U.S. patent application number 10/042527 was filed with the patent office on 2002-10-03 for therapeutic compositions and methods of treating glycolipid storage related disorders.
Invention is credited to Butters, Terence D., Dwek, Raymond A..
Application Number | 20020142985 10/042527 |
Document ID | / |
Family ID | 10851917 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020142985 |
Kind Code |
A1 |
Dwek, Raymond A. ; et
al. |
October 3, 2002 |
Therapeutic compositions and methods of treating glycolipid storage
related disorders
Abstract
A method for treating a glycolipid storage-related disorder,
comprising administering a therapeutically effective amount of an
inhibitor of glycolipid synthesis in combination with an agent
capable of increasing the rate of glycolipid degradation or in
combination with bone marrow transplantation. Inhibitors of
glycolipid synthesis include N-butyldeoxynojirimycin (NB-DNJ),
N-butyldeoxygalactonojirimycin (NB-DGJ) or N-nonyldeoxynojirimycin
(NN-DNJ). Glycolipid storage-related disorders include Gaucher
disease, Sandhoff's disease, Fabry's disease, Tay-Sach's disease,
Niemann-Pick C storage disease, GM1 gangliosidosis, genetic
disorders in which neuronal glycolipid accumulation contributes to
disease pathology.
Inventors: |
Dwek, Raymond A.; (Oxford,
GB) ; Butters, Terence D.; (Oxford, GB) |
Correspondence
Address: |
David A. Jackson
Klauber & Jackson
411 Hackensack Avenue
Hackensack
NJ
07601
US
|
Family ID: |
10851917 |
Appl. No.: |
10/042527 |
Filed: |
October 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10042527 |
Oct 19, 2001 |
|
|
|
PCT/GB00/01560 |
Apr 20, 2000 |
|
|
|
Current U.S.
Class: |
514/44A ;
514/238.8; 514/328 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 45/06 20130101; A61P 25/08 20180101; A61P 3/00 20180101; A61P
25/00 20180101; A61P 43/00 20180101; A61P 25/28 20180101 |
Class at
Publication: |
514/44 ; 514/328;
514/238.8 |
International
Class: |
A61K 048/00; A61K
031/535; A61K 031/445 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 20, 1999 |
GB |
9909066.4 |
Claims
We claim:
1. A method for treating a glycolipid storage-related disorder,
comprising administering a therapeutically effective amount of an
inhibitor of glycolipid synthesis in combination with an agent
capable of increasing the rate of glycolipid degradation.
2. The method of claim 1, wherein the inhibitor of glucosylceramide
synthesis is an imido sugar.
3. The method of claim 2, wherein the imido sugar is selected from
the group consisting of N-butyldeoxynojirimycin (NB-DNJ),
N-butyldeoxygalactonojirimycin (NB-DGN), and
N-nonyldeoxynojirimycin (NN-DNJ).
4. The method of claim 3, wherein the imido sugar is
N-butyldeoxygalactonojirimycin (NB-DGN)
5. The method of claim 1, wherein the inhibitor is selected from
the group consisting of
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP),
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or a
structurally related analogue thereof.
6. The method of claim 1, wherein the inhibitor is a nucleic acid
encoding a peptide or protein capable of inhibiting glycolipid
synthesis.
7. The method of claim 6, wherein the nucleic acid is an antisense
sequence.
8. The method of claim 6, wherein the nucleic acid is a catalytic
RNA capable of interfering with the expression of enzymes
responsible for glycolipid synthesis.
9. The method of claim 1, wherein the inhibitor of glycolipid
synthesis is an inhibitor of neuronal glycolipid synthesis.
10. The method of claim 1, wherein the agent capable of increasing
the rate of glycolipid degradation is an enzyme involved in
glycolipid degradation.
11. The method of claim 10, wherein the enzyme is selected from the
group consisting of glucocerebrosidase, lysosomal hexoseaminidase,
galactosidase, sialidase, and glucosylceramide glucosidase.
12. The method of claim 1, wherein the agent capable of increasing
the rate of neuronal glycolipid degradation is a molecule which
increases the activity of a glycolipid degrading enzyme.
13. The method of claim 1, wherein the agent capable of increasing
the rate of neuronal glycolipid degradation is a nucleic acid
sequence which encodes a neuronal glycolipid degrading enzyme.
14. The method of claim 1, wherein the glycolipid storage-related
disorder is selected from the group consisting of Gaucher disease,
Sandhoff's disease, Fabry's disease, Tay-Sach's disease,
Niemann-Pick disease, GM1 gangliosidosis, Alzheimer's disease,
stroke, and epilepsy.
15. The method of claim 1, wherein the inhibitor of glycolipid
synthesis and the agent capable of increasing the rate of
glycolipid degradation are given simultaneously, sequentially, or
separately.
16. A method for treating a glycolipid storage-related disorder,
comprising administering a therapeutically effective amount of an
inhibitor of glycolipid synthesis in combination with bone marrow
transplantation.
17. The method of claim 16, wherein the inhibitor of
glucosylceramide synthesis is an imido sugar.
18. The method of claim 17, wherein the imido sugar is selected
from the group consisting of N-butyldeoxynojirimycin (NB-DNJ),
N-butyldeoxygalactonojirimycin (NB-DGN), and
N-nonyldeoxynojirimycin (NN-DNJ).
19. The method of claim 18, wherein the imido sugar is
N-butyldeoxygalactonojirimycin (NB-DGN)
20. The method of claim 16, wherein the inhibitor is selected from
the group consisting of
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP),
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or a
structurally related analogue thereof.
21. The method of claim 16, wherein the inhibitor is a nucleic acid
encoding a peptide or protein capable of inhibiting glycolipid
synthesis.
22. The method of claim 21, wherein the nucleic acid is an
antisense sequence.
23. The method of claim 21, wherein the nucleic acid is a catalytic
RNA capable of interfering with the expression of enzymes
responsible for glycolipid synthesis.
24. The method of claim 16, wherein the inhibitor of glycolipid
synthesis is an inhibitor of neuronal glycolipid synthesis.
25. A pharmaceutical composition useful for the treatment of
glycolipid storage-related disorders, comprising a therapeutically
effective amount of an inhibitor of glycolipid synthesis, an agent
capable of increasing the rate of glycolipid degradation, and a
pharmaceutically acceptable carrier.
26. The pharmaceutical composition of claim 25, wherein the
inhibitor of glucosylceramide synthesis is an imido sugar.
27. The pharmaceutical composition of claim 26, wherein the imido
sugar is selected from the group consisting of
N-butyldeoxynojirimycin (NB-DNJ), N-butyldeoxygalactonojirimycin
(NB-DGN), and N-nonyldeoxynojirimycin (NN-DNJ).
28. The pharmaceutical composition of claim 27, wherein the imido
sugar is N-butyldeoxygalactonojirimycin (NB-DGN)
29. The pharmaceutical composition of claim 25, wherein the
inhibitor is selected from the group consisting of
1-phenyl-2-decanoylamino-3-morpholi- no-1-propanol (PDMP),
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-prop- anol or a
structurally related analogue thereof.
30. The pharmaceutical composition of claim 25, wherein the
inhibitor is a nucleic acid encoding a peptide or protein capable
of inhibiting glycolipid synthesis.
31. The pharmaceutical composition of claim 30, wherein the nucleic
acid is an antisense sequence.
32. The pharmaceutical composition of claim 30, wherein the nucleic
acid is a catalytic RNA capable of interfering with the expression
of enzymes responsible for glycolipid synthesis.
33. The pharmaceutical composition of claim 25, wherein the
inhibitor of glycolipid synthesis is an inhibitor of neuronal
glycolipid synthesis.
34. The pharmaceutical composition of claim 25, wherein the agent
capable of increasing the rate of glycolipid degradation is an
enzyme involved in glycolipid degradation.
35. The pharmaceutical composition of claim 34, wherein the enzyme
is selected from the group consisting of glucocerebrosidase,
lysosomal hexoseaminidase, galactosidase, sialidase, and
glucosylceramide glucosidase.
36. The pharmaceutical composition of claim 25, wherein the agent
capable of increasing the rate of neuronal glycolipid degradation
is a molecule which increases the activity of a glycolipid
degrading enzyme.
37. The pharmaceutical composition of claim 25, wherein the agent
capable of increasing the rate of neuronal glycolipid degradation
is a nucleic acid sequence which encodes a neuronal glycolipid
degrading enzyme.
38. The pharmaceutical composition of claim 25, wherein the
glycolipid storage-related disorder is selected from the group
consisting of Gaucher disease, Sandhoff's disease, Fabry's disease,
Tay-Sach's disease, Niemann-Pick disease, GM1 gangliosidosis,
Alzheimer's disease, stroke, and epilepsy.
Description
RELATED PATENT APPLICATIONS
[0001] This application is a continuation of PCT/GB00/01560 filed
Apr. 20, 2000, which application is herein specifically
incorporated by reference.
INTRODUCTION
[0002] The present invention relates to compounds and methods of
treatment of glycolipid storage related disorders, including such
diseases as Niemann-Pick C storage disease, Gaucher disease,
Sandhoff disease, Tay-Sach's disease, GM1 gangliosidosis,
Alzheimer's disease, stroke, epilepsy and cancers such as
glioblastoma and astrocytoma.
BACKGROUND OF THE INVENTION
[0003] The GM.sub.2 gangliosidoses are a group of glycosphingolipid
(GSL) lysosomal storage diseases which includes Tay-Sachs disease,
Sandhoff disease and GM.sub.2 activator deficiency (Gravel et al
(1995) in The Metabolic and Molecular Bases of Inherited Disease
(Scriver et al) Vol 2, pp 2839-79, 3 vols, McGraw Hill, New York).
