AU653363B2 - Methods for inducing cell differentiation using ceramides - Google Patents

Description

VEUSE ONJZJ Per~ yj )q pAge", 1/13-13113, drawing i, replacaki by new page tl4 114, due o laIte iransrilial by the rceiving 0(11cc INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 92/03129 A61 31/13 Al (43) International Publication Date: 5 March 1992 (05.03,92) (21) International Application Number: PCT/US91/05743 (72) Inventors; and Inventors/Applicants (for US only) HANNUN, Yusuf, A.

(22) International Filing Date: 13 August 1991 (13.08.91) [US/US]; 131 Summerlin Drive, Chapel Hill, NC 27514 BELL, Robert, M. [US/US]; 3705 Dairy Pond Place, Durham, NC 27705 (US).

Priority data: 566,978 13 August 1990 (13.08.90) US (74) Agents: CALDWELL, John, W. et al.; Woodcock Washburn Kurtz Mackiewicz Norris, One Liberty Place, 46th Floor, Philadelphia, PA 19103 (US).

Parent Application or Grant (63) Related by Continuation US 566,978 (CIP) (81) Designated States: AT (European patent), AU, BE (Euro- Filed on 13 August 1990 (13.08,90) pean patent), BR, CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FI, FR (European patent), GB (European pa.

(71) Applicant (for all designated States except US): DUKE UNI- tent), GR (European patent), HU, IT (European patent), VERSITY [US/US]; Office of Technology Transfer, JP, KR, LU (European patent), NL (European patent), Durham, NC 27706 NO, SE (European patent), US.

Published With international search report.

653363 ',)Title: METHODS FOR INDUCING CELL DIFFERENTIATION USING CERAMIDES H H I I R C C I I

R

2

N

CH

2

OH

R

3 R4 (57) Abstract The present invention provides methods and compositions for inducing differentiation of cells. Compositions having formula wherein RI is C, to about C 20 alkyl or alkenyl; R 2 is hydroxyl, alkoxy or H, R 3 is H or lower alkyl; R 4 is COR 5

SO

2

R

5 or CSRs, where R 5 is Ci to C 20 alkyl, alkenyl, or alkynyl, which may be substituted by one or more of the following functional groups: OH, SH, OR 6

SR

6

NR

7

R

8 COOR9, and CONRioRg, where R 6

R

7 Rg, R 9 and Rio independently are H, alkyl, aryl, alkaryl and arylalkyl using up to about 10 carbons, are administered to cells of a mammal that are capable of undergoing differentiation in amounts effective to induce differentiation of the cells. The invention also provides methods and compositions for altering the phenotype of cells and for treating diseases characterized hy hyperproliferation of cells.

*K rarl to h 1 i Ll'e N, IW&Ii$, Stiion, II) W'O 92/03129 PCT/US91/05743 -1- METHODS FOR INDUCING CELL DIFFERENTIATION USING CERAMIDES CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of copending application Serial No. 566,978 filed August 13, 1990 the disclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION The present invention relates to the field of compounds and methods for inducing cell differentiation. More particularly the present invention is concerned with the use of ceramide and derivatives to induce cell differentiation and for treatment of conditions characterized by abnormal cell proliferation.

REFERENCE TO GOVERNMENT SUPPORT The research disclosed herein was supported in part by National Institutes of Health grants ES 00155 and CA 46738.

The United States government has certain rights in the invention.

BACKGROUND OF THE XNVENTION A number of human malignant and non-malignant diseases have as one of their distinguishing features the hyperproliferation of cells. In these diseases, cells proliferate at abnormally high rates. The cells found in cancerous tumors and leukemias grow and divide uncontrollably, which accounts in part for their rapid spread in the body.

Similarly, with some non-malignant diseases such as psoriasis, the cells also grow and divide at abnormally high rates. In WO 92/03129 cr 93/US9I/5743 2 these diseases, the hyperproliferating cells are present in a relatively undifferentiated state. Undifferentiated cells are able to grow and divide. Once a cell differentiates, however, it loses the ability to proliferate. Some proposed treatments have been aimed towards inducing cell differentiation to stop cell proliferation, and thus bring the diseases under control.

Recently it has been found that sphingolipids play important roles in cell growth, oncogenesis, and differentiation (Hannun, Y.A. and Bell, R. M. (1989) Science 243: 500-507). Sphingolipid breakdown products are emerging as a novel class of cell regulatory molecules. Sphingolipid breakdown products, sphingosine and lysosphingolipids, inhibit protein kinase C, believed to be a pivotal enzyme in cell regulation and signal transduction (Hannun, Y.A. et al. (1986) J. Biol Chem. 261: 12604-12609). Sphingolipids and lysosphingolipids affect significant cellular responses and exhibit anti-tumor promoter activities in various mammalian cells (Hannun, Y.A. and Bell, R.M. (1987) Science 235: 670- 674; Hannun, Y.A. et al. (1987) J. Biol. Chem. 262: 13620- 13626; and Wilson, E. et al. (1987) Arch. Biochem. Biophys.

259: 204-214).

U.S. Patent 4,710,490 issued December 1, 1987 to Catsimpoolas discloses compositions which contain lipid containing molecules possessing angiogenic activity. The lipids are derived from mammalian sources, particularly the omentum. Mixtures of known lipids, such as gangliosides, were also found to possess angiogenic activity. The compositions stimulated the growth of blood vessels in vitro, and in vivo.

Gangliosides possessed the greatest angiogenic activity, whereas glycolipids such as ceramide derivatives had little or no activity.

U.S. Patent 4,673,667 issued June 16, 1987 to Catsimpoolas discloses plasmin inhibitory substances derived from mammalian omental extracts. The substances contain lipid components. Gangliosides exhibited the greatest plasmin inhibiting activity. Various ceramide or ceramide derivative WO 92/03129 PCT/US91/05743 3 samples exhibited no or minimal plasmin inhibiting activity.

Japanese patent application Hl-93562 published April 12, 1989 discloses sphingosine derivatives that are useful for the treatment of tumors.

U.S. patent 4,816,450 issued March 28, 1989 to Bell discloses long chain bases, generally sphingosine and sphingosine derivatives, useful for inhibiting protein kinase C. Activation of protein kinase C has been identified as fundamental to tumor promotion, cellular transformation and to understanding the inhibition by anti-tumor agents.

Interferon which induces cell differentiation has been tested for treatment of tumors. Similarly, vitamin D 3 which induces differentiation of HL-60 cells, a human myelocytic leukemia cell line, has also been tested for tumor treatment. Although vitamin D 3 is able to induce cell differentiation, the use of this compound for treating tumors is not feasible since the large amounts of vitamin D 3 needed interferes with calcium metabolism in the body to an unacceptable degree.

