose, mannitol,
salt, starch and kaolin. Wetting agents include propylene glycol
and sorbitan monostearate.
[0150] As used herein, local application or administration refers
to administration of a pharmaceutical preparation according to the
invention to the site, such as an inflamed joint, that exhibits the
painful and/or inflamed condition. Such local application includes
intrajoint, such as intra-articular application, via injection,
application via catheter or delivery as part of a biocompatible
device. Thus, local application refers to application to a discrete
internal area of the body, such as, for example, a joint, soft
tissue area (such as muscle, tendon, ligaments, intraocular or
other fleshy internal areas), or other internal area of the body.
In particular, as used herein, local application refers to
applications that provide substantially no systemic delivery and/or
systemic administration of the active agents in the present
compositions. Also, as used herein, local application is intended
to refer to applications to discrete areas of the body, that is,
other than the various large body cavities (such as, for example,
the peritoneal and/or pleural cavities).
[0151] As used herein, topical application refers to application to
the surface of the body, such as to the skin, eyes, mucosa and
lips, which can be in or on any part of the body, including but not
limited to the epidermis, any other dermis, or any other body
tissue. Topical administration or application means the direct
contact of the pharmaceutical preparation according to the
invention with tissue, such as skin or membrane, particularly the
cornea, or oral, vaginal or anorectal mucosa. Thus, for purposes
herein topical application refers to application to the tissue of
an accessible body surface, such as, for example, the skin (the
outer integument or covering) and the mucosa (the mucus-producing,
secreting and/or containing surfaces). In particular, topical
application refers to applications that provide little or
substantially no systemic delivery of the active compounds in the
present compositions. Exemplary mucosal surfaces include the
mucosal surfaces of the eyes, mouth (such as the lips, tongue,
gums, cheeks, sublingual and roof of the mouth), larynx, esophagus,
bronchus, trachea, nasal passages, vagina and rectum/anus.
[0152] For oral administration, an active ingredient can be
administered in solid dosage forms, such as capsules, tablets, and
powders, or in liquid dosage forms, such as elixirs, syrups, and
suspensions. Active component(s) can be encapsulated in gelatin
capsules together with inactive ingredients and powdered carriers,
such as glucose, lactose, sucrose, mannitol, starch, cellulose or
cellulose derivatives, magnesium stearate, stearic acid, sodium
saccharin, talcum, magnesium carbonate and the like. Examples of
additional inactive ingredients that may be added to provide
desirable color, taste, stability, buffering capacity, dispersion
or other known desirable features are red iron oxide, silica gel,
sodium lauryl sulfate, titanium dioxide, edible white ink and the
like. Similar diluents can be used to make compressed tablets. Both
tablets and capsules can be manufactured as sustained release
products to provide for continuous release of medication over a
period of hours. Compressed tablets can be sugar coated or film
coated to mask any unpleasant taste and protect the tablet from the
atmosphere, or enteric coated for selective disintegration in the
gastrointestinal tract. Liquid dosage forms for oral administration
can contain coloring and flavoring to increase patient acceptance.
To facilitate drug stability and absorption, peptides of the
invention can be released from a capsule after passing through the
harsh proteolytic environment of the stomach. Methods for enhancing
peptide stability and absorption after oral administration are well
known in the art (e.g., Mahato R I. Emerging trends in oral
delivery of peptide and protein drugs. Critical Reviews in
Therapeutic Drug Carrier Systems. 20:153-214, 2003).
[0153] Dosage forms such as lozenges, chewable tablets and chewing
gum permit more rapid therapeutic action compared to per-oral
dosage forms of the synthetic peptide amide compounds of the
invention having significant buccal absorption. Chewing gum
formulations are solid, single dose preparations with a base
consisting mainly of gum, that are intended to be chewed but not
swallowed, and contain one or more compounds of the invention which
are released by chewing and are intended to be used for local
treatment of pain and inflammation of the mouth or systemic
delivery after absorption through the buccal mucosa. See for
example, U.S. Pat. No. 6,322,828 to Athanikar and Gubler entitled:
Process for manufacturing a pharmaceutical chewing gum.
[0154] For nasal administration, the peripherally selective kappa
opioid receptor agonists can be formulated as aerosols. The term
"aerosol" includes any gas-borne suspended phase of the compounds
of the instant invention which is capable of being inhaled into the
bronchioles or nasal passages. Specifically, aerosol includes a
gas-borne suspension of droplets of the compounds of the instant
invention, as may be produced in a metered dose inhaler or
nebulizer, or in a mist sprayer. Aerosol also includes a dry powder
composition of a compound of the instant invention suspended in air
or other carrier gas, which may be delivered by insufflation from
an inhaler device, for example. See Ganderton & Jones, Drug
Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda
(1990) Critical Reviews in Therapeutic Drug Carrier Systems
6:273-313; and Raeburn et al. (1992) J. Pharmacol. Toxicol. Methods
27:143-159.
[0155] The pharmaceutical compositions of the invention can be
prepared in a formulation suitable for systemic delivery, such as
for instance by intravenous, subcutaneous, intramuscular,
intraperitoneal, intranasal, transdermal, intravaginal,
intrarectal, intrapulmonary or oral delivery. Alternatively, the
pharmaceutical compositions of the invention can be suitably
formulated for local delivery, such as, for instance, for topical,
or iontophoretic delivery, or for transdermal delivery by a patch
coated, diffused or impregnated with the formulation, and local
application to the joints, such as by intra-articular
injection.
[0156] Preparations for parenteral administration include sterile
solutions ready for injection, sterile dry soluble products ready
to be combined with a solvent just prior to use, including
hypodermic tablets, sterile suspensions ready for injection,
sterile dry insoluble products ready to be combined with a vehicle
just prior to use and sterile emulsions. The solutions may be
either aqueous or nonaqueous, and thereby formulated for delivery
by injection, infusion, or using implantable pumps. For
intravenous, subcutaneous, and intramuscular administration, useful
formulations of the invention include microcapsule preparations
with controlled release properties (R. Pwar et al. Protein and
peptide parenteral controlled delivery. Expert Opin Biol Ther.
4(8):1203-12, 2004) or encapsul-ation in liposomes, with an
exemplary form being polyethylene coated liposomes, which are known
in the art to have an extended circulation time in the vasculature
(e.g. Koppal, T. "Drug delivery technologies are right on target",
Drug Discov. Dev. 6, 49-50, 2003).
[0157] For ophthalmic administration, the present invention
provides a method of treating glaucoma or ophthalmic pain and
inflammation, comprising administering to an eye of a patient in
need thereof a therapeutically effective amount of a synthetic
peptide amide of the invention. The synthetic peptide amide can be
administered topically with an eye-compatible pharmaceutical
carrier or non-systemically using a contact lens or intraocular
implant that can optionally contain polymers that provide sustained
release of the synthetic peptide amide. Such eye-compatible
pharmaceutical carriers can include adjuvants, antimicrobial
preservatives, surfactants, and viscolyzers etc. It is known in the
art that high concentrations of many compounds are irritant to the
eye and low concentrations are less irritant; thus the formulation
is often designed to include the lowest effective concentrations of
active compound, preservative, surfactant, and/or viscolyzer, said
viscolyzer preferably having a high surface tension to reduce
irritation of the eye while increasing the retention of ophthalmic
solutions at the eye surface. Such controlled release of the
synthetic peptide amides of the invention can last 6 months to a
year for implants, or for shorter periods (3-14 days) for contact
lenses. Such implants can be osmotic pumps, biodegradable matrices,
or intraocular sustained release devices. Such topical compositions
can include a buffered saline solution with or without
liposomes.
[0158] Aqueous polymeric solutions, aqueous suspensions, ointments,
and gels can be used for topical formulations of the synthetic
peptide amides of the invention for ocular applications. The
aqueous formulations may also contain liposomes for creating a
reservoir of the synthetic peptide amide. Certain of these topical
formulations are gels which enhance pre-corneal retention without
the inconvenience and impairment of vision associated with
ointments. The eye-compatible pharmaceutical carrier can also
include a biodegradable synthetic polymer. Biodegradable
microsphere compositions approved for human use include the
polylactides: poly(lactic acid), poly(glycolic acid), and
poly(lactic-coglycolic) acid. Additional biodegradable formulations
include, but are not limited to: poly(anhydride-co-imide),
poly(lactic-glycolic acid), polyethyl-2-cyanoacrylate,
polycaprolactone, polyhydroxybutyrate valerate, polyorthoester, and
polyethylene-oxide/polybutylene teraphthalate. Intraocular
implantation or injection of sustained release compositions that
include a synthetic peptide amide of the invention can provide
long-term control (ranging from months to years) of intraocular
pressure, and thereby avoiding or reducing the need for topical
preparations. Useful methods for formulating and dispensing
ophthalmic medications are disclosed in U.S. Pat. No. 7,122,579 to
Schwartz et al, and in U.S. Pat. No. 7,105,512 to Morizono et al.
Methods for formulating ophthalmic medications in contact lenses
are disclosed by Gulsen and Chauhan, Ophthalmic drug delivery
through contact lenses. Investigative Ophthalmology and Visual
Science, (2004) 45:2342-2347.
[0159] Preparations for transdermal delivery are incorporated into
a device suitable for said delivery, said device utilizing, e.g.,
iontophoresis (Kalia Y N et al. Iontophoretic Drug Delivery. Adv
Drug Deliv Rev. 56:619-58, 2004) or a dermis penetrating surface
(Prausnitz M R. Microneedles for Transdermal Drug Delivery. Adv
Drug Deliv Rev. 56:581-7, 2004), such as are known in the art to be
useful for improving the transdermal delivery of drugs. An
electrotransport device and methods of operation thereof are
disclosed in U.S. Pat. No. 6,718,201. Methods for the use of
iontophoresis to promote transdermal delivery of peptides are
disclosed in U.S. Pat. Nos. 6,313,092 and 6,743,432.
[0160] As used herein the terms "electrotransport",
"iontophoresis", and "iontophoretic" refer to the delivery through
a body surface (e.g., skin or mucosa) of one or more
pharmaceutically active compounds by means of an applied
electromotive force to an agent containing reservoir. The compound
may be delivered by electromigration, electroporation,
electroosmosis or any combination thereof. Electroosmosis has also
been referred to as electrohydrokinesis, electro convection, and
electrically induced osmosis. In general, electroosmosis of a
compound into a tissue results from the migration of solvent in
which the compound is contained, as a result of the application of
electromotive force to the therapeutic species reservoir, such as
for instance, solvent flow induced by electromigration of other
ionic species. During the electrotransport process, certain
modifications or alterations of the skin may occur such as the
formation of transiently existing pores in the skin, also referred
to as "electroporation." Any electrically assisted transport of
species enhanced by modifications or alterations to the body
surface (e.g., formation of pores in the skin) are also included in
the term "electrotransport" as used herein. Thus, as used herein,
applied to the compounds of the instant invention, the terms
"electrotransport", "iontophoresis" and "iontophoretic" refer to
(1) the delivery of charged agents by electromigration, (2) the
delivery of uncharged agents by the process of electroosmosis, (3)
the delivery of charged or uncharged agents by electroporation, (4)
the delivery of charged agents by the combined processes of
electromigration and electroosmosis, and/or (5) the delivery of a
mixture of charged and uncharged agents by the combined processes
of electromigration and electroosmosis. Electrotransport devices
generally employ two electrodes, both of which are positioned in
close electrical contact with some portion of the skin of the body.
One electrode, called the active or donor electrode, is the
electrode from which the therapeutic agent is delivered into the
body. The other electrode, called the counter or return electrode,
serves to close the electrical circuit through the body. In
conjunction with the patient's skin, the circuit is completed by
connection of the electrodes to a source of electrical energy,
e.g., a battery, and usually to circuitry capable of controlling
current passing through the device.
[0161] Depending upon the electrical charge of the compound to be
delivered transdermally, either the anode or cathode may be the
active or donor electrode. Thus, if the compound to be transported
is positively charged, e.g., the compound exemplified in Example 1
herein, then the positive electrode (the anode) will be the active
electrode and the negative electrode (the cathode) will serve as
the counter electrode, completing the circuit. However, if the
compound to be delivered is negatively charged, then the cathodic
electrode will be the active electrode and the anodic electrode
will be the counter electrode. Electrotransport devices
additionally require a reservoir or source of the therapeutic agent
that is to be delivered into the body. Such drug reservoirs are
connected to the anode or the cathode of the electrotransport
device to provide a fixed or renewable source of one or more
desired species or agents. Each electrode assembly is comprised of
an electrically conductive electrode in ion-transmitting relation
with an ionically conductive liquid reservoir which in use is
placed in contact with the patient's skin. Gel reservoirs such as
those described in Webster (U.S. Pat. No. 4,383,529) are one form
of reservoir since hydrated gels are easier to handle and
manufacture than liquid-filled containers. Water is one liquid
solvent that can be used in such reservoirs, in part because the
salts of the peptide compounds of the invention are water soluble
and in part because water is non-irritating to the skin, thereby
enabling prolonged contact between the hydrogel reservoir and the
skin. For electrotransport, the synthetic peptides of the invention
can be formulated with flux enhancers such as ionic surfactants or
cosolvents other than water (See for example, U.S. Pat. No.
4,722,726 and European Patent Application 278,473, respectively).
Alternatively the outer layer (i.e., the stratum corneum) of the
skin can be mechanically disrupted prior to electrotransport
delivery therethrough, for example as described in U.S. Pat. No.
5,250,023.
[0162] Peripherally synthetic peptide amides that are well suited
for electrotransport can be selected by measuring their
electrotransport flux through the body surface (e.g., the skin or
mucosa), e.g., as compared to a standardized test peptide with
known electrotransport flux characteristics, e.g. thyrotropin
releasing hormone (R. Burnette et al. J. Pharm. Sci. (1986) 75:738)
or vasopressin (Nair et al. Pharmacol Res. 48:175-82, 2003).
Transdermal electrotransport flux can be determined using a number
of in vivo or in vitro methods well known in the art. In vitro
methods include clamping a piece of skin of an appropriate mammal
(e.g., human cadaver skin) between the donor and receptor
compartments of an electrotransport flux cell, with the stratum
corneum side of the skin piece facing the donor compartment. A
liquid solution or gel containing the drug to be delivered is
placed in contact with the stratum corneum, and electric current is
applied to electrodes, one electrode in each compartment. The
transdermal flux is calculated by sampling the amount of drug in
the receptor compartment. Two successful models used to optimize
transdermal electrotransport drug delivery are the isolated pig
skin flap model (Heit M C et al. Transdermal iontophoretic peptide
delivery: in vitro and in vivo studies with luteinizing hormone
releasing hormone. J. Pharm. Sci. 82:240-243, 1993), and the use of
isolated hairless skin from hairless rodents or guinea pigs, for
example. See Hadzija B W et al. Effect of freezing on iontophoretic
transport through hairless rat skin. J. Pharm. Pharmacol. 44,
387-390, 1992. Compounds of the invention for transdermal
iontophoretic delivery can have one, or typically, two charged
nitrogens, to facilitate their delivery.
[0163] Other useful transdermal delivery devices employ high
velocity delivery under pressure to achieve skin penetration
without the use of a needle. Transdermal delivery can be improved,
as is known in the art, by the use of chemical enhancers, sometimes
referred to in the art as "permeation enhancers", i.e., compounds
that are administered along with the drug (or in some cases used to
pretreat the skin, prior to drug administration) in order to
increase the permeability of the stratum corneum, and thereby
provide for enhanced penetration of the drug through the skin.
Chemical penetration enhancers are compounds that are innocuous and
serve merely to facilitate diffusion of the drug through the
stratum corneum, whether by passive diffusion or an energy driven
process such as electrotransport. See, for example, Meidan V M et
al Enhanced iontophoretic delivery of buspirone hydrochloride
across human skin using chemical enhancers. Int. J. Pharm.
264:73-83, 2003.
[0164] Pharmaceutical dosage forms for rectal administration
include rectal suppositories, capsules and tablets for systemic
effect. Rectal suppositories as used herein mean solid bodies for
insertion into the rectum which melt or soften at body temperature
releasing one or more pharmacologically or therapeutically active
ingredients. Pharmaceutically acceptable substances utilized in
rectal suppositories include bases or vehicles and agents that
raise the melting point of the suppositories. Examples of bases
include cocoa butter (theobroma oil), glycerin-gelatin, carbowax,
(polyoxyethylene glycol) and appropriate mixtures of mono-, di- and
triglycerides of fatty acids. Combinations of the various bases can
also be used. Agents that raise the melting point of suppositories
include spermaceti and wax. Rectal suppositories may be prepared
either by the compression method or by molding. Rectal
suppositories typically weigh about 2 gm to about 3 gm. Tablets and
capsules for rectal administration are manufactured using the same
pharmaceutically acceptable substance(s) and by the same methods as
for formulations for oral administration.
[0165] Pharmaceutically acceptable carriers used in parenteral
preparations include aqueous vehicles, nonaqueous vehicles,
antimicrobial agents, isotonic agents, buffers, antioxidants, local
anesthetics, suspending and dispersing agents, emulsifying agents,
sequestering or chelating agents and other pharmaceutically
acceptable substances.
[0166] Examples of aqueous vehicles include sodium chloride for
injection, Ringers solution for injection, isotonic dextrose for
injection, sterile water for injection, dextrose and lactated
Ringers solution for injection. Nonaqueous parenteral vehicles
include fixed oils of vegetable origin, cottonseed oil, corn oil,
sesame oil and peanut oil. Antimicrobial agents in bacteriostatic
or fungistatic concentrations must be added to parenteral
preparations packaged in multiple dose containers which include
phenols or cresols, mercurials, benzyl alcohol, chlorobutanol,
methyl and propyl p-hydroxybenzoic acid esters, thimerosal,
benzalkonium chloride and benzethonium chloride. Isotonic agents
include sodium chloride and dextrose. Buffers include phosphate and
citrate. Antioxidants include sodium bisulfite. Local anesthetics
include procaine hydrochloride. Suspending and dispersing agents
include sodium carboxymethylcelluose, hydroxypropyl methylcellulose
and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80
(Tween 80). A sequestering or chelating agent of metal ions such as
EDTA can also be incorporated. Pharmaceutical carriers also include
ethyl alcohol, polyethylene glycol and propylene glycol for water
miscible vehicles and the pH can be adjusted to a physiologically
compatible pH by addition of sodium hydroxide, hydrochloric acid,
citric acid or lactic acid.
[0167] The active ingredient may be administered all at once, or
may be divided into a number of smaller doses to be administered at
intervals of time, or as a controlled release formulation. The term
"controlled release formulation" encompasses formulations that
allow the continuous delivery of a synthetic peptide amide of the
invention to a subject over a period of time, for example, several
days to weeks. Such formulations may be administered subcutaneously
or intramuscularly and allow for the continual steady state release
of a predetermined amount of compound in the subject over time. The
controlled release formulation of synthetic peptide amide may be,
for example, a formulation of drug containing polymeric
microcapsules, such as those described in U.S. Pat. Nos. 4,677,191
and 4,728,721, incorporated herein by reference. The concentration
of the pharmaceutically active compound is adjusted so that
administration provides an effective amount to produce a desired
effect. The exact dose depends on the age, weight and condition of
the patient or animal, as is known in the art. For any particular
subject, specific dosage regimens can be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
formulations. Thus, the concentration ranges set forth herein are
exemplary only and are not intended to limit the scope or practice
of the claimed invention.
[0168] The unit dose parenteral preparations include packaging in
an ampoule or prepackaged in a syringe with, or without a needle
for delivery. All preparations for parenteral administration are
typically sterile, as is practiced in the art. Illustratively,
intravenous infusion of a sterile aqueous buffered solution
containing an active compound is an effective mode of
administration. In another embodiment a sterile aqueous or oily
solution or suspension containing the active material can be
injected as necessary to produce the desired pharmacological
effect.
[0169] The pharmaceutical compositions of the invention can be
delivered or administered intravenously, transdermally,
transmucosally, intranasally, subcutaneously, intramuscularly,
orally or topically (such as for example to the eye). The
compositions can be administered for prophylaxis or treatment of
individuals suffering from, or at risk of a disease or a condition.
Prophylaxis is defined as a measure designed to preserve the health
of an individual. For therapeutic applications, a pharmaceutical
composition is typically administered to a subject suffering from a
disease or condition, in an amount sufficient to inhibit, prevent,
or ameliorate the disease or condition. An amount adequate to
accomplish this is defined as a "therapeutically effective
dose."
[0170] The pharmaceutical compositions of the invention can be
administered to a mammal for prophylactic or therapeutic purposes
in any of the above-described formulations and delivery modes. The
mammal can be any mammal, such as a domesticated or feral mammal,
or even a wild mammal. The mammal can be any primate, ungulate,
canine or feline. For instance, and without limitation, the mammal
may be a pet or companion animal, such as a dog or a cat; a
high-value mammal such as a thoroughbred horse or a show animal; a
farm animal, such as a cow, a goat, a sheep or pig; or a primate
such as an ape, gorilla, orangutan, lemur, monkey or chimpanzee.
Humans are suitable mammals for prophylaxis or treatment using
pharmaceutical compositions of the invention.
[0171] The pharmaceutical compositions of the invention can be
administered to a mammal having a disease or condition treatable by
activation of the kappa opioid receptor. Alternatively, the
pharmaceutical compositions can be administered as prophylactics to
a mammal having a risk of contracting or developing a disease or
condition preventable by activation of the kappa opioid receptor.
Diseases or conditions that can be treated or prevented by
administration of the pharmaceutical compositions of the invention
include, without limitation, any condition that can be ameliorated
by activation of the kappa opioid receptor, including such
conditions as pain, inflammation, pruritis, hyponatremia,
hypokalemia, congestive heart failure, liver cirrhosis, nephrotic
syndrome, hypertension, edema, ileus, tussis and glaucoma.
[0172] In a particular embodiment, the pharmaceutical compositions
of the invention can be co-administered with or can include one or
more other therapeutic compounds or adjuvants, such as but not
limited to other opioids, cannabinoids, antidepressants,
anticonvulsants, neuroleptics, antihistamines, acetaminophen,
corticosteroids, ion channel blocking agents, non-steroidal
anti-inflammatory drugs (NSAIDs), and diuretics, many of which are
synergistic in effect with the synthetic peptide amides of the
invention.
[0173] Suitable opioids, include, without limitation, alfentanil,
alphaprodine, anileridine, bremazocine, buprenorphine, butorphanol,
codeine, conorphone, dextromoramide, dextropropoxyphene, dezocine,
diamorphine, dihydrocodeine, dihydromorphine, diphenoxylate,
dipipanone, doxpicomine, ethoheptazine, ethylketazocine,
ethylmorphine, etorphine, fentanyl, hydrocodone, hydromorphone,
ketobemidone, levomethadyl, levorphanol, lofentanil, loperamide,
meperidine (pethidine), meptazinol, methadone, morphine,
morphine-6-glucuronide, nalbuphine, nalorphine, nicomorphine,
oxycodone, oxymorphone, pentazocine, phenazocine, phenoperidine,
piritramide, propiram, propoxyphene, remifentanil, sufentanil,
tilidate, tonazocine, and tramadol.
[0174] One embodiment of the invention is co-formulation and/or
co-administration of an opioid with substantial agonist activity at
the mu opioid receptor, such as morphine, fentanyl, hydromorphone,
or oxycodone, together with a synthetic peptide amide of the
invention, for the purpose of a mu opioid dose-sparing effect,
where the dose of the mu opioid is reduced to minimize common mu
opioid side effects, particularly in opioid-naive patients. Such
side effects include constipation, nausea, vomiting, sedation,
respiratory depression, pruritis (itching), mental confusion,
disorientation and cognitive impairment, urinary retention, biliary
spasm, delirium, myoclonic jerks, and seizures. The selection of
the reduced mu opioid dose requires expert clinical judgment, and
depends on the unique characteristics of the various mu opioids, as
well as patient characteristics such as pain intensity, patient
age, coexisting disease, current drug regimen and potential drug
interactions, prior treatment outcomes, and patient preference
(McCaffery, M. and Pasero, C., Pain Clinical Manual, Second
Edition, Mosby, 1999).
[0175] Cannabinoids suitable for administration with or
incorporation into the pharmaceutical compositions of the
invention, include any natural cannabinoid, such as for instance,
tetrahydrocannabinol (THC), or a THC derivative, or a synthetic
cannabinoid, such as, for instance, levonantradol, marinol,
nabilone, rimonabant or savitex.
[0176] Suitable antidepressants that can be co-administered with or
incorporated into the pharmaceutical compositions of the invention,
include for example, tricyclic antidepressants such as imipramine,
desipramine, trimipramine, protriptyline, norttriptyline,
amitriptyline, doxepin, and clomipramine; atypical antidepressants
such as amoxapine, maprotiline, trazodone, bupropion, and
venlafaxine; serotonin-specific reuptake inhibitors such as
fluoxetine, sertraline, paroxetine, citalopram and fluvoxamine;
norepinephrine-specific reuptake inhibitors such as reboxetine; or
dual-action antidepressants such as nefazodone and mirtazapine.
