WO2009038605A2 - Flexible, polyvalent antiviral dendridic conjugates for the treatment of hiv/aids and enveloped viral infection - Google Patents

Abstract

The invention provides flexible antiviral conjugates that exhibit exceptional antiviral activity, including activity against HIV and against drug-resistant HIV strains. Conjugates of the invention include a low molecular weight, flexible scaffold and at least one binding moiety such as a human cell membrane protein which binds to one or more viral surface proteins of an enveloped virus, such as human CD4 or a small molecule ligand which binds to one or more viral surface proteins of an enveloped virus.

Description

FLEXIBLE, POLYVALENT ANTIVIRAL DENDRIDIC CONJUGATES FOR THE TREATMENT OF HIV/AIDS AND ENVELOPED VIRAL INFECTION

The present application claims the benefit of U.S. provisional application no. 60/932,464 filed May 31, 2007, which is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE

Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and/or paragraphing priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference. More generally, documents or references are cited in this text, either in a Reference List, or in the text itself; and, each of these documents or references ("herein-cited references"), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

STATEMENT OF U.S. GOVERNMENT INTEREST

Funding for the present invention was provided in part by support from the intramural research program of the National Cancer Institute, a component of the U.S. National Institutes of Health Accordingly, the Government of the United States may have certain rights in and to the invention

BACKGROUND OF THE INVENTION

Infection of target cells by enveloped viruses is initiated by binding of viral surface proteins to selected cell surface receptors. For example, human immunodeficiency virus (HIV) infection of target cells is initiated by binding of the viral envelope glycoprotein gpl20 to the cell surface receptor CD4 (1-3). This first step in the infection process is an attractive drug target because de novo infection of target cells would be blocked, preventing all subsequent stages of the viral lifecycle. No therapeutic agents that target surface protein - receptor binding are currently available, although certain small molecules are in clinical trials (reviewed in 4).

With regard to HIV and CD4, soluble monomelic CD4 (sCD4) neutralizes primary isolates poorly at pharmacologically realizable concentrations (5), and is therefore not useful as a therapeutic agent against HIV-I. Minimally passaged clinical primary isolates, which best model the in vivo virus, are unaffected by physiologically sustainable concentrations of sCD4 in infectivity assays (6). Recent reports suggest that the sCD4-induced conformational change in gpl20 (7-9) makes sCD4 binding energetically unfavorable (6).

In contrast to the soluble monomelic form, CD4 on the cell surface is thought to be clustered (10, 11). The clustered CD4 may mediate an avidity effect, so that sCD4 competes poorly with target cell CD4 for viral gpl20 (6). To mimic the clustering of cell surface CD4, Arthos et al designed a multimeric CD4 construct, D1D2-Igαtp, achieving a similar avidity effect (12). FIG. 1. D1D2-Igαtp is a multi-domain protein that comprises the two extracellular N-terminal domains of human CD4, Dl and D2, fused to the hinge, Cγ2, and Cγ3 domains of human IgGl, fused in turn to the 18 a.a. human IgA α secretory tailpiece. The α-secretory tailpiece promotes disulfide-mediated immunoglobulin multimerization. Analytical ultracentrifugation and dynamic light scattering revealed that D1D2-Igαtp molecules are typically multimeric species that carry 6-8 or more Ig2(CD4)2 units (i.e. 12-14 CD4 units) and have sizes ranging from ~600 — 1200 kDa, with a 12-nm average hydrodynamic radius (12). D1D2-Igαtp neutralizes minimally passaged primary clinical isolates of HIV-I at <3 nM IC90 values (6, 12), lower than those reported for monoclonal antibodies, polyclonal sera, or other CD4 constructs (6, 13, 14). FIG. 2

It has been hypothesized that the steric bulk of D1D2-Igαtp, which is many times larger than a monoclonal antibody, prevents approach and binding of gpl20 to target cell CD4 (12). However, the large mass of D1D2-Igαtp is also a disadvantage, as it would likely result in undesirable pharmacologic properties, such as poor distribution and immunogenicity. Some monovalent compounds, such as BMS-378806, have been developed to bind gpl20 at the CD4 binding site. A bivalent version has a 5-fold greater potency in vitro (as measured by IC90 values), but substantial increases are not seen with polyvalent species based on a rigid scaffold (27).

Thus, there remains a need for polyvalent binding of enveloped viruses using low molecular weight, flexible scaffold for the treatment of enveloped and HIV viral infection. There further remains a need for reducing the resistance of a patient to highly active antiretroviral therapy by blocking de novo infection of target and by utilizing the cellular receptor(s) of an enveloped virus (e.g., CD4 in the case of HIV) in a therapeutic modality to reduce binding to the cellular receptor(s) and entry into the cell.

SUMMARY OF THE INVENTION

The present invention provides, a flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a human cell membrane protein or a portion thereof which binds to one or more viral surface proteins of an enveloped virus.

The present invention further provides, a flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a small molecule ligand which binds to one or more viral surface proteins of an enveloped virus.

In one embodiment, the present invention provides, flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a human CD4 protein or a derivative thereof.

In another embodiment, the present invention further provides flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a small molecule ligand which binds to the viral envelope glycoprotein gpl20 of human immunodeficiency virus, simian immunodeficiency virus or simian/human immunodeficiency virus.

In a further embodiment, the present invention provides a flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a small molecule ligand which binds to one or more viral surface proteins of an enveloped virus.

In a still further embodiment, the present invention provides a flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a small molecule ligand which binds to the viral envelope glycoprotein gpl20 of human immunodeficiency virus, simian immunodeficiency virus or simian/human immunodeficiency virus.

In another embodiment, the flexible antiviral conjugate of the invention, further comprises a flexible spacer moiety linking the scaffold and the binding moiety. In some embodiments, the flexible spacer moiety is a poly(ethylene glycol), a poly(alkanediol) - including but not limited to, a poly(l,3-propanediol), a poly(l,4-butanediol), a poly(l,5- pentanediol)- or a human protein functionalized at the N-terminus with a carbodiimide moiety, a bis-imidoester moiety, a phenolate moiety, an imidazolyl moiety, an acetimidate moiety, a malonimidate moiety, a maleimide moiety or a substituted maleimide moiety. In some embodiments, the flexible spacer moiety is attached to an amine surface group of the scaffold using a heterobifunctional linker comprising a maleimide group and an N- hydroxysuccinimide ester, wherein the heterobifunctional linker is succinimidyl 4-[N- maleimidomethyl]-cyclohexane- 1 -carboxylate.

In other embodiments, the flexible spacer moiety is a heterobifunctional molecule. In some embodiments, the heterobifunctional molecule is a heterobifinctional alkane, including but not limited to, a maleimide-terminal n-hydroxysuccinimide ester-terminal alkane, a hydroxy- terminal alkane, or a mercapto-terminal alkane. In other embodiments, the flexible spacer moiety is a heterobifunctional molecule comprising a maleimide group and an n- hydroxysuccinimide ester, including, but not limited to, succinimidyl 4-[n- maleimidomethylj-cyclohexane- 1 -carboxylate, succinimidyl-4-[n-maleimidomethyl]- cyclohexane-l-carboxy-[6-amidocaproate] or a substituted maleimide moiety. In stil other embodiments, the heterobifunctional molecule is succinimidyl 4-[n-maleimidomethyl]- cyclohexane- 1 -carboxylate, or succinimidyl-4-[N-Maleimidomethyl]cyclohexane- 1 -carboxy- [6-amidocaproate], or a poly(ethylene glycol) (PEG or PEO) molecule with an n- hydroxysuccinimide ester (NHS) at one end and a maleimide (MaI) at the other, including but not limited to NHS-PEO₂-MaI, NHS-PEO₄-MaI, NHS-PEO₆-MaI, NHS-PEO₈-MaI, NHS- PEOi₀-MaI or NHS-PEOi₂-MaI.

In still other embodiments, the flexible spacer moiety is a polypeptide. In some embodiments, the the flexible spacer moiety is a human IgG Hinge Domain protein modified at the N-terminus with a maleimide or substituted maleimide moiety. In in still other embodiments the flexible spacer moiety is a polypeptide which has the sequence:

CRDSSRPEARRDVRTNRDSSRPEARRDVRTNRDSSRPEARRDVRTNK (SEQ ID NO: 1);

RDSSRPEARRDVRTN (SEQ ID NO: 2);

RDSSRPEARRDVRTNRDSSRPEARRDVRTN (SEQ ID NO: 3); RDSSRPEARRDVRTNRDSSRPEARRDVRTNRDSSRPEARRDVRTN (SEQ ID NO: 4);

CRDSSRPEARRDVRTNK (SEQ ID NO: 5);

CRDSSRPEARRDVRTNRDSSRPEARRDVRTNK (SEQ ID NO: 6); KRDSSRPEARRDVRTNC (SEQ ID NO: 7);

KRDSSRPEARRDVRTNRDSSRPEARRDVRTNC (SEQ ID NO: 8); AGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMS (SEQ ID NO: 9); CAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSK (SEQ ID NO: 10); KAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSC (SEQ ID NO: 11);

AGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSAGGAKPAPAAAEEK AAPAAAKPATTEGEFPETREKMS (SEQ ID NO: 12);

CAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSAGGAKPAPAAAEE KAAPAAAKPATTEGEFPETREKMSK (SEQ ID NO: 13);

KAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSAGGAKPAPAAAEE KAAPAAAKPATTEGEFPETREKMSC (SEQ ID NO: 14);

AGGARPAPAAAEERAAPAAARPATTEGEFPETRERMS (SEQ ID NO: 15); CAGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSK (SEQ ID NO: 16); KAGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSC (SEQ ID NO: 17);

AGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSAGGARPAPAAAEERA APAAARPATTEGEFPETRERMS (SEQ ID NO: 18);

CAGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSAGGARPAPAAAEER AAPAAARPATTEGEFPETRERMSK (SEQ ID NO: 19);

KAGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSAGGARPAPAAAEER AAPAAARPATTEGEFPETRERMSC (SEQ ID NO: 20);

KNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRC (SEQ

ID NO: 21);

NTRVDRRAEPRSSDR (SEQ ID NO: 22); NTRVDRRAEPRSSDRNTRVDRRAEPRSSDR (SEQ ID NO: 23); NTRVDRRAEPRSSDRNTRVDRRAEPRSSDRNTRVDRRAEPRSSDR (SEQ ID NO: 24);

KNTRVDRRAEPRSSDRC (SEQ ID NO: 25);

KNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRC (SEQ ID NO: 26); NTKVDKKAEPKSCDK (SEQ ID NO: 27); NTKVDKKAEPKSCDKNTKVDKKAEPKSCDK (SEQ ID NO: 28);

NTKVDKKAEPKSCDKNTKVDKKAEPKSCDKNTKVDKKAEPKSCDK (SEQ ID NO: 29);

KNTKVDKKAEPKSCDKC (SEQ ID NO: 30); KNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKC (SEQ ID NO: 31);

KNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKC (SEQ ID NO: 32);

CNTKVDKKAEPKSCDKK (SEQ ID NO:33); CNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKK (SEQ ID NO: 34);

CNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKK (SEQ ID NO: 35);

CNTRVDRRAEPRSSDRK (SEQ ID NO: 36); CNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRK (SEQ ID NO: 37);

CNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRK (SEQ ID NO: 38);

KRDSSRPEARRDVRTNRDSSRPEARRDVRTNRDSSRPEARRDVRTNC (SEQ ID NO: 39); or any part, combination or rearrangement thereof.

In still another embodiment of the invention, the flexible antiviral conjugate of the invention comprises a low molecular weight, flexible scaffold, wherein said low molecular weight, flexible scaffold is a dendrimer. In some embodiments, the dendrimer is a branched polypeptide, a branched nucleic acid, a branched polyethyleneamine, a branched polysaccharide, a branched polyamidoamine, a branched polyacrylic acid, a branched polyalcohol or a branched synthetic polymer. In still other embodiments, the dendrimer comprises a poly(amidoamine) polymer having at least one maleimide, hydroxyl, amine, or poly(ethylene glycol) surface group. In yet other embodiments, the dendrimer comprises a poly(amidoamine) polymer having 2-4 maleimide, hydroxyl, amine, or poly(ethylene glycol) surface group. In yet still other embodiments, the dendrimer comprises a poly(amidoamine) polymer having at least 4 maleimide, hydroxyl, amine, or poly(ethylene glycol) surface group.

In another embodiment of the invention the dendrimer comprises two or more poly(amidoamine) polymers each having at least one maleimide, hydroxyl, amine, or poly(ethylene glycol) surface group linked by a diaminoalkyl linker, a carbodiimide linker, a bis-imidoester linker, a phenolate linker, an imidazolyl linker, an acetimidate linker, a malonimidate linker, a maleimide linker, a substituted maleimide linker or a disulfide linker. In some embodiments, the dendrimer comprises two or more poly(amidoamine) polymers each having 2-4 maleimide, hydroxyl, amine, or poly(ethylene glycol) surface groups linked by a diaminobutyl linker. In still other embodiments, the dendrimer comprises two poly(amidoamine) polymers each having 4 maleimide, hydroxyl, amine, or poly(ethylene glycol) surface groups linked by a diaminobutyl or a diaminoalkyl linker. In still another embodiment, the each surface group is bound to a flexible spacer moiety which is bound to a binding moiety.

In some embodiments of the invention, the dendrimer comprising two or more maleimide, hydroxyl, amine, or poly(ethylene glycol) surface groups comprises only maleimide surface groups. In other embodiments of the invention, the dendrimer comprising two or more maleimide, hydroxyl, amine, or poly( ethylene glycol) surface groups comprises only hydroxyl surface groups. In yet other embodiments of the invention, a dendrimer comprising two or more maleimide, hydroxyl, amine, or poly(ethylene glycol) surface groups comprise only amine surface groups. In still other embodiments of the invention, a dendrimer comprising two or more maleimide, hydroxyl, amine, or poly(ethylene glycol) surface groups comprise only poly(ethylene glycol) surface groups.

In yet another embodiment, the flexible antiviral conjugate of the invention has the structure depicted in FIG 3.

In yet another embodiment, the flexible antiviral conjugate of the invention has the structure depicted in FIG 4.

In yet another embodiment of the invention, the flexible antiviral conjugate of the invention, optionally further comprising a flexible spacer moiety linking the scaffold and the binding moiety, wherein binding moiety is a small molecule ligand which binds to the viral envelope glycoprotein gpl20 having the formula :

wherein R represents a direct bond to to the scaffold or to the flexible spacer moiety or a linking group suitable to bind to the scaffold or flexible spacer moiety.

In yet another embodiment, the binding moiety is yet another divalent, trivalent, or multivalent conjugate of a gpl20 ligand, so that the invention is a flexible, polyvalent antiviral conjugate of a polyvalent antiviral conjugate (the terminal conjugate), which may or may not be flexible. In another embodiment, this divalent, trivalent polyvalent antiviral terminal conjugate is presents gpl20 ligands in a geometry and/or with a degree of flexibility that optimizes binding of multiple sites on a gp 120 trimer. This geometry is based on, for example, measurements from a gpl20 trimer model obtained from cryo electron tomography.

The invention further provides a pharmaceutical composition comprising at least one flexible antiviral conjugate of the invention and a pharmaceutically acceptable diluent or carrier.

In another aspect, the invention provides a method for treating an enveloped virus infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of the invention.

In another aspect, the invention provides a method for treating HIV infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of the invention.

In yet another aspect, the invention provides a method for treating HIV infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of the invention comprising a human CD4 molecule and/or a therapeutically effective amount of one or more flexible antiviral conjugates of the invention comprising small molecule ligand which binds to the viral envelope glycoprotein gpl20.

In still another aspect, the invention provides a method for reducing resistance to treatment of an enveloped virus in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of the invention.

In another aspect, the invention provides a method for reducing resistance to highly active antiretro viral therapy in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of the invention.

In yet another aspect, the invention provides a method for reducing resistance to highly active antiretroviral therapy in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of the invention comprising a human CD4 molecule and/or a therapeutically effective amount of one or more flexible antiviral conjugates of the invention comprising small molecule ligand which binds to the viral envelope glycoprotein gpl20.

In some embodiments of the invention, the invention provides the methods described herein further comprising administering a therapeutically effective amount of at least one other agent used for highly active antiretroviral therapy. In still other embodiments, the other agent or agents are nucleoside HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, HIV entry inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, or HIV integrase inhibitors or combinations thereof.

In some embodiments, the one or more flexible antiviral conjugates of the invention are administered parenterally, transdermally, mucosally, nasally, buccally, sublingually, topically or orally. In still other embodiments, the flexible antiviral conjugates of the invention comprising a viral surface protein of an enveloped virus or a human CD4 molecule are administered parenterally.

In a still another embodiment, the subject is a mammal, preferably a human. In some embodiments, the human subject is a male or a female. In still other embodiments, the human subject is an elderly individual, an adult individual or an adolescent individual. In other embodiments, the human subject is suffering from one or more symptoms of resistance to highly active antiretroviral therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Direct visualization of SIV neutralization. Cryo tomographic slices (thickness 8.2 nm, A, B, and C) and three-dimensional automatic density threshold segmentations (D and E), of (A and D) untreated, (B) MICA-IgP-treated, and (C and E) DlD2-IgP-treated SIV virions in vitreous ice, from tilt series recorded at 300 kV and reconstructed using weighted back-projection. Scale bars 100 nm.

Figure 2. Models of CD4-neutralized viral gpl20 constructed from the X-ray crystal structure coordinates of soluble CD4 (28), IgG bl2, (29), a model of the IgA α secretory tailpiece (30), and a model of the CD4- and 17b-liganded gpl20 trimer (32). A, Three-dimensional automatic density threshold segmentation of individual DlD2-Igαtp-SIV complexes in vitreous ice, from tilt series recorded at 300 kV. Tomograms of the complexes shown at upper left, upper right, lower left, and lower right are shown in Movies M5, M6, M7, and M8, respectively. Scale bar 15 nm. B, Hypothetical molecular models of intra-virion (left) and inter-virion (right) spike crosslinking by D1D2-Igαtp. CD4 domains that are not part of a D1D2-Igαtp molecule are not shown. The models were created using PyMoI (31). The gray surface represents the viral membrane.

Figure 3. A diagram of the CD4 polymer. The construct described here is a poly(amidoamine) (PAMAM), diaminobutane core dendrimer with branches that are extended by a triple repeat of the human IgG hinge region, which is in turn attached to the 183-a.a N-terminal fragment of human CD4, for a total of 8 human CD4 molecules attached to a highly flexible molecular scaffold.

Figure 4 depicts a diagram of CD4-dendrimer embodiment.

DETAILED DESCRIPTION Definitions

In order that the invention may be more readily understood, certain terms are first defined and collected here for convenience. Other definitions appear in context throughout the application.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

The term "scaffold" refers to a synthetic, three-dimensional organic molecule having one or more functional groups, including but not limited to hydroxyl, amine, or poly(ethylene glycol), said functional groups being capable of being derivatized using known synthetic processes. In some embodiments, the scaffold comprises amine groups along the backbone which can be bound to other parts of the conjugate using a heterobifunctional linker comprising a maleimide group and an N-hydroxysuccinimide ester, including, but not limited to, succinimidyl 4-[N-maleimidomethyl]-cyclohexane-l-carboxylate. In other embodiments, the scaffold comprises maleimide groups along the surface of the backbone which can be bound to other parts of the conjugate.