Despite the efforts in developing treatments for diseases characterized by cellular hyperproliferation, there is still a need for treatments for these diseases.

Accordingly, it is an object of the invention to provide methods and compositions for inducing cell differentiation.

It is also an object of the invention to provide methods and compositions for altering the phenotype of cells. It is a further object to provide methods and compositions for treating diseases characterized by hyperproliferation of cells. Yet another object is to provide compositions for prevention ana palliation of diseased or abnormal states in mammals characterized by abnormal cell differentiation. Other objects of the invention will become apparent from v review of the present specification and appended claims.

SUMMARY OF THE INVENTION The present invention provides compositions and methods for inducing cell differentiation. The invention also provides methods and compositions for altering the phenotype of cells and treating diseases characterized by hyperproliferation of cells. In the methods of the invention, compounds are administered to a mammal, usually a human patient, in therapeutically effective amounts, such compounds having formula I: H H I I

R

1 C -C CH20H I I I

R

2

N

R

3

R

4 wherein R 1 is C 1 to about C 20 alkyl or alkenyl;

R

2 is hydroxyl, alkoxy or H,

R

3 is H or lower alkyl;

R

4 is COR 5

SOR

5 or CSR 5 where R 5 is C 1 to C 20 alkyl, al-eny,, or alkylyl, which may be substituted by one or more of t',e following functional groups: OH, SH, OR6, SR6, NR 7

R

8 COORg, and CONRioR 8 where R 6

R

7

R

8 Rg, and

R

10 independently are H, alkyl, aryl, alkaryl and arylalkyl using up to about 10 carbons.

The compounds of the invention are useful in treating conditions where hyperproliferation of cells is present or there is significant disturbance in differentiation of cells. Accordingly, the compounds and pharmaceutical oe preparations of the invention are useful in the S: preparation and/or manufacture of a medicament for inducing differentiation of cells.

30 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a graph of the dose-dependence of I ceramide in response to 1,25-(OH) 2

D

3 HL-60 cells were treated with 100 nM 1,25-(OH) 2

D

3 for 2 hours. The mass of ceramide was measured using E. coli DAG kinase. The vertical axis shows ceramide concentration as per cent of control (no C 18

/C

2 ceramide). The horizontal axis shows the log of the 1, 25-(OH) 2

D

3 concentration. Bars represent 1 standard deviation. Base-line ceramide levels were 26.1 0.82 pmol/nmol of phospholipid.

Figure 2 shows a graph of the time course of mass increase of ceramide in response to 1,25-(OH) 2

D

3 cells were harvested at the indicated time points after treatment with 100 nm 1,25-(OH) 2

D

3 Ceramide mass was measured enzymatically using sn-1,2-diacylglycerol (DAG) kinase as described in Preiss, et al., J. Biol. Chem.

261:8697-8700 (1986) and Van Veldhoven, Anal. -iochem.

183:177-189 (1989). The vertical axis shows ceramide concentration in pmol/nmol phospholipids. The horizontal axis shows the elapsed time after the start of the experiment in hours. Bars represent 1 standard deviation.

Figure 3 shows a graph of the mass changes of total phospholipids phosphatidylcholine (PC) and sphingomyelin in response to 1,25-(OH) 2

D

3 treatment of cells. The levels of phospholipids, PC, and sphingomyelin in HL-60 cells were measured at the 20 indicated time points after treatment with 100 nM 1,25-

(OH)

2

D

3 as described in "Mass Measurement of Lipids" found in the Experimental section of the specification. In Figure 3A, the vertical axis shows the concentration of phospholipids in 10 14 mol/cell, and the horizontal axis shows the elapsed time in hours. In Figure 3B, the vertical axis shows the concentration of phosphatidyl choline in pmol/nmol of phospholipids and the horizontal axis shows elapsed time in hours. In Figure 3C, the vertical axis shows the concentration of sphingomyelin in 30 pmol/nmol of phospholipids and the horizontal axis shows the elapsed time in hours. The results are averages of two determinations. Bars represent 1 standard deviation for duplicate measurements. The data are representative of two different experiments.

Figure 4 shows the ability of C 18

/C

2 ceramide and 1 nM 1,25-(OH) 2

D

3 to induce HL-60 cell differentiation.

cells (2.5 X 106 cells/mi) were treated simultaneously with various concentrations of C 18

/C

2 ceramide and 1 nM 1,25-(OH) 2

D

3 Cell differentiation was judged by nitro blue tetrazolium (NBT) reducing ability (shaded) and nonspecific esterase (NSE) (unshaded) activity. The vertical axis shows the percent of NBT and NSE positive cells and the horizontal axis shows the amount in LM of C 18

/C

2 ceramide. The effects of C18/C 2 ceramide on cell growth are shown in the inset. The horizontal axis of the inset shows growth X 105 cells/ml) and the horizontal axis of the inset shows the amount of C 18

/C

2 ceramide in piM. The results were obtained from three different experiments. Bars represent 1 standard deviation for duplicate measurements.

Figure 5 shows a graph of the effects of C 18

/C

2 ceramide on HL-60 cell growth. The cells (2.5 X 105 cells/ml) were treated with various concentrations of

C

18

/C

2 ceramide. The different concentrations are represented by control (unshaded circle), 1 lM (shaded 20 diamond), 3 lM (shaded square), 6 iM (shaded circle) and (AM (shaded triangle). The vertical axis shows cell growth (X 105 cells/ml) and the horizontal axis shows elapsed time in days. The results were obtained from three determinations. Bars represent 1 standard deviation for duplicate measurements.

Figure 6 shows a graph of the effects of Clj/C% ceramide on HL-60 cell differentiation in the absence of 1,25-(OH) 2

D

3 The cells were treated with various concentrations of C 18

/C

2 ceramide for 4 days. Cell S 30 differentiation was judged by NBT (shaded) reducing ability and nonspecific esterase (NSE) (unshaded) activity. The vertical axis shows NBT and NSE positive cells and the horizontal axis shows the amount of C18/C 2 ceramide in iM. The results are averages of three determinations. Bars represent 1 standard deviation for multiple measurements.

Figure 7 shows a graph of the time course of cell differentiation induced by C 18

/C

2 ceramide. cells were treated with (shaded) or without (unshaded) 6 p.

M ceramide. Cell differentiation was judged by NBT (shaded and unshaded circles) reducing ability and nonspecific esterase (shaded and unshaded squares) activity. The vertical axis shows NBT and NSE positive cells and the horizontal axis shows elapsed time in days.