They result from mutations in the genes encoding the hexosaminidase
.alpha. subunit, .beta. subunit and GM.sub.2 activator protein
respectively. They are characterised by progressive
neurodegeneration in response to high levels of lysosomal storage
of GM.sub.2 and related GSLs, in neurones of the central nervous
system (CNS) (Gravel et al (1995) supra). There are currently no
therapies for these diseases. Potential therapeutic strategies for
Tay-Sachs and Sandhoff disease include enzyme augmentation and
substrate deprivation (Radin (1996) Glycoconj. J 13:153-7; Platt et
al (1998) Biochemical Pharmacology 56:421-30).
[0004] Enzyme augmentation could be achieved clinically through
strategies such as enzyme replacement, bone marrow transplantation,
or gene therapy.
[0005] Defects in ganglioside biosynthesis are found in most human
cancers and are thought to underlie the invasive and malignant
properties of brain tumours (Hakomori (1996) Cancer Res.
56:5309-5318, Fredman et al. (1996) Glycoconj. J. 13:391-399).
[0006] Glycolipid metabolism also plays a critical role in other
neuronal disorders, such as Alzheimer's disease and epilepsy.
Niemann-Pick Type C patient neurons present with fibrillar tangles
reminiscent of the morphology seen in Alzheimer's disease.
Interestingly, GM1 ganglioside binding by amyloid beta-protein
induces conformational changes that support its formation of
fibrous polymers, and the fibrillar deposition of this protein is
an early event in Alzheimer's disease (Yanagisawa et al (1995) Nat
Med 1: 1062-6, Choo-Smith et al (1997) Biol Chem 272:22987-90).
Thus, decreasing GM1 synthesis could inhibit the fibre formation
seen in Alzheimer's disease.
[0007] The imino sugar N-butyldeoxynojirimycin (NB-DNJ) is a potent
inhibitor of alpha-glucosidase 1 (involved in N-glycan synthesis),
and an even more potent inhibitor of glucosylceramide
glucosyltransferase. NB-DNJ is currently undergoing clinical trials
as a treatment for Gaucher and Fabry diseases, glycolipid storage
disorders resulting from mutations in glucocerebrosidase and
alpha-galactosidase A, respectively.
SUMMARY OF THE INVENTION
[0008] The present invention if based, in part, on the discovery
that NB-DNJ administered to mice together with glucocerebrosidase
(the major therapy for Gaucher Type I patients) unexpectedly does
not compromise the activity of glucocerebrosidase, and further,
provides an augmentation of enzyme activity over time due to a
protective effect of NB-DNJ on the enzyme. This result is
surprising as the efficacy of the enzyme would be expected to be
compromised in the presence of NB-DNJ, as NB-DNJ is a weak
inhibitor of glucocerebrosidase (IC.sub.50=0.52 mM). It has further
been discovered that the co-administration of NB-DNJ with bone
marrow transplantation to provide enzyme augmentation to increase
the rate of neuronal glycolipid degradation provides an unexpected
synergistic effect.
[0009] Accordingly, in one aspect, the invention features a method
for treating a glycolipid storage-related disorder, comprising
administering a therapeutically effective amount of an inhibitor of
glycolipid synthesis in combination with an agent capable of
increasing the rate of glycolipid degradation. In one embodiment,
the inhibitor of glucosylceramide synthesis is an imido sugar. In
more specific embodiments, the imido sugar is selected from the
group consisting of N-butyldeoxynojirimycin (NB-DNJ),
N-butyldeoxygalactonojirimycin (NB-DGN), and
N-nonyldeoxynojirimycin (NN-DNJ). In specific embodiments, the
inhibitor of glycolipid synthesis and the agent capable of
increasing the rate of glycolipid degradation are given
simultaneously, sequentially, or separately.
[0010] Disorders which result from accumulation/storage of
glucosylceramide-containing glycolipids include Gaucher disease,
Sandhoff's disease, Fabry's disease, Tay-Sach's disease,
Niemann-Pick C storage disease, GM1 gangliosidosis, genetic
disorders in which neuronal glycolipid accumulation contributes to
the disease pathology, e.g. mucopolysaccharidoses, neurological
disorders in which glucosylceramide-containing glycolipid
accumulation contributes to disease pathology such as Alzheimer's
disease, stroke and epilepsy, cancers of neuronal origin such as
glioblastoma and astrocytoma and cancers originating outside
neuronal tissue but presenting with neuronal metastases.
[0011] In another embodiment of the method of the invention, the
inhibitor is selected from the group consisting of
1-phenyl-2-decanoylamino-3-morph- olino-1-propanol (PDMP),
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-p- ropanol or a
structurally related analogue thereof.
[0012] In other embodiments of the method of the invention, the
inhibitor is a nucleic acid encoding a peptide or protein capable
of inhibiting glycolipid synthesis. In more specific embodiments,
the nucleic acid is an antisense sequence, or a catalytic RNA
capable of interfering with the expression of enzymes responsible
for glycolipid synthesis.
[0013] In one embodiment of the method of the invention, the agent
capable of increasing the rate of glycolipid degradation is an
enzyme involved in glycolipid degradation. In more specific
embodiments, the enzyme is selected from the group consisting of
glucocerebrosidase, lysosomal hexoseaminidase, galactosidase,
sialidase, and glucosylceramide glucosidase. In another embodiment,
the agent capable of increasing the rate of neuronal glycolipid
degradation is a molecule which increases the activity of a
glycolipid degrading enzyme.
[0014] In further embodiments, the agent capable of increasing the
rate of neuronal glycolipid degradation is a nucleic acid sequence
which encodes a neuronal glycolipid degrading enzyme.
[0015] In a second aspect, the invention features a method for
treating a glycolipid storage-related disorder, comprising
administering a therapeutically effective amount of an inhibitor of
glycolipid synthesis in combination with bone marrow
transplantation. In one embodiment, the inhibitor of
glucosylceramide synthesis is an imido sugar, and in more specific
embodiments, the imido sugar is selected from the group consisting
of N-butyldeoxynojirimycin (NB-DNJ), N-butyldeoxygalactonojiri-
mycin (NB-DGN), and N-nonyldeoxynojirimycin (NN-DNJ). In a second
embodiment, the inhibitor is selected from the group consisting of
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP),
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or a
structurally related analogue thereof. In a third embodiment of
this aspect of the invention, the inhibitor is a nucleic acid
encoding a peptide or protein capable of inhibiting glycolipid
synthesis, and may be an antisense sequence or a catalytic RNA
capable of interfering with the expression of enzymes responsible
for glycolipid synthesis.
[0016] In a third aspect, the present invention features a
pharmaceutical composition useful for the treatment of glycolipid
storage-related disorders, comprising a therapeutically effective
amount of an inhibitor of glycolipid synthesis, an agent capable of
increasing the rate of glycolipid degradation, and a
pharmaceutically acceptable carrier. In one embodiment, the
inhibitor of glucosylceramide synthesis is an imido sugar, and in
more specific embodiments, the imido sugar is selected from the
group consisting of N-butyldeoxynojirimycin (NB-DNJ),
N-butyldeoxygalactonojirimycin (NB-DGN), and
N-nonyldeoxynojirimycin (NN-DNJ). In a second embodiment, the
inhibitor is selected from the group consisting of
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP),
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or a
structurally related analogue thereof. In a third embodiment of
this aspect of the invention, the inhibitor is a nucleic acid
encoding a peptide or protein capable of inhibiting glycolipid
synthesis, and may be an antisense sequence or a catalytic RNA
capable of interfering with the expression of enzymes responsible
for glycolipid synthesis.
[0017] In one embodiment of the pharmaceutical composition of the
invention, the agent capable of increasing the rate of glycolipid
degradation is an enzyme involved in glycolipid degradation. In
more specific embodiments, the enzyme is selected from the group
consisting of glucocerebrosidase, lysosomal hexoseaminidase,
galactosidase, sialidase, and glucosylceramide glucosidase. In a
further embodiment, the agent capable of increasing the rate of
neuronal glycolipid degradation is a molecule which increases the
activity of a glycolipid degrading enzyme. In a more specific
embodiment, the agent capable of increasing the rate of neuronal
glycolipid degradation is a nucleic acid sequence which encodes a
neuronal glycolipid degrading enzyme.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a graph plotting % survival against age of
Sandhoff mice in days when treated with different agents.
[0019] FIGS. 2-5 are graphs showing the short term distribution of
radiolabelled NB-DNJ and NB-DGJ in mouse. Mice (n=5 per group) were
dissected 90 min after oral administration of [.sup.14C]-NB-DNJ
(open bars) or [.sup.3H]-NB-DGJ (filled bars).
[0020] FIG. 2=total compound in intestine and urine.
[0021] FIG. 3=total compound in organs.
[0022] FIG. 4=compound concentration in serum.
[0023] FIG. 5=compound in organs expressed as a ratio to compound
in serum. * denotes a significant difference between the NB-DNJ and
the NB-DGJ treated mice (p<0.05).
[0024] FIGS. 6-8 show glycosphingolipid depletion in mouse liver
after feeding NB-DNJ or NB-DGJ. Gangliosides were purified from
liver and separated by TLC. GM.sub.2 concentration was measured by
densitometry of the scanned TLC chromatograms.
[0025] FIG. 6=GM.sub.2 concentration in livers of mice fed 300-4800
mg/kg/day NB-DNJ (open bars) or NB-DGJ (filled bars) for 10 days,
(n=5 per group).
[0026] FIG. 7=TLC separated GM.sub.2 band of livers from mice
treated for 5 weeks with 2400 mg/kg/day.
[0027] FIG. 8=densitometry of TLC in B. * denotes significantly
lower concentration than the control concentration (p<0.05).
[0028] FIG. 9 shows the growth of mice fed NB-DNJ or NB-DGJ. Mice
were given 2400 mg/kg/day of NB-DNJ (.smallcircle.), NB-DGJ
(.circle-solid.), or a control diet (.quadrature.). N=10 per group.