The term "binding moiety" refers to a natural or synthetic molecule or ligand, which is capable of binding to one or more viral surface proteins of a virus. Such binding moieties include, but are not limited to, human cell membranes, such as CD4, and small molecule, organic or inorganic ligands, such as BMS-378806. Such binding moieties may further comprise one or more functional groups capable of being derivatized using known synthetic processes

The term "flexible spacer moiety" refers to a natural or synthetic molecule having two or more functional groups said functional groups being capable of being derivatized using known synthetic processes, such that one functional group of the flexible spacer moiety is derivatized and bound to a scaffold functional group and another functional group of the flexible spacer moiety is derivatized and bound to a binding moiety. Such flexible spacer moieties include, but are not limited to a poly(ethylene glycol), a poly(alkanediol), a polypeptide, or a human protein functionalized at the N-terminus with a carbodiimide moiety, a bis-imidoeste moiety, a phenolate moiety, an imidazolyl moiety, an acetimidate moiety, a malonimidate moiety, a maleimide moiety or a substituted maleimide moiety.

The term "alkane" refers to a straight or branched hydrocarbon chain radical, containing solely carbon and hydrogen atoms, having in the range from one up to twelve carbon atoms, and which is attached to the rest of the molecule by a single bond, such as illustratively, methyl, ethyl, n-propyl 1 -methylethyl (iso-propyl), n-butyl, n-pentyl, and 1,1- dimethylethyl (tert-butyl).

The term "enveloped virus" refers to a virus having a nucleoid, a capsid and a lipid envelope covering the viral capsid. Enveloped viruses include, but are not limited to herpes simplex virus type 1 , herpes simplex virus type 2, varicella zoster virus, toga virus, syncytial virus, paramyxovirus, myxovirus, human herpes virus-6, human immunodeficiency virus (HIV), cytomegalovirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, corona virus, influenza, Epstien-Barr virus, Ross River Virus, Eastern equine encephalitis Virus, Venezuelan equine encephalitis, Western equine encephalitis, Sindbis Virus, Semiliki Forest Virus, St. Louis encephalitis Virus, Japanese encephalitis Virus, Dengue Virus, Yellow fever Virus, Tick-borne encephalitis Virus, New Castle Disease Virus and Ebola Virus.

The term "viral surface protein" refers to a protein on the surface of an enveloped virus that associates with or binds to a binding moiety.

The term "human CD4 molecule" refers to a human CD4 protein which contains one, two, three, or four of the domains, in any sequence, or any other portion of the human CD4 molecule or its polypeptide sequence, or any derivative of the human CD4 molecule containing one or more mutations, including addition of a cysteine residue or residues, or replacement of a residue or residues by a cysteine or cysteines.

The term "HIV" refers to human immunodeficiency virus, including, but not limited to, HIV-I, HIV-2, specific treatment resistant mutations of HIV-I or HIV-2, or particular isolated strains or mutations of HIV obtained from a subject to be treated by the methods of the invention.

The term "surface group" refers to functional group found on a dendrimer which is capable of being derivatized using known synthetic processes such that the surface group can bind to a flexible spacer moiety or a binding moiety.

The term "linker" refers to a natural or synthetic molecule having two or more functional groups said functional groups being capable of being derivatized using known synthetic processes, such that each functional group on the linker is capable of being bound to functional groups on different dendrimer units.

The term "dendrimer" refers to a synthetic, three-dimensional macromolecule, built up from a monomer, with new branches added in steps until a tree-like structure is created.

A "peptide" is a sequence of at least two amino acids. Peptides can consist of short as well as long amino acid sequences, including full-length proteins.

The term "subject" refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human.

It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a peptide" includes multiple peptides, reference to "a spacer" includes two or more spacers.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

Synthesis of Conjugates

Dendrimer Synthesis

A dendrimer with reactive surface groups, such as malemide surface groups can be synthesized from commercially available dendrimers with amine surface groups, as shown in the following two-step synthesis:

Scheme I:

Dendrimer-NH

Solvent

In Scheme 1, Maleic anhydride is disolved in a solvent (for example, ethyl ether). Upon dissolution, a dendrimer having amine surface groups is added and the resulting suspension is stirred at room temperature for approximately one hour. After reflux, the suspension is cooled to 15-20° in an ice bath and the maleanilic acid product is obtained by suction filtration. The maleanilic acid is added to acetic anhydride and anhydrous sodium acetate (or fused potassium acetate) , and the resulting suspension is dissolved by swirling and heating on a steam bath for 30 minutes. The reaction mixture is cooled almost to room temperature in a cold water bath and is then poured into of ice water to precipitate.

Cell Membrane Protein Conjugates

Cell membrane protein conjugates can be prepared using the dendrimers described above. The dendrimers are bound to the cell membrane proteins by formation of an irreversible thioether linkage using known techniques. Alternatively, the dendrimers are bound to a flexible spacer moiety by formation of an irreversible thioether linkage which is in turn bound to the cell membrane protein by formation of another irreversible thioether linkage. The maleimide group described herein reacts specifically with sulfhydryl groups when the pH of the reaction mixture is between pH 6.5 and 7.5 and forms a stable thioether linkage that is not reversible. At neutral pH, maleimides react with sulfhydryls 1, 000-fold faster than with amines, but at pH >8.5, the reaction favors primary amines. Maleimides do not react with tyrosines, histidines or methionines. Hydrolysis of maleimides to a non- reactive maleamic acid can compete with thiol modification, especially above pH 8.0. Thiols must be excluded from reactions buffers used with maleimides because they will compete for coupling sites. Excess maleimides can be quenched at the end of a reaction by adding free thiols. EDTA can be included in the coupling buffer to minimize oxidation of sulfhydryls.

Small Molecule Ligand Conjugates

Small molecule ligand conjugates can be prepared using the dendrimers described above. The dendrimers are bound to the small molecule ligands by formation of an irreversible linkage betweenthe ligand and the dendrimer using known techniques such that the affitnity of the lingand for binding to the viral surface protein is not significantly altered. Alternatively, the dendrimers are bound to a flexible spacer moiety by formation of an irreversible thioether, carbamate, carbodiimide, or ether linkage, which is in turn bound to the small molecule ligand such that the affitnity of the ligand for binding to the viral surface protein is not significantly altered

Certain small molecule ligands of the invention can be synthesized using the procedures outlined in the synthesis below:

Scheme II

In Scheme II, the synthesis of a small molecule ligand as depicted is initiated by acylating 7-azaindole (1) with methyl chlorooxoacetate in the presence OfAlCl₃. Hydrolysis of the resulting ester, using a base, such as K₂CO_3j in a solvent, such as aqueous MeOH, is followed by coupling with the a benzoylated piperazine which may be further substituted by one or more protecting or stabilizing group such as a hydroxy, p-hydroxy, methoxy, or ethoxy. (The piperazine may be obtained by exposing (R)-(-)-2-methylpiperazine and substituted or unsubstituted methyl benzoate to 1 mol equiv of diethylaluminum chloride in CH₂Cl₂). The reaction is reaction mediated by 3-(diethoxyphosphoryloxy)-l ,2,3- benzotriazin-4(3H)-one in the presence of N,N-diisopropylethlyamine (/Pr₂NEt, Hunig's base) in a solvent, such as DMF, to afford 4. Incorporation of the methoxy substituent at the 4-position of the Small Molecule Ligand is accomplished by a serial process, initiated by oxidation of the nitrogen atom of the heterocycle and followed by nitration with fuming HNO₃ in CF₃CO₂H, which affords a 4-nitro N-oxide 5. Heating 5 with an excess of NaOMe in MeOH resulted an ipso displacement of nitrite which can be converted tothe methoxy group by reduction of the N-oxide using PCl₃ in EtOAc.

Conjugates Comprising One Or More Spacer Moieties

Flexible spacer moieties including, but not limited to, PEG, poly(alkanediol) linkers, synthetic polymers, as well as polypeptides of any sequence, including the human IgG hinge domain sequence or any variant thereof, can be obtained using known techniques or can be obtained commercially.

Cell membrane proteins can be bound to flexible spacer moieties using known techniques. For example, maleimide + sulfhydryl reactions can be performed by adding the sulfhydryl-containing species to the maleimide-contianing species (flexible), in neutral aqueous buffer, sometimes containing EDTA. The protein molecule may be linked to a maleimide functionalized flexible spacer or directly to a maleimide surface dendrimer using this reaction, or to an amine surface dendrimer using a heterobifunctional linker comprising a maleimide group and an N-hydroxysuccinimide ester, such as succinimidyl 4-[N- maleimidomethyl]-cyclohexane- 1 -carboxylate or Succinimidyl-4-[N-maleimidomethyl]- cyclohexane- 1 -carboxy-[6-amidocaproate] .

Small molecule ligands can be bound to a flexible spacer moiety using known techniques. For example, conjugation of the small molecule ligand can be accomplished by using p-chloromethylbenzoate instead of methylbenzoate in Scheme II above. The chloro group para to the carboxy group in the phenyl ring in the chloro-ligand could then be the site of nucleophilic aromatic substitution with a hydroxyl group from a hydroxyl-surface group, with the reaction carried out in anhydrous base, followed by aqueous acid workup. Alternatively, the nucleophilic aromatic substitution reaction can be carried out to high yield without solvent in a microwave oven, as described (43).

Nucleophilic aromatic substitution reaction can also be carried between the p- chloromethylbenzoate and the dendrimer or flexible spacer directly. This species is then reacted in Scheme II above. In this way, the small molecule ligand is synthesized as part of and not separate from the dendrimer and/or flexible spacer conjugation procedure. In another example, p-hydroxymethylbenzoate is used in place of methylbenzoate in Scheme II above, either before or after reaction between the hydroxyl group and an isocyanate- functionalized dendrimer, forming a carbamate linkage between the small molecule ligand and the dendrimer and/or flexible spacer. In another example, p-mercaptomethylbenzoate is used in place of methylbenzoate in Scheme II above, either before or after reaction between the sulfhydryl group and poly(ethylene glycol)-functionalized or hydroxyl-functionalized dendrimer using a heterobifunctional linker such as, but not limited to, (N-[p-Maleimidophenyl]isocyanate), forming a carbamate linkage between the poly(ethylene glycol) and/or hydroxyl and the heterobifunctinal linker, which in turn is linked to the small molecule ligand by a thioether linkage.