The results were obtained from three determinations. Bars represent 1 standard deviation for multiple measurements.

Figure 8 shows the effects of C 18

/C

2 ceramide on mass of sphinglmyelin in HL-60 cells. The cells were treated with 5 lM C 18

/C

2 ceramide for the indicated times.

Sphingomyelin was extracted and measured as described in the Experimental section. The vertical axis shows the percent of control (in the absence of C 18

/C

2 ceramide) and the horizontal axis shows elapsed time in hours. Bars show 1 standard deviation for multiple measurements. The I, results were obtained from two different experiments.

Figure 9 shows a graph of the effects of transient increase of C18/C 2 ceramide on HL-60 cell differentiation.

The cells were treated without or with C 18

/C

2 ceramide 1, or 2 pM) for 2 hours, washed with RPMI 1640 media three times, and then resuspended in serum-free RPMI 1640 media. At the indicated day washing out treatment, the differentiation was measured by NBT reducing ability as described in the Experimental section. The vertical axis shows of NBT positive cells and the horizontal axis shows elapsed time in days. Open circles represent 0 pLM 30 C18/C 2 ceramide. Closed triangles represent 0.5 iM Cis/C 2 ceramide. Closed squares represent 1 C 18

/C

2 ceramide.

Closed circles represent 2 P.M C 18

/C

2 ceramide. Bars represent 1 standard deviation for multiple measurements.

The results were obtained from two different experiments.

Figure 10 shows a graph of the effects of transient increase of C 18

/C

2 ceramide on HL-60 cell differentiation.

The vertical axis of shows NBT positive cells and the horizontal axis shows the time of treatment in hours. 0 M C 18

/C

2 ceramide is represented by unshaded bars. 0.5 pM

C

18

/C

2 ceramide is represented by light cross-hatched bars. 1 [LM CIS/CZ ceramide is represented by dark crosshatched bars. 2 LM C 18

/C

2 ceramide is represented by shaded bars. Bars represent 1 standard deviation for multiple measurements. The results were obtained from two different experiments.

Figure 11 shows an autoradiogram of a thin layer chromatography (TLC) plate after 24 hours exposure of the uptake and metabolism of 3

H]C

18

/C

2 ceramide into cells. Cells (5 X 106 cells/ml) were labeled with 4 pLM [3H] C 18

/C

2 ceramide (1 X 105 cpm/ml). The uptake of

C

18

/C

2 ceramide was about 0.5 hours after treatment.

The rest of the ceramide remained unchanged in the medium. Samples were taken at 0, 0.5, 1, 2, 3, 4, 6, 12, and 24 hour time points. Lipids were extracted and separated by TLC plate. A 24 hour exposure of the plate was taken. SM (sphingomyelin), SPH (sphingosine) and ceramide are identified by arrows.

Figure 12 shows a graph of the lipids extracted from cells showing the uptake and metabolism of 3

H]C

18 /CZ ceramide into HL-60 cells. Cells (5 X 106 cells/ml) were labeled with 4 plM H] C 18

/C

2 ceramide (1 X 105 cpm/ml). Lipids were extracted, separated on TLC plates, and radioactivity was counted as described in the u! Experimental section. The uptake of C 18

/C

2 ceramide was about 20% at 0.5 hours after treatment. The rest of the 30 ceramide remained unchanged in the medium. Ceramide e is represented by open circles. Sphingomyelin is represented by open squares. Sphingosine is represented by closed circles. The vertical axis shows CPM per nmol of phospholipid. The horizontal axis shows elapsed time in hours.

Figure 13 shows graphs of the effects of sphingosine on 1,25-(OH) 2

D

3 -induced HL-60 cell differentiation. cells (2.5 X 105 cells/ml) were treated with various concentrations of sphingosine for 4 days in the presence of 1 nM 1,25-(OH) 2

D

3 Cell differentiation was judged by NBT reducing ability (shaded) and NSE (unshaded) activity.

The effects of sphingosine on HL-60 cell growth are shown in the inset. The vertical axis shows NBT and NSE positive cells. The horizontal axis shows the concentration of sphingosine in .IM. The vertical axis of the inset shows growth of cells (X 105 cells/ml) and the horizontal axis shows the concentration of sphingosine in IM. The results were obtained from three determinations.

Bars represent 1 standard deviation for multiple measurements.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Applicants have discovered that administration of ceramide and derivatives of ceramide induces differentiation of HL-60 cells, a line of myelocytic leukemia cells. Administration of ceramide or derivatives of ceramide slows proliferation of the cells and induces the cells to display a differentiated phenotype indicative of normal monocyte cells. It is believed that this effect will be manifested in other types of cells as well.

The human cell line HL-60, originally isolated from a patient with acute myelocytic leukemia, is frequently used to study myeloid cell differentiation. These cells can be induced to mature into granulocytes when treated with agents such as dimethyl sulfoxide or retinoic acid, or into monocyte/macrophage-like cells upon incubation •with phorbol WO 92/03129 PCr/US91/05743 6 saters, la,25-dihydroxyvitamin D 3 or ganglioside GM 3 The mechanism by which maturation is caused by most of these compounds is not known. For a review of the characeristics of the HL-60 promyelocytic leukemia cell line and its use as a model for the study cell of differentiation see Collins, S.J. (1987) Blood 20: 1233-1244. This well-known cell model has been used to show the usefulness of the compounds of this invention for treating diseases characterized by cell hyperproliferation.

In prior work (Okazaki, T. et al, (November 1989) J. Biol. Chem. 264: 19076-19080), it was reported that differentiation of HL-60 cells with 1.25-(OH)ZD 3 was accompanied by sphingomyelin turnover. It was found that activity of a neutral sphingolyelinase, detected in extracts of HL-60 cells, was induced by 1,25-(OH) 2

D

3 treatment and was accompanied by the generation of ceramide and phosphorylcholine. Sphingomyelin, ceramide and phosphorylcholine levels returned to baseline levels within four hours, suggesting a resynthesis phase of sphingomyelin, thus completing a sphingomyelin cycle. These observations are believed to indicate the operation of a sphingomyelin cycle in which inactive parental sphingolipids are convorted to active metabolites during cell activation. Unlike the phosphatidylinositol cycle, sphingomyelin turnover occurs over a longer period and may be involved with longer term cell changes.

