U.S. patent application number 14/972301 was filed with the patent office on 2016-12-15 for carbohydrate-lipid constructs and their use in preventing or treating viral infection.
The applicant listed for this patent is KODE BIOTECH LIMITED. Invention is credited to Stephen Michael Henry.
Application Number | 20160361423 14/972301 |
Document ID | / |
Family ID | 39926214 |
Filed Date | 2016-12-15 |
United States Patent
Application |
20160361423 |
Kind Code |
A1 |
Henry; Stephen Michael |
December 15, 2016 |
CARBOHYDRATE-LIPID CONSTRUCTS AND THEIR USE IN PREVENTING OR
TREATING VIRAL INFECTION
Abstract
The invention relates to selected carbohydrate-lipid constructs
and their use as mimics of ligands for receptors expressed by a
virus. In particular, the invention relates to the use of selected
carbohydrate-lipid constructs in methods of inhibiting virus
infection and/or promoting clearance of virus from infected
subjects. Carbohydrate-lipid constructs selected for use in these
methods where the virus is Human Immunodeficiency Virus (HIV) are
provided.
Inventors: |
Henry; Stephen Michael;
(Auckland, NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KODE BIOTECH LIMITED |
Auckland |
|
NZ |
|
|
Family ID: |
39926214 |
Appl. No.: |
14/972301 |
Filed: |
December 17, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14085156 |
Nov 20, 2013 |
9226968 |
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14972301 |
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13459399 |
Apr 30, 2012 |
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14085156 |
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12451120 |
Mar 29, 2010 |
8211860 |
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PCT/NZ2008/000095 |
Apr 28, 2008 |
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13459399 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/685 20130101;
A61K 31/7028 20130101; A61K 31/70 20130101; A61P 31/12 20180101;
A61P 31/18 20180101; A61K 31/702 20130101; A61K 35/18 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 31/685 20060101 A61K031/685; A61K 35/18 20060101
A61K035/18; A61K 31/702 20060101 A61K031/702 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 27, 2007 |
NZ |
554853 |
Jul 24, 2007 |
NZ |
556736 |
Apr 24, 2008 |
NZ |
567754 |
Claims
1. A method of inhibiting infection of the cells of a subject by a
virus by administering to the subject an amount of
carbohydrate-lipid construct of the formula F-S.sub.1-S.sub.2-L
where: F is selected from the group consisting of glycotopes of
ligands for one or more receptors expressed by the virus;
S.sub.1-S.sub.2 is a spacer linking F to L; and L is a lipid
selected from the group consisting of diacyl- and
dialkyl-glycerolipids, including glycerophospholipids.
2. The method of claim 1 where the amount is effective to inhibit
binding of the receptor expressed by the virus to the cell surface
expressed ligand.
3. The method of claim 1 where the receptor is expressed by the
human immunodeficiency virus (HIV).
4. The method of claim 1 where S.sub.1 is a C.sub.2-5-aminoalkyl
selected from the group consisting of: 2-aminoethyl, 3-aminopropyl,
4-aminobutyl, or 5-aminopentyl.
5. The method of claim 1 where S.sub.2 is selected from the group
consisting of: --CO(CH.sub.2).sub.3CO--,
--CO(CH.sub.2).sub.4CO-(adipate), and --CO(CH.sub.2).sub.5CO--.
6. The method of claim 1 where L is selected from the group
consisting of: diacylglycerolipids, phosphatidate, phosphatidyl
choline, phosphatidyl, ethanolamine, phosphatidyl serine,
phosphatidyl inositol, phosphatidyl glycerol; and diphosphatidyl
glycerol derived from one or more of trans-3-hexadecenoic acid,
cis-5-hexadecenoic acid, cis-7-hexadecenoic acid,
cis-9-hexadecenoic acid, cis-6-octadecenoic acid,
cis-9-octadecenoic acid, trans-9-octadecenoic acid,
trans-11-octadecenoic acid, cis-11-octadecenoic acid,
cis-11-eicosenioic acid or cis-13-docsenoic acid.
7. The method of claim 6 where the lipid is derived from one or
more cis-destaurated fatty acids.
8. The method of claim 1 where L is selected from the group
consisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine
(DOPE) and 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine
(DSPE); and rac-1,2-dioleoylglycerol (DOG).
9. The method of claim 1 where L is a glycerophospholipid and the
construct includes the substructure: ##STR00026## where X is H or
C, * is other than H and n is an integer 2 to 5.
10. The method of claim 1 where L is a glycerophospholipid and the
construct includes the substructure: ##STR00027## where: X is H;
R.sub.1 is a C.sub.p-alkyl glycoside; R.sub.2 and R.sub.3 are
independently selected from the group consisting of:
trans-3-hexadecenal, cis-5-hexadecenal, cis-7-hexadecenal,
cis-9-hexadecenal, cis-6-octadecenal, cis-9-octadecenal,
trans-9-octadecenal, trans-11-octadecenal, cis-11-octadecenal,
cis-11-eicosenal or cis-13-docsenal; n is 2 to 5; and p is 2 or
3.
11. The method of claim 10 where the glycoside is
1-O--(O-.alpha.-D-galactopyranosyl-(I.fwdarw.4)-O-.beta.-D-galactopyranos-
yl-(1.fwdarw.4)-.beta.-D-glucopyranosyl (Gb.sub.3), n is 4 and p is
3.
12. The method of claim 1 where the carbohydrate-lipid construct
has the structure: ##STR00028## designated Gb.sub.3-sp3-Ad-DOPE (I)
and where M is typically H, but may be replaced by another
monovalent cation such as Na.sup.+, K.sup.+, NH.sub.4.sup.+ or
triethylamine ([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
13. The method of claim 1 where the carbohydrate-lipid construct
has the structure: ##STR00029## designated Gb.sub.3-sp3-Ad-DSPE
(II) and where M is typically H, but may be replaced by another
monovalent cation such as Na.sup.+, K.sup.+, NH.sub.4.sup.+ or
triethylamine ([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
14. The method of claim 1 where the carbohydrate-lipid construct
has the structure: ##STR00030## designated Gb3-sp2-Ad-DOPE (III)
and where M is typically H, but may be replaced by another
monovalent cation such as Na.sup.+, K.sup.+, NH.sub.4.sup.+ or
triethylamine ([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
15. The method of claim 1 where the carbohydrate-lipid construct
has the structure: ##STR00031## designated Gb.sub.3-sp2-Ad-DSPE
(IV) and where M is typically H, but may be replaced by another
monovalent cation such as Na.sup.+, K.sup.+, NH.sub.4.sup.+ or
triethylamine ([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
16. The method of claim 1 where the administering to the subject is
by intravascular injection.
17. The method of claim 1 where the administering is by intravenous
injection.
18. The method of claim 1 where the administering to the subject is
to provide a concentration in the plasma of the subject of greater
than 400 .mu.M.
19. The method of claim 1 where the administering to the subject is
by topical application.
20. The method of claim 1 where the administering to the subject is
by topical application as a cream or suppository.
21. A method of promoting clearance of a virus from an infected
subject by administering to the subject an amount of
carbohydrate-lipid construct of the formula F-S.sub.1-S.sub.2-L
where: F is selected from the group consisting of glycotopes of
ligands for one or more receptors expressed by the virus;
S.sub.1-S.sub.2 is a spacer linking F to L; and L is a lipid
selected from the group consisting of diacyl- and
dialkyl-glycerolipids, including glycerophospholipids.
22. The method of claim 21 where the administering to the subject
is by intravascular injection.
23. The method of claim 21 where the administering is by
intravenous injection.
24. The method of claim 21 where the amount is sufficient to
promote partitioning of the carbohydrate-lipid construct into the
membranes of cells of the vascular system.
25. The method of claim 21 where the administering to the subject
is to provide an initial concentration in the plasma of the subject
of greater than 400 .mu.M.
26. The method of claim 21 where the virus is human
immunodeficiency virus (HIV).
27. The method of claim 21 where S.sub.1-S.sub.2 is selected to
provide a carbohydrate-lipid construct that partitions into a lipid
bi-layer when a solution of the construct is contacted with the
lipid bi-layer.
28. The method of claim 21 where S.sub.1 is a C.sub.2-5-aminoalkyl
selected from, the group consisting of: 2-aminoethyl,
3-aminopropyl, 4-aminobutyl, or 5-aminopentyl.
29. The method of claim 21 where S.sub.2 is selected from the group
consisting of: --CO(CH.sub.2).sub.3CO--,
--CO(CH.sub.2).sub.4CO-(adipate), --CO(CH.sub.2).sub.5CO-- and
--CO(CH.sub.2).sub.5NHCO(CH.sub.2).sub.5CO--.
30. The method of claim 21 where L is selected from the group
consisting of: diacylglycerolipids, phosphatidate, phosphatidyl
choline, phosphatidyl ethanolamine, phosphatidyl serine,
phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl
glycerol derived from one or more of trans-3-hexadecenoic acid,
cis-5-hexadecenoic acid, cis-7-hexadecenoic acid,
cis-9-hexadecenoic acid, cis-6-octadecenoic acid,
cis-9-octadecenoic acid, trans-9-octadecenoic acid,
trans-11-octadecenoic acid, cis-11-octadecenoic acid,
cis-11-eicosenoic acid or cis-13-docsenoic acid.
31. The method of claim 30 where the lipid is derived from one or
more cis-destaurated fatty acids.
32. The method of claim 21 where L is selected from the group
consisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine
(DOPE) and 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine
(DSPE); and rac-1,2-dioleoylglycerol (DOG).
33. The method of claim 21 where L is a glycerophospholipid and the
construct includes the substructure: ##STR00032## where X is H or
C, * is other than H and n is an integer 2 to 5.
34. The method of claim 21 where L is a glycerophospholipid and the
construct includes the substructure: ##STR00033## where: X is H;
R.sub.1 is a C.sub.p-alkyl glycoside; R.sub.2 and R.sub.3 are
independently selected from the group consisting of:
trans-3-hexadecenal, cis-5-hexadecenal, cis-7-hexadecenal,
cis-9-hexadecenal, cis-6-octadecenal, cis-9-octadecenal,
trans-9-octadecenal, trans-11-octadecenal, cis-11-octadecenal,
cis-11-eicosenal or cis-13-docsenal; n is 2 to 5; and p is 2 or
3.
35. The method of claim 34 where the glycoside is
1-O--(O-.alpha.-D-galactopyranosyl-(1.fwdarw.4)-O-.beta.-D-galactopyranos-
yl-(1.fwdarw.4)-.beta.-D-glucopyranosyl (Gb.sub.3), n is 4 and p is
3.
36. The method of claim 22 where the carbohydrate-lipid construct
has the structure: ##STR00034## designated Gb.sub.3-sp3-Ad-DOPE (I)
and where M is typically H, but may be replaced by another
monovalent cation such as Na.sup.+, K.sup.+, NH.sub.4.sup.+ or
triethylamine ([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
37. The method of claim 22 where the carbohydrate-lipid construct
has the structure: ##STR00035## designated Gb.sub.3-sp3-Ad-DSPE
(II) and where M is typically H, but may be replaced by another
monovalent cation such as Na.sup.+, K.sup.+, NH.sub.4.sup.+ or
triethylamine ([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
38. The method of claim 22 where the carbohydrate-lipid construct
has the structure: ##STR00036## designated Gb.sub.3-sp2-Ad-DOPE
(III) and where M is typically H, but may be replaced by another
monovalent cation such as Na.sup.+, K.sup.+, NH.sub.4.sup.+ or
triethylamine ([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
39. The method of claim 22 where the carbohydrate-lipid construct
has the structure: ##STR00037## designated Gb.sub.3-sp2-Ad-DSPE
(IV) and where M is typically H, but may be replaced by another
monovalent cation such as Na.sup.+, K.sup.+, NH.sub.4.sup.+ or
triethylamine ([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
40. A pharmaceutical preparation for administration to a subject
comprising a receptor binding carbohydrate-lipid construct of the
formula F-S.sub.1-S.sub.2-L where: F is selected from the group
consisting of glycotopes of ligands for one or more receptors;
Si-S.sub.2 is a spacer linking F to L; and L is a lipid selected
from the group consisting of diacyl- and dialkyl-glycerolipids,
including glycerophospholipids; and pharmaceutically acceptable
formulants.
41. The pharmaceutical preparation of claim 40 where the one or
more receptors are expressed by a virus.
42. The pharmaceutical preparation of claim 40 where the one or
more receptors are expressed by the human immunodeficiency virus
(HIV).
43. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation is in the form of a cream or
suppository.
44. The pharmaceutical preparation of claim 40 where Si-S.sub.2 is
selected to provide a carbohydrate-lipid construct that partitions
into a lipid bi-layer when a solution of the construct is contacted
with the lipid bi-layer.
45. The pharmaceutical preparation of claim 40 where Si is a
C.sub.2-5-aminoalkyl selected from the group consisting of:
2-aminoethyl, 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl.