[0177] Suitable neuroleptics that can be co-administered with or
incorporated into the pharmaceutical compositions of the invention,
include any neuroleptic, for example, a compound with D2 dopamine
receptor antagonist activity such as domperidone, metaclopramide,
levosulpiride, sulpiride, thiethylperazine, ziprasidone, zotepine,
clozapine, chlorpromazine, acetophenazine, carphenazine,
chlorprothixene, fluphenazine, loxapine, mesoridazine, molindone,
perphenazine, pimozide, piperacetazine, perchlorperazine,
thioridazine, thiothixene, trifluoperazine, triflupromazine,
pipamperone, amperozide, quietiapine, melperone, remoxipride,
haloperidol, rispiridone, olanzepine, sertindole, ziprasidone,
amisulpride, prochlorperazine, and thiothixene.
[0178] Anticonvulsants such as phenobarbital, phenytoin, primidone,
carbamazepine, ethosuximide, lamotrigine, valproic acid,
vigabatrin, felbamate, gabapentin, levetiracetam, oxcarbazepine,
remacemide, tiagabine, and topiramate can also usefully be
incorporated into the pharmaceutical compositions of the
invention.
[0179] Muscle relaxants such as methocarbamol, orphenadrine,
carisoprodol, meprobamate, chlorphenesin carbamate, diazepam,
chlordiazepoxide and chlorzoxazone; anti-migraine agents such as
sumitriptan, analeptics such as caffeine, methylphenidate,
amphetamine and modafinil; antihistamines such as chlorpheniramine,
cyproheptadine, promethazine and pyrilamine, as well as
corticosteroids such as methylprednisolone, betamethasone,
hydrocortisone, prednisolone, cortisone, dexamethasone, prednisone,
alclometasone, clobetasol, clocortrolone, desonide, desoximetasone,
diflorasone, fluocinolone, fluocinonide, flurandrenolide,
fluticasone, floromethalone, halcinonide, halobetasol, loteprednol,
mometasone, prednicarbate, and triamcinolone can also be
incorporated into the pharmaceutical compositions of the
invention.
[0180] Ion channel blocking agents such as, for instance, the
sodium ion channel blocker, carbamazepine, as commonly used in the
treatment of tinnitus, arrhythmia, ischemic stroke and epilepsy can
be co-administered with or incorporated into the pharmaceutical
compositions of the invention. Alternatively, or in addition,
calcium ion channel blockers, such as ziconotide, can also be used,
as can antagonists of the ion channel associated with the NMDA
receptor, such as ketamine There is evidence that at least some of
these ion channel blockers can potentiate the analgesic effects of
the kappa agonist and thereby reduce the dose required for
affective pain relief. See for instance, Wang et al., 2000, Pain
84: 271-81.
[0181] Suitable NSAIDs, or other non-opioid compounds with
anti-inflammatory and/or analgesic activity, that can be
co-administered with or incorporated into the pharmaceutical
compositions of the invention include, but are not limited to one
or more of the following: aminoarylcarboxylic acid derivatives such
as etofenamate, meclofenamic acid, mefanamic acid, niflumic acid;
arylacetic acid derivatives such as acemetacin, amfenac,
cinmetacin, clopirac, diclofenac, fenclofenac, fenclorac, fenclozic
acid, fentiazac, glucametacin, isoxepac, lonazolac, metiazinic
acid, naproxin, oxametacine, proglumetacin, sulindac, tiaramide and
tolmetin; arylbutyric acid derivatives such as butibufen and
fenbufen; arylcarboxylic acids such as clidanac, ketorolac and
tinoridine. arylpropionic acid derivatives such as bucloxic acid,
carprofen, fenoprofen, flunoxaprofen, ibuprofen, ibuproxam,
oxaprozin, phenylalkanoic acid derivatives such as flurbiprofen,
piketoprofen, pirprofen, pranoprofen, protizinic acid and
tiaprofenic acid; pyranocarboxylic acids such as etodolac;
pyrazoles such as mepirizole; pyrazolones such as clofezone,
feprazone, mofebutazone, oxyphinbutazone, phenylbutazone, phenyl
pyrazolidininones, suxibuzone and thiazolinobutazone; salicylic
acid derivatives such as aspirin, bromosaligenin, diflusinal,
fendosal, glycol salicylate, mesalamine, 1-naphthyl salicylate,
magnesium salicylate, olsalazine and salicylamide, salsalate, and
sulfasalazine; thiazinecarboxamides such as droxicam, isoxicam and
piroxicam others such as .epsilon.-acetamidocaproic acid,
acetaminophen, s-adenosylmethionine, 3-amino-4-hydroxybutyric acid,
amixetrine, bendazac, bucolome, carbazones, cromolyn,
difenpiramide, ditazol, hydroxychloroquine, indomethacin,
ketoprofen and its active metabolite 6-methoxy-2-naphthylacetic
acid; guaiazulene, heterocylic aminoalkyl esters of mycophenolic
acid and derivatives, nabumetone, nimesulide, orgotein, oxaceprol,
oxazole derivatives, paranyline, pifoxime, 2-substituted-4,
6-di-tertiary-butyl-s-hydroxy-1,3-pyrimidines, proquazone and
tenidap, and cox-2 (cyclooxygenase II) inhibitors, such as
celecoxib or rofecoxib.
[0182] Suitable diuretics that can be co-administered with or
incorporated into the pharmaceutical preparations of the invention,
include, for example, inhibitors of carbonic anhydrase, such as
acetazolamide, dichlorphenamide, and methazolamide; osmotic
diuretics, such as glycerin, isosorbide, mannitol, and urea;
inhibitors of Na.sup.+--K.sup.+-2Cl.sup.- symport (loop diuretics
or high-ceiling diuretics), such as furosemide, bumetanide,
ethacrynic acid, torsemide, axosemide, piretanide, and tripamide;
inhibitors of Na.sup.+--Cl.sup.- symport (thiazide and thiazidelike
diuretics), such as bendroflumethiazide, chlorothiazide,
hydrochlorothiazide, hydroflumethazide, methyclothiazide,
polythiazide, trichlormethiazide, chlorthalidone, indapamide,
metolazone, and quinethazone; and, in addition, inhibitors of renal
epithelial Na.sup.+ channels, such as amiloride and triamterene,
antagonists of mineralocorticoid receptors (aldosterone
antagonists), such as spironolactone, canrenone, potassium
canrenoate, and eplerenone, which, together, are also classified as
K.sup.+-sparing diuretics. One embodiment is co-formulation and/or
co-administration of a loop or thiazide diuretic together with a
synthetic peptide amide of the invention for the purpose of a loop
or thiazide diuretic dose-sparing effect, wherein the dose of the
loop or thiazide diuretic is reduced to minimize undesired water
retention, and prevent or reduce hyponatremia, particularly in the
context of congestive heart failure, as well as other medical
conditions where decreasing body fluid retention and normalizing
sodium balance could be beneficial to a patient in need thereof.
See R M Reynolds et al. Disorders of sodium balance Brit. Med. J.
2006; 332:702-705.
[0183] The kappa opioid receptor-associated hyponatremia can be any
disease or condition where hyponatremia (low sodium condition) is
present, e.g., in humans, when the sodium concentration in the
plasma falls below 135 mmol/L, an abnormality that can occur in
isolation or, more frequently, as a complication of other medical
conditions, or as a consequence of using medications that can cause
sodium depletion.
[0184] A further embodiment is co-formulation and/or
co-administration of a potassium-sparing diuretic, e.g., a
mineralocorticoid receptor antagonist, such as spironolactone or
eplerenone, together with a synthetic peptide amide of the
invention, for the purpose of enabling a reduced dose of said
potassium-sparing diuretic, wherein the dose of said diuretic is
reduced to minimize hyperkalemia or metabolic acidosis, e.g., in
patients with hepatic cirrhosis.
[0185] In particular embodiments, the synthetic peptide amides of
the invention exhibit a long lasting duration of action when
administered in therapeutically relevant doses in vivo. For
instance, in some embodiments, the synthetic peptide amides of the
invention when administered to a mammal at a dose of 3 mg/kg of the
synthetic peptide amide maintain at least about 50% of maximum
efficacy in a kappa opioid receptor-dependent assay at 3 hours post
administration. In certain other embodiments, the synthetic peptide
amides of the invention when administered to a mammal at a dose of
0.1 mg/kg of the synthetic peptide amide maintain at least about
50% of maximum efficacy in a kappa opioid receptor-dependent assay
at 3 hours post administration. The maximum efficacy is
operationally defined as the highest level of efficacy determined
for the particular kappa opioid receptor-dependent assay for all
agonists tested.
[0186] In certain embodiments, the synthetic peptide amides of the
invention when administered to a mammal at a dose of 0.1 mg/kg
maintain at least about 75% of maximum efficacy at 3 hours post
administration. In still other embodiments, the synthetic peptide
amides of the invention when administered to a mammal at a dose of
0.1 mg/kg maintain at least about 90% of maximum efficacy at 3
hours post administration. In certain other embodiments, the
synthetic peptide amides of the invention when administered to a
mammal at a dose of 0.1 mg/kg maintain at least about 95% of
maximum efficacy at three hours post administration.
[0187] The invention further provides a method of treating or
preventing a kappa opioid receptor-associated disease or condition
in a mammal, wherein the method includes administering to the
mammal a composition containing an effective amount of a synthetic
peptide amide of the invention. The mammal can be any mammal, such
as a domesticated or feral mammal, or even a wild mammal.
Alternatively, the mammal can be a primate, an ungulate, a canine
or a feline. For instance, and without limitation, the mammal may
be a pet or companion animal, such as a high-value mammal such as a
thoroughbred or show animal; a farm animal, such as a cow, a goat,
a sheep or pig; or a primate such as an ape or monkey. In one
particular aspect, the mammal is a human.
[0188] The effective amount can be determined according to routine
methods by one of ordinary skill in the art. For instance, an
effective amount can be determined as a dosage unit sufficient to
prevent or to treat a kappa receptor-associated disease or
condition in the mammal. Alternatively, the effective amount may be
determined as an amount sufficient to approximate the EC.sub.50
concentration or an amount sufficient to approximate two or three
times or up to about five or even about ten times the EC.sub.50
concentration in a therapeutically relevant body fluid of the
mammal, for instance, where the body fluid is in direct apposition
to a target tissue, such as the synovial fluid of an inflamed joint
in a patient suffering from rheumatoid arthritis.
[0189] In one embodiment the synthetic peptide amide of the
invention is a pharmaceutical composition that includes an
effective amount of the synthetic peptide amide of the invention
and a pharmaceutically acceptable excipient or carrier. In one
aspect, the pharmaceutical composition includes a synthetic peptide
amide of the invention in an amount effective to treat or prevent a
kappa opioid receptor-associated condition in a mammal, such as a
human. In another aspect the kappa opioid receptor-associated
condition is pain, inflammation, pruritis, edema, ileus, tussis or
glaucoma.
[0190] In one embodiment the pharmaceutical composition of the
invention further includes one or more of the following compounds:
an opioid, a cannabinoid, an antidepressant, an anticonvulsant, a
neuroleptic, a corticosteroid, an ion channel blocking agent or a
non-steroidal anti-inflammatory drug (NSAID).
[0191] Pharmaceutical compositions of a synthetic peptide amide of
the invention and a pharmaceutically acceptable vehicle or carrier
can be used to treat or prevent one or more of a variety of kappa
opioid receptor-associated diseases, disorders or conditions.
[0192] The kappa opioid receptor-associated disease, disorders or
condition preventable or treatable with the synthetic peptide
amides of the invention can be any kappa opioid receptor-associated
disease, disorder or condition, including but not limited to acute
or chronic pain, inflammation, pruritis, hyponatremia, edema,
ileus, tussis and glaucoma. For instance, the kappa opioid
receptor-associated pain can be neuropathic pain, somatic pain,
visceral pain or cutaneous pain. Some diseases, disorders, or
conditions are associated with more than one form of pain, e.g.,
post-operative pain (also known as post-surgical pain) can have any
or all of neuropathic, somatic, visceral, and cutaneous pain
components, depending upon the type and extent of surgical
procedure employed.
[0193] The kappa opioid receptor-associated inflammation can be any
inflammatory disease or condition including, but not limited to
sinusitis, rheumatoid arthritis tenosynovitis, bursitis,
tendonitis, lateral epicondylitis, adhesive capsulitis,
osteomyelitis, osteoarthritic inflammation, inflammatory bowel
disease (IBD), irritable bowel syndrome (IBS), ocular inflammation,
otitic inflammation or autoimmune inflammation.
[0194] In another embodiment, the kappa opioid receptor-associated
inflammatory diseases and conditions preventable or treatable by
the methods of the present invention is an inflammatory disease or
condition characterized by elevated levels of one or more
proinflammatory cytokines, including, but not limited to tumor
necrosis factor-.alpha. (TNF-.alpha.), interleukin 1.beta.
(IL-1.beta.), interleukin 6 (IL-6) and matrix-metalloproteases
(MMPS), such as MMP-1 and MMP-3.
[0195] The invention further provides a method of prophylaxis or
treatment of a kappa opioid receptor-associated disease or
condition in a mammal, wherein a synthetic peptide amide of the
invention is co-administered with a reduced dose of a mu opioid
agonist analgesic compound to produce a therapeutic analgesic
effect, the mu opioid agonist analgesic compound having an
associated side effect, (especially respiratory depression,
sedation, euphoria, antidiuresis, nausea, vomiting, constipation,
and physical tolerance, dependence, and addiction). The reduced
dose of the mu opioid agonist analgesic compound (e.g. morphine or
fentanyl or any other opioid with substantial agonist activity at
the mu opioid receptor) administered by this method has lower
associated side effects than the side effects associated with the
dose of the compound necessary to achieve the same therapeutic
analgesic effect when administered alone.
[0196] The invention also provides a method of treating or
preventing peripheral hyperalgesia, wherein the method includes
topically applying or locally administering to a mammal in need of
the treatment, an effective amount of a composition that includes
an anti-hyperalgesically-effective amount of a synthetic peptide
amide of the invention in a vehicle formulated for topical
application or local administration.
[0197] The present invention also provides a method of treating,
preventing or inhibiting a kappa opioid receptor-associated
inflammatory disease or condition. The inflammatory disease or
condition can be any inflammatory disease or condition such as for
instance, cardiovascular inflammation, neurological inflammation,
skeletal inflammation, muscular inflammation, gastrointestinal
inflammation, ocular inflammation, otic inflammation, inflammation
due to insect bites or inflammation due to wound healing.
[0198] The cardiovascular inflammatory disease or condition
treatable or preventable by the methods of the present invention
can be any cardiovascular inflammatory disease or condition, such
as cardiovascular inflammation due to a variety of causes, such as
for instance, atherosclerosis, ischemia, restenosis or
vasculitis.
[0199] Inflammatory diseases and conditions treatable or
preventable by the methods of the present invention also include
for example and without limitation, asthma, Sjogren's syndrome,
pulmonary inflammation, bronchitis, chronic airway inflammation and
chronic obstructive pulmonary disease (COPD).
[0200] Other inflammatory diseases and conditions treatable or
preventable by the methods of the present invention also include
immune diseases and conditions such as, without limitation,
allergy, psoriasis, psoriatic arthritis, eczema, scleroderma,
atopic dermatitis and systemic lupus erythematosus.
[0201] Still other inflammatory diseases and conditions treatable
or preventable by the methods of the present invention include
arthritis, synovitis, osteomyelitis, rheumatoid arthritis,
osteoarthritis and ankylosing spondylitis. Furthermore,
inflammatory diseases and conditions treatable or preventable by
the methods of the invention also include septicemia and septic
shock.
[0202] Inflammatory diseases and conditions treatable or
preventable by the methods of the invention also include diabetes,
glucose intolerance, insulin resistance and obesity.
[0203] Other inflammatory diseases and conditions treatable or
preventable by the methods of the present invention include
colitis, ulcerative colitis, Crohn's disease, IBD (inflammatory
bowel disease) and IBS (Irritable bowel syndrome).
[0204] The present invention also provides methods of treatment or
prevention of inflammatory diseases and conditions wherein the
inflammatory disease or condition is due to tumor proliferation,
tumor metastasis or transplantation rejection.
[0205] The kappa opioid receptor-associated pruritis can be any
pruritic disease or condition such as, for instance, ocular
pruritis, e.g., associated with conjunctivitis, otitic pruritis,
pruritis associated with end-stage renal disease, where many
patients are receiving kidney dialysis, and other forms of
cholestasis, including primary biliary cirrhosis, intrahepatic
cholestasis of pregnancy, chronic cholestatic liver disease,
uremia, malignant cholestasis, jaundice, as well as dermatological
conditions such as eczema (dermatitis), including atopic or contact
dermatitis, psoriasis, polycythemia vera, lichen planus, lichen
simplex chronicus, pediculosis (lice), thyrotoxicosis, tinea pedis,
urticaria, scabies, vaginitis, anal pruritis associated with
hemorrhoids and, as well as insect bite pruritis and drug-induced
pruritis, such as mu opioid-induced pruritis.
[0206] The kappa opioid receptor-associated edema can be any
edematous disease or condition such as, for instance, edema due to
congestive heart disease or to a syndrome of inappropriate
antidiuretic hormone (ADH) secretion.
[0207] Kappa opioid receptor-associated ileus can be any ileus
disease or condition including, but not limited to, post-operative
ileus and opioid-induced bowel dysfunction.
[0208] Kappa opioid receptor-associated neuropathic pain can be any
neuropathic pain, such as, for instance, trigeminal neuralgia,
diabetic pain, viral pain such as herpes zoster-associated pain,
chemotherapy-induced pain, nerve-encroaching metastatic cancer
pain, neuropathic pain associated with traumatic injury and
surgical procedures, as well as variants of headache pain that are
thought to have a neuropathic component, e.g., migraine.
[0209] Kappa opioid-associated pain also includes ocular pain, such
as that following photo-refractive keratectomy (PRK), ocular
laceration, orbital floor fracture, chemical burns, corneal
abrasion or irritation, or pain associated with conjunctivitis,
corneal ulcers, scleritis, episcleritis, sclerokeratitis, herpes
zoster ophthalmicus, interstitisal keratitis, acute iritis,
keratoconjunctivitis sicca, orbital cellulites, orbital
pseudotumor, pemphigus, trachoma or uveitis.
[0210] Kappa opioid-associated pain also includes throat pain,
particularly associated with inflammatory conditions, such as
allergic rhinitis, acute bronchitis, the common cold, contact
ulcers, herpes simplex viral lesions, infectious mononucleosis,
influenza, laryngeal cancer, acute laryngitis, acute necrotizing
ulcerative gingivitis, peritonsillar abscess, pharyngeal burns,
pharyngitis, reflus laryngopharyngitis, acute sinusitis, and
tonsillitis.
[0211] In addition, kappa opioid receptor-associated pain can be
arthritic pain, kidney-stone, urinary tract stone, gallstone, and
bile duct stone pain, dysmenorrhea, uterine cramping,
endometriosis, mastitis, dyspepsia, post-surgical pain (such as,
for instance, from appendectomy, open colorectal surgery, hernia
repair, prostatectomy, colonic resection, gastrectomy, splenectomy,
colectomy, colostomy, pelvic laparoscopy, tubal ligation,
hysterectomy, vasectomy or cholecystecomy), post medical procedure
pain (such as, for instance, after colonoscopy, cystoscopy,
hysteroscopy or cervical or endometrial biopsy), otitic pain,
breakthrough cancer pain, and pain associated with a GI disorder
such as IBD or IBS or other inflammatory conditions, particularly
of the viscera (e.g., gastro-esophageal reflux disease,
pancreatitis, acute polynephritis, ulcerative colitis, acute
pyelo-nephritis, cholecystitis, cirrhosis, hepatic abscess,
hepatitis, duodenal or gastric ulcer, esophagitis, gastritis,
gastroenteritis, colitis, diverticulitis, intestinal obstruction,
ovarian cyst, pelvic inflammatory disease, perforated ulcer,
peritonitis, prostatitis, interstitial cystitis), or exposure to
toxic agents, such as insect toxins, or inflammation due to the
effects of drugs such as salicylates or NSAIDs.
[0212] The present invention provides a method of treating or
preventing a kappa opioid receptor-associated disease or condition
in a mammal, such as a human, wherein the method includes
administering to the mammal a composition comprising an effective
amount of a synthetic peptide amide of the invention. In another
embodiment the kappa opioid receptor-associated condition is pain,
inflammation (such as rheumatoid arthritic inflammation,
osteoarthritic inflammation, IBD inflammation, IBS inflammation,
ocular inflammation, otitic inflammation or autoimmune
inflammation), pruritis (such as atopic dermatitis,
kidney-dialysis-associated pruritis, ocular pruritis, otitic
pruritis, insect bite pruritis, or opioid-induced pruritis), edema,
ileus, tussis or glaucoma. In one aspect, the pain is a neuropathic
pain (such as trigeminal neuralgia, migraine, diabetic pain, viral
pain, chemotherapy-induced pain or metastatic cancer pain), a
somatic pain, a visceral pain or a cutaneous pain. In another
aspect the pain is arthritic pain, kidney-stone pain, uterine
cramping, dysmenorrhea, endometriosis, dyspepsia, post-surgical
pain, post medical procedure pain, ocular pain, otitic pain,
breakthrough cancer pain or pain associated with a GI disorder,
such as IBD or IBS. In another aspect the pain is pain associated
with surgery, wherein the surgery is pelvic laparoscopy, tubal
ligation, hysterectomy and cholecystecomy. Alternatively, the pain
can be pain associated with a medical procedure, such as for
instance, colonoscopy, cystoscopy, hysteroscopy or endometrial
biopsy. In a specific aspect, the atopic dermatitis can be
psoriasis, eczema or contact dermatitis. In another specific
aspect, the ileus is post-operative ileus or opioid-induced bowel
dysfunction.
[0213] Kappa opioid receptor-associated pain includes hyperalgesia,
which is believed to be caused by changes in the milieu of the
peripheral sensory terminal occur secondary to local tissue damage.
Tissue damage (e.g., abrasions, burns) and inflammation can produce
significant increases in the excitability of polymodal nociceptors
(C fibers) and high threshold mechanoreceptors (Handwerker et al.
(1991) Proceeding of the VIth World Congress on Pain, Bond et al.,
eds., Elsevier Science Publishers BV, pp. 59-70; Schaible et al.
(1993) Pain 55:5-54). This increased excitability and exaggerated
responses of sensory afferents is believed to underlie
hyperalgesia, where the pain response is the result of an
exaggerated response to a stimulus. The importance of the
hyperalgesic state in the post-injury pain state has been
repeatedly demonstrated and appears to account for a major
proportion of the post-injury/inflammatory pain state. See for
example, Woold et al. (1993) Anesthesia and Analgesia 77:362-79;
Dubner et al. (1994) In, Textbook of Pain, Melzack et al., eds.,
Churchill-Livingstone, London, pp. 225-242.
[0214] In another embodiment the kappa opioid receptor-associated
condition is pain, inflammation (such as rheumatoid arthritic
inflammation, osteoarthritic inflammation, IBD inflammation, IBS
inflammation, ocular inflammation, otitic inflammation or
autoimmune inflammation), pruritis (such as atopic dermatitis,
kidney-dialysis-associated pruritis, ocular pruritis, otitic
pruritis, insect bite pruritis, or opioid-induced pruritis), edema,
ileus, tussis or glaucoma. In one aspect, the pain is a neuropathic
pain (such as trigeminal neuralgia, migraine, diabetic pain, viral
pain, chemotherapy-induced pain or metastatic cancer pain), a
somatic pain, a visceral pain or a cutaneous pain. In another
aspect the pain is arthritic pain, kidney-stone pain, uterine
cramping, dysmenorrhea, endometriosis, dyspepsia, post-surgical
pain, post medical procedure pain, ocular pain, otitic pain,
breakthrough cancer pain or pain associated with a GI disorder,
such as IBD or IBS. In another aspect the pain is pain associated
with surgery, wherein the surgery is pelvic laparoscopy, tubal
ligation, hysterectomy and cholecystecomy. Alternatively, the pain
can be pain associated with a medical procedure, such as for
instance, colonoscopy, cystoscopy, hysteroscopy or endometrial
biopsy. In a specific aspect, the atopic dermatitis can be
psoriasis, eczema or contact dermatitis. In another specific
aspect, the ileus is post-operative ileus or opioid-induced bowel
dysfunction.