At least 300 different sphingolipids are synthesized in various mammalian cell types. Structurally, sphingolipids are composed of a long-chain sphingoid base, an amide-linked fatty acid, and a polar head group at the 1-position. Except for ceramide, which has hydroxyl at the 1-position, and for Swhich has a phosphorylcholine head group, all other sphingolipids contain carbohydrate head groups and hence are designated glycosphingolipids. These neutral lipids, contain from one (cerebrosides) to 20 or more glucose units, while acidic glycosphingolipids, contain one or more sialic acid residues (gangliosides) or sulfate monoester groups WO 92/03129 PC/US9/05743 7 (sulfatides). Most of the gangliosides and complex glycolipids are thought to reside on the outer leaflet of the cell membrane. Sphingomyelin, however, also resides in the interior of the cell.

The compounds suitable for use in the invention may be naturally occurring or synthetically produced. Compounds having formula I are suitable for use in the pharmaceutical preparations and methods of the invention.

H H R C- C CH2OH I R N

R

3

R

4 wherein R, is C, to about C 20 alkyl or alkenyl;

R

z is bydroxyl, alkoxy or H,

R

3 is H or lower alkyl;

R

4 is SOzR 5 or CSRs, where R 5 is C 1 to C 20 alkyl, alkenyl, or alkynyl, which may be substituted by one or more of the following functional groups: OH, SH, OR 6

SR

6

NR

7 yR COORg, and CONRioRB, where Re, R 7

R

8

R

9 and Rio independently are H, alkyl, aryl, alkaryl and arylalkyl using up to about carbons.

The compounds are preferably cell soluble, i.e., Meone o f able to pass through the cellwal- and enter the interior of the cell. Compounds wherein Ri and R4 taken together have from about 10 to about ?8 carbons are more preferred. The total length of the carbon chains of Ri and R 4 may be divided in aniy combination between R, and R 4 provided that Ri and R 4 each contain at least one carbon. For instance, RI could be

C

1 and R, could be C12, or R, could be Ci 0 and R 4 could be C 7 It has been found that compounds of this size are more easily able to pass through the cell membrane and enter the interior of the cell.

RI is pvrfe a c i to about C20 alkyl or alkenyl; -mere preferably C, to about C 0 alkyl or C, to Clz alkyl or WO 92/03129 PC/US91/05743 8 alkenyl. R, is preferably H, hydroxyl or alkoxy, more preferably hydroxyl or alkoxy. In one preferred embodiment

R

2 is methoxy. R 3 is preferably H or lower alkyl Ci to

C

6 alkyl), more preferably H,

R

4 is preferably COR 5

SO

2

R

5 or CSRs, where R 5 is C 1 to Co 0 alkyl, alkenyl, or alkynyl which may be substituted by one or more of the functional groups OH, SH, OR 6

SR

6

NR

7 R8, COOR, and CONRioR., where R 6

R

7 RE, R 9 and R 10 independently are H, lower alkyl (i.e C, to C 6 and C, to C 2 aryl, alkaryl, anjd arylalkyl. More preferably R 4 is CORs where R 5 is Ci to

C

20 alkyl or alkenyl. Suitable aryl include phenyl, substituted phenyl and pyridine. Suitable arylakyl include benzyl and phenethyl.

Preferred compounds include ceramide, C1,/C 2 ceramide, C18/C 6 ceramide, C 1

/C

8 ceramide and sphingosine. The nomenclature for these compounds is explained in the Experimental section of this specification.

Compounds of this invention are useful for inducing cellular differentiation. They are administered to cells in a mammal, usually a human patient, that are capable of differentiation in an amount effective to induce differentiation of the. cells. The terms cellular differentiation, differentiation of cells and similar terms are intended to refer to the biological process wherein cells mature and acquire the characteristics of a functional cell.

During differentiation the cell may, for example, acquire or ese1morphological shape or characteristics, and gain or lose the ability to bind substances or perform chemical reactions.

The term inducing differentiation is intended to refer to the acts of manipulating cells that are capable of differentiation to acquire a differentiated phenotype. Generally, mammalian cells begin as immature, undifferentiated cells that then undergo differentiation during which time they acquire the characteristics of mature, differentiated cells.

Compounds having the. structure of formula I are also useful for altering the phenotype of cells. The shape, behavior and other characteristics of a cell including r, WO '92/03129' PCT/US91/05743 9 biochemical activities are generally known as the phenotype of a cell. In this embodiment of the invention, the compounds of the invention are administered to cells having a transformed phenotype in a mammal, usually a human patient, in an amount effective to alter the phenotype of the cell to a phenotype associated with normal cells of the same kind.

The "normal" phenotype of a cell refers to a cell that appears normal by conventional criteria such as shape, markers, growth, response to environment, and regulations. Transformed cells are cells that have been derived from normal cells, either spontaneously or by manipulation, that have acquired cancer-like properties such as more immature/undifferentiated phenotype, increased growth, poor or no response to environment and to controls of cell growth, or the ability to cause tumors in animal models. Altering the phenotype of a cell thus refers to the acts of changing at least one characteristic of the cell, including the ability to bind compounds, express enzymatic activity, response to its environment and other cellular characteristics.

Because of the ability of the compounds of this invention to induce cell differentiation and alter the phenotype of cells, such compounds are expected to be useful for treatment of diseases characterized by hyperproliferation of cells, or where there is significant disturbance in differentiation of cells. Diseases characterized by hyperproliferation of cells include diseases wherein one of the consequences or manifestations of the disease is abnormal proliferation of the involved cells.

Abnormal proliferation of cells is generally manifested by an increase in the number of cells present when compared to the number of cells present in the absence of d,~ease. Hyperproliferation of cells may occur in normal, abnormal or malignant cells. Diseases that may be characterized by hyperproliferation of cells include cancerous tumors, leukemias, non-malignant tumors, psoriasis, atherosclerosis and other diseases. This list is intended to be illustrative and not exhaustive of such diseases. These W~O 92/03 CA1 O CT/US91/05743 10 diseases share in the fact that they are primarily caused by increased and abnormal proliferation of either malignant (e.g.

cancer, leukemia and lymphoma), premalignant myelodysplasia), or benign lymphoproliferative, benign tumors, and psoriasis) cells. Inhibition of cell proliferation by compounds in accordance with this invention may slow the growth of affected cells in these diseases yielding a significant therapeutic and potentially curative effect.

Many disorders of this type are also characterized by having undifferentiated cells. Undifferentiated cells or undifferentiated phenotype refers to immature cells that are usually unable to function as mature cells because they lack the necessary biochemical and physiological machinery characteristic of mature cells. During the process of cell differentiaticn, immature cells begin to express the biochemical and physiological characteristics of mature cells.

