WO1994010991A1

WO1994010991A1 - Ceramide derivatives

Info

Publication number: WO1994010991A1
Application number: PCT/US1993/010223
Authority: WO
Inventors: Richard E. Pagano; Anne G. Rosenwald
Original assignee: Carnegie Institution Of Washington
Priority date: 1992-11-12
Filing date: 1993-10-29
Publication date: 1994-05-26
Also published as: AU5411094A

Abstract

The present invention relates to a method of inhibiting glycoprotein processing and secretion and to ceramide derivatives suitable for use in such a method.

Description

CERAMIDE DERIVATIVES

TECHNICAL FIELD

The present invention relates, in general, to a method of inhibiting glycoprotein processing and secretion and to ceramide derivatives suitable for use in such a method. More specifically, the invention relates to methods of inhibiting viral glycoprotein processing, and thereby inhibiting release of infectious virions, to methods of

inhibiting cell growth and proliferation, and to methods of inhibiting cell secretions. BACKGROUND

The Golgi complex, found in all nucleated cells, plays a central role in directing protein traffic within cells. In addition, it is the major site of synthesis of sphingolipids, which serve a variety of functions in cells and are thought to play a central role in cellular physiology and pathology.

Sphingolipids are synthesized at the Golgi complex from a common endogenous precursor, ceramide (or N- acyl-sphingosine). Derivatives of ceramide labeled with radioactive or fluorescent tags have been added to cells from exogenous sources and have been found to be readily incorporated into cellular membranes and, subsequently, utilized by the

cellular biosynthetic and transport machinery. One striking property of (fluorescent) ceramide

derivatives is that they accumulate at the Golgi apparatus of living cells and can be easily

visualized by fluorescence microscopy. The Golgi apparatus-associated ceramide derivatives are metabolized to several different sphingolipid end- products and the latter are transported to the cell surface via the "secretory pathway" which is

utilized by newly-synthesized plasma membrane and secretory proteins.

Since newly-synthesized proteins en route to the cell surface must first travel through compartments containing the sphingolipid

biosynthetic enzymes, and since sphingolipids comprise a significant fraction of cellular membrane lipids, tests were undertaken to determine whether transport of proteins through the secretory pathway and sphingolipid biosynthesis might be coupled to one another. Initial experiments conducted to study lipid and protein trafficking through the cell were carried out using an inhibitor which blocks the further metabolism of ceramide to certain

sphingolipids (Rosenwald et al, Biochemistry 31:3581 (1992)). The effect of this inhibitor on protein transport through the Golgi complex was studied using cells infected with vesicular stomatitis virus (VSV). Modification of the glycan structure of VSV component "G protein" and the intracellular sites where those modifications occur are well-documented in the literature; thus the modifications of VSV-G protein were used to determine through which

intracellular sites the protein moved.

The results of these initial studies indicated that transport of both sphingolipids and proteins were affected by this drug. In subsequent studies, other inhibitors were tested which block other steps of sphingolipid metabolism, but none duplicated the results obtained with the above inhibitor. The present invention resulted, at least in part, from the realization that the effect of the inhibitor might be attributable to an increase in endogenous ceramide. This possibility, which was tested directly by examining the effects of

exogenously supplied ceramide derivatives on VSV-G protein transport, was found to be the case.

SUMMARY OF THE INVENTION

The present invention relates to a method of inhibiting the processing and secretion of glycoproteins in eucaryotic (for example, mammalian) cells. The inhibition can be effected by

administering to the mammal a ceramide (Cer)

derivative of the formula:

|

wherein:

R_o is (C₁-C₂₀)alkyl or (C₁-C₂₀)alkenyl,

R₁ is hydroxyl, (C₁-C₄)alkoxy, or H,

R₂ is hydroxyl, (C₁-C₄ alkoxy, or H,

R₃ is H or (C₁-C₄)alkyl, and

R₄ is COR₅,

wherein:

R₅ is (C₁-C₂₀)alkyl, (C₁-C₂₀)alkenyl, or

(C₁-C₂₀)alkynyl, which may be substituted with one or more of the following: H, OH, SH, NH₃, halogeno. (C₁-C₄)alkyl, aryl, (C₁-C₄)alkylaryl, aryl(C₁-C₄) alkyl, or

The derivatives can be used to inhibit processing and secretion of, for example, viral glycoproteins. In addition, the derivatives can be used to inhibit cell growth and proliferation by inhibiting processing and secretion of cellular glycoproteins.