[0215] In another embodiment the kappa opioid receptor-associated
condition is a kappa opioid receptor-associated condition
preventable or treatable by sodium and potassium-sparing diuresis,
also known as aquaresis. An example of such kappa opioid
receptor-associated conditions preventable or treatable by
administering a synthetic peptide amide of the invention includes
edema. The edema may be due to any of a variety of diseases or
conditions, such as congestive heart disease or syndrome of
inappropriate ADH secretion.
[0216] In another embodiment the kappa opioid receptor-associated
condition is hyponatremia or other edematous disease. The kappa
opioid receptor-associated hyponatremia or edema can be any
hyponatremic or edematous disease or condition such as, for
instance, hyponatremia and edema associated with congestive heart
failure or to a syndrome of inappropriate antidiuretic hormone
(ADH) secretion, or hyponatremia that is associated with intensive
diuretic therapy with thiazides and/or loop diuretics. The
synthetic peptide amides of the invention exhibit a significant
sodium-sparing and potassium-sparing aquaretic effect, which is
beneficial in the treatment of edema-forming pathological
conditions associated with hyponatremia and/or hypokalemia.
Accordingly, the synthetic peptide amides of the invention also
have utility in methods of treating or preventing
hyponatremia-related conditions, examples of which are provided
below. Hyponatremia-related conditions can be categorized according
to volume status as hypervolemic, euvolemic, or hypovolemic.
[0217] Hypervolemic hyponatremia is usually caused by an increase
in total body water level as may be observed in cases of congestive
heart failure, nephrotic syndrome and hepatic cirrhosis.
[0218] Euvolemic hyponatremia is often found in the syndrome of
inappropriate antidiuretic hormone (ADH) secretion and may also be
associated with pneumonia, small-cell lung cancer, polydipsia,
cases of head injury, and organic causes (e.g., use of certain
drugs, such as haloperidol) or a psychogenic cause.
[0219] Hypovolemic hyponatremia is due to a relative decrease in
total body sodium level and may be associated with, for instance
and without limitation, diuretic use, cases of interstitial
nephritis or excessive sweating.
[0220] These forms of hyponatremia can be further classified
according to the concentration of sodium in the urine (i.e.,
whether the concentration is greater than or less than thirty
millimoles per liter. See: R M Reynolds et al. Disorders of sodium
balance, Brit. Med. J. 2006; 332:702-705.
[0221] The kappa opioid receptor-associated hyponatremia can be any
disease or condition where hyponatremia (low sodium condition) is
present, e.g., in humans, when the sodium concentration in the
plasma falls below 135 mmol/L, an abnormality that can occur in
isolation or, more frequently, as a complication of other medical
conditions, or as a consequence of using medications that can cause
sodium depletion.
[0222] In addition to these conditions, numerous other conditions
are associated with hyponatremia including, without limitation:
neoplastic causes of excess ADH secretion, including carcinomas of
lung, duodenum, pancreas, ovary, bladder, and ureter, thymoma,
mesothelioma, bronchial adenoma, carcinoid, gangliocytoma and
Ewing's sarcoma; infections such as: pneumonia (bacterial or
viral), abscesses (lung or brain), cavitation (aspergillosis),
tuberculosis (lung or brain), meningitis (bacterial or viral),
encephalitis and AIDS; vascular causes such as: cerebrovascular
occlusions or hemorrhage and cavernous sinus thrombosis; neurologic
causes such as: Guillan-Barre syndrome, multiple sclerosis,
delirium tremens, amyotrophic lateral sclerosis, hydrocephalus,
psychosis, peripheral neuropathy, head trauma (closed and
penetrating), CNS tumors or infections and CNS insults affecting
hypothalamic osmoreceptors; congenital malformations including:
agenesis of corpus callosum, cleftlip/palate and other midline
defects; metabolic causes such as: acute intermittent porphyria,
asthma, pneurothorax and positive-pressure respiration; drugs such
as thiazide diuretics, acetaminophen, barbiturates, cholinergic
agents, estrogen, oral hypoglycemic agents, vasopressin or
desmopressin, high-dose oxytocin, chlorpropamide, vincristine,
carbamezepine, nicotine, phenothiazines, cyclophosphamide,
tricyclic antidepressants, monoamine oxidase inhibitors and
serotonin reuptake inhibitors; administration of excess hypotonic
fluids, e.g., during hospitalization, surgery, or during or after
athletic events (i.e., exercise-associated hyponatremia), as well
as use of low-sodium nutritional supplements in elderly
individuals. See for example, Harrison's Principles of Internal
Medicine, 16th Ed. (2005), p. 2102.
[0223] Other conditions associated with hyponatremia include renal
failure, nephrotic syndrome (membranous nephropathy and minimal
change disease), cachexia, malnutrition, rhabdomyolysis, surgical
procedures, elective cardiac catheterization, blood loss, as well
as hypercalcemia, hypokalemia, and hyperglycemia with consequent
glycosuria leading to osmotic diuresis.
[0224] The invention also provides a method of treating or
preventing a neurodegenerative disease or condition in a mammal,
such as a human, wherein the method includes administering to the
mammal a composition that includes an effective amount of a
synthetic peptide amide as described above. The neurodegenerative
disease or condition can be any neurodegenerative disease or
condition, such as for instance, ischemia, anoxia, stroke, brain
injury, spinal cord injury or reperfusion injury. Alternatively,
the neurodegenerative disease or condition can be a
neurodegenerative disease of the eye. Particular neurodegenerative
diseases of the eye treatable or preventable by the method of the
invention include glaucoma, macular degeneration, retinal ischemic
disease and diabetic neuropathy.
[0225] In certain embodiments the invention provides methods of
prevention or treatment of certain neuronal diseases and
conditions, such as diseases and conditions having a
neurodegenerative component. Synthetic peptide amides of the
invention can be administered in an amount effective to protect
neuronal cells against the effects of pathology or injury that
would lead to neurodegeneration and/or neuronal cell death of the
untreated cells. For example, several diseases or conditions of the
eye that have a neurodegenerative component can be prevented or
treated by administration of an effective amount of the synthetic
peptide amides of the invention. Such diseases and conditions of
the eye include glaucoma, macular degeneration, retinal ischemic
disease and diabetic neuropathy. Progression of these diseases and
conditions is believed to involve neurodegeneration or neuronal
cell death, for example by programmed cell death (apoptosis) in
which the neuronal cells are committed to a pathway that without
intervention would lead to cell death. It has been found that
development or progression of these diseases and conditions can be
prevented, or at least slowed, by treatment with kappa opioid
receptor agonists. This improved outcome is believed to be due to
neuroprotection by the kappa opioid receptor agonists. See for
instance, Kaushik et al. "Neuroprotection in Glaucoma" (2003) J.
Postgraduate Medicine vol. 49 (1): pp. 90-95.
[0226] In the case of glaucoma it is believed that prophylaxis and
treatment by administration of kappa opioid receptor agonists is
mediated by at least two distinct activities induced by activation
of the kappa opioid receptor: neuroprotection and reduction of
intraocular pressure (IOP). While not wishing to be bound by
theory, it is believed that neuroprotection is due, at least in
part, to induction of atrial natriuretic peptide (ANP) in the eye,
leading to protection against oxidative damage and other
insults.
[0227] Abnormally high intraocular pressure is also believed to be
a factor leading to the development of glaucoma. Elevated
intraocular pressure can also be prevented or treated by
administration of kappa opioid receptor agonists by three separate
activities triggered by activation of the receptor: reduction in
secretion of aqueous humor, increased outflow of aqueous humor and
aquaresis (sodium- and potassium-sparing diuresis, resulting in
loss of water).
[0228] The invention also provides a method of treating or
preventing a kappa-receptor-associated disease or condition of the
eye of a mammal, such as high intraocular pressure (IOP). The
method includes administering to the mammal a composition that
includes an effective amount of a synthetic peptide amide as
described above. In one aspect of the invention, the synthetic
peptide amide is administered topically. In another aspect, the
synthetic peptide amide is administered as an implant.
[0229] In other embodiments the invention provides methods of
prevention or treatment of certain cardiovascular diseases and
conditions having a cellular degenerative component. Synthetic
peptide amides of the invention can be administered in an amount
effective to protect myocardial cells against the effects of
pathology or injury that would lead to degeneration and/or cell
death of the untreated cells. For example, several cardiovascular
diseases or conditions can be prevented or treated by
administration of an effective amount of the synthetic peptide
amides of the invention. Such cardiovascular diseases and
conditions include, without limitation, coronary heart disease,
ischemia, cardiac infarct, reperfusion injury and arrhythmia. See
for example, Wu et al. "Cardioprotection of Preconditioning by
Metabolic Inhibition in the Rat Ventricular Myocyte--Involvement of
kappa Opioid Receptor" (1999) Circulation Res vol. 84: pp.
1388-1395. See also Yu et al. "Anti-Arrythmic Effect of kappa
Opioid Receptor Stimulation in the Perfused Rat Heart: Involvement
of a cAMP-Dependent Pathway" (1999) J Mol Cell Cardiol. vol.
31(10): pp. 1809-1819.
[0230] Diseases and conditions of other tissues and organs that can
be prevented or treated by administration of an effective amount of
the synthetic peptide amides of the invention include, but are not
limited to ischemia, anoxia, stroke, brain or spinal cord injury
and reperfusion injury.
[0231] Another form of kappa opioid receptor-associated pain
treatable or preventable with the synthetic peptide amides of the
invention is hyperalgesia. In one embodiment, the method includes
administering an effective amount of a synthetic peptide amide of
the invention to a mammal suffering from or at risk of developing
hyperalgesia to prevent, ameliorate or completely alleviate the
hyperalgesia.
[0232] The synthetic peptide amides of the invention can be
administered by methods disclosed herein for the treatment or
prevention of any hyperalgesic condition, such as, but without
limitation, a hyperalgesic condition associated with allergic
dermatitis, contact dermatitis, skin ulcers, inflammation, rashes,
fungal irritation and hyperalgesic conditions associated with
infectious agents, burns, abrasions, bruises, contusions,
frostbite, rashes, acne, insect bites/stings, skin ulcers,
mucositis, gingivitis, bronchitis, laryngitis, sore throat,
shingles, fungal irritation, fever blisters, boils, Plantar's
warts, surgical procedures or vaginal lesions. For instance, the
synthetic peptide amides of the invention can be administered
topically to a mucosal surface, such as the mouth, esophagus or
larynx, or to the bronchial or nasal passages. Alternatively, the
synthetic peptide amides of the invention can be administered
topically to the vagina or rectum/anus.
[0233] Moreover, the synthetic peptide amides of the invention can
be administered by methods disclosed herein for the treatment or
prevention of any hyperalgesic condition associated with burns,
abrasions, bruises, abrasions (such as conical abrasions),
contusions, frostbite, rashes, acne, insect bites/stings, skin
ulcers (for instance, diabetic ulcers or a decubitus ulcers),
mucositis, inflammation, gingivitis, bronchitis, laryngitis, sore
throat, shingles, fungal irritation (such as athlete's foot or jock
itch), fever blisters, boils, Plantar's warts or vaginal lesions
(such as vaginal lesions associated with mycosis or sexually
transmitted diseases). Methods contemplated for administration of
the synthetic peptide amides of the invention for the treatment or
prevention of hyperalgesia include those wherein the compound is
topically applied to a surface in the eyes, mouth, larynx,
esophagus, bronchial, nasal passages, vagina or rectum/anus.
[0234] Hyperalgesic conditions associated with post-surgery
recovery can also be addressed by administration of the synthetic
peptide amides of the invention. The hyperalgesic conditions
associated with post-surgery recovery can be any hyperalgesic
conditions associated with post-surgery recovery, such as for
instance, radial keratectomy, tooth extraction, lumpectomy,
episiotomy, laparoscopy and arthroscopy.
[0235] Hyperalgesic conditions associated with inflammation are
also addressable by administration of the synthetic peptide amides
of the invention. Periodontal inflammation, orthodontic
inflammation, inflammatory conjunctivitis, hemorrhoids and venereal
inflammations can be treated or prevented by topical or local
administration of the synthetic peptide amides of the
invention.
[0236] The invention also provides a method of inducing diuresis in
a mammal in need thereof. The method includes administering to the
mammal a composition comprising an effective amount of a synthetic
peptide amide of the invention as described above.
[0237] The invention further provides a method of inducing
prolactin secretion in a mammal. The method includes administering
to the mammal a composition comprising an effective amount of a
synthetic peptide amide of the invention as described above. The
method of inducing prolactin secretion is suitable for treating a
mammal, such as a human suffering from insufficient lactation,
inadequate lactation, sub-optimal lactation, reduced sperm
motility, an age-related disorder, type I diabetes, insomnia or
inadequate REM sleep. In a particular aspect, the method includes
co-administering the synthetic peptide amide with a reduced dose of
a mu opioid agonist analgesic compound to produce a therapeutic
analgesic effect, the compound having an associated side effect,
wherein the reduced dose of the compound has a lower associated
side effect than the side effect associated with the dose of the mu
opioid agonist analgesic compound necessary to achieve the
therapeutic analgesic effect when administered alone.
[0238] The present invention also provides a method of binding a
kappa opioid receptor in a mammal, the method includes the step of
administering to the mammal a composition containing an effective
amount of a synthetic peptide amide of the present invention. The
effective amount can be determined according to routine methods by
one of ordinary skill in the art. For instance, the effective
amount can be determined as a dosage unit sufficient to bind kappa
opioid receptors in a mammal and cause an antinociceptive effect,
an anti-inflammatory effect, an aquaretic effect, or an elevation
of serum prolactin levels or any other kappa opioid
receptor-responsive effect. Alternatively, the effective amount may
be determined as an amount sufficient to approximate the EC.sub.50
in a body fluid of the mammal, or an amount sufficient to
approximate two or three, or up to about five or even about ten
times the EC.sub.50 in a therapeutically relevant body fluid of the
mammal.
EXAMPLES
General Experimental Synthetic Methods
[0239] Amino acid derivatives and resins were purchased from
commercial providers (Novabiochem, Bachem, Peptide International
and PepTech Corporation). Other chemicals and solvents were
purchased from Sigma-Aldrich, Fisher Scientific and VWR. The
compounds herein were synthesized by standard methods in solid
phase peptide chemistry utilizing both Fmoc and Boc methodology.
Unless otherwise specified, all reactions were performed at room
temperature.
[0240] The following standard references provide guidance on
general experimental setup, and the availability of required
starting material and reagents: Kates, S. A., Albericio, F., Eds.,
Solid Phase Synthesis, A Practical Guide, Marcel Dekker, New York,
Basel, (2000); Bodanszky, M., Bodanszky, A., Eds., The Practice of
Peptide Synthesis, Second Edition, Springer-Verlag, (1994);
Atherton, E., Sheppard, R. C., Eds., Solid Phase Peptide Synthesis,
A Practical Approach, IRL Press at Oxford University Press, (1989);
Stewart, J. M., Young, J. D., Solid Phase Synthesis, Pierce
Chemical Company, (1984); Bisello, et al., J. Biol. Chem. 273,
22498-22505 (1998); and Merrifield, R. B., J. Am. Chem. Soc. 85,
2149-2154 (1963).
[0241] Abbreviations used herein: ACN: acetonitrile; Aloc:
allyloxy-carbonyl; Boc: tert-butoxycarbonyl; BOP:
benzotriazole-1-yl-oxy-tris(dimethylamino)-phosphonium
hexafluorophosphate; Cbz: Benzyloxycarbonyl; Cbz-OSu:
N.alpha.-(Benzyloxy-carbonyloxy) succinimide; DBU:
1,8-diazabicyclo[5.4.0]undec-7-ene; DCM: Dichloro-methane; Dde:
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; DIC:
N,N'-diiso-propylcarbodiimide; DIEA: N,N-diisopropylethylamine;
DMF: N,N-dimethyl-formamide; Fmoc: 9-fluorenylmethoxycarbonyl;
HATU: 2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate; HBTU:
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate; HOBt: 1-hydroxybenzotriazole; HPLC: high
performance liquid chromatography; i: iso; ivDde:
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl; NMM:
4-methyl morpholino; NMP: N-methylpyrrolidinone; All: allyl;
o-NBS--Cl: o-nitrobenzenesulfonyl chloride; Pbf:
2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl; PyBOP:
benzotriazole-1-yloxy-tris-pyrrolidino-phosphonium
hexafluorophosphate; RP: reversed phase; TBTU:
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate; TEAP: triethylammonium phosphate; TFA:
trifluoroacetic acid; TIS: triisopropylsilane; TMOF: trimethyl
orthoformate; TMSOTf: trimethylsilyl trifluoromethanesulfonate;
Trt: trityl.
[0242] Peptides synthesized by Fmoc methodology were cleaved with a
mixture of TFA/TIS/H.sub.2O (v/v/v=95:2.5:2.5). The cleavage step
in the Boc methodology was accomplished either with a mixture of
HF/anisole (v/v=9:1) or with a mixture of TMSOTf/TFA/m-cresol
(v/v/v=2:7:1).
[0243] Coupling reactions in peptide chain elongation were carried
out either manually or on a peptide synthesizer and mediated by
coupling reagents with a 2 to 4-fold excess amino acid derivatives.
The coupling reagents used in the synthesis of the various
compounds of the invention were chosen from the following
combinations: DIC/HOBt, HATU/DIEA, HBTU/DIEA, TBTU/DIEA,
PyBOP/DIEA, and BOP/DIEA.
[0244] Deprotection of the side chain of amino acid in position No.
4 (designated Xaa.sub.4 in the final synthetic peptide amide
product) of resin bound peptides was achieved as follows: Peptides
were assembled starting from Xaa.sub.4 and progressively adding
Xaa.sub.3, then Xaa.sub.2 and finally, Xaa.sub.1. The side chain
protecting groups of the diamino acid introduced at Xaa.sub.4 were
selectively removed as follows: (i) N-Dde or N-ivDde groups were
removed by 2-4% hydrazine in DMF. See Chabra, S. R., et al.,
Tetrahedron Lett. 39:1603-1606 (1998) and Rohwedder, B., et al.,
Tetrahedron Lett., 39: 1175 (1998); (ii) N-Aloc: removed by 3 eq.
(Ph.sub.3P).sub.4Pd in CHCl.sub.3/AcOH/NMM (v/v/v=37:2:1). See
Kates, S. A., et al. in "Peptides Chemistry, Structure and Biology,
Proc. 13.sup.th American Peptide Symposium", Hodges, R. S. and
Smith, J. A. (Eds), ESCOM, Leiden, 113-115 (1994).
[0245] When peptides were assembled with Boc protection
methodology, the side chain protecting group of the diamino acids
introduced at Xaa.sub.4 was N-Fmoc, which was removed by 20-30%
piperidine in DMF.
[0246] Isopropylation of the terminal nitrogen on the side chain of
amino acid at Xaa.sub.4 of resin bound peptides was achieved as
follows: After deprotection, the resin bound peptide with the free
.omega.-amino function at Xaa.sub.4 was reacted with a mixture of
acetone and NaBH(OAc).sub.3 in TMOF producing the resin bound
N-.omega.-isopropyl peptide.
[0247] Monomethylation of the terminal nitrogen on the side chain
of amino acid at Xaa.sub.4 of resin bound peptides: To synthesize
resin bound N-w-methyl peptides, the free .omega.-amino function
was first derivatized with o-nitrobenzene-sulfonyl chloride
(o-NBS--Cl; Biron, E.; Chatterjee, J.; Kessler, H. Optimized
selective N-methylation of peptides on solid support. J. Pep. Sci.
12:213-219 (2006). The resulting sulfonamide was then methylated
with a mixture of dimethylsulphate and
1,8-diaza-bicyclo[5.4.0]undec-7-ene in NMP. The o-NBS protecting
group was subsequently removed by a mixture of mercaptoethanol and
1,8-diazabicyclo[5.4.0]undec-7-ene in NMP.
[0248] Guanylation of the terminal nitrogen on the side chain of
amino acid at Xaa.sub.4 of resin bound peptides: After
deprotection, the resin bound peptide with the free .omega.-amino
function in position No. 4 was reacted with a mixture of
1H-pyrazole-1-carboxamidine hydrochloride (Bernatowicz, M. S., et
al., J. Org. Chem. 57, 2497-2502 (1992) and DIEA in DMF producing
the resin bound N-w-guanidino peptide.
[0249] Peptides were purified by preparative HPLC in
triethylammonium phosphate (TEAP) or trifluoroacetic acid (TFA)
buffers. When required, the compounds were finally converted to
trifluoroacetate or acetate salts using conventional HPLC
methodology. Fractions with purity exceeding 97% were pooled and
lyophilized. Purity of the synthesized peptides was determined by
analytical RP-HPLC.
[0250] Analytical RP-HPLC was performed on a Waters 600
multisolvent delivery system with a Waters 486 tunable absorbance
UV detector and a Waters 746 data module. HPLC analyses of peptides
were carried out using a Vydac C.sub.18 column (0.46.times.25 cm, 5
.mu.m particle size, 300 .ANG. pore size) at a flow rate of 2.0
ml/min Solvents A and B were 0.1% TFA in H.sub.2O and 0.1% TFA in
80% ACN/20% H.sub.2O, respectively. Retention times (t.sub.R) are
given in minutes. Preparative RP-HPLC was accomplished using a
Vydac C.sub.18 preparative cartridge (4.7.times.30 cm, 15-20 .mu.m
particle size, 300 .ANG. pore size) at a flow rate of 100 ml/min,
on a Waters Prep LC 2000 preparative chromatograph system with a
Waters 486 tunable absorbance UV detector and a Servogor 120 strip
chart recorder. Buffers A and B were 0.1% TFA in H.sub.2O and 0.1%
TFA in 60% ACN/40% H.sub.2O, respectively. HPLC analysis of the
final compound was performed on a Hewlett Packard 1090 Liquid
Chromatograph using a Phenomenex Synergi MAX-RP C.sub.12 column
(2.0.times.150 mm, 4 .mu.m particle size, 80 .ANG. pore size) at a
flow rate of 0.3 ml/min at 40.degree. C. Buffers A and B were 0.01%
TFA in H.sub.2O and 0.01% TFA in 70% ACN/30% H.sub.2O,
respectively. The identity of the synthetic peptide amides was
confirmed by electrospray mass spectrometry. Mass spectra were
recorded on a Finnigan LCQ mass spectrometer with an ESI
source.
Synthetic Peptide Amides (1)-(103):
##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039##
##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044##
##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049##
##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054##
##STR00055## ##STR00056## ##STR00057##
[0251] Example 1
Synthesis of compound (1)
D-Phe-D-Phe-D-Leu-(.epsilon.-Me)D-Lys-[4-amidinohomopiperazine
amide]
[0252] The amino acid derivatives used were Cbz-D-Phe-OH,
Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, and Fmoc-D-Lys(Dde)-OH. The fully
protected resin bound peptide was synthesized manually starting
from p-nitro-phenylcarbonate Wang resin (5.0 g, 4.4 mmol;
Novabiochem). The attachment of homopiperazine to the resin was
achieved by mixing it with a solution of homopiperazine (8.7 g, 87
mmol; Acros Organics) in DCM (100 mL) overnight at room
temperature. The resin was washed with DMF and DCM and dried in
vacuo. The resulting homopiperazine carbamate Wang resin (5.1 g;
homopiperazine-[carbamate Wang resin]) was split into several
portions and a portion of 1.5 g (1.3 mmol) was used to continue the
peptide synthesis. DIC/HOBt mediated single couplings were
performed with a 3-fold excess of amino acid derivatives. The Fmoc
group was removed with 25% piperidine in DMF. Upon completion of
peptide chain elongation, the resin was treated with 4% hydrazine
in DMF for 3.times.3 min for Dde removal. The resin was washed with
DMF and DCM and dried in vacuo. The resulting peptide resin (2.4 g;
Cbz-D-Phe-D-Phe-DLeu-DLys-homopiperazine-[carbamate Wang resin])
was split again and a portion of 0.6 g (0.3 mmol) was used for
subsequent derivatization (N-methylation).
[0253] Methylation of the .omega.-amino function of D-Lys at
Xaa.sub.4 was carried out in three steps: (i) [o-NBS Protection]:
The resin-bound peptide (0.3 mmol) was first treated with a
solution o-NBS--Cl (0.4 g, 2 mmol) and collidine (0.7 ml, 5 mmol)
in NMP (7 ml) at room temperature for 30 minutes. The resin was
then washed with NMP. (ii) [N-Methylation]: The resin-bound o-NBS
protected peptide was then reacted with a solution of
1,8-diazabicyclo[5.4.0]undec-7-ene (0.5 ml, 3 mmol) and
dimethylsulfate (1.0 ml, 10 mmol; Aldrich) in NMP (7 ml) at room
temperature for 5 minutes. The resin was then washed with NMP and
the washing process was repeated once. (iii) [o-NBS Deprotection]:
The peptide resin was treated with a solution of mercaptoethanol
(0.7 ml, 10 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.8 ml, 5
mmol) in NMP (7 ml) at room temperature for 5 minutes. The resin
was then washed with NMP and the washing process was repeated
once.
