U.S. patent application number 10/988427 was filed with the patent office on 2005-06-23 for glucoimidazole and polyhydroxycyclohexenyl amine derivatives to treat gaucher disease.
This patent application is currently assigned to Amicus Therapeutics, Inc.. Invention is credited to Fan, Jian-Qiang, Sheth, Kamlesh, Zhu, Xiaoxiang.
Application Number | 20050137223 10/988427 |
Document ID | / |
Family ID | 34590422 |
Filed Date | 2005-06-23 |
United States Patent
Application |
20050137223 |
Kind Code |
A1 |
Fan, Jian-Qiang ; et
al. |
June 23, 2005 |
Glucoimidazole and polyhydroxycyclohexenyl amine derivatives to
treat gaucher disease
Abstract
The present invention provides glucoimidazole (GIZ) and
polyhydroxycyclohexenyl amine (PHCA) derivatives, methods of making
them, and methods of use where the GIZ and PHCA derivatives have a
short, flexible linker emanating from the corresponding position of
the ring oxygen in a pyranose; and a lipophilic moiety connected to
the linker and pharmaceutically acceptable salts thereof. More
particularly, the present invention further provides a method for
treating individuals having Gaucher disease by administering the
novel GIZ or PHCA derivatives as "active-site specific chaperones"
for the mutant glucocerebrosidase associated with the disease.
Inventors: |
Fan, Jian-Qiang; (Demarest,
NJ) ; Zhu, Xiaoxiang; (North Brunswick, NJ) ;
Sheth, Kamlesh; (North Brunswick, NJ) |
Correspondence
Address: |
DARBY & DARBY P.C.
P. O. BOX 5257
NEW YORK
NY
10150-5257
US
|
Assignee: |
Amicus Therapeutics, Inc.
|
Family ID: |
34590422 |
Appl. No.: |
10/988427 |
Filed: |
November 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60519496 |
Nov 12, 2003 |
|
|
|
Current U.S.
Class: |
514/303 ;
514/44R; 546/118; 564/123 |
Current CPC
Class: |
A61P 7/06 20180101; A61P
19/00 20180101; A61P 25/00 20180101; C07D 487/04 20130101; A61P
1/16 20180101; A61P 3/06 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/303 ;
546/118; 564/123; 514/044 |
International
Class: |
C07D 471/02; A61K
048/00; A61K 031/4745 |
Claims
What is claimed:
1. A compound of the Formula I: 12wherein B is hydrogen, hydroxy,
N-acetamino, or halogen; R.sup.1 and R.sup.2 are optionally
present, are flexible linkers with a linear length of about 6 .ANG.
to about 12 .ANG.; L.sup.1 and L.sup.2 are lipophilic groups having
3-12 carbon atoms comprising a substituted or unsubstituted: alkyl,
alkenyl, alkynyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl;
heteroaryl; heterocyclic; heterocycloalkyl; or heteroarylalkyl; and
R.sup.5 is a hydrogen, hydroxy, or hydroxymethyl.
2. A compound of the Formula I: 13wherein B is hydrogen, hydroxy,
N-acetamino, or halogen; R.sup.1 and R.sup.2 are independently
C.sub.2-C.sub.6 substituted or unsubstituted: alkyl, alkenyl, or
alkynyl, which are optionally interrupted by one or more moieties
chosen from the group consisting of NH, NHCOO, NHCONH, NHCSO,
NHCSNH, CONH, NHCO, NR.sup.3, O, S, S(O).sub.m and --S(O).sub.m
NR.sup.3; L.sup.1 and L.sup.2 are lipophilic groups having 3-12
carbon atoms comprising a substituted or unsubstituted: alkyl,
alkenyl, alkynyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl;
heteroaryl; heterocyclic; heterocycloalkyl; or heteroarylalkyl;
wherein R.sup.1-L.sup.1 or R.sup.2-L.sup.2 can be a hydrogen, if
either R.sup.2-L.sup.2 or R.sup.1-L.sup.1 is other than a hydrogen;
R.sup.3 is independently selected from each occurrence a hydrogen,
or a substituted or unsubstituted: alkyl, alkenyl; alknyl;
cycloalkyl, cycloalkenyl; aryl; arylalkyl; heteroaryl;
heterocyclic; heterocyclyalkyl; or heteroarylalkyl, or --C(O)
attached to a C.sub.1-C.sub.6 substituted or unsubstituted alkyl; m
is 1 or 2; and R.sup.5 is a hydrogen, hydroxy, or hydroxymethyl;
and pharmaceutically acceptable salts and prodrugs thereof.
3. A method for synthesizing a compound of the Formula III:
14wherein R.sup.1 and R.sup.2 are independently C.sub.2-C.sub.6
substituted or unsubstituted: alkyl, alkenyl, or alkynyl, which are
optionally interrupted by one or more moieties chosen from the
group consisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH, CONH, NHCO,
NR.sup.3, O, S, S(O).sub.m and --S(O).sub.m NR.sup.3; L.sup.1 and
L.sup.2 are lipophilic groups having 3-12 carbon atoms comprising a
substituted or unsubstituted: alkyl, alkenyl, alkynyl; cycloalkyl,
cycloalkenyl; aryl; arylalkyl; heteroaryl; heterocyclic;
heterocycloalkyl; or heteroarylalkyl; wherein R.sup.1-L.sup.1 or
R.sup.2-L.sup.2 can be a hydrogen, if either R.sup.2-L.sup.2 or
R.sup.1-L.sup.1 is other than a hydrogen; R.sup.3 is independently
selected from each occurrence a hydrogen, or a substituted or
unsubstituted: alkyl, alkenyl; alknyl; cycloalkyl, cycloalkenyl;
aryl; arylalkyl; heteroaryl; heterocyclic; heterocyclyalkyl; or
heteroarylalkyl, or --C(O) attached to a C.sub.1-C.sub.6
substituted or unsubstituted alkyl; m is 1 or 2; and
pharmaceutically acceptable salts and prodrugs thereof; and
comprising the steps of: a) reacting a compound of the Formula IV:
15 wherein P.sub.1, P.sub.2, P.sub.3 and P.sub.4 are O-protection
groups, with N-iodo-succinimide in a polar aprotic solvent or
additional reactions for selective removal of 3-iodo group from
Formula VI to yield compounds of the Formula V, VI, or VII: 16b)
reacting a compound of the Formula V, VI or VII with a compound of
the Formula VIII or IX: 17 wherein L is a L.sup.1 or L.sup.2 and R
is R.sup.1 or R.sup.2, in the presence of palladium catalyst in a
polar aprotic solvent to yield a compound of the Formula X: 18
deprotecting the O-protection groups to yield formula III: 19
4. The compound according to claim 2 wherein R.sup.2-L.sup.2 is
hydrogen.
5. The compound according to claim 2 wherein B is hydroxy.
6. The compound according to claim 2 wherein R.sup.5 is
hydroxy.
7. The compound according to claim 2 wherein R.sup.5 is
hydroxymethyl.
8. The compound according to claim 4 wherein R.sup.1-L.sup.1 is
unsubstituted C.sub.1-C.sub.12 alkyl.
9. The compound according to claim 8 wherein R.sup.1-L.sup.1 is
unsubstituted C.sub.6-C.sub.12 alkyl.
10. The compound according to claim 9 wherein R.sup.1-L.sup.1 is
unsubstituted C.sub.8 alkyl.
11. The compound according to claim 8 wherein and R.sup.1-L.sup.1
is unsubstituted branched C.sub.3-12 alkyl.
12. The compound according to claim 11 wherein R.sup.1-L.sup.1 is
unsubstituted branched C.sub.6-12 alkyl.
13. The compound according to claim 12 wherein R.sup.1-L.sup.1 is
unsubstituted branched C.sub.6 alkyl.
14. The compound according to claim 4 wherein R.sup.1-L.sup.1 is
3,3-dimethylbutyl.
15. A compound according to claim 2 comprising
(5R,6R,7S,8S)-5-hydroxymeth-
yl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-triol.
16. A compound according to claim 2 comprising
(5R,6R,7S,8S)-5-hydroxymeth-
yl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-tr-
iol.
17. A compound of the Formula II: 20wherein B is hydrogen, hydroxy,
N-acetamino, or halogen; R.sup.1 is a hydrogen, substituted or
unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
aryl, arylalkyl, heteroaryl, heterocyclic, heterocyclyalkyl, or
heteroarylalkyl; --C(O)R.sup.3 or --S(O).sub.mR.sup.3; R.sup.2
optionally present, is a flexible linker with a linear length of
about 6 .ANG. to about 12 .ANG.; L is a lipophilic group having
3-12 carbon atoms comprising a substituted or unsubstituted: alkyl,
alkenyl, alkynyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl;
heteroaryl; heterocyclic; heterocycloalkyl; or heteroarylalkyl;
R.sup.3 is independently selected from each occurrence a hydrogen,
or a substituted or unsubstituted: alkyl, alkenyl; alknyl;
cycloalkyl, cycloalkenyl; aryl; arylalkyl; heteroaryl;
heterocyclic; heterocyclyalkyl; or heteroarylalkyl, or --C(O)
attached to a C.sub.1-C.sub.6 substituted or unsubstituted alkyl; m
is 1 or 2; and R.sup.5 is a hydrogen, hydroxy, or hydroxymethyl,
and pharmaceutically acceptable salts and prodrugs thereof.
18. A compound of the Formula II: 21wherein B is hydrogen, hydroxy,
N-acetamino, or halogen; R.sup.1 is a hydrogen, substituted or
unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
aryl, arylalkyl, heteroaryl, heterocyclic, heterocyclyalkyl, or
heteroarylalkyl; --C(O)R.sup.3 or --S(O).sub.mR.sup.3; R.sup.2,
optionally present, is a C.sub.2-C.sub.6 substituted or
unsubstituted: alkyl, alkenyl, or alkynyl, which is optionally
interrupted by one or more moieties chosen from the group
consisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH, CONH, NHCO,
NR.sup.3, O, S, S(O).sub.m and --S(O).sub.m NR.sup.3; L is a
lipophilic group having 3-12 carbon atoms comprising a substituted
or unsubstituted: alkyl, alkenyl, alkynyl; cycloalkyl,
cycloalkenyl; aryl; arylalkyl; heteroaryl; heterocyclic;
heterocycloalkyl; or heteroarylalkyl; R.sup.3 is independently
selected from each occurrence a hydrogen, or a substituted or
unsubstituted: alkyl, alkenyl; alknyl; cycloalkyl, cycloalkenyl;
aryl; arylalkyl; heteroaryl; heterocyclic; heterocyclyalkyl; or
heteroarylalkyl, or --C(O) attached to a C.sub.1-C.sub.6
substituted or unsubstituted alkyl; m is 1 or 2; and R.sup.5 is a
hydrogen, hydroxy, or hydroxymethyl; and pharmaceutically
acceptable salts and prodrugs thereof.
19. The compound according to claim 18 wherein R.sup.5 is
hydroxy.
20. A method of inhibiting glucocerebrosidase, comprising
contacting a glucocerebrosidase with a compound as set forth in
claim 1, in an amount effective to inhibit the
glucocerebrosidase.
21. The method of claim 20, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyr-
idine-6,7,8-triol.
22. The method of claim 20, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimi-
dazo[1,2-a]pyridine-6,7,8-triol.
23. The method of 20, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethy-
l-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-triol.
24. The method of claim 20, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-phenethyl-5,6,7,8-tetrahydroimidazo[1,2-a-
]pyridine-6,7,8-triol.
25. A method of inhibiting glucocerebrosidase comprising contacting
glucocerebrosidase with a compound as set forth in claim 18, in an
amount effective to inhibit the glucocerebrosidase.
26. A method of inhibiting glucocerebrosidase in a mammalian cell
comprising contacting the cell with an inhibitory amount of a
compound as set forth in claim 1.
27. The method of claim 26, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyr-
idine-6,7,8-triol.
28. The method of claim 26, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimi-
dazo[1,2-a]pyridine-6,7,8-triol.
29. The method of claim 26, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,-
7,8-triol.
30. The method of claim 26, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-phenethyl-5,6,7,8-tetrahydroimidazo[1,2-a-
]pyridine-6,7,8-triol.
31. A method of inhibiting glucocerebrosidase in a mammalian cell
comprising contacting the cell with an inhibitory amount of a
compound as set forth in claim 18.
32. A method of enhancing in a mammalian cell the activity of
glucocerebrosidase, comprising contacting the cell with a compound
as set forth in claim 1 in an amount effective to enhance the
activity of glucocerebrosidase, and wherein the effective amount
does not inhibit glucocerebrosidase activity.
33. The method of claim 32, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyr-
idine-6,7,8-triol.
34. A method of enhancing in a mammalian cell the activity of
glucocerebrosidase, comprising contacting the cell with a compound
as set forth in claim 18 in an amount effective to enhance the
activity of glucocerebrosidase, and wherein the effective amount
does not inhibit glucocerebrosidase activity.
35. A method of stabilizing glucocerebrosidase, comprising
contacting the glucocerebrosidase with a compound as set forth in
claim 1, wherein the compound binds reversibly to the
glucocerebrosidase, in an amount effective to stabilize the
glucocerebrosidase.
36. The method of claim 35, wherein the compound is
(5R,6R,7S,8S)-5-Hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyr-
idine-6,7,8-triol.
37. The method of claim 35, wherein the compound is
(5R,6R,7S,8S)-5-Hydroxymethyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimi-
dazo[1,2-a]pyridine-6,7,8-triol.
38. The method of claim 35, wherein the compound is
(5R,6R,7S,8S)-5-Hydroxymethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,-
7,8-triol.
39. The method of claim 35, wherein the compound is
(5R,6R,7S,8S)-5-Hydroxymethyl-2-phenethyl-5,6,7,8-tetrahydroimidazo[1,2-a-
]pyridine-6,7,8-triol.
40. A method of stabilizing glucocerebrosidase, comprising
contacting the glucocerebrosidase with a compound as set forth in
claim 18, wherein the compound binds reversibly to the
glucocerebrosidase, in an amount effective to stabilize the
glucocerebrosidase.
41. A pharmaceutical composition comprising a compound as set forth
in claim 1 and a pharmaceutically acceptable carrier.
42. The method of claim 41, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyr-
idine-6,7,8-triol.
43. The method of claim 41, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimi-
dazo[1,2-a]pyridine-6,7,8-triol.