Accordingly, it is an object of the invention to provide a method of inhibiting

glycoprotein processing and secretion. It is another object of the invention to provide Cer derivatives suitable for use in such a method.

Other objects and advantages of the invention will be clear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. Effect of 25 μM N-(hexanoyl)- D-erythro-sphingosine (C₆Cer) on transport of vesicular stomatitis virus-G (VSV-G) protein through the secretory pathway. FIGURE 1A shows transport of VSV-G protein through the medial Golgi in the presence or absence of 25 μM C₆Cer; and FIGURE 1B shows transport of VSV-G protein through the trans Golgi. For FIGURES 1A and B, control (●) and

+ 25 μM C₆Cer (♡). FIGURE 2: Effect of increasing amounts of C₆Cer on transport of VSV-G protein through the trans Golgi. 0 μM (●); 5 μM (O); 10 μM (▲); 15 μM (Δ); 20 μM (■); and 25 μM (♡). FIGURE 3: Production of infectious VSV particles in the presence C₆Cer. FIGURE 3A shows release of particles over time in the presence of 0 or 25 μM C₆Cer; and FIGURE 3B shows release of particles in the presence of increasing C₆Cer after 8 h of infection. In FIGURE 3A, each point is the average of duplicate determinations, 0 μM (●) and 25 μM C₆Cer(♡). In FIGURE 3B, each point is the average of triplicate determinations, bars show the standard deviation. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in general, to a method of inhibiting glycoprotein processing, and secretion in eucaryotic (for example, mammalian) cells. Specific embodiments relate to methods of inhibiting viral glycoprotein processing and thereby inhibiting release of infectious virions, to methods of inhibiting cell secretions and to methods of inhibiting cell growth and proliferation. Each of these methods involves the use of Cer derivatives, wherein the term "derivatives" as used herein includes "analogs".

In general terms, Cer derivatives suitable for use in the present method include molecules in which: i) the chain length of the fatty acid moiety attached to the sphingosine or

phytosphingosine backbone is altered; ii) the stereochemistry of the sphingosine or

phytosphingosine backbone is altered; iii) chemical substitutions are introduced on the fatty acid moiety; and iv) chemical modifications are

introduced on the sphingosine or phytosphingosine backbone. With respect to (i) above, "short chain" fatty acids (i.e., fatty acids of 2-14 carbon atoms) are preferred under conditions where increased water solubility is advantageous. Where targeting of the Cer derivative to a particular organ is required, however, long chain molecules (i.e., fatty acids of 16 carbons or greater) are preferred as such

molecules can be expected to remain at the target site longer than short chain counterparts. As for (iii) and (iv) above, substitutions on the fatty acid and/or sphingosine or phytosphingosine backbone can be expected to improve the efficacy of a

particular derivative by decreasing the rate at which it is metabolized. In addition, specific substitutions of the fatty acid and/or backbone can be expected to result in a more potent inhibitory effect on secretion. The derivatives can be

labelled with a detectable "tag", for example, the derivatives can be fluorescent.