For example, in vivo, stem cells differentiated into granulocytes, and monocytes. The inability of undifferentiated cells to change into more differentiated cells having a healthier phenotype, contributes to the lack of normal function. The ability of compounds in accordance with this invention to induce differentiation should also help in attenuating the increased proliferation of these cells and in allowing the cells to acquire the necessary biochemical and phenotypic character ivsics that allow them to function as normal cells. For example, in the case of psoriasis, the compounds of of this invention are believed to induce differentiation of the abnormal proliferating cells in psoriasis which should allow the cells to differentiate into healthy skin. Similarly, in milodysplasias, these compounds are believed to cause differentiation of early and undifferentiated myeloid cells which may play a significant role in combating the main health hazards from these disorders, i.e. the decreased numbers of normal, welldifferentiated blood cells. Leukemia, lymphoma, and other forms of cancer may also be treated by increasing the differentiation of those malignant cells. Since WO 92/013129 (PC/ US91/05743 11 differentiated cells are usually unable to divide, this helps in treating those diseases since the individual cells will no longer be able to replenish the malignant clone and will not be able to metastasize.

The utility of the compounds in accordance with this invention versus a wide array of neoplastic disease is also strongly supported by the observations that both tumor necrosis factor (TNF) and gamma interferon elevate the levels of ceramide and that ceramide may mediate the effects of these agents on HL60 cell differentiation. Therefore, these compounds are expected to be useful in cancer treatment by inducing tumor necrosis and tumor regression.

Since ceramide and derivatives are able to slow the growth of lymphocytes, the compounds of this invention are also believed to be useful in inducing immunosuppression in mammals, particularly humans. Other agents that suppress the growth of lymphocytes such as steroids, anti-lymphocyte antibodies, and others play important roles in inducing immunosuppression. Immunosuppression is very important in organ rejection such as occurs in renal transplant, heart transplant, liver transplant and other organ transplant.

Also, since steroids increase ceramide, ceramide may mimic the effects of steroids as immunosuppressants. The slowing of the growth of cells, particularly lymphocytos, refers to retarding or inhibiting the normal rate of growth and cell division of the cells. Thus compounds that slow the growth of cells have the effect of slowing the rate of growth and normal function of those cells.

Auto-immune disorders are characterized by increased activity and proliferation of self-reactive lymphocyte The ability of ceramide to potentially suppress growth of lymphocytes is expected to significantly contribute to suppressing manifestations of autoimmune disorders. Since corticosteroids increase the levels of ceramide, and corticosteroids have a therapeutic role in autoimmune disorders, it is now believed that ceramide and Jther compounds in accordance with this invention may mediate the NVO 92/0129 PCT/US91/05743 12 action of steroids in these disorders.

Obesity may also be characterized by increased proliferation and metabolism of fat cells in the body. The compounds of this invention may slow the growth of these cells and thus contribute to the reduction of obesity. A major connection arises from the fact that tumor necrosis factor increases the levels of ceramide. TNF is postulated to play a role in inducing cachexia and has been implicated as a potential therapy for obesity. It is now believed that ceramide may be useful for treatment of obesity.

Atherosclerosis is another disease characterized by increased and possibly abnormal proliferation of smooth muscle cells and endothelial cells. It is expected that slowing the growth of these cells by treatment with the compounds of this invention will contribute to the control of atherosclerosis.

The compounds of this invention are also expected to be useful as an anti-skin aging treatment. Retinoic acid, which is known for use in anti-skin aging treatments, elevates the levels of ceramide in skin cells. Since retinoic acid elevates the levels of ceramide, ceramide may mediate the action of retinoic acid and be useful in anti-aging skin treatments.

The present invention may also be useful in chemoprevention, i.e. the treatment of cells to prevent or slow the change of the cells from a normal phenotype to a transformed malignant phenotype. Ceramide is able to induce differentiation of malignant cells and otherwise undifferentiated cells, thus the compounds of the invention are believed to be useful in inducing and slowing the proliferation of early (clinically undetectable) malignant cells. This would constitute a strong chemopreventive agent.

Similarly, retinoic acid may be useful as a chemopreventive agent, thus since retinoic acid elevate levels of ceramide, the compounds of the invention may also be useful for chemoprevention along this route.

The compounds of the present invention and/or their pharmaceutically active salts may be formulated into WO 92/03129 PCT/US91/05743 13 pharmaceutical compositions or medicaments which may be used to treat mammals such as man, which are afflicted with the various conditions described herein and others which are caused by defective differentiation processes. The pharmaceutical compositions or medicaments preferably contain therapeutically effective amounts of the compounds of the present invention and/or their pharmaceutically acceptable salts. The compounds of the invention may be administered to a mammal having the disease, or suspected of having the disease, singly or in combination with other compounds of the invention or other therapeutic or palliative agents. The compositions of the present invention may be administered in any mode, such as orally, parenterally, intradermally, intramuscularly, intravenously, subcutaneously or topically.

The actual mode can readily be determined by analogy to known methodologies and will depend on the particular disease state being treated, its severity, and the age and condition of the patient. They may be administered orally in tablet, capsule, or elixir form, or parenterally in the form of a solution or suspension. For injection purposes, the medium used is preferably a sterile liquid. As an injection medium, it is preferred to use water which contains the stabilizing agents, solubilizing agents and/or buffers conventional in the case of injection solutions. Desirable additives include, for example, tartrate and borate buffers, ethanol, dimethylsulfoxide, complex forming agents (for example, ethylenediaminetetraacetic acid) high molecular weight polymers (for example polyethylene oxide) for viscosity regulation or polyethylene derivatives of sorbitan anhydrides.

The total routine daily, weekly, monthly, etc.) dose of the compounds according to the present invention will be that effective to result in differentiation of the affected cells, a reduction in cell proliferation, or an improvement or stabilization of the condition being treated.

One of skill in the art can readily ascertain the optimum therapeutically effective dosage to use for a particular case, using as a starting point the range delineated above.

WO 92/03129 PCV/US91/05743 14 When a composition for the treatment of a disease is prepared or manufactured, a compound or a physiologically acceptable salt of a compound according to this invention or a mixture thereof may be shaped together with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavoring, and/or additive, into a unit dosage form. Typical examples of additives that can be used in tablets and capsules are binders such as tragacanth gum, gum arabic, corn starch and gelatin; excipients such microcrystalline cellulose, sealing agents such as corn starch, pre-gelatinized starch and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, and aspartase; and flavorings such as peppermint. Other additives include edible oil as a liquid carrier such as in capsules, shellac, sugar and combinations thereof such as in tablet coating).

Parenteral injection may employ, as a vehicle to dissolve or suspend the active ingredient, water, natural vegetable oils such as sesame oil, coconut oil, peanut oil and cottonseed oil, and synthetic oils such as ethyl oleate, and may contain buffering agents, preservatives and antioxidants as required.