* denotes a significant difference compared to control weights
(p<0.01).
[0029] FIG. 10 shows the lymphoid organ size in mouse after NB-DNJ
or NB-DGJ treatment. Wet weight of thymus and spleen was determined
at dissection after 5 weeks of treatment with 2400 mg/kg/day of
NB-DNJ (open bars). NB-DGJ (filled bars), or a control diet (dashed
bars). N=4 per group. * denotes a significant difference compared
to control weights (p<0.001).
[0030] FIG. 11 shows the inhibition of lactase activity by NB-DNJ,
NB-DGJ, DNJ, and DGJ. Lactase activity expressed as % of control
activity at different concentrations of NB-DNJ (.smallcircle.),
NB-DDJ (.circle-solid.), DNJ (.quadrature.), and DGJ
(.box-solid.).
DETAILED DESCRIPTION OF THE INVENTION
[0031] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to
particular methods, compositions, and experimental conditions
described, as such methods and compounds may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only the appended claims.
[0032] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus for example,
references to "an inhibitor of glucosylceramide synthesis" includes
mixtures of such inhibitors, reference to "the formulation" or "the
method" includes one or more formulations, methods, and/or steps of
the type described herein and/or which will become apparent to
those persons skilled in the art upon reading this disclosure and
so forth.
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and described the methods and/or materials in
connection with which the publications are cited.
[0034] Definitions
[0035] In the context of the present invention, the term
"inhibitor" is intended to include inhibitors which inhibit
glucosylceramide synthesis. It includes molecules such as
N-butyldeoxynojirirnycin, N-butyldeoxygalactonojirimycin,
N-nonyldeoxynojirinycin and other imino sugar-structured inhibitors
of glucosylceramide synthesis. It also includes other inhibitors of
glycolipid synthesis, especially glucosylceramide synthesis,
including agents such as
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP),
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and
structurally related analogues thereof. Further, inhibition can
also be achieved by the use of genetic approaches, based on the
introduction of nucleic acid coding for proteins or peptides
capable of inhibiting glycolipid synthesis or antisense sequences
or catalytic RNA capable of interfering with the expression of
enzymes responsible for glycolipid and especially glucosylceramide
synthesis (e.g. glucosylceramide synthase). A combination of any of
the above inhibitors can be used.
[0036] Furthermore, inhibition can also be achieved by the use of
genetic approaches, based on the introduction of nucleic acid
coding for proteins or peptides capable of inhibiting
glucosylceramide synthesis or antisense sequences or catalytic RNA
capable of interfering with the expression of enzymes responsible
for glucosylceramide synthesis (e.g. glucosylceramide synthase). A
combination of any of the above approaches can be used.
[0037] The term "substantially pure," when referring to a
polypeptide, means a polypeptide that is at least 60%, by weight,
free from the proteins and naturally-occurring organic molecules
with which it is naturally associated. A substantially pure
glucosylceramide synthesis inhibitor is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by
weight, glucosylceramide synthesis inhibitor. A substantially pure
glucosylceramie synthesis inhibitor such as N-butyldeoxynojirimycin
(NB-DNJ), can be obtained, for example, by chemical synthesis or by
isolation from natural sources. Purity can be measured by any
appropriate method, e.g., column chromatography, polyacrylamide gel
electrophoresis, or HPLC analysis.
[0038] "Treatment" refers to the administration of medicine or the
performance of medical procedures with respect to a patient, for
either prophylaxis (prevention) or to cure the infirmity or malady
in the instance where the patient is afflicted.
[0039] A "therapeutically effective amount" is an amount of a
reagent sufficient to achieve the desired treatment effect.
General Aspects of the Invention
[0040] Potential therapeutic strategies for disorders of diseases
such as Tay-Sachs and Sandhoff diseases include enzyme replacement,
bone marrow transplantation, or gene therapy. Intravenous
administration of mannose-terminated glucocerebrosidase
(.beta.-D-glycosyl-N-acylsphingosin- e glucohydrolase, EC 3.2.1.45)
is an effective therapy for type 1 Gaucher disease, which is a
non-neurological GSL storage disease (Grabowski et al (1995) Ann.
Intern. Med. 122:33-39; Beutler et al (1991) Blood 78:1183-9).
However, as glycoprotein enzymes fail to cross the blood-brain
barrier, this is not a suitable approach for disease involving GSL
storage in the CNS. Bone marrow transplantation has the potential
to increase enzyme levels in the periphery, and to a limited extent
in the CNS due to secretion of enzyme from cells of bone marrow
origin, including microglia (Krivit et al (1995) Cell-Transplant
4:385-392). Results of bone marrow transplantation in GSL lysosomal
storage diseases involving storage in the CNS have been mixed
(Hoogerbrugge et al (1995) Lancet 345:1398-1402). Partial success
was recently reported in a mouse model of Sandhoff disease given
syngeneic wild type bone marrow (Norfus et al (1998) J. Clin.
Invest. 101:1881-8). This led to increased survival of the mice and
improved neurological function. Gene therapy also has promise for
treating these diseases, although this is currently experimental
(Salvetti et al (1995) Br. Med. Bull 51: 106-122). Substrate
deprivation is a potentially generic pharmacological approach for
treating the GSL storage diseases (Platt et al (1998) Biochemical
Pharmacology 56: 421-30), including the GM.sub.2 gangliosidoses.
This strategy is based upon partial inhibition of the ceramide
specific glucosyltransferase (glucosylceramide synthase,
UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80)
which catalyses the first step in GSL biosynthesis (Sandhoff et al
(1998) Adv. Lipid Res. 26:119-142). This would reduce the levels of
GSLs synthesised so they could be catabolised fully by the residual
enzyme activity present in the cells.
[0041] Substrate deprivation, utilising the GSL biosynthesis
inhibitor N-butyldeoxynojirimycin (NB-DNJ), has previously been
tested in an in vitro model of Gaucher disease and shown to prevent
storage (Platt et al (1994) J. Biol. Chem. 269:8362-6). NB-DNJ has
also been evaluated in an asymptomatic mouse model of Tay-Sachs
disease and shown to reduce GM.sub.2 accumulation in the brain and
prevent the neuropathology associated with its storage (Platt et al
(1997) Science 276:428-31). NB-DNJ is currently under clinical
evaluation in type 1 Gaucher disease.
[0042] The galactose analogue of NB-DNJ,
N-butyldeoxygalactonojirimycin (NB-DGJ), is known to inhibit GSL
synthesis in vitro as effectively as NB-DNJ, but is more specific
in that it does not inhibit .alpha.-glucosidase I and II or
.beta.-glucocerebrosidase (Platt et al, (1994) J Biol Chem 269(43):
27108-14). It is known that only approximately 10% of the serum
level of NB-DNJ is present in the cerebrospinal fluid. Accordingly,
high systemic doses of NB-DNJ may have to be administered in order
to achieve therapeutic levels in the CNS, and may have to be
administered for the duration of a patients life. High
concentrations of NB-DNJ in humans causes diarrhoea and in mice it
causes weight loss and reduces the size of lymphoid organs. Thus,
it would be advantageous to have an inhibitor of glucosylceramide
synthesis which does not have these disadvantages of NB-DNJ.
[0043] We have now shown that, when administered to healthy mice,
the distribution of NB-DGJ in vivo is equivalent or superior to
that of NB-DNJ and inhibited GSL synthesis. In addition and
significantly, NB-DGJ does not appear to cause the side effects
associated with NB-DNJ.
[0044] Thus, one specific embodiment, the invention provides a
pharmaceutical composition of N-butyldeoxygalactonojirimycin and an
agent capable of increasing the rate of glycolipid degradation for
use in the treatment of a disorder which has at least a component
based on glycolipid storage. The inhibitor of glycolipid synthesis
and the agent capable of increasing the rate of glycolipid
degradation may be provided as a combined preparation or separately
for simultaneous, sequential or separate use in the treatment of a
disorder which has at least a component based on glycolipid
storage.
[0045] For example, it is envisaged that an inhibitor of glycolipid
synthesis, such as NB-DNJ, can be administered to a patient with a
glycolipid storage disease in order to maintain low levels of
glycolipids. If the dosage of NB-DNJ is incorrect for any reason,
an agent for increasing the rate of glycolipid degradation can be
administered to restore the low levels of glycolipids.
[0046] Methods and processes for the production of
N-butyldeoxynojirimycin can be found for example in U.S. Pat. Nos.
4,182,767; 4,266,025; 4,405,714; and 5,151,519; and in EPO
B-0012278, and A-0624652.
[0047] Pharmaceutical Compositions and Methods of
Administration
[0048] The invention provides methods of treatment comprising
administering to a subject an effective amount of an agent of the
invention. In a preferred aspect, the compound is substantially
purified (e.g., substantially free from substances that limit its
effect or produce undesired side-effects). The subject is
preferably an animal, including but not limited to animals such as
cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a
mammal, and most preferably human. In one specific embodiment, a
non-human mammal is the subject. In another specific embodiment, a
human mammal is the subject.
[0049] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid are described
above; additional appropriate formulations and routes of
administration are described below.
[0050] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction can be enteral or parenteral and include
but are not limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
The compounds may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compositions of the invention into the central
nervous system by any suitable route, including intraventricular
and intrathecal injection; intraventricular injection may be
facilitated by an intraventricular catheter, for example, attached
to a reservoir, such as an Ommaya reservoir. Pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent.
[0051] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved, for example, and
not by way of limitation, by local infusion during surgery, topical
application, e.g., by injection, by means of a catheter, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers. In one embodiment, administration can be by
direct injection by aerosol inhaler.