The following Cer derivatives are suitable for use in the present method:

wherein:

R₀ is (C₁-C₂₀)alkyl or (C₁-C₂₀)alkenyl,

R₁ is hydroxyl, (C₁-C₄) alkoxy, or H,

R₂ is hydroxyl, (C₁-C₄) alkoxy, or H,

R₃ is H or (C₁-C₄)alkyl, and

R₄ is COR₅,

wherein:

Rs is (C₁-C₂₀)alkyl, (C₁-C₂₀)alkenyl, or

(C₁-C₂₀)alkynyl, which may be substituted with one or more of the following: H, OH, SH, NH₃, halogeno, (C₁-C₄)alkyl, aryl, (C₁-C₄)alkylaryl, aryl(C₁-C₄)alkyl, or

Possible aryl groups include phenyl, substituted phenyl and pyridine. Suitable arylalkyl groups include benzyl and phenethyl. Possible halogeno groups include B and F.

In a preferred embodiment of the present invention, the Cer derivative is of the formula I wherein R₀ is (C₁₃-C₁₇)alkenyl, R₁ and R₂ are,

independently, hydroxyl, (C₁-C₄) alkoxy or hydrogen; R₃ is hydrogen or methyl, and R₅ is pentyl,

unsubstituted, or substituted with

In a more preferred embodiment, R₀ is (C₁₅) alkenyl, R₁ is hydrogen; R₂ is hydroxyl; R₃ is hydrogen; and R₅ is pentyl, unsubstituted.

In another preferred embodiment of the present invention, the Cer derivative is of the formula II wherein R₀ is (C₁₂-C₁₆)alkyl or (C₁₂- C₁₆) alkenyl; R₂ is hydroxyl or (C₁-C₄) alkoxy; R₃ is hydrogen or methyl; and R₅ is pentyl, unsubstituted or substituted with

In a more preferred embodiment, R₀ is (C₁₄)alkyl, R₂ is hydroxyl, R₃ is hydrogen and R₅ is pentyl,

unsubstituted.

In a further preferred embodiment, the Cer derivative is of the formula III wherein R₀ is (C₁₂- C₁₆)alkyl or (C₁₂-C₁₆) alkenyl; R₃ is hydrogen or methyl; and R₅ is pentyl, unsubstituted or substituted with

In a more preferred embodiment, R₀ is (C₁₄)alkyl, R₃ is hydrogen and R₅ is pentyl, unsubstituted.

(See also WO 92/03129).

Two specifically preferred compounds are

One skilled in the art will appreciate from a reading of this disclosure that it may be advantageous to use isolated stereoisomers of the Cer derivatives described above. Preferred isomers are the D-erythro and D-threo isomers.

Cer derivatives to which the invention relates can be synthesized as outlined by Pagano and Martin (Biochemistry 27:4439 (1988)). Alternative routes have been described by Kishimoto (Chem. Phys. Lipids 15:33 (1975)) and Schwarzman and Sandhoff (Methods Enzymol. 138:319 (1987)). Certain Cer derivatives are commercially available.

One skilled in the art will appreciate from a reading of this disclosure that, using the protocol set forth by Pagano and Martin

(Biochemistry 27:4439 (1988) and Rosenwald et al (Biochemistry 31:3581 (1992)), Cer derivatives appropriate for use in the present invention can be selected (see also the Examples that follows). One skilled in the art will also appreciate that no undue experimentation is involved in applying the protocols described to test derivatives.

The Cer derivatives of the invention can be formulated into a pharmaceutical composition. In one embodiment, that composition takes the form of a complex with defatted serum albumin as described by Pagano and Martin (Biochemistry 27:4439 (1988)). In another embodiment, the Cer derivative is

incorporated into a liposome as, for example, described by Lipsky and Pagano (Proc. Natl. Acad. Sci. USA 80:2608 (1983)). Cer can be incorporated into liposomes formed from a variety of lipid types, including but not limited to various mixtures of natural or synthetic phospholipids, sphingolipids, and cholesterol. In addition, liposomes of various sizes and lamellarity are also suitable. Cer can be incorporated into such liposomes over a wide range of concentrations, up to as much as 30-40 mol%.

