WO2012100835A1 - Methods and compositions for the treatment of aids - Google Patents

Abstract

The application relates to a method for the treatment of a disease caused by HIV infection by administering to a patient in need thereof of a specific inhibitor of the dehydroceramide desaturase. The invention relates as well to compositions comprising a Des-1inhibitor and one or more drugs suitable for the treatment of diseases caused by HIV infection as well as to methods for the identification of compounds suitable for the treatment of said diseases.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF AIDS

FIELD OF THE INVENTION

The invention relates to the field of therapeutics and, more in particular, to agents useful for the treatment of diseases caused by HIV infection based on preventing the entry of the virus to the target cell.

BACKGROUND OF THE INVENTION

Within the last decades HIV-1 (human immunodeficiency virus type 1) infection spread around the globe, so that today more than 40 million people throughout the world are infected by the virus. HIV-1 infection leads to a substantial loss of CD4+ T lymphocytes, resulting in the destruction of the cellular immune system and, ultimately, in the development of acquired immunodeficiency syndrome (AIDS).

Antiviral drug therapy can effectively suppress HIV-1 replication, and thereby preserve immune functions and prolong survival of HIV-infected patients, but drug toxicity and viral resistance limit the long-term therapeutic efficacy. Thus, as a vaccine is not in sight, novel therapeutic approaches are urgently needed.

Fusion of viral and target cell membranes initiates HIV-I infection. Conformational changes in gpl20 that accompany its binding to receptor (CD4) and co- receptor (e.g. CCR5 or CXCR4) lead to dissociation of gpl20 from gp41 and a cascade of refolding events in the latter. See Harrison S, et al, Adv. Virus Res. 2005; 64:231- 261. In the course of these rearrangements, the N-terminal "fusion peptide" of gp41 translocates and inserts into the target cell membrane. A proposed extended conformation of the gp41 ectodomain, with its fusion peptide thus inserted and the transmembrane anchor still in the viral membrane has been called the "prehairpin intermediate". See Chan D, Kim P, Cell 1998; 93:681-684.

The prehairpin intermediate is the target of various fusion inhibitors, including Fuzeon/T-20/Enfuvirtide, the first approved fusion-inhibiting antiviral drug. See Kilby J, Eron J, N. Engl. J. Med. 2003; 348:2228-2238. Fuzeon is a 36-residue C-peptide that binds to the N-trimer groove, but not the pocket. See Wild C, et al, Proc. Natl. Acad. Sci. USA 1994; 91 :9770-9774 and Rimsky D, et al, J. Virol. 1998; 72:986-993. Although a significant breakthrough, Fuzeon has several serious limitations that have hampered its widespread clinical adoption, including high dosing requirements (90 mg, twice daily via injection), high cost (greater than $25,000 per patient per year), and the emergence of resistant strains both in vitro and in patients. See Rimsky, 1998, supra and Wei X, et al, Antimicrob. Agents Chemother. 2002; 46(6): 1896-1906. These problems have limited Fuzeon's clinical use to patients with multidrug resistant HIV-I (salvage therapy).

However, at present, all of these inhibitors suffer from limited potency and/or toxicity in standard viral infectivity or cell-cell fusion assays. Accordingly, there is a need for additional inhibitors of HIV entry functioning at preventing the entry of the virus in the cell which do not suffer from the drawbacks of the compounds known to date.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a method for the treatment of a disease caused by HIV infection in a subject in need thereof comprising the administration to a subject of a dihydroceramide desaturase (Des-1) inhibitor.

In a second aspect, the invention relates to a composition or kit-of-parts comprising, together or separately, at least a Des-1 inhibitor and at least a drug used for the treatment of a disease caused by HIV infection.

In another aspect, the invention relates to a method for the treatment of a disease caused by HIV infection in a subject in need thereof comprising the administration to said subject of a composition or kit-of-parts according to the invention.

In another aspect, the invention relates to an in vitro method for inhibiting HIV infection which comprises contacting target cells with a Des-1 inhibitor under conditions effective to provoke an increase of DHSM in the membranes of said cells.

In yet another aspect, the invention relates to a method for the identification of compounds suitable for the treatment of a disease caused by HIV-1 infection which comprises:

(i) contacting a candidate compound with a preparation containing Des-1 and

(ii) determining the activity o f Des- 1 , wherein a decrease in the activity of Des-1 with respect to a control preparation is indicative that the candidate compound is suitable for the treatment of a disease caused by HIV- 1 infection.

In another aspect, the invention relates to a method for the identification of compounds suitable for the treatment of a disease caused by HIV-1 infection which comprises:

(i) contacting a candidate compound with a cell population,

(ii) isolating a sphingolipid-enriched detergent-resistant membrane from the cell population, and

(iii) d et e rminin g th e r e l ativ e amo unt o f di hy dro c er ami d e o r dihydrosphingosine with respect to ceramide or sphingosine, wherein an increase in the ratios of dihydroceramide to ceramide and/or dihydrosphingosine to sphingosine with respect to a sphingolipid-enriched detergent- resistant membrane preparation isolated from a control cell population is indicative that the candidate compound is suitable for the treatment of a disease caused by HIV-1 infection.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. DHSM forms gel- like domains in GUV. (A) Temperature variation of the GP of Laurdan embedded in phospholipid bilayers with indicated lipid compositions. Mean values ± SEM are shown (n = 3); in some cases, error bars are smaller than symbol size. (B) Representative confocal images (equatorial sections) of PC:PE:SM:Chol and PC:PE:DHSM:Chol GUV (1 : 1 : 1 : 1 , mol ratio) stained with NBD- Cer (left) and Dil probes (right). Bar = 10 fm. (C) Ratio of NBD-Cer fluorescence intensity measured in the Dil dark to bright regions of SM- and DHSM-GUV as in (B). Mean values ± SEM (n = 12). (D) Representative confocal images (equatorial section) showing di-4-ANEPPDHQ probe distribution in /₀ and Id fluid domains of PC:PE:SM:Chol and PC:PE:DHSM:Chol GUV. Individual channels and merge images are shown. Bar = 10 fm. (E,F) Emission spectra from /₀ and Id domains of PC :PE : SM : Chol (E) PC :PE :DHSM : Chol (F) GUV, represented as normalized fluorescence intensity at the different wavelengths. (G) DSC thermograms of the quaternary lipid mixtures (PC:PE:Chol:SM or DHSM, 1 : 1 : 1 : 1 mol ratio) dispersed in the form of MLV. The endotherm observed with the DHSM-MLV corresponds to a thermotropic phase transition with ΔΗ = 0.49 kcal/mol DHSM.

Figure 2. Des-1 inhibition replaces SM with DHSM in cell membranes. (A)

Chemical structure of the GT1 1 and GTl lpyr inhibitors used. (B) Effect of GTl lpyr doses indicated on viable HEK-293 cell number determined by trypan blue exclusion; determinations were performed by triplicate for each condition (n = 3). (C) Effect of GTl lpyr on cell cycling determined by propidium iodide. A representative experiment of three is shown. (D) GTl 1 (open symbols) and GTl lpyr (solid symbols) inhibition of Des-1 activity in whole cell lysates of HEK-293 (black) and TMZ-b l (red) using DHCer6-NBD as substrate. (E) Representative chromatograms obtained by selecting the exact masses of C 16-SM [(SM+H)⁺: 703.5749 amu; chromatograms b, d) and C16- DHSM [(DHSM+H)⁺: 705.591 1 amu; chromatograms a, c) in data acquired with UPLC/TOF from lipid extracts of vehicle- (chromatograms a, b) or GTl lpyr-treated cells (chromatograms c, d). Retention times (min) and peak areas are indicated. (F) GTl lpyr- induced dose-dependent changes in the DHSM/SM ratio calculated from chromatograms in (B). Data are the mean ± SD of three replicates.

Figure 3. Increased DHSM levels by Des-1 inhibition reduces HIV-1 infection. (A) TZM-bl cells pretreated with indicated GTl lpyr doses (24 h) were exposed to luciferase (luc)-encoding HIV-1 viruses pseudotyped with R5, X4 and VSV- G envelopes. Luc activity was recorded to quantify cell infection. (B) TZM-bl cells were incubated with GT1 1 (0.1 to 3 μΜ) for 24 h (solid symbols) or 5 days (open symbols), and then infected with replication-competent HIV- INFN SX viruses. Cells were assayed for luciferase activity 48 h later. Values indicate mean ± SEM of a representative experiment. * p<0.02, **p<0.001 unpaired Student's t-test.

Figure 4. Des-1 silencing inhibits HIV-1 infection. (A) TZM-bl cells were transfected with vehicle (mock), mismatched or Des-1 -specific siRNA; after 4 days, Des-1 protein levels were analyzed in cell extracts (20 μg/lane). The same membranes were probed with an anti- -actin antibody as loading control. A representative experiment is shown (n = 3). (B) Des-1 activity was assayed in total cell extracts of TZM-bl cells as in (A), using DHCer6-NBD as substrate. (C) The same cells as in (A) were pulsed with HIV- INFN SX virus (10 ng p24) and infectivity recorded by luciferase activity; each point represents the mean ± SEM of four replicates (n = 2) . * p<0.02 unpaired Student's t-test.

Figure 5. GTllpyr treatment does not affect HIV-1 receptor clustering. (A)

The same gradient fractions used in Fig. 2E were analyzed in immunoblot with the indicated antibodies. The relative abundance of the proteins in DRM (lane 1 ) and soluble (lane 5) fractions is indicated. A representative experiment is shown (n = 3). (B) Colocalization of CD4, CXCR4 and GFP-GPI (all stained in green) after antibody- induced co-patching (12°C) with the cholera toxin (CTx)- subunit (red) in HEK-293 cells. Representative merged images are shown (n = 15 for each protein). (C-E) Antibody induced co-clustering of gpl20Env IIIB (X4 strain, red) with CD4 (green) and CXCR4 (blue) in PBMC. Representative cells are shown (C, n = 10). Images were processed with ImageJ particle analyzer to determine the number (D) and area (E) of the clusters formed. Bar = 5 μιη.

Figure 6. gp41 insertion is impaired in DHSM-rich membranes. (A) HIVc- induced fusion of PC:PE:Chol:SM or DHSM (1 : 1 : 1 : 1 mol ratio) LUV. Intervesicular lipid mixing is shown as a function of the peptide-to-lipid ratio at 25°C (solid symbols and line) and 50°C (open symbols, dashed lines). Data points are the increase in NBD fluorescence (% after 30 min) measured at 530 nm (excitation at 465 nm) with a cut-off filter at 5 15 nm. Lipid concentration in all assays was 100 μΜ. (B) HEK-293-CD4 target cells were pretreated with GTl 1 at the indicated concentrations before co-culture with gpl60IIIB-expressing effector cells. Cell-cell fusion was quantified by measurement of luc activity in cell lysates. Values are mean ± SEM of triplicates in one representative experiment (n = 4). DETAILED DESCRIPTION OF THE INVENTION

Therapeutic methods of the invention

The authors of the present invention have found that, surprisingly, increasing DHSM levels in lipid membranes resulting from the inhibition of the activity of dihydroceramide desaturase (Des-1) results in an inhibition of cell infection by replication-competent and -deficient HIV-1. See example 3. As shown in example 5 of the present invention, the rigidification of the membranes as a consequence of the increase in the relative levels DHSM decreases the gp41 insertion into the membranes. This finding opens the possibility for improved therapies for HIV infections based on Des-1 inhibitors.