Treatment of Viruses and Reduction of Incidence of Resistance

The invention thus provides methods of treatment against enveloped virus infections, which methods in general comprise administration of a therapeutically effective amount of one or more conjugates of the invention to a mammal, particularly a human, suffering from or susceptible to a viral infection or disease otherwise associated with a virus.

Conjugates of the invention will be useful to treat cells infected with a virus capable of causing an immunodeficiency disease, particularly in a human. Compounds of the invention will be particularly useful to treat retroviral infection in cells and in a human, particularly HIV infected human cells. Specific examples of enveloped virus infections which may be treated in accordance with the invention include human retroviral infections such as HIV-I, HIV-2, and herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, toga virus, syncytial virus, paramyxovirus, myxovirus, human herpes virus-6, cytomegalovirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, corona virus, influenza, Epstien-Barr virus, Ross River Virus, Eastern equine encephalitis Virus, Venezuelan equine encephalitis, Western equine encephalitis, Sindbis Virus, Semiliki Forest Virus, St. Louis encephalitis Virus, Japanese encephalitis Virus, Dengue Virus, Yellow fever Virus, Tick-borne encephalitis Virus, New Castle Disease Virus, and Ebola Virus infections.

The invention also provides methods of reducing the resistance of a subject to enveloped viral therapy or highly active antiretroviral therapy associated with a virus such as HIV-I, HIV-2, and herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, toga virus, syncytial virus, paramyxovirus, myxovirus, human herpes virus-6, cytomegalovirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, corona virus, influenza, Epstien-Barr virus, Ross River Virus, Eastern equine encephalitis Virus, Venezuelan equine encephalitis, Western equine encephalitis, Sindbis Virus, Semiliki Forest Virus, St. Louis encephalitis Virus, Japanese encephalitis Virus, Dengue Virus, Yellow fever Virus, Tick-borne encephalitis Virus, New Castle Disease Virus or Ebola Virus.

As discussed above, particularly preferred conjugates of the invention are active against drug-resistant viral strains, and it is believed that conjugates of the invention are highly active against virus strains that are resistant to nucleoside HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, or HIV integrase inhibitors or combinations thereof.

Without wishing to be bound by theory, it is believed the conjugates of the invention reduce the number of viral replication cycles occurring in a patient, thereby reduing the probability of escape mutations against ofther highly active antiretroviral therapy drugs including nucleoside HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, or HIV integrase inhibitors or combinations thereof.

In certain embodiments, a conjugate of the invention is administered to a mammal, preferably, a human concurrently with one or more other biologically active agents, or with one or more other compounds of the invention, or with both. By "concurrently" it is meant that a conjugate of the invention and the other agent are administered to a mammal in a sequence and within a time interval such that the compound of the invention can act together with the other agent to provide an increased or synergistic benefit than if they were administered otherwise. For example, each component may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently closely in time so as to provide the desired treatment effect. Preferably, all components are administered at the same time, and if not administered at the same time, preferably, they are all administered from about 6 hours to about 12 hours apart from one another.

When used in combination with other therapeutic agents, the conjugates of the invention and the therapeutic agent can act additively or, more preferably, synergistically. In one embodiment, a conjugate or a composition of the invention is administered concurrently with another therapeutic agent in the same pharmaceutical composition. In another embodiment, a conjugate or a composition of the invention is administered concurrently with another therapeutic agent in separate pharmaceutical compositions. In still another embodiment, a conjugate or a composition of the invention is administered prior or subsequent to administration of another therapeutic agent. As many of the disorders for which the compounds and compositions of the invention are useful in treating are chronic disorders, in one embodiment combination therapy involves alternating between administering a compound or a composition of the invention and a pharmaceutical composition comprising another therapeutic agent, e.g., to minimize the toxicity associated with a particular drug. In certain embodiments, when a composition of the invention is administered concurrently with another therapeutic agent that potentially produces adverse side effects including, but not limited to toxicity, the therapeutic agent can advantageously be administered at a dose that falls below the threshold that the adverse side effect is elicited.

Conjugates of the invention may be used in combination with other medicaments such as nucleoside HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, or HIV integrase inhibitors or combinations thereof. Without being so limited, conjugates of the invention may be used in therapy in coηjuction with reverse transcriptase inhibitors such as a dideoxynucleoside including AZT, ddl, ddC, d4T, 3TC or 1592U89; TAT antagonists such as Ro 3-3335 and Ro 24-7429; protease inhibitors such as saquinavir, ritonavir, indinavir or AHGl 343 (Viracept); and other agents such as 9-(2-hydroxyethoxymethyl)guanine (acyclovir), ganciclovir or pencyclovir, interferon, e.g., alpha-interon or interleukin II, or in conjunction with other immune modulation agents including bone marrow or lymphocyte transplants or other medications such as levamisol or thymosin which would increase lymphocyte numbers and/or function as is appropriate.

Conjugates of the invention can be administered or formulated in combination with antibiotics. For example, they can be formulated with a macrolide (e.g., tobramycin (Tobi®)), a cephalosporin (e.g., cephalexin (Keflex®), cephradine (Velosef®), cefuroxime (Ceftin®), cefprozil (Cefzil®), cefaclor (Ceclor®), cefixime (Suprax®) or cefadroxil (Duricef®)), a clarithromycin (e.g., clarithromycin (Biaxin®)), an erythromycin (e.g., erythromycin (EMycin®)), a penicillin (e.g., penicillin V (V-Cillin K® or Pen Vee K®)) or a quinolone (e.g., ofloxacin (Floxin®), ciprofloxacin (Cipro®) or norfloxacin (Noroxin®)), aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicol antibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin), carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem and imipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g., cefbuperazone, cefinetazole, and cefininox), monobactams (e.g., aztreonam, carumonam, and tigemonam), oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamccillin, penethamate hydriodide, penicillin o-benethamine, penicillin 0, penicillin V, penicillin V benzathine, penicillin V hydrabamine, penimepicycline, and phencihicillin potassium), lincosamides (e.g., clindamycin, and lincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride), quinolones and analogs thereof (e.g., cinoxacin,, clinafloxacin, flumequine, and grepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine, benzylsulfamide, noprylsulfamide, phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone, glucosulfone sodium, and solasulfone), cycloserine, mupirocin and tuberin.

Conjugates of the invention can also be administered or formulated in combination with an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxyperndyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine, tropisetron, and mixtures thereof.

Conjugates of the invention can be administered or formulated in combination with an antidepressant. Suitable antidepressants include, but are not limited to, binedaline, caroxazone, citalopram, dimethazan, fencamine, indalpine, indeloxazine hydrocholoride, nefopam, nomifensine, oxitriptan, oxypertine, paroxetine, sertraline, thiazesim, trazodone, benmoxine, iproclozide, iproniazid, isocarboxazid, nialamide, octamoxin, phenelzine, cotinine, rolicyprine, rolipram, maprotiline, metralindole, mianserin, mirtazepine, adinazolam, amitriptyline, amitriptylinoxide, amoxapine, butriptyline, clomipramine, demexiptiline, desipramine, dibenzepin, dimetacrine, dothiepin, doxepin, fluacizine, imipramine, imipramine N-oxide, iprindole, lofepramine, melitracen, metapramine, nortriptyline, noxiptilin, opipramol, pizotyline, propizepine, protriptyline, quinupramine, tianeptine, trimipramine, adrafinil, benactyzine, bupropion, butacetin, dioxadrol, duloxetine, etoperidone, febarbamate, femoxetine, fenpentadiol, fluoxetine, fluvoxamine, hematopoφhyrin, hypericin, levophacetoperane, medifoxamine, milnacipran, minaprine, moclobemide, nefazodone, oxaflozane, piberaline, prolintane, pyrisuccideanol, ritanserin, roxindole, rubidium chloride, sulpiride, tandospirone, thozalinone, tofenacin, toloxatone, tranylcypromine, L-tryptophan, venlafaxine, viloxazine, and zimeldine.

Conjugates of the invention can be administered or formulated in combination with an antifungal agent. Suitable antifungal agents include but are not limited to amphotericin B, itraconazole, ketoconazole, fluconazole, intrathecal, flucytosine, miconazole, butoconazole, clotrimazole, nystatin, terconazole, tioconazole, ciclopirox, econazole, haloprogrin, naftifine, terbinafine, undecylenate, and griseofuldin.

Conjugates of the invention can be administered or formulated in combination with an anti-inflammatory agent. Useful anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory drugs such as salicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal, salsalate, olsalazine, sulfasalazine, acetaminophen, indomethacin, sulindac, etodolac, mefenamic acid, meclofenamate sodium, tolmetin, ketorolac, dichlofenac, ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen, flurbinprofen, oxaprozin, piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, tenoxicam, nabumetome, phenylbutazone, oxyphenbutazone, antipyrine, aminopyrine, apazone and nimesulide; leukotriene antagonists including, but not limited to, zileuton, aurothioglucose, gold sodium thiomalate and auranofϊn; steroids including, but not limited to, alclometasone diproprionate, amcinonide, beclomethasone dipropionate, betametasone, betamethasone benzoate, betamethasone diproprionate, betamethasone sodium phosphate, betamethasone valerate, clobetasol proprionate, clocortolone pivalate, hydrocortisone, hydrocortisone derivatives, desonide, desoximatasone, dexamethasone, flunisolide, flucoxinolide, flurandrenolide, halcinocide, medrysone, methylprednisolone, methprednisolone acetate, methylprednisolone sodium succinate, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebuatate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, and triamcinolone hexacetonide; and other anti-inflammatory agents including, but not limited to, methotrexate, colchicine, allopurinol, probenecid, sulfinpyrazone and benzbromarone.