The method of this invention may be carried out by directly contacting in effective amount of a composition according to the invention with cells. However, it is also possible, and within the scope of the invention, to carry out the method indirectly, by administering a compound or composition which has an in vivo activity of inducing production of one of the compounds of this invention.

Further, pro-drug precursors which are converted in vivo to a compound of the invention are also within the scope of the invention.

EXPERIMENTAL

Some of the compounds referred to in the present Specification are named by a convenient shorthand to reflect the total number of carbon atoms in the carbon chains of the compounds of formula I. For example, C18/C 2 ceramide refers WO '92/03129 PC'i/US91/05743 15 to the compound of formula I wherein R, is C15 alkenyl, R 2 is hydroxyl, R 3 is H, R 4 is COR 5 and R 5 is methyl. The double bond in Ri in C 18

/C

2 ceramide is in the same position as the double bond in ceramide. Thus there are 18 carbons in the first mentioned chain which chain contains the carbons in R 1 the carbon to which R 2 is attached, the carbon to which N is attached and the CHIOH group at the end of the chain, and there are 2 carbons in the second-mentioned chain, the amide carbon of R 4 and the methyl carbon of R 5 The compounds are referred to in this manner, with the first-mentioned carbon chain comprising R 1 and the carbons to which R 2 N, and OH are attached, and the second-mentioned carbon chain referring to the carbons in R. Similarly Ci 8

/C

6 ceramide refers to the compound of formula I wherein Ri is C 15 alkenyl,

R

2 is hydroxyl, R3 is H, R 4 is COR 5 and R 5 is pentanyl. C 11

/C

s ceramide refers to the compound wherein Ri is C 8 alkenyl, R 2 is hydroxyl, R 3 is H, R 4 is COR 5 and R 5 is heptanyl. In each of these examples, the double bond in R, is in the same position as in ceramide although other sites of unsaturation may be used. 3-0-methyl sphingosine refers to the compound of formula I wherein R, is C 15 alkenyl, R 2 is methoxy, R 3 is H,

R

4 is COR 5 and R 5 is methyl.

16,25-dihydroxyvitamin D 3 (1,25-(OH) 2

D

3 was obtained from Hoffman-LaRoche, Nutley, New Jersey. SM and PC were purchased from Avanti Polar Lipids while ceramide was purchased from Supelco. Insulin, transferrin, NBT and anaphthyl acetate were purchased from Sigma Chemical Co., St.

Louis, Missouri.

PREPARATION OF CERAMIDE AND SPHINGOSINE ANALOGS N-acetyl and H] N-acetylsphingosine were synthesized as described in Gaver, R.C. ard Sweely, J.

Amer. Chem. Soc. 88:3643-3647 (1966). Briefly, Nacetylsphingosine (C 18

/C

2 ceramide) and N-hexanoylsphingosine

(C

18 ceramide) were prepared by acylation of neutral sphingosine by acetic anhydride and caproic anhydride, respectively (yield N-acetyl sphingosine (specific activity 2.5 X 104 cpm/nmol) was prepared by WO 92/03129 PCT/US91/05743 16 reduction of 3-oxo-l-hydroxy-2-acetamide-4-octadecene, which was obtained by oxidation of cold N-acetylsphingosine with dry chromium anhydride in pyridine/benzene (yield 80% after TLC preparation), with [3H]NaBH 4 (yield =40% after TLC preparation). N-ethylsphingosine was prepared by reduction of N-acetylspingosine with LiBH 4 and purified by preparative TLC (yield C 1

/C

8 ceramide was prepared according to the method of Liotta et al. Tt A Letters 29: 3037 (1988). All structures were verified by NMR, and purity was established by TLC and estimated to exceed 97%. These compounds were dissolved with ethanol and delivered in media (final concentration of ethanol was less than PREPARATION OF 3-O-METHYLSPHINGOSINE 3-O-methylsphingosine was synthesized as described in Carter, et al., Journal of Biochemistry 192: 197-207 (1951). 100 mg of beef brain cerebrosides (Catalog No. A-46, Serdary Research Labs, London, Ontario, Canada) was dissolved in 112g1 concentrated sulfuric acid/2.3ml methanol in a round bottom flask. The mixture was heated and refluxed while stirring for 6 hours. Following heating, the reaction mixture was cooled on ice for approximately 15 minutes. Precipitates of fatty acids and methyl esters were removed by filtering thorugh #1 Whatman paper on a Bchner funnel. Filtrate was extracted four times with 1 ml petroleum ether (each extraction) to remove remaining fatty acids and esters. Ether was removed under vacuum for approximately 15 minutes. The solution was neutralized with 4N Methanolic KOH ml judging by pH paper) and precipitated potassium sulfate was filtered off on #1 Whatman paper on a Buchner funnel. The filtered solution was stored at 4 C (refrigeration) overnight and filtered to remove additional potassium sulfate precipitate which formed overnight. 6N NaOH was added to the solution to pH 10 judging by pH paper. The solution was then extracted with diethyl ether two times. The ether extracts were combined, washed once with water and dried over sodium sulfate, under vacuum. Dried extracts were dissolved in a chloroform:methanol WO 92/03129 PCT/US91/05743 17 The sample was purified by thin layer chromatography (TLC) as described in Sambasivaroco and McCluer, Journal of Lipid Research 4:106-108 (1963). A preparative TLC plate was used to separate the product using chloroform:methanol:2N ammonium hydroxide (40:10:1) as the solvent, and sphingosine as a marker. A small portion of the plate was visualized using ninhydrin spray. 3-O-methyl-spingosine runs high on the plate and was found to be the fastest migrating component.

The silica band corresponding to 3-O-methylsphingosine was scraped from the plate and 3-0-methylsphingosine was eluted from the silica with chloroform:methanol The elutant was spun in a centrifuge (IEC) at 2,000 rpm for approximately minutes and the supernatant was drawn off into a clean tube.

Elution of 3-0-methylsphingosine from the silica was repeated with chloroform:methanol and the chloroform:methanol supernatants were combined. The combined supernatants were dried under vacuum and resuspended in ethanol for a total yield of 11mg of CELL CULTURE Human myelocytic leukemia HL-60 cells (45 passages) were obtained and grown in RPMI 1640 medium (Sigma Chemical Co., St. Louis, Missouri) containing 10% fetal calf serum at 37 0 C in 5% CO 2 incubator. The cells were washed twice with phosphate buffered saline (PBS) and resuspended in serumfree media containing insulin (5mg/liter) and transferrin mg/liter) before treatment with various compounds.