One skilled in the art will appreciate that the amount of derivative administered will depend on the specific result sought to be achieved. The derivatives of the invention can be

administered, for example, orally, by injection or topically in an appropriate formulation as described above. The Cer derivatives of the invention are expected to be useful in treating infections

involving enveloped viruses (while the invention is specifically exemplified below with reference to vesicular stomatitis virus, one skilled in the art will appreciate that, in practice, infections resulting from enveloped viruses such as influenza or rhinoviruses can be treated in accordance with the present invention). This expectation results from the fact that the protein making up the coat of such viruses is modified during transport through the secretory pathway of cells and Cer derivatives appear to exert their effect by altering protein (and perhaps lipid) transport along the secretory pathway.

The derivatives of the invention, as noted above, are also expected to be useful as

antiproliferative agents. Any cells, including tumor cells, that require rapid and extensive transport of proteins through the secretory pathway can be expected to be adversely affected by Cer derivatives of the invention. One example of a malignancy that can be expected to be susceptible to Cer derivative treatment is B cell leukemias which secrete immunoglobulin.

Certain aspects of the invention are described in greater detail by reference to the non- limiting Examples that follows. The Examples demonstrate that short-chain derivatives of Cer, N- (hexanoyl)-D-erythro-sphingosine (hexanoyl Cer or

C₆Cer), N-(acetyl)-D-erythro-sphingosine (acetyl Cer or C₂Cer), N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosine (C₅- DMB-Cer), and N- [7- (4-nitrobenzo-2-oxa-1 , 3- diazole)]-6-aminocaproyl-D-erythro-sphingosine (C₆- NBD-Cer), affect the transport of the model plasma membrane protein, VSV-G protein, through the secretory pathway. The effects of these derivatives were immediate and all of the effects seen were dependent on the concentration of Cer. There are at least two possible explanations for these results. First, Cer may signal changes in the phosphorylation of key regulatory proteins modulating protein traffic. Cer stimulates an okadaic acid- inhibitable, cytosolic protein phosphatase activity (Dobrowsky et al, J. Biol. Chem. 267:5048 (1992)) and treatment of cells with okadaic acid induces fragmentation of the Golgi apparatus and arrest of intracellular transport (Lucocq et al, J. Cell

Science 100:753 (1991)). In addition, a Cer- activated, membrane-bound protein kinase activity has been described (Dressier et al. Science 255:1715 (1992); Mathias et al, Proc. Natl. Acad. Sci, USA 88:10009 (1991)). A second possibility arises from observations regarding the spatial distribution of sphingolipid metabolism. It was previously found that although C₆-NBD-Cer (and presumably other short- chain Cer analogs, including C₆Cer) targets to the trans aspects of the Golgi complex (Pagano et al, J. Cell Biol. 109:2067 (1989)), the Cer utilizing enzymes, SM synthase and GlcCer synthase, are found in the cis aspects (Coste et al, Biochim. Biophys. Acta 814:1 (1985); Jeckel et al, FEBS Lett, 261:155 (1990); Futerman et al, Biochem. J. 280:295 (1991); Futerman et al, J. Biol. Chem. 265:8650 (1990)).

These results imply that under normal conditions, some transport of Cer from trans to cis Golgi compartments occurs to deliver Cer to the synthases, although this is the opposite direction from that taken by proteins along the secretory pathway

(Dunphy et al. Cell 42:13 (1985); Rothman et al, FASEB J. 4:1460 (1990); Rothman et al. Nature

355:409 (1992)). In the presence of large amounts of Cer, as documented below, transport may be driven nearly exclusively in the retrograde direction at the expense of anterograde transport.