46. The pharmaceutical preparation of claim 40 where S.sub.2 is
selected from the group consisting of: --CO(CH.sub.2).sub.3CO--,
--CO(CH.sub.2).sub.4CO-- (adipate), and
--CO(CH.sub.2).sub.5CO--.
47. The pharmaceutical preparation of claim 40 where L is selected
from the group consisting of: diacylglycerolipids, phosphatidate,
phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl
serine, phosphatidyl inositol, phosphatidyl glycerol, and
diphosphatidyl glycerol derived from one or more of
trans-3-hexadecenoic acid, cis-5-hexadecenoic acid,
cis-7-hexadecenoic acid, cis-9-hexadecenoic acid,
cis-6-octadecenoic acid, cis-9-octadecenoic acid,
trans-9-octadecenoic acid, trans-11-octadecenoic acid,
cis-11-octadecenoic acid, cis-11-eicosenoic acid or
cis-13-docsenoic acid.
48. The pharmaceutical preparation of claim 47 where the lipid is
derived from one or more cis-destaurated fatty acids.
49. The pharmaceutical preparation of claim 40 where L is selected
from the group consisting of:
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and
1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE); and
rac-1,2-dioleoylglycerol (DOG).
50. The pharmaceutical preparation of claim 40 where L is a
glycerophospholipid and the construct includes the substructure:
##STR00038## where X is H or C, * is other than H and n is an
integer 2 to 5.
51. The pharmaceutical preparation of claim 40 where L is a
glycerophospholipid and the construct includes the substructure:
##STR00039## where: X is H; R.sub.1 is a C.sub.p-alkyl glycoside;
R.sub.2 and R.sub.3 are independently selected from the group
consisting of: trans-3-hexadecenal, cis-5-hexadecenal,
cis-7-hexadecenal, cis-9-hexadecenal, cis-6-octadecenal,
cis-9-octadecenal, trans-9-octadecenal, trans-11-octadecenal,
cis-11-octadecenal, cis-11-eicosenal or cis-13-docsenal; n is 2 to
5; and p is 2 or 3.
52. The pharmaceutical preparation of claim 51 where the glycoside
is
1-O--(O-.alpha.-D-galactopyranosyl-(1.fwdarw.4)-O-.beta.-D-galactopyranos-
yl-(1.fwdarw.4)-.beta.-D-glucopyranosyl (Gb.sub.3), n is 4 and p is
3.
53. The pharmaceutical preparation of claim 40 where the
carbohydrate-lipid construct has the structure: ##STR00040##
designated Gb.sub.3-sp3-Ad-DOPE (I) and where M is typically H, but
may be replaced by another monovalent cation such as Na.sup.+,
K.sup.+, NH.sub.4.sup.+ or triethylamine
([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
54. The pharmaceutical preparation of claim 40 where the
carbohydrate-lipid construct has the structure: ##STR00041##
designated Gb.sub.3-sp3-Ad-DSPE (II) and where M is typically H,
but may be replaced by another monovalent cation such as Na.sup.+,
K.sup.+, NH.sub.4.sup.+ or triethylamine
([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
55. The pharmaceutical preparation of claim 40 where the
carbohydrate-lipid construct has the structure: ##STR00042##
designated Gb.sub.3-sp2-Ad-DOPE (III) and where M is typically H,
but may be replaced by another monovalent cation such as Na.sup.+,
K.sup.+, NH.sub.4.sup.+ or triethylamine
([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
56. The pharmaceutical preparation of claim 40 where the
carbohydrate-lipid construct has the structure: ##STR00043##
designated Gb.sub.3-sp2-Ad-DSPE (IV) and where M is typically H,
but may be replaced by another monovalent cation such as Na.sup.+,
K.sup.+, NH.sub.4.sup.+ or triethylamine
([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
57. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation is formulated for administration by
intravascular injection.
58. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation is formulated for administration by
intravenous injection.
59. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation is formulated as an aqueous
formulation.
60. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation comprises a suspension of red blood
cells of the subject modified to incorporate the receptor binding
carbohydrate-lipid construct.
61. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation is formulated as an aqueous formulation
for administration by intravenous injection and identified for use
in inhibiting HIV infection and/or promoting clearance of HIV from
infected subjects.
62. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation is formulated for administration as a
cream or suppository.
63. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation is formulated for administration as a
cream.
64. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation is formulated as an aqueous
formulation.
65. The pharmaceutical preparation of claim 40 where the
pharmaceutical preparation is formulated as an aqueous formulation
for administration as a cream and identified for use in inhibiting
or preventing HIV infection.
66. The carbohydrate-lipid construct of the structure: ##STR00044##
designated Gb.sub.3-sp3-Ad-DOPE (I) and where M is typically H, but
may be replaced by another monovalent cation such as Na.sup.+,
K.sup.+, NH.sub.4.sup.+ or triethylamine
([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
67. The carbohydrate-lipid construct of the structure: ##STR00045##
designated Gb.sub.3-sp3-Ad-DSPE (II) and where M is typically U,
but may be replaced by another monovalent cation such as Na.sup.+,
K.sup.+, NH.sub.4.sup.+ or triethylamine
([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
68. The carbohydrate-lipid construct of the structure: ##STR00046##
designated Gb.sub.3-sp2-Ad-DOPE (III) and where M is typically H,
but may be replaced by another monovalent cation such as Na.sup.+,
K.sup.+, NH.sub.4.sup.+ or triethylamine
([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
69. The carbohydrate-lipid construct of the structure: ##STR00047##
designated Gb.sub.3-sp2-Ad-DSPE (IV) and where M is typically H,
but may be replaced by another monovalent cation such as Na.sup.+,
K.sup.+, NH.sub.4.sup.+ or triethylamine
([NH(CH.sub.2CH.sub.3).sub.3].sup.+).
70. The use of at least one of the carbohydrate-lipid constructs of
claim 66 in the manufacture of a pharmaceutical preparation for the
inhibition of HIV infection of the cells of a subject.
71. The use of at least one of the carbohydrate-lipid constructs of
claim 66 in the manufacture of a pharmaceutical preparation for
promoting clearance of HIV from an infected subject.
72. A pharmaceutical preparation for use in the prevention of HIV
infection by topical application comprising a carbohydrate-lipid
construct selected from the group consisting of:
Gb.sub.3-sp3-Ad-DOPE (I); Gb.sub.3-sp3-Ad-DSPE (II);
Gb.sub.3-sp2-Ad-DOPE (III); and Gb.sub.3-sp2-Ad-DSPE (IV).
Description
TECHNICAL FIELD
[0001] The invention relates to selected carbohydrate-lipid
constructs and their use as mimics of ligands for receptors
expressed by a virus.
[0002] In particular, the invention relates to the use of selected
carbohydrate-lipid constructs in methods of inhibiting viral
infection and/or promoting clearance of virus from infected
subjects.
[0003] Carbohydrate-lipid constructs selected for use in these
methods where the virus is Human Immunodeficiency Virus (HIV) are
provided.
BACKGROUND ART
[0004] Infection with HIV and Acquired Immune Deficiency Syndrome
(AIDS) continues to increase worldwide, despite intense research to
control its spread. Furthermore, the emergence of new viral
infections presents additional challenges to public health.
[0005] Therapies to treat infection that target viruses may be
limited in efficacy due to resistance and genetic variance of the
virus.
[0006] HIV infection is mediated by the viral fusion glycoprotein
gp120-gp41 binding the cell surface expressed receptor CD4. This
binding is the basis of the viral targeting of T lymphocytes and
monocyte macrophages. The receptor gp120 shows an affinity in vitro
for several glycosphingolipids (GSLs) (Bhat et al (1993); Fantini
et al (1998); Mylvaganam and Lingwood (1999a)).
[0007] A need exists for glycolipid mimics that are dispersible in
biocompatible media and can be used to modify the interaction
between naturally occurring membrane incorporated glycoconjugates,
such as GSLs, and the receptors expressed by a virus. Such water
soluble glycolipid mimics have been recognized as having potential
for use in the preventative treatment of individuals at risk of
infection from viruses such as HIV.
[0008] Lund et al (2006) have investigated the effect of the water
soluble glycolipid mimic adamantylGb.sub.3 on HIV infection of
cells in culture. In previous studies adamantylGb.sub.3 had been
demonstrated to be a superior ligand for the receptor gp120
(Mahfoud et al (2002)).
[0009] A dose dependent inhibition of infection of Jurkat T cells
by HIV-1 pre-incubated with adamantylGb.sub.3 has been demonstrated
in vitro (Lund et al 2006). The in vivo inhibition of infection by
HIV-1 was not reported, but the water soluble glycolipid mimic was
indicated to have no effect on Jurkat T cell viability. Transient
changes in CD4 surface expression were observed. Lund et al (2006)
attributed the dose dependent inhibition of infection to an
inhibition of attachment of the pre-treated HIV-1 to the Jurkat T
cells. The adamantylGb.sub.3 treated virus remained non-host cell
attached and virions could not be found within the Jurkat
cells.
[0010] Further studies on HIV-1 infection of primary lymphoid cells
in vitro provided results consistent with those observed for Jurkat
T cells as host cells. Infection by both wild type and drug
resistant HIV-1 infection was inhibited by the pre-treatment of the
water soluble glycolipid mimic adamantylGb.sub.3. However,
pre-incubation of cells with adamantylGb.sub.3 was ineffective.
[0011] Lund et al (2006) noted the effective concentration range
required to inhibit HIV-1 infection would be difficult to maintain
clinically, but suggested the formulation of adamantylGb.sub.3
within a cream might provide a topical ointment for the prevention
of mucosal HIV infection.
[0012] It is an object of the invention to provide receptor binding
carbohydrate-lipid constructs that are effective to inhibit viral
infection of the cells of a subject.
[0013] It is a further object of the invention to provide receptor
binding carbohydrate-lipid constructs that are effective to promote
clearance of virus from an infected subject.
[0014] These objects are to be read disjunctively with the object
of to at least provide a useful choice.
[Followed by page 4]
DISCLOSURE OF INVENTION
[0015] In a first aspect the invention provides a method of
inhibiting infection of the cells of a subject by a virus by
administering to the subject an amount of carbohydrate-lipid
construct of the formula F-S.sub.1-S.sub.2-L where: [0016] F is
selected from the group consisting of glycotopes of ligands for one
or more receptors expressed by the virus; [0017] S.sub.1-S.sub.2 is
a spacer linking F to L; and [0018] L is a lipid selected from the
group consisting of diacyl- and dialkyl-glycerolipids, including
glycerophospholipids.
[0019] Preferably, the amount is effective to inhibit binding of
the receptor expressed by the virus to a cell surface expressed
ligand.
[0020] Preferably, the receptor is expressed by the human
immunodeficiency virus (HIV).
[0021] S.sub.1-S.sub.2 is selected to provide a carbohydrate-lipid
construct that is dispersible in water.
[0022] Preferably, S.sub.1 is a C.sub.2-5-aminoalkyl selected from
the group consisting of: 2-aminoethyl; 3-aminopropyl; 4-aminobutyl;
and 5-aminopentyl.
[0023] Preferably, S.sub.2 is selected from the group consisting
of: --CO(CH.sub.2).sub.3CO--; --CO(CH.sub.2).sub.4CO-- (adipate);
and --CO(CH.sub.2).sub.5CO--.
[0024] Preferably, L is selected from the group consisting of:
diacylglycerolipids, phosphatidate, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl
inositol, phosphatidyl glycerol, and diphosphatidyl glycerol
derived from one or more of trans-3-hexadecenoic acid,
cis-5-hexadecenoic acid, cis-7-hexadecenoic acid,
cis-9-hexadecenoic acid, cis-6-octadecenoic acid,
cis-9-octadecenoic acid, trans-9-octadecenoic acid,
trans-11-octadecenoic acid, cis-11-octadecenoic acid,
cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably,
the lipid is derived from one or more cis-desaturated fatty acids.
Most preferably, L is selected from the group consisting of:
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and
1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE); and
rac-1,2-dioleoylglycerol (DOG).
[0025] Preferably, L is a glycerophospholipid and the construct
includes the substructure:
##STR00001##
where X is H or C, * is other than H and n is an integer 2 to
5.
[0026] More preferably, L is a glycerophospholipid and the
construct includes the substructure:
##STR00002##
where: [0027] X is H; [0028] R.sub.1 is a C.sub.p-alkyl glycoside,
[0029] R.sub.2 and R.sub.3 are independently selected from the
group consisting of: trans-3-hexadecenal, cis-5-hexadecenal,
cis-7-hexadecenal, cis-9-hexadecenal, cis-6-octadecenal,
cis-9-octadecenal, trans-9-octadecenal, trans-1-octadecenal,
cis-11-octadecenal, cis-11-eicosanal and cis-13-docsenal; [0030] n
is 2 to 5; and [0031] p is 2 or 3.
[0032] Most preferably, the glycoside is
1-O--(O-.alpha.-D-galactopyranosyl-(1.fwdarw.4)-O-.beta.-D-galactopyranos-
yl-(1.fwdarw.4)-.beta.-D-glucopyranosyl (Gb.sub.3), n is 4 and p is
3.