'~SS MEASUREMENTS OF LIPIDS After harvesting the cells at the indicated times, the lipids were extracted by the method of Bligh and Dyer, Can. J. Biochem. Physiol. 37:911-917 (1959). Thk samples were dried down under nitrogen gas and dissolved with 0.1 ml chloroform: 40 Al was applied to a thin layer chromatography (TLC) plate (Merck); and 40 pl was used for measurement of phospholipid phosphate (duplicate measurement). Phosphate was measured as described in Van Veldhoven, P. and Mammaerts, Ann. Biochem. 161:45-48 (1987). To identify sphingomyelin (SM) and phosphorylcholine TLC plates were developed in WO 92/03129 PCT/US91/05743 18 chloroform/methanol/acetic acid/H0a (50/30/8/5) (solvent A) or chloroform/methanol/2N NHO4H (60/35/5) (solvent The combination of solvents A ard B were used for two-dimensional TLC. After staining the plates with iodine vapor, the spots corresponding to SM and PC were scraped, extracted with chloroform/methanol dried down under nitrogen, and phospholipid phosphate was measured.

CERAMIDE MEASUREMENTS The mass of ceramide was measured enzymatically using sn-l,2-diacylglycerol (DAG) kinase as described in Preiss et al., J. Biol. Chem. 261:8697-8700 (1986) and Van Veldhoven, Anal. Biochem. 183: 177-189 (1989). To confirm the conversion of ceramide in HL-60 cells to ceramide 1-phosphate, ceramide phosphate and phosphatidic acid were separated on TLC by different solvent systems such as chloroform/methanol /acetic acid (65/15/5), chloroform/pyridine/formic acid (60/30/8), and chloroform/acetone/methanol/acetic acid/H 2 0 Rf values were compared witi'. those of reference standard ceramide. Additionally, cerautide 1-phosphate was converted to sphingosine 1-phosphate by alkaline hydrolysis twenty hours in IN NaOH) and the Rf value was compared with that of reference sphingosine 1-phosphate in the solvent containing butanol/acetic acid/H 2 0 or chloroform/ acetone/methanol/acetic acid/H 2 0 (10/4/2/2/1) and found to be identical at 0.45 or 0.42.

ANALYS16 OF HL-60 CELL GROWTH AND DIFFERENTIATION Cell growth was quantified using a hemocytometer.

Cell viability was judged by the ability to exclude tryan blue. Viability was always more than 80% unless otherwise described. Nitro blue tetrazolium (NBT) reducing ability was used as a marker for both macrophage/monocyte and granulocyte lineage and non-specific esterase was used as a marker for macrophacge/monocyte lineage. Both markers were measured as described in Okazaki, T. et al., J. Cell Physiol. 131:50-57 (1987).

UPTAKE A'J) METABOLISM OF [H]C 18

C

2

CERAMIDE:

After resuspending in serum-free media, HL-60 cells WO 92/03129 PCT/US91/05743 19 were labeled with [3H]C18/C 2 ceramide (1 x 10 s cpm/ml), harvested at the indicated titaes, and washed three times with PBS. Lipids were then extracted by the method of Bligh and Dyer, supra. The dried-down samples were dissolved with 100 pl chloroform; 20 il were applied on TLC plates and 40 pl were used for measuring phospholipid phosphate. The plates were developed in the solvent containing chloroform/methanol/2N (40/10/1) to separate C 1 8

/C

2 ceramide, SM and sphingosine (SPH). The spots were scraped and counted in Safety Solve (Research Products International Corp.) in a LKB scintillation counter (LKB).

DOSE AND TIME DEPENDENCE OF CERAMIDE FORMATION IN RESPONSE TO 25-(OH)zD3: The mass of ceramide was measured by adapting the diacylglycerol-kinase assay previously developed to quantitate diacylglycerol (see Van Veldhove et al supra). The E. coli DAG kinase is able to quantitatively convert ceramide to ceramide 1-phosphate. Cellular ceramide was identified following conversion to ceramide 1-phosphate by comigration with standard ceramide phosphate on thin layer chromatography (TLC). Cellular and reference ceramide phosphate showed identical Rf values when TLC plates were developed in 4 different solvent systems. Further identification was achieved by alkaline hydrolysis of ceramide phosphate. This resulted in the formation of sphingosine 1-phosphate which comigrated (Rf 0.45) with standard on TLC developed in butanol/acetic acid/H 2 0 solvent system.

As shown in Figure 1, treatment of HL-60 cells with 13,25-(OH) 2

D

3 resulted in a dose-dependent elevation in ceramide levels at 2 hours following treatment. The data are shown as of control (in the absence of C 1

,/C

2 ceramide).

Bars represent one standard derivation for duplicate measurements. Baseline ceramide levels were 26.1 0.82 nmol/nmol phospholipid. The results are representative of three different experiments.

Maximal ceramide elevations occurred at 100 nM 1,25-

(OH)

2 Da. At this concentration of 1,25-(OH) 2

D

3 there was a WO 92/03129 PCT/US91/05743 0 41% increase in ceramide mass over control levels. The dosedependence of ceramide formation on 1,25-(OH) 2

D

3 clsely paralleled the dose-dependence of HL-60 cell differentiation on 1,25-(OH) 2

D

3 which peaks at 100-300 nM. These results, therefore, suggest a quantitative relationship between ceramide formation and cell differentiation.

The time dependence of ceramide levels in response to the action of 1,25-(OH) 2

D

3 on HL-60 cells was also investigated. As shown in Figure 2, HL-60 cells were harvested at the indicated time points after treatment with 100 nM CIB/Cz ceramide. Ceramide mass was measured as described in the Experimental Procedures. The results were obtained from two determinations. Bars represent one standard deviation. Data are representative of three different experiments when HL-60 cells were treated with 100 nM 1,25- (OH)2r/ 3 (an optimal concentration from the dose-response illustrated in Fig. ceramide levels progressively increased over the first 2 hours and then returned to baseline. The earliest increase was detected at 30 min following 1,25-(OH) 2

D

3 treatment over baseline) and peaked at 2 hours with a 41% increase (Fig. 2).

These studies show that 1,25-(OH) 2

D

3 incudes a timeand dose-dependent transient increase in ceramide levels which clearly precedes the onset of differentiation of HL-60 cells (peak eramiide formation occurs at 2 hours with phenotypic changes of differentiation occurring at 2-4 days.). Since ceramide formation appcars to be one of the earliest biochemical changes in response to 1,25-(OH)D, 3 these studies suggest a role for ceramide as a lipid mediator.