44. The method of claim 41, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,-
7,8-triol.
45. The method of claim 41, wherein the compound is
(5R,6R,7S,8S)-5-hydroxymethyl-2-phenethyl-5,6,7,8-tetrahydroimidazo[1,2-a-
]pyridine-6,7,8-triol.
46. A pharmaceutical composition comprising a compound as set forth
claim 18 and a pharmaceutically acceptable carrier.
47. A method of inhibiting a .beta.-glucosidase, comprising
contacting the .beta.-glucosidase with a compound as set forth in
claim 1, wherein the compound binds reversibly to the
13-glucosidase, in an amount effective to inhibit the
.beta.-glucosidase.
48. A method of inhibiting a .beta.-glucosidase, comprising
contacting the .beta.-glucosidase with a compound as set forth in
claim 18, wherein the compound binds reversibly to the
.beta.-glucosidase, in an amount effective to inhibit the
.beta.-glucosidase.
49. A method for treating Gaucher disease comprising administering
to an individual in need of such treatment a pharmaceutical
composition as set forth in claim 1 in an amount effective to
enhance the activity of glucocerebrosidase.
50. The method of claim 49, wherein the treatment further comprises
administering to the individual a functional glucocerebrosidase
enzyme.
51. The method of claim 49, wherein the treatment further comprises
administering to the individual a vector functional
glucocerebrosidase gene.
52. A method for treating Gaucher disease comprising administering
to an individual in need of such treatment a pharmaceutical
composition as set forth in claim 18 in an amount effective to
enhance the activity of glucocerebrosidase.
53. The method of claim 52, wherein the treatment further comprises
administering to the individual a functional glucocerebrosidase
enzyme.
54. The method of claim 52, wherein the treatment further comprises
administering to the individual a vector functional
glucocerebrosidase gene.
55. A glucoimidazole derivative having a short flexible linker
emanating from the corresponding ring oxygen position in a pyranose
and a lipophilic moiety connected to the linker; and
pharmaceutically acceptable salts of such derivatives.
56. A polyhydroxycyclohexyl amine derivatives having a short
flexible linker emanating from the corresponding ring oxygen
position in a pyranose and a lipophilic moiety connected to the
linker; and pharmaceutically acceptable salts of such derivatives.
Description
[0001] This application claims priority to provisional application
60/519,496 filed Nov. 12, 2003, herein incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention provides novel glucoimidazole (GIZ)
and polyhydroxy-cyclohexenyl amine (PHCA) derivatives having a
short, flexible linker emanating from the corresponding position of
the ring oxygen in a pyranose; and a lipophilic moiety connected to
the linker and pharmaceutically acceptable salts thereof. The
present invention further provides a method for treating
individuals having Gaucher disease by administering the novel GIZ
or PHCA derivatives as "active-site specific chaperones" for the
mutant glucocerebrosidase associated with the disease.
BACKGROUND OF THE INVENTION
[0003] Protein Folding
[0004] Proteins are synthesized in the cytoplasm, and the newly
synthesized proteins are secreted into the lumen of the endoplasmic
reticulum (ER) in a largely unfolded state. In general, protein
folding is governed by the principle of self assembly. Newly
synthesized polypeptides fold into their native conformation based
on their amino acid sequences (Anfinsen et al., Adv. Protein Chem.
1975; 29: 205-300). In vivo, protein folding is complicated,
because the combination of ambient temperature and high protein
concentration stimulates the process of aggregation, in which amino
acids normally buried in the hydrophobic core interact with their
neighbors non-specifically. To avoid this problem, protein folding
is usually facilitated by a special group of proteins called
molecular chaperones which prevent nascent polypeptide chains from
aggregating, and bind to unfolded protein such that the protein
refolds in the native conformation (Hartl, Nature 1996; 381:
571-580).
[0005] Molecular chaperones are present in virtually all types of
cells and in most cellular compartments. Some are involved in the
transport of proteins and permit cells to survive under stresses
such as heat shock and glucose starvation. Among the molecular
chaperones (Gething et al., Nature 1992; 355: 33-45; Caplan, Trends
Cell. Biol. 1999; 9: 262-268; Lin et al., Mol. Biol. Cell. 1993; 4:
109-1119; Bergeron et al., Trends Biochem. Sci. 1994; 19:124-128),
Bip (immunoglobulin heavy-chain binding protein, Grp78) is the best
characterized chaperone of the ER (Haas, Curr. Top. Microbiol.
Immunol. 1991; 167: 71-82). Like other molecular chaperones, Bip
interacts with many secretory and membrane proteins within the ER
throughout their maturation, although the interaction is normally
weak and short-lived when the folding proceeds smoothly. Once the
native protein conformation is achieved, the molecular chaperone no
longer interacts with the protein. Bip binding to a protein that
fails to fold, assemble or be properly glycosylated, becomes
stable, and is usually followed by degradation of the protein
through the ER-associated degradation. This process serves as a
"quality control" system in the ER, ensuring that only those
properly folded and assembled proteins are transported out of the
ER for further maturation, and improperly folded proteins are
retained for subsequent degradation (Hurtley et al., Annu. Rev.
Cell. Biol. 1989; 5: 277-307).
[0006] Certain missense mutations result in amino acid
substitutions that alter the native and proper folding of the
protein. To correct these misfoldings, investigations have
attempted to use various molecules as artificial chaperones. High
concentrations of glycerol, dimethylsulfoxide, trimethylamine
N-oxide, or deuterated water have been shown to stabilize the
mutant protein and increase the intracellular trafficking of mutant
protein in several diseases (Brown et al., Cell Stress Chaperones
1996; 1: 117-125; Burrows et al., Proc. Natl. Acad. Sci. USA. 2000;
97: 1796-801). These compounds are considered non-specific chemical
chaperones to improve the general protein folding, although the
mechanism of the function is still unknown. The high dosage of this
class of compounds required for efficacy makes them difficult or
inappropriate to use clinically, although they are useful for the
biochemical examination of folding defect of a protein
intracellularly. They also lack specificity.
[0007] Active Site Specific Chaperones for Enzymes
[0008] Co-owned U.S. Pat. Nos. 6,274,597, and 6,774,135 which are
incorporated herein by reference, disclose a novel therapeutic
strategy for Fabry disease, a lysosomal storage disorder (LSD)
caused by a deficiency in lysosomal .alpha.-galactosidase A
(.alpha.-Gal A) activity. .alpha.-Gal A deficiency often results
from mutations in the gene that encode mutant proteins that result
in folding defects. It was discovered that administration of
1-deoxygalactonojirimycin (DGJ), a potent competitive inhibitor of
.alpha.-Gal A, effectively increased in vitro stability of a mutant
.alpha.-Gal A (R301Q) at neutral pH. These results were also
observed in lymphoblasts established from Fabry patients with the
R301Q or Q279E mutations. Surprising, cultivation of the cells with
DGJ at sub-inhibitory concentrations resulted in a substantial
increase of residual enzyme activity. Furthermore, oral
administration of DGJ to transgenic mice overexpressing a mutant
(R301Q) .alpha.-Gal A substantially elevated the enzyme activity in
major organs (Fan et al., Nat. Med. 1999; 5: 112-115).
[0009] The principle of this strategy is as the follows. Since the
mutant enzyme appears to fold improperly in the ER where pH is
neutral, as evidenced by its instability at pH 7.0 in vitro (Ishii
et al., Biochem. Biophys. Res. Comm. 1993; 197: 1585-1589), the
enzyme would be retarded in the normal transport pathway from the
ER to the Golgi apparatus and subjected to rapid degradation. If a
mutant enzyme could be efficiently transported to the lysosomes, it
may retain normal or near normal kinetic properties and would
remain active, because the mutant enzyme is sufficiently stable
below pH 5.0. The goal, therefore, was to induce the mutant enzyme
to adjust the proper conformation in the ER. In particular, a
compound that can induce a stable molecular conformation of the
enzyme could serve as an "active-site specific chaperone" (ASSC) or
"pharmacological chaperone" to stabilize the mutant enzyme in a
proper conformation for transport to the lysosomes. In the case of
enzymes, such a compound unexpectedly was discovered to be a
competitive inhibitor of the enzyme. Competitive inhibitors of an
enzyme are known to occupy the catalytic site of the properly
folded enzyme, resulting in stabilization of its correct
conformation in vitro. It was found that they also serve as ASSCs
or pharmacological chaperones to induce the proper folding of
enzyme in vivo, thus rescuing the mutant enzyme from the ER quality
control system.
[0010] Co-owned U.S. Pat. Nos. 6,583,158, 6,589,964, 6,599,919, and
U.S. application Ser. No. 10/304,395 to Fan et al., exemplify the
ASSC strategy with numerous other lysosomal storage diseases,
including Gaucher disease. These findings demonstrate that this
therapeutic strategy of using potent competitive inhibitors as
ASSCs to increase the residual enzyme activity in the patient's
cells is not limited to Fabry disease, and can be applied to enzyme
deficiency diseases of this sort, and particularly to lysosomal
storage disorders. In general, effective ASSCs of specific enzymes
associated with particular diseases are potent competitive
inhibitors of the enzyme. Unexpectedly, a more potent inhibitor of
the enzyme acts as a better ASSC for the mutant enzyme (Fan, Trends
Pharmacol Sci. 2003; 24: 355-60).
[0011] Potent Inhibitors of .beta.-glucocerebrosidase
[0012] .beta.-Glucocerebrosidase (GCase, or acid
.beta.-glucosidase) is a lysosomal hydrolase that catalyzes the
hydrolytic cleavage of glucose from glucosylceramide (Brady et al.,
Biochem. Biophys. Res. Commun. 1965; 18: 221-225). The deficiency
of the enzyme activity results in progressive accumulation of
glucosylceramide, a normal intermediate in the catabolism of
globoside and gangliosides, in lysosomes of macrophages, leading to
Gaucher disease, the most common lysosomal storage disorder
(Beutler et al., in The Metabolic and Molecular Bases of Inherited
Disease, 8th ed., McGraw-Hill, New York 2001, 3635-3668). Details
regarding the disease and therapeutic treatment will be described
below.
[0013] Sawkar et al have reported that the addition of an inhibitor
of GCase to a fibroblast culture medium leads to a 2-fold increase
in the activity of N370S GCase, indicating that a potent inhibitor
of GCase may be of therapeutic interest in the treatment of Gaucher
disease, although the particular inhibitor was not sufficient
enough as a therapeutic agent because of high cytotoxocity (Sawkar
A. R. et al., Proc Natl Acad Sci USA. 2002; 99(24): 15428-33).
Therefore, effort has been taken to design and synthesize potent
inhibitors for GCase.
[0014] The catalytic mechanism of .beta.-glycosidases is believed
to proceed via a covalent glycosyl-enzyme intermediate with a
positive charge generated at the anomeric position (Ichikawa et
al., J. Am. Chem. Soc. 1998; 120: 3007-3018; Heightman et al.,
1999; Angew. Chem. Int. Ed. 1999; 38: 750-770). Ichikawa et al.
have designed a class of potent inhibitors for .beta.-glycosidases,
1-N-iminosugars in which a nitrogen atom is at the anomeric
position of a monosaccharide (Structure 1A, Isofagomine or
hydroxy-piperidine). In a preliminary study, D-glucose-type
1-N-iminosugar (isofagomine, or hydroxypiperidine 1) has been shown
to be a potent inhibitor of GCase (U.S. Pat. No. 6,583,158 to Fan
et al.). N-alkyl derivatives of 1-deoxynojirimycin (DNJ) are also
potent inhibitors of GCase, particularly those have longer alkyl
group (greater than C.sub.6 alkyl chain), although DNJ itself and
those N-alkyl DNJ with shorter chains are not inhibitory (Structure
1B, N-nonyl 1-deoxynojirimycin) (U.S. Pat. No. 6,583,158). However,
these inhibitors are either not specific enough or not potent
enough towards to the GCase and not suitable for the treatment of
Gaucher disease.
[0015] Based on these findings, it was realized that GCase may
contain two substrate binding sites in the catalytic domain: one
which recognizes the glucosyl residue; the other which recognizes
the ceramide moiety (Structure 1C, 6-nonyl hydroxypiperidine, RD-1:
recognition domain 1; RD-2: recognition domain 2.). Determination
of the crystal structure of GCase revealed an annulus of
hydrophobic residues surrounding the entrance to the monosaccharide
binding site (Dvir et al., EMBO reports 2003; 4:1-6), suggesting
that a hydrophobic moiety attached to a sugar residue with a long
alkyl chain is required for the interaction to the hydrophobic
amino acid residues. 1
[0016] Among many .beta.-glucosidase inhibitors, isofagomine (IFG)
and N-octyl valienamine were found to be the most potent inhibitors
of GCase, with IC.sub.50 values of approximately 50 nM, or 300 nM,
respectively (PCT/JP02/08882 to Ogawa et al; Ogawa et al., Bioorg.
Med. Chem. Lett. 1996; 6: 929-932).
[0017] In addition, tetrahydroimidazopyridines or glucoimidazole
(GIZ) derivatives were also found to be potent inhibitors of
.beta.-glucosidases (Panday et al., Helvetica Chimica Acta 2000;
83: 58-75). Although the inhibitors described by Panday were
potent, they lack specificity for GCase.
[0018] Thus, there remains a need in the art to design or identify
specific competitive inhibitors of enzymes, and evaluate them for
their ability to act as chaperones for the corresponding mutant
enzymes that are associated with numerous LSDs, particularly
inhibitors of GCase associated with Gaucher disease, and other
disorders resulting from misfolded proteins.
SUMMARY OF THE INVENTION
[0019] The present invention provides a compound of the Formula I:
2
[0020] wherein B, R.sup.1, R.sup.2, R.sup.5, L.sup.1, and L.sup.2
are described herein below. The present invention also provides
salts, esters and prodrugs of the compounds of Formula I.
[0021] The present invention also provides a compound of the
Formula II: 3
[0022] wherein B, R.sup.1, R.sup.2, R.sup.5, and L are described
herein below. The present invention also provides salts, esters and
prodrugs of the compounds of Formula II.
[0023] Additionally, the present invention described methods of
synthesizing compounds according to Formula I.
[0024] The present invention also provides salts, esters and
prodrugs of the compounds of Formula I and Formula II.