[0033] In specific embodiments of the first aspect of the invention
the carbohydrate-lipid construct has the structure:
##STR00003##
designated Gb.sub.3-sp3-Ad-DOPE (I); the structure:
##STR00004##
designated Gb.sub.3-sp3-Ad-DSPE (II); the structure:
##STR00005##
designated Gb.sub.3-sp2-Ad-DOPE (III); or the structure:
##STR00006##
designated Gb.sub.3-sp2-Ad-DSPE (IV).
[0034] In a first embodiment of the first aspect of the invention
the administering to the subject is by intravascular injection.
Preferably, the administering is by intravenous injection.
[0035] Preferably, the administering to the subject is to provide a
concentration in the plasma of the subject of greater than 400
.mu.M.
[0036] In a second embodiment of the first aspect of the invention
the administering to the subject is by topical application.
Preferably, the administering to the subject is by topical
application as a cream or suppository.
[0037] In a second aspect the invention provides a method of
promoting clearance of a virus from an infected subject by
administering to the subject an amount of carbohydrate-lipid
construct of the formula F-S.sub.1-S.sub.2-L where: [0038] F is
selected from the group consisting of glycotopes of ligands for one
or more receptors expressed by the virus; [0039] S.sub.1-S.sub.2 is
a spacer linking F to L; and [0040] L is a lipid selected from the
group consisting of diacyl- and dialkyl-glycerolipids, including
glycerophospholipids.
[0041] Preferably, the administering to the subject is by
intravascular injection. More preferably, the administering is by
intravenous injection.
[0042] Preferably, the amount is sufficient to promote partitioning
of the carbohydrate-lipid construct into the membranes of cells of
the vascular system.
[0043] Preferably, the administering to the subject is to provide
an initial concentration in the plasma of the subject of greater
than 400 .mu.M.
[0044] Preferably, the receptor is expressed by the human
immunodeficiency virus (HIV).
[0045] S.sub.1-S.sub.2 is selected to provide a carbohydrate-lipid
construct that is dispersible in water.
[0046] Preferably, S.sub.1-S.sub.2 is selected to provide a
carbohydrate-lipid construct that partitions into a lipid bi-layer
when a solution of the construct is contacted with the lipid
bi-layer.
[0047] Preferably, S.sub.1 is a C.sub.2-5-aminoalkyl selected from
the group consisting of: 2-aminoethyl, 3-aminopropyl, 4-aminobutyl,
or 5-aminopentyl.
[0048] Preferably, S.sub.2 is selected from the group consisting
of: --CO(CH.sub.2).sub.3CO--, --CO(CH.sub.2).sub.4CO-- (adipate),
--CO(CH.sub.2).sub.5CO-- and
--CO(CH.sub.2).sub.5NHCO(CH.sub.2).sub.5CO--.
[0049] Preferably, L is selected from the group consisting of:
diacylglycerolipids, phosphatidate, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl
inositol, phosphatidyl glycerol, and diphosphatidyl glycerol
derived from one or more of trans-3-hexadecenoic acid,
cis-5-hexadecenoic acid, cis-7-hexadecenoic acid,
cis-9-hexadecenoic acid, cis-6-octadecenoic acid,
cis-9-octadecenoic acid, trans-9-octadecenoic acid,
trans-11-octadecenoic acid, cis-11-octadecenoic acid,
cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably,
the lipid is derived from one or more cis-destaurated fatty acids.
Most preferably, L is selected from the group consisting of:
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and
1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE); and
rac-1,2-dioleoylglycerol (DOG).
[0050] Preferably, L is a glycerophospholipid and the construct
includes the substructure:
##STR00007##
where X is H or C, * is other than H and n is an integer 2 to
5.
[0051] More preferably, L is a glycerophospholipid and the
construct includes the substructure:
##STR00008##
where: [0052] X is H; [0053] R.sub.1 is a C.sub.p-alkyl glycoside,
[0054] R.sub.2 and R.sub.3 are independently selected from the
group consisting of: trans-3-hexadecenal, cis-5-hexadecenal,
cis-7-hexadecenal, cis-9-hexadecenal, cis-6-octadecenal,
cis-9-octadecenal, trans-9-octadecenal, trans-11-octadecenal,
cis-11-octadecenal, cis-11-eicosenal or cis-13-docsenal; [0055] n
is 2 to 5; and [0056] p is 2 or 3.
[0057] Most preferably, the glycoside is
1-O--(O-.alpha.-D-galactopyranosyl-(1.fwdarw.4)-O-.beta.-D-galactopyranos-
yl-(1.fwdarw.4)-.beta.-D-glucopyranosyl (Gb.sub.3), n is 4 and p is
3.
[0058] In specific embodiments of the second aspect of the
invention the carbohydrate-lipid construct has the structure:
##STR00009##
designated Gb.sub.3-sp3-Ad-DOPE (I); the structure:
##STR00010##
designated Gb.sub.3-sp3-Ad-DSPE (II); the structure:
##STR00011##
designated Gb.sub.3-sp2-Ad-DOPE (III); or the structure:
##STR00012##
designated Gb.sub.3-sp2-Ad-DSPE (IV).
[0059] In a third aspect the invention provides a pharmaceutical
preparation for administration to a subject comprising a receptor
binding carbohydrate-lipid construct of the formula
F-S.sub.1-S.sub.2-L where: [0060] F is selected from the group
consisting of glycotopes of ligands for a receptor; [0061]
S.sub.1-S.sub.2 is a spacer linking F to L; and [0062] L is a lipid
selected from the group consisting of diacyl- and
dialkyl-glycerolipids, including glycerophospholipids; and
pharmaceutically acceptable formulants.
[0063] Preferably, the receptor is expressed by a virus. More
preferably, the receptor is expressed by the human immunodeficiency
virus (HIV).
[0064] Preferably, the pharmaceutical preparation is in the form of
a cream or suppository.
[0065] S.sub.1-S.sub.2 is selected to provide a carbohydrate-lipid
construct that is dispersible in water.
[0066] Preferably, S.sub.1-S.sub.2 is selected to provide a
carbohydrate-lipid construct that partitions into a lipid bi-layer
when a solution of the construct is contacted with the lipid
bi-layer.
[0067] Preferably, S.sub.1 is a C.sub.2-5-aminoalkyl selected from
the group consisting of: 2-aminoethyl, 3-aminopropyl, 4-aminobutyl,
or 5-aminopentyl.
[0068] Preferably, S.sub.2 is selected from the group consisting
of: --CO(CH.sub.2).sub.3CO--; --CO(CH.sub.2).sub.4CO-- (adipate);
and --CO(CH.sub.2).sub.5CO--.
[0069] Preferably, L is selected from the group consisting of:
diacylglycerolipids, phosphatidate, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl
inositol, phosphatidyl glycerol, and diphosphatidyl glycerol
derived from one or more of trans-3-hexadecenoic acid,
cis-5-hexadecenoic acid, cis-7-hexadecenoic acid,
cis-9-hexadecenoic acid, cis-6-octadecenoic acid,
cis-9-octadecenoic acid, trans-9-octadecenoic acid,
trans-11-octadecenoic acid, cis-11-octadecenoic acid,
cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably,
the lipid is derived from one or more cis-destaurated fatty acids.
Most preferably, L is selected from the group consisting of:
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and
1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE); and
rac-1,2-dioleoylglycerol (DOG).
[0070] Preferably, L is a glycerophospholipid and the construct
includes the substructure:
##STR00013##
where X is H or C, * is other than H and n is an integer 2 to
5.
[0071] More preferably, L is a glycerophospholipid and the
construct includes the substructure:
##STR00014##
where: [0072] X is H; [0073] R.sub.1 is a C.sub.p-alkyl glycoside,
[0074] R.sub.2 and R.sub.3 are independently selected from the
group consisting of: trans-3-hexadecenal, cis-5-hexadecenal,
cis-7-hexadecenal, cis-9-hexadecenal, cis-6-octadecenal,
cis-9-octadecenal, trans-9-octadecenal, trans-11-octadecenal,
cis-11-octadecenal, cis-11-eicosenal or cis-13-docsenal; [0075] n
is 2 to 5; and [0076] p is 2 or 3.
[0077] Most preferably, the glycoside is
1-O--(O-.alpha.-D-galactopyranosyl-(1.fwdarw.4)-O-.beta.-D-galactopyranos-
yl-(1.fwdarw.4)-.beta.-D-glucopyranosyl (Gb.sub.3), n is 4 and p is
3.
[0078] In specific embodiments of the third aspect of the invention
the carbohydrate-lipid construct has the structure:
##STR00015##
designated Gb.sub.3-sp3-Ad-DOPE (I); the structure:
##STR00016##
designated Gb.sub.3-sp3-Ad-DSPE (II); the structure:
##STR00017##
designated Gb.sub.3-sp2-Ad-DOPE (III); or the structure:
##STR00018##
designated Gb.sub.3-sp2-Ad-DSPE (IV).
[0079] In a first embodiment of the third aspect of the invention
the pharmaceutical preparation is formulated for administration by
intravascular injection. Preferably, the pharmaceutical preparation
is formulated for administration by intravenous injection. More
preferably, the pharmaceutical preparation is formulated as an
aqueous formulation. Yet more preferably, the pharmaceutical
preparation is a suspension of red blood cells of the subject
modified to incorporate the receptor binding carbohydrate-lipid
construct. Most preferably, the pharmaceutical preparation is
identified for use in inhibiting HIV infection and/or promoting
clearance of HIV from infected subjects.
[0080] In a third embodiment of the third aspect of the invention
the pharmaceutical preparation is formulated for administration as
a cream or suppository. Preferably, the pharmaceutical preparation
is formulated for administration as a cream. More preferably, the
pharmaceutical preparation is formulated as an aqueous formulation.
Most preferably, the pharmaceutical preparation is identified for
use in inhibiting or preventing HIV infection.
[0081] In the description and claims of the specification the
following terms and phrases have the meaning provided:
[0082] "Carbohydrate-lipid construct" means a synthetic molecule
used as a glycolipid mimic.
[0083] "Gb3" means the carbohydrate portion of the ganglioside Gb3
(Chemical Abstract Service (CAS) REGISTRY number 71965-57-6)
[0084] "C.sub.p-alkyl glycoside" means an alkyl glycoside
consisting of an unbranched chain of p methylene units attached to
the carbohydrate via a glycosidic linkage as exemplified by the
propyl glycoside (p is 3) of the structure:
##STR00019##
designated Gb.sub.3-sp3.
[0085] "Dispersible in water" means a stable, single phase
dispersion of the carbohydrate-lipid construct may be prepared in
water at a concentration of up to at least 1000 .mu.M in the
absence of organic solvents or detergents.
[0086] "Glycotope" means the portion of the carbohydrate moiety of
a ligand that associates with the binding site of a receptor.
[0087] "Ligand" means any molecule or portion of a molecule that
binds to one or more macromolecules, such as surface expressed
antigens.
[0088] "Pharmaceutically acceptable formulants" means ingredients
included in the formulation of a pharmaceutical composition.
[0089] "Receptor" means a macromolecule or portion of a
macromolecule such as a surface expressed antigen that binds to one
or more ligands.
[0090] "Vascular system" means the system of vessels that convey
fluids such as blood or lymph, or provide for the circulation of
such fluids.
[0091] In the context of administering to the subject to provide a
specified concentration in the plasma of the subject the
administering may be by repeated administration to maintain the
specified concentration in the plasma.
[0092] From the structures and substructures of the
carbohydrate-lipid constructs it will be recognised that M is
typically H, but may be replaced by another monovalent cation such
as Na.sup.+, K.sup.+, NH.sub.4.sup.+ and triethylamine
([NH(CH.sub.2CH.sub.3).sub.3].sup.+), and the secondary amino
functions of the carbohydrate-lipid construct may be protonated.
The carbohydrate-lipid constructs may be prepared as a range of
pharmaceutically acceptable salts.
[0093] Where the suffix "-al" is employed in respect of the
substituents R.sub.2 and R.sub.3, an aldehyde structure is intended
as exemplified by cis-9-octadecenal of the structure:
##STR00020##
[0094] The invention will now be described in detail with reference
to examples and the Figures of the accompanying drawings pages that
are indicative of the utility of the subject matter claimed in the
treatment of human subjects.
BRIEF DESCRIPTION OF FIGURES
[0095] FIG. 1. .sup.1H-NMR data for the carbohydrate-lipid
construct designated Gb.sub.3-sp3-Ad-DOPE (I).
[0096] FIG. 2. Effect of 250 .mu.M Gb.sub.3-sp3-Ad-DOPE (I) on
VSV/HIV infection in Jurkat Cells (RLU): A--Control; B--AZT;
C--VSV/HIV; and D--250 .mu.M Gb.sub.3-sp3-Ad-DOPE (I).
[0097] FIG. 3. Inhibition of infection of Jurkat cells by
pre-incubation of X4 HIV-1.sub.IIIB with the carbohydrate-lipid
construct designated Gb.sub.3-sp3-Ad-DOPE (I) (p24 pg/mL)(r=4):
A--Control; B--50 .mu.M; C--100 .mu.M; D--200 .mu.M; E--400 .mu.M;
F--600 .mu.M; G--800 .mu.M; and H--1000 .mu.M.