[0254] To protect the resulting N-methyl secondary amine of D-Lys
at Xaa.sub.4, the resin-bound methylated peptide was reacted with a
solution of Cbz-OSu (6 mmol) in DMF (7 ml). The resin was washed
with DMF and DCM and dried in vacuo. The peptide was then cleaved
from the resin by treatment with a solution of TFA/DCM (15 ml,
v/v=1:1) at room temperature for 2 hours. The resin was then
filtered and washed with TFA. The filtrate was evaporated in vacuo
and the crude peptide (0.3 mmol;
Cbz-D-Phe-D-Phe-D-Leu-D-Lys(Me,Cbz)-[homopiperazine amide]) was
precipitated from diethyl ether.
[0255] For guanylation of the homopiperazine at the C-terminus, the
above peptide (0.3 mmol) was treated with a solution of
1H-Pyrazole-1-carboxamidine hydrochloride (0.4 g, 3.0 mmol) and
DIEA (0.5 ml, 6 mmol) in DMF (3 ml) overnight at room temperature.
Acetic acid and H.sub.2O were added to quench the reaction and the
solution was frozen and dried on a lyophilizer to give the desired
protected peptide,
Cbz-D-Phe-D-Phe-D-Leu-D-Lys(Me,Z)-[4-Amidinohomopiperazine amide]
(0.6 g).
[0256] For final deprotection/hydrolysis, the above peptide (0.6 g)
was treated with a mixture of TMSOTf/TFA/m-cresol (10 ml,
v/v/v=2:7:1) at room temperature for two hours. The mixture was
evaporated and the crude synthetic peptide amide (0.6 g) was
precipitated from diethyl ether.
[0257] For purification, the above-derived crude synthetic peptide
amide (0.6 g) was dissolved in 0.1% TFA in H.sub.2O (50 ml) and the
solution was loaded onto an HPLC column and purified using TFA
buffer system (buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60%
ACN/40% H.sub.2O). The compound was eluted with a linear gradient
of buffer B, 25% B to 75% B over 30 min, t.sub.R=37% B. The
fractions with purity exceeding 97% were pooled, frozen, and dried
on a lyophilizer to give the purified synthetic peptide amide as
white amorphous powder (153 mg). HPLC analysis: t.sub.R=14.41 min,
purity 99.8%, gradient 5% B to 25% B over 20 min; MS (MH.sup.+):
expected molecular ion mass 692.5, observed 692.5.
Example 2
Synthesis of compound (2):
D-Phe-D-Phe-D-Leu-D-Lys-[.omega.(4-aminopiperidine-4-carboxylic
acid)]-OH
[0258] See Biron et al., Optimized selective N-methylation of
peptides on solid support. J. Peptide Science 12: 213-219 (2006).
The amino acid derivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH,
Fmoc-D-Leu-OH, Fmoc-D-Lys(Dde)-OH, and
N-Boc-amino-(4-N-Fmoc-piperidinyl) carboxylic acid. HPLC and MS
analyses were performed as described in the synthesis of compound
(1) described above.
[0259] The fully protected resin-bound peptide was synthesized
manually starting from 2-Chlorotrityl chloride resin (1.8 g, 0.9
mmol; Peptide International). Attachment of
N-Boc-amino-(4-N-Fmoc-piperidinyl) carboxylic acid followed by
peptide chain elongation and deprotection of Dde in D-Lys(Dde) at
Xaa.sub.4 was carried out according to the procedure described in
the synthesis of compound (1). See above. The resulting peptide
resin (0.9 mmol;
Boc-D-Phe-D-Phe-D-Leu-D-Lys-(N-Boc-amino-4-piperidinylcarboxylic
acid)-[2-Cl-Trt resin]) was split and a portion of 0.3 mmol was
used for subsequent cleavage. The peptide resin (0.3 mmol) was then
treated with a mixture of TFA/TIS/H.sub.2O (15 ml,
v/v/v=95:2.5:2.5) at room temperature for 90 minutes. The resin was
then filtered and washed with TFA. The filtrate was evaporated in
vacuo and the crude synthetic peptide amide (0.3 mmol;
D-Phe-D-Phe-D-Leu-D-Lys-[.omega.(4-aminopiperidine-4-carboxylic
acid)]-OH) was precipitated from diethyl ether.
[0260] For purification, the crude synthetic peptide amide (0.3
mmol) was dissolved in 2% acetic acid in H.sub.2O (50 ml) and the
solution was loaded onto an HPLC column and purified using TEAP
buffer system with a pH 5.2 (buffers A=TEAP 5.2 and B=20% TEAP 5.2
in 80% ACN). The compound was eluted with a linear gradient of
buffer B, 7% B to 37% B over 60 minutes. Fractions with purity
exceeding 95% were pooled and the resulting solution was diluted
with two volumes of water. The diluted solution was then loaded
onto an HPLC column for salt exchange and further purification with
a TFA buffer system (buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA
in 80% ACN/20% H.sub.2O) and a linear gradient of buffer B, 2% B to
75% B over 25 minutes. Fractions with purity exceeding 97% were
pooled, frozen, and dried on a lyophilizer to yield the purified
synthetic peptide amide as white amorphous powder (93 mg). HPLC
analysis: t.sub.R=16.43 min, purity 99.2%, gradient 5% B to 25% B
over 20 min; MS (MH.sup.+): expected molecular ion mass 680.4,
observed 680.3.
[0261] Compound (2) was also prepared using the following amino
acid derivatives: Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH,
and Boc-4-amino-1-Fmoc-(piperidine)-4-carboxylic acid.
[0262] The fully protected resin-bound peptide was synthesized
manually starting from 2-Chlorotrityl chloride resin (PS 1% DVB,
500 g, 1 meq/g). The resin was treated with
Boc-4-amino-1-Fmoc-4-(piperidine)-4-carboxylic acid (280 g, 600
mmol) in a mixture of DMF, DCM and DIEA (260 mL of each) was added.
The mixture was stirred for 4 hours and then the resin was capped
for 1 h by the addition of MeOH (258 mL) and DIEA (258 mL).
[0263] The resin was isolated and washed with DMF (3.times.3 L).
The resin containing the first amino acid was treated with
piperidine in DMF (3.times.3 L of 35%), washed with DMF (9.times.3
L) and Fmoc-D-Lys(Boc)-OH (472 g) was coupled using PyBOP (519 g)
in the presence of HOBt (153 g) and DIEA (516 mL) and in DCM/DMF
(500 mL/500 mL) with stirring for 2.25 hours. The dipeptide
containing resin was isolated and washed with DMF (3.times.3.6 L).
The Fmoc group was removed by treatment with piperidine in DMF
[0264] (3.times.3.6 L of 35%) and the resin was washed with DMF
(9.times.3.6 L) and treated with Fmoc-D-Leu-OH (354 g), DIC (157
mL) and HOBt (154 g) in DCM/DMF (500 mL/500 mL) and stirred for 1
hour. Subsequent washing with DMF (3.times.4.1 L) followed by
cleavage of the Fmoc group with piperidine in DMF (3.times.4.2 L of
35%) and then washing of the resin with DMF (9.times.4.2 L)
provided the resin bound tripeptide. This material was treated with
Fmoc-D-Phe-OH (387 g), DIC (157 mL) and HOBt (153 g) in DCM/DMF
(500 mL/500 mL) and stirred overnight. The resin was isolated,
washed with DMF (3.times.4.7 L) and then treated with piperidine in
DMF (3.times.4.7 L of 35%) to cleave the Fmoc group and then washed
again with DMF (9.times.4.7 L). The tetrapeptide loaded resin was
treated with Fmoc-D-Phe-OH (389 g), DIC (157 mL) and HOBt (154 g)
in DCM/DMF (500 mL/500 mL) and stirred for 2.25 hours. The resin
was isolated, washed with DMF (3.times.5.2 L) and then treated
piperidine (3.times.5.2 L of 35%) in DMF. The resin was isolated,
and washed sequentially with DMF (9.times.5.2 L) then DCM
(5.times.5.2 L). It was dried to provide a 90.4% yield of protected
peptide bound to the resin. The peptide was cleaved from the resin
using TFA/water (4.5 L, 95/5), which also served to remove the Boc
protecting groups. The mixture was filtered, concentrated (1/3) and
then precipitated by addition to MTBE (42 L). The solid was
collected by filtration and dried under reduced pressure to give
crude synthetic peptide amide.
[0265] For purification, the crude synthetic peptide amide was
dissolved in 0.1% TFA in H.sub.2O and purified by preparative
reverse phase HPLC (C18) using 0.1% TFA/water--ACN gradient as
mobile phase. Fractions with purity exceeding 95% were pooled,
concentrated and lyophilized to provide pure synthetic peptide
amide (>95.5% pure). Ion exchange was conducted using a Dowex
ion exchange resin, eluting with water. The aqueous phase was
filtered (0.22 .mu.m filter capsule) and freeze-dried to give the
acetate salt of the synthetic peptide amide (2) with overall yield,
71.3%, >99% purity.
[0266] Hydrochloride, hydrobromide and fumarate counterions were
evaluated for their ability to form crystalline salts of synthetic
peptide amide (2). Approximately 1 or 2 equivalents (depending on
desired stoichiometry) of hydrochloric acid, hydrobromic acid or
fumaric acid, as a dilute solution in methanol (0.2-0.3 g) was
added to synthetic peptide amide (2) (50-70 mg) dissolved in
methanol (0.2-0.3 g). Each individual salt solution was added to
isopropyl acetate (3-5 mL) and the resulting amorphous precipitate
was collected by filtration and dried at ambient temperature and
pressure. Crystallization experiments were carried out by
dissolving the 10-20 mg of the specific amorphous salt obtained
above in 70:30 ethanol-water mixture (0.1-0.2 g) followed by the
addition of ethanol to adjust the ratio to 90:10 (.about.0.6-0.8
mL). Each solution was then seeded with solid particles of the
respective precipitated salt. Each sample tube was equipped with a
magnetic stir bar and the sample was gently stirred at ambient
temperature. The samples were periodically examined by
plane-polarized light microscopy. Under these conditions, the mono-
and dihydrochloride salts, the di-hydrobromide salt and the
mono-fumarate salt crystallized as needles of 20 to 50 .mu.m in
length with a thickness of about 1 .mu.m.
Example 3
Synthesis of compound (3):
D-Phe-D-Phe-D-Leu-(.epsilon.-Me)D-Lys-[.omega.(4-aminopiperidine-4-carbox-
ylic acid)]-OH
[0267] The synthesis was started with 0.3 mmol of the peptide
resin:
Boc-D-Phe-D-Phe-D-Leu-D-Lys-(N-Boc-amino-4-piperidinylcarboxylic
acid)-[2-Cl-Trt resin], which was prepared during the synthesis of
compound (2) as described below. HPLC and MS analyses were also
performed as described in the synthesis of compound (2) above.
[0268] For the methylation of the .omega.-amino function of D-Lys
at Xaa.sub.4, a three-step procedure as described in the synthesis
of compound (1) was followed. See description above. The
resin-bound methylated peptide
(Boc-D-Phe-D-Phe-D-Leu-(.epsilon.-Me)D-Lys-(N-Boc-amino-4-piperidinylcarb-
oxylic acid)-[2-Cl-Trt resin]) was then treated with a mixture of
TFA/TIS/H.sub.2O (15 ml, v/v/v=95:2.5:2.5) at room temperature for
90 minutes. The resin was then filtered and washed with TFA. The
filtrate was evaporated in vacuo and the crude synthetic peptide
amide (0.3 mmol;
D-Phe-D-Phe-D-Leu-(.epsilon.-Me)D-Lys-[.omega.(4-amino-piperidine-4-carbo-
xylic acid)]-OH) was precipitated from diethyl ether.
[0269] The crude synthetic peptide amide (0.3 mmol) was purified by
preparative HPLC according to the protocol described in the
synthesis of compound (2). See above. Fractions with purity
exceeding 97% were pooled, frozen, and dried on a lyophilizer to
yield the purified synthetic peptide amide as white amorphous
powder (185 mg). HPLC analysis: t.sub.R=16.93 min, purity 99.2%,
gradient 5% B to 25% B over 20 min; MS (MH.sup.+): expected
molecular ion mass 694.4, observed 694.4.
Example 4
Synthesis of compound (4):
D-Phe-D-Phe-D-Leu-D-Lys-[N-(4-piperidinyl)-L-proline]-OH
[0270] The amino acid derivatives used were Boc-D-Phe-OH,
Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Dde)-OH, and
N-(1-Fmoc-piperidin-4-yl)-L-proline. HPLC and MS analyses were
performed as described in the synthesis of compound (1). See
detailed description above. Couplings were mediated by
HATU/DIEA.
[0271] The fully protected resin-bound peptide was synthesized
manually starting from 2-Chlorotrityl chloride resin (3.2 g, 2.4
mmol; NeoMPS). The attachment of the first amino acid to the resin
was achieved by treatment with a mixture of
N-(1-Fmoc-piperidin-4-yl)-L-proline (2.0 g, 4.8 mmol) and DIEA (3.3
ml, 19.2 mmol) in DCM (40 ml) and DMF (10 ml) at room temperature
for 4 hours. The resin was washed with 3.times.DCM/MeOH/DIEA
(v/v/v=17:2:1) and 3.times.DCM and dried in vacuo. The resulting
resin (3.7 g; N-(4-piperidinyl)-L-proline-[2-Cl-Trt resin]) was
split into several portions and a portion of 1.9 g (1.2 mmol) was
used to continue the peptide synthesis. HATU/DIEA-mediated single
couplings were performed with a 3-fold excess of amino acid
derivatives. The Fmoc group was removed with 25% piperidine in DMF.
Upon completion of peptide chain elongation, the resin was treated
with 4% hydrazine in DMF three times for 3 min each to remove Dde.
The resin was washed with DMF and DCM and dried in vacuo. The
resulting peptide resin (2.1 g;
Boc-D-Phe-D-Phe-D-Leu-D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trt
resin]) was split again and a portion of 0.7 g (0.4 mmol) was used
for subsequent cleavage. The peptide resin was treated with a
mixture of TFA/TIS/H.sub.2O (15 ml, v/v/v=95:2.5:2.5) at room
temperature for 90 minutes. The resin was filtered and washed with
TFA. The filtrate was evaporated in vacuo and the crude peptide
(220 mg, D-Phe-D-Phe-D-Leu-D-Lys-[N-(4-piperidinyl)-L-proline]-OH)
was precipitated from diethyl ether.
[0272] For purification, the above crude peptide (220 mg) was
dissolved in 0.1% TFA in H.sub.2O (50 ml) and the solution was
loaded onto an HPLC column and purified using TFA buffer system
(buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60% ACN/40%
H.sub.2O). The compound was eluted with a linear gradient of buffer
B, 25% B to 75% B over 25 min, t.sub.R=43% B. Fractions with purity
exceeding 97% were pooled, frozen, and dried on a lyophilizer to
give the purified synthetic peptide amide as white amorphous powder
(89 mg). HPLC analysis: t.sub.R=18.22 min, purity 99.5%, gradient
5% B to 25% B over 20 min; MS (MH.sup.+): expected molecular ion
mass 734.5, observed 734.4.
Example 5
Synthesis of compound (5):
D-Phe-D-Phe-D-Leu-D-Har-[N-(4-piperidinyl)-L-proline]-OH
[0273] The peptide-resin:
Boc-D-Phe-D-Phe-D-Leu-D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trt
resin], which was prepared during the synthesis of compound (4)
described above, was used as the starting material. HPLC and MS
analyses were performed as described in the synthesis of compound
(1) above.
[0274] For guanylation of the .omega.-amino function of D-Lys at
Xaa.sub.4, the peptide resin (0.7 g, 0.4 mmol) was treated with a
mixture of 1H-Pyrazole-1-carboxamidine hydrochloride (0.6 g, 4.0
mmol) and DIEA (0.7 ml, 4.0 mmol) in DMF (15 ml) overnight at room
temperature. The resin was washed with DMF and DCM and dried in
vacuo. The peptide was then cleaved from the resin by treatment
with a mixture of TFA/TIS/H.sub.2O (15 ml, v/v/v=95:2.5:2.5) at
room temperature for 90 minutes. The resin was then filtered and
washed with TFA. The filtrate was evaporated in vacuo and the crude
synthetic peptide amide (170 mg;
D-Phe-D-Phe-D-Leu-D-Har-[N-(4-piperidinyl)-L-proline]-OH) was
precipitated from diethyl ether.
[0275] For purification, the above crude synthetic peptide amide
(170 mg) was dissolved in 0.1% TFA in H.sub.2O (50 ml) and the
solution was loaded onto an HPLC column and purified using a TFA
buffer system (buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60%
ACN/40% H.sub.2O). The compound was eluted with a linear gradient
of buffer B, 25% B to 75% B over 25 min, t.sub.R=46% B. Fractions
with purity exceeding 97% were pooled, frozen, and lyophilized to
yield the purified synthetic peptide amide as white amorphous
powder (81 mg). HPLC analysis: t.sub.R=19.42 min, purity 100%,
gradient 5% B to 25% B over 20 min; MS (MH.sup.+): expected
molecular ion mass 776.5, observed 776.5.
Example 6
Synthesis of compound (6):
D-Phe-D-Phe-D-Leu-(.epsilon.-Me)D-Lys-[N-(4-piperidinyl)-L-proline]-OH
[0276] Synthesis was initiated with 0.7 g (0.4 mmol) of the peptide
resin,
Boc-D-Phe-D-Phe-D-Leu-D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trt
resin], which was prepared during the synthesis of compound (4) as
described above. HPLC and MS analyses were performed as described
in the synthesis of compound (1) above. In this case, the
Xaa.sub.1-Xaa.sub.4 peptide was pre-synthesized and coupled.
[0277] For the methylation of the .omega.-amino function of D-Lys
at Xaa.sub.4, a three-step procedure was followed as described in
the synthesis of compound (1) above. The resin-bound methylated
peptide
(Boc-D-Phe-D-Phe-D-Leu-(.epsilon.-Me)D-Lys-N-(4-piperidinyl)-L-proline-[2-
-Cl-Trt resin]) was then treated with a mixture of TFA/TIS/H.sub.2O
(15 ml, v/v/v=95:2.5:2.5) at room temperature for 90 minutes. The
resin was filtered and washed with TFA. The filtrate was evaporated
in vacuo and crude peptide (200 mg;
D-Phe-D-Phe-D-Leu-(.epsilon.-Me)D-Lys-[N-(4-piperidinyl)-L-proline]OH)
precipitated from diethyl ether.
[0278] For purification, the above crude peptide (200 mg) was
dissolved in 0.1% TFA in H.sub.2O (50 ml) and the solution loaded
onto an HPLC column and purified using a TFA buffer system (buffers
A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60% ACN/40% H.sub.2O). The
compound was eluted with a linear gradient of 25% to 75% buffer B,
over 30 min, t.sub.R=42% B. Fractions with purity exceeding 97%
were pooled, frozen, and dried on a lyophilizer to yield the
purified synthetic peptide amide as white amorphous powder (41 mg).
HPLC analysis: t.sub.R=18.66 min, purity 98.1%, gradient 5% B to
25% B over 20 min; MS (MH.sup.+): expected molecular ion mass
748.5, observed 748.5.
Example 7
Synthesis of (7): D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazine
amide]
[0279] The amino acid derivatives used were Boc-D-Phe-OH,
Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, and Fmoc-D-Arg(Pbf)-OH. HPLC and MS
analyses were performed as in the synthesis of compound (1)
described above. The fully protected resin bound peptide was
synthesized on a SYMPHONY Multiple Synthesizer (Protein Technology
Inc.) starting from the homopiperazine carbamate Wang resin (0.35
mmol; homopiperazine-[carbamate Wang resin]) that was prepared
during the synthesis of compound (1). HBTU/DIEA mediated single
couplings with a 4-fold excess of amino acid derivatives were
performed. The Fmoc group was removed with 25% piperidine in DMF.
Upon completion of the automated synthesis, the peptide resin
(Boc-D-Phe-D-Phe-D-Leu-D-Arg(Pbf)-[homopiperazine amide]) was
transferred into a manual peptide synthesis vessel and treated with
a mixture of TFA/TIS/H.sub.2O (15 ml, v/v/v=95:2.5:2.5) at room
temperature for 90 minutes. The resin was filtered and washed with
TFA. The filtrate was evaporated in vacuo and the crude peptide
(380 mg; D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazine amide]) was
precipitated from diethyl ether.
[0280] For purification, the above crude peptide (380 mg) was
dissolved in 0.1% TFA in H.sub.2O (50 ml) and the solution was
loaded onto an HPLC column and purified using a TFA buffer system
(buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60% ACN/40%
H.sub.2O). The compound was eluted with a linear gradient of buffer
B, 25% B to 75% B over 25 min, t.sub.R=36% B. Fractions with purity
exceeding 97% were pooled, frozen, and lyophilized to give the
purified synthetic peptide amide as white amorphous powder (222
mg). HPLC analysis: t.sub.R=16.75 min, purity 100%, gradient 2% B
to 22% B over 20 min; MS (MH.sup.+): expected molecular ion mass
664.4, observed 664.5.
Example 8
Synthesis of compound (8):
D-Phe-D-Phe-D-Leu-D-Har-[.omega.(4-aminopiperidine-4-carboxylic
acid]-OH
[0281] This compound was prepared essentially according to the
procedure described above for the synthesis of compound (5) except
that N-Boc-amino-(4-N-Fmoc-piperidinyl) carboxylic acid was
substituted for N-(1-Fmoc-piperidin-4-yl)-L-proline in the
attachment to 2-Cl-Trt resin. Final purified synthetic peptide
amide: amorphous powder, 85 mg in yield in a synthesis scale of 1
mmol HPLC analysis: t.sub.R=17.87 min, purity 100%, gradient 5% B
to 25% B over 20 min; MS (MH.sup.+): expected molecular ion mass
722.4, observed 722.5.
Example 9
Synthesis of compound (9):
D-Phe-D-Phe-D-Leu-(.epsilon.-iPr)D-Lys-[.omega.(4-aminopiperidine-4-carbo-
xylic acid)]-OH
[0282] Synthesis was initiated from 0.15 mmol of the peptide resin,
Boc-D-Phe-D-Phe-D-Leu-D-Lys-(N-Boc-amino-4-piperidinylcarboxylic
acid)-[2-Cl-Trt resin]), which was prepared during the synthesis of
compound (2) above. For isopropylation of the .omega.-amino
function of D-Lys at Xaa.sub.4, the peptide resin was treated with
a mixture of sodium triacetoxyborohydride (3 mmol) and acetone (6
mmol) in TMOF (10 mL) for 4 h at room temperature. Subsequent
cleavage and purification steps were carried out according to the
procedure described in the synthesis of compound (2). Final
purified synthetic peptide amide: amorphous powder, 67 mg in yield.
HPLC analysis: t.sub.R=19.29 min, purity 98.4%, gradient 5% B to
25% B over 20 min; MS (MH.sup.+): expected molecular ion mass
722.5, observed 722.5.
Example 10
Synthesis of compound (10):
D-Phe-D-Phe-D-Leu-(.beta.-amidino)D-Dap-[.omega.(4-aminopiperidine-4-carb-
oxylic acid)]-OH
[0283] The amino acid derivatives used were Boc-D-Phe-OH,
Boc-D-Phe-OH, Boc-D-Leu-OH, Boc-D-Dap(Fmoc)-OH, and
N-Fmoc-amino-(4-N-Boc-piperidinyl) carboxylic acid. HPLC and MS
analyses were performed as described in the synthesis of compound
(1). The fully protected resin-bound peptide was synthesized
manually starting with 4-Fmoc-hydrazinobenzoyl AM NovaGel resin (3
mmol; Novabiochem). The Fmoc protecting group on the starting resin
was first removed by 25% piperidine in DMF and the resin was then
treated with a mixture of N-Fmoc-amino-(4-N-Boc-piperidinyl)
carboxylic acid (7.5 mmol), PyBOP (7.5 mmol), and DIEA (15 mmol) in
DMF overnight at room temperature. The Fmoc group on the attached
amino acid was replaced by o-NBS in two steps: (i) Fmoc removal by
25% piperidine in DMF. (ii) o-NBS protection according the
procedure described in the synthesis of compound (1). The resulting
peptide resin, N-o-NBS-amino-(4-N-Boc-piperidinyl) carboxylic
acid-[hydrazinobenzoyl AM NovaGel resin], was split into several
portions and a portion of 1 mmol was used to continue the peptide
synthesis. PyBOP/DIEA mediated single couplings were performed with
a 3-fold excess of amino acid derivatives. The Boc group was
removed with 30% TFA in DCM. Upon completion of peptide chain
elongation, the resin was treated with 2% DBU in DMF for 2.times.8
min for Fmoc removal, followed by guanylation of the .omega.-amino
function of D-Dap at Xaa.sub.4. The final o-NBS deprotection was
carried out according to the procedure described in the synthesis
of compound (1).
