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.

TECHNICAL FIELD

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 viral 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.

BACKGROUND ART

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.

Therapies to treat infection that target viruses may be limited in efficacy due to resistance and genetic variance of the virus.

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)).

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.

Lund et al (2006) have investigated the effect of the water soluble glycolipid mimic adamantylGb3 on HIV infection of cells in culture. In previous studies adamantylGb3 had been demonstrated to be a superior ligand for the receptor gp120 (Mahfoud et al (2002)).

A dose dependent inhibition of infection of Jurkat T cells by HIV-1 pre-incubated with adamantylGb3 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 adamantylGb3 treated virus remained non-host cell attached and virions could not be found within the Jurkat cells.

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 adamantylGb3. However, pre-incubation of cells with adamantylGb3 was ineffective.

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 adamantylGb3 within a cream might provide a topical ointment for the prevention of mucosal HIV infection.

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.

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.

These objects are to be read disjunctively with the object of to at least provide a useful choice.

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-S1-S2-L where: F is selected from the group consisting of glycotopes of ligands for one or more receptors expressed by the virus; S1-S2 is a spacer linking F to L; and L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids.

Preferably, the amount is effective to inhibit binding of the receptor expressed by the virus to a cell surface expressed ligand.

Preferably, the receptor is expressed by the human immunodeficiency virus (HIV).

S1-S2 is selected to provide a carbohydrate-lipid construct that is dispersible in water.

Preferably, S1 is a C2-5-aminoalkyl selected from the group consisting of: 2-aminoethyl; 3-aminopropyl; 4-aminobutyl; and 5-aminopentyl.

Preferably, S2 is selected from the group consisting of: —CO(CH2)3CO—; —CO(CH2)4CO— (adipate); and —CO(CH2)5CO—.

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).

Preferably, L is a glycerophospholipid and the construct includes the substructure:

where X is H or C, * is other than H and n is an integer 2 to 5.

More preferably, L is a glycerophospholipid and the construct includes the substructure:

where: X is H; R1 is a Cp-alkyl glycoside, R2 and R3 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 and cis-13-docsenal; n is 2 to 5; and p is 2 or

Most preferably, the glycoside is 1-O—(O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl (Gb3), n is 4 and p is 3.

In specific embodiments of the first aspect of the invention the carbohydrate-lipid construct has the structure:

designated Gb3-sp3-Ad-DOPE (I); the structure:

designated Gb3-sp3-Ad-DSPE (II); the structure:

designated Gb3-sp2-Ad-DOPE (III); or the structure:

designated Gb3-sp2-Ad-DSPE (IV).

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.

Preferably, the administering to the subject is to provide a concentration in the plasma of the subject of greater than 400 μM.

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.

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-S1-S2-L where: F is selected from the group consisting of glycotopes of ligands for one or more receptors expressed by the virus; S1-S2 is a spacer linking F to L; and L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids.

Preferably, the administering to the subject is by intravascular injection. More preferably, the administering is by intravenous injection.

Preferably, the amount is sufficient to promote partitioning of the carbohydrate-lipid construct into the membranes of cells of the vascular system.

Preferably, the administering to the subject is to provide an initial concentration in the plasma of the subject of greater than 400 μM.

Preferably, the receptor is expressed by the human immunodeficiency virus (HIV).

S1-S2 is selected to provide a carbohydrate-lipid construct that is dispersible in water.

Preferably, S1-S2 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.

Preferably, S1 is a C2-5-aminoalkyl selected from the group consisting of: 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl.

Preferably, S2 is selected from the group consisting of: —CO(CH2)3CO—, —CO(CH2)4CO— (adipate), —CO(CH2)5CO— and —CO(CH2)5NHCO(CH2)5CO—.

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).

Preferably, L is a glycerophospholipid and the construct includes the substructure:

where X is H or C, * is other than H and n is an integer 2 to 5.

More preferably, L is a glycerophospholipid and the construct includes the substructure:

where: X is H; R1 is a Cp-alkyl glycoside, R2 and R3 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.

Most preferably, the glycoside is 1-O—(O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl (Gb3), n is 4 and p is 3.

In specific embodiments of the second aspect of the invention the carbohydrate-lipid construct has the structure:

designated Gb3-sp3-Ad-DOPE (I); the structure:

designated Gb3-sp3-Ad-DSPE (II); the structure:

designated Gb3-sp2-Ad-DOPE (III); or the structure:

designated Gb3-sp2-Ad-DSPE (IV).

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-S1-S2-L where: F is selected from the group consisting of glycotopes of ligands for a receptor; S1-S2 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.

Preferably, the receptor is expressed by a virus. More preferably, the receptor is expressed by the human immunodeficiency virus (HIV).

Preferably, the pharmaceutical preparation is in the form of a cream or suppository.

S1-S2 is selected to provide a carbohydrate-lipid construct that is dispersible in water.

Preferably, S1-S2 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.

Preferably, S1 is a C2-5-aminoalkyl selected from the group consisting of: 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl.

Preferably, S2 is selected from the group consisting of: —CO(CH2)3CO—; —CO(CH2)4CO— (adipate); and —CO(CH2)5CO—.

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).

Preferably, L is a glycerophospholipid and the construct includes the substructure:

where X is H or C, * is other than H and n is an integer 2 to 5.

More preferably, L is a glycerophospholipid and the construct includes the substructure:

where: X is H; R1 is a Cp-alkyl glycoside, R2 and R3 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.

Most preferably, the glycoside is 1-O—(O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl (Gb3), n is 4 and p is 3.

In specific embodiments of the third aspect of the invention the carbohydrate-lipid construct has the structure:

designated Gb3-sp3-Ad-DOPE (I); the structure:

designated Gb3-sp3-Ad-DSPE (II); the structure:

designated Gb3-sp2-Ad-DOPE (III); or the structure:

designated Gb3-sp2-Ad-DSPE (IV).

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.

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.

In the description and claims of the specification the following terms and phrases have the meaning provided:

“Carbohydrate-lipid construct” means a synthetic molecule used as a glycolipid mimic.

“Gb3” means the carbohydrate portion of the ganglioside Gb3 (Chemical Abstract Service (CAS) REGISTRY number 71965-57-6)

“Cp-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:

designated Gb3-sp3,

“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 μM in the absence of organic solvents or detergents.

“Glycotope” means the portion of the carbohydrate moiety of a ligand that associates with the binding site of a receptor.

“Ligand” means any molecule or portion of a molecule that binds to one or more macromolecules, such as surface expressed antigens.

“Pharmaceutically acceptable formulants” means ingredients included in the formulation of a pharmaceutical composition.

“Receptor” means a macromolecule or portion of a macromolecule such as a surface expressed antigen that binds to one or more ligands.

“Vascular system” means the system of vessels that convey fluids such as blood or lymph, or provide for the circulation of such fluids.

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.

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+, K+, NH4+ and triethylamine ([NH(CH2CH3)3]+), 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.

Where the suffix “-al” is employed in respect of the substituents R2 and R3, an aldehyde structure is intended as exemplified by cis-9-octadecenal of the structure:

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.

FIG. 1. 1H-NMR data for the carbohydrate-lipid construct designated Gb3-sp3-Ad-DOPE (I).

FIG. 2. Effect of 250 μM Gb3-sp3-Ad-DOPE (I) on VSV/HIV infection in Jurkat Cells (RLU): A—Control; B—AZT; C—VSV/HIV; and D—250 μM Gb3-sp3-Ad-DOPE (I).