[0098] FIG. 4. Inhibition of infection of Jurkat cells by
pre-incubation of X4 HIV-1.sub.IIIB with the carbohydrate-lipid
construct designated Gb.sub.3-sp3-Ad-DOPE (I) (p24 pg/mL)(r=3):
A--Control; B--50 .mu.M; C--100 .mu.M; D--200 .mu.M; E--400 .mu.M;
F--600 .mu.M; G--800 .mu.M; and H--1000 .mu.M.
[0099] FIG. 5. Inhibition of infection of peripheral blood
mononuclear cells by pre-incubation of R5 HIV-1.sub.Ba-L with the
carbohydrate-lipid construct designated Gb.sub.3-sp2-Ad-DOPE (III)
(p24 pg/mL) (r=4).
[0100] FIG. 6. Inhibition of infection of peripheral blood
mononuclear cells by pre-incubation of X4 HIV-1.sub.IIIB with the
carbohydrate-lipid construct designated Gb.sub.3-sp2-Ad-DOPE (III)
(p24 pg/mL)(r=4).
[0101] FIG. 7. Infection of Jurkat cells by pseudoenvelope-typed
VSV-G/NL4-3lucHIV-1 (luciferase assay).
[0102] FIG. 8. Infection of NIH3T3 cells by pseudoenvelope-typed
VSV-G/NL4-3lucHIV-1 (luciferase assay).
[0103] FIG. 9. Infection of (a) Jurkat cells and (b) NIH3T3 cells
by pseudoenvelope-typed VSV-G/NL4-3lucHIV-1 (luciferase assay).
[0104] FIG. 10. Infection of (a) NIH3T3 cells and (b) Jurkat cells
by pseudoenvelope-typed VSV-G/NL4-3lucHIV-1 (PCR).
[0105] FIG. 11. Inhibition of infection of rectal mucosa by VSV/HIV
by application of a carbopol-based gel containing 3 mM the
carbohydrate-lipid construct designated Gb3-sp2-Ad-DOPE (III)(copy
number HIV-1 cDNA) (n=4).
[0106] FIG. 12. Inhibition of infection of vaginal mucosa by
VSV/HIV by application of a carbopol-based gel containing 3 mM the
carbohydrate-lipid construct designated Gb.sub.3-sp2-Ad-DOPE
(III)(copy number HIV-1 cDNA) (n=4).
[0107] FIG. 13. Inhibition of infection of rectal mucosa by VSV/HIV
by direct application of a 3 mM solution of the carbohydrate-lipid
construct designated Gb.sub.3-sp2-Ad-DOPE (III)(copy number HIV-1
cDNA)(n=4).
[0108] FIG. 14. Inhibition of infection of vaginal mucosa by
VSV/HIV by direct application of a 3 mM solution of the
carbohydrate-lipid construct designated Gb.sub.3-sp2-Ad-DOPE (III)
(copy number HIV-1 cDNA)(n=4).
DETAILED DESCRIPTION
[0109] The specification accompanying international application no.
PCT/NZ2005/000052 (publication no. WO 2005/090368) describes the
preparation and use of water soluble carbohydrate-lipid constructs.
In one example of the use of these constructs, qualitative and
quantitative changes in the surface antigen expression of red blood
cells (RBCs) is effected to provide quality control cells (e.g.
SECURACELL.TM.) for use in validation of blood grouping.
[0110] Naturally occurring glycoconjugates, such as GSLs are not
readily dispersible in water. Furthermore, it has been recognized
that isolated GSLs do not always retain the binding characteristics
of the membrane-bound glycolipid. In fact it is stated in the
specification accompanying international application no.
PCT/CA97/00877 (publication no. WO 98/23627) that solubilised GSLs
may have little or no binding affinity for compounds which bind
strongly to the membrane bound GSL.
[0111] Mylvaganam and Lingwood (1999d) stated in the context of
binding between the GSL globotriaosyl ceramide (Gb.sub.3) and the
bacterial toxin verotoxin that the reduction in binding affinity
may be attributed to conformational changes influenced by the
aglycone moiety. When incorporated in the plasma membrane
conformational changes (favourable orientations) of the glycone
moiety may be restricted by the plane of the membrane. The
development of water soluble glycolipid mimics was pursued
resulting in adamantyl conjugates which retained affinity for the
verotoxin receptor.
[0112] The carbohydrate-lipid constructs described in the
specification accompanying international application no.
PCT/NZ2005/000052 are dispersible in water and spontaneously
incorporate into cell membranes as demonstrated by their use in the
preparation of quality control cells.
[0113] The present invention provides selected carbohydrate-lipid
constructs that are glycolipid mimics, but are dispersible in
aqueous or biocompatible media. The constructs may therefore be
used in methods of preventing infection of cells by viruses in
vivo.
[0114] The selected carbohydrate-lipid constructs are of the
general formula F-S.sub.1-S.sub.2-L where the alkylglycoside
portion (F-S.sub.1) is selected to provide a ligand for a receptor
expressed by a virus, and the spacer portion (S.sub.1-S.sub.2) is
selected to provide a dispersible construct.
[0115] The constructs may function to inhibit both: [0116] 1.
natural ligand-receptor binding (including "multivalent" binding
(Schengrund (2003)); and [0117] 2. post-binding events essential to
infection of the target host cell and subsequent replication of the
virus.
[0118] As noted by Lund et al (2006) HIV targeting of CD4 and
chemokine co-receptor expressing lymphoid and monocytic cells has
long been appreciated as the major mechanism of HIV-host cell
interaction.
[0119] The gp120 receptor has also been shown to have an affinity
in vitro for a number of GSLs including galactosyl ceramide,
sulphogalactosyl ceramide and GM3 ganglioside (Feng et al (1996);
Bhat et al (1993); Fantini et al (1998)). This binding affinity is
characterized at least in part by the nature of the carbohydrate
moiety (glycotope) of the GSL ligand. The receptor-GSL binding
facilitates a post-CD4 binding event to allow the host cell entry
of diverse HIV strains (Nehete et al (2002)).
[0120] Whilst not wishing to be bound by theory it is believed that
inhibiting the receptor-GSL binding event with a water soluble
carbohydrate-lipid construct will inhibit host cell entry and viral
infection of the cells. Furthermore, it is believed that inhibiting
the post-CD4 binding event in situ, i.e. at the co-receptor
expressing surface of lymphoid and monocytic cells will promote
clearance of virus from an infected subject.
[0121] The in situ inhibition of the post-CD4 binding event may
occur when the water soluble carbohydrate-lipid construct is
incorporated into the cell membrane of the lymphoid and monocytic
cells. The formation of carbohydrate-lipid construct enriched lipid
microdomains on the host cell surface may be central to both
inhibiting viral infection and promoting clearance of virus from an
infected subject.
[0122] The methods of the invention may be effective against a
plurality of types of HIV, including types X4 and R5). The ability
of the carbohydrate-lipid constructs to inhibit infection of cells
by type R5 HIV-1 is of particular significance as this is a strain
of virus that initially infects susceptible subjects.
[0123] The carbohydrate-lipid constructs selected for use in the
methods of the invention are water soluble constructs that will
partition, i.e. incorporate, into cell membranes. Furthermore, the
preparation of these synthetic constructs excludes the use of
substrates or reagents derived from zoological sources. The
carbohydrate-lipid constructs therefore provide advantages over
semi-synthetic water soluble glycolipid mimics such as the
adamantylGb.sub.3 conjugates.
[0124] A number of receptor binding carbohydrate-lipid constructs
may be effective to inhibit infection or promote clearance of virus
from infected subjects. In addition to carbohydrate-lipid
constructs including a Gb.sub.3 carbohydrate moiety, constructs
including the glycotope of the GM3 carbohydrate moiety may also
prove effective inhibitors of HIV infection and promote clearance
of the virus from an infected subject. Methods comprising the
administration of two or more water soluble carbohydrate-lipid
constructs are contemplated.
[0125] The use of the carbohydrate-lipid constructs in the methods
of the invention is believed to be particularly advantageous
because of the ability of the constructs to incorporate
non-specifically into the membranes of cells in vivo. The
non-specific modification of cells in vivo may permit multivalent
binding and the adherence of the virus to cells in which the virus
is unable to replicate.
[0126] Adherence to the cell surface of a cell via the carbohydrate
portion of the carbohydrate-lipid construct may also result in the
virus being trapped at the cell surface (cf. Asher et al (2005)).
The ability of the immune system to recognise and respond to the
presence of virus may therefore be augmented.
[0127] Although discussed with reference to the prevention and
treatment of subjects with HIV infection, it will be recognised
that a number of viral infections are initiated by the adherence of
the virus to carbohydrates expressed at the surface of cells.
[0128] Schengrund (2003) and others have reviewed the development
of saccharides as pharmacologic agents. As noted by this author,
where it is determined that the expression of glycosphingolipids is
necessary for infection (Fantini et al (1993); Hanada (2005);
Karlsson (1995); Isa et al (1997); Matrosovicha et al (1997);
Miller-Podraza et al (2000); Suzuki (1994); Connor et al (1994);
Matrosovich et al (1999); Willoughby et al (1990)), the opportunity
arises to interfere with the adherence of the virus (e.g. influenza
virus, rotavirus) to the surface of target cells.
[Followed by page 26]
EXPERIMENTAL
[0129] The carbohydrate-lipid constructs designated
Gb.sub.3-sp3-Ad-DOPE (I) and Gb.sub.3-sp2-Ad-DOPE (III) may be
prepared and characterized in accordance with the methods described
mutatis mutandis in the specification accompanying international
application number PCT/NZ2005/000052 (publication no. WO
2005/090368) and summarized in Schemes I, II and IV.
[0130] The carbohydrate-lipid construct designated
Gb.sub.3-sp2-Ad-DOPE (III) may also be prepared by the method
described below and summarized in Schemes III and IV.
[0131] Scheme I:
[0132] (a) Cl.sub.3CNN, DBU, CH.sub.2Cl.sub.2, -5.degree. C., 64%;
(b) Cl(CH.sub.2).sub.3OH, BF.sub.3*Et.sub.2O, MS-4A,
CH.sub.2Cl.sub.2, -5.degree. C., 65%; (c) NaN.sub.3, DSMO,
80.degree. C., 20 h, 91%; (d) i) NaOMe/MeOH, 80%, ii) DMT, p-TsOH,
DMF, 63%; (e) NaH, BnBr, 0.degree. C., DMF, 87%; (f) NaCNBH.sub.3,
HCl*Et.sub.2O, MS-3A, -5.degree. C., THF, 73%; (g) i) PPh.sub.3,
H.sub.2O, THF, ii) MeOCOCF.sub.3, Et.sub.3N, THF, 84%
[0133] Scheme II:
[0134] (a) Cl.sub.3CNN, K.sub.2CO.sub.3, CH.sub.2Cl.sub.2, 60%; (b)
TMSOTf, MS-4A, CH.sub.2Cl.sub.2, 72% (c) H.sub.2, 10% Pd/C, MeOH;
(d) Ac.sub.2O/Py, 90% (e) MeONa/MeOH (f) NaOH/H.sub.2O, 96%
[0135] Scheme III:
[0136] (a) Br.sub.2, CH.sub.2Cl.sub.2, +4.degree. C., 100%; (b)
AgOTf, MS-4A, CH.sub.2Cl.sub.2, 78%; (c)
MeONa/MeOH--CH.sub.2Cl.sub.2; (d) H.sub.2, 10% Pd/C, MeOH,
Boc.sub.2O, 70%; (e) CF.sub.3COOH (95%), 96%
[0137] Scheme IV:
[0138] (a), (b) DMF/CH.sub.2Cl.sub.2, Et.sub.3N, 90-95%
##STR00021##
##STR00022## ##STR00023##
##STR00024##
##STR00025##
Materials and Methods
[0139] TLC was performed on Silica gel 60 (Merck, Germany)
precoated plates. Spots were visualized by treating with 5% aqueous
orthophosphoric acid and subsequent heating to 150.degree. C. in
the case of carbohydrates or by soaking in ninhydrin solution (3
g/l in 30:1 (v/v) butanol-acetic acid) in the case of amines.
[0140] Column chromatography was carried out on Silica gel 60
(0.040-0.063 mm, Merck, Germany). Gel chromatography was performed
on Sephadex LH-20 (Pharmacia, Sweden). Solvents were removed in
vacuo at 30 to 40.degree. C.
[0141] All solvents were from KhimMed (Russia). Molecular sieves
(MS 3 .ANG. and 4 .ANG.), trimethylsilyl trifluoromethanesulfonate,
and triphenylphosphine were from Aldrich (Germany). All hydrides,
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), and trichloroacetonitrile
were from Merck (Germany).