[0284] For oxidative cleavage, the dried peptide resin was mixed
with a mixture of Cu(OAc).sub.2 (1 mmol), pyridine (4 mmol), and
DBU (2 mmol) in 5% H.sub.2O in DMF and let air bubble through the
resin for 6 h at room temperature. The resin was filtered and
washed with DMF and the filtrated was evaporated in vacuo. The
residue,
Boc-D-Phe-D-Phe-D-Leu-(.beta.-amidino)D-Dap-[.omega.(4-aminopiperidine-4--
carboxylic acid)]-OH, was treated with 95% TFA in H.sub.2O for Boc
removal. The solution was evaporated in vacuo and the crude peptide
(1 mmol;
D-Phe-D-Phe-D-Leu-(.beta.-amidino)D-Dap-[.omega.(4-aminopiperidine--
4-carboxylic acid)]-OH) was precipitated from diethyl ether.
[0285] Purification of the above crude peptide was achieved
according to the protocol described in the synthesis of compound
(2). The purified synthetic peptide amide was an amorphous powder
(16 mg). HPLC analysis: t.sub.R=16.97 min, purity 99.9%, gradient
5% B to 25% B over 20 min; MS (MH.sup.+): expected mass 680.4,
observed 680.4.
Examples 11-23
Synthesis of Compounds (11)-(23)
[0286] These compounds were prepared according to the procedure
described in the synthesis of compound (10), except that
appropriate Boc-amino acid derivatives were substituted for
Boc-D-Dap(Fmoc)-OH in the coupling at Xaa.sub.4. See U.S. Pat. No.
7,713,937.
Example 24
Synthesis of compound (24):
D-Phe-D-Phe-D-Leu-D-Arg-[4-Amidinohomopiperazine amide]
[0287] The compound was prepared by guanylation of the
homopiperazine at C-terminus of
Cbz-D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazine amide], which was
synthesized according to the procedure described in the synthesis
of compound (7), described above. Subsequent cleavage and
purification were carried out according to the procedure described
in the synthesis of compound (1), above. Final purified peptide:
amorphous powder, 102 mg in yield in a synthesis scale of 0.3 mmol
HPLC analysis: t.sub.R=17.34 min, purity 98.4%, gradient 2% B to
22% B over 20 min; MS (MH.sup.+): expected molecular ion mass
706.5, observed 706.5.
Example 25
Synthesis of compound (25):
D-Phe-D-Phe-D-Leu-D-Lys-[2,8-diazaspiro[4,5]decan-1-one amide]
[0288] To a suspension of Boc-D-Phe-OH intermediate I-1 (7.96 g,
30.0 mmol), D-Leu-OBn p-TsOH intermediate I-2 (11.80 g, 30.0 mmol),
HOBt monohydrate (4.46 g, 33.0 mmol) and DIEA (8.53 g, 66.0 mmol)
in anhydrous THF (250 mL) cooled in an ice-water bath was added
EDCI (6.33 g, 33.0 mmol) in four portions over 20 minutes with 5
minutes between each addition. The suspension was stirred overnight
from a starting temperature of 0.degree. C. to room temperature.
After evaporation of THF, the residue was dissolved in ethyl
acetate and washed sequentially with 10% citric acid, saturated
NaHCO.sub.3 and water. The organic phase was dried over sodium
sulfate and evaporated under reduced pressure. The residue was
dissolved in DCM, passed through a silica gel plug and eluted with
20% ethyl acetate in hexanes. Eluant was evaporated to give the
pure Boc-D-Phe-D-Leu-OBn, intermediate I-3 (12.40 g, 88%) as a
clear oil. LC-MS: m/z=469 (MH).
[0289] Intermediate I-3 (12.40 g, 26.5 mmol) was dissolved in DCM
(50 mL). TFA (25 mL) was added and the solution was stirred at room
temperature for 2 hours. After evaporation of DCM and TFA, the
residue was azeotroped with toluene twice to give the TFA salt of
D-Phe-Leu-OBn, intermediate I-4. This crude dipeptide was suspended
in THF, to which Boc-D-Phe-OH (6.36 g, 24 mmol), HOBt monohydrate
(4.04 g, 26.4 mmol) and DIEA (8.7 mL, 50.0 mmol) was added at
0.degree. C. EDCI (6.33 g, 6.4 mmol) was added in four portions
over 20 minutes with 5 minutes between each addition. The
suspension was stirred from 0.degree. C. to room temperature
overnight. After evaporation of THF, the residue was dissolved in
ethyl acetate and washed sequentially with 10% citric acid,
saturated NaHCO.sub.3 and water. The organic phase was dried over
sodium sulfate and evaporated under reduced pressure. The residue
was recrystalized from 400 mL acetone/hexanes (1:3) to give 9.1 g
pure product. The mother liquor was evaporated and again
recrystalized from acetone/hexanes (1:3) to give 2.0 g product. The
total yield was 11.1 g (68% for two steps). LC-MS: m/z=616
(MH).
[0290] In a flask flushed with nitrogen was added wet palladium on
carbon (1.8 g) and a solution of Boc-D-Phe-D-Phe-D-Leu-OBn,
intermediate I-5 (11.1 g, 18.05 mmol) in methanol (50 mL). The
mixture was stirred under a hydrogen balloon overnight. After
filtration through celite, methanol was evaporated under reduced
pressure. The residue was dissolved in acetone (20 mL) and slowly
added to 500 mL water with 25 mL of 1N HCl under vigorous stirring.
Pure product Boc-D-Phe-D-Phe-D-Leu-OH, intermediate I-6 was
obtained by filtration 9.4 g (99%). LC-MS: m/z=526 (MH).
[0291] To a solution of intermediate I-6 (2.06 g, 3.90 mmol),
D-Lys(Boc)-OAll hydrochloride (1.26 g, 3.90 mmol) and DIEA (1.7 ml,
9.8 mmol) in DMF was added TBTU (1.56 g, 4.88 mmol) in three
portions over 15 min at 0.degree. C. After stirring overnight from
a starting temperature of 0.degree. C. to room temperature, DMF was
evaporated under high vacuum. The crude reaction mixture was
precipitate in 400 ml ice water and filtered to collect the
precipitate, Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OAll intermediate I-7
(2.60 g), which was used without further purification for the next
step.
[0292] To a solution of intermediate I-7 (2.60 g, 3.3 mmol) in MeCN
(75 mL) was added pyrrolidine (1.1 ml, 13.3 mmol) and palladium
tetrakis(triphenylphosphine) (400 mg, 0.35 mmol) at 0.degree. C.
The reaction mixture was stirred at room temperature for 3 hours
and evaporated to dryness. Residue was purified by reverse phase
column chromatography with 30% MeCN/water to 90% MeCN/water to give
pure acid, intermediate I-8 (2.0 g, 80%) after evaporation of
acetonitrile/water. LC-MS: m/z=754 (MH.sup.+).
[0293] To a solution of the acid, intermediate I-8 (150 mg, 0.20
mmol), the amine HNR.sub.aR.sub.b, 2,8-diazaspiro [4,5]decan-1-one
(57 mg, 0.30 mmol) and DIEA (175 ul, 1.0 mmol) in DMF (5 mL) was
added HBTU (11.3 mg, 0.3 mmol) at 0.degree. C. After stirring
overnight from a starting temperature of 0.degree. C. to room
temperature, DMF was evaporated under reduced pressure. The residue
was stirred with 4N HCl in 1,4-dioxane (2.0 mL) at room temperature
for 1 hour. After removal of dioxane, the residue was dissolved in
water and purified by reverse phase column chromatography with a
gradient of 10% MeCN/water to 60% MeCN/water in 30 minutes to give
pure synthetic peptide amide, compound (25) (108 mg, 78% yield for
the two steps) after evaporation of solvent. LC-MS: m/z=690
(MW).
Examples 26
37: Synthesis of Compounds (26)-(37)
[0294] These compounds were prepared according to the procedure
described in the synthesis of compound (26) above except for the
substitution of the appropriate amine used in the final step as
described in detail in U.S. Pat. No. 7,713,937.
Example 38
Synthesis of compound (38):
D-Phe-D-Phe-D-Leu-D-Orn-[4-(2-aminoethyl)-1-carboxymethyl-piperazine]-OH
[0295] The amino acid derivatives used were Boc-D-Phe-OH,
Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, Fmoc-D-Orn(Boc)-OH, and
Fmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine dihydrochloride.
The fully protected resin-bound peptide was synthesized on a
SYMPHONY Multiple Synthesizer (Protein Technology Inc.) starting
from 2-Chlorotrityl chloride resin (0.4 mmol; Novabiochem). The
attachment of the first amino acid to the resin was achieved by
treatment with a mixture of
Fmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine dihydrochloride
(0.24 g, 0.5 mmol; Chem-Impex International Inc.) and DIEA (0.35
mL, 2 mmol) in DMF (7 ml) at room temperature for 4 hours. The
resin was washed with 3.times.DCM/MeOH/DIEA (v/v/v=17:2:1) and
3.times.DCM. The subsequent peptide chain elongation was achieved
by HBTU/DIEA mediated single couplings with a 3-fold excess of
amino acid derivatives. The Fmoc group was removed by 25%
piperidine in DMF. For cleavage, the final peptide resin was
treated with a mixture of TFA/TIS/H.sub.2O (15 ml,
v/v/v=95:2.5:2.5) at room temperature for 90 minutes. The resin was
filtered and washed with TFA. The filtrate was evaporated in vacuo
and the crude peptide (0.2 g,
D-Phe-D-Phe-D-Leu-D-Orn-[4-(2-aminoethyl)-1-carboxymethyl-piperazine]-OH)
was precipitated from diethyl ether.
[0296] For purification, the above crude peptide (0.2 g) was
dissolved in 0.1% TFA in H.sub.2O (50 ml) and the solution was
loaded onto an HPLC column and purified using a TFA buffer system
(buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60% ACN/40%
H.sub.2O). The compound was eluted with a linear gradient of buffer
B, 25% B to 75% B over 30 min, t.sub.R=35% B. The fractions with
purity exceeding 97% were pooled, frozen, and dried on a
lyophilizer to yield the purified synthetic peptide amide as a
white amorphous powder (101 mg). HPLC analysis: t.sub.R=16.24,
purity 100%, gradient 5% B to 25% B over 20 min; MS (MH.sup.+):
expected molecular ion mass 709.4, observed 709.4.
Examples 39-48
Synthesis of Compounds (39)-(48)
[0297] These compounds were prepared according to the procedure
described in the synthesis of compound (38) using the appropriate
amino acid derivatives as detailed in U.S. Pat. No. 7,713,937.
Example 49
Synthesis of compound (49):
D-Phe-D-Phe-D-Leu-D-Orn-[4-(2-aminoethyl)-1-carboxymethyl-piperazine]-NH.-
sub.2
[0298] The amino acid derivatives used were Boc-D-Phe-OH,
Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, Fmoc-D-Orn(Boc)-OH, and
Fmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine dihydrochloride.
The fully protected resin-bound peptide was synthesized on a
SYMPHONY Multiple Synthesizer (Protein Technology Inc.) starting
from Rink Amide AM resin (0.3 mmol; Novabiochem). The attachment of
the first amino acid to the resin was achieved by treatment with a
mixture of Fmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine
dihydrochloride (0.48 g, 1 mmol; Chem-Impex International Inc.),
HBTU (0.38 g, 1 mmol) and DIEA (0.53 mL, 3 mmol) in DMF (7 ml) at
room temperature for 3 hours. The resin was washed with three times
in DMF.
[0299] Subsequent peptide chain elongation was achieved by
HBTU/DIEA mediated single couplings with a 3-fold excess of amino
acid derivatives. The Fmoc group was removed by 25% piperidine in
DMF. For cleavage, the final peptide resin was treated with a
mixture of TFA/TIS/H.sub.2O (15 ml, v/v/v=95:2.5:2.5) at room
temperature for 90 minutes. The resin was filtered and washed with
TFA. The filtrate was evaporated in vacuo and the crude synthetic
peptide amide (0.1 g,
D-Phe-D-Phe-D-Leu-D-Orn-[4-(2-aminoethyl)-1-carboxymethyl-piperazine]-NH.-
sub.2) was precipitated from diethyl ether.
[0300] For purification, the above crude synthetic peptide amide
(0.1 g) was dissolved in 0.1% TFA in H.sub.2O (50 ml) and the
solution was loaded onto an HPLC column and purified using a TFA
buffer system (buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60%
ACN/40% H.sub.2O). The compound was eluted with a linear gradient
of buffer B, 25% B to 75% B over 30 min, t.sub.R=38% B. The
fractions with purity exceeding 97% were pooled, frozen, and dried
on a lyophilizer to yield the purified synthetic peptide amide as a
white amorphous powder (36 mg). HPLC analysis: t.sub.R=16.59,
purity 99.5%, gradient 2% B to 22% B over 20 min; MS (MH.sup.+):
expected mass 708.5, observed 708.3.
Examples 50 and 51
Synthesis of Compounds (50) and (51)
[0301] These compounds were prepared according to the procedure
described in the synthesis of compound (49) For compounds (50) and
(51) N-(1-Fmoc-piperidin-4-yl)-L-proline (NeoMPS) and
Fmoc-4-amino-1-carboxymethyl-piperidine (NeoMPS) were respectively
substituted for Fmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine
dihydrochloride in the attachment to Rink Amide AM resin.
Example 52
Synthesis of compound (52):
D-Phe-D-Phe-D-Leu-D-Orn-[4-(N-methyl)amidinohomopiperazine
amide]
[0302] Compound (52) was prepared by guanylation of the
homopiperazine at C-terminus of
Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine amide], which was
synthesized according to the procedure described below.
[0303] The guanylation reagent S-Methyl-N-methylisothiourea
hydroiodide was prepared by reacting 1,3-dimethyl-2-thiourea with
methyl iodide in anhydrous methanol. See McKay, A. F.; Hatton, W.
G. Synthesis of Cyclic Guanidino Acids. J. Am. Chem. Soc. (1955),
78, 1618-1620 and Kennedy, K. J., et al. A Facile Route to Cyclic
and Acyclic Alkyl-Arginines. Synthetic Communications, (1998), 28,
741-746.
[0304] For synthesis of
Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine amide], the amino
acid derivatives used were Z-D-Phe-OH, Fmoc-D-Phe-OH,
Fmoc-D-Leu-OH, and Fmoc-D-Orn(Cbz)-OH. The fully protected resin
bound peptide was synthesized manually starting from
p-nitrophenylcarbonate Wang resin (5.0 g, 4.4 mmol; Novabiochem).
The attachment of homopiperazine to the resin was achieved by
mixing a solution of homopiperazine (8.7 g, 87 mmol; Acros
Organics) in DCM (100 ml) overnight at room temperature. The resin
was washed with 3.times.DMF and 3.times.DCM. The subsequent peptide
chain elongation was achieved by HBTU/DIEA-mediated single
couplings with a 3-fold excess of amino acid derivatives. The Fmoc
group was removed by 25% piperidine in DMF. For cleavage, the final
synthetic peptide amide resin was treated with a mixture of TFA/DCM
(100 mL, v/v=1:1) at room temperature for 2 hours. The resin was
filtered and washed with DCM. The filtrate was evaporated in vacuo
and the residue was dissolved in 0.1% TFA in 60% ACN/40% H.sub.2O.
The solution was frozen, and dried on a lyophilizer to give the
crude peptide intermediate
Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-homopiperazine (4.4 g). For
purification, the crude peptide (4.4 g) was divided into two
portions and each portion was dissolved in 0.1% TFA in 30% ACN (100
ml). Each solution was loaded onto an HPLC column and purified
using a TFA buffer system (buffers A=0.1% TFA in H.sub.2O and
B=0.1% TFA in 60% ACN/40% H.sub.2O). The compound was eluted with a
linear gradient of buffer B, 40% B to 100% B over 25 min,
t.sub.R=87% B. The fractions with purity exceeding 97% were pooled,
frozen, and dried on a lyophilizer to yield the purified peptide
intermediate as a white amorphous powder (3.0 g).
[0305] For guanylation of the homopiperazine at C-terminus, the
above peptide intermediate (210 mg, 0.3 mmol) was treated with a
mixture of S-Methyl-N-methylisothiourea hydroiodide (1.4 g, 6 mmol)
and DIEA (1.0 mL, 12 mmol) in DMF (4 mL) at room temperature for 18
days. The mixture was evaporated in vacuo and the residue was
dissolved in 0.1% TFA in 30% ACN/70% H.sub.2O and the solution was
loaded onto an HPLC column and purified using a TFA buffer system
(buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60% ACN/40%
H.sub.2O). The compound was eluted with a linear gradient of buffer
B, 70% B to 100% B over 30 min, t.sub.R=85% B. The fractions with
purity exceeding 97% were pooled, frozen, and dried on a
lyophilizer to yield the purified peptide intermediate,
Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[N-methylhomopiperazine-1-carboximidamid-
e amide], as white amorphous powder (100 mg).
[0306] For final deprotection/hydrolysis, the above purified
peptide (100 mg) was treated with a mixture of TMSOTf/TFA/m-cresol
(10 ml, v/v/v=2:7:1) at room temperature for 2 hours. The mixture
was evaporated in vacuo and the crude peptide (100 mg) was
precipitated from diethyl ether.
[0307] For purification, the above crude peptide (100 mg) was
dissolved in 0.1% TFA in H.sub.2O (50 ml) and the solution was
loaded onto an HPLC column and purified using TFA buffer system
(buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60% ACN/40%
H.sub.2O). The compound was eluted with a linear gradient of buffer
B, 25% B to 75% B over 25 min, t.sub.R=43% B. The fractions with
purity exceeding 97% were pooled, frozen, and dried on a
lyophilizer to yield the purified synthetic peptide amide as a
white amorphous powder (53 mg). HPLC analysis: t.sub.R=17.99 min,
purity 99.4%, gradient 2% B to 22% B over 20 min; MS (MH.sup.+):
expected molecular ion mass 678.4, observed 678.5.
Example 53
Synthesis of compound (53):
D-Phe-D-Phe-D-Leu-D-Orn-[4-amidinohomopiperazine amide]
[0308] Synthesis was initiated from 0.2 mmol of the peptide
intermediate, Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine
amide], which was prepared during the synthesis of compound (52).
For guanylation of the homopiperazine at C-terminus, the peptide
was treated with a solution of 1H-Pyrazole-1-carboxamidine
hydrochloride (0.4 g, 3.0 mmol) and DIEA (0.5 ml, 6 mmol) in DMF (3
ml) overnight at room temperature. Acetic acid and H.sub.2O were
added to quench the reaction and the solution was frozen and dried
on a lyophilizer to give the desired protected peptide,
Cbz-DPhe-DPhe-DLeu-DOrn(Cbz)-[4-Amidinohomopiperazine amide] (0.2
mmol). The subsequent deprotection/hydrolysis and HPLC purification
were carried out according to the procedure described in the
synthesis of compound (52). Final purified synthetic peptide amide:
amorphous powder, 74 mg in yield. HPLC analysis: t.sub.R=10.10 min,
purity 98.7%, gradient 10% B to 30% B over 20 min; MS (MH.sup.+):
expected mass 664.4, observed 664.5.
Example 54
Synthesis of compound (54):
D-Phe-D-Phe-D-Leu-D-Orn-[4-(4,5-dihydro-1H-imidazol-2-yl)homopiperazine
amide]
[0309] Synthesis was initiated from 0.2 mmol of the peptide
intermediate, Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine
amide], which was prepared during the synthesis of compound (52).
For guanylation of the homopiperazine at C-terminus, the peptide
was treated with a solution of 2-Methylthio-2-imidazoline
hydroiodide (730 mg, 3.0 mmol; Aldrich) and DIEA (0.5 ml, 6 mmol)
in DMF (3 ml) for four days at room temperature. Acetic acid and
H.sub.2O were added to quench the reaction and the solution was
frozen and dried on a lyophilizer to give the desired protected
peptide,
Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[4-(4,5-dihydro-1H-imidazol-2-yl)homopip-
erazine amide] (0.2 mmol). The subsequent deprotection/hydrolysis
and HPLC purification were carried out according to the procedure
described in the synthesis of compound (52). The final purified
synthetic peptide amide was an amorphous powder, 46 mg in yield.
HPLC analysis: t.sub.R=10.89 min, purity 100%, gradient 10% B to
30% B over 20 min; MS (MH.sup.+): expected molecular ion mass
690.4, observed 690.5.
Example 55
Synthesis of compound (55):
D-Phe-D-Phe-D-Leu-D-Orn-[4-ethylhomopiperazine amide]
[0310] Synthesis was initiated from 0.3 mmol of the peptide
intermediate, Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine
amide], which was prepared during the synthesis of compound (52).
For ethylation of the homopiperazine at C-terminus, the peptide was
treated with a solution of iodoethane (0.4 mmol; Aldrich) and DIEA
(0.5 ml, 6 mmol) in DMF (3 ml) for 1 day at room temperature. The
subsequent deprotection/hydrolysis and HPLC purification were
carried out according to the procedure described in the synthesis
of compound (52). Final purified synthetic peptide amide: amorphous
powder, 75 mg in yield. HPLC analysis: t.sub.R=10.43 min, purity
98.4%, gradient 10% B to 30% B over 20 min; MS (MH.sup.+): expected
mass 650.4, observed 650.3.
Example 56
Synthesis of compound (56): D-Phe-D-Phe-D-Leu-D-Orn-[homopiperazine
amide]
[0311] Synthesis was initiated from 0.3 mmol of the peptide
intermediate, Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine
amide], which was prepared during the synthesis of compound (52).
The peptide was hydrolyzed with a mixture of TMSOTf/TFA/m-cresol
(10 ml, v/v/v=2:7:1) and the crude product was purified by
preparative HPLC according to the procedure described in the
synthesis of compound (52). Final purified synthetic peptide amide:
amorphous powder, 225 mg in yield. HPLC analysis: t.sub.R=16.43
min, purity 100%, gradient 2% B to 22% B over 20 min; MS
(MH.sup.+): expected molecular ion mass 622.4, observed 622.4.
Example 57
Synthesis of compound (57):
D-Phe-D-Phe-D-Leu-(.delta.-Me)D-Orn-[4-amidinohomopiperazine
amide]
[0312] The amino acid derivatives used were Z-D-Phe-OH,
Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, and Fmoc-D-Orn(Aloc)-OH. The fully
protected resin bound peptide was synthesized manually starting
from p-nitrophenyl-carbonate Wang resin (5.0 g, 4.4 mmol;
Novabiochem). The attachment of homo-piperazine to the resin was
achieved by mixing it with a solution of homopiperazine (8.7 g, 87
mmol; Acros Organics) in DCM (100 mL) overnight at room
temperature. The resin was washed with DMF and DCM and dried in
vacuo. The resulting homopiperazine carbamate Wang resin (5.1 g;
homopiperazine-[carbamate Wang resin]) was split into several
portions and a portion of 1.1 g (1 mmol) was used to continue the
peptide synthesis. DIC/HOBt mediated single couplings were
performed with a 3-fold excess of amino acid derivatives. The Fmoc
group was removed with 25% piperidine in DMF. Upon completion of
peptide chain elongation, the resin was treated with
Pd(PPh.sub.3).sub.4 (3.5 g, 3.0 mmol; Aldrich) in a mixture of
CHCl.sub.3/AcOH/NMM (60 ml, v/v/v=37:2:1) under Argon atmosphere at
room temperature for 3 h for Aloc removal. The resin was washed
with DMF and DCM and dried in vacuo. The resulting peptide resin
(1.8 g; Z-D-Phe-D-Phe-D-Leu-D-Orn-homopiperazine-[carbamate Wang
resin]) was split again and a portion of 0.9 g (0.5 mmol) was used
for subsequent derivatization (N-methylation).
[0313] Methylation of the .omega.-amino function of D-Orn at
Xaa.sub.4 was carried out in three steps: (i) [o-NBS Protection]:
The resin-bound peptide (0.5 mmol) was first treated with a
solution o-NBS--Cl (0.4 g, 2 mmol) and collidine (0.7 ml, 5 mmol)
in NMP (7 ml) at room temperature for 30 minutes. The resin was
then washed with NMP. (ii) [N-Methylation]: The resin-bound o-NBS
protected peptide was then reacted with a solution of
1,8-diazabicyclo[5.4.0]undec-7-ene (0.5 ml, 3 mmol) and
dimethylsulfate (1.0 ml, 10 mmol; Aldrich) in NMP (7 ml) at room
temperature for five minutes. The resin was then washed with NMP
and the N-methylation process was repeated once. (iii) [o-NBS
Deprotection]: The peptide resin was treated with a solution of
mercaptoethanol (0.7 ml, 10 mmol) and
1,8-diazabicyclo[5.4.0]undec-7-ene (0.8 ml, 5 mmol) in NMP (7 ml)
at room temperature for five minutes. The resin was then washed
with NMP and the deprotection process was repeated once.