[0052] In another embodiment, the compound can be delivered in a
vesicle, in particular a liposome (see Langer (1990) Science
249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.)
[0053] In yet another embodiment, the compound can be delivered in
a controlled release system. In one embodiment, a pump may be used
(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng.
14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989)
N. Engl. J. Med. 321:574). In another embodiment, polymeric
materials can be used (see Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida
(1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger
and Peppas, J. (1983) Macromol. Sci. Rev. Macromol. Chem. 23:61;
see also Levy et al. (1985) Science 228:190; During et al. (1989)
Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In
yet another embodiment, a controlled release system can be placed
in proximity of the therapeutic target, i.e., the airways, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release (1984) supra, vol. 2,
pp. 115-138). Other suitable controlled release systems are
discussed in the review by Langer (1990) Science 249:1527-1533.
[0054] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of an agent, and a pharmaceutically acceptable
carrier. In a particular embodiment, 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. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic 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 and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the subject. The formulation should suit the mode
of administration.
[0055] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lidocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0056] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0057] The amount of the compound of the invention which will be
effective in the treatment of glycolipid storage related disorders
can be determined by standard clinical techniques based on the
present description. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each subject's circumstances. However, suitable dosage ranges for
intravenous administration are generally about 20-500 micrograms of
active compound per kilogram body weight. Suitable dosage ranges
for intranasal administration are generally about 0.01 pg/kg body
weight to 1 mg/kg body weight. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0058] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0059] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects (a) approval by the agency of manufacture,
use or sale for human administration, (b) directions for use, or
both.
[0060] Therapeutic Uses of Glucosylceramide Synthesis
Inhibitors
[0061] The invention provides for treatment or prevention of
glucosylceramide-containing glycolipid storage diseases and
disorders, such as Gaucher disease, Sandhoff's disease, Fabry's
disease, Tay-Sach's disease, Niemann-Pick C storage disease, GM1
gangliosidosis, and other genetic disorders, by administration of a
therapeutic agent capable of inhibiting glycolipid synthesis and a
glycolipid degrading enzyme or in combination with bone marrow
transplantation. Agents capable of inhibiting glycolipid or
glucosylceramide synthesis include but are not limited to: imide
sugars such as N-butyldeoxynojirimycin,
N-butyldeoxygalactonojirimycin, and N-nonyldeoxynojirimycin;
compounds such as 1-phenyl-2-decanoylamino-3-morpholino-1-propanol
(PDMP), D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol
and structurally related analogues thereof; nucleic acids encoding
a peptide or protein inhibitor of glucosylcermide synthesis; an
antisense sequence or catalytic RNA capable of interfering with the
expression of one or more enzymes required for glucosylceramide
synthesis, such as, glucosylceramide synthase.
[0062] The change in glycolipid synthesis, in particular,
gluycosylceramide synthesis, due to the administration of such
compounds can be readily detected, e.g., by obtaining a biopsy
sample, or by assaying in vitro the levels of activities of enzymes
involved in glucosylceramide synthesis, or the levels of mRNAs
encoding such enzymes, or any combination of the foregoing., Such
assays can be performed before and after the administration of the
compound as described herein.
[0063] In one embodiment, a nucleic acid comprising a sequence
encoding a peptide or protein inhibitor of glucosylceramide
synthesis is administered. In another embodiment, a nucleic acid
sequence encoding an agent capable of increasing the rate of
neuronal glycolipid degradation, e.g., a glucosylceramide
glucosidase, is administered. Any suitable methods for
administering a nucleic acid sequence available in the art can be
used according to the present invention.
[0064] Methods for administering and expressing a nucleic acid
sequence are generally known in the area of gene therapy. For
general reviews of the methods of gene therapy, see Goldspiel et
al. (1993) Clinical Pharmacy 12:488-505; Wu and Wu (1991)
Biotherapy 3:87-95; Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol.
32:573-596; Mulligan (1993) Science 260:926-932; and Morgan and
Anderson (1993) Ann. Rev. Biochem. 62:191-217; May (1993) TIBTECH
11(5): 155-215. Methods commonly known in the art of recombinant
DNA technology which can be used in the present invention are
described in Ausubel et al. (eds.), 1993, Current Protocols in
Molecular Biology, John Wiley & Sons, NY; and Kriegler (1990)
Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
[0065] In a particular aspects, the compound comprises a nucleic
acid encoding a peptide or protein inhibitor of glucosylceramide
synthesis or encoding an enzyme required for neuronal glycolipid
degradation, such nucleic acid being part of an expression vector
that expresses a the peptide or protein in a suitable host. In
particular, such a nucleic acid has a promoter operably linked to
the coding region, said promoter being inducible or constitutive
(and, optionally, tissue-specific). In another particular
embodiment, a nucleic acid molecule is used in which the coding
sequences and any other desired sequences are flanked by regions
that promote homologous recombination at a desired site in the
genome, thus providing for intrachromosomal expression of the
nucleic acid (Koller and Smithies (1989) Proc. Natl. Acad. Sci. USA
86:8932-8935; Zijlstra et al. (1989) Nature 342:435-438).
[0066] Delivery of the nucleic acid into a subject may be direct,
in which case the subject is directly exposed to the nucleic acid
or nucleic acid-carrying vector; this approach is known as in vivo
gene therapy. Alternatively, delivery of the nucleic acid into the
subject may be indirect, in which case cells are first transformed
with the nucleic acid in vitro and then transplanted into the
subject, known as "ex vivo gene therapy".
[0067] In another embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any of numerous methods known
in the art, e.g., by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that it
becomes intracellular, e.g., by infection using a defective or
attenuated retroviral or other viral vector (see U.S. Pat. No.
4,980,286); by direct injection of naked DNA; by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); by
coating with lipids, cell-surface receptors or transfecting agents;
by encapsulation in liposomes, microparticles or microcapsules; by
administering it in linkage to a peptide which is known to enter
the nucleus; or by administering it in linkage to a ligand subject
to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J.
Biol. Chem. 262:4429-4432), which can be used to target cell types
specifically expressing the receptors. In another embodiment, a
nucleic acid-ligand complex can be formed in which the ligand
comprises a fusogenic viral peptide to disrupt endosomes, allowing
the nucleic acid to avoid lysosomal degradation. In yet another
embodiment, the nucleic acid can be targeted in vivo for cell
specific uptake and expression, by targeting a specific receptor
(see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992 (Wu et
al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316
dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22,
1993 (Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
USA 86:8932-8935; Zijlstra et al. (1989) Nature 342:435-438).
[0068] In a further embodiment, a viral vector that contains a
nucleic acid encoding a glycolipid degrading enzyme is used, for
example, a retroviral vector can be used (see Miller et al. (1993)
Meth. Enzymol. 217:581-599). These retroviral vectors have been
modified to delete retroviral sequences that are not necessary for
packaging of the viral genome and integration into host cell DNA.
The nucleic acid encoding the enzyme to be used in gene therapy is
cloned into the vector, which facilitates delivery of the gene into
a subject. More detail about retroviral vectors can be found in
Boesen et al. (1994) Biotherapy 6:291-302, which describes the use
of a retroviral vector to deliver the mdr1 gene to hematopoietic
stem cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of retroviral
vectors in gene therapy are:, Clowes et al. (1994) J. Clin. Invest.
93:644-651; Kiem et al. (1994) Blood 83:1467-1473; Salmons and
Gunzberg (1993) Human Gene Therapy 4:129-141; and Grossman and
Wilson (1993) Curr. Opin. in Genetics and Devel. 3:110-114.
[0069] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson (1993) Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al. (1994) Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.
(1991) Science 252:431-434; Rosenfeld et al. (1992) Cell
68:143-155; Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234;
PCT Publication WO94/12649; and Wang, et al. (1995) Gene Therapy
2:775-783. Adeno-associated virus (AAV) has also been proposed for
use in gene therapy (Walsh et al. (1993) Proc. Soc. Exp. Biol. Med.
204:289-300; U.S. Pat. No. 5,436,146).
[0070] Another suitable approach to gene therapy involves
transferring a gene to cells in tissue culture by such methods as
electroporation, lipofection, calcium phosphate mediated
transfection, or viral infection. Usually, the method of transfer
includes the transfer of a selectable marker to the cells. The
cells are then placed under selection to isolate those cells that
have taken up and are expressing the transferred gene. Those cells
are then delivered to a subject.
[0071] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr (1993) Meth. Enzymol. 217:599-618; Cohen et
al. (1993) Meth. Enzymol. 217:618-644; Cline (1985) Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the nucleic acid to the
cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny.
[0072] The resulting recombinant cells can be delivered to a
subject by various methods known in the art. In a preferred
embodiment, epithelial cells are injected, e.g., subcutaneously. In
another embodiment, recombinant skin cells may be applied as a skin
graft onto the subject; recombinant blood cells (e.g.,
hematopoietic stem or progenitor cells) are preferably administered
intravenously. The amount of cells envisioned for use depends on
the desired effect, the condition of the subject, etc., and can be
determined by one skilled in the art.
[0073] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to neuronal cells, glial
cells (e.g., oligodendrocytes or astrocytes), epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood or fetal liver. In a
preferred embodiment, the cell used for gene therapy is autologous
to the subject that is treated.
[0074] In an embodiment in which recombinant cells are used in gene
therapy, a nucleic acid encoding a peptide or protein inhibitor of
glucosylceramide synthesis, or an agent capable of increasing the
rate of neuronal glycolipid degradation is introduced into the
cells such that it is expressible by the cells or their progeny,
and the recombinant cells are then administered in vivo for
therapeutic effect. In a specific embodiment, stem or progenitor
cells are used. Any stem or progenitor cells which can be isolated
and maintained in vitro can be used in accordance with this
embodiment of the present invention (see e.g. PCT Publication WO
94/08598, dated Apr. 28, 1994; Stemple and Anderson (1992) Cell
71:973-985; Rheinwald (1980) Meth. Cell Bio. 21A:229; and Pittelkow
and Scott (1986) Mayo Clinic Proc. 61:771).