[0142] Anhydrous tetrahydrofuran (THF) and diethyl ether
(Et.sub.2O) were obtained by distillation from lithium aluminium
hydride (H.sub.4AlLi). Dichloromethane for glycoside synthesis was
dried by distillation from phosphorous pentoxide and calcium
hydride, and stored over molecular sieves MS 4 .ANG.. Solid
reagents were dried for 2 h in vacuo (0.1 mm Hg) at 20 to
40.degree. C.
[0143] Deacetylation was performed according to Zemplen in
anhydrous methanol. The solution of the acetylated compound was
treated with 2 M sodium methylate in methanol up to pH 9. When the
reaction was completed, Na.sup.+ ions were removed with cation
exchange resin Dowex 50X-400 (H.sup.+) (Acros, Belgium). The
solution was concentrated in vacuo.
[0144] Optical rotation was measured on a Jasco DIP-360 digital
polarimeter at 25.degree. C.
[0145] Mass spectra were recorded on a Vision-2000 (Thermo
Bioanalysis, UK) MALDI-TOF mass spectrometer using dihydroxybenzoic
acid as a matrix.
[0146] .sup.1H NMR spectra were recorded on a Bruker WM
spectrometer (500 MHz) at 25.degree. C. Chemical shifts (.delta.,
ppm) were recorded relative to D.sub.2O (.delta.=4.750), CDCl.sub.3
(.delta.=7.270), and CD.sub.3OD (.delta.=3.500) as internal
standards. The values of coupling constants (Hz) are provided. The
signals in the .sup.1H NMR spectra were assigned by suppression of
spin-spin interaction (double resonance) and 2D-1H,1H-COSY
experiments.
Preparation of
(2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranosyl)-(1.fwdarw.4)-2,3,6-tri-
-O-acetyl-.alpha.-D-glucopyranosyl trichloroacetimidate (1)
[0147] Trichloroacetonitrile (12.1 ml, 121 mmol) and DBU (0.45 ml,
3 mmol) were added to a solution of 1a (7.68 g, 12.1 mmol) in dry
dichloromethane (150 ml) at -5.degree. C. The reaction mixture was
stirred at -5.degree. C. for 3.5 h and concentrated in vacuo.
[0148] Flash chromatography (2:1 to 1:2 (0.1% Et.sub.3N)
toluene-ethyl acetate) of the residue provided 1 (6.01 g, 63.9%) as
a light yellow foam, R.sub.f 0.55 (2:1 toluene-acetone).
[0149] .sup.1H NMR, CDCl.sub.3: 1.95-2.2 (7s, 21H, 7Ac), 4.49 (d,
1H, J.sub.1,2=8.07, H-1b), 4.91 (dd, 1H, J.sub.3,2=10.3,
J.sub.3,4=2.8, H-3b), 5.05 (dd, 1H, J.sub.2,1=3.5, J.sub.2,3=9.3,
H-2a), 5.12 (dd, 1H, J.sub.2,1=8.07, J.sub.2,3=10.3, H-2b), 5.32
(d, 1H, J.sub.4,3=3, J.sub.4,5<1, H-4b), 5.52 (t, 1H,
J.sub.3,2=J.sub.3,4=9.29, H-3a), 6.48 (d, 1H.sub.A. J.sub.1,2=3.5,
H-1a), 8.64 (s, 1H, HN.dbd.CCCl.sub.3).
Preparation of
3-chloropropyl-(2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranosyl)-(1.fwda-
rw.4)-2,3,6-tri-O-acetyl-.beta.-D-glucopyranoside (2)
[0150] A mixture of 2.94 g (3.8 mmol) of trichloroacetimidate 1,
0.66 ml (7.5 mmol) 3-chloropropanol, 50 ml dichloromethane, and 3 g
of molecular sieves MS 4 .ANG. was cooled to -5.degree. C. An 8%
solution of BF.sub.3.Et.sub.2O (0.4 mmol) in anhydrous
dichloromethane was added drop wise with stirring.
[0151] After 30 min, the reaction mixture was filtered, diluted
with chloroform (500 ml), and washed with water, saturated sodium
hydrocarbonate solution, and water to pH 7. The washed reaction
mixture was dried by filtration through a cotton layer and
concentrated in vacuo.
[0152] Column chromatography on Silica gel (elution with 2.5:1
(v/v) toluene-ethyl acetate) resulted in 1.75 g (65%) of lactose
derivative (2) as white foam. R.sub.f 0.54 (2:1 toluene-acetone),
R.sub.f 0.50 (4:2:1 hexane-chloroform-isopropanol), [.alpha.].sub.D
-4.degree. (c 1.0, CHCl.sub.3), m/z 712.2 (M.sup.+).
[0153] .sup.1H NMR, CDCl.sub.3: 1.95 (br. s, 5H, Ac, --CH.sub.2--),
2.0-2.2 (6s, 18H, 6Ac), 3.52 (m, 2H, --CH.sub.2Cl), 3.63 (m, 1H,
H-5a), 3.68 (m, 1H, OCHH--), 3.79 (t, 1H, J=9.3, H-4a), 3.88 (m,
1H, H-5b), 3.93-3.98 (m, 1H, OCHH--), 4.05-4.15 (m, 3H, H-6a',
H-6b, H-6b'), 4.45 (d, 2H, H-1a, H-1b, J.sub.2,1=7.83) 4.47 (m, 1H,
H-6a), 4.89 (dd, 1H, J.sub.2,3=9.3, J.sub.2,1=7.82, H-2a), 4.96
(dd, 1H, J.sub.3,2=10.5, J.sub.3,4=3.42, H-3b), 5.11 (dd, 1H,
J.sub.2,3=10.5, J.sub.2,1=7.83, H-2b), 5.21 (t, 1H, J=9.3, H-3a),
5.35 (dd, 1H, J.sub.4,3=3.42, J.sub.4,5<1).
Preparation of 3-azidopropyl
(2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranosyl)-(1.fwdarw.4)-2,3,6-tri-
-O-acetyl-.beta.-D-glucopyranoside (3)
[0154] A mixture of 2.15 g (3 mmol) of trichloropropylglycoside 2,
0.59 g (9 mmol) NaN.sub.3, and 30 ml DMSO was maintained at
80.degree. C. with stirring for 20 h. The mixture was then diluted
with chloroform (500 ml), washed with water (4.times.100 ml), dried
by filtration through a cotton layer, and concentrated in
vacuo.
[0155] Column chromatography on Silica gel (elution with 8:2:1
hexane-chloroform-isopropanol) resulted in 1.96 g (91%) of
glycoside (3) as a white foam, R.sub.f 0.54 (2:1 (v/v)
toluene-acetone), R.sub.f 0.50 (4:2:1 (v/v/v)
hexane-chloroform-isopropanol), [.alpha.].sub.D -5.40 (c 1.0,
CHCl.sub.3), m/z 718.8 (M.sup.+).
[0156] .sup.1H NMR, CDCl.sub.3: 1.85 (m, 2H, --CH.sub.2--),
1.98-2.2 (7s, 21H, 7Ac), 3.36 (m, 2H, --CH.sub.2N.sub.3), 3.61 (m,
2H, H-5a, OCHH--CH.sub.2--), 3.8 (t, 1H, J.sub.3,4=J.sub.4,5=9.29,
H-4a), 3.85-3.94 (m, 2H, OCHH--CH.sub.2; H-5b), 4.05-4.17 (m, 3H,
H-6a, H-6a', H-6b), 4.49 (d, 1H, J.sub.1,2=8.07, H-1a), 4.5 (m, 1H,
H-6b'), 4.51 (d, 1H, J.sub.1,2=8.07, H-1b), 4.9 (dd, 1H,
J.sub.2,1=8.07, J.sub.2,3=9.29, H-2a), 4.97 (dd, 1H,
J.sub.3,2=10.27, J.sub.3,4=3, H-3b), 5.12 (dd, 1H, J.sub.2,1=8.07,
J.sub.2,3=10.27, H-2b), 5.2 (t, 1H, J.sub.3,2=J.sub.3,4=9.29,
H-3a), 5.36 (dd, 1H, J.sub.4,3=3, J.sub.4,5<1).
Preparation of 3-azidopropyl
(4,6-O-benzylidene-.beta.-D-galactopyranosyl)-(1.fwdarw.4)-.beta.-D-gluco-
pyranoside (4)
[0157] The lactoside 3 (1.74 g, 2.4 mmol) was deacetylated
according to Zemplen and co-evaporated with toluene (2.times.30
ml). The residue was treated with .alpha.,.alpha.-dimethoxytoluene
(0.65 ml, 3.6 mmol) and p-toluenesulfonic acid (50 mg, to pH 3) in
DMF (20 ml) for 3 h. The reaction mixture was then quenched with
pyridine, concentrated, and co-evaporated with o-xylene.
[0158] Column chromatography on Silica gel (elution with 9:1 (v/v)
chloroform-isopropanol) and recrystallization (chloroform-methanol)
resulted in 0.756 mg (62%) of benzylidene derivative (4). R.sub.f
0.6 (5:1 chloroform-isopropanol), [.alpha.].sub.D -25.7.degree. (c
1.0, methanol), m/z 513.4 (M.sup.+).
[0159] .sup.1H NMR, CD.sub.3OD: 2.06 (m, 2H, --CH.sub.3--), 3.45
(dd, 1H, J.sub.2,1=J.sub.2,3'=9, H-2a), 3.61 (m, 1H, H-5a), 3.64
(m, 2H, --CH.sub.2N.sub.3), 3.74-3.9 (m, 6H, OCHH--; H-3a, H-4a;
H-2b, H-3b, H-5b), 4.08-4.18 (m, 3H, H-6, H-6a', OCHH--), 4.34-4.44
(m, 3H, H-6b, H-6b', H-4b), 4.5 (d, 1H, J.sub.1,2=7.9, H-1a), 4.68
(d, 1H, J.sub.1,2=8, H-1b), 5.82 (s, 1H, CHPh), 7.55-7.72 (m, 5H,
CHPh).
Preparation of 3-azidopropyl
(4,6-O-benzylidene-3-O-benzyl-.beta.-D-galactopyranosyl)-(1.fwdarw.4)-2,3-
,6-tri-O-benzyl-.beta.-D-glucopyranoside (5)
[0160] Sodium hydride in mineral oil (290 mg, 12 mmol) was slowly
added in 4 to 5 portions to a solution of 4 (726 mg, 1.5 mmol) in
DMF (15 ml) at 0.degree. C. with stirring. After 1 h, the ice bath
was removed and benzyl bromide was added drop wise. The mixture was
stirred overnight. 10 ml of methanol was then added. After 1 h, the
mixture was diluted with chloroform (500 ml), and washed with water
(3.times.200 ml), dried by filtration through a cotton layer,
concentrated, and co-evaporated in vacuo with o-xylene.
[0161] Column chromatography on Silica gel (elution with 10:1
toluene-ethyl acetate) resulted in 1.24 g (87%) of lactose
derivative 5 as white foam, R.sub.f 0.56 (5:3 (v/v) hexane-ethyl
acetate), [.alpha.].sub.D +10.8.degree. (c 1.0, CHCl.sub.3), m/z
963.8 (M.sup.+).
[0162] .sup.1H NMR, CDCl.sub.3: 1.85 (m, 2H, --CH.sub.2--), 2.91
(m, 1H, H-5b), 3.33 (m, 1H, H-5a), 3.34-3.42 (m, 4H, H-2a, H-3b,
--CH.sub.2N.sub.3), 3.55-3.62 (m, 2H, OCHH--; H-3a), 3.73 (dd, 1H,
J.sub.2,1=8, J.sub.2,3=10, H-2b), 3.92-3.97 (m, 2H, H-4a, OCHH--),
4.0 (br. d, 1H, J.sub.4,3=3.6, H-4b), 4.34 (d, 1H, J.sub.1,2=7.9,
H-1a), 4.42 (d, 1H, J.sub.1,2=8, H-1b), 5.43 (s, 1H, CH(Bd),
7.14-7.50 (m, 30H; Ph).
Preparation of 3-azidopropyl
(2,3,6-O-tri-O-benzyl-.beta.-D-galactopyranosyl)-(1.fwdarw.4)-2,3,6-tri-O-
-benzyl-.beta.-D-glucopyranoside (6)
[0163] Hydrogen chloride in diethyl ether was added to a mixture of
5 (1.24 g, 1.3 mmol), sodium cyanoborohydride (0.57 g, 9.1 mmol),
and freshly activated molecular sieves MS 3 .ANG. (33 g) in
anhydrous THF (20 ml) until the evolution of gas ceased.
[0164] The mixture was stirred for 2 h, diluted with chloroform
(300 ml), washed with water, saturated sodium hydrocarbonate
solution, and water to pH 7. The washed mixture was dried by
filtration through a cotton layer and concentrated in vacuo.
[0165] Column chromatography on Silica gel (elution with 20:1 to
7:3 (v/v) toluene-ethyl acetate) resulted in 0.91 g (65%) of
lactose derivative 6 as a white foam, R.sub.f 0.42 (9:1 (v/v)
toluene-acetone), [.alpha.].sub.D +17.80 (c 1.0, CHCl.sub.3), m/z
965.8 (M.sup.+).