[0314] To protect the resulting N-methyl secondary amine of D-Orn
at Xaa.sub.4, the resin-bound methylated peptide was reacted with a
solution of Cbz-OSu (6 mmol) in DMF (7 ml). The resin was washed
with DMF and DCM and dried in vacuo. The peptide was then cleaved
from the resin by treatment with a solution of TFA/DCM (15 ml,
v/v=1:1) at room temperature for 2 hours. The resin was then
filtered and washed with TFA. The filtrate was evaporated in vacuo
and the crude peptide (0.5 mmol;
Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Me,Cbz)-[homopiperazine amide]) was
obtained as an oil.
[0315] For guanylation of the homopiperazine at the C-terminus, a
portion of the above peptide (0.3 mmol) was treated with a solution
of 1H-Pyrazole-1-carboxamidine hydrochloride (0.4 g, 3.0 mmol) and
DIEA (0.5 ml, 6 mmol) in DMF (3 ml) overnight at room temperature.
Acetic acid and H.sub.2O were added to quench the reaction and the
solution was frozen and dried on a lyophilizer to give the desired
protected peptide,
Z-D-Phe-D-Phe-D-Leu-D-Orn(Me,Z)-[4-Amidinohomopiperazine amide]
(0.3 mmol).
[0316] For final deprotection/hydrolysis, the above peptide (0.3
mmol) was treated with a mixture of TMSOTf/TFA/m-cresol (10 ml,
v/v/v=2:7:1) at room temperature for 2 hours. The mixture was
evaporated affording the crude peptide (0.3 mmol) as an oil. The
preparative HPLC purification of the crude peptide was carried out
according to the procedure as described in the synthesis of
compound (38). Final purified synthetic peptide amide: amorphous
powder, 183 mg in yield. HPLC analysis: t.sub.R=17.12 min, purity
98.9%, gradient 2% B to 22% B over 20 min; MS (MH.sup.+): expected
mass 678.4, observed 678.5.
Example 58
Synthesis of compound (58):
D-Phe-D-Phe-D-Leu-(.delta.-iPr)D-Orn-[.omega.(4-aminopiperidine-4-carboxy-
lic acid)]-OH
[0317] The amino acid derivatives used were Boc-D-Phe-OH,
Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, Fmoc-D-Orn(Aloc)-OH, and
N-Boc-amino-(4-N-Fmoc-piperidinyl) carboxylic acid. The fully
protected resin bound peptide was synthesized manually starting
from 2-Chlorotrityl chloride resin (0.8 mmol). The attachment of
N-Boc-amino-(4-N-Fmoc-piperidinyl) carboxylic acid to resin and
subsequent couplings were carried out according to the procedure
described in the synthesis of compound (38). The assembled peptide
resin,
Boc-D-Phe-D-Phe-D-Leu-D-Orn-.omega.(4-Aminopiperidine-4-carboxylic
acid)-[2-Cl-Trt resin], was treated with Pd(PPh.sub.3).sub.4 (4.5
mmol; Aldrich) in a mixture of CHCl.sub.3/AcOH/NMM (80 ml,
v/v/v=37:2:1) under Argon atmosphere at room temperature for 3 h
for Aloc removal. Subsequent N-isopropylation was carried out
according the procedure described in the synthesis of compound
(59). Final cleavage and preparative HPLC purification were
accomplished according to the procedure described in the synthesis
of compound (38). Final purified synthetic peptide amide: amorphous
powder, 336 mg in yield. HPLC analysis: t.sub.R=18.88 min, purity
98.9%, gradient 5% B to 25% B over 20 min; MS (MH.sup.+): expected
molecular ion mass 708.4, observed 708.4.
Example 59
Synthesis of compound (59):
D-Phe-D-Phe-D-Leu-(.delta.-iPr)D-Orn-[4-amidinohomopiperazine
amide]
[0318] Synthesis was initiated from 0.9 g (0.5 mmol) of the
peptide-resin: Z-D-Phe-D-Phe-D-Leu-D-Orn-homopiperazine-[carbamate
Wang resin], which was prepared during the synthesis of compound
(57) described above.
[0319] For isopropylation of the .omega.-amino function of D-Orn at
Xaa.sub.4, the peptide resin was treated with a mixture of sodium
triacetoxyborohydride (3 mmol) and acetone (6 mmol) in TMOF (10 mL)
over night at room temperature. The peptide resin was then treated
with a solution of Cbz-OSu (6 mmol) in DMF (7 ml) for Cbz
protection. The resin was washed with DMF and DCM and dried in
vacuo. The peptide was then cleaved from the resin by treatment
with a solution of TFA/DCM (15 ml, v/v=1:1) at room temperature for
2 hours. The resin was filtered and washed with TFA. The filtrate
was evaporated in vacuo and the crude peptide (0.5 mmol;
Cbz-D-Phe-D-Phe-D-Leu-D-Orn(iPr,Cbz)-[homopiperazine amide]) was
obtained as an oil.
[0320] A portion of the above peptide (0.3 mmol) was continued for
subsequent guanylation, cleavage and purification steps, which were
carried out according to the procedure described in the synthesis
of compound (57). Final purified synthetic peptide amide: amorphous
powder, 166 mg in yield. HPLC analysis: t.sub.R=18.71 min, purity
99.4%, gradient 2% B to 22% B over 20 min; MS (MH.sup.+): expected
mass 706.5, observed 706.5.
Example 60
Synthesis of compound (60):
D-Phe-D-Phe-D-Leu-(.delta.-Me)D-Orn-[homopiperazine amide]
[0321] Synthesis was initiated from 0.2 mmol of the peptide
intermediate, Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Me,Cbz)-[homopiperazine
amide], which was prepared during the synthesis of compound (57).
The peptide was hydrolyzed with a mixture of TMSOTf/TFA/m-cresol
(10 ml, v/v/v=2:7:1) and the crude product was purified by
preparative HPLC according to the procedure described in the
synthesis of compound (52). Final purified synthetic peptide amide:
amorphous powder, 98 mg in yield. HPLC analysis: t.sub.R=16.38 min,
purity 99.6%, gradient 2% B to 22% B over 20 min; MS (MH.sup.+):
expected molecular ion mass 636.4, observed 636.5.
Example 61
Synthesis of compound (61):
D-Phe-D-Phe-D-Leu-(.delta.-iPr)D-Orn-[homopiperazine amide]
[0322] Synthesis was initiated from 0.2 mmol of the peptide
intermediate, Cbz-D-Phe-D-Phe-D-Leu-D-Orn(iPr,Cbz)-[homopiperazine
amide], which was prepared during the synthesis of compound (59).
The peptide was hydrolyzed with a mixture of TMSOTf/TFA/m-cresol
(10 ml, v/v/v=2:7:1) and the crude product was purified by
preparative HPLC according to the procedure described in the
synthesis of compound (52). Final purified synthetic peptide amide:
amorphous powder, 87 mg in yield. HPLC analysis: t.sub.R=18.41 min,
purity 100%, gradient 2% B to 22% B over 20 min; MS (MH.sup.+):
expected molecular ion mass 664.5, observed 664.5.
Example 62
Synthesis of compound (62):
D-Phe-D-Phe-D-Leu-D-Lys-[1,3-dioxolan-2-yl)methanamine amide]
[0323] Intermediate I-8 was prepared as described in Example 25. To
a solution of the acid, intermediate I-8 (150 mg, 0.20 mmol), the
amine HNR.sub.aR.sub.b, (1,3-dioxolan-2yl)methanamine (31 mg, 0.30
mmol) and DIEA (175 ul, 1.0 mmol) in DMF (5 mL) was added HBTU (113
mg, 0.3 mmol) at 0.degree. C. After stirring overnight from a
starting temperature of 0.degree. C. to room temperature, DMF was
evaporated under reduced pressure. The residue was stirred with 4N
HCl in 1,4-dioxane (2.0 mL) at room temperature for 1 hour. After
removal of dioxane, the residue was dissolved in water and purified
by RP HPLC with a gradient of 10% MeCN/water to 60% MeCN/water in
30 minutes to give pure synthetic peptide amide, compound (62) (44
mg, 44% yield for the two steps) after evaporation of solvent.
LC-MS: m/z=639 (MW).
Examples 63-75
Synthesis of Compounds (63)-(75)
[0324] Compounds (63)-(75) were prepared essentially as described
in Example 62, by substituting the appropriate amide in the
coupling step as detailed in U.S. Pat. No. 7,713,937. For example,
for compound (63) 2-(piperazin-1-yl)pyrimidine was used in the
amide coupling step.
Example 76
Synthesis of Compound (76)
##STR00058##
[0326] The amino acid derivatives, Boc-D-Phe-OH, Fmoc-D-Phe-OH,
Fmoc-D-Leu-OH, Boc-D-Leu-OH, Fmoc-D-Orn(Boc)-OH, and
Fmoc-Lys(Dde)-OH were used in this synthesis. The fully protected
resin bound peptide was synthesized manually starting from
2-chlorotrityl chloride resin (0.3 mmol; Peptide International).
The attachment of the first amino acid to the resin was achieved by
treatment with a mixture of Fmoc-Lys(Dde)-OH (0.29 g, 0.5 mmol;
Novabiochem) and DIEA (0.35 mL, 2 mmol) in DCM (7 ml) at room
temperature for 4 hours. The resin was washed with
3.times.DCM/MeOH/DIEA (v/v/v=17:2:1) and then treated with 25%
piperidine in DMF for Fmoc removal. The subsequent peptide chain
elongation was achieved by PyBOP/DIEA mediated single couplings
with a 3-fold excess of amino acid derivatives, Fmoc-D-Orn(Boc)-OH
and Boc-D-Leu-OH. The resulting peptide resin,
Boc-D-Leu-D-Orn(Boc)-Lys(Dde)-[2-Cl-Trt resin], was treated with 4%
hydrazine in DMF three times for 3 min each to remove Dde. The
subsequent peptide chain elongation was achieved by PyBOP/DIEA
mediated single couplings with a 3-fold excess of amino acid
derivatives, Fmoc-D-Orn(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, and
Boc-D-Phe-OH. The Fmoc group was removed with 25% piperidine in
DMF. The fully assembled peptide was cleaved from the resin by
treatment with a mixture of TFA/TIS/H.sub.2O (15 ml,
v/v/v=95:2.5:2.5) at room temperature for 90 minutes. The resin was
filtered and washed with TFA. The filtrate was evaporated in vacuo
and the crude peptide (0.3 mmol;
D-Phe-D-Phe-D-Leu-D-Orn-[eLys(D-Orn-D-Leu-H)]--OH) was precipitated
from diethyl ether.
[0327] For purification, the crude peptide (0.3 mmol) was dissolved
in 2% acetic acid in H.sub.2O (50 ml) and the solution was loaded
onto an HPLC column and purified using TEAP buffer system with a pH
5.2 (buffers A=TEAP 5.2 and B=20% TEAP 5.2 in 80% ACN). The
compound was eluted with a linear gradient of buffer B, 10% B to
40% B over 60 minutes. Fractions with purity exceeding 95% were
pooled and the resulting solution was diluted with two volumes of
water. The diluted solution was then loaded onto an HPLC column for
salt exchange and further purification with a TFA buffer system
(buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 80% ACN/20%
H.sub.2O) and a linear gradient of buffer B, 2% B to 75% B over 25
minutes. Fractions with purity exceeding 97% were pooled, frozen,
and dried on a lyophilizer to yield the purified peptide as white
amorphous powder (396 mg). HPLC analysis: t.sub.R=13.63 min, purity
99.7%, gradient 10% B to 30% B over 20 min; MS (MH.sup.+): expected
mass 895.5, observed 895.6.
Example 77
Synthesis of Compound (77)
##STR00059##
[0329] For the synthesis of compound (77) an additional amino acid
residue D-Phe in the peptide resin intermediate,
Boc-D-Phe-D-Leu-D-Orn(Boc)-Lys(Dde)-[2-Cl-Trt resin]. The resin
intermediate was prepared by attachment of Fmoc-Lys(Dde)-OH to
2-Chlorotrityl chloride resin followed by Fmoc removal and
couplings of amino acid derivatives Fmoc-D-Orn(Boc)-OH,
Fmoc-D-Leu-OH, and Boc-D-Phe-OH. Final purified peptide: amorphous
powder, 508 mg in yield in a synthesis scale of 0.3 mmol HPLC
analysis: t.sub.R=18.90 min, purity 100%, gradient 10% B to 30% B
over 20 min; MS (MH.sup.+): expected molecular ion mass 1042.4,
observed 1042.7.
Examples 78-89
Synthesis of Compounds (78)-(89)
[0330] These compounds, (78)-(89), listed above, can be synthesized
by methods analogous to those used in the synthesis of compounds
(76) and (77) described above.
Example 90
Synthesis of compound (90)
D-Phe-D-Phe-D-Leu-D-Orn-[R/S-2-carboxymorpholine]-OH
[0331] The amino acid derivatives used were Boc-D-Phe-OH,
Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, Fmoc-D-Orn(Boc)-OH, and
(R,S)-Fmoc-2-carboxymorpholine. The fully protected resin bound
peptide was synthesized on a SYMPHONY Multiple Synthesizer (Protein
Technology Inc.) starting from 2-Chlorotrityl chloride resin (0.4
mmol; Novabiochem). The attachment of the first amino acid to the
resin was achieved by treatment with a mixture of
(R,S)-Fmoc-2-carboxymorpholine (0.18 g, 0.5 mmol; NeoMPS) and DIEA
(0.35 mL, 2 mmol) in DCM (7 ml) at room temperature for 4 hours.
The resin was washed with 3.times.DCM/MeOH/DIEA (v/v/v=17:2:1) and
3.times.DCM. The subsequent peptide chain elongation was achieved
by HBTU/DIEA mediated single couplings with a 3-fold excess of
amino acid derivatives. The Fmoc group was removed by 25%
piperidine in DMF. For cleavage, the final peptide resin was
treated with a mixture of TFA/TIS/H2O (15 ml, v/v/v=95:2.5:2.5) at
room temperature for 90 minutes. The resin was filtered and washed
with TFA. The filtrate was evaporated in vacuo and the crude
peptide (0.15 g, DPhe-DPhe-DLeu-DOrn-[R/S-2-carboxymorpholine]-OH)
was precipitated from diethyl ether.
[0332] For purification, the above crude peptide (0.15 g) was
dissolved in 0.1% TFA in H.sub.2O (50 ml) and the solution was
loaded onto an HPLC column and purified using TFA buffer system
(buffers A=0.1% TFA in H.sub.2O and B=0.1% TFA in 60% ACN/40%
H.sub.2O). The compound was eluted with a linear gradient of buffer
B, 25% B to 75% B over 30 min, t.sub.R=45% B. The fractions with
purity exceeding 97% were pooled, frozen, and dried on a
lyophilizer to give the purified peptide as white amorphous powder
(84 mg). The compound was a mixture of diastereoisomers as no
attempt was made to separate the two isomers,
DPhe-DPhe-DLeu-DOrn-[R-2-carboxymorpholine]-OH and
DPhe-DPhe-DLeu-DOrn-[S-2-carboxymorpholine]-OH. HPLC analysis:
t.sub.R=16.93 min (49.6%) and 17.34 min (50.4%), combined purity
100%, gradient 10% B to 30% B over 20 minutes.
##STR00060##
Synthesis of Compounds (91)-(103)
[0333] Compounds (91)-(103) can be prepared by the above-described
synthetic methods and by other synthetic methods well known in the
art.
Example 104
Synthesis of Compound (104)
##STR00061##
[0335] Step 1: To a solution of H-D-Phe-DLeu-OBn, I-4 (2.0 g, 4.27
mmol), Boc-D-tert-Leu-OH (924 mg, 4.0 mmol) or
Boc-D-tert-Butyl-D-Ala-OH (980 mg, 4.0 mmol) and DIEA (1.0 mL, 6.0
mmol) in DMF was added HBTU (1.52 g) in three portions at 0.degree.
C. over a 10 minute period. The mixture was stirred from 0.degree.
C. to room temperature overnight. After evaporation of DMF, the
residue was extracted between ethyl acetate and saturated sodium
bicarbonate. The organic layer was dried over sodium sulfate and
evaporated to dryness. The residue was purified by column
chromatography with 10% to 60% ethyl acetate/hexanes to give pure
product. (96% yield for R=tert-butyl and 90% yield for
R=neopentyl). The dipeptide benzyl ester was hydrogenated with
palladium on carbon (10%, 300 mg) with a hydrogen balloon in MeOH
for 3 hours. The mixture was filtered through celite and evaporated
to give the tripeptide product (91% yield for R=tert-butyl and 100%
yield for R=neopentyl).
[0336] Step 2: To a solution of Fmoc-D-Lys(Boc)-OH (468 mg, 1.0
mmol), benzyl 4-(benzyloxycarbonylamino)piperidine-4-carboxylate
(368 mg, 1.0 mmol) and DIEA (520 uL, 3.0 mmol) in DMF was added
HBTU (474 mg, 1.25 mmol) in three portions at 0.degree. C. over 10
minutes. The mixture was stirred from 0.degree. C. to room
temperature overnight. After evaporation of DMF, the residue was
extracted between ethyl acetate and saturated sodium bicarbonate.
The organic layer was dried over sodium sulfate and evaporated to
dryness. The residue was purified by column chromatography with 30%
to 60% ethyl acetate/hexanes to give pure product (760 mg, 93%,
m/e=819 (MH).sup.+). The intermediate obtained (760 mg) was
dissolved in DCM and then pyrrolidine (4 mL) was added. After
stirring at room temperature for 1 hour, the DCM was evaporated
from the mixture. The residue was dissolved in MeOH and purified by
a C18 column with 5% to 60% MeCN/water in 30 mins. Pure fractions
were combined and lyophilized overnight to give the desired product
(580 mg).
[0337] Step 3: To a solution of the tripeptide obtained in step 1
(R=neopentyl, 152 mg, 0.30 mmol), the product obtained in step 2
(179 mg, 0.30 mmol) and DIEA (170 uL, 1.0 mmol) in DMF was added
HBTU (152 mg, 0.40 mmol) at 0.degree. C. The mixture was stirred
from 0.degree. C. to room temperature for 2 hours. After
evaporation of DMF, the residue was dissolved in MeOH and purified
by a C18 column chromatography with a gradient of 30% to 100%
MeCN/water in 40 minutes. Pure fractions were combined and
lyophilized overnight to give the desired product (180 mg, 55%,
m/e=1084 (M+H)).
[0338] Step 4: A solution of the product obtained in step 3 (82 mg)
in MeOH was hydrogenated with palladium on carbon (10%, 20 mg)
under a hydrogen balloon for 1 hour. The mixture was filtered
through celite and evaporated to dryness. The residue obtained was
stirred with TFA/DCM (4 mL, 1:1 v/v) at room temperature for 1
hour. After evaporation of solvent, the residue was purified by C18
column chromatography by elution with a gradient of 0% to 100%
MeCN/water in 40 minutes. Pure fractions were combined and
lyophilized overnight to give compound (104) (39 mg, 78% yield,
LC-MS: m/z=661 (MH).sup.+).
Example 105
Synthesis of Compound (105)
##STR00062##
[0340] Step 1: To a solution of H-D-tert-Leu-D-Phe-D-Leu-OH
synthesized according to step 1 in the synthesis of compound (104)
(R=tert-butyl, 491 mg, 1.0 mmol), D-Lys(Boc)-OAll (323 mg, 1.0
mmol) and DIEA (440 uL, 2.5 mmol) in DMF was added HBTU (400 mg,
0.40 mmol) at 0.degree. C. The mixture was stirred from 0.degree.
C. to room temperature for 3 hours. After evaporation of DMF, the
residue was precipitate in water (200 mL) and filtered to give the
tetrapeptide intermediate (0.79 g, 99% yield, m/z=761, (MH).sup.+).
To a solution of the tetrapeptide allyl ester (0.79 g, 1.04 mmol)
in MeCN was added palladium tetrakis-(triphenylphosphine) (120 mg)
and pyrrolidine (345 uL) at 0.degree. C. After stirring at room
temperature overnight, the acetonitrile was evaporated from the
mixture. The residue was purified by C18 column chromatography with
a gradient of 30% to 90% MeCN/water. Pure fractions were combined
and lyophilized to give the desired product (0.41 g, 57%), m/z=720
(MH).sup.+.
[0341] Step 2: To a solution of the tetrapeptide obtained in Step 1
(180 mg, 0.25 mmol), benzyl
4-(benzyloxycarbonyl-amino)piperidine-4-carboxylate (101 mg, 0.25
mmol) and DIEA (250 uL, 1.4 mmol) in DMF was added HBTU (140 mg,
0.37 mmol) at 0.degree. C. The mixture was stirred from 0.degree.
C. to room temperature overnight. After evaporation of DMF, the
residue was purified purified by C18 column chromatography with a
gradient of 25% to 80% MeCN/water. Pure fractions were combined and
lyophilized to give the desired product (0.37 g, 100%), m/z=1071
(MH).sup.+.
[0342] Step 3: A solution of the product obtained in Step 2 (0.37
g) in TFA/DCM was stirred at room temperature for 1 hour. After
evaporation of solvent, the residue was extracted with ethyl
acetate and saturated sodium bicarbonate. The organic layer was
dried over sodium sulfate and evaporated to dryness. The residue
(0.20 g) was hydrogenated with palladium on carbon (10%, 50 mg) in
MeOH under a hydrogen balloon for 1 hour. The mixture was filtered
through celite and evaporated to dryness. The residue was dissolved
in MeOH and purified by C18 column chromatography with a gradient
of 5% to 60% MeCN/water. Pure fractions were combined and
lyophilized to give compound (105) (50 mg, 27%), m/z=647
(MH).sup.+.
Example 106
Inhibition of cAMP Production by Stimulation of Endogenous Mouse
Kappa-Opioid Receptor in R1.G1 Cells
[0343] Potency of the synthetic peptide amides as kappa-opioid
receptor agonists was determined by measuring the inhibition of
forskolin-stimulated adenylate cyclase activity. R1.G1 cells (a
mouse thymoma cell line that expresses only the kappa-opioid
receptor and no other opioid receptor subtype) were first exposed
to forskolin (to induce cAMP) plus the synthetic peptide amide at
the test concentration. After incubation, the cAMP level in the
challenged R1.G1 cells was determined using a time resolved
fluorescence resonance energy transfer (TR-FRET)-based cAMP
immunoassay (LANCE.TM., Perkin Elmer). The detailed method is
described below:
[0344] Mouse R1.G1 cells (ATCC, Manassas, Va.) were grown in
suspension in high glucose-DMEM (Dulbecco's Modified Eagle's
Medium, Cellgro, Herndon, Va.) containing 10% horse serum and 2%
glutaMax (Invitrogen, Carlsbad. Calif.) without added antibiotics.
On the day of the experiment, cells were spun at 1,000 rpm for 5
minutes at room temperature and then washed once with HBSS (Hanks'
Balanced Saline Solution, Invitrogen, Carlsbad, Calif.). Cells were
then spun again and resuspended in stimulation buffer (HBSS with
0.05% FAF-BSA (Fatty acid-free bovine serum albumin, Roche Applied
Science, Indianapolis, Ind.), 5 mM HEPES) to 2 million cells per
ml. Antibody supplied with the LANCE.TM. cAMP immunoassay kit was
then added to the cells according to the manufacturer's
instructions, and 12,000 cells per well were then added to the
wells containing forskolin to a predetermined fixed final
concentration (typically about 2.5 uM) and the previously
determined amount of the synthetic peptide amide to be tested.
[0345] The synthetic peptide amides were tested in a range of
concentrations to determine potency. Cells were incubated with the
synthetic peptide amide plus forskolin for about 20 minutes at room
temperature. After incubation, cells are lysed by adding 12 ul of
detection mix as supplied with the LANCE.TM. kit, followed by
incubation for one hour at room temperature. Time resolved
fluorescence was read using a 330-380 nm excitation filter, a 665
nm emission filter, dichroic mirror 380, and Z=1 mm A standard
curve for cAMP concentration in this assay permitted determination
of the amount of cAMP present in each well. A curve was produced by
plotting synthetic peptide amide concentration against cAMP levels
in the test cells, and subjected to non-linear regression using a
four-parameter curve fitting algorithm to calculate the EC.sub.50,
the concentration of the synthetic peptide amide required to
produce 50% of the maximal suppression of cAMP production by the
synthetic peptide amide.