Thus, in a first aspect, the invention relates to a method for the treatment of disease a caused by HIV infection a subject in need thereof which comprises the administration to said patient a dihydroceramide desaturase inhibitor.

The term "dhydroceramide desaturase" , a s us e d herein , i s utilized interchangeably with "Des-1 ", "DEGS" or "sphingolipid delta4 desaturase" (IPI of human DEGS: IPI00021 147.1). Human Des-1 is located on chromosome lq42.12. Des- 1 converts dihydroceramide into ceramide in the sphingosine-ceramide pathway. The corresponding gene encodes a member of the membrane fatty acid desaturase family which is responsible for inserting double bonds into specific positions in fatty acids. The protein is predicted to be a multiple membrane-spanning protein localized to the endoplasmic reticulum.

A related enzyme dihydroceramide desaturase 2 (Des-2) has bifunctional delta-4 desaturase and hydroxylase activities. In a preferred embodiment, the inhibitors for use according to the present invention are Des-1 specific.

The term "dihydroceramide desaturase (Des-1) inhibitor", as used herein, refers to any molecule or combination of molecules which results in a decrease in the activity of Des-1 by either decreasing the cellular levels of the mRNA encoding Des-1 or by decreasing the levels of Des- 1 protein or by decreasing the enzymatic activity of Des- 1.

Compounds leading to reduced Des-1 mRNA levels can be identified using standard assays for determining mRNA expression levels such as RT-PCR, RNA protection analysis, Northern blot, in situ hybridization or microarray technology.

Compounds leading to reduced Des-1 protein levels can be identified using standard assays for determining protein expression levels such as Western-blot or Western transfer, ELISA (enzyme- linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS- ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, based on the use of protein biochips or microarrays which include specific antibodies or assays based on colloidal precipitation in formats such as dipsticks. Compounds leading to reduced Des-1 enzymatic activity can be identified by using assays based on the quantification of the fluorescent fatty acid released from 12- (4-nitro-benzo-2-oxa-l,3-diazolo)dodecylsphingosine (Cer-C12NBD). See Nikolova- Karakashian M, et al., Methods Enzymol. 2000; 311 : 194-201 and Bedia C, et al., Org. Biomol. Chem. 2005; 3:3707-3712. The released fatty acid can be separated from the substrate by either HPLC or ion exchange chromatography.

Preferred Des-1 inhibitors for use according to the present invention include, without limitation, the molecules depicted in Table 1.

Table I : DON- I inh ibitors su itable for use in the present iin ent ion

Cyclopropenylceramide as desaturase inhibitors as described in EP1354870A characterised by the general formula I:

wherein

P i and R₂ that can be the same or different, represent aryl, heteroaryl, alkyl or acyl groups with one or more unsaturations in the chain that can also be branched or straight and can have or not have substitutions;

P 3 can be an alkyl, alkenyl, alkynyl, aryl or any heterocyclic group and

n is an integer that can have any value including 0 and can be branched or straight, with or without substitutions and contain unsaturated carbons on the chain.

Preferably, Ri is H or -CH3;

R₂ is selected from the group consisting of:

- CO(CH₂)₁_₇CH₃

- CO(CH₂)_!_₇NC₅H₅ Table I : Dcs- I inhibitors suitable for use in the present iin ent ion

-COCOC₆H₅ or

-CO-(CH₂)₄-CH₃

-CO-(CH₂)₈-CH₃ and/or

Also preferably, Ri and R₂ are independently selected from the group consisting of:

- CO(CH₂)_!_₇CH₃

- CO(CH₂)₁_₇NC₅H₅

-COO(CH₂)_!_₇CH₃

-COCOC₆H₅ or

-CO-(CH₂)₄-CH₃

-CO-(CH₂)₈-CH₃

More preferably, the compound of general formula I is selected from the group consisting of GT1 1 and GT1 lpyr having the structures: Table .1 : Dcs-1 inhibitors suitable for use in the present invention

More prefera y, the compound of general formula I are GTl l derivatives as described by Triola G, et al, J. Org. Chem. 2003; 68:9924-9932 wherein:

Ri is H, R₂ is -COCO(CH₂)₄CH₃, R3 is - (CH₂)i_i₂CH₃ and n=0

(n-hexanoyl derivative) and

Ri is H, R₂ is -COCO(CH₂)₈CH₃, R3 is - (CH₂)i_i₂CH₃ and n=0 (N-decanoyl derivative),

II A compound having the structure:

as described by Brodesser S, Kolter T, J. Lipids 2001 ; Article ID 724015 (doi: 10.1155/2011/724015)

III 4-HPR (also known as N-(4-hydroxyphenyl)retinamide or fenretinide) (CAS 65646-68-6) as described by Zheng L, et al., Biochim. Biophys. Acta. 2006; 1758: 1864-1884 and Kraveka J, et al, J. Biol. Chem. 2009; 282: 16718-16728,

IV Resveratrol as described by Szkudelski T, et al, Eur. J. Pharmacol. 2006;

552: 176-181,

V N-acetylcysteine as described by Michel C, et al, J. Biol. Chem. 1997;

272:22432-22437, Table .1 : Dcs-1 inhibitors suitable for use in the present invention

VI Thiol reagents and, in particular, DTT, as described by Michel, 1997, supra,

VII XM462 as described by Munoz Olaya J, et al, ChemMedChem 2008; 3, 946-953 and having the structure:

CH3 C

VIII Celecoxib (4-(5-(4-Methylphenyl)-3-(trifluoromethyl)-lH-pyrazol - 1 yl)benzenesulfonamide or CAS 169590-42-5) as described by Schiffmann S, et al, J. Lipid. Res. 2008; 50:32-40 having the structure:

IX 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole as described by French K, et al, Cancer Res. 2003; 63:5962-5969,

X 3-(4-hydroxyphenyl)-2H-l-benzopyran-7-ol (also known as phenoxodiol or

idronoxil) (CAS 81267-65-4) and its analogs, Table .1 : Dcs-1 inhibitors suitable for use in the present invention

XI Vitamin E analogs having the general structure:

wherein

Rl is a hydrogen atom or a methyl group

R2 is H

R3 and R4 are identical or not, and are each independently a hydrogen, a hydroxyl, a C1-C4 alkyl, a C1-C4 alkoxy or a C1-C4 alkanoloxy

Q is

CH₃

Preferably, the vitamin E analog corresponds γ-tocopherol which corresponds to the above structure wherein Rl and R2 and H, and R3 and R4 are methyl groups having the structure:

XII An inhibitory antibody capable of specifically binding and inhibiting the activity of Des-1,

XIII A ribozyme or a DNA enzyme specific for the sequence of a Des-1, Table .1 : Dcs-1 inhibitors suitable for use in the present invention

XIV An interfering RNA specific for the sequence of a Des-1, such as the siR A described in EP1786442-A2, having a sequence selected from the group of:

5 ' -UGUGGAAUCGCUGGUUUGG-3 ' (SEQ ID NO: i )

5' -GUUAUCAAUACCGUGGCAC-3' (SEQ ID NO: 2)

5' -CCUUCAAUGUGGGUUAUCAtt-3' (SEQ ID NO: 3),

XV An inhibiting peptide specific for a dihydroceramide desaturase or

XVI A dihydroceramide desaturase-specific antisense nucleic acid.

Small organic compounds (I to XI)

Des-1 inhibitors for use according to the present invention as defined in positions I to XI of Table 1 are compounds wherein the active agent is a small organic compound.

The terms "active agent", "pharmacologically active agent" and "drug" are used in the present invention to include the active drug compound as well as any of its pharmaceutically acceptable salts, esters, amides, prodrugs, metabolites, analogs, solvates, hydrates, enantiomers, polymorphs, and derivatives.

The term "small organic compound", as used herein, refers to a compound of molecular weight less than about 5000, usually less than about 2500, usually, less than about 2000, more usually, less than about 1500, preferably about 100 to about 1000, more preferably about 300 to about 600. The small organic compounds, as the term is used herein, include also peptides, oligonucleotides, polysaccharides, fatty acids and lipids having a molecular weight less than about 5000. The term refers as well to isomers, homologs, analogs, derivatives, enantiomers of the above and/or functionally equivalent compositions thereof.

"Analog" as used herein, refers to a small organic compound, a nucleotide, a protein, or a polypeptide that possesses similar or identical activity or function(s) as the compound, nucleotide, protein or polypeptide or compound having the desired activity and therapeutic effect of the present invention.

The terms "salt" or "pharmaceutically acceptable salt" as used herein refer to salts which are known to be non-toxic and are commonly used in pharmaceutical practice. Such pharmaceutically acceptable salts include but are not limited to metal salts, salts with organic bases and salts with basic amino acids. Metal salts include, for example, alkali metal salts, such as sodium and potassium salts, and alkaline earth metal salts, such as calcium, magnesium and barium salts. Salts with organic bases include, for example, salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine and N,N-dibenzylethylenediamine groups. Salts with basic amino acids include, for example, salts with arginine and lysine. Acid addition salts such as hydrochloride, sulfate, and succinate salts, and similar salts are also included within the scope of the present invention.

The term "alkyl", as used herein, denotes a saturated or unsaturated, branched or unbranched and optionally substituted hydrocarbon radical of 1-24 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl and tetracosyl, as well as cycloalkyl groups such as cyclopentyl and cyclohexyl.

The term "alkenyl" as used herein refers to a branched or unbranched hydrocarbon group typically although not necessarily containing 2 to about 24 carbon atoms and at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, and the like. Generally, although not necessarily, alkenyl groups herein contain 2 to about 12 carbon atoms. The term "lower alkenyl" refers to an alkenyl group of 2 to 4 carbon atoms.

The term "alkynyl" as used herein refers to a branched or unbranched hydrocarbon group typically although not necessarily containing 2 to about 24 carbon atoms and at least one triple bond, such as ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, octynyl and decynyl.

In general, the terms aryl and heteroaryl, as used herein, refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. The term heteroaryl, as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl and isoquinolinyl.

The term "acyl" as used herein refers to a group having the general formula - C(0)R, where R is alkyl, alkenyl, alkynyl, aryl, carbocyclic, heterocyclic, or aromatic heterocyclic.