[0025] The present invention further provides a method of enhancing
in a mammalian cell the activity of GCase by contacting the cell
with a compound of Formula I or Formula II in an amount effective
to enhance the activity of GCase, i.e., a non-inhibitory
amount.
[0026] The present invention also provide a method of stabilizing
in mammalian cell the GCase by contacting the cell with a compound
of Formula I or Formula II in an amount effective to stabilize the
GCase.
[0027] The present invention also provides compositions comprising
a compound of Formula I and Formula II and a pharmaceutically
acceptable carrier.
[0028] Also provided is a method for treating Gaucher disease by
administering to an individual in need of such treatment a
pharmaceutical composition comprising a compound of Formula I or
Formula II in an amount effective to enhance the activity of
GCase.
[0029] In addition, the present invention provides a method of
inhibiting GCase in a mammalian cell when used at an inhibitory
concentrations, by contacting the cell with a compound of Formula I
or Formula II.
[0030] The present invention provides a method of inhibiting GCase
in vitro, when used at an inhibitory concentrations, by contacting
the cell with a compound of Formula I or Formula II.
[0031] The present invention provides a method of inhibiting
.beta.-glucosidases in a mammalian cell when used at an inhibitory
concentrations, by contacting the cell with an inhibitory
concentration of a compound of Formula I or Formula II.
[0032] The present invention provides a method of inhibiting
.beta.-glucosidase in vitro, when used at an inhibitory
concentrations, by contacting the cell with a compound of Formula I
or Formula II.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1. Structures of GCase-specific compounds. (a) DNJ,
1-deoxynojirimycin; (b) IFG, isofagomine; (c) GIZ, glucoimidazole;
(d) 2-PE-GIZ, 2-phenylethyl glucoimidazole; (e) 2-octyl GIZ,
2-octyl glucoimidazole, and (f) 2-DMB GIZ, 2-dimethylbutyl GIZ.
[0034] FIG. 2. Synthesis of glucoimidazole derivatives.
(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-iodo-5,6,7,8--
tetrahydroimidazo[1,2-.alpha.]pyridine (1) was prepared according
to the published procedure (Panday et al., Helv. Chim. Acta, 2000,
83: 58-79 and 1997, 80, 979-987). Iodo compound (1) was coupled
with phenylacetylene, octyne, or 3,3-dimethyl-1-butyne in the
presence of Pd(PPh.sub.3).sub.4 to afford compound (2), (3), or (4)
respectively. Hydrogenation of compound (3), filtration and
evaporation of the solvent gave a mixture with a reduced triple
bond. Treatment of the mixture with 1 M solution of BCl.sub.3 in
CH.sub.2Cl.sub.2 gave compound (6). Compounds (5) and (7) were
prepared by the same procedure starting from compound (2) or
(4).
[0035] FIG. 3. Synthesis of various glucoimidazole derivatives.
Various GIZ derivatives can be synthesized according the synthetic
scheme shown here, using iodination of protected GIZ, followed by
coupling of iodo residues with an alkyne or alkene reagents.
[0036] FIG. 4. Inhibitory activity of glucoimidazole derivatives
against GCase. The enzyme activity was performed as indicated in
the text. .circle-solid.=glucoimidazole (GIZ);
.smallcircle.=2-phenylethyl glucoimidazole; and
.tangle-solidup.=2-octyl glucoimidazole.
DETAILED DESCRIPTION
[0037] The present invention provides the design and synthesis of a
novel class of potent competitive inhibitors of GCase,
glucimidazole (GIZ), and polyhydroxy-cyclohexyl amine (PHCA)
derivatives of glucose, and demonstrates their ability to increase
the residual enzyme activity in fibroblasts from Gaucher patients
with N370S mutation.
[0038] Gaucher Disease
[0039] Gaucher disease is a lysosomal storage disorder resulting
from the deficient activity of .beta.-glucocerebrosidase
(hereinafter referred to as GCase) and the accumulation of its
undegraded substrate, glucosylceramide (glucocerebroside), a normal
intermediate in the catabolism of globoside and gangliosides
(Beutler et al., The Metabolic and Molecular Bases of Inherited
Disease, 8th ed. 2001; Scriver, C. R., Beaudet, A. L., Sly, W. S.
and Valle, D., ed.) pp. 3635-3668, McGraw-Hill, New York). In some
cases, the deficient activity of GCase is caused by mutations in
the GCase gene, resulting in misfolding and subsequent degradation
of the gene product in the ER. On the basis of the extent, and age
of onset of primary neurological involvement, three clinical
phenotypes are generally distinguished: (i) the non-neuronopathic
variant (type 1, or adult form); (ii) the acute neuronopathic
variant (type 2, or infantile form); and (iii) the subacute
neuronopathic variant (type 3 or juvenile form). Type 1 Gaucher
disease characterized by hepatosplenomegaly, secondary
hypersplenism, and skeletal involvement is the most prevalent form
and the severity and clinical course of this variant is
particularly heterogeneous, ranging from early onset to no clinical
manifestations (Grabowski, Gaucher disease: Enzymology, genetics,
and treatment. 1993, Plenum Press, New York). In contrast, patients
with the neurologic forms (types 2 and 3) are rare. The correlation
of clinical severity and genotypes indicates that mild mutations
presenting residual enzyme activity often result in type 1 disease,
whereas severe or null mutations cause type 3 or type 2
disease.
[0040] Gaucher patients have been found from all regions of the
world. Particularly, the disease is most common in the Ashkenazi
Jewish population, where the frequency of Gaucher disease-causing
alleles is approximately 0.0343 (Beutler et al., supra). The
incidence is estimated as 1:4,000 (Mathoth et al., Am J Med Genet.
1987; 27: 561-5). Approximately 97% of GCase mutations in Ashkenazi
Jews and 75% of the GCase mutations in non-Jewish populations can
be detected by screening for the five most common mutations. Of
many mutations that have now been documented, the N370S mutation
which results in exclusively type 1 Gaucher disease is the most
common mutation and is reported to be present in about 6% of the
Ashkenazi Jewish population (Beutler et al., Blood 1992; 79:
1662-6). The L444P mutation which causes type 3 disease among
homozygotes, exists at polymorphic levels in northern Sweden (Dahl
et al., Am J Hum Genet. 1990; 47: 275-8). An insertion of a G at
nucleotide position 84 of the cDNA is the second common Jewish
mutation. It is found in approximately 0.6% of the Jewish
population. This mutation results in a frameshift even before the
N-terminus of the mature protein, and an allele bearing this
mutation produces no enzyme activity and results in type 2 disease
(Beutler et al., Proc Natl Acad Sci USA. 1991; 88: 10544-7).
[0041] The gene coding for GCase has been mapped to chromosome 1 at
q21 (Ginns et al., Proc Natl Acad Sci USA 1985; 82: 7101-5). The
gene for GCase is approximately 7 kb in length and contains 11
exons. GCase cDNA is about 2 kb in length, and active enzyme can be
produced from in vivo translation of the cDNA in a variety of
eukaryotic cells, including COS cells and insect cells infected
with baculovirus (Grabowski et al., Enzyme 1989; 41: 131-42). Human
GCase is a homomeric glycoprotein. The mature polypeptide is 497
amino acids with a calculated molecular mass of 55,575. The
glycosylated enzyme from placenta has a molecular weight of about
65 kD. Saposin C activates GCase in vitro in the presence of
negatively charged phospholipids.
[0042] Current Treatment.
[0043] Enzyme replacement therapy is currently available to type 1
Gaucher patients. Intravenous infusion of human placental enzyme
GCase or recombinant enzymeGCase (modified to expose covered
mannose residues) has been shown to be effective at reversing many
characteristic clinical manifestations in type 1 Gaucher patients
(Kay et al., Trans Assoc. Am. Phys. 1991; 104: 258-264; and
Grabowski et al., Pediatr. Res. 1993; 33: 139A). For the type 2 or
type 3 patients having neurological involvement, the enzyme
replacement therapy (ERT) is less effective, since the enzymes do
not cross the blood brain barrier after intravenous infusion.
[0044] Another approach to the treatment of Gaucher disease is the
use of inhibitors of glucocerebroside synthetase GCase to lower the
levels of glucosylceramide and glycolipids (Inokuchi et al., Lipid
Res. 1987; 28: 565-71; Platt et al., Biochem. Pharmacol. 1998; 56:
421-430; and Radin et al., Glycoconjugate J. 1996; 13: 153-157).
This is known as substrate reduction therapy (SRT). A modest
improvement of clinical symptoms in patients was observed after
one-year treatment (Cox et al., Lancet 2000; 355: 1481-1485) with
small molecule glucose derivatives. SRT, which uses small molecule
inhibitors to prevent the synthesis of pathogenic substrates, is
under evaluation for several LSDs, and N-butyl 1-deoxynojirimycin
(NB-DNJ) has conditional marketing approval in Europe and the U.S.
for the treatment of Gaucher disease (Butters et al., Philos Trans
R Soc Lond B Biol Sci. 2003; 358: 927-945.). One advantage of this
as compared to ERT is that the small molecule inhibitors may
potentially cross the blood brain barrier and prevent substrate
accumulation in the brain. The most frequent adverse effect was
diarrhea, which occurred in 79% of patients shortly after the start
of the treatment. It is uncertain whether the long-term reduction
of glycolipids will have other adverse effects.
[0045] As discussed above, another small molecule approach recently
developed is known as active site specific chaperone (ASSC) therapy
(Fan et al Nat Med. 1999; 5: 112-115; Fan, Trends Pharmacol Sci.
2003; 24: 355-360). ASSC uses low concentrations of potent enzyme
inhibitors, which are specific for the mutant (or wild type)
enzyme, to enhance the folding and activity of the mutant proteins
in patients with LSDs. Since the active site inhibitors used in
ASSC are specific for the disease-causing enzyme, the therapy is
targeted to a single protein and a particular metabolic pathway,
unlike SRT which inhibits an entire synthetic pathway. Like SRT,
the small molecule inhibitors for ASSC have the potential of
crossing the blood brain barrier and therefore could be used to
treat neurological LSD forms.
[0046] Design and Synthesis of Potent Inhibitors for GCase and
Effective ASSCs for Gaucher Disease
[0047] A more potent inhibitor of an enzyme acts as a better ASSC
for the mutant enzyme (Fan, Trends Pharmacol Sci. 2003; 24:355-60).
Accordingly, the present invention designed and synthesized a novel
class of potent inhibitors for GCase, and used these inhibitors as
ASSCs for the enhancement of residual enzyme activity in cells
derived from Gaucher patients.
[0048] Fan et al. have also determined that ASSC therapy can be
used to treat Gaucher disease using hydroxypiperidine (HP)
derivatives, which may be administered to individuals having
Gaucher disease as "active-site specific chaperones" for the mutant
glucocerebrosidase associated with the disease. (U.S. patent
application entitled Hydroxy Piperidine Derivatives to Treat
Gaucher Disease, filed Nov. 12, 2004).
[0049] In addition to enhancing the activity of the mutant (or wild
type) enzymes associated with the LSDs, the ASSCs have also been
demonstrated to enhance the activity of the corresponding wild-type
enzyme (see U.S. Pat. No. 6,589,964 to Fan et al.), thus having
utility as co-therapy for enzyme replacement therapy and gene
therapy in LSD patients.
[0050] The present invention also contemplates the inhibition of
.beta.-glucocerebrosidase (GCase). Such inhibition is useful, for
example, for studying the effects of a lack of b-glucocerebrosidase
in animal models. Since targeted deletion of
.beta.-glucocerebrosidase in mammals is lethal, since the knock-out
mice cannot be generated for the purposes of evaluating the Gaucher
disease state. The present invention contemplates administering an
inhibitory amount of the .beta.-glucosidase inhibitors to animals
to mimic the Gaucher disease phenotype.
Definitions
[0051] As used herein, the following terms have the following
definitions.
Chemical
[0052] The term `alkyl` refers to a straight or branched
C.sub.1-C.sub.20 hydrocarbon group consisting solely of carbon and
hydrogen atoms, containing no unsaturation, and which is attached
to the rest of the molecule by a single bond, e.g., methyl, ethyl,
n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl,
1,1-dimethylethyl (t-butyl). The alkyls used herein are preferably
C.sub.1-C.sub.8 alkyls.
[0053] The term "alkenyl" refers to a C.sub.2-C.sub.20 aliphatic
hydrocarbon group containing at least one carbon-carbon double bond
and which may be a straight or branched chain, e.g., ethenyl,
1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1-propenyl,
1-butenyl, 2-butenyl.
[0054] The term "alkynyl" refers to a C.sub.2-C.sub.20 straight or
branched chain hydrocarbon radicals having at least one
carbon-carbon triple bond, e.g. ethynyl, propynyl, butnyl.
[0055] The term "cycloalkyl" denotes an saturated, non-aromatic
mono- or multicyclic hydrocarbon ring system such as cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl. Examples of multicyclic
cycloalkyl groups include perhydronapththyl, adamantyl and
norbornyl groups bridged cyclic group or sprirobicyclic groups,
e.g., spiro (4,4) non-2-yl.
[0056] The term "cycloalkenyl" an unsaturated, non-aromatic mono-
or multicyclic hydrocarbon ring system containing 3 to about 14
carbon atoms, system such as cyclopropenyl, cyclobutenyl,
cyclopentenyl, cyclohexenyl.
[0057] The term "cycloalkalkyl" refers to a cycloalkyl as defined
above directly attached to an alkyl group as defined above, that
results in the creation of a stable structure such as
cyclopropylmethyl, cyclobutylethyl, cyclopentylethyl.
[0058] The term "aryl" refers to aromatic radicals having in the
range of about 6 to about 14 carbon atoms such as phenyl, naphthyl,
tetrahydronapthyl, indanyl, biphenyl.
[0059] The term "arylalkyl" refers to an aryl group as defined
above directly bonded to an alkyl group as defined above, e.g.,
--CH.sub.2C.sub.6H.sub.5, and --C.sub.2H.sub.4C.sub.6H.sub.5.