Example 107
Potency of Synthetic Peptide Amides on the Human Kappa Opioid
Receptor
[0346] Human Embryonic Kidney cells (HEK-293 cells, ATCC, Manassas,
Va.) in 100 mm dishes were transfected with transfection reagent,
Fugene6 (Roche Molecular Biochemicals) and DNA constructs in a 3.3
to 1 ratio. The DNA constructs used in the transfection were as
follows: (i) an expression vector for the human kappa opioid
receptor, (ii) an expression vector for a human chimeric G-protein,
and (iii) a luciferase reporter construct in which luciferase
expression is induced by the calcium sensitive transcription factor
NFAT.
[0347] The expression vector containing the human kappa opioid
receptor was constructed as follows: The human OPRK1 gene was
cloned from human dorsal root ganglion total RNA by PCR and the
gene inserted into expression vector pcDNA3 (Invitrogen, Carlsbad,
Calif.) to construct human OPRK1 mammalian expression vector
pcDNA3-hOPRK1.
[0348] To construct the human chimeric G-protein expression vector,
the chimeric G-protein G.alpha.qi5 was first constructed by
replacing the last 5 amino acids of human G.alpha.q with the
sequence of the last 5 amino acids of G.alpha.i by PCR. A second
mutation was introduced to this human G.alpha.qi5 gene at amino
acid position 66 to substitute a glycine (G) with an aspartic acid
(D) by site-directed mutagenesis. This gene was then subcloned into
a mammalian expression vector pcDNA5/FRT (Invitrogen) to yield the
human chimeric G-protein expression vector,
pcDNA5/FRT-hGNAq-G66D-i5.
[0349] To prepare the luciferase reporter gene construct, synthetic
response elements including 3 copies of TRE
(12-O-tetradecanoylphorbol-13-acetate-responsive elements) and 3
copies of NFAT (nuclear factor of activated T-cells) were
incorporated upstream of a c-fos minimal promoter. This response
element and promoter cassette was then inserted into a luciferase
reporter gene vector pGL3-basic (Promega) to construct the
luciferase reporter gene plasmid construct
pGL3b-3TRE-3NFAT-cfos-Luc.
[0350] The transfection mixture for each plate of cells included 6
micrograms pcDNA3-hOPRK1, 6 micrograms of pcDNA5/FRT-hGNAq-G66D-i5,
and 0.6 micrograms of pGL3b-3TRE-3NFAT-cfos-Luc. Cells were
incubated for one day at 37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2 following transfection, and plated in opaque
96-well plates at 45,000 cells per well in 100 microliters of
medium. The next day, test and reference compounds were added to
the cells in individual wells. A range of concentrations of test
compounds was added to one set of wells and a similar range of
concentrations of reference compounds was added to a set of control
wells. The cells were then incubated for 5 hours at 37.degree. C.
At the end of the incubation, cells were lysed by adding 100
microliters of detection mix containing luciferase substrate (AMP
(22 ug/ml), ATP (1.1 mg/ml), dithiothreitol (3.85 mg/ml), HEPES (50
mM final concentration), EDTA (0.2 mg/ml), Triton N-101 (4 ul/ml),
phenylacetic acid (45 ug/ml), oxalic acid (8.5 ug/ml), luciferin
(28 ug/ml), pH 7.8). Plates were sealed and luminescence read
within 30 minutes. The concentration of each of the compounds was
plotted against luminescence counts per second (cps) and the
resulting response curves subjected to non-linear regression using
a four-parameter curve-fitting algorithm to calculate the EC.sub.50
(the concentration of compound required to produce 50% of the
maximal increase in luciferase activity) and the efficacy (the
percent maximal activation compared to full induction by any of the
well-known kappa opioid receptor agonists, such as asimadoline
(EMD-61753: See Joshi et al., 2000, J. Neurosci. 20(15):5874-9), or
U-69593: See Heidbreder et al., 1999, Brain Res.
616(1-2):335-8).
[0351] The EC.sub.50 values were obtained from the cAMP inhibition
assay with synthetic peptide amides synthesized according to the
present invention and tested on mouse kappa opioid receptor (mKOR
EC.sub.50 values from 0.005-100 nM) and on the human kappa opioid
receptor (hKOR EC.sub.50 values from 0.001-1 nM) as described
above.
[0352] Synthetic peptide amides of the invention were tested in a
similar assay for potency on the human mu opioid receptor. Each
compound tested had an EC.sub.50 for the human mu opioid receptor
greater than or equal to 1 .mu.M. The synthetic peptide amide (53)
was tested in a similar assay for potency on the human mu opioid
receptor. The compound had an EC.sub.50 for the human mu opioid
receptor greater than or equal to 1 .mu.M.
Example 108
Membrane Permeability of the Synthetic Peptide Amides
[0353] The Caco-2 cell line is a human colon adenocarcinoma cell
line that differentiates in culture and is used to model the
epithelial lining of the human small intestine. Compounds of the
present invention can be tested in a membrane permeability assay
using the TC7 subclone of Caco-2 in a standard assay (Cerep,
Seattle, Wash.). Briefly, the apparent permeability coefficient
(P.sub.app) can be determined in the apical-to-basolateral (A-B)
direction across cell monolayers cultured on 96-well polycarbonate
membrane filters.
[0354] Compounds were tested at a concentration of 10 .mu.M at pH
6.5 in 1% DMSO, with the recipient side maintained at pH 7.4. The
assay plate was incubated for 60 minutes at 37.degree. C. with
gentle shaking. Samples were taken at time zero from the donor side
and at the end of the incubation period from both the donor and
recipient sides. Samples were analyzed by HPLC-MS/MS. The
P.sub.app-value (expressed as 10.sup.-6 cm/sec) was then calculated
based on the appearance rate of compound in the recipient side. The
P.sub.app was calculated with the equation:
P app = 1 S C 0 ( Q T ) ##EQU00001##
where P.sub.app is the apparent permeability; S is the membrane
surface area, C.sub.0 is the donor concentration at time 0, and
dQ/dt is the amount of drug transported per time. Four reference
compounds (labetalol, propranolol, ranitidine, and vinblastine)
were concurrently tested to ensure the validity of the assay, as
well as asimadoline, which is purported to be a peripherally acting
kappa opioid. Results are shown in Table I (below).
[0355] Compounds that exhibit low permeability in this type of
assay are believed to have reduced potential for crossing the
blood-brain barrier in vivo, since high passive permeability
appears to be a key feature of CNS-acting drugs (Mahar Doan et al.
Passive permeability and P-glycoprotein-mediated efflux
differentiate central nervous system (CNS) and non-CNS marketed
drugs. J Pharmacol Exp Ther. 2002; 303:1029-37).
TABLE-US-00001 TABLE I Membrane permeability Mean Permeability
Compound (cm.sup.-6/sec) (1) <0.10 (3) <0.02 (6) <0.02
Asimadoline 37.5 Labetalol 9.9 Propranolol 53.8 Ranitidine 0.5
Vinblastine <0.2
Example 109
Inhibition of Cytochrome P.sub.450 Oxidases
[0356] Inhibition of cytochrome P.sub.450 oxidase isozymes CYP1A,
CYP2C9, CYP2C19, CYP2D6 and CYP3A4 by synthetic peptide amide
compounds of the invention was determined according to the
following methods performed by Cerep (Seattle, Wash.):
[0357] In the cytochrome P.sub.450 CYP1A assay, human liver
microsomes (0.2 mg/ml protein) were incubated for 15 minutes at
37.degree. C. with 10 .mu.M test compound, 1 .mu.M ethoxyresorufin,
1.3 mM NADP, 3.3 mM glucose-6-phosphate and 0.4 U/ml
glucose-6-phosphate dehydrogenase. In the absence of test compound,
the ethoxyresorufin added as substrate is oxidized to resorufin,
and in the presence of an inhibitor of the CYP isozyme, the amount
of resorufin produced is reduced. Furafylline was used as a
reference inhibitor.
[0358] The cytochrome P.sub.450 CYP2C9 assay reaction mixture
containing human liver microsomes (0.2 mg/ml protein) was incubated
for 15 minutes at 37.degree. C. with 10 .mu.M test compound, 10
.mu.M tolbutamide, 1.3 mM NADP, 3.3 mM glucose-6-phosphate and 0.4
U/ml glucose-6-phosphate dehydrogenase. In the absence of test
compound, the tolbutamide is oxidized to 4-hydroxytolbutamide, and
in the presence of an inhibitor of the CYP isozyme, the amount of
4-hydroxytolbutamide produced is reduced. Sulfaphenazole
(IC.sub.50: 0.35 .mu.M) was the reference inhibitor.
[0359] For the cytochrome P.sub.450 CYP2C19 assay, human liver
microsomes (0.2 mg/ml protein) were incubated for 15 minutes at
37.degree. C. with 10 .mu.M test compound, 10 .mu.M omeprazole, 1.3
mM NADP, 3.3 mM glucose-6-phosphate and 0.4 U/ml
glucose-6-phosphate dehydrogenase. In the absence of test compound,
the omeprazole is oxidized to 5-hydroxy-omeprazole, and in the
presence of an inhibitor of the CYP isozyme, the amount of
5-hydroxy-omeprazole produced is reduced. Oxybutinin (IC.sub.50:
7.1 .mu.M) was the reference inhibitor.
[0360] The cytochrome P.sub.450 CYP2D6 assay reaction containing
human liver microsomes (0.2 mg/ml protein) was incubated for 15
minutes at 37.degree. C. with 10 .mu.M test compound, 5 .mu.M
dextromethorphan, 1.3 mM NADP, 3.3 mM glucose-6-phosphate and 0.4
U/ml glucose-6-phosphate dehydrogenase. In the absence of test
compound, the dextromethorphan is oxidized, and in the presence of
an inhibitor of the CYP isozyme, the amount of oxidation product is
reduced. Quinidine (IC.sub.50: 0.093 .mu.M) was the reference
inhibitor.
[0361] For the cytochrome P.sub.450 CYP2C19 assay, human liver
microsomes (0.2 mg/ml protein) were incubated for 20 minutes at
37.degree. C. with 10 .mu.M test compound, 5 .mu.M midazolam, 1.3
mM NADP, 3.3 mM glucose-6-phosphate and 0.4 U/ml
glucose-6-phosphate dehydrogenase. In the absence of test compound,
the midazolam is oxidized, and in the presence of an inhibitor of
the recombinant isozyme, the amount of oxidation product is
reduced. The oxidation product is determined from the area under
the curve after HPLC-MS/MS separation. Ketoconazole (IC.sub.50:
0.55 .mu.M) was the reference inhibitor.
[0362] In each assay, the percent inhibition of the cytochrome
P.sub.450 CYP P.sub.450 isozyme was determined as one hundred times
the ratio of (1-minus the amount of product in the sample in the
presence of the test compound) divided by the amount of product in
the sample containing untreated isozyme. The results of duplicate
assays (expressed as percent remaining CYP activity) are shown in
Table II.
TABLE-US-00002 TABLE II Percent Activity of Cytochrome P.sub.450
CYP Isozymes Compound (54) (54) P.sub.450 isozyme (1) (3) (6) Expt
1 Expt 2 CYP1A 89.8 93.1 89.5 97.1 95.8 CYP2C9 93.2 97.4 92.1 99.2
98.2 CYP2C19 98.5 103.2 97.2 94.1 97.8 CYP2D6 96.0 99.5 93.9 98.4
98.2 CYP3A4 92.5 94.3 93.6 94.7 95.9
Example 110
Pharmacokinetics of Compound (2) in Rat
[0363] To determine brain to plasma concentration ratios of
compound (2), a group of 6 conscious jugular vein catheterized rats
were administered 3 mg/kg of peptide over a 5 minute infusion
period into the jugular vein catheter. Thirty, 60 and 180 minutes
following the start of infusion, blood samples were collected from
two animals at each time point by terminal cardiac puncture and
whole brains were rapidly removed. Plasma was isolated by
centrifugation. Tandem liquid chromatography mass spectrometry
(LC-MS/MS) was used to quantify the concentration of drug in rat
plasma and brain. Results are shown in FIG. 1.
Example 111
Pharmacokinetics of Compound (6) in Mice
[0364] A single bolus of the synthetic peptide amide compound was
administered by subcutaneous injection to ICR mice (n=6, males,
body wt 23-37 g, Charles River, Wilmington, Mass.) and plasma
samples taken at 5, 10, 15, 20, 30 60, 90, 120, and 180 minutes
post-injection. FIG. 2 shows the results obtained after
subcutaneous injection of a 1 mg/kg dose of compound (6) in ICR
mice. The "half life" for this study was determined as the time
required for the plasma concentration to fall by 50% after maximum
concentration in the plasma was achieved; the computed elimination
half-life, based on the elimination rate constant of the slowest
elimination phase, is expected to be longer. See Table III
below.
Example 112
Pharmacokinetics of Synthetic Peptide Amide Compounds (52) and (6)
in Cynomolgus Monkeys
[0365] A single bolus of the synthetic peptide amide compound was
administered by intravenous injection to cynomolgus monkeys (n=4
males, body wt 3-5 kg, SNBL USA, Ltd., Everett, Wash.), and plasma
samples taken at 5, 10, 15, 20, 30 60, 90 120, and 180 minutes
post-injection. The "half life" was determined as the time required
for the plasma concentration to fall by 50% after maximum
concentration in the plasma was achieved. Cynomolgus monkeys were
injected with a single intravenous dose of synthetic peptide amide
compound (52) according to the protocol detailed in Example 52, and
the half life of plasma concentration determined.
[0366] Results are shown in Table III.
TABLE-US-00003 TABLE III In vivo half life of synthetic peptide
amide compounds (6) and (52) Cynomolgus Monkeys (6) (52)
Administration Route intravenous intravenous Half Life (min) 58.6
69
[0367] Persistence of compound (3) in the plasma of cynomolgus
monkeys after intravenous administration of a bolus of 0.56 mg/kg
is shown in FIG. 3.
Example 113
Acetic Acid-Induced Writhing Assay in Mice
[0368] This test identifies compounds which exhibit analgesic
activity against visceral pain or pain associated with activation
of low pH-sensitive nociceptors [see Barber and Gottschlich (1986)
Med. Res. Rev. 12: 525-562; Ramabadran and Bansinath (1986) Pharm.
Res. 3: 263-270]. Intraperitoneal administration of dilute acetic
acid solution causes a writhing behaviour in mice. A writhe is
defined as a contraction of the abdominal muscles accompanied by an
extension of the forelimbs and elongation of the body. The number
of writhes observed in the presence and absence of test compounds
is counted to determine the analgesic activity of the
compounds.
[0369] Each day a writhing assay was performed, a vehicle control
group of mice (n=6-8) that were treated identically to the test
group (except that test compound was omitted from the injection
dose) was always included and the average total number of writhes
in this group used as the absolute reference point defining 0%
decrease in pain perception for all other mice receiving a test
compound on that day. Specifically, the total number of writhes of
each mouse receiving the test compound was converted to % decrease
in pain perception according to the following equation:
% decrease in pain perception = ( W v - W c ) W v .times. 100
##EQU00002##
[0370] Where W.sub.v is the mean number of writhes in
vehicle-treated group and W.sub.c is the number of writhes in
compound-treated mouse. The data were analyzed using the
2-parameter Hill's equation (a.k.a. Emax model), where Emax is
assumed to be 100% antinociperception (i.e., no writhes over the 15
min post-acetic acid administration).
[0371] Male ICR mice, 23-37 grams in weight, were weighed and
placed in individual observation chamber (usually a 4000 ml glass
beaker) with a fine layer of SANI-CHIPS rodent bedding at the
bottom. To determine the activity and potency of test compounds,
different doses of the compound solution or vehicle were injected
subcutaneously in the back of the neck 15 or 180 minutes prior to
administration of acetic acid solution. After administration of the
compound or vehicle control, mice were returned to their individual
observation chambers awaiting the intraperitoneal administration of
acetic acid solution. Fifteen minutes or three hours later,
according to the interval time defined in each experiment between
compound delivery and acetic acid injection, a dose corresponding
to 10 ml/kg of a 0.6% (v/v) acetic acid solution was then injected
intraperitoneally (i.p.) into the right lower quadrant of the
abdomen Immediately after the injection, the mouse was returned to
its observation chamber and the recording of the number of writhes
begun immediately. The number of writhes was counted over a 15-min
period starting from the time of acetic acid injection, the data
being collected over three separate 5 minute time periods (0-5 min,
5-10 min, and 10-15 min).
[0372] The data were reported as ED.sub.50, and Hill coefficient.
The ED.sub.50 is expressed either as mean.+-.standard error of the
mean (sem) (ED.sub.50+/-sem) or as geometric mean with 95%
confidence intervals (95% CI) using t-scores. The Hill coefficient
is expressed as the arithmetic mean.+-.sem calculated from the
values obtained from the animals. Results for compound (2) are
shown in FIG. 4 (solid circles).
[0373] For dose-response analysis, raw data were converted to %
maximum possible effect (% MPE) using the formula: % MPE=((test
score-vehicle-treated score)/(0-vehicle-treated score)).times.100.
Raw data were analyzed using a one-way ANOVA followed by Dunnett's
post-tests. The dose which elicited 50% attenuation of
hypersensitivity (ED.sub.50) was determined using linear regression
analysis. Compounds were administered by the intravenous route.
Table IV summarizes the results of these experiments.
TABLE-US-00004 TABLE IV Effects of Compounds (2) and (5) on Acetic
Acid-Induced Writhing in Mice. ED.sub.50 % MPE % MPE % MPE Com-
(mg/kg, iv, (180 min (240 min (300 min pound 15 min post-dose)
post-ED.sub.90) post-ED.sub.90) post-ED.sub.90) (2) 0.07 (0.06-0.1)
77 .+-. 5% 81 .+-. 4% 84 .+-. 4% (5) 0.01 (0.01-0.02) 54 .+-. 10%
NT NT NT = not tested
[0374] A dose response for compound (2) in the acetic acid-induced
writhing model in mice was generated using 0.01, 0.03, 0.1 and 0.3
mg/kg administered intravenously as described above. Using the
above method a linear dose response relationship was determined for
compound (2) for doses ranging from 0.01 mg/kg to 0.3 mg/kg, as
shown in FIG. 5.
Example 114
Inhibition of Locomotion in Mice to Measure Sedation by Compounds
after Subcutaneous Injection (Locomotion Reduction Assay)
[0375] Compounds which exhibit sedative activity inhibit the
spontaneous locomotion of mice in a test chamber. To determine the
potential sedative effect of test compounds, the extent of
locomotion after the administration of the test compound or vehicle
control can be determined and compared with a specialized apparatus
designed for this purpose (Opto-Varimex Activity Meter). At the
start of each experiment, each mouse was weighed and examined to
determine good health. To determine the activity and potency of
compounds, different doses of the compound solution or vehicle were
injected subcutaneously 15 or 180 minutes prior to initiation of
data collection. The subcutaneous injection was performed in the
back of the neck of the mouse, pinched in a "tent" to allow proper
access for the syringe needle. After injection, each animal was
placed individually in Plexiglas boxes (43 cm.times.43 cm) inside
the Opto-Varimex Activity Meter apparatus. Before the animal was
placed in the apparatus, a thin layer of SANI-CHIPS rodent bedding
was placed on the bottom of the Plexiglas box to provide a
comfortable environment. Each Opto-Varimex Activity Meter apparatus
was then turned on and data acquisition begun by the ATM3
Auto-Track System. The data were processed and results expressed in
the same way as described for the writhing assay data above.
Example 115
Analgesic vs. Sedative Effects of Synthetic Peptide Amide (54):
D-Phe-D-Phe-D-Leu-D-Orn-[4-(4,5-dihydro-1H-imidazol-2-yl)homopiperazine
amide]
[0376] Inhibition of acetic acid-induced writhing is an indication
of an analgesic effect (also called an antinociceptive effect). A
reduction in locomotion can be used as a measure of a general
sedative effect. The ED.sub.50 determined in the acetic
acid-induced writhing assay in IRC mice was 74 .mu.g/kg [with a 95%
confidence interval of 49-99 .mu.g/kg] when synthetic peptide amide
(54) was delivered subcutaneously. The ED.sub.50 value determined
in the inhibition of locomotion assay was 3172 .mu.g/kg [with a 95%
confidence interval of 1810-4534 .mu.g/kg] for the same synthetic
peptide amide delivered subcutaneously. See FIG. 6. The therapeutic
ratio of the analgesic effect over the sedative effect is the fold
higher ED.sub.50 required to achieve a sedative effect as compared
to the ED.sub.50 required to achieve an analgesic effect. Thus,
compound (54) exhibits a (3172/74) fold ratio, i.e. 42.86 fold.
Thus, the therapeutic ratio for compound (54) is approximately 43
fold.
Example 116
Analgesic Vs. Sedative Effects of Synthetic Peptide Amide (3)
[0377] Inhibition of acetic acid-induced writhing by a compound is
an indication of an analgesic effect (also called an
antinociceptive effect). Similarly, a reduction in locomotion
caused by administration of the compound can be used as a measure
of its general sedative effect.
[0378] The ED.sub.50 determined in the acetic acid-induced writhing
assay in ICR mice was 52 .mu.g/kg when synthetic peptide amide (3)
was administered subcutaneously as described in Example 113 and
shown in FIG. 4 (solid circles). The ED.sub.50 value determined in
the inhibition of locomotion assay as described in Example 114 was
2685 .mu.g/kg for the same synthetic peptide amide administered
subcutaneously. See FIG. 4 (solid squares). The therapeutic ratio
of the analgesic effect over the sedative effect is the fold higher
ED.sub.50 required to achieve a sedative effect as compared to the
ED.sub.50 required to achieve an analgesic effect. Thus, compound
(3) exhibits a (2685/52) fold ratio, i.e. 51.6 fold. Thus, the
therapeutic ratio is approximately 52 fold for compound (3).
Example 117
Synthetic Peptide Amide Pharmacokinetcs in Monkeys
[0379] Samples were administered to male monkeys, Macaca
fascicularis (SNBL USA, Ltd., Everett, Wash., purpose-bred
cynomolgus monkeys. Closely related to humans, both
phylogenetically and physiologically) aged 3-7 years and weighing
3-5 kilograms. Samples were administered in a superficial vein of
the arm or leg (e.g. brachial, or saphenous) in 0.9% saline for
injection, USP (Baxter Healthcare, Deerfield, Ill.) as follows: A
cassette sample containing 0.4 mg of compound (52) of the present
invention and 0.4 mg of each of nine other compounds (for a total
dose of 4 mg) was prepared in 2 ml 0.9% saline for injection,
providing a concentration of 0.2 mg/ml of each of the ten
compounds. Exactly 2 ml was administered as an intravenous bolus to
the test animal, resulting in a total dose level of 0.8 to 1.3
mg/kg depending on the body weight of the individual animal. The
intravenous injection was followed by a 1 ml flush with 0.9% saline
for injection. Blood samples of 0.6 ml were collected by
venipuncture from a peripheral vein at 2, 5, 10, 15 and 30 minutes
post dose injection, and then at 1, 2 and 4 hours. Each sample was
placed in a pre-chilled glass test tube containing lithium heparin
and immediately chilled on ice. Plasma was collected after
centrifugation at 2,000 g for fifteen minutes at 2-8.degree. C. The
plasma layers of each sample were transferred to polypropylene
tubes and stored frozen at -60.degree. C. or lower until assayed.
One hundred microliter aliquots of thawed plasma were spiked with 5
microliters of a 400 ng/ml solution of an appropriate internal
standard (in this case a known synthetic peptide amide compound) in
0.1% TFA, and the proteins were precipitated with 100 microliters
of 0.1% TFA in acetonitrile. The samples were centrifuged at
1000.times.g for 5 minutes and the supernatants analyzed by LC-MS.
LC-MS analysis was performed on a Finnigan LCQ Deca mass
spectrometer interfaced to a Surveyor HPLC system (Thermo Electron
Corporation, Waltham, Mass., USA). HPLC analysis was performed on
2.1.times.150 mm C18 reversed phase columns with a gradient of
0.01% TFA in acetonitrile in 0.01% TFA in water. Mass detection was
performed in the selected reaction monitoring mode (SRM).
Quantitation was performed against a calibration curve of the
analyte in blank cynomolgus monkey plasma using the same internal
standard. Data analysis and the extraction of pharmacokinetic
parameters were performed with the program PK Solutions 2.0 (Summit
Research Services, Ashland, Ohio, USA). Results for compound (52):
D-Phe-D-Phe-D-Leu-D-Orn-[4-(N-methyl)amidino-homopiperazine amide]
are shown in FIG. 7.