Example 1

Effects of a Short-chain Cer Analog, N-hexanoyl-D- erythro-sphingosine (hexanoyl Cer), on the Transport of a Model Plasma Membrane Protein

CHO cells, passaged as previously described (Rosenwald et al. Biochemistry 31:3581 (1992)) were grown to confluence on 35 mm culture dishes and were infected with VSV in medium

containing 5% fetal bovine serum (FBS) for a total of 4 h. The cells were then starved for methionine by incubation in methionine-free (serum-free) medium (GIBCO/BRL, Bethesda, MD) for 15 min at 37°C. VSV proteins were labeled by incubation for 5 min at 37°C in the same medium, but containing 5 μCi/ml

[³⁵S]methionine (>1000 Ci/mmol; cell-labelling grade; Amersham, Arlington Heights, IL). Labeling medium was replaced with chase medium (Rosenwald et al, Biochemistry 31:3581 (1992)) with either 25 μM defatted bovine serum albumin (BSA) or 25 μM

C₆Cer/BSA (Pagano and Martin, Biochemistry 27:4439 (1988)) and incubated at 37°C in chase medium for up to 80 min. Cells were harvested by washing the monolayers twice with cold chase medium without BSA and without C₆Cer, then scraping in lysis buffer as described by Rosenwald et al (1992). Samples were treated with endoglycosidase H as described by

Rosenwald et al (1992), except the enzyme

concentration was 2.5 mU/ml. After digestion, samples were prepared for electrophoresis and analyzed as described by Rosenwald et al (1992). Densitometry was performed as described (Rosenwald et al (1992)), but using a Molecular Dynamics 300A computing densitometer (Sunnyvale, CA). The results presented in FIGURES 1A and B show that incubation of VSV-infected Chinese hamster ovary (CHO) cells with 25 μM C₆Cer slowed vesicular stomatitis virus (VSV-G protein) transport through the medial Golgi compartment (as measured by

acquisition of resistance to the endoglycosidase, endo H), increasing the t½ for transit nearly 2- fold. Transport through the trans compartment

(measured by a change in R₁, indicative of

sialylation), was more severely affected by

treatment with C₆Cer, increasing the t½ at least 6- fold (see FIGURES 1A and B).

Example 2

VSV-G Protein Transport Through the trans Golgi Cells infected with VSV were starved, labeled, _{and chased as in Example 1, but with 0-} 25 μM C₆Cer present in the chase medium (BSA was 25 μM in all samples). Chases were terminated after washing the monolayers two times as in Example 1, then scraped directly into SDS-PAGE sample buffer as described (Rosenwald et al (1992)). The

concentrations used were: 0 μM (●), 5 μM (O), 10 μM (▲), 15 μM (Δ), 20 μM (■), and 25 μM (♡).

The data presented in FIGURE 2 show that the rate of VSV-G transport through the trans Golgi decreased with increasing concentrations of C₆Cer. Similar results were seen with three other analogs, acetyl Cer, and the fluorescent analogs, C₆-NBD-Cer (Lipsky and Pagano, Proc. Natl. Acad. Sci., USA 80:2608 (1983), Lipsky and Pagano, J. Cell Biol.

100:27 (1985)), and C₃-DMB-Cer (Pagano et al, J. Cell Biol. 113:1267 (1991)). Interestingly, the acetyl Cer analog did not appear to be as effective as the C₆Cer and C₆-NBD-Cer analogs. Cer, rather than one of its metabolites- appeared to be responsible for inhibition of VSV-G transport . Sphingosine, an intracellular signal in several systems (Hannun et al, Science 243:500

(1989); Kolesnick, Prog. Lipid Res. 30:1 (1991);

Koval et al Biochim. Biophys. Acta 1082:193 (1991); Merrill et al, Biochim. Biophys. Acta 1044:1

(1990)), had no effect on VSV-G transport through the trans Golgi at concentrations where C₆Cer had a marked effect. In addition, transport was

unaffected by incubation of cells with the ceramide synthase inhibitor, Fumonisin B, (Wang et al, J.