[0166] .sup.1H NMR, CDCl.sub.3: 1.85 (m, 2H, --CH.sub.2--), 2.39
(d, 1H, J=2.2, OH), 4.04 (br. s, 1H, H-4b), 4.34 (d, 1H,
J.sub.1,2=7.9, H-1a), 4.42 (d, 1H, J.sub.1,2=8, H-1b), 7.14-7.50
(m, 30H, Ph).
[0167] .sup.1H NMR of acetylated analytical probe 6a, CDCl.sub.3:
1.85 (m, 2H, --CH.sub.2--), 4.34 (d, 1H, J.sub.1,2=7.9, H-1a), 4.42
(d, 1H, J.sub.1,2=8, H-1b), 5.5 (br. d, 1H, J.sub.4,3=3.43, H-4b),
7.14-7.50 (m, 30H, Ph).
Preparation of 3-trifluoroacetamidopropyl
(2,3,6-O-tri-O-benzyl-.beta.-D-galactopyranosyl)-(1.fwdarw.4)-2,3,6-tri-O-
-benzyl-.beta.-D-glucopyranoside (7)
[0168] A mixture of derivative 6 (0.914 g, 0.94 mmol),
triphenylphosphine (0.5 g, 1.9 mmol) and THF (10 ml) was stirred
for 0.5 h, 100 .mu.l of water added, and the mixture stirred
overnight. The reaction mixture was then concentrated and
co-evaporated with methanol. The residue was dissolved in methanol
(15 ml) and triethylamine (30 .mu.l) and methyl trifluoroacetate
(0.48 ml, 4.7 mmol) added. The solution was held for 30 min and
then concentrated.
[0169] Column chromatography on Silica gel (elution with 5:1 to 1:1
(v/v) hexane-acetone) resulted in 0.87 g (84%) of lactose
derivative 7 as white foam, R.sub.f 0.49 (9:1 (v/v)
hexane-acetone), [.alpha.].sub.D-17.degree. (c 1.0, CHCl.sub.3),
m/z 1060.1 (M.sup.++Na).
[0170] .sup.1H NMR, CDCl.sub.3: 1.88 (m, 2H, --CH.sub.2--), 2.40
(br. s, 1H, OH), 4.05 (br. s, 1H, H-4b), 4.36 (d, 1H,
J.sub.1,2=7.8, H-1a), 4.40 (d, 1H, J.sub.1,2=7.6, H-1b), 7.10-7.35
(m, 30H, Ph).
Preparation of 2,3,4,6-tetra-O-benzyl-.beta.-D-galactopyranosyl
trichloroacetimidate (9)
[0171] A mixture of galactose derivative 8 (2 g, 3.65 mmol),
trichloroacetonitrile (1.75 ml, 17.55 mmol), anhydrous potassium
carbonate (2 g, 14.6 mmol), and dichloromethane (4 ml) was stirred
for 22 h at room temperature under argon. The mixture was then
filtered through a Celite layer and concentrated in vacuo. Column
chromatography on Silica gel (elution with 4:1 (v/v) hexane-ethyl
acetate (1% Et.sub.3N) resulted in 1.5 g (60%) of 9 as white foam,
R.sub.f 0.47 (7:3 (v/v) hexane-ethyl acetate containing 1%
Et.sub.3N) and 0.46 g (0.8 mmol, 23%) of the starting derivative 8,
R.sub.f 0.27 (7:3 (v/v) hexane-ethyl acetate containing 1%
Et.sub.3N).
[0172] .sup.1H NMR (CDCl.sub.3): 3.60-3.70 (m, 3H, H-3, H-6, H-6'),
3.75 (t, 1H, J.sub.5,6=6.30, H-5), 3.98 (d, 1H, J.sub.4,3=2.19,
H-4), 4.08 (dd, 1H, J.sub.2,3=9.73, J.sub.2,1=7.95, H-2), 4.42 and
4.47 (ABq, 2H, J=12.00, PhCH.sub.2), 4.63 and 4.95 (ABq, 2H,
J=11.51, PhCH.sub.2), 4.72 (s, 2H, PhCH.sub.2), 4.80 and 4.90 (ABq,
2H, J=10.95, PhCH.sub.2), 5.74 (d, 1H, J.sub.1,2=7.95, H-1),
7.22-7.35 (m, 20H, ArH), 8.62 (s, 1H, NH).
Preparation of 3-trifluoroacetamidopropyl
(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-(1.fwdarw.4)-(2,3,6-t-
ri-O-benzyl-.beta.-D-galactopyranosyl)-(1.fwdarw.4)-2,3,6-tri-O-benzyl-.be-
ta.-D-glucopyranoside (10)
[0173] A mixture of lactose derivative 7 (158 mg, 0.153 mmol),
trichloroacetimidate 9 (120 mg, 0.175 mmol), molecular sieves MS 4
.ANG. (0.5 g), and dichloromethane (5 ml) was stirred for 30 min at
room temperature under argon. 0.1 ml of a 1% (v/v) solution of
trimethylsilyl trifluoromethanesulfonate in dichloromethane was
then added. After 2 h, another 50 mg (0.073 mmol)
trichloroacetimidate 9 and 30 .mu.l of a 1% (v/v) solution of
trimethylsilyl trifluoromethanesulfonate in dichloromethane were
added. The reaction mixture was stirred overnight at +4.degree. C.,
quenched with triethylamine (5 .mu.l), filtered, and concentrated
in vacuo.
[0174] Column chromatography on Silica gel (elution with 12:1 to
1:1 (v/v) toluene-ethyl acetate) resulted in 170 mg (72%) of
trisaccharide 10; R.sub.f 0.56 (4:1 (v/v) toluene-ethyl acetate);
[.alpha.].sub.D +30.8.degree. (c 1.0, CHCl.sub.3).
[0175] .sup.1H NMR, CDCl.sub.3: 1.78-1.89 (m, 2H, --CH.sub.2--),
4.34 (d, 1H, J.sub.1,2=7.8, H-1a), 4.43 (d, 1H, J.sub.1,2=7.4,
H-1b), 5.06 (d, 1H, J.sub.1,2=3.0, H-1c), 7.14-7.48 (m, 50H,
Ph).
Preparation of 3-trifluoroacetamidopropyl
(2,3,4,6-tetra-O-acetyl-.alpha.-D-galactopyranosyl)-(1.fwdarw.4)-(2,3,6-t-
ri-O-acetyl-.beta.-D-galactopyranosyl)-(1.fwdarw.4)-2,3,6-tri-O-acetyl-.be-
ta.-D-glucopyranoside (11)
[0176] The catalyst 10% Pd/C (10 mg) was added to a solution of the
protected oligosaccharide 10 (73 mg, 0.047 mmol) in methanol (7
ml), the mixture degassed, and the flask filled with hydrogen. The
reaction mixture was stirred for 1 h, filtered off from the
catalyst through a Celite layer, and concentrated in vacuo. The dry
residue was dissolved in pyridine (2 ml), acetic anhydride (1 ml)
added, and the mixture held for 3 h. The solvents were then
evaporated and residue co-evaporated with toluene (4.times.2
ml).
[0177] Column chromatography on Silica gel (elution with 2:1
hexane-acetone) resulted in 43.5 mg (90%) of trisaccharide 11 as a
white foam, R.sub.f 0.52 (2:1 hexane-acetone), [.alpha.].sub.D
+30.40 (c 1.0, CHCl.sub.3).
[0178] .sup.1H NMR, CDCl.sub.3: 1.87 (2H, m, CH.sub.2); 1.99, 2.05,
2.05, 2.06, 2.07, 2.07, 2.09, 2.09, 2.12, and 2.14 (10.times.3H, 10
s, 10 Ac); 3.37 and 3.52 (2.times.1H, 2 m, 2 CHN); 3.63 (1H, ddd,
J.sub.4,5=9.8, J.sub.5,6=4.9, J.sub.5,6=2.0, H-5a); 3.72 (1H, m,
OCH); 3.77 (1H, ddd.apprxeq.br. T, J.sub.4,5<1, J.sub.5,6=6.8,
J.sub.5,6=6.1, H-5b); 3.79 (1H, dd, J.sub.3,4=9.3, J.sub.4,5=9.8,
H-4a); 3.87 (1H, m, OCH); 4.02 (1H, dd.apprxeq.br. d,
J.sub.3,4=2.5, J.sub.4,5<1, H-4b); 4.09 (1H, dd, J.sub.5,6=4.9,
J.sub.6,6=12.0, H-6a); 4.12 (1H, dd, J.sub.5,6=5.6,
J.sub.6,6'=10.8, H-6c); 4.14 (1H, dd, J.sub.5,6=6.8,
J.sub.6,6'=11.0, H-6b); 4.17 (1H, dd, J.sub.5,6'=8.6,
J.sub.6,6'=10.8, H-6'c); 4.45 (1H, dd, J.sub.5,6'=6.1,
J.sub.6,6'=11.0, H-6'b); 4.49 (1H, ddd .quadrature. br. T,
J.sub.4,5<1, J.sub.5,6=5.6, J.sub.5,6'=8.6, H-5c); 4.50 (1H, d,
J.sub.1,2=7.8, H-1a); 4.55 (1H, d, J.sub.1,2=7.8, H-1b); 4.59 (1H,
dd, J.sub.5,6'=2.0, J.sub.6,6'=12.0, H-6'a); 4.76 (1H, dd,
J.sub.2,3=10.8, J.sub.3,4=2.5, H-3b); 4.86 (1H, dd, J.sub.1,2=8.1,
J.sub.2,3=9.5, H-2a); 4.10 (1H, d, J.sub.1,2=3.4, H-1c); 5.12 (1H,
dd, J.sub.1,2=7.8, J.sub.2,3=10.8, H-2b); 5.19 (1H, dd,
J.sub.1,2=3.4, J.sub.2,3=11.0, H-2c); 5.22 (1H, dd.apprxeq.T,
J.sub.2,3=9.5, J.sub.3,4=9.3, H-3a); 5.40 (1H, dd, J.sub.2,3=11.0,
J.sub.3,4=3.4, H-3c); 5.59 (1H, dd.apprxeq.br. d, J.sub.3,4=3.4,
J.sub.4,5<1, H-4c); 7.09 (1H, m, NHCOCF.sub.3).
Preparation of 3-aminopropyl
.alpha.-D-galactopyranosyl-(1.fwdarw.4)-.beta.-D-galactopyranosyl-(1.fwda-
rw.4)-.beta.-D-glucopyranoside (Gb.sub.3-sp3) (12)
[0179] Sodium methylate (30 .mu.l of 2 M solution in methanol) was
added to a solution of trisaccharide (11) (43 mg, 0.042 mmol) in
anhydrous methanol (3 ml) and held for 2 h. The solution was then
concentrated in vacuo, water (3 ml) added, and the mixture held for
3 h. The mixture was then applied to a column (10.times.50 mm) with
Dowex 50X4-400 (H.sup.+) cation exchange resin.
[0180] The target compound was eluted with 1 M aqueous ammonia and
the eluant concentrated in vacuo. Lyophilization from water
provided trisaccharide 12 (23 mg, quant.) as a colorless powder.
R.sub.f 0.3 (100:10:10:10:2 (v/v/v/v/v)
ethanol-n-butanol-pyridine-water-acetic acid), [.alpha.].sub.D
+42.degree. (c 1; water), m/z 584.9 (M.sup.++Na).
[0181] .sup.1H NMR, D.sub.2O: 1.98-2.05 (m, 2H, --CH.sub.2--), 3.17
(m, 2H, --CH.sub.2NH.sub.2), 3.33-3.35 (m, 1H, H-2a), 4.36 (m, 1H,
H-5c), 4.53 (d, 2H, J=7.8, H-1a, H-1b), 4.97 (d, 1H,
J.sub.1,2=3.67, H-1c).
Preparation of 2-azidoethyl (3,4-di-O-acetyl-2,
6-di-O-benzyl-.alpha.-D-galactopyranosyl)-(1.fwdarw.4)-(2,
3,6-tri-O-benzyl-.beta.-D-galactopyranosyl)-(1.fwdarw.4)-2,3,6-tri-O-benz-
yl-.beta.-D-glucopyranoside (13)
[0182] To the solution of ethyl
3,4-di-O-acetyl-2,6-di-O-benzyl-1-thio-.beta.-D-galactopyranoside
(550 mg, 1.11 mmol) in dichlorometnane (10 ml) was added Br.sub.2
(57 .mu.l, 1.11 mmol). The mixture was held for 20 min at room
temperature, then concentrated in vacuo at room temperature and
co-evaporated with anhydrous benzene (3.times.30 ml). The crude
3,4-di-O-acetyl-2,6-di-O-benzyl-.alpha.-D-galactopyranosylbromide
(14) was used for glycosylation without purification.