Example 118
Spinal Nerve Ligation (SNL) Model
[0380] The SNL model (Kim and Chung 1992) was used to induce
chronic neuropathic pain. The rats were anesthetized with
isoflurane, the left L5 transverse process was removed, and the L5
and L6 spinal nerves were tightly ligated with 6-0 silk suture. The
wound was then closed with internal sutures and external staples.
Fourteen days following SNL, baseline, post-injury and
post-treatment values for non-noxious mechanical sensitivity were
evaluated using 8 Semmes-Weinstein filaments (Stoelting, Wood Dale,
Ill., USA) with varying stiffness (0.4, 0.7, 1.2, 2.0, 3.6, 5.5,
8.5, and 15 g) according to the up-down method (Chaplan et al.
1994). Animals were placed on a perforated metallic platform and
allowed to acclimate to their surroundings for a minimum of 30
minutes before testing. The mean and standard error of the mean
(SEM) were determined for each paw in each treatment group. Since
this stimulus is normally not considered painful, significant
injury-induced increases in responsiveness in this test are
interpreted as a measure of mechanical allodynia. The dose which
elicited 50% attenuation of mechanical hypersensitivity (ED.sub.50)
was determined using linear regression analysis. Compound (2) was
administered by the intravenous route. FIG. 8 summarizes the
results of these experiments. The calculated ED.sub.50 for compound
(2) in this model was 0.38 mg/kg (0.31-0.45; 95% confidence
interval).
Example 119
Ocular Analgesia Induced by Compounds (2), (3) and (4)
[0381] Ocular analgesia was evaluated by instilling five volumes of
the test compound, 50 microliters each in physiological saline, at
the concentration to be tested into the right eye of naive albino
New Zealand strain rabbits within a period of twenty minutes.
Fifteen minutes after the last instillation of the test compound,
each animal was administered a single instillation of 30
microliters of 10 mg/ml capsaicin (33 mM) in the treated eye.
Capsaicin is known to induce corneal pain. Corneal pain was
evaluated by measurement of the palpebral opening measured in
millimeters using a transparent ruler over the treated and
untreated eyes. In this animal model, the reduction in size of the
palpebral opening after instillation of capsaicin is an indication
of the degree of ocular pain. Thus, any observed restoration
(increase) in size of the palpebral opening after treatment with
test compound is taken as a measure of relief from
capsaicin-induced ocular pain.
[0382] These evaluations were performed before treatment with the
test compound (pre-test), immediately prior to the instillation of
capsaicin, and then 1, 5, 10, 15, 20, 25, 30, 40, 50 and 60 minutes
following the instillation of capsaicin. Table V shows the mean of
palpebral opening measurements (relative to the untreated eye
expressed as percent of control) averaged over the period from
10-30 minutes after capsaicin instillation in rabbits pre-instilled
with a kappa opioid agonist of the invention and after
preinstillation with a standard concentration of diltiazem, a
benzothiazepine calcium channel blocker with local anesthetic
effects. See Gonzalez et al., (1993) Invest. Ophthalmol. Vis. Sci.
34: 3329-3335.
TABLE-US-00005 TABLE V Effect of compounds (2), (3) and (4) in
reducing ocular pain Time Mean SEM Compound (post capsaicin) (%
Control) (% Control) None (Saline) 10-30 min. 61.2 6.5 Diltiazem at
10 mM 10-30 min. 74.6 5.5 (2) at 10 mg/ml 10-30 min. 82.7 5.3 (3)
at 10 mg/ml 10-30 min. 76.0 5.2 (4) at 10 mg/ml 10-30 min. 56.7 8.4
Mean is of five animals; SEM: Standard error of the mean
Example 120
Dose Response of Compound (2) in Capsaicin-Induced Ocular Pain
[0383] Ocular analgesia induced by Compound (2) at several
concentrations instilled into the right eye of naive albino New
Zealand strain rabbits was evaluated as described above. Results
were compared with analgesia induced by 10 mg/ml morphine (a
non-selective opioid agonist) as a systemic active control, and
with 10 mM diltiazem as a topical active-control in the same
experiment and under the same conditions. Table VI below shows the
accumulated results.
TABLE-US-00006 TABLE VI Dose-Response of Compound (2) in
Capsaicin-Induced Ocular Pain Time Mean SEM Compound (post
capsaicin) (% Control) (% Control) Morphine at 10 mg/ml 10-30 min.
74.8 11.1 Diltiazem at 10 mM 10-30 min. 77.6 7.4 (2) at 1 mg/ml
10-30 min. 60.5 9.9 (2) at 10 mg/ml 10-30 min. 56.6 9.3 (2) at 25
mg/ml 10-30 min. 75.5 7.1 (2) at 50 mg/ml 10-30 min. 87.5 4.8 Mean
is of ten animals; SEM: Standard error about the mean
Example 121
Effect of Compound (2) in a Rat Pancreatitis Model
[0384] Chronic pancreatic inflammation was induced in rats by
intravenous administration of dibutyltin dichloride (DBTC, Aldrich
Milwaukee, Wis.) dissolved in 100% ethanol at a dose of 8 mg/kg
under isofluorane anesthesia (2-3 liters/min, 4%/vol until
anesthetized, then 2.5%/vol throughout the procedure. Control
animals received the same volume of vehicle (100% ethanol) alone.
Pancreatitis pain was assessed by determination of abdominal
sensitivity to probing the abdomen of rats with a calibrated von
Frey filament (4 g). Rats were allowed to acclimate in suspended
wire-mesh cages for 30 min before testing. A response was indicated
by the sharp withdrawal of the abdomen, licking of abdominal area,
or whole body withdrawal. A single trial consisted of 10
applications of von Frey filament applied once every 10 s to allow
the animal to cease any response and return to a relatively
inactive position. The mean occurrence of withdrawal events in each
trial is expressed as the number of responses to 10 applications.
Rats without inflammation of the pancreas typically display
withdrawal frequencies to probing with von Frey filament of 0-1.
The animals were allowed to recover for 6 days after DBTC
administration prior to any pharmacological manipulations. Animal
not demonstrating sufficient abdominal hypersensitivity (i.e., rats
with less than 5 positive responses out of a possible 10) were
excluded from the study.
[0385] The number of positive responses, following abdominal
probing (out of a possible 10), were recorded at each time point.
Data are presented as average number of withdrawals (.+-.SEM) for
each dosing group at each corresponding time point. For
dose-response analysis, raw data were converted to % maximum
possible effect (% MPE) using the formula: % MPE=((test score-post
DBTC score)/(pre DBTC score-post DBTC score))*100. Raw data were
analyzed using a two-way repeated measures ANOVA followed by
Bonferroni post-tests. The dose which elicited 50% attenuation of
hypersensitivity (ED.sub.50) was determined using linear regression
analysis. Compounds were administered by the intraperitoneal route.
FIG. 9 summarizes the results of these experiments. The calculated
ED.sub.50 for compound (2) in this model was 0.03 mg/kg
(0.006-0.14; 95% confidence interval).
[0386] To determine if the efficacy of Compound (2) (1 mg/kg) is
mediated via activation of peripheral kappa opioid receptors,
groups of eight rats were pretreated with either the selective
kappa opioid receptor antagonist nor-BNI (1 mg/kg), or with a
non-selective opioid receptor antagonist, naloxone methiodide (10
mg/kg), which does not cross the blood-brain barrier, prior to
treatment with compound (2). FIG. 10 summarizes the results of
these studies.
Example 122
Pruritus Model in Mice
[0387] Groups of 10 (and in one case, 11) male Swiss Webster mice
(25-30 g) were used. Each animal was weighed and allowed to
acclimate for at least one hour in individual, rectangular
observation boxes. The tails of mice were immersed for 30 seconds
in warm water to dilate tail veins and the animals then received an
intravenous injection of either vehicle (saline) or compound (2)
(0.01, 0.03, 0.10 and 0.30 mg of free base/kg). Fifteen minutes
later, each mouse was given either GNTI dihydrochloride (Tocris)
(0.30 mg/kg; 0.25 ml/25 g) or compound 48/80 (Sigma) (50 .mu.g in
0.10 ml saline) subcutaneously behind the neck. The animals were
then observed in pairs (occasionally in threes) and the number of
hind leg scratching movements directed at the neck was counted for
30 minutes. The mean percent inhibition of scratching caused by
compound (2) was plotted and the dose associated with 50%
inhibition was obtained by linear regression analysis
(PharmProTools). Table VII summarizes the results of these
experiments.
TABLE-US-00007 TABLE VII Effects of Compound (2) on Pruritus
Induced by Either Compound 48/80 or GNTI in Mice. Compound 48/80
Model GNTI Model ED.sub.50 (mg/kg, iv, ED.sub.50 (mg/kg, iv,
Compound # 15 min post-dose) 15 min post-dose) (2) 0.08 (0.04-0.2)
0.05 (0.02-0.1)
Example 123
CFA Model for Inflammatory Pain in Rats
[0388] Induction of inflammatory pain: Local inflammation was
induced in male Sprague-Dawley rats (weighing 200-250 grams) by 50
.mu.L complete Freund's adjuvant (CFA; Sigma, Mycobacterium
tuberculosis 1 mg/mL) injected subcutaneously into the plantar
surface of the left hind paw. Test compounds were administered and
evaluated for efficacy 24 hr following CFA administration.
[0389] Assessment of inflammatory pain: Paw withdrawal thresholds
(PWTs) in response to a noxious mechanical stimulus were measured
using the Randall-Selitto paw pressure apparatus (Ugo Basile). This
apparatus generates a linearly increasing mechanical force. The
stimulus is applied to the dorsal surface of the hind paws by a
dome-shaped plastic tip placed between the 3rd and 4th metatarsus.
To avoid tissue damage, a cut-off pressure was set at 390 grams.
PWTs were defined as the force in grams at the first obvious pain
behavior, which includes paw withdrawal, struggle, and/or
vocalization. Rats were tested prior to, and 24 hr following, CFA
administration. After about 30 minutes, vehicle or test compound
was administered and PWT tests were carried out over a period of 2
hours. See Table VIII (below).
[0390] Animals not demonstrating sufficient mechanical
hypersensitivity following CFA administration (i.e., rats with
PWTs>150 g) were excluded from the study. All behavioral
assessments were carried out under blinded conditions. Intravenous
administration of Compound (2) produced a dose- and time-dependent
reversal of mechanical hypersensitivity produced by unilateral
CFA-induced inflammatory pain.
[0391] The two-way repeated measures ANOVA revealed significant
main effects of Dose and Time; F(4,217)=152.1, p<0.0001 and
F(6,217)=60.8, p<0.0001, respectively. Further the
Dose.times.Time interaction was statistically significant;
F(24,217)=16.7, p<0.0001. Compound (2) produced a marked
increase in PWTs of the injured hind paw, elevating thresholds
above pre-CFA levels. Further, the anti-hyperalgesic actions of
Compound (2) persisted for 90 min at the 0.1 mg/kg and at least 120
min at doses of 0.3 mg/kg and above. The calculated ED.sub.50 for
Compound (2) was 0.09 mg/kg (0.07-0.12; 95% CI) 30 min following
intravenous administration.
TABLE-US-00008 TABLE VIII Dose-dependent attenuation of CFA-induced
mechanical hyperalgesia by intravenous compound (2) Time Vehicle
Compd (2) Compd (2) Compd (2) Compd (2) (min) alone (0.03 mg/kg)
(0.1 mg/kg) (0.3 mg/kg) (1 mg/kg) Naive 249 .+-. 4 243 .+-. 11 239
.+-. 8 215 .+-. 6 216 .+-. 8 CFA 112 .+-. 6 119 .+-. 9 122 .+-. 8
115 .+-. 6 116 .+-. 6 15 107 .+-. 6 130 .+-. 5 182 .+-. 20*** 261
.+-. 17*** 244 .+-. 22*** 30 111 .+-. 5 141 .+-. 9 197 .+-. 8***
328 .+-. 24*** 328 .+-. 19*** 60 102 .+-. 8 118 .+-. 9 178 .+-.
8*** 212 .+-. 6*** 275 .+-. 10*** 90 105 .+-. 4 100 .+-. 9 171 .+-.
11*** 219 .+-. 15*** 239 .+-. 15*** 120 110 .+-. 5 110 .+-. 14 118
.+-. 6 241 .+-. 15*** 263 .+-. 8*** Values are reported as mean
PWTs .+-. SEM; ***denotes p < 0.001 vs. Vehicle (Bonferroni
tests).
Example 124
Carrageenan Model for Inflammation in Rats
[0392] Induction of inflammation: Hindpaw swelling was induced by a
single intraplantar injection of .lamda.-carrageenan (0.1 mL, 2%
w/v in deionized water).
[0393] Assessments of hind paw inflammatory swelling (edema): Paw
volume measurements were obtained prior to, and 3 hours following,
unilateral carrageenan administration using a plethysmometer (Ugo
Basile). Vehicle (saline) was administered in one hindpaw and test
compound was administered in the lateral hindpaw 30 minutes prior
to carrageenan injection. Rats were gently restrained having their
ankles stabilized. Once stabilized, a hind paw was submerged (up to
the ankle joint) for approximately 1 second into a known volume of
buffer and the total fluid displaced was recorded. This procedure
was repeated for both hind paws.
[0394] Intravenous administration of Compound (2) also resulted in
a significant, dose-related suppression of carrageenan-induced paw
swelling. Thirty minutes prior to carrageenan administration,
Compound (2) was administered intravenously, then paw volumes were
assessed 3.5 hr post treatment (i.e., 3 hr post-carrageenan
injection). Compound (2) produced a marked inhibition of paw
swelling, achieving an equivalent effect to ibuprofen (Table 3).
The minimum effective anti-inflammatory dose of Compound (2)
determined in this model was 0.3 mg/kg.
TABLE-US-00009 TABLE X Intravenous Compound (2) dose-dependently
attenuates carrageenan- induced hind paw swelling Vehicle Ibuprofen
Compd (2) Compd (2) Compd (2) alone (100 mg/kg) (0.1 mg/kg) (0.3
mg/kg) (1 mg/kg) 1.4 .+-. 0.1 0.8 .+-. 0.1* 1.3 .+-. 0.2 0.9 .+-.
0.1* 0.9 .+-. 0.1* Data are expressed as change in paw volume
(inflamed - non-inflamed) in mL .+-. SEM. *denotes p < 0.05 vs.
Vehicle (Dunnett's tests)
Example 125
Synoviocyte Division Model of Human Rheumatoid Arthritis
[0395] General methods for synoviocyte cell culture are well known
in the art. See for example: Seki et al., Arthritis Rheum.
41:1356-1364 (1998) and U.S. Pat. No. 7,244,573 to Carman et al.
and U.S. Pat. No. 7,348,162 to Saris et al.
[0396] Human synovium was removed from the joint of a rheumatoid
arthritis subject, cut into small pieces and then digested with
collagenase while shaking at 37.degree. C. The resulting cells were
washed twice and plated without any further isolation
procedures.
[0397] Two days after plating the cells were replated in 48 well
plates in cell culture media containing fetal bovine serum. The
cells were incubated with test or reference compound after a
further 2 days of plating. Following Giemsa staining at two weeks
after incubation with test or reference compound, the synoviocyte
cell numbers were counted directly in each well by microscopy. FIG.
11 shows the inhibition of synoviocyte cell proliferation over a
range of concentrations of Compound (2) from 0.01 nM to 50 nM. The
apparent EC.sub.50 for inhibition by Compound (2) is approximately
0.16 nM. For comparison, parallel assays with the
glucocorticosteroid, Budesonide used as an anti-inflammatory in the
treatment of rheumatoid arthritis, exhibited an EC.sub.50 of
approximately 0.21 nM for inhibition of synoviocyte
proliferation.
Example 126
Effect of Compound (2) in the Rat Monoiodoacetate Model of
Osteoarthritis Knee Pain
[0398] Injection of monoiodoacetate (MIA) into the knee joints of
rats evokes a painful response that measured as a weight bearing
difference between the MIA-injected knees and saline-injected knees
and is used as a model for human osteoarthritis (OA).
[0399] The right knees of approximately 8 week old male Lewis rats
were injected with 0.3 mg MIA in 50 uL of saline through the
patella ligament into the synovial joint space and left knees were
injected with 50 uL of saline as control. MIA injected rats were
dosed sub-cutaneously on day 12 after MIA injection with either
vehicle (sterile water for injection), or compound (2) at 0.1
mg/kg, 0.3 mg/kg, or 1 mg/kg in a volume of 1 mL/kg (six rats per
group). Incapacitance testing was measured as the difference in
hind paw weight bearing between the MIA and saline-injected knees.
Three separate measurements each over a second were averaged. Data
is shown as mean with standard error about the mean. Groups were
compared using Dunnet's test and data with a P value of less than
0.05 was accepted as significant.
[0400] Results: Subcutaneous injection of vehicle (water) alone
gave the base-line difference between right and left hind paw
weight bearing measured on day 19 post MIA-injection, was 22 g.
Animals receiving 0.1 mg/kg compound (2) exhibited a mean
difference between right and left hind paw weight bearing of 15 g
with a P<0.0002 (n=6) and those receiving 0.3 mg/kg compound (2)
exhibited a mean difference between right and left hind paw weight
bearing of 13 g with a P<0.0001 (n=4).
Example 127
Rat Dural Plasma Protein Extravasation as a Migraine Model
[0401] Plasma protein extravasation from the dura of laboratory
animals following electrical stimulation of the trigeminal ganglion
is used as a model to test migraine therapies. Protein
extravasation from the dura of electrically stimulated rats was
used to test compound (2) as a treatment candidate for migraine.
Sumatriptan was used as a positive control. Male Sprague-Dawley
rats (250-350 g) were injected subcutaneously with water,
sumatriptan or compound (2) at 30, 3, 1 and 0.3 mg/kg, each in 1
mL/kg approximately 20 mins. Before being anesthetized with
Nembutal (60 mg/kg, ip) and placed in a stereotactic frame (David
Kopf Instruments) with the incisor bar set at -2.5 mm A midline
sagital scalp incision was made and two pairs of bilateral holes
were drilled through the skull (3.2 mm posterioirly, 1.8 and 3.8 mm
laterally, coordinates being referenced to bregma. Pairs of
stainless steel electrodes (Rhodes Med. Systems Inc.) insulated
except at the tip were lowered through the holes in both
hemispheres to a depth of 9.2 mm below the dura. After 28 mins
After dosing a solution of fluoroscein isothiocyanate-labelled
bovine serum albumin (FITC-BSA, Sigma A977 20 mg/kg) was injected
into the femoral vein. After 2 mins. The left trigeminal ganglion
was stimulated, 1.0 mA, 5 Hz, 5 mins duration with a Model S48
Grass Instrument Stimulator. Rats were then exsanguinated with 40
mL saline. The top of the skull was removed and the dura collected.
Membrane samples were removed from both hemispheres and rinsed with
water and spread on microscope slides. The slides were dried at
room temperature and covered with cover slips and a 70%
glycerol/water solution.
[0402] Slides were quantified with a fluorescence microscope
(Zeiss) equipped with a grating monochromator and a
spectrophotometer for quantifying FTIC-BSA in each dura sample.
Data were collected on a personal computer from the motorized stage
reading fluorescence measurements at 25 points in 500 .mu.m steps
on each sample. The extravasation ratio (fluorescence from the
stimulated side compared to the unstimulated side) was calculated.
Vehicle alone or ineffective treatments resulted in an
extravasation ratio of approximately 2, whereas totally effective
treatments resulted in an extravasation ratio of approximately 1.
Results wee expressed as mean values with standard errors of the
mean. Statistical evaluations using ANOVA were followed by
comparison to control groups by Dunnet's test. P<0.05 was
accepted as significant.
[0403] Vehicle alone gave an extravasation ratio of approximately
1.9 and sumatriptan gave an extravasation ratio of approximately
1.1; Compound (2) at 0.3, 1, 3 and 30 mg/kg yielded extravasation
ratios of approximately 1.8 (P>0.05), 1.6 (P>0.05), 1.0
(P<0.05) and 1.2 (P<0.05) respectively.
Example 128
Effect of Compound (2) on Human Macrophage Cytokine Production
[0404] The anti-inflammatory efficacy of a candidate molecule was
measured as reduction of cytokine production after
lipopolysaccharide (LPS) and IFN-.gamma. induction of human
monocytes. The cytokines monitored were IL-1.beta., IL-8,
TNF.alpha. and GM-CSF. The anti-inflammatory cytokine, IL-10 was
used as positive control.
[0405] Monocyte isolation and incubation: Monocytes were isolated
from an individual female Caucasian subject by density gradient
centrifugation in percoll and plated in 48-well plates at
2.times.10.sup.5 cells/ml/well. Cells were cultured in Iscove's
medium (invitrogen, UK) supplemented with penicillin (100
units/ml)/streptomycin (100 ug/ml), nonessential; lx amino acids, 1
mM sodium pyruvate, 25 mM HEPES, 10% FBS (Invitrogen, UK), 2 ng/ml
GM-CSF (PeproTech, UK). After 7 days in culture cells were exposed
to on of the following: (i) 0.1 nM, 1 nM, 3 nM, 10 nM or 30 nM
compound (2); (ii) 10 ng/ml IL-10; for 4 hours prior to addition of
1 ng/ml LPS+10 ng/ml IFN-.gamma. (PeproTech, UK) for 18 hours.
Assays were performed in triplicate.
[0406] Analysis of cytokines: Bead-based assays for each cytokine
were analysed on a Luminex-100 according to the manufacturer's
instructions. Statistical comparisons were by ANOVA with Dunnet's
and Bonferroni's post analysis test for concentration-response
curves and effects of nor-BNI respectively, using Graph Pad Prism
software. Statistical significance was accepted at the 0.05
level.
TABLE-US-00010 TNF: Percent of Untreated Level (approximate)
Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 TNF.alpha. % 100 95 35 55
70 55 45 20
TABLE-US-00011 IL-1.beta.: Percent of Untreated Level (approximate)
Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 IL-1.beta. % 100 80 10 5
25 -10 -25 40
TABLE-US-00012 IL-8: Percent of Untreated Level (approximate)
Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 IL-8 % 100 90 75 70 60 45
40 25
TABLE-US-00013 GM-CSF: Percent of Untreated Level (approximate)
Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 GM-CSF % 100 20 40 -30 -5
-55 -60 -40
Example 129
Effect of Compound (2) on Human Synoviocyte Cytokine Production
[0407] Synoviocytes were prepared as described in Example 125
above, two days later the cells were replated in tissue culture
treated 96-well plates. After a further two days cells were
incubated with compound (2) at 0.1, 0.3, 1, 3, 10 and 30 nM at 4
hours before stimulus. Cytokine production was stimulated with
IFN.gamma. (10 ng/mL, PeproTech, UK) and Mab CD40 (1 .mu.g/mL,
eBiosciences, UK) for 4 hours before assaying MMPs and 48 hours
before TNF.alpha. was assayed. Cells stimulated with IFN.gamma. and
Mab CD40 were used as standards. Budesonide (1 .mu.M) was added 1
hour before stimulus as positive control. TNF.alpha. and MMP-1 and
MMP-3 assays were analysed on a Luminex 100 according to the
manufacturer's instructions. Statistical comparisons were by ANOVA
with Dunnet's test for multiple comparisons using Graph Pad Prism
ver. 5 software. Statistical significance was accepted at the
P<0.05 level.
TABLE-US-00014 TNF: Percent of Untreated Level (approximate)
Compound (2) nM 0 0.1 0.3 1 3 10 30 Budesonide TNF.alpha. % 100 20
-25 -25 -20 -30 -45 -30
TABLE-US-00015 MMP-1: Percent of Untreated Level (approximate)
Compound (2) nM 0 0.1 0.3 1 3 10 30 Budesonide MMP-1 % 100 10 -40
-250 -220 -120 -200 -240
TABLE-US-00016 MMP-3: Percent of Untreated Level (approximate)
Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 MMP-3 % 100 -25 0 -15 -15
-20 -30 -35
[0408] Elevated levels of the pro-inflammatory cytokines
TNF.alpha., IL1.beta., IL-6, IL8 and IL-10 have been linked to
propensity for glucose intolerance, insulin resistance, diabetes
and obesity. Development of these conditions can be prevented or at
least inhibited by the administration of the synthetic peptide
amides of the present invention. Treatments according to the
present invention are useful as adjunct therapies for these
conditions.
[0409] The specifications of each of the U.S. patents and published
patent applications, and the texts of the literature references
cited in this specification are herein incorporated by reference in
their entireties. In the event that any definition or description
contained found in one or more of these references is in conflict
with the corresponding definition or description herein, then the
definition or description disclosed herein is intended.
[0410] The examples provided herein are for illustration purposes
only and are not intended to limit the scope of the invention, the
full breadth of which will be readily recognized by those of skill
in the art.
* * * * *