The term "heterocycle," "heterocyclyl," or "heterocyclic," refers to a stable 5- to 7- membered monocyclic or stable 8- to 1 1-membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The term heterocycle or heterocyclic includes heteroaryl moieties.

In preferred embodiments, n is an integer having a value of 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2, 1 or 0.

In a preferred embodiment the dihydroceramide desaturase inhibitor is a cyclopropene-containing sphingolipid as defined in Table 1 (I). More preferably, the Des-1 inhibitor is a compound having the general structure:

wherein R_{l s} R₂ and R₃ are as follows:

'^* Numbering as in Triola G, et al , J. Org. Chem. 2003; 68:9924-9932.

The invention also provides the use of variants of the general formula I wherein both Rl and R2 are independently selected from the group consisting of:

- -(CH₂)₆-CH₃

-CONH-(CH₂)₇-CH₃

-CSNH-(CH₂)₇-CH₃

-COCO-CH₂-CH₃

-COCO-(CH₂)₅-CH₃

-COCO-C₆H₅

-CO-(CH₂)₄-CH₃

-CO-(CH₂)₈-CH₃

Anti Des-1 inhibitory antibodies (IX)

Antibodies against Des-1 may effectively inhibit the function of this protein and, therefore, can be used in the methods according to the present invention. An "inhibitory antibody", as used herein, refers to antibodies which are capable of inhibiting at least partially the biological activities of the dihydroceramide desaturase.

The term "antibody of the invention" includes, for example, polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.

In general all the resultant molecules retain almost one variable domain of an antibody which gives the high specificity and affinity characteristic of the antigen- antibody binding. Some examples of these constructions are:

Chimeric antibodies: These refers to antibodies constructed with variable regions from an antibody of some species (normally a mammal in which the mAb was generated) and constant regions of other species (the one in which the chimeric antibody is to be used);

Humanized antibodies: By "humanized antibody" is meant an antibody derived from a non-human antibody, typically a murine antibody, that retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans.

Primatized antibodies: A next step in this approach of making an antibody more similar at humans' are the so called primatized antibodies, i.e. a recombinant antibody which has been engineered to contain the variable heavy and light domains of a monkey (or other primate) antibody, in particular, a cynomolgus monkey antibody, and which contains human constant domain sequences, preferably the human immunoglobulin gamma 1 or gamma 4 constant domain (or PE variant). See Newman D, et al, Biotechnology 1992; 10: 1458-1460; Newman R, et al, US 5,658,570 and Anderson D, et al, US 6,1 13,898. These antibodies have been reported to exhibit a high degree of homology to human antibodies (i.e. 85-98%) display human effector functions, have reduced immunogenicity, and may exhibit high affinity to human antigens. Another highly efficient means for generating recombinant antibodies is disclosed by Newman, 1992, supra. - Human antibodies: By "human antibody" is meant an antibody containing entirely human light and heavy chain as well as constant regions, produced by any of the known standard methods. As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g. mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

Antibody fragments: An antibody fragment is a fragment of an antibody such as, for example, Fab, F(ab')2, Fab' and scFv. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies but more recently these fragments can be produced directly by recombinant host cells. In other embodiments, the antibody of choice is a single chain Fv (scFv) fragment which additionally may be monospecific or bispecific. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, which name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen- binding sites and is still capable of cross-linking antigen. "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH- VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab')Z antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. See Rosenburg M and Moore G, Eds., "Pharmacology of Monoclonal Antibodies", Vol. 113, p. 269-315 (Springer- Verlag, New York, NY, USA 1994). The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. See Whitlow M, et al, WO 1993011161 and Hollinger P, et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448.

Bispecific antibodies: Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression o f two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities. See Millstein C, et al, Nature 1983; 305:537-539. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. See Wabl M, et al, WO 1993008829 and Traunecker A, et al, EMBO J. 1991; 10:3655-3659.

Pes- 1 -specific ribozymes (X) Ribozyme molecules designed to catalytically cleave a target mRNA transcripts can also be used to prevent the translation of dihydroceramide desaturase mRNAs. Accordingly, in another embodiment, the compositions of the invention comprise ribozymes specifically directed to the dihydroceramide desaturase mRNA. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA and include, without limitation, hammerhead ribozymes, RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA). See Rossi J, Curr. Biol. 1994; 4:469-471 ; Zaug A, et al, Science 1984; 224:574-578 ; Zaug A, et al, Science 1986; 231 :470-475; Zaug A, et al, Nature 1986; 324:429-433, Cech T, et al, WO 198804300 and Been M, et al, Cell 1986; 47:207-216.

Pes- 1 -specific RNA interference (RNAi) (XI)

In another embodiment, the inhibitors of the dihydroceramide desaturase that form part of the compositions of the invention are RNAi which are capable of knocking down the expression of a dihydroceramide desaturase or any component gene necessary for a dihydroceramide desaturase function.

The double stranded oligonucleotides used to attain RNAi are preferably less than 30 base pairs in length and, more preferably, comprise about 25, 24, 23, 22, 21, 20, 19, 18 or 17 base pairs of ribonucleic acid. Optionally the dsRNA oligonucleotides of the invention may include 3' overhang ends.

The specific sequence utilized in design of the oligonucleotides may be any contiguous sequence of nucleotides contained within the expressed gene message of the target. Programs and algorithms, known in the art, may be used to select appropriate target sequences. In addition, optimal sequences may be selected utilizing programs designed to predict the secondary structure of a specified single stranded nucleic acid sequence and allowing selection of those sequences likely to occur in exposed single stranded regions of a folded mRNA. Methods and compositions for designing appropriate oligonucleotides are known in the art. See Shannon K, et al, US 6,251,588.

Several different types of molecules have been used effectively in the RNAi technology including: Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology.

MicroRNA (miRNA) are a related class of gene regulatory small RNAs, typically 21-23 nt in length. They typically differ from siRNA because they are processed from single stranded RNA precursors and show only partially complementary to mRNA targets.

Short hairpin RNA (shRNA) is yet another type of RNA that may be used to effect RNAi. It is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression. shRNA is transcribed by RNA polymerase III.

Illustrative, non-limitative, examples of interfering RNA specific for the sequence of a dihydroceramide desaturase include, without limitation, the interfering RNAs having the sequence selected from the group consisting of:

5' -UGUGGAAUCGCUGGUUUGG-3' (SEQ ID NO: l)

5' -GUUAUCAAUACCGUGGCAC-3' (SEQ ID NO: 2)

5' -CCUUCAAUGUGGGUUAUCAtt-3' (SEQ ID NO: 3)

Dihydroceramide desaturase specific-inhibiting peptides (XII)

A "diydroceramide desaturase specific-inhibiting peptide" as used herein, relates to a peptide that biologically mimics regions of the protein sequence of the dhydroceramide desaturase that are involve in the dhydroceramide desaturase activity and thus they would inhibit the physiological activity of the dhydroceramide desaturase.

The screening and design of a dhydroceramide desaturase specific-inhibiting peptide have been described in the art. See Olson G, et al., J. Med. Chem. 1993; 36: 3039-3049 and Gillespie P, et al, Biopolymers 1997; 43: 191-217.

Dihydroceramide desaturase -specific antisense nucleic acids (XIII)

In a further embodiment, the invention relates to the use of the isolated "antisense" nucleic acids to inhibit Des-1 expression (e.g. by inhibiting the transcription and/or translation of the dihydroceramide desaturase coding nucleic acids). The antisense nucleic acids may bind to the potential drug target by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, these methods refer to the range of techniques generally employed in the art, and include any methods that rely on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a dhydroceramide desaturase polypeptide. Alternatively, the antisense construct is an oligonucleotide probe, which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of a target nucleic acid. Such oligonucleotide probes are preferably modified oligonucleotides, resistant to endogenous nucleases, such as for example, exonucleases and endonucleases, and are, therefore, stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are DNA phosphoramidate, phosphothioate and methylphosphonate analogs. See Hogan M, Kessler D, US 5,176,996; Matteucci M, US 5,264,564 and Froehler B, US 5,256,775. Additionally, general approaches to constructing oligomers useful in antisense therapy have been widely reviewed. See Van der Krol A, et ah, BioTechniques 1988; 6:958-976 and Stein C, et ah, Cancer Res. 1988; 48:2659-2668.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxytiethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl- 2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2- fluoroarabinose, xylulose, and hexose. The antisense oligonucleotide can also contain a neutral peptide-like backbone. Such molecules are termed peptide nucleic acid (PNA)- oligomers. See Perry-O'Keefe X, et al, Proc. Natl. Acad. Sci. USA 1996; 93: 14670- 14675. One advantage of PNA oligomers is their capability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of the DNA. In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or an analog thereof.

In yet a further embodiment, the antisense oligonucleotide is an alpha-anomeric oligonucleotide. An alpha-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual antiparallel orientation, the strands run parallel to each other. See Gautier C, et al, Nucl. Acids Res. 1987; 15:6625-6641. The oligonucleotide could be a 2'-0-methylribonucleotide or a chimeric RNA-DNA analog. See Inoue H, et al, Nucl. Acids Res. 1987; 15:6131-6148 and Inoue H, et al, FEBS Lett. 1987; 215: 327-330.

In certain embodiments, the antisense oligonucleotides are morpholino antisenses.

The Des-1 inhibitors for use in the methods of the present invention are used then in amounts which are effective to achieve a therapeutic effect in a disease caused by HIV infection.

"Therapeutic effect" as used herein means the amount of a composition that alone, or together with an additional therapeutic agent(s) (for example nucleoside/nucleotide reverse transcriptase inhibitors, a non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion/entry inhibitors or integrase inhibitors) induces the desired response. In reference to the treatment of HIV, a therapeutic effect refers to one or more of the following: 1) reduction in the number of infected cells; 2) reduction in the number of virions present in serum; 3) inhibition (i.e., slowing to some extent, preferably completely) the rate of HIV replication; 4) relieving or reducing to some extent one or more of the symptoms associated with HIV; and 5) relieving or reducing the side effects associated with the administration of other antiretro viral agents. In one example, a desired response is to inhibit HIV replication in a cell to which the therapy is administered. HIV replication does not need to be completely eliminated for the composition to be effective. For example, a composition can decrease HIV replication by a desired amount, for example by at least 10 percent, at least 20 percent, at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, at least 95 percent, at least 98 percent, or even at least 100 percent (elimination of HIV), as compared to HIV replication in the absence of the composition. In another example, a desired response is to inhibit HIV infection. The HIV infected cells do not need to be completely eliminated for the composition to be effective. For example, a composition can decrease the number of HIV infected cells by a desired amount, for example by at least 10 percent, at least 20 percent, at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, at least 95 percent, at least 98 percent, or even at least 100 percent (elimination of detectable HIV infected cells), as compared to the number of HIV infected cells in the absence of the composition.