[0060] The term "heterocyclic ring" or "heterocycyl" refer to a
stable 3- to 15-membered ring radical which consists of carbon
atoms and from one to five heteroatoms selected from the group
consisting of nitrogen, phosphorus, oxygen and sulfur. For purposes
of this invention, the heterocyclic ring radical may be a
monocyclic, bicyclic or tricyclic ring system, which may include
fused, bridged or spiro ring systems, and the nitrogen, phosphorus,
carbon, oxygen or sulfur atoms in the heterocyclic ring radical may
be optionally oxidized to various oxidation states. In addition,
the nitrogen atom may be optionally quaternized; and the ring
radical may be partially or fully saturated (i.e., heteroaromatic
or heteroaryl aromatic). Examples of such heterocyclic ring
radicals include, but are not limited to, azetidinyl, acridinyl,
benzodioxolyl, benzodioxanyl, benzofurnyl, carbazolyl, cinnolinyl,
dioxolanyl, indolizinyl, naphthyridinyl, perhydroazepinyl,
phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pyridyl,
pteridinyl, purinyl, quinazolinyl, quinoxalinyl, quinolinyl,
isoquinolinyl, tetrazoyl, imidazolyl, tetrahydroisouinolyl,
piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,
2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, pyrrolyl,
4-piperidonyl, pyrrolidinyl, pyrazinyl, pyrimidinyl, pyridazinyl,
oxazolyl, oxazolinyl, oxasolidinyl, triazolyl, indanyl, isoxazolyl,
isoxasolidinyl, morpholinyl, thiazolyl, thiazolinyl, thiazolidinyl,
isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, isoindolyl,
indolinyl, isoindolinyl, octahydroindolyl, octahydroisoindolyl,
quinolyl, isoquinolyl, decahydroisoquinolyl, benzimidazolyl,
thiadiazolyl, benzopyranyl, benzothiazolyl, benzooxazolyl, furyl,
tetrahydrofurtyl, tetrahydropyranyl, thienyl, benzothienyl,
thiamorpholinyl, thiamorpholinyl sulfoxide thiamorpholinyl sulfone,
dioxaphospholanyl, oxadiazolyl, chromanyl, isochromanyl.
[0061] The heterocyclic ring radical may be attached to the main
structure at any heteroatom or carbon atom that results in the
creation of a stable structure.
[0062] The term "heteroarylalkyl" refers to heteroaryl ring radical
as defined above directly bonded to alkyl group. The
heteroarylalkyl radical may be attached to the main structure at
any carbon atom from alkyl group that results in the creation of a
stable structure.
[0063] The term "heterocyclylalkyl" refers to a heterocylic ring
radical as defined above directly bonded to alkyl group. The
heterocyclylalkyl radical may be attached to the main structure at
carbon atom in the alkyl group that results in the creation of a
stable structure.
[0064] The substituents in the `substituted alkyl`, `substituted
alkenyl` `substituted alkynyl` `substituted cycloalkyl`
`substituted cycloalkalkyl` `substituted cyclocalkenyl`
`substituted arylalkyl` `substituted aryl` `substituted
heterocyclic ring`, `substituted heteroaryl ring,` `substituted
heteroarylalkyl`, or `substituted heterocyclylalkyl ring`, may be
the same or different with one or more selected from the groups
hydrogen, hydroxy, halogen, carboxyl, cyano, amino, nitro, oxo
(.dbd.O), thio (.dbd.S), or optionally substituted groups selected
from alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,
aryl, heteroaryl, heteroarylalkyl, heterocyclic ring, --COOR.sup.x,
--C(O)R.sup.x, --C(S)R.sup.x, --C(O)NR.sup.xR.sup.y,
--C(O)ONR.sup.xR.sup.y, --NR.sup.xCONR.sup.yR.sup.z,
--N(R.sup.x)SOR.sup.y, --N(R.sup.x)SO.sub.2R.sup.y,
--(.dbd.N--N(R.sup.x)R.sup.y), --NR.sup.xC(O)OR.sup.y,
--NR.sup.xR.sup.y, --NR.sup.xC(O)R.sup.y--,
--NR.sup.xC(S)R.sup.y--NR.sup.xC(S)NR.sup.yR.sup- .z,
--SONR.sup.xR.sup.y--, --SO.sub.2NR.sup.xR.sup.y--, --OR.sup.x,
--OR.sup.xC(O)NR.sup.yR.sup.z, --OR.sup.xC(O)OR.sup.y--,
--OC(O)R.sup.x, --OC(O)NR.sup.xR.sup.y, --R.sup.xNR.sup.yR.sup.z,
--R.sup.xR.sup.yR.sup.z- , --R.sup.xCF.sub.3,
--R.sup.xNR.sup.yC(O)R.sup.z, --R.sup.xOR.sup.y,
--R.sup.xC(O)OR.sup.y, --R.sup.xC(O)NR.sup.yR.sup.z,
--R.sup.xC(O)R.sup.x, --R.sup.xOC(O)R.sup.y, --SR.sup.x,
--SOR.sup.x, --SO.sub.2R.sup.x, --ONO.sub.2, wherein R.sup.x,
R.sup.y and R.sup.z in each of the above groups can be hydrogen
atom, substituted or unsubstituted alkyl, haloalkyl, substituted or
unsubstituted arylalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkalkyl substituted or unsubstituted
heterocyclic ring, substituted or unsubstituted heterocyclylalkyl,
substituted or unsubstituted heteroaryl or substituted or
unsubstituted heteroarylalkyl.
[0065] The term "lipophilic" refers to a functional residue capable
of interacting with hydrophobic amino acid residues. Examples of
liphophilic groups include but are not limited to C.sub.1-C.sub.12
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted cycloalkyl; substituted or unsubstituted
cycloalkenyl; substituted or unsubstituted aryl; substituted or
unsubstituted arylalkyl; substituted or unsubstituted heteroaryl;
substituted or unsubstituted heterocyclic; substituted or
unsubstituted heterocycloalkyl; and substituted or unsubstituted
heteroarylalkyl.
[0066] The term "halogen" refers to radicals of fluorine, chlorine,
bromine and iodine.
[0067] The term "hydrogenatable protecting group" refers to
protecting groups capable of removal by hydrogenating conditions,
for example, benzyl and 4-methoxybenzyl.
[0068] The term "acid" refers to Bronsted or Lewis acid such as
sulfuric acid, hydrochloride or hydrochloride generated by reaction
of acid chloride with alcohol.
[0069] The term "organic acid" refers to organic to Bronsted or
Lewis acid such as p-toluenesulfonic acid, camphorsulfonic
acid.
[0070] The term "crown ether" refers to large ring compounds
containing several oxygen atoms in a regular pattern such as
12-crown-4,15-crown-5,1- 8-crown-6, dicyclohexano-18-crown-6.
[0071] The term "organometallic reagent" refers to a compound that
contains a bond between a carbon and a metal atom, such as
organolithium, organozinc, organocopper, Grignard reagents.
[0072] The term "hydrogenolysis conditions" refers to catalytic
hydrogenolysis using catalyst such as, but not limited to,
Pd(OH).sub.2/C, Pd/C, Pt or Raney nickel in the presence of a
hydrogen source.
[0073] The term "hydrogenation" refers to a chemical reaction in
which unsaturated bond between carbon atoms are reduced by
attaching a hydrogen atom to each other using catalyst such as, but
not limited to, Pd(OH).sub.2/C, Pd/C, Pt or Raney nickel in the
presence of a hydrogen source.
[0074] The term "reducing agent" refers to an agent converting a
functional group in a molecule from one oxidation state to lower
one, such as LiAlH.sub.4, NaBH.sub.4, Zn(BH.sub.4).sub.2,
i-Bu.sub.2AlH and Li-s-Bu.sub.3H.
[0075] The term "leaving group" refers to a group that can be
substituted by nucleophilic reagent, such as TsO--, TfO--, MsO--,
Cl, Br, I.
[0076] The term "N-protecting group" refers to groups temporarily
protecting amine or imine group to avoid further sites of reaction,
such as, but not limited to, 4-methoxybenzyl, benzyl,
tert-butyloxycarbonyl, benzyloxycarbonyl.
[0077] The term "O-protecting group" refers to groups temporarily
protecting an hydroxy to avoid further sites of reaction, such as,
but not limited to, 4-methoxybenzyl, benzyl, and
trimethylsilyl.
[0078] The term "base" refers to organic Lewis base, such as
pyridine, triethylamine, diisopropylethylamine.
[0079] The term "nucleophilic moiety and nucleophilic reagent"
refer to a reagent that forms a bond to its reaction partner (the
electrophile) by donating both bonding electrons, such as metal
salt of an alcohol, thiol, or 1-alkyne.
[0080] The term "a short flexible linker" refers to linkers with
linear length of about 6 .ANG. to about 12 .ANG., preferably about
9 .ANG..
Biological
[0081] As used herein, the term "active site specific chaperone"
(ASSC) refers to any molecule including but not limited to a
protein, peptide, nucleic acid, carbohydrate, that specifically
interacts reversibly with an active site of a protein and enhances
formation of a stable molecular conformation. As used herein,
"ASSC" does not include endogenous general chaperones present in
the ER of cells such as Bip, calnexin or calreticulin, or general,
non-specific chemical chaperones such as deuterated water, DMSO, or
TMAO.
[0082] As used herein, the term "active site" refers to the region
of a protein that binds a substrate or binding partner and
contributes the amino acid residues that directly participate in
the making and breaking of chemical bonds. According to the present
invention, the active site encompasses the catalytic domain of the
GCase.
[0083] The term "wild-type activity" refers to the normal
physiological function of a GCase in a cell. Such functionality can
be tested by any means known to establish functionality of a
protein, specifically, an enzyme. Certain tests may evaluate
attributes of a protein that may or may not correspond to its
actual in vivo function, but nevertheless are aggregate surrogates
of protein functionality, and wild-type behavior in such tests is
an acceptable consequence of the protein folding rescue techniques
of the invention. One such activity in accordance with the
invention is appropriate transport from the endoplasmic reticulum
to the particular destination of GCase in the cell, i.e., the
lysosome.
[0084] The term "functional GCase protein" refers to a GCase
protein that has the ability to fold in a proper conformation,
achieve its native location in the cell, and have catabolic
activity towards glucocerebroside and other lipid substrates. A
functional GCase protein includes wild-type GCase proteins (see
definitions below), e.g., as depicted in SEQ ID NO: 2.
[0085] As used herein, the term "mutant GCase" refers to a GCase
translated from a gene containing one or more genetic mutations
that result in an altered protein sequence that does not achieve
its native conformations under the conditions normally present in
the ER. The failure to achieve this conformation results in the
GCase being degraded, rather than being transported through its
normal pathway in the protein transport system to its proper
location within the cell.
[0086] Some specific embodiments of such GCase mutations are the
N370S mutation and the L444P mutation.
[0087] The term "enhancing the activity" of GCase means stabilizing
a proper conformation of a mutant GCase protein in the ER so that
it folds in a proper conformation, achieves its native location in
the cell, and has catabolic activity towards cerebroside, its lipid
substrate). This term also refers to increasing the wild-type
activity of an exogenously administered GCase protein, i.e., by
increasing the stability and extending the in vivo half-life of
wild-type GCase, thus, prolonging its activity.
[0088] The term "stabilize a proper conformation" refers to the
ability of a compound of the invention to induce or stabilize the
conformation of a mutated GCase, regardless whether in the ER or
other cellular compartments, that is functionally identical to the
conformation of the wild type enzyme. By "functionally identical",
the invention means that there may be minor variations in the
conformation (indeed almost all proteins exhibit some
conformational flexibility in their physiological state) but that
conformational flexibility does not result in (1) aggregation, (2)
elimination through the endoplasmic reticulum-associated
degregation, (3) impairment of enzyme function, and/or (4) improper
transport within the cell. This term also refers to the ability of
a compound to stabilize a proper conformation of wild-type GCase in
viov following exogenously added GCase, or an vitro in a
formulation of enzyme.
[0089] A "wild-type GCase gene" refers to nucleic acid sequences
which encode an ASM protein capable of having functional biological
activity in vivo. The wild-type GCase nucleic acid sequence may
contain nucleotide changes that differ from the known, published
sequence, e.g., SEQ ID NO: 1, as long as the changes result in
amino acid substitutions that have little or no effect on the
biological activity. As used herein, the term wild-type may also
include GCase nucleic acid sequences engineered to encoding a GCase
protein capable of increased or enhanced activity relative to the
endogenous or native GCase protein.
[0090] A "wild-type GCase protein" refers to any protein encoded by
a wild-type gene that is capable of having functional biological
activity when expressed or introduced in vivo.
Molecular Biology
[0091] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization; B. D. Hames & S. J. Higgins eds. (1985);
Transcription And Translation (B. D. Hames & S. J. Higgins,
eds. 1984); Animal Cell Culture (R. I. Freshney, ed. 1986);
Immobilized Cells And Enzymes; IRL Press, (1986); B. Perbal, A
Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, Inc. (1994).
[0092] As used herein, the term "isolated" means that the
referenced material is removed from the environment in which it is
normally found. Thus, an isolated biological material can be free
of cellular components, i.e., components of the cells in which the
material is found or produced. In the case of nucleic acid
molecules, an isolated nucleic acid includes a PCR product, an
isolated mRNA, a cDNA, or a restriction fragment. In another
embodiment, an isolated nucleic acid is preferably excised from the
chromosome in which it may be found, and more preferably is no
longer joined to non-regulatory, non-coding regions, or to other
genes, located upstream or downstream of the gene contained by the
isolated nucleic acid molecule when found in the chromosome. In yet
another embodiment, the isolated nucleic acid lacks one or more
introns. Isolated nucleic acid molecules include sequences inserted
into plasmids, cosmids, artificial chromosomes, and the like. Thus,
in a specific embodiment, a recombinant nucleic acid is an isolated
nucleic acid. An isolated protein, may be associated with other
proteins or nucleic acids, or both, with which it associates in the
cell, or with cellular membranes if it is a membrane-associated
protein. In a specific embodiment, an isolated GCase protein is a
recombinant GCase protein expressed from an expression vector. An
isolated material may be, but need not be, purified.
[0093] The term "purified" as used herein refers to material that
has been isolated under conditions that reduce or eliminate
unrelated materials, i.e., contaminants. For example, a purified
GCase protein is preferably substantially free of other proteins or
nucleic acids with which GCase is normally associated in a cell. As
used herein, the term "substantially free" is used operationally,
in the context of analytical testing of the material. Preferably,
purified ASM substantially free of contaminants is at least 50%
pure; more preferably, at least 90% pure, and more preferably still
at least 99% pure. Purity can be evaluated by chromatography, gel
electrophoresis, immunoassay, composition analysis, biological
assay, and other methods known in the art.