Conjugates of the invention can be administered or formulated Ln combination with an immunomodulatory agent. Immunomodulatory agents include, but are not limited to, methothrexate, leflunomide, cyclophosphamide, cyclosporine A, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), T cell receptor modulators, and cytokine receptor modulators, peptide mimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments or epitope binding fragments), nucleic acid molecules (e.g., antisense nucleic acid molecules and triple helices), small molecules, organic compounds, and inorganic compounds. Examples of T cell receptor modulators include, but are not limited to, anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH IH (Ilex)), anti-CD2 antibodies, anti-CDl la antibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g., IDEC-114 (IDEC)) and CTLA4-immunoglobulin. Examples of cytokine receptor modulators include, but are not limited to, soluble cytokine receptors (e.g., the extracellular domain of a TNF-. alpha, receptor or a fragment thereof, the extracellular domain of an IL-I. beta, receptor or a fragment thereof, and the extracellular domain of an IL-6 receptor or a fragment thereof), cytokines or fragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF- . alpha., interferon (IFN)-. alpha., IFN-.beta., IFN-. gamma., and GM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibodies), anti-cytokine antibodies (e.g., anti-IFN antibodies, anti-TNF-. alpha, antibodies, anti-IL-l.beta. antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8 (Abgenix)), and anti-IL-12 antibodies).

Conjugates of the invention can be administered or formulated in combination with cytokines. Examples of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin- 10 (IL-10), interleukin- 12 (IL-12), interleukin 15 (IL-15), interleukin 18 (IL-18), platelet derived growth factor (PDGF), erythropoietin (Epo), epidermal growth factor (EGF), fibroblast growth factor (FGF), granulocyte macrophage stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), prolactin, and interferon (IFN), e.g., IFN-alpha, and IFN-gamma). Conjugates of the invention can be administered or formulated in combination with hormones. Examples of hormones include, but are not limited to, luteinizing hormone releasing hormone (LHRH), growth hormone (GH), growth hormone releasing hormone, ACTH, somatostatin, somatotropin, somatomedin, parathyroid hormone, hypothalamic releasing factors, insulin, glucagon, enkephalins, vasopressin, calcitonin, heparin, low molecular weight heparins, heparinoids, synthetic and natural opioids, insulin thyroid stimulating hormones, and endorphins.

Conjugates of the invention can be administered or formulated in combination with .beta.-interferons which include, but are not limited to, interferon beta- Ia and interferon beta- Ib.

Conjugates of the invention can be administered or formulated in combination with an alkylating agent. Examples of alkylating agents include, but are not limited to nitrogen mustards, ethylenimines, methylmelamines, alkyl sulfonates, nitrosoureas, triazenes, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, hexamethylmelaine, thiotepa, busulfan, carmustine, streptozocin, dacarbazine and temozolomide.

Formulation and Administration

The invention also provides a pharmaceutical composition, comprising an effective amount a conjugate described herein and a pharmaceutically acceptable diluent or carrier. In certain embodiments, the conjugate is administered to the subject in a pharmaceutically- acceptable formulation. In certain embodiments, the pharmaceutical compositions are suitable for topical, intravenous, parental, or oral administration. The methods of the invention further include administering to a subject a therapeutically effective amount of a conjugate in combination with another pharmaceutically active compound. Pharmaceutically active compounds that may be used can be found in Harrison 's Principles of Internal Medicine, Thirteenth Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; and the Physicians Desk Reference 50th Edition 1997, Oradell New Jersey, Medical Economics Co., the complete contents of which are expressly incorporated herein by reference.

The phrase "pharmaceutically acceptable" refers to conjugates of the present invention, compositions containing such conjugates, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically-acceptable carrier" includes pharmaceutically- acceptable material, composition or vehicle, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

Methods of preparing these compositions include the step of bringing into association a conjugate with the carrier and, optionally, one or more accessory ingredients. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

Regardless of the route of administration selected, the conjugates, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Formulations are provided to a subject in an effective amount. The term "effective amount" includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result. An effective amount of conjugate may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response.

The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. As a rule, the dosage for in vivo therapeutics or diagnostics will vary. Several factors are typically taken into account when determining an appropriate dosage. These factors include age, sex and weight of the patient, the condition being treated, and the severity of the condition.

Suitable dosages and formulations of immune modulators can be empirically determined by the administering physician. Standard texts, such as Remington: The Science and Practice of Pharmacy, 17th edition, Mack Publishing Company, and the Physician's Desk Reference, each of which are incorporated herein by reference, can be consulted to prepare suitable compositions and doses for administration. A determination of the appropriate dosage is within the skill of one in the art given the parameters for use described herein. Standard texts, such as Remington: The Science and Practice of Pharmacy, 17th edition, Mack Publishing Company, incorporated herein by reference, can be consulted to prepare suitable compositions and formulations for administration, without undue experimentation. Suitable dosages can also be based upon the text and documents cited herein. A determination of the appropriate dosages is within the skill of one in the art given the parameters herein.

In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of an enveloped virus infection, HIV infection, AIDS or the symptoms thereof. A therapeutically effective amount can be provided in one or a series of administrations. In terms of an adjuvant, an effective amount is one sufficient to enhance the immune response to the immunogen. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art.

As a rule, the dosage for in vivo therapeutics or diagnostics will vary. Several factors are typically taken into account when determining an appropriate dosage. These factors include age, sex and weight of the patient, the condition being treated, the severity of the condition and the form of the antibody being administered.

The dosage of the conjugates can vary from about 0.01 mg to about 5,000 mg per day, preferably about 1 mg to about 2,500 mg per day, more preferably about 10 mg to about 1 ,200 mg per day. Ascertaining dosage ranges is well within the skill of one in the art. The dosage of conjugates can range from about 0.0 to 70 mg/kg of body weight. Such dosages may vary, for example, depending on whether multiple administrations are given, tissue type and route of administration, the condition of the individual, the desired objective and other factors known to those of skill in the art. Administrations can be conducted infrequently, or on a regular weekly basis until a desired, measurable parameter is detected, such as diminution of disease symptoms. Administration can then be diminished, such as to a biweekly or monthly basis, as appropriate.

A therapeutically effective amount can be administered in one or more doses. The term "administration" or "administering" includes routes of introducing the compound(s) to a subject to perform their intended function. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), oral, inhalation, rectal and transdermal.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration," "administered systemically", "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound(s), drug or other material, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

Such dosages may vary, for example, depending on whether multiple administrations are given, tissue type and route of administration, the condition of the individual, the desired objective and other factors known to those of skill in the art. Available routes of administration include subcutaneous, intramuscular, intraperitoneal, intradermal, oral, intranasal, intrapulmonary (i.e., by aerosol), intravenously, intramuscularly, subcutaneously, intracavity, intrathecally or transdermally, alone or in combination with other pharmaceutical agents.

Oral Dosage Forms

Conjugates of the invention and compositions comprising them that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Typical oral dosage forms of the invention are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions of the invention is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103.TM and Starch 1500 LM.

Disintegrants are used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms of the invention. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, specifically from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W. R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Piano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated.

Parenteral and Intravascular Dosage Forms

Parenteral and intravascular dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection and constant infusion), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral and intravascular dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products (including, but not limited to lyophilized powders, pellets, and tablets) ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms of the invention.

For intravascular administration, for instance by direct injection into the blood vessel, or surrounding area, it may be desirable to administer the compositions locally to the area in need of treatment. This can be achieved, for example, by local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers. A suitable such membrane is Gliadel® provided by Guilford Pharmaceuticals Inc.

Transdermal, Topical, And Mucosal Dosage Forms

Transdermal, topical, and mucosal dosage forms of the invention include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes or as oral gels. Further, transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal, topical, and mucosal dosage forms encompassed by this invention are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane- 1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are non-toxic and pharmaceutically acceptable. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990).

Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients of the invention. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery-enhancing or penetration-enhancing agent. Different salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, preferred methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.

Kits

This invention therefore encompasses kits which, when used by the medical practitioner, can simplify the administration of appropriate amounts of conjugates of the invention to a patient.

A typical kit of the invention comprises one or more unit dosage forms of a conjugate of the invention, and instructions for use.

Kits of the invention can further comprise devices that are used to administer flexible antiviral conjugates of the invention. Examples of such devices include, but are not limited to, intravenous cannulation devices, syringes, drip bags, patches, topical gels, pumps, containers that provide protection from photodegredation, autoinjectors, and inhalers.

Kits of the invention can further comprise pharmaceutically acceptable vehicles that can be used to administer one or more active ingredients. For example, if an active ingredient is provided in a solid form that must be reconstituted for parenteral administration, the kit can comprise a sealed container of a suitable vehicle in which the active ingredient can be dissolved to form a particulate-free sterile solution that is suitable for parenteral administration. Examples of pharmaceutically acceptable vehicles include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

The invention is further described by way of the following non-limiting examples.

EXAMPLES

In order that the invention may be more fully understood, the following examples are provided. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any way. Analytical methods:

The following methods may be used to characterize the structures of the various embodiments of this invention and their effect on the structures of HIV and enveloped viruses.

Preparation of Purified SIV — Simian immunodeficiency virus (SIV) strain mac239 that are purified (15) and CD45-depleted to remove microvesicles (16) and inactivated with Aldrithiol-2, which preserves the envelope proteins (17).