Effective amounts of the dihydroceramide desaturase inhibitor will depend upon the mode of administration, frequency of administration, nature of the treatment, age and condition of the individual to be treated, and the type of pharmaceutical composition used to deliver the compound into a living system. Effective levels of dhydroceramide desaturase blocker may range from 50 nM to 5 μΜ (given to experimental animals as 20-30 mg/kg twice daily for ten days), depending upon the compound, system, experimental and clinical endpoints, and toxicity thresholds. While individual doses vary, optimal ranges of effective amounts may be determined by one of ordinary skill in the art. For dehydroceramide desaturase inhibitors that are involved in clinical trials for other indications, the safe and effective dosages identified in such trials can be considered when selecting dosages for treatments according to the present invention.

The term disease caused by HIV-1 infection, as used herein, refers to any disease which results from the infection by HIV of one or more cellular types. Most preferably, the disease is selected from the group consisting of Acquired Immune Deficiency Syndrome (AIDS) or the HIV- or HAA T-associated disorders HIV-associated dementia (HAD), Immune Reconstitution Disease (IRD) and lipodystrophy.

All forms of HIV-I are potentially treatable with compounds of the present invention. The compounds of the present invention are useful for treating protease inhibitor resistant HIV, reverse transcriptase inhibitor resistant HIV, and entry/fusion inhibitor resistant HIV. These compounds are also useful in the treatment of HIV groups M, N, and O; HIV-I subtypes, including the Al, A2, B, C, D, Fl, F2, G, H, and J subtypes; and circulating recombinant HIV forms. The compounds of the present invention are useful for treating CCR5 tropic HIV strains as well as CXCR4 tropic HIV strains.

The dihydroceramide desaturase inhibitor used according to the methods of the present invention can be administered alone or as a pharmaceutical composition, which includes the compound(s) and a pharmaceutically-acceptable carrier. The pharmaceutical composition can also include suitable excipients, or stabilizers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions. Typically, the composition will contain from about 0.01 to 99 percent, preferably from about 5 to 95 percent of active compound(s), together with the carrier.

The dihydroceramide desaturase inhibitor, when combined with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers, whether in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions, can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocular ly, intraarterially, intralesionally, transdermally, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes (i.e. inhalation).

For most therapeutic purposes, the dihydroceramide desaturase inhibitor can be administered orally as a solid or as a solution or suspension in liquid form, via injection as a solution or suspension in liquid form, or via inhalation of a nebulized solution or suspension. The solid unit dosage forms can be of the conventional type. The solid form can be a capsule, such as an ordinary gelatin type containing the compounds of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.

For injectable dosages, solutions or suspensions of these materials can be prepared in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose, and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

For use as aerosols, the compound in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.

For transdermal routes, the compound is present in a carrier which forms a composition in the form of a cream, lotion, solution, and/or emulsion. The composition can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.

Compounds of the present invention can be administered topically, that is by non-systemic administration. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin such as gels, liniments, lotions, creams, ointments or pastes. Gels for topical or transdermal administration of compounds of the present invention can include a mixture of volatile solvents, nonvolatile solvents, and water. There are several optional ingredients which can be added to the topical composition.

The composition may be formulated as an ointment, cream, gel, suppository, liquid for vaginal irrigation, intravaginal ring or concentrate for preparation of the same. For example, the topical administration forms disclosed in Cheng S, et ah, US 5,795,911 or Chen S, et al, US 5,968,973 may be used in the context of the present invention. Vaseline, for instance, may be used as a basis for an ointment.

In one embodiment of the invention, the composition is formulated for topical administration to the vagina of a woman before and/or after sexual intercourse. Other methods of topical administration are possible, such as administration to the penis (e.g. formulated as a lubricant), and may also depend on sexual practices.

Compositions of the invention

The present invention also contemplates that the administration of the dihydroceramide desaturase inhibitor can be carried out in combination with other suitable therapeutic treatments which are useful for treating a disease caused by infection by HIV. Thus, in another aspect, the invention relates to a composition or kit- of-parts according comprising a Des-1 inhibitor and one or more drugs used for the treatment of a disease caused by HIV infection.

The term "composition", as used herein, relates to a mixture of ingredients.

As used herein, the term "kit-of-parts" shall encompass an entity of physically separated components, which are intended for individual use, but in functional relation to each other.

The term "Des-1 inhibitor" has been described in detail in relation to the therapeutic method of the invention and is applied herein in the same meaning.

In a preferred embodiment, the drug used for the treatment of a disease caused by HIV infection is selected from the group consisting of a HIV protease inhibitor, a HIV reverse transcriptase and a HIV entry inhibitor.

The term "protease inhibitor" as used herein means inhibitors of the HIV-1 protease, an enzyme required for the proteolytic cleavage of viral polyprotein precursors (e.g. viral GAG and GAG Pol polyproteins), into the individual functional proteins found in infectious HIV-1. Suitable protease inhibitors that can be combined with the Des-1 inhibitors according to the invention is selected from the group consisting of ritonavir, lopinavir, saquinavir, amprenavir, fosamprenavir, nelfmavir, tipranavir, indinavir, atazanavir, TMC-126, darunavir, mozenavir (DMP-450), JE-2147 (AG1776), L-756423, KNI-272, DPC-681, DPC-684, telinavir (SC-52151), BMS 186318, droxinavir (SC- 55389a), DMP-323, KNI-227, l-[(2-hydroxyethoxy)methyl]-6- (phenylthio)-thymine, AG- 1859, RO-033-4649, R-944, DMP-850, DMP-851, and brecanavir (GW640385). Preferred protease inhibitors for use in combination with a compound of the present invention include saquinavir, ritonavir, indinavir, nelfhavir, amprenavir, lopinavir, atazanavir, darunavir, brecanavir, fosamprenavir, and tipranavir. Particularly useful such combinations include, for example, AZT+3TC; TDF+3TC; TDF+FTC; ABC+3TC; and Abacavir+3TC.

The term "reverse transcriptase inhibitors" as used herein, refer to any compound which inhibits the activity of HIV- 1 reverse transcriptase, the enzyme which catalyzes the conversion of viral genomic HIV-1 RNA into pro viral HIV-1 DNA. Suitable reverse transcriptase inhibitors for use in the compositions according to the present invention is one or more compounds selected from the group consisting of emtricitabine, capravirine, tenofovir, lamivudine, zalcitabine, delavirdine, nevirapine, didanosine, stavudine, abacavir, alovudine, zidovudine, racemic emtricitabine, apricitabine, emivirine, elvucitabine, TMC-278, DPC-083, amdoxovir, (-)-beta-D-2,6- diamino -purine dioxolane, MIV-210 (FLG), DFC (dexelvucitabine), dioxolane thymidine, Calanolide A, etravirine (TMC-125), L697639, atevirdine (U87201E), MIV- 150, GSK- 695634, GSK-678248, TMC-278, KP1461, KP-1212, lodenosine (FddA), 5- [(3,5-dichlorophenyl)thio]-4-isopropyl-l-(4-pyridylmethyl)imidazole-2 -methanol carbamic acid, (-)-P-D-2,6-diaminopurine dioxolane, AVX-754, BCH-13520, BMS- 56 190 ((4 S)-6-chloro-4-[(lE)-cyclopropylethenyl]-3,- 4-dihydro-4-trifluoromethyl-2 (lH)-quinazolinone), TMC-120, and L697639, where the compounds are present in amounts effective for treatment of HIV when used in a combination therapy.

The term "viral entry inhibitor", as used herein, refers to any compound capable of interfering with the entry of viruses into cells. In some embodiments, the viral entry inhibitor is a fusion inhibitor, a CD4 receptor binding inhibitor, is a CD4 mimic or a gpl20 mimic. In some further embodiments, the viral entry inhibitor is a gp41 antagonist, a CD4 monoclonal antibody or a CCR5 antagonist, including CCR5 antagonist sub-classes such as, for example, zinc finger inhibitors. In yet another embodiment, the viral entry inhibitor is a CXCR4 co-receptor antagonist.

Additionally, the compositions according to the present invention may further comprise an antiretro viral agent selected from the group consisting of vaccines, gene therapy treatments, cytokines, TAT inhibitors, and imrnunomodulators in amounts effective for treatment of HIV when used in a combination therapy.

Additionally, the compositions according to the present invention may further comprise an antiinfective agent selected from the group consisting of antifungals, antibacterials, anti-neoplasties, anti-protozoals, DNA polymerase inhibitors, DNA synthesis inhibitors, anti-HIV antibodies, HIV antisense drugs, IL-2 agonists, I±- glucosidase inhibitors, purine nucleoside phosphorylase inhibitors, apoptosis agonists, apoptosis inhibitors, and cholinesterase inhibitors, where the compounds are present in amounts effective for treatment of HIV when used in a combination therapy.

Additionally, the compositions according to the present invention may further comprise an immunomodulator, which is selected from the group consisting of pentamidine isethionate, autologous CD8+ infusion, I± -interferon immunoglobulins, thymic peptides, IGF-I, anti-Leu3A, auto vaccination, biostimulation, extracorporeal photophoresis, cyclosporin, rapamycin, FK-565, FK-506, GCSF, GM-CSF, hyperthermia, isopinosine, rVIG, HIVIG, passive immunotherapy and polio vaccine hyperimmunization, where the compounds are present in amounts effective for treatment of HIV when used in a combination therapy.

Some embodiments of the present invention comprise a compound of the present invention and a secondary pharmaceutical agent selected from the group consisting of antifungals, antibacterials, anti-neoplasties, anti-protozoals, ceragenins, DNA polymerase inhibitors, DNA synthesis inhibitors, anti-HIV antibodies, HIV antisense drugs, IL-2 agonists, I±-glucosidase inhibitors, purine nucleoside phosphorylase inhibitors, apoptosis agonists, apoptosis inhibitors, and cholinesterase inhibitors in amounts effective for treatment of HIV when used in a combination therapy.

In another embodiment, the invention relates to a method for the treatment of a disease caused by HIV infection in a subject in need thereof comprising the administration to said subject of a composition or kit-of-parts according to the present invention.

The person skilled in the art shall understand, in the context of the present invention, that the medicament for the combined administration of a Des-1 inhibitor and the agent or agents useful in the treatment of diseases caused by HIV infection may be prepared as a single dosing form or in separate dosing forms. "Combined administration" is understood to mean that the compounds which form part of the kit-of-parts according to the invention may be administered jointly or separately, simultaneously, at the same time or sequentially in the treatment of the pathologies previously mentioned in any order. For example, the administration of the Des-1 inhibitor may be performed first, followed by the administration of one or more therapeutic agents useful in the treatment of diseases caused by HIV infection; or the administration of the Des-1 inhibitor may be performed at the end, preceded by the administration of one or more therapeutic agents useful in the treatment of diseases caused by HIV infection; or the administration of the Des-1 inhibitor may be performed at the same time as the administration of one or more therapeutic agents useful in the treatment diseases caused by HIV infection.