[0094] The terms "about" and "approximately" shall generally mean
an acceptable degree of error for the quantity measured given the
nature or precision of the measurements. Typical, exemplary degrees
of error are within 20 percent (%), preferably within 10%, and more
preferably within 5% of a given value or range of values.
Alternatively, and particularly in biological systems, the terms
"about" and "approximately" may mean values that are within an
order of magnitude, preferably within 10- or 5-fold, and more
preferably within 2-fold of a given value. Numerical quantities
given herein are approximate unless stated otherwise, meaning that
the term "about" or "approximately" can be inferred when not
expressly stated.
[0095] The term "host cell" means any cell of any organism that is
selected, modified, transformed, grown, or used or manipulated in
any way, for the production of a substance by the cell, for example
the expression by the cell of a mutant or functional mammalian
GCase gene, including a DNA or RNA sequence, or the GCase enzyme.
Host cells can further be used for preliminary evaluation of the
ASSC concept other assays. A "recombinant DNA molecule" is a DNA
molecule that has undergone a molecular biological manipulation or
engineering.
[0096] A "gene" is a sequence of nucleotides which code for a "gene
product". Generally, a gene product is a protein. However, a gene
product can also be another type of molecule in a cell, such as an
RNA (e.g., a tRNA or a rRNA). For the purposes of the present
invention, a gene product also refers to an mRNA sequence which may
be found in a cell. As used herein, a gene refers to the nucleotide
sequences encoding wild-type or mutant GCase.
[0097] The term "express" and "expression" means allowing or
causing the information in a GCase gene or DNA sequence to become
manifest, for example producing RNA (such as rRNA or mRNA) or a
GCase protein by activating the cellular functions involved in
transcription and translation of a corresponding GCase gene or DNA
sequence, i.e., sequences encoding GCase. A GCase DNA sequence is
expressed by a cell to form an "expression product" such as a GCase
RNA (e.g., an mRNA or an rRNA) or a GCase protein. The expression
product itself, e.g., the resulting GCase RNA or protein, may also
said to be "expressed" by the cell.
[0098] The term "transfection" means the introduction of a foreign
nucleic acid into a cell. The term "transformation" also means the
introduction of a "foreign" (i.e., extrinsic or extracellular)
gene, DNA or RNA sequence into a host cell so that the host cell
will express the introduced gene or sequence to produce a desired
substance, in this invention typically an RNA coded by the
introduced gene or sequence, but also a protein or an enzyme coded
by the introduced gene or sequence. The introduced gene or sequence
may also be called a "cloned" or "foreign" gene or sequence, may
include regulatory or control sequences (e.g., start, stop,
promoter, signal, secretion or other sequences used by a cell's
genetic machinery). The gene or sequence may include nonfunctional
sequences or sequences with no known function. A host cell that
receives and expresses introduced DNA or RNA has been "transformed"
and is a "transformant" or a "clone". The DNA or RNA introduced to
a host cell can come from any source, including cells of the same
genus or species as the host cell or cells of a different genus or
species. As used herein, transfection or transformation will
include introduction of sequences encoding functional GCase in
individuals having mutated endogenous GCase genes.
[0099] The terms "vector", "cloning vector", and "expression
vector" mean the vehicle by which a GCase DNA or RNA sequence
(e.g., a foreign gene) can be introduced into a host cell so as to
transform the host and promote expression (e.g., transcription and
translation) of the introduced sequence. Vectors include any
genetic element, such as a plasmid, phage, transposon, cosmid,
chromosome, virus, virion, etc., which is capable of replication
when associated with the proper control elements and which can
transfer GCase gene sequences between cells. Thus, the term
includes cloning and expression vehicles, as well as viral
vectors.
[0100] The term "expression system" means a host cell and
compatible vector under suitable conditions, e.g. for the
expression of a GCase protein coded for by foreign DNA carried by
the vector and introduced to the host cell. Common expression
systems include E. coli host cells and plasmid vectors, insect host
cells such as Sf9, Hi5 or S2 cells and Baculovirus vectors and
expression systems, and mammalian host cells and vectors.
[0101] The term "gene therapy" refers to a method of changing the
expression of an endogenous gene by exogenous administration of a
gene, i.e., a GCase gene. As used herein, gene therapy also refers
to the replacement of a defective GCase gene, or replacement of a
missing GCase gene, by introducing a functional gene corresponding
to the defective or missing GCase gene into somatic or stem cells
of an individual in need. Gene therapy can be accomplished by "ex
vivo" methods, in which differentiated or somatic stem cells are
removed from the individual's body followed by the introduction of
a normal copy of the defective gene into the explanted cells using
a viral vector as the gene delivery vehicle. In addition, in vivo
direct gene transfer is gene transfer into cells in the individual
in situ using a broad range of viral vectors, liposomes, protein
DNA complexes, or naked DNA in order to achieve a therapeutic
outcome.
[0102] The term "recombinant protein" refers to a GCase protein
(gene product) encoded by a therapeutic GCase gene carried on a
vector. Generally, the cell receiving the vector will lack
expression and/or activity of any endogenous GCase protein
corresponding to the recombinant protein, or if there is expression
of such an endogenous GCase protein, it is of a mutant or at a very
low level. In one embodiment, the recombinant protein is produced
by a cell in tissue culture for experimental and therapeutic
purposes. In another embodiment, the recombinant protein is
produced in vivo from cells transformed with vector, wherein the
vector or the cells comprising the vector have been administered to
a subject, i.e., gene therapy. The recombinant GCase protein will
likely be indistinguishable from wild-type protein in normal
individuals, i.e., individuals who are not deficient in the protein
or do not have Gaucher disease.
Therapeutic and Administration
[0103] A "subject" or "patient" is a human or an animal that has
developed, or is likely to develop Gaucher disease, more
particularly a mammal, preferably a rodent or a primate, and most
preferably a human. In one embodiment, the patient is a member of
the Ashkenazi Jewish population who has been diagnosed with, or who
has been identified as having an increased risk of developing
Gaucher disease due inherited mutations in the GCase gene. However,
the term "subject" encompasses anyone in the world having, or
genetically at risk of developing, Gaucher disease.
[0104] The term "prevention" refers to the prevention of the onset
of the disease, which means to prophylactically interfere with a
pathological mechanism that results in the disease, e.g., Gaucher
disease. In the context of the present invention, such a
pathological mechanism can be an increase in mutant protein folding
and expression of GCase.
[0105] The term "treatment" means to therapeutically intervene in
the development of a disease in a subject showing a symptom of this
disease. In the context of the present invention, these symptoms
can include but are not limited to accumulation of GCase in
lysosomes, hepatosplenomegaly, psychomotor retardation, pulmonary
abnormalities degeneration of bones and joints, and progressive
neurodegeneration.
[0106] The term "therapeutically effective amount" is used herein
to mean an amount or dose of the GIZ or PHCA derivatives of the
present invention that is sufficient to increase the level of
mutant GCase expression, e.g., to about 3-5%, preferably by about
10%, and more preferably by about 30% of the level found in normal
cells, i.e., cells from an individual not having Gaucher disease.
Preferably, a therapeutically effective amount can ameliorate or
prevent a clinically significant deficit GCase activity in the
subject. Alternatively, a therapeutically effective amount is
sufficient to cause an improvement in a clinically significant
condition in the subject, e.g., amelioration of progressive
neurodegeneration in Types 2 and 3 Gaucher patients.
[0107] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, and dizziness, when administered to a human.
Preferably, as used herein, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly,
in humans.
[0108] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil.
Water or aqueous solution saline solutions and aqueous dextrose and
glycerol solutions are preferably employed as carriers,
particularly for injectable solutions. Suitable pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by
E. W. Martin.
Novel Compounds and Synthesis Compounds
[0109] According to the present invention, the ASSC is a
glucoimidazole derivatives as defined in Formula I. In a specific
embodiment, the ASSC is
(5R,6R,7S,8S)-5-hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]-
pyridine-6,7,8-triol. The ASSC also can be a C-derivative (and
optionally, additionally N-alkylated) of polyhydroxycyclohexyl
amine having (i) all hydroxy groups on the cyclohexyl ring in the
same configurations as a glucose, providing the amine is attached
to the carbon corresponding the anomeric carbon of a glucose; (ii)
a short, flexible linker emanating from the corresponding position
of the ring oxygen in a pyranose; and (iii) a lipophilic moiety
connected to the linker. Since the C-6 position of the glucose
residue is not recognized by the most of GCase and
.beta.-glucosidases (De Bruyne et al. Eur. J. Biochem. 1979;
102:257-67), the substitutes at the C-6 position can be a hydrogen,
hydroxy or hydroxymethyl.
[0110] The present invention provides a compound of the Formula I:
4
[0111] wherein B is hydrogen, hydroxy, N-acetamino, or halogen;
[0112] R.sup.1 and R.sup.2 optionally present are short, flexible
linkers linear length of about 6 .ANG. to about 12 .ANG.,
preferably about 9 .ANG.. R.sup.1 and R.sup.2 can also be
independently C.sub.2-C.sub.6 substituted or unsubstituted: alkyl,
alkenyl, or alkynyl, which are optionally interrupted by one or
more moieties chosen from the group consisting of NH, NHCOO,
NHCONH, NHCSO, NHCSNH, CONH, NHCO, NR.sup.3, O, S, S(O).sub.m and
--S(O).sub.m NR.sup.3;
[0113] whereas m is 1 or 2, and
[0114] R.sup.3 is independently selected from each occurrence a
hydrogen, or a substituted or unsubstituted: alkyl, alkenyl;
alknyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl; heteroaryl;
heterocyclic; heterocyclyalkyl; or heteroarylalkyl, or --C(O)
attached to a C.sub.1-C.sub.6 substituted or unsubstituted
alkyl;
[0115] and pharmaceutically acceptable salts and prodrugs
thereof.
[0116] In addition, R.sup.1-L.sup.1 or R.sup.2-L.sup.2 can be a
hydrogen, if R.sup.2-L.sup.2 or R.sup.1-L.sup.1 is other than a
hydrogen, respectively.
[0117] R.sup.5 represents a hydrogen, hydroxy, or hydroxymethyl. In
a preferred embodiment, R.sup.5 is hydroxymethyl.
[0118] L.sup.1 and L.sup.2 are lipophilic groups having 3-12 carbon
atoms comprising a substituted or unsubstituted: alkyl, alkenyl,
alkynyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl; heteroaryl;
heterocyclic; heterocycloalkyl; or heteroarylalkyl.
[0119] In a preferred embodiment, R.sup.1-L.sup.1 is unsubstituted
C.sub.1-C.sub.12 alkyl, or more preferably an unsubstituted
C.sub.6-C.sub.12 alkyl, or more preferably an unsubstituted C.sub.8
alkyl. In another embodiment, R.sup.1-L.sup.1 is an unsubstituted
branched C.sub.3-12 alkyl, or an unsubstituted branched C.sub.6-12
alkyl, or more preferably an unsubstituted branched C.sub.6 alkyl.
In one embodiment, R.sup.1-L.sup.1 is 3,3-dimethylbutyl.
[0120] A preferred compound of the invention is
(5R,6R,7S,8S)-5-hydroxymet-
hyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-triol.
Other preferred compounds of the invention include
(5R,6R,7S,8S)-5-hydroxymethy-
l-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-tri-
ol,
(5R,6R,7S,8S)-5-hydroxymethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-
-6,7,8-triol, and
(5R,6R,7S,8S)-5-hydroxymethyl-2-phenethyl-5,6,7,8-tetrah-
ydroimidazo[1,2-a]pyridine-6,7,8-triol.
[0121] Collectively, the compounds of Formula I are referred to as
"glucoimidazole (GIZ) derivatives."
[0122] The present invention also provides a compound of the
Formula II: 5
[0123] wherein B is hydrogen, hydroxy, N-acetamino, or halogen;
[0124] R.sup.1 is a hydrogen, substituted or unsubstituted: alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, arylalkyl,
heteroaryl, heterocyclic, heterocyclyalkyl, or heteroarylalkyl;
--C(O)R.sup.3 or --S(O).sub.mR.sup.3;
[0125] R.sup.2, optionally present is a short, flexible linker
linear length of about 6 .ANG. to about 12 .ANG., preferably about
8 .ANG.-10 .ANG., and more preferrably about 9 .ANG.. R.sup.2 can
aso be group having 2-6 carbon atoms comprising a substituted or
unsubstituted: alkyl, alkenyl, or alkynyl, which are optionally
interrupted by one or more moieties chosen from the group
consisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH, CONH, NHCO,
NR.sup.3, O, S, S(O).sub.m and --S(O).sub.mNR.sup.3;
[0126] whereas m is 1 or 2, and R.sup.3 is independently selected
from each occurrence a hydrogen, or a substituted or unsubstituted:
alkyl, alkenyl; alknyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl;
heteroaryl; heterocyclic; heterocyclyalkyl; or heteroarylalkyl, or
--C(O) attached to a C.sub.1-C.sub.6 substituted or unsubstituted
alkyl; and
[0127] L is a lipophilic group having 3-12 carbon atoms comprising
a substituted or unsubstituted: alkyl, alkenyl, alkynyl;
cycloalkyl, cycloalkenyl; aryl; arylalkyl; heteroaryl;
heterocyclic; heterocycloalkyl; or heteroarylalkyl; and
[0128] pharmaceutically acceptable salts and prodrugs thereof.
[0129] In addition, R.sup.2-L can be a hydrogen.
[0130] R.sup.5 represents a hydrogen, hydroxy, or hydroxymethyl. In
a preferred embodiment, R.sup.5 is hydroxymethyl.
[0131] Collectively, the compounds of Formula II are referred to as
"PHCA derivatives."
[0132] Compounds of the present invention include pharmaceutically
acceptable salts and pro-drugs of Formula I and Formula II.