Cryo-Electron Tomography — Purified viral suspensions are mixed with either a solution of Tris-buffered saline, pH 7.4 (TBS) or an equal volume of an equal concentration of the specific embodiment or conjugate of the invention, immediately and without incubation deposited on Quantifoil grids (Quantifoil Inc., Jena, Germany) and plunge-frozen using a Vitrobot device (FEI Company, OR). Grids are imaged at liquid nitrogen temperatures and 6- μm defocussed using a Polara field emission gun electron microscope (FEI Company, OR) operated at 300 kV, equipped with a GIF 2000 2K x 2K CCD placed at the end of a Gatan energy filter (Gatan, Inc., Pleasanton, CA). All tilt series for tomographic reconstruction are acquired using the FEI tomography software package Xplore3D (FEI Company, OR) and are reconstructed without fiducial markers using Protomo (18). Visualization is carried out using software tools implemented in the program Amira (TGS, San Diego, CA).

Solution Atomic Force Microscopy — A solution of a conjugate of the invention in TBS is deposited on freshly-cleaved mica coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO), incubated for 15 min, rinsed 3^χ with TBS, and imaged in TBS in TappingMode on a Digital Instruments MultiMode atomic force microscope (Veeco, Santa Barbara, CA) with a Veeco NP-S scanning probe.

Conventional Electron Microscopy — Uranyl acetate-stained samples are prepared by deposition of 3 μL of a 0.071 mg/mL solution of of a conjugate of the invention in 50 mM Tris-Cl, pH 7.4 (TBA), on a carbon-coated copper 300-mesh grid (Electron Microscopy Supplies, Fort Washington, PA), followed by mixing with 10 μL of 1 % uranyl acetate (Electron Microscopy Sciences, Hatfield, PA) and drying using filter paper. Images were acquired at 120 kV and 1-μm defocus on a Tecnai 12 thermionic filament electron microscope (FEI Company, OR) equipped with a 4K x 4K CCD operated at 2K x 2K without an energy filter.

Air Atomic Force Microscopy — An IgM solution of 3.3 μg/mL protein in 50 μL of 50 mM Tris-Cl, pH 7.4, (TBA) is incubated on PLL-coated mica for 2 minutes at room temperature, followed by rinsing with 2 mL 0. IX TBA and drying under nitrogen. A solution of a conjugate of the invention of 4.73 μg/mL protein in 50 μL PBS is incubated on PLL-coated mica at room temperature for 10 min, followed by rinsing with 10 mL Milli-Q water and drying under nitrogen. Uranyl acetate (UA) pre-treatment is performed by incubation of 50 μL of protein in TBA on freshly-cleaved mica for 2 minutes at room temperature, followed by rinsing twice with 1 mL of 0. IX TBA. This is followed by mixing of the meniscus with 100 μL of 1% UA. The sample is dried to a thin film by application of the inverted sample to a piece of filter paper, followed by drying under nitrogen. Images re acquired in air TappingMode on a Digital Instruments MultiMode atomic force microscope (Veeco, Santa Barbara, CA) with a SuperSharp silicon scanning probe (NanoSensors, Neuchatel, Switzerland).

Synthetic Examples

Synthesis of a Flexible Linker-Functionalized Dendrimer

To a solution of commercially available Generation 1 poly(amidoamine) (PANAM) dendrimer with amine surface groups (Dendritic Nanotechnologies) in 100 mM PBS, 50 mM EDTA, pH 7.2, is added an 8:1 molar excess Of NHS-PEOi₂-MaI (Pierce), solid, followed by stirring at room temperature for 30 min. Excess unreacted NHS-PEOi₂-MaI is neutralized by addition of a 1 : 1 molar ratio of ethanolamine, methamine, ethamine, or any other primary amine, solid. The dendrimer is now functionalized with an extended, highly flexible linker bearing maleimide surface groups.

Synthesis of a Small Molecule gpl20 Ligand.

The synthesis of a small molecule ligand of the invention is initiated by acylating 7- azaindole with methyl chlorooxoacetate in the presence Of AlCl₃. Hydrolysis of the resulting ester, using K₂CO₃ in aqueous MeOH, was followed by coupling with thep- mercaptobenzoylated piperazine (in turn derived from reaction of piperazine with p- mercaptomethylbenzoate), a reaction mediated by 3-(diethoxyphosphoryloxy)- 1,2,3- benzotriazin-4(3H)-one in the presence of VV,N-diisopropylethlyaniine (¹Pr₂NEt, Ηunig's base) in DMF, to afford an overall yield of 57%. Incorporation of the methoxy substituent at the 4- position of the product is accomplished by a serial process, initiated by oxidation of the nitrogen atom of the heterocycle and followed by nitration with fuming HNO₃ in CF₃CO₂H, which affords the 4-nitro N-oxide . Heating the N-Oxide with an excess of NaOMe in MeOH resulted an ipso displacement of nitrite, setting the stage for the final transformation to the small molecule ligand. This is accomplished in a straightforward fashion by reduction of the N-oxide using PCl₃ in EtOAc. Synthesis of Soluble Recombinant Human CD4

Using Invitrogen Clone ORFEOME Human HORFOl IQHl 1860. human CD4 in vector pENTR(tm)221 (Price $765.00) and standard molecular biological techniques, engineer and express the D1D2 fragment with an additional C-terminal cysteine (a.a. 184). The expression, purification, and verification of activity is carried out in E. coli exactly as described (36), except that 50 mM EDTA is added to all buffers to prevent disulfide formation.

Synthesis of a Small Molecule gpl20 Ligand Conjugate of the Invention

To a solution of commercially-available Generation 2 poly(amidoamine) (PANAM) dendrimer with [poly(ethylene glycol)] ₁₂ surface groups (Dendritic Nano technologies) in 100 mM EDTA, pH 8.5, is added a 16:1 molar excess of (N-[/?-Maleimidophenyl]isocyanate), solid, followed by stirring at room temperature for 15 min. The pH is lowered to 7.2 by the addition of H₄EDTA_, followed by addition of a 16:1 molar excess of the product created in "Synthesis of a Flexible Linker-Functionalized Dendrimer". The small molecule gpl20 ligand conjugate of the invention is synthesized by leaving this solution to react at room temperature for 30 min, and isolate using low-molecular- weight-cutoff (e.g., small pore size) size exclusion chromatography.

Synthesis of Human CD4 Conjugate of the Invention

Human CD4 expressed as described above ("Synthesis of Soluble Recombinant Human CD4") is added in an 8: 1 molar excess to the product solution of the "Synthesis of a Flexible Linker-Functionalized Dendrimer" reaction. The solution is left at room temperature for 10 minutes. The CD4-coηjugate of the invention is isolated using a size exclusion chromatography column, with retention of fractions containing species approximately 100-200 kDa.

Viral Assays

Example A: Neutralization Assay

Conjugates of the invention are tested Neutralization assays against various strains of HIV-I, Clades M, N, O, HIV-2, or SIV, as described in Binley, J. M., Wrin, T., Korber, B., Zwick, M. B., Wang, M., Chappey, C, Stiegler, G., Kunert, R., Zolla-Pazner, S., Katinger, H., Petropoulos, C. J., and Burton, D. R. (2004) J. Virol 78(23), 13232-13252 Example B: HIV Proliferation Assay

Conjugates of the invention are tested in a plaque-forming assay of HIV-I proliferation in a human CD4+lymphocytic cell line (HT-4) by the procedures described in K.Y. Hostetler et al., J. Biol. Chem., 265:6112-6117 (1990); and K.Y. Hostetler et al., J. Biol. Chem., 266:11714-11717 (1991).

Example C: Activity against AZT-resistant and PFA-resistant HIV strains

Selected conjugates of the invention, PFA (Foscarnet) and AZT are tested for activity against three HIV infected cell lines: 1) LAI E89K, a PFA-resistant HIV cell line this strain also has been referred to as 89LAI-Lys; see G. Tachedijian et al., Virology, 70:7171-7181 (1996)); 2) A018-post, an AZT-resistant HIV cell line; and 3) LAl, a wild-type HIV-I cell line (not resistant). The assays are conducted by procedures as disclosed in K. Y. Hostetler et al., J. Biol. Chem., 265:6112-6117 (1990); and K. Y. Hostetler et al., J. Biol. Chem., 266:11714-11717 (1991).

Example D: Animal Models

Conjugates of the invention are tested in animal models to ascertain the toxicity and drug distribution characteristics of the conjugates.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended with be encompassed by the following claims.

References

1. Dalgleish, A. G., Beverley, P. C, Clapham, P. R., Crawford, D. H., Greaves, M. F., and Weiss, R. A. (1984) Nature 312(5996), 763-767

2. Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham, P. R., Weiss, R. A., and Axel, R. (1986) Cell 47(3), 333-348

3. Turner, B. G., and Summers, M. F. (1999) J. MoI. Biol. 285(1), 1-32

4. Kadow, J., Wang, H. G., and Lin, P. F. (2006) Curr. Opin. Investig. Drugs 7(8), 721- 726

5. Daar, E. S., and Ho, D. D. (199I) Am. J. Med. 90(4A), 22S-26S

6. Kwong, P. D., Doyle, M. L., Casper, D. J., Cicala, C, Leavitt, S. A., Majeed, S., Steenbeke, T. D., Venturi, M., Chaiken, I., Fung, M., Katinger, H., Parren, P. W., Robinson, J., Van Ryk, D., Wang, L., Burton, D. R., Freire, E., Wyatt, R., Sodroski, J., Hendrickson, W. A., and Arthos, J. (2002) Nature 420(6916), 678-682

7. Chen, B., Vogan, E. M., Gong, H., Skehel, J. J., Wiley, D. C, and Harrison, S. C. (2005) Nature 433(7028), 834-841

8. Kwong, P. D. (2005) Nature 433(7028), 815-816

9. Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., and Hendrickson, W. A. (1998) Nature 393(6686), 648-659