The compositions or kit-of-parts of the invention may be administered using any suitable route including intravascular, intratumoral, intracranial, intraperitoneal, intrasplenic, intramuscular, subretinal, subcutaneous, mucosal, topical and oral route. In a preferred embodiment, the composition or kit-of-parts is administered topically.

In vitro method for inhibiting HIV infection

The identification by the authors of the present invention of the ability of Des-1 inhibitors to prevent HIV-1 infection of cells also allows the use of Des-1 inhibitors for in vitro preventing HIV infection of target cells. Thus, in another aspect, the invention relates to an in vitro method for inhibiting HIV infection which comprises contacting cells with a Des-1 inhibitor under conditions effective to provoke an increase in the relative amounts of dihydroceramide or dihydroesphingosine with respect to ceramide or sphingosine SM in the membranes of said cells.

The term "Des-1" inhibitor has been explained in detail above.

The term "target cells", as used herein, refers to cells that are permissive for infection by HIV. In a more preferred embodiment of the present invention, the target cell is capable of expressing a CD4 receptor and either CCR5 or CXCR4, or both CCR5 and CXCR4. Use of a target cell capable of expressing CCR5 and CXCR4 is especially preferable when the tropism of the HIV strain from a clinical sample is unknown.

The target cells are preferably mammalian cells, and more preferably human cells. In one embodiment, target cells endogenously expressing human CD4 receptor are used in the assay of the present invention. Alternatively, a nucleic acid sequence encoding human CD4 can be introduced into the target cell by transfection such as for example using lipofectamine, vaccinia virus or other conventional method for introducing a nucleic acid encoding CD4 into the target cells. Moreover, the target cells of the present invention also express one or more endogenous or recombinant coreceptors, such as a chemokine receptor necessary for fusion of the viral envelope protein of interest to the target cell. Expression of CD4 and the chemokine receptor(s) must be sufficient to allow target cells to fuse with the effector cells. Examples of suitable promoters for use in expressing CD4 and/or a chemokine receptor are cytomegalovirus (CMV), and Rous sarcoma virus (RSV).

In a preferred embodiment, the target cells are CD4- and/or CXCR4-positive cells.

The expression "conditions effective to provoke an increase in the relative amounts of dihydroceramide or dihydroesphingosine with respect to ceramide or sphingosine SM in the membranes of said cells" refers to those conditions which result in the effective replacement of SM by DHSM and/or of ceramide by dihydroceramide. Methods adequate for determining whether conditions result in the replacement of SM by DHSM include any method known in the art adequate for determining the lipid composition of a sample such as ultra performance liquid chromatography (UPLC) coupled to mass spectrometry, thin-layer chromatography or the like.

Methods for the identification of compounds suitable for the treatment of a disease caused by HIV infection

The identification of the effect of Des-1 inhibitors in preventing the infection of target cells by HIV allows the development of methods for the identification of anti- HIV compounds based on their ability of said compounds to inhibit the activity of Des- 1. Thus, in another aspect, the invention relates to a method for the identification of compounds suitable for the treatment of a disease caused by HIV-1 infection (first identification method of the invention) which comprises

(i) contacting a candidate compound with a preparation containing cellular membranes and Des-1, and

(ii) determining the activity o f Des- 1 , wherein a decrease in the activity of Des-1 with respect to a control preparation is indicative that the candidate compound is suitable for the treatment of a disease caused by HIV- 1 infection.

In a first step, the first identification method of the invention includes the step of contacting a candidate compound with a preparation containing cellular membranes and Des-1. The term "preparation containing cellular membranes and Des-1" refers to any membrane preparation that contains the membranes wherein the Des-1 protein is natively found, namely, ER membranes. In a preferred embodiment, the preparation containing membranes and Des-1 is a microsomal preparation. The term microsome, as used herein, is defined operationally as the particulate fraction obtained from a tissue homogenate by ultra centrifugation after the nuclear and mitochondrial fractions have been removed by low speed centrifugation.

In a still more preferred embodiment, the microsomal preparation is a microsomal preparation derived from liver cells. The term "liver microsomes" refers to closed vesicles of fragmented endoplasmic reticulum created when liver cells or tissue are disrupted by homogenization. Liver microsomes can be obtained using any method known in the art. Preferably, the rat liver microsomes are prepared using differential centrifugation. See Buhs R, et al, US 4,333,925; Smith W, et al, US 4,471,058, Hoffman W, et al, US 4,851,436; Madhok T, DeLuca H, Biochem. J. 1979; 184:491- 499 and Coughtrie M, et al, Clin. Chem. 1991; 37:739-742. The intactness of microsomes can be assessed, for instance, by determining mannose-6-phosphate activity.

In a second step, the first identification method of the invention involves the determination of the activity of Des-1. The activity of Des-1 in the membranes can be determined using suitable methods known in the art. See Nikolova-Karakashian, 2000, supra and Bedia, 2005, supra.

Once the activity of Des-1 is determined in the treated preparation, the compound is identified as suitable for the treatment of a disease caused by HIV-1 infection if the Des-1 activity is lower than that observed in a control preparation. A control preparation, as used herein, refers to a preparation which has been left untreated or which has been treated with the vehicle used for the treatment with the candidate compound. In yet another aspect, the invention relates to a method for the identification of compounds suitable for the treatment of a disease caused by HIV-1 infection (second identification method of the invention) which comprises:

(i) contacting a candidate compound with a cell population,

(ii) isolating a sphingolipid-enriched detergent-resistant membrane from the cell population, and

(iii) determining the relative amount of dihydroceramide or dihydrosphingosine with respect to ceramide or sphingosine, wherein an increase in the ratios of dihydroceramide to ceramide and/or dihydrosphingosine to sphingosine with respect to a sphingolipid-enriched detergent- resistant membrane preparation isolated from a control cell population is indicative that the candidate compound is suitable for the treatment of a disease caused by HIV-1 infection.

In a first step, the second identification method of the invention involves the contacting of a cell population with a candidate compound. According to the invention, "putting in contact" a cell with the candidate compound includes any possible way of taking the candidate compound inside the cell expressing the DNA construct. Thus, in the event that the candidate compound is a molecule with low molecular weight, it is enough to add said molecule to the culture medium. In the event that the candidate compound is a molecule with a high molecular weight (e.g. biological polymers such as a nucleic acid or a protein), it is necessary to provide the means so that this molecule can access the cell interior. In the event that the candidate molecule is a nucleic acid, conventional transfection means can be used, as described previously for the introduction of the DNA construct. In the event that the candidate compound is a protein, the cell can be put in contact with the protein directly or with the nucleic acid encoding it coupled to elements allowing its transcription/translation once they are in the cell interior. To that end, any of the aforementioned methods can be used to allow its entrance in the cell interior. Alternatively, it is possible to put the cell in contact with a variant of the protein to be studied which has been modified with a peptide which can promote the translocation of the protein to the cell interior, such as the Tat peptide derived from the HIV-1 TAT protein, the third helix of the Antennapedia homeodomain protein from D. melanogaster, the VP22 protein of the herpes simplex virus and arginine oligomers. See Lindgren A, et al, Trends Pharmacol. Sci 2000; 21 :99-103; Schwarze S, et al, Trends Pharmacol. Sci. 2000; 21 :45-48; Lundberg M, et al, Mol. Therapy 2003; 8: 143-150 and Snyder E, Dowdy S, Pharm. Res. 2004; 21 :389-393.

The compound to be assayed is preferably not isolated but forms part of a more or less complex mixture derived from a natural source or forming part of a library of compounds. Examples of libraries of compounds which can be assayed according to the method of the present invention include, but are not limited to, libraries of peptides including both peptides and peptide analogs comprising D-amino acids or peptides comprising non-peptide bonds, libraries of nucleic acids including nucleic acids with phosphothioate type non-phosphodiester bonds or peptide nucleic acids, libraries of antibodies, carbohydrates, compounds with a low molecular weight preferably organic molecules and peptide mimetics. In the event that a library of organic compounds with a low molecular weight is used, the library can have been preselected so that it contains compounds which can access the cell interior more easily. The compounds can thus be selected based on certain parameters such as size, lipophilicity, hydrophilicity, and capacity to form hydrogen bonds.

The compounds to be assayed can alternatively form part of an extract obtained from a natural source. The natural source can be an animal or plant source obtained from any environment, including but not limited to extracts of land, air and marine media.

In a second step, the second identification method of the invention involves isolating a sphingolipid-enriched detergent-resistant membrane from the cell population. The preparation of the sphingolipid-enriched detergent-resistant membrane can be carried out by lysis of the cells followed by fractionation. In a preferred embodiment, the sphingolipid-enriched detergent-resistant membrane is isolated and its cells are lysed in a detergent-containing buffer followed by fractionation in a density gradient. See Manes S, et al, EMBO Rep. 2000; 1 : 190-196.

In a third step, the second identification method of the invention involves determining the relative amount of dihydroceramide or dihydrosphingosine with respect to ceramide or sphingosine. The determination of the relative amount of dihydroceramide or dihydrosphingosine with respect to ceramide or sphingosine can be carried out by any method known in the art for determining the lipid composition of a sample containing lipids including thin-layer chromatography and ultra performance liquid chromatography coupled to mass spectrometry.

Once the relative levels of dihydroceramide or dihydrosphingosine with respect to ceramide or sphingosine are determined, a compound is considered as suitable for the treatment of a disease caused by HIV-1 infection when the dihydroceramide/ceramide or dihydrosphingosine/sphingosine ratios in the sphingolipid-enriched detergent- resistant membrane preparation isolated from the cell population treated with the candidate compound are higher those in a preparation isolated from control cells. The term "control cells", as used herein, refers to cells which have been left untreated or which have been treated with the vehicle wherein the candidate compound is dissolved.

The term "increase", as used herein refers to an increase in the relative levels of dihydroceramide or dihydrosphingosine with respect to ceramide or sphingosine of at least by at least 10 percent, at least 20 percent, at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, at least 95 percent, at least 98 percent, or even at least 100 percent.

In the event that the candidate compound forms part of a more or less complex mixture, the first identification method of the invention additionally comprises one or several steps (iv) of fractioning said mixture and the repetition of steps (i), (ii) and (iii) of the method of the invention a variable number of times until the compound of the mixture responsible for the transcription promoting activity is isolated. Methods for fractioning the compounds present in a mixture include but are not limited to chromatography (thin layer, gas, gel molecular exclusion, affinity chromatography) crystallization, distillation, filtration, precipitation, sublimation, extraction, evaporation, centrifugation, mass spectroscopy and adsorption.