Pharmaceutically acceptable salts forming part of this invention
include salts derived from inorganics such as L1, Na, K, Ca, Mg,
Fe, Cu, Zn, Mn; salts of organic bases such as
N,N'-diacetylethylenediamine, glucamine, triethylamine, choline,
hydroxide, dicyclohexylamine, metformin, benzylamine,
trialkylamine, thiamine; chiral bases like alkylphenylamine,
glycinol, phenyl glycinol, salts of natural amino acids such as
glycine, alanine, valine, leucine, isoleucine, norleucine,
tyrosine, cystine, cysteine, methionine, proline, hydroxy proline,
histidine, ornithine, lysine, arginine, serine; non-natural amino
acids such as D-isomers or substituted amino acids; guanidine,
substituted guanidine wherein the substituents are selected from
nitro, amino, alkyl, alkenyl, alkynyl, ammonium or substituted
ammonium salts and aluminum salts. Salts may include acid addition
salts where appropriate which are, sulphates, nitrates, phosphates,
perchlorates, borates, hydrohalides, acetates, tartrates, maleates,
citrates, succinates, palmoates, methanesulphonates, benzoates,
salicylates, benzenesulfonates, ascorbates, glycerophosphates,
ketoglutarates. Pharmaceutically acceptable solvates may be
hydrates or comprise other solvents of crystallization such as
alcohols.
[0133] Prodrugs are compounds which are converted in vivo to active
forms (see, e.g., R. B. Silverman, 1992, "The Organic Chemistry of
Drug Design and Drug Action", Academic Press, Chp. 8). Prodrugs can
be used to alter the biodistribution (e.g., to allow compounds
which would not typically enter the reactive site of the protease)
or the pharmacokinetics for a particular compound. For example, a
carboxylic acid group can be esterified, e.g., with a methyl group
or an ethyl group, to yield an ester. When the ester is
administered to a subject, the ester is cleaved, enzymatically or
non-enzymatically, reductively, oxidatively, or hydrolytically, to
reveal the anionic group. An anionic group can be esterified with
moieties (e.g., acyloxymethyl esters) which are cleaved to reveal
an intermediate compound which subsequently decomposes to yield the
active compound.
[0134] Examples of prodrugs and their uses are well known in the
art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J.
Pharm. Sci. 66: 1-19). The prodrugs can be prepared in situ during
the final isolation and purification of the compounds, or by
separately reacting the purified compound with a suitable
derivatizing agent. For example hydroxy groups can be converted
into esters via treatment with a carboxilic acid in the presence of
a catalyst. Examples of cleavable alcohol prodrug moieties include
substituted and unsubstituted, branched or unbranched lower alkyl
ester moieties, (e.g., ethyl esters), lower alkenyl esters,
di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl
ester), acylamino lower alkyl esters, acyloxy lower alkyl esters
(e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester),
aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g.,
with methyl, halo, or methoxy substituents) aryl and aryl-lower
alkyl esters, amides, lower-alkyl amides, di-lower alkyl amides,
and hydroxy amides.
Synthesis
[0135] Additionally, the present invention describes a method for
the synthesis of compounds according to Formula III: 6
[0136] R.sup.1 and R.sup.2 optionally present are short, flexible
linkers having a linear length of about 6 .ANG. to about 12 .ANG.,
preferably about 9 .ANG.. R.sup.1 and R.sup.2 can also be
independently C.sub.2-C.sub.6 substituted or unsubstituted: alkyl,
alkenyl, or alkynyl, which are optionally interrupted by one or
more moieties chosen from the group consisting of NH, NHCOO,
NHCONH, NHCSO, NHCSNH, CONH, NHCO, NR.sup.3, O, S, S(O).sub.m and
--S(O).sub.m NR.sup.3;
[0137] whereas m is 1 or 2, and R.sup.3 is independently selected
from each occurrence a hydrogen, or a substituted or unsubstituted:
alkyl, alkenyl; alknyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl;
heteroaryl; heterocyclic; heterocyclyalkyl; or heteroarylalkyl.
[0138] In addition, R.sup.1-L.sup.1 or R.sup.2-L.sup.2 can be a
hydrogen, if either R.sup.2-L.sup.2 or R.sup.1-L.sup.1 is other
than a hydrogen, respectively.
[0139] L.sup.1 and L.sup.2 are lipophilic groups having 3-12 carbon
atoms comprising a substituted or unsubstituted: alkyl, alkenyl,
alkynyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl; heteroaryl;
heterocyclic; heterocycloalkyl; or heteroarylalkyl.
[0140] Also, pharmaceutically acceptable salts and prodrugs thereof
may be synthesized.
[0141] This synthesis comprises the steps of:
[0142] a) reacting a compound of the Formula IV: 7
[0143] wherein P.sub.1, P.sub.2, P.sub.3 and P.sub.4 are
O-protection groups, with N-iodo-succinimide in a polar aprotic
solvent or additional reactions for selective removal of 3-iodo
group from Formula VI to afford compounds of the Formula V, VI, or
VII: 8
[0144] b) reacting a compound of the Formula V, VI or VII with a
compound of the Formula VIII or IX: 9
[0145] wherein L is a L.sup.1 or L.sup.2 and R is R.sup.1 or
R.sup.2, in the presence of Palladium catalyst such as
Pd(PPh.sub.3).sub.4 in a polar aprotic solvent to afford a compound
of the Formula X: 10
[0146] c) deprotection of O-protection groups affords Formula III:
11
[0147] Similarly, compounds of corresponding to Formula I and
Formula II where R.sup.5 is hydrogen or hydroxy can be formed by a
similar synthesis with modification of the starting material, using
standard organic techniques.
[0148] Therapeutic Applications
[0149] The present invention further provides a method for the
prevention or treatment of Gaucher disease, which method comprises
increasing the expression or activity of the mutant GCase, or by
increasing or stabilizing the activity of recombinant, wild-type
replacement GCase (i.e., ERT or gene therapy), in a subject or
patient in need of such treatment.
[0150] According to the present invention, a "therapeutically
effective amount" also means an amount of the GIZ or PHCA
derivative that enhances without inhibiting the activity of the
GCase protein, i.e., an effective amount enhances more than it
inhibits so the net effect is an enhancement. This will generally
fall somewhere below the IC.sub.50 value of that inhibitor for
GCase intracellularly, or below about 50 .mu.M in culture
medium.
[0151] The small molecule analogue that increases GCase expression
or activity is advantageously formulated in a pharmaceutical
composition, with a pharmaceutically acceptable carrier. In this
context, the GIZ or PHCA derivative is the active ingredient or
therapeutic agent.
[0152] The concentration or amount of the active ingredient depends
on the desired dosage and administration regimen, as discussed
below. Suitable dose ranges of the small molecule analogue may
include from about 10 .mu.g/kg to about 100 mg/kg of body weight
per day.
Combination Therapy
GIZ or PHCA Chaperones and Protein Replacement
[0153] The pharmaceutical compositions of the invention may also
include other biologically active compounds in addition to GIZ or
PHCA derivatives of the present invention. For example, in one
embodiment, the small molecule may be administered in solution with
the replacement, wild-type (or otherwise functional) recombinant
GCase during enzyme infusion in replacement therapy. Protein
replacement therapy increases the amount of protein by exogenously
introducing wild-type or biologically functional protein by way of
infusion. This therapy has been developed for many genetic
disorders including Gaucher disease. The wild-type enzyme is
purified from a recombinant cellular expression system (e.g.,
mammalian cells or insect cells-see U.S. Pat. No. 5,580,757 to
Desnick et al.; U.S. Pat. Nos. 6,395,884 and 6,458,574 to Selden et
al.; U.S. Pat. No. 6,461,609 to Calhoun et al.; U.S. Pat. No.
6,210,666 to Miyamura et al.; U.S. Pat. No. 6,083,725 to Selden et
al.; U.S. Pat. No. 6,451,600 to Rasmussen et al.; U.S. Pat. No.
5,236,838 to Rasmussen et al.; and U.S. Pat. No. 5,879,680 to Ginns
et al.), human placenta, or animal milk (see U.S. Pat. No.
6,188,045 to Reuser et al.).
[0154] After the infusion, the exogenous GCase is expected to be
taken up by tissues through non-specific or receptor-specific
mechanism. In general, the uptake efficiency is not high, and the
circulation time of the exogenous protein is short. In addition,
the exogenous GCase is unstable and subject to rapid intracellular
degradation. Accordingly, it is expected that co-administration
with the GIZ or PHCA compounds of the present invention, which act
as chaperones for the enzyme, will improve the stability and
prevent the degradation of the exogenously administered GCase.
[0155] In another embodiment, the small molecule analogue also may
be administered in conjunction with, but not necessarily the same
composition, as the recombinant wild-type, or otherwise functional,
GCase protein. In this embodiment, the replacement GCase protein
and the GIZ or PHCA compounds of the present invention are
formulated in separate compositions. The GIZ or PHCA derivatives
and the replacement GCase may be administered according to the same
route, e.g., intravenous infusion, or different routes, e.g.,
intravenous infusion for the replacement protein, and oral
administration for the GIZ or PHCA compound.
GIZ or PHCA Derivatives and Gene Therapy
[0156] In addition, the GIZ or PHCA compositions of the present
invention may be administered in conjunction with a recombinant
vector encoding a wild-type, or otherwise functional GCase gene,
i.e., in association with gene therapy. Recently, recombinant gene
therapy methods are in clinical or pre-clinical development for the
treatment of lysosomal storage disorders, see, e.g., U.S. Pat. No.
5,658,567, for recombinant alpha-galactosidase A therapy for Fabry
disease; U.S. Pat. No. 5,580,757, for Cloning and Expression of
Biologically Active .alpha.-galactosidase A as a Fusion Protein;
U.S. Pat. No. 6,066,626, for Compositions and method for treating
lysosomal storage disease; U.S. Pat. No. 6,083,725, for Transfected
human cells expressing human alpha-galactosidase A protein; U.S.
Pat. No. 6,335,011, for Methods for delivering DNA to muscle cells
using recombinant adeno-associated virus virions to treat lysosomal
storage disease; Bishop, D. F. et al., Proc. Natl. Acad. Sci. USA
1986; 83: 4859-4863; Medin, J. A. et al., Proc. Natl. Acad. Sci.
USA 1996; 93: 7917-7922; Novo, F. J., Gene Therapy 1997; 4:
488-492; Ohshima, T. et al., Proc. Natl. Acad. Sci. USA 1997; 94:
2540-2544; Sugimoto Y. et al., Human Gene Therapy 1995; 6: 905-915;
Sly et al., Proc. Natl. Acad. Sci. USA 2002; 99(9): 5760-2; Raben
et al., Curr. Mol. Med 2002; 2(2): 145-66; Eto et al., Curr. Mol.
Med. 2002; 2(1): 83-9; Vogler et al., Pediatr. Dev. Pathol. 2001;
4(5): 421-33; Barranger et al., Expert Opin. Biol. Ther. 2001;
1(5): 857-67; Yew et al., Curr. Opin. Mol. Ther. 2001; 3(4):
399-406; Caillaud et al., Biomed. Pharmacother. 2000; 54(10):
505-12 and Ioannu et al., J. Am. Soc. Nephrol. 2000; 11(8):
1542-7.
[0157] It is important to note that in addition to stabilizing the
expressed GCase enzyme, the GIZ or PHCA derivative will also
stabilize and enhance expression of any endogenous mutant GCase
that is deficient as a result of mutations that prevent proper
folding and processing in the ER.
[0158] Formulations and Administration
[0159] According to the invention, the pharmaceutical composition
of the invention, e.g., the GIZ or PHCA derivative, can be
introduced parenterally, transmucosally, e.g., orally (per os),
nasally, or rectally, or transdermally. Parental routes include
intravenous, intra-arteriole, intramuscular, intradermal,
subcutaneous, intraperitoneal, intraventricular, and intracranial
administration.
[0160] With respect to combination therapy with protein
replacement, in the embodiment where the GIZ or PHCA derivative is
administered in the same composition with the replacement GCase
enzyme, the formulation is preferably suitable for parenteral
administration, including intravenous subcutaneous, and
intraperitoneal, however, formulations suitable for other routes of
administration such as oral, intranasal, or transdermal are also
contemplated.
[0161] In one embodiment, transdermal administration is achieved by
liposomes. Lipid bilayer vesicles are closed, fluid-filled
microscopic spheres which are formed principally from individual
molecules having polar (hydrophilic) and non-polar (lipophilic)
portions. The hydrophilic portions may comprise phosphate,
glycerylphosphato, carboxy, sulfato, amino, hydroxy, choline or
other polar groups. Examples of lipophilic groups are saturated or
unsaturated hydrocarbons such as alkyl, alkenyl or other lipid
groups. Sterols (e.g., cholesterol) and other pharmaceutically
acceptable adjuvants (including anti-oxidants such as
alpha-tocopherol) may also be included to improve vesicle stability
or confer other desirable characteristics.
[0162] Liposomes are a subset of these bilayer vesicles and are
comprised principally of phospholipid molecules that contain two
hydrophobic tails consisting of fatty acid chains. Upon exposure to
water, these molecules spontaneously align to form spherical,
bilayer membranes with the lipophilic ends of the molecules in each
layer associated in the center of the membrane and the opposing
polar ends forming the respective inner and outer surface of the
bilayer membrane(s). Thus, each side of the membrane presents a
hydrophilic surface while the interior of the membrane comprises a
lipophilic medium. These membranes may be arranged in a series of
concentric, spherical membranes separated by thin strata of water,
in a manner not dissimilar to the layers of an onion, around an
internal aqueous space. These multilamellar vesicles (MLV) can be
converted into Unilamellar Vesicles (UV) with the application of a
shearing force.
[0163] The pharmaceutical formulations suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. In all cases, the
form must be sterile and must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and polyethylene glycol), suitable mixtures thereof, and
vegetable oils. The proper fluidity can be maintained, for example,
by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The preventions of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, and sorbic acid. In many
cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monosterate and gelatin. The advantage of using liposomes to
deliver the ceramide and sphyingomyelin analogues according to the
method of the present invention is that liposomes cross the
blood-brain barrier. Since Types 2 and 3 Gaucher disease are
characterized by neurodegeneration due to an accumulation of
glucoceramide, effective targeting to the brain is critical for any
therapeutic.
[0164] Sterile injectable solutions are prepared by incorporating
the purified GCase and GIZ or PHCA derivative in the required
amount in the appropriate solvent with various of the other
ingredients enumerated above, as required, followed by filter or
terminal sterilization. Generally, dispersions are prepared by
incorporating the various sterilized active ingredients into a
sterile vehicle which contains the basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and the freeze-drying technique which yield a powder of the active
ingredient plus any additional desired ingredient from previously
sterile-filtered solution thereof.