Biol. Chem. 266:14466 (1991)). However, the

glycosphingolipid synthesis inhibitor, 1-phenyl-2- decanoylamino-3-morpholino-1-propanol, which

increases intracellular Cer concentrations (Felding- Habermann et al, Biochemistry 29:6314 (1990);

Inokuchi et al, J. Lipid Res. 28:565 (1987); Okada et al, FEBS Lett. 25 (1988); Shayman et al, J. Biol. Chem. 266:22968 (1991)), slows VSV-G transport

(Rosenwald et al. Biochemistry 31:3581 (1992)). To explore the possibility that other metabolites of ceramide, like Cer-1-phosphate (Dressier and

Kolsnick, J. Biol. Chem. 265:14917 (1990); Kolesnick and Hemmer, J. Biol. Chem. 265:18803 (1990)), might be the signal in this system, experiments were performed with the fluorescent analog, C₆-NBD-Cer, whose metabolism can be easily assessed (Lipsky and Pagano, Proc. Natl. Acad. Sci., USA 80:2608 (1983); Lipsky et al, J. Cell Biol. 100:27 (1985)). After incubation with 25 μM C₆-NBD-Cer for 30 min at 37°C, 80% was unmodified, less than 1% was converted to Cer-1-phosphate, and the remaining 20% was

metabolized to the corresponding fluorescent analogs of sphingomyelin (C₆-NBD-SM) and glucosylceramide

(C₆-NBD-GlcCer). However, when infected cells were incubated with 25 μM C₆-NBD-Cer, C₆-NBD-SM, or C6- NBD-GlcCer, only C₆-NBD-Cer markedly affected transport of VSV-G through the trans Golgi.

Example 3

Effect of C₆Cer on Release of Infectious VSV Particles

CHO cells on 60 mm dishes were infected with VSV as in Examples 1 and 2, except the

infection was performed in serum-free media. After incubation for 30 min at 37°C, the monolayers were washed twice with medium containing 5% fetal bovine serum and twice with serum-free medium to remove unadsorbed virions. The cells were then incubated for up to 24 h in serum-free medium containing 0- 25 μM C₆Cer (all samples contained 25 μM BSA). At the appropriate times (2-25 h for FIGURE 3A; 8 h for FIGURE 3B), aliquots of this medium were removed and stored at -70°C. To determine the number of

particles released, the samples were thawed and diluted in serum-containing medium. Dilutions were incubated with confluent monolayers of CHO cells for 30-60 min at 37°C to allow virions to attach to cells. This viral inoculum was removed and replaced with growth medium containing 2% fetal bovine serum and 0.75% agarose. This mixture was allowed to harden at room temperature, then incubated at 37°C for 18-28h. Following this incubation, the agarose was removed, then the monolayers were washed and stained with 1% methylene blue in 20% ethanol

(Henneman and Kohn in Tissue Culture Association Manual, V.J. Evans, V.P. Perry and M.M. Vincent,

Eds. (Tissue Culture Association Biomedical Research Institute, Rockville, MD) vol. 2 pp. 103-104 (1976)) to reveal viral plaques (evident as clear spots on a blue background). The plaques were counted, and calculations performed to determine the number of plaque-forming units in the initial aliquot. The results presented in FIGURE 3A show that the number of plaque forming units released over time in the presence 25 μM C₆Cer was decreased relative to untreated cells throughout the time course, especially within the first 10 h of

infection, where a 50-200 fold reduction in the number of infectious particles was seen. At the latest time point (25 h), a 2-fold reduction was seen (Fig. 3A; (see thesis of D. Larsen, Johns

Hopkins University (1983)). The number of

infectious particles released decreased with

increasing concentration of C₆Cer (Fig. 3B). After 8 h of infection, the presence of 5μM C₆Cer resulted in a 3-fold reduction in plaque forming units and the presence of 25 μM C₆Cer resulted in a 200-fold reduction. These results were due to the release of fewer particles and to the release of non-infectious particles. First, supernatants from cells treated with C₆Cer contained at least 2-3 fold fewer viral particles than control supernatants at both early