10. Krummel, M. F., Sjaastad, M. D., Wulfing, C, and Davis, M. M. (2000) Science 289(5483), 1349-1352

11. Singer, 1. 1., Scott, S., Kawka, D. W., Chin, J., Daugherty, B. L., DeMartino, J. A., DiSalvo, J., Gould, S. L., Lineberger, J. E., Malkowitz, L., Miller, M. D., Mitnaul, L., Siciliano, S. J., Staruch, M. J., Williams, H. R., Zweerink, H. J., and Springer, M. S. (200I) J. Virol. 75(8), 3779-3790

12. Arthos, J., Cicala, C, Steenbeke, T. D., Chun, T. W., DeIa Cruz, C, Hanback, D. B., Khazanie, P., Nam, D., Schuck, P., Selig, S. M., Van Ryk, D., Chaikin, M. A., and Fauci, A. S. (2002) J. Biol. Chem. 277(13), 11456-11464

13. Binley, J. M., Wrin, T., Korber, B., Zwick, M. B., Wang, M., Chappey, C, Stiegler, G., Kunert, R., Zolla-Pazner, S., Katinger, H., Petropoulos, C. J., and Burton, D. R. (2004) J. Virol. 78(23), 13232-13252

14. Trkola, A., Pomales, A. B., Yuan, H., Korber, B., Maddon, P. J., Allaway, G. P., Katinger, H., Barbas, C. F., 3rd, Burton, D. R., and Ho, D. D. (1995) J. Virol. 69(11), 6609-6617

15. Chertova, E., Bess, J. W., Jr., Crise, B. J., Sowder Ii, R. C, Schaden, T. M., Hilburn, J. M., Hoxie, J. A., Benveniste, R. E., Lifson, J. D., Henderson, L. E., and Arthur, L. O. (2002) J. Virol. 76(11), 5315-5325

16. Trubey, C. M., Chertova, E., Coren, L. V., Hilburn, J. M., Hixson, C. V., Nagashima, K., Lifson, J. D., and Ott, D. E. (2003) J. Virol. 77(23), 12699-12709

17. Rossio, J. L., Esser, M. T., Suryanarayana, K., Schneider, D. K., Bess, J. W., Jr., Vasquez, G. M., Wiltrout, T. A., Chertova, E., Grimes, M. K., Sattentau, Q., Arthur, L. O., Henderson, L. E., and Lifson, J. D. (1998) J. Virol. 72(10), 7992-8001

18. Winkler, H., and Taylor, K. A. (2006) Ultramicroscopy 106(3), 240

19. Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J. H., Lanier, L. L., and Spies, T. (1999) Science 285(5428), 727-729

20. Kolar, G. R., and Capra, J. D. (2003) Immunoglobulins: structure and function. In: Paul, W. E. (ed). Fundamental Immunology, 5th Ed., Lippincott, Williams, and Wilkins, Philadelphia 21. Hansma, H. G., and Hoh, J. H. (1994) Annu. Rev. Biophys. Biomol. Struct. 23, 115- 139

22. Zhu, P., Liu, J., Bess, J., Jr., Chertova, E., Lifson, J. D., Grise, H., Ofek, G. A., Taylor, K. A., and Roux, K. H. (2006) Nature 441(7095), 847-852

23. Zanetti, G., Briggs, J. A., Grunewald, K., Sattentau, Q. J., and Fuller, S. D. (2006) PLoSPathog. 2(8)

24. Zhu, P., Chertova, E., Bess, J., Jr., Lifson, J. D., Arthur, L. O., Liu, J., Taylor, K. A., and Roux, K. H. (2003) Proc. Natl. Acad. ScL U. S. A. 100(26), 15812-15817

25. Fazel, S., Wiersma, E. J., and Shulman, M. J. (1997) Int. Immunol. 9(8), 1149-1158

26. Zhu, P., Olson, W. C, and Roux, K. H. (2001) J Virol 75(14), 6682-6686

27. Wang, J., Le, N., Heredia, A., Song, H., Redfield, R., and Wang, L. X. (2005) Org. Biomol. Chem. 3(9), 1781-1786

28. Wang, J., Yan, Y., Garrett, T. P. J., Liu, J., Rodgers, D. W., Garlick, R. L., Tarr, G. E., Husain, Y., Reinherz, E. L., and Harrison, S. C. (1990) Nature 348(6300), 411

29. Saphire, E. O., Parren, P. W. H. L, Pantophlet, R., Zwick, M. B., Morris, G. M., Rudd, P. M., Dwek, R. A., Stanfield, R. L., Burton, D. R., and Wilson, I. A. (2001) Science 293(5532), 1155-1159

30. Furtado, P. B., Whitty, P. W., Robertson, A., Eaton, J. T., Almogren, A., Kerr, M. A., Woof, J. M., and Perkins, S. J. (2004) Journal of Molecular Biology 338(5), 921

31. Delano, W. L. (2006) PyMoI. http://pymol.sourceforge. net/

32. Kwong, P. D., Wyatt, R., Sattentau, Q. J., Sodroski, J., and Hendrickson, W. A. (200O) J. Virol. 74(4), 1961-1972

33. Kong, R., Tan, J. J., Ma, X. H., Chen, W. Z., and Wang, C. X. (2006) Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 1764(4), 766

34. Wang, T. et al. J. Med. Chem., 46 (20), 4236 -4239, 2003.

35. Lee, S. C, Parthasarathy, R., Botwin, K., Kunneman, D., Rowold, E., Lange, G., Klover, J., Abegg, A., Zobel, J., Beck, T., Miller, T., Hood, W., Monahan, J., McKearn, J. P., Jansson, R., and Voliva, C. F. (2004) Biomedical Microdevices 6(3), 191

36. Osburne, M. S., Neidhardt, E. A., Godoy, J. E., van Schravendijk, M. R., and Grossman, T. H. (1999) Journal of Immunological Methods 224(1-2), 19

37. N. E. Searle (to E. I. du Pont de Nemours and Co., Inc.) U.S. pat. 2,444,536 (1948) [C. A., 42, 7340c (1948)].

38. A. Michael and J. F. Wing. Am. Chem. J, 7, 278 (1885).

39. R. Anschutz and Q. Wirtz, Ann., 239, 140, 142 (1887).

40. K. Auwers, Ann., 309, 346 (1899).

41. A. E. Kretov and N. E. Kul'chitskaya, Zhur. Obshchei Khim., 26, 208 (1956) [C.A., 50, 13771g (1956)].

42. W. R. Roderick, J. Am. Chem. Soc, 79, 1710 (1957).

43. Hashemi, M. M., and Akhbari, M. (2004) Synthetic Communications 34(15), 2783- 2787

Claims

We claim:

1. A flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a human cell membrane protein or a portion thereof which binds to one or more viral surface proteins of an enveloped virus.

2. A flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a small molecule ligand which binds to one or more viral surface proteins of an enveloped virus.

3. A flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a small molecule ligand which binds to one or more viral surface proteins of an enveloped virus.

4. A flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a human CD4 protein or a derivative thereof.

5. A flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a small molecule ligand which binds to the viral envelope glycoprotein gpl20 of human immunodeficiency virus, simian immunodeficiency virus or simian/human immunodeficiency virus.

6. A flexible antiviral conjugate comprising, a low molecular weight, flexible scaffold; and at least one binding moiety wherein said binding moiety is a small molecule ligand which binds to the viral envelope glycoprotein gpl20 of human immunodeficiency virus, simian immunodeficiency virus or simian/human immunodeficiency virus.

7. The flexible antiviral conjugate of any one of claims 1-6, further comprising a flexible spacer moiety linking the scaffold and the binding moiety.

8. The flexible antiviral conjugate of claim 7, wherein the flexible spacer moiety is a poly(ethylene glycol), a poly(alkanediol) or a human protein functionalized at the N-terminus with a carbodiimide moiety, a bis-imidoeste moiety, a phenolate moiety, an imidazolyl moiety, an acetimidate moiety, a malonimidate moiety, a maleimide moiety or a substituted maleimide moiety.

9. The flexible antiviral conjugate of claim 8, wherein the a poly(alkanediol) is a poly(l,3-propanediol), a poly(l,4-butanediol), or a poly(l,5-pentanediol).

10. The flexible antiviral conjugate of claim 7, wherein the flexible spacer moiety is a attached to an amine surface group of the scaffold using a heterobifunctional linker comprising a maleimide group and an N-hydroxysuccinimide ester.

11. The flexible antiviral conjugate of claim 10, wherein the heterobifunctional linker is succinimidyl 4-[N-maleimidomethyl] -cyclohexane- 1 -carboxylate.

12. The flexible antiviral conjugate of claim 7, wherein the flexible spacer moiety is a heterobifunctional molecule.

13. The flexible antiviral conjugate of claim 12, wherein the heterobifunctional molecule is a maleimide-terminal alkane, a n-hydroxiysuccinimide ester-terminal alkane, a hydroxy- terminal alkane, or a mercapto-terminal alkane.

14. The flexible antiviral conjugate of claim 12, wherein the heterobifunctional molecule comprises a maleimide group and an n-hydroxysuccinimide ester; a poly(ethyleneglycol) comprising an n-hydroxysyccinimide ester at one end; or a substituted maleimide moiety.

15. The flexible antiviral conjugate of claim 14, wherein the heterobifunctional molecule is succinimidyl 4-[n-maleimidomethyl]-cyclohexane-l -carboxylate; succinimidyl-4-[N- Maleimidomethyl]cyclohexane-l-carboxy-[6-amidocaproate], NHS-PEO₂-MaI, NHS-PEO₄- MaI, NHS-PEO₆-MaI, NHS-PEO₈-MaI, NHS-PEO₁₀-MaI or NHS-PEOi₂-MaL.

16. The flexible antiviral conjugate of claim 7, wherein the flexible spacer moiety is a polypeptide

17. The flexible antiviral conjugate of claim 16, wherein the polypeptide is a human IgG Hinge Domain protein modified at the N-terminus with a maleimide or substituted maleimide moiety.