[0075] In another embodiment, the nucleic acid to be introduced for
purposes of gene therapy may comprise an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0076] Direct injection of a DNA coding for a peptide or protein
inhibitor of glucosylceramide synthesis or an agent capable of
increasing the rate of neuronal glycolipid degradation may also be
performed according to, for example, the techniques described in
U.S. Pat. No. 5,589,466. These techniques involve the injection of
"naked DNA", i.e., isolated DNA molecules in the absence of
liposomes, cells, or any other material besides a suitable carrier.
The injection of DNA encoding a protein and operably linked to a
suitable promoter results in the production of the protein in cells
near the site of injection and the elicitation of an immune
response in the subject to the protein encoded by the injected
DNA.
[0077] In one embodiment of the invention, NPC is treated or
prevented by administration of a compound that inhibits the
expression of one or more enzymes responsible for glucosylceramide
synthesis. Compounds useful for this purpose may include antibodies
directed to glucosylceramide synthesis enzymes (and fragments and
derivatives containing the binding region thereof), and antisense
or ribozyme nucleic acids.
[0078] In a further embodiment, the expression of an enzyme
involved in neuronal glucosylceramide synthesis is inhibited by use
of antisense nucleic acids. The present invention provides the
therapeutic or prophylactic use of nucleic acids comprising at
least six nucleotides that are antisense to a gene or cDNA encoding
an enzyme involved in glucosylceramide synthesis or a portion
thereof. As used herein, an "antisense" nucleic acid refers to a
nucleic acid capable of hybridizing by virtue of some sequence
complementarity to a portion of an RNA (preferably mRNA) encoding
an enzyme involved in glucosylceramide synthesis. The antisense
nucleic acid may be complementary to a coding and/or noncoding
region of an mRNA encoding an enzyme involved in glucosylceramide
synthesis. Such antisense nucleic acids have utility as compounds
that inhibit expression of an enzyme involved in glucosylceramide
synthesis, and can be used in the treatment or prevention of
neurological disorder.
[0079] The antisense nucleic acids of the invention are
double-stranded or single-stranded oligonucleotides, RNA or DNA or
a modification or derivative thereof, and can be directly
administered to a cell or produced intracellularly by transcription
of exogenous, introduced sequences.
[0080] The invention further provides pharmaceutical compositions
comprising a therapeutically effective amount of an antisense
nucleic acid which inhibits the expression of an enzyme involved in
glucosylceramide synthesis, and a pharmaceutically-acceptable
carrier, vehicle or diluent. The antisense nucleic acids are of at
least six nucleotides and are preferably oligonucleotides ranging
from 6 to about 50 oligonucleotides. In specific aspects, the
oligonucleotide is at least 10 nucleotides, at least 15
nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
The oligonucleotides can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof and can be single-stranded
or double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone. The oligonucleotide
may include other appended groups such as peptides; agents that
facilitate transport across the cell membrane (see, e.g., Letsinger
et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et
al. (1987) Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No.
WO 88/09810, published Dec. 15, 1988) or blood-brain barrier (see,
e.g., PCT Publication No. WO 89/10134, published Apr. 25, 1988);
hybridization-triggered cleavage agents (see, e.g., Krol et al.
(1988) BioTechniques 6:958-976) or intercalating agents (see, e.g.,
Zon (1988) Pharm. Res. 5:539-549). In a particular aspect of the
invention, a antisense oligonucleotide is provided, preferably of
single-stranded DNA. The oligonucleotide may be modified at any
position on its structure with substituents generally known in the
art.
[0081] The antisense oligonucleotide may comprise any suitable of
the following modified base moieties, e.g., 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine,
and other base analogs.
[0082] In another embodiment, the oligonucleotide comprises at
least one modified sugar moiety, e.g., one of the following sugar
moieties: arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0083] In yet another embodiment, the oligonucleotide comprises at
least one of the following modified phosphate backbones: a
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, a formacetal, or an analog of formacetal.
[0084] In yet another embodiment, the oligonucleotide is an,
.alpha.-anomeric oligonucleotide. An, .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual, .beta.-units,
the strands run parallel to each other (Gautier et al. (1987) Nucl.
Acids Res. 15:6625-6641).
[0085] The oligonucleotide may be conjugated to another molecule,
e.g., a peptide, hybridization triggered cross-linking agent,
transport agent, or hybridization-triggered cleavage agent.
[0086] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g., by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(1988) Nucl. Acids Res. 16:3209, and methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. USA
85:7448-7451).
[0087] In another embodiment, the antisense nucleic acid of the
invention is produced intracellularly by transcription from an
exogenous sequence. For example, a vector can be introduced in vivo
such that it is taken up by a cell, within which cell the vector or
a portion thereof is transcribed, producing an antisense nucleic
acid (RNA) of the invention. Such a vector would contain a sequence
encoding the antisense nucleic acid. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such vectors can
be constructed by recombinant DNA technology standard in the art.
Vectors can be plasmid, viral, or others known in the art, used for
replication and expression in mammalian cells. Expression of the
sequence encoding the antisense RNA can be by any promoter known in
the art to act in mammalian, preferably human, cells. Such
promoters can be inducible or constitutive. Examples of such
promoters are outlined above.
[0088] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a gene encoding an enzyme involved in glucosylceramide
synthesis, preferably a human gene encoding an enzyme involved in
glucosylceramide synthesis, however, absolute complementarity,
although preferred, is not required. A sequence "complementary to
at least a portion of an RNA," as referred to herein, means a
sequence having sufficient complementarity to be able to hybridize
under stringent conditions (e.g., highly stringent conditions
comprising hybridization in 7% sodium dodecyl sulfate (SDS), 1 mM
EDTA at 65.degree. C. and washing in 0.1.times. SSC/0.1% SDS at
68.degree. C., or moderately stringent conditions comprising
washing in 0.2.times. SSC/0.1% SDS at 42.degree. C. with the RNA,
forming a stable duplex; in the case of double-stranded antisense
nucleic acids, a single strand of the duplex DNA may thus be
tested, or triplex formation may be assayed. The ability to
hybridize will depend on both the degree of complementarity and the
length of the antisense nucleic acid. Generally, the longer the
hybridizing nucleic acid, the more base mismatches with an RNA
encoding an enzyme involved in glucosylceramide synthesis it may
contain and still form a stable duplex (or triplex, as the case may
be). One skilled in the art can ascertain a tolerable degree of
mismatch by use of standard procedures to determine the melting
point of the hybridized complex.
[0089] Pharmaceutical compositions of the invention, comprising an
effective amount of an antisense nucleic acid of the invention in a
pharmaceutically acceptable carrier, vehicle or diluent can be
administered to a subject having neurological disorder. The amount
of antisense nucleic acid which will be effective in the treatment
of a neurological disorder can be determined by standard clinical
techniques.
[0090] In a specific embodiment, pharmaceutical compositions
comprising one or more antisense nucleic acids to an enzyme
involved in glucosylceramide synthesis are administered via
liposomes, microparticles, or microcapsules. In various embodiments
of the invention, such compositions may be used to achieve
sustained release of the antisense nucleic acids.
[0091] Inhibitory Ribozyme and Triple Helix Approaches
[0092] In another embodiment, symptoms of a glycolipid
storage-related disorder may be ameliorated by decreasing the level
of an enzyme involved in glucosylceramide synthesis by using gene
sequences encoding the an enzyme involved in glucosylceramide
synthesis in conjunction with well-known gene "knock-out," ribozyme
or triple helix methods to decrease gene expression of an enzyme
involved in glucosylceramide synthesis. In this approach ribozyme
or triple helix molecules are used to modulate the activity,
expression or synthesis of the gene encoding the enzyme involved in
glucosylceramide synthesis, and thus to ameliorate the symptoms of
the disorder. Such molecules may be designed to reduce or inhibit
expression of a mutant or non-mutant target gene. Techniques for
the production and use of such molecules are well known to those of
skill in the art.
[0093] Ribozyme molecules designed to catalytically cleave gene
mRNA transcripts encoding an enzyme involved in glucosylceramide
synthesis can be used to prevent translation of target gene mRNA
and, therefore, expression of the gene product. (See, e.g., PCT
International Publication WO90/11364, published Oct. 4, 1990;
Sarver et al. (1990) Science 247:1222-1225).
[0094] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. (For a review, see Rossi (1994)
Current Biology 4, 469-471). The mechanism of ribozyme action
involves sequence specific hybridization of the ribozyme molecule
to complementary target RNA, followed by an endonucleolytic
cleavage event. The composition of ribozyme molecules must include
one or more sequences complementary to the target gene mRNA, and
must include the well known catalytic sequence responsible for mRNA
cleavage. For this sequence, see, e.g., U.S. Pat. No. 5,093,246,
which is incorporated herein by reference in its entirety.
[0095] While ribozymes that cleave mRNA at site specific
recognition sequences can be used to destroy mRNAs encoding an
enzyme involved in glucosylceramide synthesis, the use of
hammerhead ribozymes is preferred. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes
is well known in the art and is described more fully in Myers
(1995) Molecular Biology and Biotechnology: A Comprehensive Desk
Reference, VCH Publishers, New York, (see especially FIG. 4, page
833) and in Haseloff and Gerlach (1988) Nature, 334, 585-591, each
of which is incorporated herein by reference in its entirety.
[0096] Preferably the ribozyme is engineered so that the cleavage
recognition site is located near the 5' end of the mRNA encoding
the enzyme involved in glucosylceramide synthesis, i.e., to
increase efficiency and minimize the intracellular accumulation of
non-functional mRNA transcripts.