(4 h) and late (24 h) times after infection as seen by electron microscopy. Second, when viral

particles from [³⁵S]methionine-labeled cells were obtained from supernatants from equal numbers of cells by high-speed centrifugation and analyzed by gel electrophoresis, less radioactivity was found in samples from treated cells than from untreated cells (~50% after 2 h of chase). Finally, the ratio of VSV-G to internal VSV proteins was ~4 fold lower in virion samples from treated cells than from control cells after 2 h of chase, indicating the particles released were aberrantly assembled. Thus, the effects of short-chain Cer analogs on release of infectious viral particles indicate that these agents can be used to control infections stemming from enveloped viruses, since this treatment

resulted in the delayed release of infectious particles, decrease in the number of particles, and in particles that were assembled incorrectly.

Example 4

Toxicity of Short-Chain Cer Derivatives The short-chain CER derivatives were not toxic to cells as measured by two criteria. First, protein synthesis was unaffected by C₆Cer. Cells treated with up to 25 μM C₆Cer for 30 min

incorporated the same amount of [³⁵S]methionine into protein as did control cells. Second, the cells appeared to maintain viability in the presence of the short-chain Cer analogs. Uninfected cells treated for up to 24 h with 25 μM C₆Cer in serum- free media maintained the ability to exclude the vital dye, trypan blue (>95% for control and treated samples), although the cell number did not increase under these incubation conditions. However, if the Cer-containing medium was removed and replaced with serum-containing medium, the cells doubled over the subsequent 24 h, demonstrating cells remained viable. Cessation of cell growth in the presence of Cer was previously demonstrated in HL-60 cells

(Okazaki et al, J. Biol. Chem. 265:15823 (1990)).

One skilled in the art will appreciate from a reading of the foregoing disclosure that various changes can be made in form and detail without departing from the true scope of the

invention.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting glycoprotein processing in a cell and secretion of said

glycoprotein from said cell comprising contacting said cell with a ceramide derivative of the formula: | | | 2

4

wherein:

R₀ is (C₁-C₂₀)alkyl or (C₁-C₂₀) alkenyl,

R₁ is hydroxyl, (C₁-C₄) alkoxy, or H,

R₂ is hydroxyl, (C₁-C₄) alkoxy, or H,

R₃ is H or (C₁-C₄)alkyl, and

R₄ is COR₅,

wherein:

R₅ is (C₁-C₂₀ ) alkyl , (C₁-C₂₀ ) alkenyl , or

(C₁-C₂₀)alkynyl, which may be substituted with one or more of the following: H, OH, SH, NH₃, halogeno, (C₁-C₄)alkyl, aryl, (C₁-C₄)alkylaryl, aryl(C₁-C₄)alkyl,

under conditions such that said processing and secretion are inhibited.

2. The method according to claim 1 wherein said glycoprotein is a viral glycoprotein.

3. A method of treating a viral

infection comprising contacting cells infected with said virus with a ceramide derivative of the

formula:

wherein:

R₀ is (C₁-C₂₀)alkyl or (C₁-C₂₀) alkenyl,

R₁ is hydroxyl, (C₁-C₄) alkoxy, or H,

R₂ is hydroxyl, (C₁-C₄) alkoxy, or H,

R₃ is H or (C₁-C₄)alkyl, and

R₄ is COR₅,

wherein:

R, is (C₁-C₂₀)alkyl, (C₁-C₂₀) alkenyl, or (C₁-C₂₀)alkynyl, which may be substituted with one or more of the following: H, OH, SH, NH₃, halogeno, (C₁-C₄)alkyl, aryl, (C₁-C₄)alkylaryl, aryl(C₁-C₄)alkyl, or

under conditions such that said treatment is effected.

4. The method according to claim 3 wherein said virus is an enveloped virus.

5. The method according to claim 4 wherein said virus is influenza or rhinovirus.

6. The method according to claim 1 or 3 wherein the derivative is

7. The method according to claim 1 or 3 wherein the derivative is