The assays described herein may be conducted as high-throughput assays.

Techniques for performing high-throughput assays include use of microtiter plates or pico-, nano- or micro-liter arrays. The assays of the invention are designed to permit high throughput screening of large compound libraries, like by, for example, automating the assay steps and providing candidate viral entry inhibitors from any source to assay. Assays which are mn in parallel on a solid support (e.g. microtiter formats on microtiter plates in robotic assays) are well known. Automated systems and methods for detecting and measuring changes in optical detection (or signal) are known. See Pham A, et al., US 6,171,780; Stylli C, et al, US 5,985,214 and Akong M, et al, US 6,057,114.

All the publications mentioned herein are incorporated hereby in their entirety by reference.

The invention is described herein by of the following examples which are merely illustrative but not limitative of the scope of the invention.

EXAMPLES EXPERIMENTAL PROCEDURES

Materials. Egg phosphatidylcholine (PC), egg phosphatidyl ethanolamine (PE) and egg diacylglycerol (DAG) were purchased from Lipid Products (South Nutfield, UK). Egg SM and Choi were from Avanti Polar Lipids (Alabaster, AL, USA). Ι,Γ- dioctadecyl-3,3,3 ^'3 ^'-tetramethylindo carbo cyanine perchlorate (Dil) and di- ANEPPDHQ were from Invitrogen (Eugene, OR, USA). The fluorescent probes N-(7- nitro-benz-2-oxa-l,3-diazol-4-yl)phosphatidylethanolamine (N-NBD-P E ) a n d N- (lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). HIV p24 ELISA from Innogenetics (Gent, BE), rifampicin from Boehringer (Ingelheim, DE), Optiprep (Nycomed Pharma, Oslo, NO); CellTiter-Glo Luminescent Cell Viability Assay, MTS, BrightGLo Luciferase System and Dual Luciferase system were from Promega (Madison, WI, USA). The rabbit polyclonal anti-Des-1 antibody was a gift of J. M. Kraveka (Medical University of South Carolina, Charleston, SC, USA); anti-transferrin receptor (Zymed Laboratories, San Francisco, CA, USA), anti- -actin (Abeam Inc., Cambridge, UK), and anti-CD4 and anti-caveolin-1 antibodies (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sequence DKWASLWNWFNITNWLWYIK (SEQ ID NO: 4) (HIVc or preTM), representing the pretransmembrane stretch of HIV-1 (BH10 isolate) gp41 was synthesized by solid-phase synthesis using Fmoc chemistry, as C- terminal carboxamides and purified by HPLC at the Proteomics Unit of the University Pompeu-Fabra (Barcelona, ES). Peptide stock solutions were prepared in dimethylsulfoxide (DMSO) (spectroscopy grade) and concentrations determined by the bicinchoninic acid microassay (Pierce, Rockford, IL, USA). Chemical synthesis. N-(7-nitro-benz-2-oxa-l,3-diazol-4-yl) ceramide (NBD- Cer) was synthesized in our facilities. DHSM was obtained by hydrogenation of sphingomyelin. 6-[N-(7-nitro-2, 1 ,3-benzoxadiazol-4-yl)amino] hexanoylsphinganine (DHCer(C6)-NBD) and GT11 were prepared as described in Munoz-Olaya, 2008, supra.

GTl lpyr was synthesized from the aminodiol. 8-bromooctanic acid (11.8 mg, 0.053 mmol) dissolved in dichloromethane (DCM, 2 ml) was added to a solution of l-ethyl-3- (3-dimethylaminopropyl)carbodiimide (10.2 mg, 0.053 mmol) and N- hydroxybenzotriazole (8.1 1 mg, 0.053 mmol) in DCM, and the solution stirred (25°C, 10 min). In parallel, triethylamine (10 μΐ, 0.072 mmol) was added dropwise at 25°C to a solution of the cyclopropenylaminodiol (15 mg, 0.048 mmol) in DCM (2 mL). The activated acid was added and the mixture stirred (25°C, 12 h). The organic layer was then washed with brine, dried (MgS0₄), the solvent evaporated, and the residue purified by flash chromatography. Elution with methylene chloride/methanol (40: 1) afforded the bromoacyl analog of GT11 (20 mg, 0.038 mmol) with a yield of 80%.

The GT11 bromoacyl analog (20 mg, 0.038 mmol) was dissolved in toluene (2 mL) and, after addition of pyridine (2 mL), was heated at 90°C with stirring (2 h). Solvents were removed under vacuum, the residue thoroughly washed with hexane and diethylether, and then dissolved in methanol and filtered. Evaporation of methanol afforded GT11-pyr (15 mg, 29 mmol, 75% yield) as a white solid.

Dihydroceramide desaturase activity. 6-[N-(7-nitro-2,l,3-benzoxadiazol-4- yl)amino] hexanoylsphinganine (DHCerC6NBD 50 μΜ in ethanol), alone or with inhibitors, was incubated (3 h, 37°C) with the cells. After cell lysis by hypotonic shock and sonication, the lysates were diluted with methanol (900 μΐ) and centrifuged (17,000 g, 3 min). The supernatant was analyzed by HPLC coupled to a fluorescence detector, using a reversed-phase column eluted with 20% H₂0 and 80% acetonitrile, both with a 0.1% of trifluoroacetic acid. Fluorescence emission was recorded at 530 nm (excitation at 465 nm). A total of 100 μΐ was injected and each sample was run for up to 15 min.

Lipid phase studies. The appropriate lipid mixtures in organic solvent contained 0.2 mol % Dil, 0.4 mol % NBD-Cer, or 0.1 mol % Laurdan as needed. Multilamellar (MLV), large unilamellar (LUV) and giant unilamellar (GUV) vesicles were prepared as described. See Sot J, et ah, FEBS Lett. 2008; 582:3230-3236. Excitation wavelengths were 430 nm for NBD-Cer and 561 nm for Dil. Images were collected through two channels using band-pass filters of 515 ± 15 nm for NBD-Cer and 593 ± 20 nm for Dil. The generalized polarization (GP) of Laurdan 829 was measured in a SLM-AMINCO 8100 spectra fluorometer, equipped with thermoregulated cell holders. The excitation GP_EX parameter was calculated according to

GPEX = (I440-I490) / (I440+I490)

where I440 and I490 are the emission intensities obtained at 440 and 490 nm, respectively, exciting at 360 nm. The final probe/lipid molar ratio was 1/1000.

GUV were stained with di-4-ANEPPDHQ (10 μΜ) dissolved in buffer, and images analyzed in an inverted confocal fluorescence microscope with a high efficiency spectral detector (Leica TCS SP5, Leica Microsystems CMS GmbH, Mannheim, DE). The excitation wavelength was 488 nm. Images were collected simultaneously in the 500-550 nm and 650-730 nm channels and analyzed using the LAS AF software (Leica Microsystems CMS GmbH, Mannheim, DE). Differential scanning calorimetry (DSC) measurements were performed on MLV as described. See Sot, 2008, supra.

Cell lines and cytotoxic assays. Human embryonic kidney (HEK)-293, HEK- 293 CD4 (a subclone stably expressing CD4 receptor) and TZM-bl cells were cultured as described. See Manes, 2000, supra. Cell viability after 24 h incubation with GT1 1 (not shown) or GT1 lpyr was assessed by trypan blue staining and MTS, and compared with vehicle-treated cells. The cell cycle was analyzed by FACS after propidium iodide staining.

DRM isolation. GTl lpyr-incubated HEK-293CD4 cells (24 h, 37°C) were lysed and DRM isolated. See Manes, 2000, supra. Five fractions were collected from the top to the bottom of the gradient, the first corresponding to the DRM fraction. Half was precipitated with TCA and analyzed by western blot with indicated antibodies, and half processed for lipid composition analysis, using UPLC coupled to an orthogonal acceleration time-of- flight mass spectrometer with an electrospray ionization interface (LCT Premier Waters, Millford, MA, USA). Data were acquired using positive ionisation mode over a mass range of m/z 50 to 1500 in W-mode. A scan time of 0.15s and interscan delay of 0.01s were used at a nominal instrument resolution of 1 1500 (FWHM). Leucine enkephalin was used as the lock spray calibrant. Accurate masses used for correction were MH m/z 556.2771 and [M-H] m z 554.2615. These conditions preclude the use of two standards flanking the mass spectrometry region of interest.

Immunofluorescence analyses. For antibody-induced co-patching, HEK- 293CD4 cells or HEK-293 transfected with GFP-GPI (raft marker) were incubated (30 min, 12°C) with a mouse monoclonal anti-CD4 (HP2.6, See Del Real G, et al., J. Exp. Med. 2002; 196(3):293-301) or anti-CXCR4 (K1046, Santa Cruz) antibodies, and biotinylated cholera toxin β-subunit, followed by incubation with appropriate Cy2 secondary antibodies and Cy3-streptavidin (Jackson ImmunoResearch, West Grove, PA, USA), fixed with paraformaldehyde (3.7%, 5 min, 4°C) and methanol (5 min, - 20°C) and analyzed by confocal microscopy. Images were acquired in an Olympus- F1000 confocal microscope and processed with Image J.

For gpl20-induced co-patching, PBMC were sequentially incubated (30 min, 12°C) with recombinant gpl20 (T cell line-adapted X4 virus, isolate IIIB, AIDS Research and Reference Reagent Program, NIAID, NIH, Bethesda, MD, USA), and a rabbit polyclonal anti-gpl20 raised in our laboratory in combination with FITC-HP2.6, and biotinylated CXCR4 (FAB 172, R&D Systems, Minneapolis, MN, USA) antibodies. Finally, anti-rabbit or streptavidin-Cy5 were added (30 min, 12°C), fixed and analyzed by confocal microscopy.

Env-mediated cell-cell fusion assays. HEK-293CD4 cells (and HEK-293 cells as negative control) were transfected with pSCluc containing the firefly luciferase gene under the vaccinia virus 7.5 promoter, and pNull promoterless renilla luciferase plasmids, and then incubated with GTl 1 C8 (24 h, 37°C). HIV-len m_B was introduced into effector HEK-293 cells by infection with a recombinant vaccinia virus (1 h, 37°C); 12 h post-infection, effector cells cultured in rifampicin (100 μg/mL) were mixed (6 h, 37°C) with GTl 1 -treated HEK-293CD4 cells (1 :2 ratio), and firefly and renilla activity quantified in cell lysates. Relative light units (RLU) were calculated as the quotient of firefly and renilla activity values, and were indicative of effector:target cell fusion.