[0097] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one that occurs naturally in Tetrahymena thermophila (known as the
IVS, or L-19 IVS RNA) and that has been extensively described by
Thomas Cech and collaborators (Zaug, et al. (1984) Science, 224,
574-578; Zaug and Cech (1986) Science, 231, 470-475; Zaug, et al.
(1986) Nature, 324, 429-433; published International patent
application No. WO 88/04300 by University Patents Inc.; Been and
Cech (1986) Cell, 47, 207-216). The Cech-type ribozymes have an
eight base pair active site which hybridizes to a target RNA
sequence whereafter cleavage of the target RNA takes place. The
invention encompasses those Cech-type ribozymes which target eight
base-pair active site sequences that are present in the gene
encoding the enzyme involved in glucosylceramide synthesis.
[0098] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g., for improved stability,
targeting, etc.) and should be delivered to cells that express the
enzyme involved in glucosylceramide synthesis in vivo. A preferred
method of delivery involves using a DNA construct "encoding" the
ribozyme under the control of a strong constitutive pol III or pol
II promoter, so that transfected cells will produce sufficient
quantities of the ribozyme to destroy endogenous mRNA encoding the
enzyme involved in glucosylceramide synthesis and inhibit
translation. Because ribozymes, unlike antisense molecules, are
catalytic, a lower intracellular concentration is required for
efficacy.
[0099] Endogenous expression of an enzyme involved in
glucosylceramide synthesis can also be reduced by inactivating or
"knocking out" the gene encoding an enzyme involved in
glucosylceramide synthesis, or the promoter of such a gene, using
targeted homologous recombination (e.g., see Smithies et al. 1985)
Nature 317:230-234; Thomas and Capecchi (1987) Cell 51:503-512;
Thompson et al. (1989) Cell 5:313-321; and Zijlstra et al. (1989)
Nature 342:435-438, each of which is incorporated by reference
herein in its entirety). For example, a mutant gene encoding a
non-functional an enzyme involved in glucosylceramide synthesis (or
a completely unrelated DNA sequence) flanked by DNA homologous to
the endogenous gene (either the coding regions or regulatory
regions of the gene encoding an enzyme involved in glucosylceramide
synthesis) can be used, with or without a selectable marker and/or
a negative selectable marker, to transfect cells that express the
target gene in vivo. Insertion of the DNA construct, via targeted
homologous recombination, results in inactivation of the target
gene. Such approaches are particularly suited in the agricultural
field where modifications to ES (embryonic stem) cells can be used
to generate animal offspring with an inactive target gene. However,
this approach can be adapted for use in humans provided the
recombinant DNA constructs are directly administered or targeted to
the required site in vivo using appropriate viral vectors.
[0100] Alternatively, the endogenous expression of a gene encoding
an enzyme involved in glucosylceramide synthesis can be reduced by
targeting deoxyribonucleotide sequences complementary to the
regulatory region of the gene (i.e., the gene promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the gene encoding an enzyme involved in
glucosylceramide synthesis in target cells in the body. (See
generally, Helene (1991) Anticancer Drug Des. 6(6), 569-584; Helene
et al. (1992) Ann. N.Y. Acad. Sci., 660, 27-36; and Maher (1992)
Bioassays 14(12), 807-815).
[0101] Nucleic acid molecules to be used in triplex helix formation
for the inhibition of transcription in the present invention should
be single stranded and composed of deoxynucleotides. The base
composition of these oligonucleotides must be designed to promote
triple helix formation via Hoogsteen base pairing rules, which
generally require sizeable stretches of either purines or
pyrimidines to be present on one strand of a duplex. Nucleotide
sequences may be pyrimidine-based, which will result in TAT and CGC
+triplets across the three associated strands of the resulting
triple helix. The pyrimidine-rich molecules provide base
complementarity to a purine-rich region of a single strand of the
duplex in a parallel orientation to that strand. In addition,
nucleic acid molecules may be chosen that are purine-rich, for
example, contain a stretch of G residues. These molecules will form
a triple helix with a DNA duplex that is rich in GC pairs, in which
the majority of the purine residues are located on a single strand
of the targeted duplex, resulting in GGC triplets across the three
strands in the triplex.
[0102] Alternatively, the potential sequences that can be targeted
for triple helix formation may be increased by creating a so called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3', 3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizeable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
[0103] In one embodiment, wherein the antisense, ribozyme, or
triple helix molecules described herein are utilized to inhibit
mutant gene expression, it is possible that the technique may so
efficiently reduce or inhibit the transcription (triple helix) or
translation (antisense, ribozyme) of mRNA produced by normal gene
alleles of an enzyme involved in glucosylceramide synthesis that
the situation may arise wherein the concentration of such an enzyme
involved in glucosylceramide synthesis present may be lower than is
necessary for a normal phenotype. In such cases, to ensure that
substantially normal levels of activity of a gene encoding an
enzyme involved in glucosylceramide synthesis are maintained, gene
therapy may be used to introduce into cells nucleic acid molecules
that encode and express an enzyme involved in glucosylceramide
synthesis that exhibit normal gene activity and that do not contain
sequences susceptible to whatever antisense, ribozyme, or triple
helix treatments are being utilized. Alternatively, in instances
whereby the gene encodes an extracellular protein, a normal enzyme
can be co-administered in order to maintain the requisite level of
activity.
[0104] Antisense RNA and DNA, ribozyme, and triple helix molecules
of the invention may be prepared by any method known in the art for
the synthesis of DNA and RNA molecules, as discussed above. These
include techniques for chemically synthesizing
oligodeoxyribonucleotides and oligoribonucleotides well known in
the art such as for example solid phase phosphoramidite chemical
synthesis. Alternatively, RNA molecules may be generated by in
vitro and in vivo transcription of DNA sequences encoding the
antisense RNA molecule. Such DNA sequences may be incorporated into
a wide variety of vectors that incorporate suitable RNA polymerase
promoters such as the T7 or SP6 polymerase promoters.
Alternatively, antisense cDNA constructs that synthesize antisense
RNA constitutively or inducibly, depending on the promoter used,
can be introduced stably into cell lines.
EXAMPLES
[0105] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the methods and compositions of
the invention, and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers used (e.g., amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
Example 1
Materials and Methods
[0106] Animals.
[0107] Female C57BL/6 mice were housed under standard non-sterile
conditions. The mice were provided with water ad libitum and prior
to drug administration were fed pelleted chow (expended Rat and
Mouse Chow 1, SDS Ltd., Witham, Essex, UK). All experiments were
performed on age-matched animals.
[0108] Treatment of Mice with NB-DNJ and NB-DGJ.
[0109] The mice (6 weeks old) were fed a diet of powdered chow
(expended Rat and Mouse Chow 3, ground, SDS Ltd.) or diet
containing NB-DNJ or NB-DGJ. The diet and compound (both as dry
solids) were mixed thoroughly, stored at room temperature, and used
within 7 days of mixing. The mice were maintained on NB-DNJ or
NB-DGJ at doses of 300-4800 mg/kg/day for 10 days, or 2400
mg/kg/day for 5 weeks.
[0110] Radiolabelling of NB-DGJ.
[0111] A galactose oxidase/Na[.sup.3H].sub.4B method was used to
radiolabel the C6-carbon of NB-DGJ. A solution of NB-DGJ (1.3 mg),
galactose oxidase (80 units), and catalase (37000 units) in 200
.mu.l 10 mM sodium phosphate buffer was incubated for 24 h at room
temperature whilst stirring. The reaction was stopped by heating
the solution to 95.degree. C. for 5 min. After centrifuging (10
mins, 13000 rpm), 1M NaOH was added to the supernatant until pH
10-12 was achieved. Na[.sup.3H].sub.4B (4.3 mCi) was added and the
solution incubated for 2h at 30.degree. C., after which NaBD.sub.4
(1 mg) was added and the solution incubated for 1 h at 30.degree.
C. The solution was neutralised with 1M acetic acid and then dried
down. After removing borate by washing with acidified methanol
(0.6% glacial acetic acid in methanol) 5-10 times, the
[.sup.3H]-NB-DGJ mixture was resuspended in water, added to an
AG50-column (equilibrated with water) and eluted with 1-4 M
NH.sub.3. [.sup.3H]-NB-DGJ was further purified on HPLC (Dionex
CS10 hpcec chromatography, isocratic elution with 50 mM
Na.sub.2SO.sub.4, 2.5 mM H.sub.2SO.sub.4, 2.5 mM H.sub.2SO.sub.4,
and 5% ACN), and finally the AG50-column step was repeated.
[0112] Short-Term Distribution of [.sup.14C]-NB-DNJ and
[.sup.3H]-NB-DGJ in Mice.
[0113] Mice were orally gavaged with 100 .mu.l water containing 25
.mu.g (106 cpm) [.sup.14C]-NB-DNJ or [.sup.3H]-NB-DGJ and 1 mg
non-radiolabelled NB-DNJ or NB-DGJ, respectively. Urine and faeces
were collected over 90 min. After 90 min the mice were killed and
the serum, organs, and any additional urine and faeces were
collected. Organs were homogenized in a four fold volume of water
and faeces in a ten fold volume. Aliquots of 500 .mu.l homogenate,
100 .mu.l urine, or 50 .mu.l serum were mixed with 4 ml
scintillation fluid and [.sup.14C] or [.sup.3H] counts measured.
The quenching by the different tissues of both isotopes was
determined by measuring the counts of known amounts of
radiolabelled compound added to tissue homogenates, and the results
were corrected accordingly.
[0114] Glycosphingolipid Analysis of Mouse Liver.
[0115] Liver samples were homogenised in water and lyophilised.