[0183] The mixture of lactose derivative 15 (Sun et al (2006)) (500
mg, 0.525 mmol), 1,1,3,3-tetramethylurea (300 .mu.l), molecular
sieves MS 4 .ANG. (1 g), and dichloromethane (25 ml) was stirred
for 30 min at room temperature. Silver trifluoromethanesulfonate
(285 mg, 1.11 mmol), molecular sieves MS 4 .ANG. (0.5 g), and the
freshly prepared galactopyranosylbromide (14) in dichloromethane
(15 ml) were then added. The reaction mixture was stirred
overnight, filtered, and concentrated in vacuo.
[0184] Column chromatography on Silica gel (elution with 3:1 to 1:1
(v/v) hexane-ethyl acetate) resulted in 570 mg (79%) of
trisaccharide 13, R.sub.f 0.25 (2:1 (v/v) hexane-ethyl acetate);
[.alpha.].sub.D +32.degree. (c 0.8, CHCl.sub.3)
[0185] .sup.1H NMR, CDCl.sub.3: 1.88, 1.94 (2s, 2Ac), 3.00 (dd, 1H,
J.sub.5,6=4.9, J.sub.6',6=8.4, H-6a), 3.19 (dd, J.sub.1,2=8.5,
J.sub.2,3=8.9, H-2a), 3.30-3.36 (m, 2H, --CHHN.sub.3, H-6'a),
3.38-3.47 (m, 4H, H-5a, H-5b, H-2b, H-6b), 3.48-3.54 (m, 1H,
--CHHN.sub.3), 3.61 (dd, 1H, J.sub.2,3=8.9, J.sub.3,4=9.2, H-3a),
3.69-3.75 (m, 3H, H-6'b, H-6c, --OCHH--), 3.85 (dd, 1H,
J.sub.5,6=4.6, J.sub.6,6'=11.0, H-6c), 3.89 (dd, 1H, J.sub.1,2=3.4,
J.sub.2,3=10.8, H-2c), 3.95 (dd, 1H, J.sub.3,4=9.2, J.sub.4,5=9.5,
H-4a), 4.0-4.1 (m, 4H, --OCHH--, H-4b, CH.sub.2Ph), 4.25, 4.29,
4.32, 4.39 (4 d, 4.times.1H, J.sub.AB=12, 4-CHPh), 4.43 (d, 1H,
J.sub.1,2=7.6, H-1), 4.48 (d, 1H, J.sub.1,2=7.6, H-1), 4.54-4.62
(m, 5H, 4-CHPh, H-5c), 4.71-4.84 (m, 4H, 4-CHPh), 4.89, 4.91, and
5.09 (3 d, 3.times.1H, 3 4-CHPh), 5.15 (d, 1H, J.sub.1,2=3.0,
H-1c), 5.39 (dd, 1H, J.sub.2,3=10.8, J.sub.3,4=3.4, H-3c), 5.56
(dd, 1H, J.sub.3,4=3.4, J.sub.4,5=0.9, H-4c), 7.14-7.48 (m, 40H,
Ph).
Preparation of 2-aminoethyl
.alpha.-D-galactopyranosyl-(1.fwdarw.4)-.beta.-D-galactopyranosyl-(1.fwda-
rw.4)-.beta.-D-glucopyranoside (Gb.sub.3-sp2) (16)
[0186] Sodium methylate (100 .mu.l of 2 M solution in methanol) was
added to a suspension of trisaccharide (13) (500 mg, 0.363 mmol) in
anhydrous methanol (50 ml). The mixture was stirred overnight at
room temperature, quenched with acetic acid, and concentrated in
vacuo.
[0187] Column chromatography on Silica gel (elution with 2:1 to 1:1
(v/v) hexane-ethyl acetate) resulted in 470 mg of trisaccharide
(17), R.sub.f 0.5 (1:1 (v/v) hexane-ethyl acetate), [.alpha.].sub.D
+36.degree. (c 0.5, CHCl.sub.3).
[0188] To a solution of trisaccharide (17) and Boc.sub.2O ((150 mg,
0.91 mmol) in anhydrous methanol (50 ml) was added the catalyst 10%
Pd/C (500 mg). The mixture was degassed and the flask filled with
hydrogen. The reaction mixture was stirred for 3 h, filtered off
from the Pd/C, and concentrated in vacuo.
[0189] Column chromatography on Silica gel (elution with 6:5:1
(v/v/v) chloroform-ethanol-water) resulted in 160 mg (68%) of
trisaccharide 18 R.sub.f 0.3 (6:5:1 (v/v/v)
dichloromethane-ethanol-water). .sup.1H NMR, D.sub.2O: 1.45 (s, 9H,
(CH.sub.3).sub.3COCO--), 4.53 (d, 1H, J.sub.1,2=7.8, H-1b), 4.58
(d, 1H, J.sub.1,2=7.4, H-1b), 4.98 (d, 1H, J.sub.1,2=3.0,
H-1c).
[0190] The trisaccharide 18 was then treated with 95% CF.sub.3COOH
(5 ml, 10 min). Upon completion, the mixture was concentrated in
vacuo, co-evaporated with toluene, and applied to a column
(10.times.100 mm) of Dowex 50X4-400 (H') cation exchange resin. The
target compound was eluted with 1 M aqueous ammonia and the eluant
was concentrated in vacuo. Lyophilization from water provided
trisaccharide 16 (135, quant.) as a colorless powder. R.sub.f 0.35
(100:10:10:10:2 (v/v/v/v/v) ethanol-n-butanol-pyridine-water-acetic
acid), [.alpha.].sub.D +250 (c 0.2; water).
[0191] .sup.1H NMR, D.sub.2O: 3.32 (m, 2H, --CH.sub.2NH.sub.2),
3.40-3.45 (m, 1H, H-2a), 3.63 (dd, 1H, J.sub.1,2=7.9,
J.sub.2,3=10.3, H-2b), 3.66-3.78 (m, 5H, H-5a, H-3a, H-4a, H-6c,
H-6'c), 3.8 (dd, 1H, J.sub.3,4=3.1, J.sub.3,2=10.3, H-3b), 3.84 (m,
2H, J.sub.5,6=4.4, J.sub.5,6'=7.9, H-5b), 3.88-3.92 (m, 3H, H-2c,
H-6b, --OCHH--), 3.96 (dd, 1H, J.sub.3,4=3.3, J.sub.3,2=10.3,
H-3c), 3.98-4.03 (m, 2H, H-6a, H-6'b), 4.06 (dd, 1H, J.sub.5,6=2.2,
J.sub.6,6'=12.3, H-6'a), 4.08 (dd, 1H, J.sub.3,4=3.3,
J.sub.4,5=0.9, H-4c), 4.09 (d, 1H, J.sub.3,4=3.1, H-4b), 4.17-4.21
(m, 1H, --OCHH--), 4.41 (m, 1H, H-5c), 4.56 (d, 1H, J=7.9, H-1b),
4.60 (d, 1H, J=8.1, H-1a), 5.00 (d, 1H, J.sub.1,2=3.9, H-1c).
Preparation of activated
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (Ad-DOPE)
(19)
[0192] A solution of DOPE (40 .mu.mol) in chloroform (1.5 ml) and
triethylamine (7 .mu.l) were added to a solution of
bis(N-hydroxysuccinimidyl) adipate (200 .mu.mol) in dry
N,N-dimethylformamide (1.5 ml). The mixture was kept for 2 h at
room temperature, quenched with acetic acid, and partially
concentrated in vacuo.
[0193] Gel filtration on Sephadex LH-20 (1:1 (v/v)
chloroform-methanol containing 0.2% acetic acid) of the residue
yielded the activated lipid (37 mg, 95%) as a colorless syrup;
R.sub.f 0.5 (6:3:0.5 (v/v/v) chloroform-methanol-water).
[0194] .sup.1H NMR (2:1 CDCl.sub.3-CD.sub.3OD): 5.5 [m, 4H,
2.times.(--CH.dbd.CH--)], 5.39 (m, 1H,
--OCH.sub.2--CHO--CH.sub.2O--), 4.58 (dd, 1H, J=3.67, J=11.98,
--CCOOHCH--CHO--CH.sub.2O--), 4.34 (dd, 1H, J=6.61, J=11.98,
--CCOOHCH--CHO--CH.sub.2O--), 4.26 (m, 2H,
PO--CH.sub.2--CH.sub.2--NH.sub.2), 4.18 (m, 2H, --CH.sub.2--OP),
3.62 (m, 2H, PO--CH.sub.2--CH.sub.2--NH.sub.2), 3.00 (s, 4H,
ONSuc), 2.8 (m, 2H, --CH.sub.2--CO (Ad), 2.50 [m, 4H,
2.times.(--CH.sub.2--CO)], 2.42 [m, 2H, --CH.sub.2--CO (Ad)], 2.17
[m, 8H, 2.times.(--CH.sub.2--CH.dbd.CH--CH.sub.2--)], 1.93 (m, 4H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 1.78 [m, 4H,
2.times.(COCH.sub.2CH.sub.2--)], 1,43, 1.47 (2 br. s, 40H, 20
CH.sub.2), 1.04 (m, 6H, 2 CH.sub.3).
Preparation of Gb.sub.3-sp3-Ad-DOPE (I) and Gb.sub.3-sp2-Ad-DOPE
(III)
[0195] To a solution of activated DOPE (19) (10.5 .mu.mol) in
dichloromethane (300 .mu.l) was added (12) or (16) (10 .mu.mol) in
DMF (0.5 ml) and then triethylamine (3 .mu.l). The mixture was kept
for 2 h at room temperature. Gel filtration on Sephadex LH-20 (1:1
(v/v) chloroform-methanol) of the mixture yielded (I) or (III)
(90-95%).
[0196] Gb.sub.3-sp3-Ad-DOPE (I) was determined to have a molecular
weight (MW) of 1415.7 and .sup.1H NMR (CDCl.sub.3/CD.sub.3OD, 2:1),
.delta.: 5.5 (m, 4H, 2.times.(--CH.dbd.CH--), 5.43-5.39 (m, 1H,
--OCH.sub.2--CHO--CH.sub.2O--), 5.13 (d, 1H, J=3.6, H-1 Gal),
4.61-4.58 (m, 2H; J=7.1, H-1 (Gal); J=3.7, J=12.1,
--CCOOHCH--CHO--CH.sub.2O--), 4.46 (d, J=7.9, H-1 Gal), 2.53-2.48
(m, 4H, 2.times.(--CH.sub.2--CO), 2.42-2.37 (m, 4H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.21-2.16 (m, 8H,
2.times.(--CH.sub.2--CH.dbd.CH--CH.sub.2--), 2.00-1.95 (m, 2H,
O--CH.sub.2CH.sub.2CH.sub.2--NH), 1.78 (m, 8H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO and
2.times.(COCH.sub.2CH.sub.2--), 1.50, 1.47 (2 bs, 40H, 20
CH.sub.2), 1.05 (m, 6H, 2 CH.sub.3) (FIG. 1).
[0197] Gb.sub.3-sp2-Ad-DOPE (III) was determined to have a
molecular weight (MW) of 1415.7 and .sup.1H NMR
(CDCl.sub.3/CD.sub.3OD, 2:1), .delta.: 5.5 (m, 4H,
2.times.(--CH.dbd.CH--), 5.43-5.39 (m, 1H,
--OCH.sub.2--CHO--CH.sub.2O--), 5.13 (d, 1H, J=3.6, H-1 Gal),
4.61-4.58 (m, 2H; J=7.1, H-1 (Gal); J=3.7, J=12.1,
--CCOOHCH--CHO--CH.sub.2O--), 4.46 (d, J=7.9, H-1 Gal), 2.53-2.48
(m, 4H, 2.times.(--CH.sub.2--CO), 2.42-2.37 (m, 4H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.21-2.16 (m, 8H,
2.times.(--CH.sub.2--CH.dbd.CH--CH.sub.2--), 1.78 (m, 8H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO and
2.times.(COCH.sub.2CH.sub.2--), 1.50, 1.47 (2 bs, 40H, 20
CH.sub.2), 1.05 (m, 6H, 2 CH.sub.3).
In Vitro Studies
[0198] Inhibition of Infection of Jurkat Cells with a
Pseudoenvelope-Typed HIV
[0199] The ability of Gb.sub.3-sp3-Ad-DOPE (I) to inhibit infection
by a pseudoenvelope-typed HIV having an outer envelope derived from
the mouse vesticular stomatitis virus (VSV) and having an ENV-minus
modified HIV genome derived from the X4 HIV-1 NL4-3 virus was
evaluated.
[0200] The methods described mutatis mutandis in the publication of
Lund et al. (2006) were used to evaluate the ability of 250 .mu.M
Gb.sub.3-sp3-Ad-DOPE (I) to inhibit infection of Jurkat cells by
the pseudoenvelope-typed HIV. The carbohydrate-lipid construct was
demonstrated to inhibit infection by the VSV pseudoenvelope-typed
virus (FIG. 2).
[0201] The ability of AZT to inhibit infection was used as a
positive control.
Dose Response for Inhibition of Infection with a
Pseudoenvelope-Typed HIV
[0202] HIV-1.sub.IIIB, an X4 type, T-cell-tropic HIV virus, was
sourced from the National Institutes of Health AIDS Research and
Reference Reagent Program. The virus was handled in a Level III
biocontainment facility. A multiplicity of infection (m.o.i) of 0.7
was used.