HIV-1 infectivity assays. Single-round infection experiments were performed as described. See Del Real, 2002, supra. Replication-deficient HIV-1 viruses were generated by cotransfecting pNL4.31ucR-E- (AIDS Research and Reference Reagent Program, NIAID, NIH, Bethesda, MD, USA) and either ADA, NL4.3 or VSVG envelopes in HEK-293T cells. Equal amounts (45 ng of p24) of replication-deficient HIV-1 viruses (HEK-293T cells supernatants at 48 h posttransfection) were added to vehicle- or GT1 lpyr-pretreated TZM-bl cells (24h, 37°C); after 48 h, cell infection was determined by luciferase activity measurement in cell lysates. For infection with replication-competent HIV- I_{NFN SX} virus, TZM-bl cells (2.5x10⁵) seeded in 96-well plates were incubated (200 μΐ) with GT11 (0.1-8 μΜ) for 24 h or 5 days, and virus (10 ng of p24) added 24 h later. Cells were assayed for luciferase activity 48 h later (BrightGLo Luciferase System, Promega, WI, USA) in a Fluoroskan Ascent FL luminometer. Cell viability was assayed in parallel with CellTiter-Glo Luminescent Cell Viability Assay (Promega, WI, USA). The percentage of inhibition was calculated as described. See Buzon M, et al, Antivir. Ther. 2008; 13:881-893. For knockdown experiments, TZM-bl cells were transfected with vehicle (mock), siRNA control (Ambion Silencer siRNA control; 40 nM) or Des-1 -specific siRNA (5'- CCUUCAAUGUGGGUUAUCAtt-3^*; 40 nM) (SEQ ID NO: 3) using Lipofectamine 2000 (Invitrogen, Boulder, CO, USA); after 4 days, cells were assayed for Des-1 levels by Western blot, Des-1 activity, or exposed to HIV- I_{NFN SX} (2, 10 or 50 ng p24) and infectivity analyzed as above.

Intervesicular lipid mixing. Membrane lipid mixing was monitored in a Jobin Yvon Fluoromax-3 spectrofluorometer using a resonance energy transfer (RET) assay. The assay is based on the dilution of N-NBD-PE and N-Rh-PE. See Saez-Cirion A, et al, J. Biol. Chem. 2002; 277:25649-25659.

Example 1

DHSM rigidifies l₀ domains in GUV formed by ternary mixtures

Studies in artificial membranes support the idea that DHSM-containing liposomes form with Choi more condensed /₀ domains than those containing SM. The generalized polarization (GP) of Laurdan in bilayers formed by SM, DHSM and Choi was studied initially to evaluate the role of DHSM in biological membranes. As predicted, DHSM:Chol bilayers showed higher Laurdan GP values than those consisting of SM:Chol in the 15-50°C temperature range; temperature increase fluidified the bilayer and GP decreased accordingly. SM replacement with different amounts of acyl- matched DHSM promoted a dose-dependent increase in GP values at all temperatures tested, indicating rigidification of the bilayer. See Fig. 1 A. GUV composed of PC :PE :Chol and SM or DHSM were stained with the fluorescent probes Dil and NBD-Cer. Dil partitions preferentially in I_d domains, whereas the NBD-Cer probe binds similarly to /₀ and domains, but is excluded from s₀ phases. See Sot, 2008 , supra. The Dil probe stained PC :PE : SM : Chol and PC:PE:DHSM:Chol vesicles similarly, indicating that DHSM did not alter /_d phase formation. See Fig. IB. Since a change in bilayer composition could change Dil partitioning into the l l₀ domains, control experiments using the /₀ phase probe Laurdan confirmed that Dil specifically stained I_d domains in both lipid mixtures (not shown). See Bagatolli L, Biochim. Biophys. Acta 2006; 1758: 1541-1556. In contrast, NBD-Cer stained PC:PE:DHSM:Chol GUV more weakly compared to PC:PE:SM:Chol GUV. See Fig. IB. This reduction in NBD-Cer staining was evident in the region from which Dil was excluded. Indeed, the ratio of NBD-Cer fluorescence intensity in the /₀ to the phases was clearly lower in DHSM-containing GUV (SM-GUV: 1.06 ± 0.16; DHSM- GUV : 0.42 ± 0. 13 ; Fig. 1 C) . This suggests the presence of s₀ phase domains interspersed in the /₀ phase of PC:PE:DHSM:Chol bilayers . Three-dimensional reconstructions of DHSM-GUV show two main domains, circular in shape, limiting one I_d and one /₀ macrodomain. Circular boundaries are an indication of liquid-liquid domain coexistence. See Bagatolli, 2006, supra. This supports our previous assertion that there is not a single, macro scopically separated s₀ domain, but rather a number of s₀ microdomains interspersed within the /₀ phase.

The presence of rigid, gel-like domains in bilayers containing DHSM was further studied using di-4-ANEPPDHQ. Emission fluorescence of this probe shifts towards red when inserted in I_d and towards green when incorporated in /₀ environments. See Jin L, et ah , Biophys. J. 2006; 90: 2563-2575. Confocal analysis of SM-containing GUV showed regions predominantly in the I_d phase (yellow in merge image) mixed with smaller /₀ domains (green). In contrast, substantial unstained areas in DHSM-containing vesicles were observed, suggesting the presence of more rigid domains. See Fig. ID. Quantification of emission spectra showed a notable reduction in the area below the /₀ spectrum of DHSM- compared to SM-containing GUV; however, I_d spectra were comparable in both. See Figs. IE and IF. The results suggest that DHSM-induced s₀ domains are formed essentially at the expense of the /₀ phase. See Sot, 2008, supra. Differential scanning calorimetry (DSC) data supported decisively the presence of gel domains in DHSM-containing bilayers. When a gel phase melts cooperatively into a fluid domain, a DSC endotherm is observed, whereas l₀- thermal transitions are not detected. A clear endotherm extending from 20°-50°C in DHSM- but not in SM- containing GUV was found indicating that the gel domain melted under these conditions. See Fig. 1G. In summary, the DHSM in the bilayer imposed a change from fluid /₀ to s₀ domains. This is in agreement with previous observations of a rigidifying effect of DHSM, although evidence of s₀ domain formation was lacking. Example 2

Chemical inhibition of Des-1 increases DHSM levels

in l₀ domains of living cells

Since replacement of only 30% of SM by DHSM induced a clear change in bilayer rigidity, the effect of increasing DHSM levels in the plasma membrane of living cells was analyzed. Inhibition of Des-1 , the main enzyme introducing unsaturation into dihydroceramides (DHCer), seemed an attractive means of increasing DHSM levels in cultured cells. See Fig. 1A. The compound GT1 1 effectively inhibits Des-1 activity in neurons. See Triola, 2005, supra. Toxicity studies in HEK-293 cells indicated that 24 h incubation with GTl lpyr (a hydrophilic derivative of GT1 1 ; See Fig . 2A) at concentrations <30 μΜ affected minimally cell growth. See Fig. 2B. Cell cycle analyses indicated a dose-dependent increase in the percentage of GT1 lpyr-treated cells in the S- phase peak, with a concomitant decrease in the G2/M peak. At the doses tested, GTl lpyr did not affect the subGo/Gi peak compared to controls. See Fig. 2C. Short incubation (<24 h) with GT1 lpyr does not cause cell death, although it might slow cell cycle progression.

Then, the relative potency of GTl lpyr and GT1 1 in inhibiting Des-1 activity was compared. Lysates of HEK-293 cells and TZM-bl , a CD4- and CCR5 -expressing HeLa subclone, were incubated with different amounts of GT1 1 and GT1 lpyr, and Des- 1 activity determined by HPLC using DHCerC6NBD as substrate. See Munoz-Olaya, 2008, supra. GT1 1 and GT1 lpyr inhibited Des-1 activity in a dose-dependent manner with similar potency. See Fig. 2D. To determine whether Des-1 inhibition led to an effective replacement of SM by DHSM in the cell membrane, sphingolipid-enriched detergent-resistant membrane (DRM) fractions were isolated from HEK-293 cells. These membranes were untreated or treated with GTl lpyr for 24 h, and their lipid composition was analyzed subsequently by ultra performance liquid chromatography (UPLC) coupled to mass spectrometry, using C16 SM as a probe (the most abundant SM in this cell type). Representative chromatograms of vehicle and GTl lpyr (30 μM)-treated cells are shown. See Fig. 2E. GTl lpyr promoted a dose-dependent decrease in the relative amount of C16 SM, which paralleled an increase in the amount of DHSM species in the DRM fraction. The DHSM/SM ratio reached a maximum of 0.3 at 30 μΜ GT 1 lpyr. See Fig. 2F. A comparable variation in the DHSM/SM ratio for C16:0, C24:0 and C24: l species after GTl lpyr treatment was found. GTl lpyr also increased the amount of DHCer while decreasing Cer levels in DRM in a dose-dependent manner. However, Des-1 inhibition did not affect the glucosylceramide or lactosylceramide levels or the SM/glycosphingolipid ratio. These results indicate that GTl lpyr- induced inhibition of Des-1 causes the partial replacement of SM and Cer by DHSM and DHCer in living cells. It should be noted, nonetheless, that Cer and DHCer levels are much lower than those for SM and DHSM in this cell type. Example 3

Increased DHSM levels inhibit HIV-1 infection

To analyze whether decreasing Des-1 activity affects HIV-1 infection of target cells, TZM-bl cells were incubated with subtoxic doses of GTl lpyr (15-30 μΜ), followed by an exposure to equal amounts of replication-deficient pNL4.31ucR-E viruses pseudotyped with ADA (R5 strain), NL4.3 (X4 strain) or vesicular stomatitis virus (VSV)-G (control) envelopes. See He J, et al, J. Virol. 1995; 69:6705-6711. GTl lpyr reduced cell infection by ADA- and NL4.3 -pseudotyped viruses up to 60%, without affecting entry of VSV-G virions. See Fig. 3A. Since VSV-G-pseudotyped viruses effectively infected GTl lpyr-treated cells, the inhibitory effect of this compound is HIV Env-specific and is not due to a cytotoxic effect. Independent infection experiments using the replication-competent HIV- I_{NFN SX} strain in TZM-bl cells treated with the hydrophobic inhibitor GT11 showed a time- and dose-dependent inhibitory effect on HIV-1 infection at non-toxic GT11 doses (not shown). See Fig. 3B. The time-dependent inhibitory effect at very low GT11 doses might reflect slow replacement of SM by DHSM at suboptimal inhibitory concentrations, although possible "long-term" pleiotropic effects of the compound cannot be ruled out. These results indicate that GT11 and GT1 lpyr inhibit specifically cell infection by X4- and R5 HIV-1 strains.