Dried homogenates were extracted twice in chloroform: methanol
(2:1, v/v), first overnight at 4.degree. C. and then for 3h at room
temperature, pooled and dried under nitrogen. The extracts were
resuspended in 500 .mu.l chloroform: methanol (1:1, v/v),
base-treated by adding 83 .mu.l of 0.35 M NaOH in methanol and
digested for 90 min at room temperature and partitioned by adding
83 .mu.l water: methanol (9:1, v/v), 166.5 .mu.l water and 416
.mu.l chloroform. The upper phase containing the gangliosides was
separated from the lower phase after mixing and low speed
centrifugation, and the lower phase was washed twice with Folsh
(chloroform: methanol: 0.47% KCl, 3:48:47, v/v). Upper phases were
combined, dried down to half volume under nitrogen, dialysed
against water, lyophilised and resuspended in chloroform: methanol
(2:1, v/v). An equivalent of 5 mg dry weight of tissue was
separated by TLC chloroform: methanol: 0.22% CaCl.sub.2, 60:35:8,
v/v). The TLC plate was air-dried, sprayed with orcinol: sulphuric
acid (0.2% (w/v): 2N), and heat-treated (90.degree. C. for 10 min).
The intensity of bands was quantified by scanning densitometry.
[0116] Determination of NB-DNJ and NB-DGJ Concentrations in Serum
and Liver.
[0117] Serum and supernatant of liver homogenate (130 mg/ml in 10%
methanol) were centrifuged three times through a Millipore
Ultrafree filter, after an internal standard (NB-pentylDNJ) had
been added to the samples. The pooled filtrates were purified on an
HCl preconditioned SCX column, eluted with 1% NH.sub.3 in MeOH,
dried down, resuspended in water, further purified on a C18 column
(MeOH preconditioning, H2O wash, and MeOH elution), and finally
quantified by HPLC (Dionex CS10 hpcec chromatography, isocratic
elution with 50 mM Na.sub.3SO.sub.4, 2.5 mM H.sub.3SO.sub.4, and 5%
ACN).
[0118] Purification of Disaccharidases and Measurement of Sucrase,
Maltase and Lactase Activity.
[0119] The enzymes sucrase-isomaltase (EC 3.2.1.10/48) and
lactase-phlorizin hydrolase (EC 3.2.1.62/108) were purified from
porcine intestine at 4.degree. C. as follows. The intestine (100 g)
was cut into small pieces, washed by stirring in 250 ml of 150 mM
NaCi/10 mM KCl for 30 min, and extracted twice with 125 ml of 2M
urea, 50 mM EDTA, and 50 mM KCl at pH 7. The urea extracts were
combined and homogenised (Waring blender), the homogenate was
centrifuged at 60,000 g for 75 min, and the pellet was resuspended
in 50 ml of a solution containing 10 mM EDTA and 10 mM
L-cysteine-HCl in 50 mM potassium phosphate buffer at pH 7.5
(pre-equilibrated to 37.degree. C.). After addition of papain (15
units/ml), the mixture was incubated for 30 min at 37.degree. C.,
and centrifuged at 105000 g for 60 min. The supernatant was removed
and precipitated in 75 ml of ethanol at -20.degree. C. for 1 h. The
precipitate was recovered by centrifugation at 5000 g for 10 min,
dissolved in 5-10 ml of 10 mM potassium phosphate buffer at pH 7.5,
and the solution was centrifuged at 30000 g for 60 min. The
supernatant was removed and stored at 4.degree. C. in the presence
of 0.02% sodium azide. Sucrase, maltase and lactase activity were
determined in the enzyme preparation (diluted to a suitable
concentration) by incubating 50 .mu.l enzyme. 125 .mu.l sodium
citrate buffer (60 mM, pH 6), and 125 .mu.l disaccharide substrate
at 37.degree. C. for 30 min, heating to 100.degree. C. for 3 min to
inactivate the enzyme centrifuging the mixture at 13000 g for 10
min, and determining the glucose concentration by adding 50 .mu.l
of the supernatant to 1 ml trinder reagent (Sigma) and reading the
absorbance at 505 nm after 18 min.
[0120] Statistical Analysis.
[0121] Conventional statistical methods were employed to calculate
mean values and standard errors of the mean (S.E.M.). Differences
between groups of mice were tested for significance using Student's
t-test for unpaired observations. Results in the text and tables
are presented as means .+-.S.E.M.
Example 2
Co-Administration of Ceredase.TM. and NB-DNJ
[0122] A group of mice were treated with NB-DNJ at 4800 mg/kg/day
for 5 weeks. After a low intravenous dose (5-10 U/kg) of
Ceredase.TM. (Genzyme Corporation) administered as a single
injection via the tail vein, serum enzyme activity was measured by
taking sequential serum samples from the tail vein to monitor
enzyme activity over time. Cerdase.TM. is a modified form of .beta.
glucocerebrosidase. The results are shown in Table 1.
1TABLE 1 Effect of NB-DNJ on circulatory activity and half life of
Ceredase .TM. Mouse Peak Activity T.sub.1/2 (min) Control 1 5.8 4.2
2 7.9 3.3 3 8.0 1.5 4 6.8 1.8 5 30.0 1.4 6 2.8 2.0 7 13.6 1.2 8
17.6 1.2 Mean .+-. sem 11.6 .+-. 3.1 2.1 .+-. 0.4 NB-DNJ 1 13.9 1.7
2 32.1 4.9 3 24.1 5.3 4 13.1 3.0 5 21.0 3.5 6 68.3 2.4 7 19.2 2.8
Mean .+-. sem 27.4 .+-. 7.2 3.4 .+-. 0.5
[0123] Ceredase activity and serum half lives appeared to be
increased in mice treated with NB-DNJ, suggesting a protective
effect of the compound to enzyme clearance. It was concluded that
(a) co-administration of NB-DNJ with Ceredase.TM. does not
compromise activity and (b) there is a surprising augmentation of
enzyme activity over time due to a protective effect of the
compound on the enzyme.
Example 3
Co-administration of NB-DNJ and Bone Marrow Transplantation in a
Mouse Model of Sandhoff Disease
[0124] Sandhoff mice were bone marrow transplanted at two weeks of
age and drug therapy initiated at 9.5-11 weeks of age (600
mg/kg/day). Survival curves were plotted for each group of animals
with each point on the graph representing a death (FIG. 1). The
untreated (no BMT, no drug) survived (longest survivor) until 140
days (filled circles), NB-DNJ only (no BMT) survived until 170
days, BMT only (no NB-DNJ) survived until 200 days, and NB-DNJ plus
BMT had extended survival from 200-280 days. The data show synergy
approximately 13% above additive.
Example 4
Short-Term Distribution of [.sup.3H]-NB-DGJ and [.sup.14C]-NB-DNJ
in Mice
[0125] The short-term distribution of NB-DGJ and NB-DNJ in mice was
determined by giving the compounds to mice by oral gavage, as
described in Example 1. The radioactive counts in organs, serum,
faeces and urine were measured after 90 min. The concentration of
NB-DNJ was 28% higher than that of NB-DGJ in the total urine
collected while in the intestine there was 77% more NB-DGJ than
NB-DNJ (FIG. 2). This suggests that NB-DGJ passed more slowly out
of the gastrointestinal (GI) tract relative to NB-DNJ. There
appeared to be no difference in distribution of the two compounds
in other tissue (FIG. 3). The serum concentration however differed
significantly with a lower level of NB-DGJ relative to NB-DNJ (FIG.
4), possibly reflecting the slower uptake of NB-DGJ from the GI
tract. When adjusted for differential serum levels NB-DGJ was
distributed to the tissue more efficiently than NB-DNJ (FIG.
5).
Example 5
Long Term Distribution of NB-DGJ and NB-DNJ in Mouse Serum and
Liver
[0126] To assay the steady state levels of the compounds when
administered long term via the oral route, the concentrations of
NB-DGJ and NB-DNJ in serum and liver were determined by HPLC after
treating mice with 2400 mg/kg/day of NB-DNJ or NB-DGJ
(non-radiolabelled) for 5 weeks . The experiments were conducted as
described in Example 1 above. The results are shown in Table 2.
[0127] Both serum and liver concentration of drug were higher in
NB-DGJ treated mice compared to NB-DNJ treated (66.+-.3.1 .mu.M
compared to 51.+-.13.3 .mu.M for serum, and 207.+-.30.6 .mu.M
compared to 103.+-.21.2 for liver). The level of NB-DGJ in liver
compared to that of NB-DNJ suggests that NB-DGJ is selectively
taken up into the liver as compared to NB-DNJ. Thus, NB-DGJ may
enter tissues more efficiently and persist longer than NB-DNJ.
2TABLE 2 Concentration of NB-DGJ and NB-DNJ in serum and liver:
Mice were treated with 2400 mg/kg/day of NB-DGJ or NB-DNJ for 5
weeks (n = 2), and the compound concentration in serum and liver
was then determined by duplicate runs on HPLC. Compound
concentration (.mu.M) Serum Liver NB-DGJ 60 .+-. 3.1 207 .+-. 30.6
NB-DNJ 51 .+-. 13.3 103 .+-. 21.2
Example 6
Depletion of GSL by NB-DGJ and NB-DNJ
[0128] The degree of GSL depletion in liver after 10 days or 5
weeks of treatment was compared between mice administered NB-DGJ or
NB-DNJ, using the methods described in Example 1. The livers were
chloroform: methanol-extracted, gangliosides were analysed by thin
layer chromatography and the GM.sub.2 band intensity was
quantitated by densitometry. The relative GM.sub.2 concentrations
(compared to control mice) in livers of mice treated with a range
of NB-DGJ or NB-DNJ doses (300-4800 mg/kg/day) for 10 days show a
dose-dependent response to both compounds (FIG. 6). There was no
significant difference between the GM.sub.2 depletion achieved by
the two compounds at any of the concentrations tested. After longer
treatment (2400 mg/kg/day for 5 weeks), the GM.sub.2 concentrations
in livers of mice treated with NB-DNJ or NB-DGJ were reduced to
35.+-.4% and 26.+-.11%, respectively, in relation to the
concentration in control livers (FIGS. 7 and 8).