[0203] Gb.sub.3-sp3-Ad-DOPE (I) in powdered form was dissolved in
phosphate buffered saline to provide a stock solution of 6 mM. The
stock solution was diluted to 2 mM to provide a working
concentration.
[0204] HIV-1.sub.IIIB was incubated with Gb.sub.3-sp3-Ad-DOPE (I)
at concentrations of 50 at 1000 .mu.M for 1 hour at 37.degree. C.
prior to incubation with Jurkat cells. Incubations were in a total
volume of 100 .mu.L.
[0205] A suspension of Jurkat cells at a density of
5.times.10.sup.5 per mL in 100 .mu.L complete RPMI1640 medium was
incubated with a solution of untreated or treated (pre-incubated
with Gb.sub.3-sp3-Ad-DOPE (I) virus for 1 hour at 37.degree. C.
[0206] Incubated cells were washed four times with phosphate
buffered saline without MgCl.sub.2/CaCl.sub.2 and then cultured in
2 mL of complete RPMI1640 medium for a total of 5 days. On days 0,
3, 4 and 5 500 .mu.L aliquots of culture supernatant were
taken.
[0207] Aliquots of culture supernatant were stored at -80.degree.
C. A determination of the level of HIV p24 core protein was
conducted by ELISA (Coulter) for the supernatant of the Day 4
supernatant.
[0208] Results for quadruplicate experiments (r=4) are presented in
Table 1 and FIG. 3.
[0209] Results for triplicate experiments (r=3) are presented in
Table 2 and FIG. 4.
Dose Response for Inhibition of Infection of Peripheral Blood
Mononuclear Cells with an R5 Type Monocyte-Tropic HIV Virus
[0210] HIV-1.sub.Ba-L, an R5 type, monocyte-tropic HIV virus, was
pre-incubated with Gb.sub.3-sp3-Ad-DOPE (I) at the concentrations
provided in FIG. 5 for 1 hour. PHA/IL-2-activated human-peripheral
blood mononuclear cells (PBMCs) obtained from a healthy volunteer
donor were then infected by incubation with the pre-treated virus
for 1 hour (n=4).
[0211] p24 antigen expression, a measure of productive HIV
infection, was monitored 12 days after infection.
Gb.sub.3-sp3-Ad-DOPE (I) was observed to inhibit infection by
HIV-1.sub.Ba-L at 400 .mu.M (p<0.05) with a half-maximal
inhibitory activity (IC.sub.50) of circa 200 .mu.M.
Dose Response for Inhibition of Infection of Jurkat C Cells with X4
HIV-1 Virus
[0212] HIV-1.sub.IIIB was pre-incubated with Gb.sub.3-sp3-Ad-DOPE
(I) at the concentrations provided in FIG. 6 for 1 hour. Jurkat C
cells were then infected by incubation with the pre-treated virus
for 1 hour (n=4).
[0213] p24 antigen expression, a measure of productive HIV
infection, was monitored 5 days after infection.
Gb.sub.3-sp3-Ad-DOPE (I) was observed to inhibit infection by
HIV-1.sub.IIIB at 400 .mu.M (p<0.001) with a half-maximal
inhibitory activity (IC.sub.50) of circa 200 .mu.M.
In Vivo Studies
Mouse Model
[0214] A mouse model of HIV infection of the rectal and vaginal
mucosa was used for in vivo evaluation of Gb.sub.3-sp2-Ad-DOPE
(III). A pseudoenvelope-typed replication-deficient VSV-G/NL4-3luc
HIV-1 virus (VSV/HIV) approved for use in level 2 biocontainment
was used to validate the mouse model.
[0215] Replication deficient, VSV-G enveloped HIV-1 luciferase
containing recombinant virions were prepared by co-transfection of
293T cells with plasmids containing the VSV-G envelope and the
HIV-1 genome lacking env and with the luciferase gene inserted into
the nef gene.
[0216] 10 .mu.g of plasmid containing the envelope and 15 .mu.g of
plasmid containing the HIV genome were mixed and added drop wise to
2.5.times.10.sup.6 293T cells plated 24 hours previously. Plates
were incubated for 72 hours at 37.degree. C.
[0217] Viral supernatant was collected, centrifuged for 10 min,
filtered through a 0.45 .mu.m filter and ultracentrifuged in 8 mL
aliquots over 400 .mu.L 20% glucose for 1 hour at 19,000 rpm.
Pelleted virions were resuspended in 800 .mu.L TNE buffer and
stored at -80.degree. C. until further use. Virion content was
determined by p24 ELISA.
[0218] To determine infectivity of virus, 2.times.10.sup.5 Jurkat C
cells were plated in a 96 well tray in triplicate in 100 .mu.L
complete RPMI media lacking phenol red. 20 .mu.L volumes of virus
plus media up to a total volume of 200 .mu.L per well was then
added.
[0219] Cells were incubated for 48 hours, then lysed using Promega
cell culture lysis solution. 100 .mu.L of luciferase assay
substrate was added to 20 .mu.L of the lysed cells and luciferase
activity was measured using a luminometer.
[0220] To determine if the virus infected a mouse epithelial cell
line, 1.times.10.sup.6 NIH3T3 cells were infected with 25 and 75
.mu.L aliquots of virus and incubated in DMEM for 2 hours. DNA was
isolated and subjected to PCR as described below.
[0221] To determine if the virus infected mouse mucosal tissue,
male and female CD1 mice were challenged rectally and vaginally
with 25 .mu.L of virus administered via pipette to the rectum and
vaginal cavities of euthanized mice for 2 hours. Rectal and vaginal
tissue was then removed. DNA was isolated via the Qiagen DNEasy
Isolation Kit for 50 mg of tissue.
[0222] Primers used for PCR amplification of HIV cDNA were forward:
LTR 5'-GGGACTGGAAGGGCTAATTC-3' and reverse:
L15'-AGGCAAGCTTTATTGAGGCT TAAGC-3'. Primers used for nested PCR
were forward: L2 5'-CTGTGGATCTACCACACACA AGGCTAC-3' and reverse:
LTR U3 5'-CTCCCT GGAAAGTCCCCAGC-3'. Real time PCR was conducted
using the Roche LightCycler 2.0 using the FastStart DNA Master Plus
SYBR Green I Kit.
[0223] The pseudoenvelope-typed replication-deficient
VSV-G/NL4-3luc HIV-1 virus (VSV/HIV) was demonstrated to infect
both Jurkat and NIH3T3 cells by luciferase assay (FIGS. 7 and 8)
and PCR (FIGS. 9a and 9b).
[0224] The pseudoenvelope-typed replication-deficient
VSV-G/NL4-3luc HIV-1 virus (VSV/HIV) was demonstrated to infect
both rectal and vaginal mucosa (FIGS. 10a and 10b).
Inhibition of Rectal Infection by VSV/HIV by Gel Formulation of
Gb.sub.3-Sp3-Ad-DOPE (III)
[0225] A preliminary trial of a carbopol-based gel was performed. A
gel containing 3 mM Gb.sub.3-sp2-Ad-DOPE (III) was applied to
rectal mucosa of CD1 mice for 30 minutes before a one and one half
hour challenge with VSV/HIV. Tissue was removed and quick frozen in
liquid nitrogen. A gel containing only PBS was used as a control
(n=4).
[0226] DNA was isolated from the tissue using the Qiagen tissue
kit. Quantitative real time PCR (qPCR) using the Roche Lightcycler
was performed to determine copy number of HIV-1 genomes using
primers for the LTR region of HIV-1 and compared to a quantified
HIV-1 cDNA standard curve.
[0227] Detection of HIV cDNA copies indicated successful viral
entry and reverse transcription of the HIV genome (FIG. 11).
Inhibition of Vaginal Infection by VSV/HIV by Gel Formulation of
Gb.sub.3-Sp2-Ad-DOPE (III)
[0228] A preliminary trial of a carbopol-based gel was performed. A
gel containing 3 mM Gb.sub.3-sp2-Ad-DOPE (III) was applied to
vaginal mucosa of CD1 mice for 30 minutes before a one and one half
hour challenge with VSV/HIV. Tissue was removed and quick frozen in
liquid nitrogen. A gel containing only PBS was used as a control
(n=4).
[0229] DNA was isolated from the tissue using the Qiagen tissue
kit. Quantitative real time PCR (qPCR) using the Roche Lightcycler
was performed to determine copy number of HIV-1 genomes using
primers for the LTR region of HIV-1 and compared to a quantified
HIV-1 cDNA standard curve.
[0230] Detection of HIV cDNA copies indicated successful viral
entry and reverse transcription of the HIV genome (FIG. 12).
Inhibition of Rectal Infection by VSV/HIV Direct Application of
Gb.sub.3-Sp2-Ad-DOPE (III)
[0231] 3 mM Gb.sub.3-sp2-Ad-DOPE (III) was applied directly to
rectal mucosa of CD1 mice for 30 minutes before a one and one half
hour challenge with VSV/HIV. Tissue was removed and quick frozen in
liquid nitrogen (n=4).
[0232] DNA was isolated from the tissue using the Qiagen tissue
kit. Quantitative real time PCR (qPCR) using the Roche Lightcycler
was performed to determine copy number of HIV-1 genomes using
primers for the LTR region of HIV-1 and compared to a quantified
HIV-1 cDNA standard curve.
[0233] Detection of HIV cDNA copies indicated successful viral
entry and reverse transcription of the HIV genome (FIG. 13).
Inhibition of Vaginal Infection by VSV/HIV Direct Application of
Gb.sub.3-Sp2-Ad-DOPE (III)
[0234] 3 mM Gb.sub.3-sp2-Ad-DOPE (III) was applied directly to
vaginal mucosa of CD1 mice for 30 minutes before a one and one half
hour challenge with VSV/HIV. Tissue was removed and quick frozen in
liquid nitrogen (n=4).
[0235] DNA was isolated from the tissue using the Qiagen tissue
kit. Quantitative real time PCR (qPCR) using the Roche Lightcycler
was performed to determine copy number of HIV-1 genomes using
primers for the LTR region of HIV-1 and compared to a quantified
HIV-1 cDNA standard curve.
[0236] Detection of HIV cDNA copies indicated successful viral
entry and reverse transcription of the HIV genome (FIG. 14).
[0237] Although the invention has been described by way of examples
indicative of its utility in the treatment of human subjects it
should be appreciated that variations and modifications may be made
to the claimed methods with out departing from the scope of the
invention. It will be understood that for a non-specific
interaction, such as the interaction between the diacyl- or
dialkyl-glycerolipid portion of the carbohydrate-lipid constructs
and a membrane, structural and stereo-isomers of naturally
occurring lipids can be functionally equivalent.
[0238] Where known equivalents exist to specific features, such
equivalents are incorporated as if specifically referred to in this
specification. For example, it is contemplated that diacylglycerol
2-phosphate could be substituted for phosphatidate (diacylglycerol
3-phosphate) and that the absolute configuration of phosphatidate
could be either R or S.
[Followed by page 54]
TABLE-US-00001 TABLE 1 Inhibition of infection of Jurkat cells by
pre-incubation of X4 HIV-1 IIIB with carbohydrate- lipid construct
designated Gb.sub.3-sp3-Ad-DOPE (I) at the concentrations indicated
(r = 4). control 50 .mu.M 100 .mu.M 200 .mu.M 400 .mu.M 600 .mu.M
800 .mu.M 1000 .mu.M 1 52.438 235.054 200.097 140.632 89.202 1.008
1.008 1.209 2 229.629 220.79 224.205 14.267 2.213 1.008 0.205 1.008
3 50.027 211.147 237.264 76.345 83.175 0.205 3.62 1.611 4 203.513
47.415 28.531 221.393 2.816 0.807 128.578 0.205 mean 133.9018
178.6015 172.5243 113.1593 44.3515 0.757 33.35275 1.00825 SD
96.05684 88.00731 97.22216 88.70224 48.37245 0.380003 63.50024
0.591275 SEM 48.02842 44.00366 48.61108 44.35112 24.18623 0.190001
31.75012 0.295638
TABLE-US-00002 TABLE 2 Inhibition of infection of Jurkat cells by
pre-incubation of X4 HIV-1 IIIB with carbohydrate- lipid construct
designated Gb.sub.3-sp3-Ad-DOPE (I) at the concentrations indicated
(r = 3). control 50 .mu.M 100 .mu.M 200 .mu.M 400 .mu.M 600 .mu.M
800 .mu.M 1000 .mu.M 1 52.438 235.054 200.097 140.632 2.213 1.008
1.008 1.209 2 229.629 220.79 224.205 76.345 83.175 1.008 0.205
1.008 3 203.513 211.147 237.264 221.393 2.816 0.807 3.62 0.205 mean
161.86 222.3303 220.522 146.1233 29.40133 0.941 1.611 0.807333 SD
95.65768 12.0271 18.85523 72.67975 46.57034 0.116047 1.785371
0.531229 SEM 55.22799 6.944197 10.88607 41.96168 26.8874 0.067
1.0309 0.306705
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