[0165] Preferably the formulation contains an excipient.
Pharmaceutically acceptable excipients which may be included in the
formulation are buffers such as citrate buffer, phosphate buffer,
acetate buffer, and bicarbonate buffer, amino acids, urea,
alcohols, ascorbic acid, phospholipids; proteins, such as serum
albumin, collagen, and gelatin; salts such as EDTA or EGTA, and
sodium chloride; liposomes; polyvinylpyrollidone; sugars, such as
dextran, mannitol, sorbitol, and glycerol; propylene glycol and
polyethylene glycol (e.g., PEG-4000, PEG-6000); glycerol; glycine
or other amino acids; and lipids. Buffer systems for use with the
formulations include citrate; acetate; bicarbonate; and phosphate
buffers. Phosphate buffer is a preferred embodiment.
[0166] The formulation also preferably contains a non-ionic
detergent. Preferred non-ionic detergents include Polysorbate 20,
Polysorbate 80, Triton X-100, Triton X-114, Nonidet P-40, octyl
.alpha.-glucoside, octyl .beta.-glucoside, Brij 35, Pluronic, and
Tween 20.
[0167] For lyophilization of GCase and GIZ or PHCA preparations,
the enzyme concentration can be 0.1-10 mg/mL. Bulking agents, such
as glycine, mannitol, albumin, and dextran, can be added to the
lyophilization mixture. In addition, possible cryoprotectants, such
as disaccharides, amino acids, and PEG, can be added to the
lyophilization mixture. Any of the buffers, excipients, and
detergents listed above, can also be added.
[0168] Formulations of GIZ or PHCA compound (with or without the
GCase) for inhalation administration may contain lactose or other
excipients, or may be aqueous solutions which may contain
polyoxyethylene-9-lauryl ether, glycocholate or deoxycocholate. A
preferred inhalation aerosol is characterized by having particles
of small mass density and large size. Particles with mass densities
less than 0.4 gram per cubic centimeter and mean diameters
exceeding 5 .mu.m efficiently deliver inhaled therapeutics into the
systemic circulation. Such particles are inspired deep into the
lungs and escape the lungs' natural clearance mechanisms until the
inhaled particles deliver their therapeutic payload (Edwards et
al., Science 1997; 276: 1868-1872). Replacement protein
preparations of the present invention can be administered in
aerosolized form, for example by using methods of preparation and
formulations as described in, U.S. Pat. Nos. 5,654,007, 5,780,014,
and 5,814,607, each incorporated herein by reference. Formulation
for intranasal administration may include oily solutions for
administration in the form of nasal drops, or as a gel to be
applied intranasally.
[0169] Formulations for topical administration to the skin surface
may be prepared by dispersing the composition with a dermatological
acceptable carrier such as a lotion, cream, ointment, or soap.
Particularly useful are carriers capable of forming a film or layer
over the skin to localize application and inhibit removal. For
topical administration to internal tissue surfaces, the composition
may be dispersed in a liquid tissue adhesive or other substance
known to enhance adsorption to a tissue surface. Alternatively,
tissue-coating solutions, such as pectin-containing formulations
may be used.
[0170] In preferred embodiments, the formulations of the invention
are supplied in either liquid or powdered formulations in devices
which conveniently administer a predetermined dose of the
preparation; examples of such devices include a needle-less
injector for either subcutaneous or intramuscular injection, and a
metered aerosol delivery device. In other instances, the
preparation may be supplied in a form suitable for sustained
release, such as in a patch or dressing to be applied to the skin
for transdermal administration, or via erodable devices for
transmucosal administration. In instances where the formulation,
e.g., the GIZ or PHCA is orally administered in tablet or capsule
form, the preparation might be supplied in a bottle with a
removable cover or as blister patches.
[0171] In the embodiment where the GIZ or PHCA derivative is
administered separately than the GCase (or a vector comprising a
GCase gene), is administered alone as monotherapy, the compound can
be in a form suitable for any route of administration, including
but not limited to all of the forms described above. Alternatively,
in a preferred embodiment, the small molecule analogue can be
formulated for oral administration in the form of tablets or
capsules prepared by conventional means with pharmaceutically
acceptable excipients such as binding agents (e.g., pregelatinized
maize starch, polyvinylpyrrolidone or hydroxypropyl
methylcellulose); fillers (e.g., lactose, microcrystalline
cellulose or calcium hydrogen phosphate); lubricants (e.g.,
magnesium stearate, talc or silica); disintegrants (e.g., potato
starch or sodium starch glycolate); or wetting agents (e.g., sodium
lauryl sulphate). The tablets may be coated by methods well known
in the art.
[0172] Liquid preparations for oral administration of GIZ or PHCA
derivative may take the form of, for example, solutions, syrups or
suspensions, or they may be presented as a dry product for
constitution with water or other suitable vehicle before use. Such
liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. Preparations for oral administration may be
suitably formulated to give controlled release of the active
compound.
[0173] The small molecule analogue may also be formulated in rectal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0174] In addition to the formulations described above, the GIZ or
PHCA derivative may also be formulated as a depot preparation. Such
long acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the GIZ or PHCA derivative may be
formulated with suitable polymeric or hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0175] Timing. When the replacement GCase protein and GIZ or PHCA
derivative are in separate formulations, administration may be
simultaneous, or the GIZ or PHCA derivative may be administered
prior to, or after the GCase replacement protein. For example,
where the replacement protein is administered intravenously, the
GIZ or PHCA derivative may be administered during a period from 0 h
to 6 h later. Alternatively, the GIZ or PHCA derivative may be
administered from 0 to 6 h prior to the protein.
[0176] In a preferred embodiment, where the GIZ or PHCA derivative
and replacement protein are administered separately, and where the
has a short circulating half-life (e.g., small molecule), the GIZ
or PHCA derivative may be orally administered continuously, such as
daily, in order to maintain a constant level in the circulation.
Such constant level will be one that has been determined to be
non-toxic to the patient, and optimal regarding interaction with a
target replacement protein during the time of administration to
confer a non-inhibitory, therapeutic effect.
[0177] In another embodiment, the GIZ or PHCA derivative is
administered during the time period required for turnover of the
replacement GCase protein (which will be extended by administration
of the small molecule analogue).
[0178] Regardless of the timing, the administration must be such
that the concentrations of the GCase and GIZ or PHCA derivative
must be such that the small molecule stabilizes, but does not
prevent or inhibit the protein's activity in vivo. This also
applies where the replacement protein and small molecule are
administered in the same formulation.
[0179] With respect to the timing of the GIZ or PHCA derivative and
gene therapy combination therapy, administration of the small
molecule according to the present invention will generally follow
delivery of the GCase gene, to allow for expression of the
recombinant enzyme by the target cells/tissue. Since the expression
of the GCase gene will be sustained for a period of time, for as
long as the gene is expressible, the GIZ or PHCA derivative will be
remained effective as a chaperone and stabilizer for the
recombinant enzyme. Therefore, administration of the chaperone
molecule will be necessary for the same period as the gene is
expressed.
[0180] In a preferred embodiment, since the GIZ or PHCA derivative
may have a short circulating half-life, it is preferred that it
will be orally administered frequently, such as daily, in order to
maintain a constant level in the circulation. Such a constant level
will be one that has been determined to be non-toxic to the
patient, and optimal regarding interaction with the protein, which
will be continuously produced, to confer a non-inhibitory,
therapeutic effect.
[0181] According to the present invention, since that the
therapeutic GCase gene supplements inadequate activity of an
endogenous mutant GCase gene, the timing of the small molecule
analogue delivery becomes less significant since the effective
amount can enhance the activity of the endogenous mutant GCase as
well as increase the efficiency of the therapeutic GCase gene
product.
[0182] The presence of an small molecule chaperone, e.g., the GIZ
or PHCA derivatives of the present invention, for the GCase encoded
by the administered GCase gene will have the benefit of improving
the efficiency of protein processing during synthesis in the ER
(i.e., by preventing aggregation), and prolonging in the
circulation and tissue the half-life of the GCase, thereby
maintaining effective levels over longer time periods. This will
result in increased expression in clinically affected tissues. This
confers such beneficial effects to the Gaucher patient as enhanced
relief, reduction in the frequency of treatment, and/or reduction
in the amount of GCase gene administered. This will also reduce the
cost of treatment.
[0183] Dosages
[0184] The amount of the GIZ or PHCA compound effective to
stabilize the administered GCase protein and/or endogenous GCase
mutant protein can be determined by those skilled in the art.
Pharmacokinetics and pharmacodynamics such as half-life
(t.sub.1/2), peak plasma concentration (C.sub.max), time to peak
plasma concentration (t.sub.max), exposure as measured by area
under the curve (AUC), and tissue distribution for both the
replacement GCase protein and the small molecule analogue as well
as data for the small molecule analogue-replacement GCase protein
binding (affinity constants, association and dissociation
constants, and valency), can be obtained using ordinary methods
known in the art to determine compatible amounts required to
stabilize the replacement GCase protein, without inhibiting its
activity, and thus confer a therapeutic effect.
[0185] Toxicity and therapeutic efficacy of the composition can be
determined by standard pharmaceutical procedures, for example in
cell culture assays or using experimental animals to determine the
LD.sub.50 and the ED.sub.50. The parameters LD.sub.50 and ED.sub.50
are well known in the art, and refer to the doses of a compound
that is lethal to 50% of a population and therapeutically effective
in 50% of a population, respectively. The dose ratio between toxic
and therapeutic effects is referred to as the therapeutic index and
may be expressed as the ratio: LD.sub.50/ED.sub.50.
[0186] A therapeutically effective dose may be initially estimated
from cell culture assays and formulated in animal models to achieve
a circulating concentration range that includes the IC.sub.50. The
IC.sub.50 concentration of a compound is the concentration that
achieves a half-maximal inhibition of symptoms (e.g., as determined
from the cell culture assays). Appropriate dosages for use in a
particular individual, for example in human patients, may then be
more accurately determined using such information.
[0187] Measures of compounds in plasma may be routinely measured in
an individual such as a patient by techniques such as high
performance liquid chromatography (HPLC) or gas chromatography.
[0188] The particular dosage used in any treatment may vary within
this range, depending upon factors such as the particular dosage
form employed, the route of administration utilized, the conditions
of the individual (e.g., patient), and so forth.
[0189] According to current methods, the concentration of
replacement GCase protein is between 0.05-5.0 mg/kg of body weight,
typically administered weekly or biweekly. The protein can be
administered at a dosage ranging from 0.1 .mu.g/kg to about 10
mg/kg, preferably from about 0.1 mg/kg to about 2 mg/kg. Regularly
repeated doses of the protein are necessary over the life of the
patient. Subcutaneous injections maintain longer term systemic
exposure to the drug. The GCase is preferably administered
intravenously, e.g., in an intravenous bolus injection, in a slow
push intravenous injection, or by continuous intravenous injection.
Continuous IV infusion (e.g., over 2-6 hours) allows the
maintenance of specific levels in the blood.
[0190] The optimal concentrations of the GIZ or PHCA derivative
will be determined according to the amount required to stabilize
the recombinant GCase protein in vivo, in tissue or circulation,
without preventing its activity, bioavailability of the small
molecule analogue in tissue or in circulation, and metabolism of
the small molecule analogue in tissue or in circulation. For
example, the concentration of the 2-Octyl GIZ may be determined by
calculating the IC.sub.50 value of the 2-Octyl GIZ for GCase.
Taking into consideration bioavailability and metabolism of the
compound, concentrations around the IC50 value or slightly over the
IC.sub.50 value can then be evaluated based on effects on GCase
activity, e.g., the amount of small molecule analogue needed to
increase the amount of GCase activity or prolong activity of
replacement GCase.
EXAMPLES
EXAMPLE 1
Synthesis of GIZ Derivatives
[0191] a.
(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-(1-o-
ctynyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine
[0192]
(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-iodo-5,-
6,7,8-tetrahydroimidazo[1,2-a]pyridine as described by Panday in
Helv. Chim. Acta., 83, 58-79 (2000), (300 mg, 0.437 mmol) is
combined with [Pd(PPh.sub.3).sub.4] (0.252 mg, 0.22 mmol),
Et.sub.3N (0.27 ml), CuI (8 mg, 0.04 mmol), and 1-octynye (0.19 ml,
1.31 mmol) in DMF (7.5 ml) and is stirred at 80.degree. C. for 7
hours. The reaction mixture is diluted with ether (100 ml) and
washed with water and then aq. sat. NaCl. The organic layer is
dried over Na.sub.2SO.sub.4 and evaporated using a rotovap.
Purification by flash chromatography using CH.sub.2Cl.sub.2 as the
eluent gives the title compound as brown syrup. .sup.1H NMR (300
MHz, CDCl.sub.3): .delta.7.43-7.14 (m, 21H), 5.15 (d, 1H, J=12 Hz),
4.86-4.75 (m, 4H), 4.62 (d, 1H, J=11.4 Hz), 4.50-4.44 (m, 3H),
4.20-4.16 (m, 1H), 4.10-4.07 (m, 1H), 3.84-3.78 (m, 2H), 3.73-3.70
(m, 1H), 2.39 (t, 2H, J=7.5 Hz), 1.46-1.28 (m, 8H), 0.92-0.88 (m,
3H). MS (ES+): 669 [M+1], 691 [M+Na].
[0193] b.
(5R,6R,7S,8S)-5-Hydroxymethyl-2-n-octyl-5,6,7,8-tetrahydroimidaz-
o[1,2-a]pyridine-6,7,8-triol
[0194] A solution of
(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)met-
hyl]-2-(1-octynyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine (120
mg, 0.179 mmol) in THF/EtOH (2:1) (3 ml) is rapidly stirred with
Pd(OH).sub.2/C (0.1 g) under an atmosphere of hydrogen for 14 h.