[0129] Thus, both analogues (NB-DNJ and NB-DGJ) were shown to be
potent inhibitors of GSL biosynthesis in vivo. After 10 days of
treatment, dose-dependent GSL depletion was seen in livers of mice
fed either NB-DNJ or NB-DGJ. The lowest dose causing GSL depletion
was 600 mg/kg/day (25% reduction). The highest dose evaluated (4800
mg/kg/day) caused 60-70% depletion. Similar data were obtained with
both compounds. Although there is a two fold higher concentration
of NB-DGJ in liver this was not observed when GSL depletion was
measured, where both compounds gave comparable inhibition of
GM.sub.2 biosynthesis. This may reflect differential cellular
uptake of the compounds into hepatocytes, endothelial cells and
Kuppfer cells as GM.sub.2 may be primarily the product of one cell
type whereas the compound could be sequestered in non-GM.sub.2
synthesising cells. GSL depletion after longer treatment at a
dosage of 2400 mg/kg/day was also determined. After 5 weeks of
feeding, the GM.sub.2 concentration was reduced by 74% by NB-DGJ
and 65% by NB-DNJ. The drug distribution and GM.sub.2 depletion
suggest treatment of GSL storage disorders should be as effective
with NB-DGJ, since it has been shown that NB-DNJ reduces storage in
mouse models of these diseases and NB-DGJ is slightly superior to
NB-DNJ in inhibiting GSL biosynthesis in vivo.
Example 6
Effects of NB-DGJ and NB-DNJ on Growth and Lymphoid Organ Size
[0130] To examine the overall well being of the mice treated with
NB-DGJ or NB-DNJ (2400 mg/kg/day for 5 weeks) the mice were
monitored 2-3 times per week, body weights recorded, and the
effects of NB-DGJ and NB-DNJ on growth rates determined (FIG. 9).
The NB-DNJ treated mice grew more slowly than untreated control
mice, while NB-DGJ treated mice showed no difference in growth
rates relative to the untreated controls. After 5 weeks of
treatment, the NB-DNJ mice weighed 25% less than control and the
NB-DGJ mice. Thymuses and spleens removed from NB-DNJ mice were
smaller than those of control or NB-DGJ mice (FIG. 10), while the
weights of other organs such as liver and kidney were unaffected.
Treatment with NB-DNJ reduced the thymus weight by 61.+-.2% and
spleen weight by 62.+-.3% compared to organs from control mice. In
contrast, NB-DGJ had no effect on lymphoid organ weight. The loss
of body weight in NB-DNJ mice did not account for the large
reduction in lymphoid organ size. If expressed as a ratio to body
weight, the organ weights were still reduced significantly (thymus
to body weight ratio was reduced by 45.+-.5% and spleen to body
weight ratio by 48.+-.4% in NB-DNJ mice compared to controls). It
was observed that NB-DNJ treated mice had less fat associated with
their organs (kidney, spleen etc.) and lacked subcutaneous fat
compared to control or NB-DGJ treated mice (data not shown).
[0131] The fact that loss of body weight and reduction of lymphoid
organ size is caused by NB-DNJ but not by NB-DGJ suggests that
these effects are a function of glucosidase inhibition (or an as
yet unidentified activity) by NB-DNJ, not GSL biosynthesis
inhibition (an activity shared by both compounds). The effect of
NB- DNJ in the present study on the inhibition of glycogen
breakdown could provide a possible explanation for at least part of
the weight loss observed in NB-DNJ treated mice. It was shown that,
after 12 h of starvation, when the control and NB-DGJ treated mice
had depleted most of their glycogen, NB-DNJ treated mice still had
a significant amount of glycogen in their livers. Both following
starvation and between episodes of feeding, the mouse would
normally break down glycogen to provide the brain, muscles and
other tissues of the body with glucose. However, if glycogenoloysis
was partial inhibited, as in the NB-DNJ treated mice, the mouse
would have to use other fuel sources, such as fat, to meet its
energy demand. The store of adipose tissue would decrease with time
resulting in reduced body weight. This hypothesis fits with the
observation that the NB-DNJ treated mice (both fed and starved) had
very little subcutaneous fat compared to normal or NB-DGJ treated
mice. The inhibition of glycogenolysis by NB-DGJ is probably due to
inhibition of the glycogen debranching enzyme
(4-.alpha.-glucanotransfera- se, EC 2.4.1.25 and
.alpha.-1,6-glucosidase, EC 3.2.1.33). Although never reported for
NB-DNJ, inhibition of the .alpha.-1,6-glucosidase activity of this
enzyme has previously been observed for other DNJ-derivatives (Arai
et al. (1998) Circulation 97(13): 1290-7; Bollen et al.
Eur-J-Biochem 181(3): 775-80). If this is also the case for NB-DNJ,
over prolonged treatment periods this could cause (pathological)
glycogen storage. If this does occur however, it is exceeding slow
storage as animals on drug for prolonged periods in excess of six
months show no overt signs of pathology (data not shown). What may
be occurring is that the basal level of glycogen is increased due
to partial enzyme inhibition, but that this remains relatively
constant over time at the doses of inhibitor used in this
study.
[0132] NB-DNJ treated mice had consistently smaller lymphoid
organs. However, NB-DGJ did not show this effect, again implying
that this is not the result of GSL biosynthesis inhibition in
animals treated with NB-DNJ.
Example 6
Inhibition of Disaccharidases In Vitro
[0133] NB-DGJ, NB-DNJ and the parental non-alkylated compound DNJ
were assessed for their capacities to inhibit the sucrase and
maltase activities of the enzyme sucrase-isomaltase (which has
disaccharidase activities for the breakdown of sucrose, maltose and
isomaltose). Methods were as described in Example 1. Inhibition of
this enzyme by DNJ has previously been reported (Hanozet et al.
(1981) J. Biol. Chem 256:3703-3711). Both substrate and inhibitor
concentrations were varied and the K.sub.i calculated (Table 3).
NB-DNJ and DNJ were found to be potent inhibitors of both sucrase
and maltase (K.sub.i (sucrase)=0.03 .mu.M and K.sub.i
(maltase)=0.07 .mu.M for DNJ, and K.sub.i (sucrase)=0.26 .mu.M and
K.sub.i (maltase)=0.37 .mu.M for NB-DNJ), while NB-DGJ was less
potent (K.sub.i (sucrase)=2 mM, (maltase) non-inhibitor at 2
mM).
[0134] NB-DNJ, DNJ, NB-DGJ and DGJ were also tested for their
capacity to inhibit lactase (FIG. 11 and Table 4). DNJ, NB-DGJ and
DGJ all inhibited lactase (K.sub.i of 13 .mu.M, 30 .mu.M and 85
.mu.M for DNJ, DGJ and NB-DGJ, respectively). Lactase inhibition by
NB-DNJ was very weak (K.sub.i=4 mM).
3TABLE 3 K.sub.1s for the inhibition of sucrase and maltase by DNJ,
NB-DNJ and NB-DGJ. NI (non-inhibitory at 2 mM). K.sub.1(.mu.M)
Sucrase Maltase DNJ 0.03 0.07 NB-DNJ 0.26 0.37 NB-DGJ 2000 NI
[0135]
4TABLE 4 K.sub.1s for the inhibition of lactase by DNJ, NB-DNJ, DGJ
and NB-DGJ. K.sub.1 (.mu.M) DNJ 13 NB-DNJ 4000 DGJ 30 NB-DGJ 85
[0136] The primary side effect of NB-DNJ has been observed to be
osmotic diarrhoea. The diarrhoea is thought to be caused by
inhibition of disaccharidases in the intestine, which means that
sugars like sucrose and maltose cannot be catabolised and absorbed
from the digestive system. Sucrose consists of one glucose and one
fructose residue, and maltose of two glucose residues. It is
therefore not surprising that the results in this example show that
the glucose analogues NB-DNJ and DNJ are very potent inhibitors of
the sucrase and maltase activity while the galactose analogue
NB-DGJ is not inhibitory. It was found that DNJ, NB-DGJ and DGJ all
inhibited lactase, but the K.sub.is were at least 10.sup.2 times
higher than for sucrase and maltase inhibition by the glucose
analogues. NB-DNJ, however, was not a good inhibitor of lactase
(K.sub.i 4 mM).
[0137] In practical terms this means that NB-DGJ might be best
tolerated on a lactose-free diet, but should not interfere with the
digestion of other carbohydrates. The lack of side effects
associated with NB-DGJ in vivo may have important implications for
the potential treatment of infants and young children where these
side effects could reduce tolerability to a greater extent than
those experienced in adults.
[0138] Thus it can be seen that NB-DGJ has been shown to deplete
GSL in vivo and to exhibit far fewer in vitro and in vivo enzyme
inhibitory properties than NB-DNJ, making this a more selective
compound. Of the activities listed below in Table 5, lactase
inhibition is the only one associated with NB-DGJ and is probably
the simplest to overcome by restricting dietary intake of
lactose.
5 TABLE 5 NB-DNJ NB-DGJ GSL Biosynthesis + + Weight loss + -
Lymphoid organ reduction + - ER .alpha.-glucosidase I and II
inhibition* + - Sucrase and maltase inhibition** + - Lactase
inhibition*** - + *Platt et al (1994) J Biol Chem 269(43): 27108-14
**K.sub.1 (sucrase) = 0.26 .mu.M, K.sub.1 (maltase) = 0.37 .mu.M
for NB-DNJ ***K.sub.1 (lactase) = 85 .mu.M for NB-DGJ samples
(shown in parentheses).
* * * * *