18. The flexible antiviral conjugate of claim 16, wherein the polypeptide has the sequence:

CRDSSRPEARRDVRTNRDSSRPEARRDVRTNRDSSRPEARRDVRTNK (SEQ ID NO: 1);

RDSSRPEARRDVRTN (SEQ ID NO: 2); RDSSRPEARRDVRTNRDSSRPEARRDVRTN (SEQ ID NO: 3);

RDSSRPEARRDVRTNRDSSRPEARRDVRTNRDSSRPEARRDVRTN (SEQ ID NO: 4);

CRDSSRPEARRDVRTNK (SEQ ID NO: 5);

CRDSSRPEARRDVRTNRDSSRPEARRDVRTNK (SEQ ID NO: 6); KRDSSRPEARRDVRTNC (SEQ ID NO: 7);

KRDSSRPEARRDVRTNRDSSRPEARRDVRTNC (SEQ ID NO: 8); AGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMS (SEQ ID NO: 9); CAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSK (SEQ ID NO: 10); KAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSC (SEQ ID NO: 11);

AGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSAGGAKPAPAAAEEK AAPAAAKPATTEGEFPETREKMS (SEQ ID NO: 12);

CAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSAGGAKPAPAAAEE KAAPAAAKPATTEGEFPETREKMSK (SEQ ID NO: 13);

KAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSAGGAKPAPAAAEE KAAPAAAKPATTEGEFPETREKMSC (SEQ ID NO: 14);

AGGARPAPAAAEERAAPAAARPATTEGEFPETRERMS (SEQ ID NO: 15); CAGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSK (SEQ ID NO: 16); KAGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSC (SEQ ID NO: 17);

AGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSAGGARPAPAAAEERA APAAARPATTEGEFPETRERMS (SEQ ID NO: 18);

CAGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSAGGARPAPAAAEER AAPAAARPATTEGEFPETRERMSK (SEQ ID NO: 19); KAGGARPAPAAAEERAAPAAARPATTEGEFPETRERMSAGGARPAPAAAEER AAPAAARPATTEGEFPETRERMSC (SEQ ID NO: 20);

KNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRC (SEQ ID NO: 21);

NTRVDRRAEPRSSDR (SEQ ID NO: 22); NTRVDRRAEPRSSDRNTRVDRRAEPRSSDR (SEQ ID NO: 23);

NTRVDRRAEPRSSDRNTRVDRRAEPRSSDRNTRVDRRAEPRSSDR (SEQ ID NO: 24);

KNTRVDRRAEPRSSDRC (SEQ ID NO: 25);

KNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRC (SEQ ID NO: 26); NTKVDKKAEPKSCDK (SEQ ID NO: 27); NTKVDKKAEPKSCDKNTKVDKKAEPKSCDK (SEQ ID NO: 28);

NTKVDKKAEPKSCDKNTKVDKKAEPKSCDKNTKVDKKAEPKSCDK (SEQ ID NO: 29);

KNTKVDKKAEPKSCDKC (SEQ ID NO: 30); KNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKC (SEQ ID NO: 31);

KNTKVDKKAEPKSCDKNTKVOKKAEPKSCDKNTKVDKKAEPKSCDKC (SEQ ID NO: 32);

CNTKVDKKAEPKSCDKK (SEQ ID NO:33); CNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKK (SEQ ID NO: 34);

CNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKNTKVDKKAEPKSCDKK (SEQ ID NO: 35);

CNTRVDRRAEPRSSDRK (SEQ ID NO: 36); CNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRK (SEQ ID NO: 37);

CNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRNTRVDRRAEPRSSDRK (SEQ ID NO: 38);

KRDSSRPEARRDVRTNRDSSRPEARRDVRTNRDSSRPEARRDVRTNC (SEQ ID NO: 39); or any part, combination or rearrangement thereof.

19. The flexible antiviral conjugate of any one of claims 1 -6, wherein the low molecular weight, flexible scaffold is a dendrimer.

20. The flexible antiviral conjugate of claim 19, wherein the dendrimer is a branched polypeptide, a branched nucleic acid, a branched polyethyleneamine, a branched polysaccharide, a branched polyamidoamine, a branched polyacrylic acid, a branched polyalcohol or a branched synthetic polymer.

21. The flexible antiviral conjugate of claim 20, wherein the dendrimer comprises a poly(amidoamine) polymer having at least one maleimide, hydroxyl, amine, or poly(ethylene glycol) surface group.

22. The flexible antiviral conjugate of claim 21, wherein the dendrimer comprises a poly(amidoamine) polymer having 2-4 maleimide, hydroxyl, amine, or poly(ethylene glycol) surface group.

23. The flexible antiviral conjugate of claim 22, wherein the dendrimer comprises a poly(amidoamine) polymer having at least 4 maleimide, hydroxyl, amine, or poly(ethylene glycol) surface group.

24. The flexible antiviral conjugate of claim 19, wherein the dendrimer comprises two or more poly(amidoamine) polymers each having at least one maleimide surface group linked by a diaminoalkyl linker, a carbodiimide linker, a bis-imidoester linker, a phenolate linker, an imidazolyl linker, an acetimidate linker, a malonimidate linker, a maleimide linker, a substituted maleimide linker or a disulfide linker.

25. The flexible antiviral conjugate of claim 24, wherein the dendrimer comprises two or more poly(amidoamine) polymers each having 2-4 maleimide, hydroxyl, amine, or poly(ethylene glycol) surface groups linked by a diaminobutyl or diaminoalkyl linker.

26. The flexible antiviral conjugate of claim 25, wherein the comprises two poly(amidoamine) polymers each having 4 maleimide, hydroxyl, amine, or poly( ethylene glycol) surface groups linked by a diaminobutyl or diaminoalky linker.

27. The flexible antiviral conjugate of claim 26, wherein each maleimide, hydroxyl, amine, or poly(ethylene glycol) surface group is bound to a flexible spacer moiety which is bound to a binding moiety.

28. The flexible antiviral conjugate of claim 5, optionally further comprising a flexible spacer moiety linking the scaffold and the binding moiety, wherein binding moiety is a small molecule ligand which binds to the viral envelope glycoprotien gpl20 having the formula :

wherein R represents a direct bond to to the scaffold or to the flexible spacer moiety or a linking group suitable to bind to the scaffold or flexible spacer moiety.

29. A pharmaceutical composition comprising at least one flexible antiviral conjugate of claim 1 and a pharmaceutically acceptable diluent or carrier.

30. A pharmaceutical composition comprising at least one flexible antiviral conjugate of claim 2 and a pharmaceutically acceptable diluent or carrier.

31. A pharmaceutical composition comprising at least one flexible antiviral conjugate of claim 3 and a pharmaceutically acceptable diluent or carrier.

32. A pharmaceutical composition comprising at least one flexible antiviral conjugate of claim 4 and a pharmaceutically acceptable diluent or carrier.

33. A pharmaceutical composition comprising at least one flexible antiviral conjugate of claim 5 and a pharmaceutically acceptable diluent or carrier.

34. A pharmaceutical composition comprising at least one flexible antiviral conjugate of claim 6 and a pharmaceutically acceptable diluent or carrier.

35. A pharmaceutical composition comprising at least one flexible antiviral conjugate of claim 7 and a pharmaceutically acceptable diluent or carrier.

36. A pharmaceutical composition comprising at least one flexible antiviral conjugate of any one of claims 8-28 and a pharmaceutically acceptable diluent or carrier.

37. A method for treating an enveloped virus infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of claim 1 or claim 2.

38. A method for treating HIV infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of claim 3.

39. A method for treating HIV infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of any one of claims 4-6.

40. A method for treating HIV infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of claim 3 and a therapeutically effective amount of one or more flexible antiviral conjugates of any one of claims 4-6.

41. A methof for reducing resistance to treatment of an enveloped virus in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of claim 1 or 2.

42. A method for reducing resistance highly active antiretro viral therapy in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of claim 3.

43. A method for reducing resistance to highly active antiretroviral therapy in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of any one of claims 4-6.

44. A method for reducing resistance to highly active antiretroviral therapy therapy in a subject comprising administering to a subject in need thereof a therapeutically effective amount of one or more flexible antiviral conjugates of claim 3 and a therapeutically effective amount of one or more flexible antiviral conjugates of any one of claims 4-6.

45. The method of any one of claims 37-44 further comprising administering a therapeutically effective amount of at least one other agent used for highly active antiretroviral therapy.

46. The method of claim 45, wherein the other agent or agents are nucleoside HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, HIV entry inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, or HIV integrase inhibitors or combinations thereof.

47. The method of claim 37, wherein one or more flexible antiviral conjugates are administered parenterally, transdermally, mucosally, nasally, buccally, sublingually, topically or orally.

48. The method of any one of claims 38 or 42, wherein one or more flexible antiviral conjugates are administered parenterally.

49. The method of any one of claims 39 or 43, wherein one or more flexible antiviral conjugates are administered parenterally, transdermally, mucosally, nasally, buccally, sublingually, topically or orally.

50. The method of any one of claims 40 or 44, wherein one or more flexible antiviral conjugates of claim 3 are administered parenterally; and one or more flexible antiviral conjugates of claim 4 are administered parenterally transdermally, mucosally, nasally, buccally, sublingually, topically or orally.

51. The method of any one of claims 38-40 or 42-44 wherein the therapeutically effective amount is from about 0.01 mg to about 5,000 mg per day.

52. The method of claim 51 wherein the therapeutically effective amount is from about 1 mg to about 2,500 mg per day.

53. The method of claim 52 wherein the therapeutically effective amount is from about 10 mg to about 1,200 mg per day.

54. The method of any one of claims 38-40 or 42-44, wherein the subject is a human.

55. The method of claim 54, wherein the human is showing one or more symptoms of resistance to highly active antiretroviral therapy.