After filtration of the catalyst, the organic solution is
concentrated on a rotovap and the residue is dissolved in
CH.sub.2Cl.sub.2 (10 ml). The solution is cooled in an acetone-dry
ice bath and a solution of BCl.sub.3 (1.0 M) in CH.sub.2Cl.sub.2 is
slowly added. The reaction mixture is warmed room temperature and
stirred for 3 hours. The reaction mixture is cooled in an ice-water
bath, water added and for 0.5 hour. Most of the solvent is removed
using a rotovap and the crude product is purified by chromatography
(CHCl.sub.3/MeOH/H.sub.2O 64:25:4). Lyophilization from water gives
the title compound as white foam. .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta.7.22 (s, 1H), 4.56 (d, 1H, J=8 Hz), 4.20-4.16
(m, 1H), 3.98-3.93 (m, 2H), 3.83 (t, 1H, J=8.4 Hz), 3.70 (dd, 1H,
J=8.8 Hz and 10 Hz), 2.60 (t, 2H, J=7.2 Hz), 1.67-1.63 (m, 2H),
1.35-1.30 (m, 10H), 0.90 (t, 3H, J=6.8 Hz). MS (ES+): 313
[M+1].
[0195] c.
(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-(3,3-
-dimethylbyt-1-ynyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine
[0196] In a similar manner to that described in Example 1a,
(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-iodo-5,6,7,8--
tetrahydroimidazo[1,2-a]pyridine is converted to the title
compound. .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.7.41-7.14 (m,
20H), 7.13 (s, 1H), 5.10 (d, 1H, J=11.4 Hz), 4.81-4.66 (m, 3H),
4.62 (d, 1H, J=11.4 Hz), 4.49-4.42 (m, 3H), 4.18-4.13 (m, 1H),
4.11-4.06 (m, 2H), 3.84-3.77 (m, 2H), 3.71 (dd, 1H, J=4.8 Hz and
10.5 Hz), 1.31 (s, 9H). MS (ES+): 641 [M+1].
[0197] d.
(5R,6R,7S,8S)-5-Hydroxymethyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetr-
ahydro-imidazo[1,2-a]pyridine-6,7,8-triol
[0198] In a similar manner to that described in Example 1b,
(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-(3,3-dimethyl-
byt-1-ynyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyridine was converted
to the title compound. .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
6.97 (s, 1H), 4.41 (d, 1H, J=8 Hz), 4.09 (dd, 1H, J=2.4 Hz and 12
Hz), 3.86 (dd, 1H, J=4.0 Hz and 12 Hz), 3.79-3.71 (m, 2H), 3.61
(dd, 1H, J=8.4 Hz and 9.2 Hz), 2.48-2.44 (m, 2H), 1.50-1.47 (m,
2H), 0.89 (s, 9H). MS (ES+): 285 [M+1].
EXAMPLE 2
Inhibitory Activities of Glucoimidazole Derivatives Against
GCase
[0199] Methods. The enzyme activity was assayed with
4-methylumbelliferyl .beta.-glucoside (final concentration 3 mM) as
substrate in Reaction Buffer consisting 0.25% sodium taurocholate,
0.1% Triton X-100 in McIlvaine buffer (0.1M citrate and 0.2M
phosphate buffer) at pH 5.2. The compounds were added to each
reaction mixture individually at the final concentration indicated.
After incubation with the diluted human GCase at 37.degree. C. for
30 min, the reaction was stopped by addition of 0.2 M glycine
buffer, pH 10.8, the released 4-methylumbelliferone was determined
by a fluoremeter at excitation wavelength of 355 nm and emission
wavelength at 460 nm, respectively. The relative enzyme activity
was calculated as a percentage to those of reactions without
inhibitors. IC.sub.50 was calculated using Prism sigmoid plot.
[0200] Results. GIZ and 2-PE-GIZ were reported to be potent
inhibitors for .beta.-glucosidases (Panday et al., Helvetica
Chimica Acta 2000; 83: 58-75). The proposed inhibitory mechanism of
these compounds concerns the "shape" or conformation of these
molecules and its similar to the transition state of the substrate
(Heightman et al., Angew. Chem. Int. Ed. 1999; 38:750-770). 2-Octyl
GIZ and 2-DMB-GIZ ((5R,6R,7S,8S)-5-Hydroxy-
methyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,-
8-triol) was synthesized (see synthetic scheme in FIG. 1) and the
inhibitory activities against human GCase were shown in FIG. 4 and
Table 1. The IC50 values of GIZ and 2-PE-GIZ against GCase were 87
nM and 1.1 nM, respectively, indicating that they are potent
inhibitors of GCase. It was found that replacing the phenylethyl
group with octyl or dimethlbutyl groups at the same position in
2-PE-GIZ increased the inhibitory potency approximately 15-fold or
37-fold, supporting the present findings that a lipophilic moiety
with a short, flexible chain significantly contributes the
inhibitory activity toward GCase.
1TABLE 1 Inhibitory activity against GCase. Inhibitors Entry
IC.sub.50 (nM) Glucoiminozole (GIZ) 87 2-Octyl GIZ 6 0.07
2-Phenylethyl GIZ 5 1.1 2-Dimethylbutyl GIZ 7 0.03
EXAMPLE 3
Chaperone Activity of GIZ for GCase in Gaucher Cells
[0201] Methods. Fibroblasts established from Gaucher patients with
N370S/N370S mutation were cultured in DMEM medium supplemented with
10% fetal bovine serum and antibiotics at 37.degree. C. under 5%
CO.sub.2. The GIZ was added into the culture medium at the final
concentrations for 4 days prior to the assay. After washing the
cells with phosphate-buffered saline, the cells were harvested and
homogenized in the presence of 0.25% (w/v) sodium taurocholate and
0.1% (v/v) Triton X-100 in McIlvaine buffer (pH 5.2, Reaction
Buffer), and 10 .mu.l of the lysate was used for the determination
of residual enzyme activity. The activity of the GCase was
determined with 3 mM 4-MU-.beta.-Glc in the Reaction Buffer at
37.degree. C. for 1 hr as conduritol B epoxide (CBE)-sensitive
activity in parallel assays without or with pre-incubation with CBE
at 1.25 mM for 30 min at room temperature. Protein concentrations
in the cell lysates were determined using micro BCA protein assay
kit from Pierce.
[0202] Results. In order to examine the ability of the novel
compounds to rescue mutant enzyme activity from degradation in the
ER, GIZ was added at various concentrations in the culture medium
of fibroblasts established from Gaucher patient with homozygous
N370S mutation. The residual enzyme activity in patient cells
cultivated with inhibitors were shown to be increased approximately
2-fold. The optimal concentration for the maximum enhancement was
approximately at 300 .mu.M. Incubation of the cells with GIZ at
higher concentrations results in the reduction of the residual
enzyme activity, presumably caused by inhibition of GIZ at those
concentrations. It has been indicated that optimal concentration
for chaperone activity is dependent on the potency of inhibitory
activity and the bioavailability (Fan et al., Trends Pharmacol Sci.
2003; 24: 355-60). The combination of the highest inhibitory
activity and good permeability may contribute the lower optimal
concentration as a chaperone for the GCase activity in Gaucher
disease.
Sequence CWU 1
1
2 1 516 PRT Homo sapiens 1 Met Ala Gly Ser Leu Thr Gly Leu Leu Leu
Leu Gln Ala Val Ser Trp 1 5 10 15 Ala Ser Gly Ala Arg Pro Cys Ile
Pro Lys Ser Phe Gly Tyr Ser Ser 20 25 30 Val Val Cys Val Cys Asn
Ala Thr Tyr Cys Asp Ser Phe Asp Pro Pro 35 40 45 Thr Phe Pro Ala
Leu Gly Thr Phe Ser Arg Tyr Glu Ser Thr Arg Ser 50 55 60 Gly Arg
Arg Met Glu Leu Ser Met Gly Pro Ile Gln Ala Asn His Thr 65 70 75 80
Gly Thr Gly Leu Leu Leu Thr Leu Gln Pro Glu Gln Lys Phe Gln Lys 85
90 95 Val Lys Gly Phe Gly Gly Ala Met Thr Asp Ala Ala Ala Leu Asn
Ile 100 105 110 Leu Ala Leu Ser Pro Pro Ala Gln Asn Leu Leu Leu Lys
Ser Tyr Phe 115 120 125 Ser Glu Glu Gly Ile Gly Tyr Asn Ile Ile Arg
Val Pro Met Ala Ser 130 135 140 Cys Asp Phe Ser Ile Arg Thr Tyr Thr
Tyr Ala Asp Thr Pro Asp Asp 145 150 155 160 Phe Gln Leu His Asn Phe
Ser Leu Pro Glu Glu Asp Thr Lys Leu Lys 165 170 175 Ile Pro Leu Ile
His Arg Ala Leu Gln Leu Ala Gln Arg Pro Val Ser 180 185 190 Leu Leu
Ala Ser Pro Trp Thr Ser Pro Thr Trp Leu Lys Thr Asn Gly 195 200 205
Ala Val Asn Gly Lys Gly Ser Leu Lys Gly Gln Pro Gly Asp Ile Tyr 210
215 220 His Gln Thr Trp Ala Arg Tyr Phe Val Lys Phe Leu Asp Ala Tyr
Ala 225 230 235 240 Glu His Lys Leu Gln Phe Trp Ala Val Thr Ala Glu
Asn Glu Pro Ser 245 250 255 Ala Gly Leu Leu Ser Gly Tyr Pro Phe Gln
Cys Leu Gly Phe Thr Pro 260 265 270 Glu His Gln Arg Asp Leu Ile Ala
Arg Asp Leu Gly Pro Thr Leu Ala 275 280 285 Asn Ser Thr His His Asn
Val Arg Leu Leu Met Leu Asp Asp Gln Arg 290 295 300 Leu Leu Leu Pro
His Trp Ala Lys Val Val Leu Thr Asp Pro Glu Ala 305 310 315 320 Ala
Lys Tyr Val His Gly Ile Ala Val His Trp Tyr Leu Asp Phe Leu 325 330
335 Ala Pro Ala Lys Ala Thr Leu Gly Glu Thr His Arg Leu Phe Pro Asn
340 345 350 Thr Met Leu Phe Ala Ser Glu Ala Cys Val Gly Ser Lys Phe
Trp Glu 355 360 365 Gln Ser Val Arg Leu Gly Ser Trp Asp Arg Gly Met
Gln Tyr Ser His 370 375 380 Ser Ile Ile Thr Asn Leu Leu Tyr His Val
Val Gly Trp Thr Asp Trp 385 390 395 400 Asn Leu Ala Leu Asn Pro Glu
Gly Gly Pro Asn Trp Val Arg Asn Phe 405 410 415 Val Asp Ser Pro Ile
Ile Val Asp Ile Thr Lys Asp Thr Phe Tyr Lys 420 425 430 Gln Pro Met
Phe Tyr His Leu Gly His Phe Ser Lys Phe Ile Pro Glu 435 440 445 Gly
Ser Gln Arg Val Gly Leu Val Ala Ser Gln Lys Asn Asp Leu Asp 450 455
460 Ala Val Ala Leu Met His Pro Asp Gly Ser Ala Val Val Val Val Leu
465 470 475 480 Asn Arg Ser Ser Lys Asp Val Pro Leu Thr Ile Lys Asp
Pro Ala Val 485 490 495 Gly Phe Leu Glu Thr Ile Ser Pro Gly Tyr Ser
Ile His Thr Tyr Leu 500 505 510 Trp Arg Arg Gln 515 2 1792 DNA Homo
sapiens 2 ccttccagag aggaatgtcc caagcctttg agtagggtaa gcatcatggc
tggcagcctc 60 acaggattgc ttctacttca ggcagtgtcg tgggcatcag
gtgcccgccc ctgcatccct 120 aaaagcttcg gctacagctc ggtggtgtgt
gtctgcaatg ccacatactg tgactccttt 180 gaccccccga cctttcctgc
ccttggtacc ttcagccgct atgagagtac acgcagtggg 240 cgacggatgg
agctgagtat ggggcccatc caggctaatc acacgggcac aggcctgcta 300
ctgaccctgc agccagaaca gaagttccag aaagtgaagg gatttggagg ggccatgaca
360 gatgctgctg ctctcaacat ccttgccctg tcaccccctg cccaaaattt
gctacttaaa 420 tcgtacttct ctgaagaagg aatcggatat aacatcatcc
gggtacccat ggccagctgt 480 gacttctcca tccgcaccta cacctatgca
gacacccctg atgatttcca gttgcacaac 540 ttcagcctcc cagaggaaga
taccaagctc aagatacccc tgattcaccg agcactgcag 600 ttggcccagc
gtcccgtttc actccttgcc agcccctgga catcacccac ttggctcaag 660
accaatggag cggtgaatgg gaaggggtca ctcaagggac agcccggaga catctaccac
720 cagacctggg ccagatactt tgtgaagttc ctggatgcct atgctgagca
caagttacag 780 ttctgggcag tgacagctga aaatgagcct tctgctgggc
tgttgagtgg ataccccttc 840 cagtgcctgg gcttcacccc tgaacatcag
cgagacttaa ttgcccgtga cctaggtcct 900 accctcgcca acagtactca
ccacaatgtc cgcctactca tgctggatga ccaacgcttg 960 ctgctgcccc
actgggcaaa ggtggtactg acagacccag aagcagctaa atatgttcat 1020
ggcattgctg tacattggta cctggacttt ctggctccag ccaaagccac cctaggggag
1080 acacaccgcc tgttccccaa caccatgctc tttgcctcag aggcctgtgt
gggctccaag 1140 ttctgggagc agagtgtgcg gctaggctcc tgggatcgag
ggatgcagta cagccacagc 1200 atcatcacga acctcctgta ccatgtggtc
ggctggaccg actggaacct tgccctgaac 1260 cccgaaggag gacccaattg
ggtgcgtaac tttgtcgaca gtcccatcat tgtagacatc 1320 accaaggaca
cgttttacaa acagcccatg ttctaccacc ttggccattt cagcaagttc 1380
attcctgagg gctcccagag agtggggctg gttgccagtc agaagaacga cctggacgca
1440 gtggcattga tgcatcccga tggctctgct gttgtggtcg tgctaaaccg
ctcctctaag 1500 gatgtgcctc ttaccatcaa ggatcctgct gtgggcttcc
tggagacaat ctcacctggc 1560 tactccattc acacctacct gtggcgtcgc
cagtgatgga gcagatactc aaggaggcac 1620 tgggctcagc ctgggcatta
aagggacaga gtcagctcac acgctgtctg tgactaaaga 1680 gggcacagca
gggccagtgt gagcttacag cgacgtaagc ccaggggcaa tggtttgggt 1740
gactcacttt cccctctagg tggtgccagg ggctggaggc ccctagaaaa ag 1792
* * * * *