To confirm the potential role of Des-1 in HIV-1 infection, the virus infectivity in TZM-bl cells trans fected with Des-1 -specific small interference (si)R A was analyzed. Des-1 knockdown reduced Des-1 protein levels and activity without affecting cell viability. Des-1 silencing also increased the DHCer/Cer ratio (35-40%) compared to mock- (2-4%) or mismatched siRNA-transfected cells (5-12%). HIV-1_NFN-_SX infection (10 ng p24) in TZM-bl cells trans fected with Des-1 -specific siRNA was significantly reduced compared to mock- or mismatched siRNA-transfected cells. See Figs. 4A, 4B and 4C. Similar inhibition of infection was observed at higher (50 ng) and lower (2 ng) viral doses (Fig. 4D). Pharmacological and genetic interference with Des-1 activity thus inhibited HIV-1 infection of target cells.

Example 4

Increased DHSM levels do not affect HIV-1 receptor clustering Previous reports indicate that gpl20-induced receptor clustering is sensitive to lipid modulation. Cholesterol depletion prevents gpl20 triggering of CD4-coreceptor colocalization and decreases CCR5 diffusion. See Manes, 2000, supra and Steffens C, Hope T, J. Virol. 2004; 78 :9573-9578. Cell treatment with acid sphingomyelinase (SMase), which transforms SM to Cer, also restricts lateral CD4 mobility. See Finnegan C, et al, Proc. Natl. Acad. Sci. 2004; 101 : 15452-15457 and Finnegan C, et al, J. Virol. 2007; 81 :5294-5304. Ceramides tend to form s₀ domains in artificial membranes since DHSM also rigidities lipid bilayers. GT1 lpyr- induced inhibition of HIV-1 infection might be explained by impaired clustering of viral receptors. See Cremesti A, et al, FEBS Lett. 2002; 531 :47-53 and Megha L, J. Biol. Chem. 2004; 279:9997-10004.

Analysis of the protein composition of the same DRM fractions in which the maximal DHSM/SM ratio was detected, showed no differences in CD4 partitioning between DRM and soluble fractions in vehicle- and GT1 lpyr-treated cells. See Figs. 2F and 5 A. Des-1 inhibition did not alter the unique partitioning of either caveolin or the transferrin receptor, used as controls for DRM- associated and -excluded membrane proteins, respectively. See Fig. 5A. There were also no differences between vehicle- and GT1 lpyr-treated cells in the co- localization of CD4 and CXCR4 HIV-1 receptors with ganglioside GM1. See Fig. 5B.

Next, the effect of GTl lpyr on the formation of higher order molecular complexes between gpl20, CD4 and CXCR4 was analyzed. Receptor over-expression to overcome the lipid-modulatory effect of gpl20-induced CD4 and/or co-receptor clustering was reported. See Finnegan, 2007, supra and Viard M, et ah, J. Virol. 2002; 76: 11584-11595. To avoid misleading effects due to receptor overexpression, resting peripheral blood mononuclear cells (PBMC) was used in co-clustering experiments; assays were carried out at 12°C to avoid receptor internalization. The gpl20 protein induced the formation of large CD4- and CXCR4-containing clusters, which did not differ in number or size in vehicle- or GT1 lpyr-treated PBMC. See Figs. 5C, 5D and 5E. The results suggest that the adverse effect of GT1 lpyr in HIV-1 infection is not a result of impaired lateral mobility of HIV-1 receptors.

Example 5

DHSM-induced rigidification inhibits gp41 insertion and fusion The effect of Des-1 inhibitors on HIV-1 infection might alternatively be explained by the regulation of gp41 -mediated fusion of viral and cell membranes. DHSM stabilizes the lamellar phase towards the inverted hexagonal phase, but no more so than SM; lamellar phase stabilization by DHSM thus cannot explain any inhibitory effect on HIV-1 fusion.

Lipids could nonetheless impair the interaction between HIV-1 gp41 and the host cell membrane. SM and Choi promote surface aggregation of the gp41 pretransmembrane sequence (LWYIK) into /₀ membranes. See Saez-Cirion, 2002, supra. To study whether the DHSM-imposed change in physical properties of the bilayer prevented fusion peptide insertion, an intervesicular lipid mixing to quantitate fusion of large unilamellar vesicles (LUV) induced by the pretransmembrane stretch of gp41 (HIVc) was used. DHSM in the LUV greatly decreased the extent of HIVc- induced intervesicular fusion at 25°C; this inhibitory effect was lost at temperatures that induce melting of s₀ domains (50°C). See Figs. I F and 6A. The results indicate that gp41 fusion peptide insertion is impaired in membranes with high DHSM levels at temperatures at which s₀ domains are present. The gpl60-induced cell-cell fusion was analyzed subsequently in GT 11 -treated cells. Only HEK-293-CD4 target cells were treated with the compound (24 h), which was removed before coincubation with HEK- 293-gpl60 cells. The dose-dependent inhibition of gpl60-induced cell-cell fusion was observed in GT 11 -treated cells. See Fig. 6B. Together, these results suggested that blockade of Des-1 activity inhibits HIV-1 infection by decreasing gp41 insertion into the rigidified microdomains formed by the concurrent increase in DHSM levels.

Claims

A method for the treatment of a disease caused by HIV infection in a subject in need thereof comprising the administration to a subject of a dihydroceramide desaturase (Des-1) inhibitor.

A method according to claim 1, wherein the Des-1 inhibitor is selected from the group consisting of the compounds of Table 1.

A method according to claim 2, wherein the Des-1 inhibitor is a compound having the following structure:

wherein

Ri and R₂ that can be the same or different, represent aryl, heteroaryl, alkyl or acyl groups with one or more unsaturations in the chain that can also be branched or straight and can have or not have substitutions,

R3 can be an alkyl, alkenyl, alkynyl, aryl or any heterocyclic group, and

n is an integer that can have any value and can be branched or straight, with or without substitutions and contain unsaturated carbons on the chain.

A method as defined in claim 3, wherein n is 0 to 8.

5. A method as defined in claim 4, wherein n is 0 to 2. A method according to claim 5, wherein Rl is H, R₃ is - (CH₂)i_i₂CH₃, n is 0 and R₂ is selected from the group consisting of:

-CO-(CH₂)₆-CH₃,

-CONH-(CH₂)₇-CH₃,

-CSNH-(CH₂)₇-CH₃,

-COCO-CH₂-CH₃,

-COCO-(CH₂)₅-CH₃,

-CO-(CH₂)₄-CH₃, and

-CO-(CH₂)₈-CH₃.

A method according to any of claims 1 to 6, wherein the inhibitor is administered topically.

8. Composition or kit-of-parts comprising, together or separately, at least a Des-1 inhibitor and at least a drug used for the treatment of a disease caused by HIV infection.

Composition or kit-of-parts according to claim 8, wherein the Des-1 inhibitor is selected from the group consisting of the compounds of Table 1.

Composition or kit-of-parts according to claim 9, wherein the Des-1 inhibitor is a compound having the following structure:

OH OH

wherein

Ri and R₂ that can be the same or different, represent aryl, heteroaryl, alkyl or acyl groups with one or more unsaturations in the chain that can also be branched or straight and can have or not have substitutions,

R₃ can be an alkyl, alkenyl, alkynyl, aryl or any heterocyclic group, and

n can have any value and can be branched or straight, with or without substitutions and contain unsaturated carbons on the chain.

A method as defined in claim 10, wherein n is 0 to 8. A method as defined in claim 11, wherein n is 0 to 2.

Composition or kit-of-parts according to claim 12, wherein Ri is H, R₃ is - (CH₂)i_ i₂CH₃, n is 0 and R₂ is selected from the group consisting of:

-CO-(CH₂)₆-CH_3,

-CONH-(CH₂)₇-CH_3,

-CSNH-(CH₂)₇-CH_3,

-COCO-CH₂-CH_3,

-COCO-(CH₂)₅-CH_3,

-CO-(CH₂)₄-CH_3, and

-CO-(CH₂)₈-CH₃.

Composition or kit-of-parts according to any of claims 8 to 13, wherein the drug used for the treatment of a disease caused by HIV infection is selected from the group consisting of a HIV protease inhibitor, a HIV reverse transcriptase and a HIV entry inhibitor. A method for the treatment of a disease caused by HIV infection in a subject in need thereof comprising the administration to said subject of a composition or kit- of-parts according to any of claims 8 to 14.

16. A method as defined in claim 15, wherein the Des-1 inhibitor and the drug used for the treatment of a disease caused by HIV infection are administered together, simultaneously or separately.

17. A method as defined in any of claims 15 or 16, wherein the inhibitor is composition or kit-of-parts is administered topically.

18. An in vitro method for inhibiting HIV infection which comprises contacting target cells with a Des-1 inhibitor under conditions effective to provoke an increase of DHSM in the membranes of said cells.

19 A method according to claim 18, wherein the Des-1 inhibitor is selected from the group consisting of the compounds of Table 1. 0 A method according to claim 19, wherein the Des-1 inhibitor is a compound having the following structure:

wherein

Ri and R₂ that can be the same or different, represent aryl, heteroaryl, alkyl or acyl groups with one or more unsaturations in the chain that can also be branched or straight and can have or not have substitutions,

R₃ can be an alkyl, alkenyl, alkynyl, aryl or any heterocyclic group, and n can have any value and can be branched or straight, with or without substitutions and contain unsaturated carbons on the chain.

21. A method as defined in claim 20, wherein n is 0 to 8.

22. A method as defined in claim 21, wherein n is 0 to 2.

23. A method according to claim 22, wherein Ri is H, R₃ is - (CH₂)i_i₂CH₃, n is 0 and R₂ is selected from the group consisting of:

-CO-(CH₂)₆-CH_3,

-CONH-(CH₂)₇-CH_3,

-CSNH-(CH₂)₇-CH_3,

-COCO-CH₂-CH_3,

-COCO-(CH₂)₅-CH_3,

-CO-(CH₂)₄-CH_3, and

-CO-(CH₂)₈-CH₃

24. A method as defined in claim 18 to 23, wherein the target cells are CD4 and/or CXCR4-positive cells.

25. A method for the identification of compounds suitable for the treatment of a disease caused by HIV-1 infection which comprises:

(i) contacting a candidate compound with a preparation containing Des-1 and

(ii) determining the activity o f Des- 1 ,

wherein a decrease in the activity of Des-1 with respect to a control preparation is indicative that the candidate compound is suitable for the treatment of a disease caused by HIV-1 infection. A method according to claim 25, wherein the preparation containing Des-1 is liver microsomal preparation.

A method for the identification of compounds suitable for the treatment of a disease caused by HIV-1 infection which comprises:

(i) contacting a candidate compound with a cell population,

(ii) isolating a sphingolipid-enriched detergent-resistant membrane from the cell population, and

(iii) determining the relative amount of dihydroceramide or dihydrosphingosine with respect to ceramide or sphingosine,

wherein an increase in the ratios of dihydroceramide to ceramide and/or dihydrosphingosine to sphingosine with respect to a sphingolipid-enriched detergent-resistant membrane preparation isolated from a control cell population is indicative that the candidate compound is suitable for the treatment of a disease caused by HIV-1 infection.