DERIVATION OF CERAMIDE FROM SPHINGOMYELIN: In previous studies, okazaki, T. et upra, it was shown that 1,25-(OH) 2

D

3 induced hydrolysis of sphingomyelin with simultaneous changes in phosphorylcholine and ceramide levels; followed by resynthesis of sphingomyelin to baseline levels. To confirm that ceramide was quantitatively generated from sphingomyelin, the mass of hydrolyzed sphingomyelin was measured and compared to the WO 9 2/03129 rCr/JS91 /05743 21 mass of generated ceramide. As seen from Figure 2, the mass of ceramide peaked at 2 hours with a net formation of 134-2 pmol ceraiaide per nmol phospholipid. Ceramide levels then returned to baseline at four hours. AE, shown in Figure 3, the total levels of phospholipids (Fig. 3A) and phophat Idyl chol ine (Fig. 3B) did not significantly change over the first 4 houro following l,25-(OH) 2

D

3 treatment. However, 9phingomyelin levels decreased from 51±6 pmol/nmol phospholipid to 34+2 pmol/nmol phospholipid (Fig. 3C) at 2 hours. The net decrease in sphingomyelin mass of 17±4 pmol/nmol phospholipid is very closa to the net incrvoase in ceramide mass levels of 13±pmol/nmol phospholipid. These results strongly suggest that ceramide is generate d from sphingomyelin breakdown in response to 1,25-(OH) 2 D, action on HL-60 cells.. '1.abeling of sphingoJlipid precursors with [3H~palmitate showed that no other sphircqolipid underwent significant changes in resppnze to 10,25- (OH) ?D3 Ouring this time interval and the sphingomyelin pool constituted the largest labeled pool aimong the, various sphingolipids. Therefore, it is unlikely that ceramide could be derivcd from the hydrolys,'.s of sphingolipids other than sphingomyelin, These studies, however, do not rule out the possibility of de novo synthesis of ceramide in response tu 1,25-(OH) 2 D, although this is unlikely in the face of significant and commensurate hydrolysis of sphingomyelin.

These data also show that total phospholipids in HLcells were -5.6+1.0 fmol/cell with PC and SM accounting for 53.3±-2.3% and 541+0.6% of total phospholirids, respectivaly, These are very close to previouc data showing a SM/PC,-r-.c of 0.05 0.10 using 3 H) choline to label the two lipids.

Okazaki, T. et al., J. Blol. Chem. 264:19076t-19080 (1989).

These results also explain why no significant change in total phospholipid was detect A since the net decrease in SM levels corresponds to only 1.5 2.04 change in total phospholipid levels.

C1&dC 2 -CERAHIDE POTENTIAT'ES THE EFFECTS OP 1,25- (OI) 2

D

3 ON HL- CELL DUV~ERENTIATIO4: It has been sho1n, that the exogenous applitcation of WO 92/03129 PC/US91/05743 22 bacterial sphingomyelinase, which induces hydrolysis of membrane sphingomyelin and the formation of ceramide, potentiates the ability of 1,25-(OH) 2

D

3 to induce HL-60 cell differentiation. Okazaki, T. et al., supra, The use of bacterial sphingomyelinase, however, was complicated by the hydrolysis of membrane phosphatidyl-choline (PC) at higher concentrations of sphingomyelinase, thus limiting interpretations.

To overcome this probleim and to directly test whether ceramide can mimic the action of bacterial spingomyelinase, a synthetic cell permeable ceramide, C 18

/C

2 ceramide, having an acetate in amide linkage was prepared.

Compared to nattirally occurring ceramides, CI,/C 2 ceramide has 14-16 less carbons, and therefore, displays higher water solubility. Under similar conditions, naturally occurring ceramide with long N-acyL chains, at 50 AM, did not affect cell growth or cell differentiation consistent with the poor uptake of long-chain N-acyl ueramides.

This is analogous to cell--permeable DAG analogs such as dioctanoylglycerol and oleoylacetylglycerol which have shorter acyl chains than naturally occurring DAGs. When cells wore treated simultaneously with suboptimal concentrations of 1,25-(OH) 2

D

3 (1 nM which is 100-fold lower than the optimal concentration and various concentrations of

CI~/C

2 ceramide), enhancement of cell differentiation was observed.

cells (2.5 x 10 5 cells/ml) were treated simultaneously with various concentrations of C 18

/C

2 ceramide and 1 nM 1,25-(OH) 2 D3. Cell differentiation was judged by NBT reducing ability (shaded bars) and NSE activity (unshaded bars). The effects of C 1 8

/C

2 ceramide on cell growth are shown in the inset. The results were obtained from three different experiments. Bars represent one standard deviation.

As shown in Figure 4, C18/C 2 ceramide showed a dosedependent enhancement of 1,25-(OH) 2 D differentiation with peak effect occurring at 1 AM. On day 4 following treatment, 1 IM C 18 ceramide caused an increase of NBT reducing WO 92/03129 PC'/US91/05743 23 activity and non-specific asterase (NSE) activity from 11.9+2.9% to 57.8+1.4% and from 2.2+0.9 to 39.2+7.2%, respectively. Over the same concentration range, C 18

/C

2 ceramide induced mild inhibition of cell growth (Fig 4, inset) without significant effects on cell viability. Cell viability was always greater than 80%. Although there was mild inhibition of cell growth, the absolute numbers of NBT and NSE positive cells induced by 1 /M of C 18

/C

2 ceramide increased from 0.9 x 105 cell/ml to 2.54 x 10 5 cells/ml and from 0.16 x 105 cells/ml to 1.72 x 10 cells/ml on day 4, compared to control.

CELL PERMEABLE CERAMIDES INDUCE HL-60 CELL DIFFERENTIATION INDEPENDENT OF 1,25-(OH) 2

D

3 The strong synergy between subthreshold concentrations of 1,25-'C!H) 2

D

3 and low concentrations of

C

1

I/C

2 ceramide (10C nM-1 M) suggested that higher concentrations of Cj 1

/C

2 ceramide may induce r.fferentiation independent of 1,25-(OH) 2

D

3 addition. The effects of synthetic C18/Cz ceramide on cell growth and differentiation were therefore examined. Treatment of HL-60 cells with increasing concentrations of C 18 /Cz ceramide resulted in a dose-dependent inhibition of cell growth as shown in Figure The various concentrations of Ci8/C 2 ceramide are represented as follows: 0 Control; closed diamond 1 CM; closed square 3 M: closed circle -6 AM and closed triangle 10 AM. The results were obtained from three determinations.

Bars represent one standard deviation. Ten AM of C, 1

/C

2 ceramide caused severe loss of cells by day 7. The loss of cells may be, at least in part, due to the induction of cell differentiation by C 18

/C

2 ceramide rather than to simple toxicity since more than 30% of cells were induced to differentiate by day 2 of treatment.

As shown in Figure 6, increasing concentrations of

CU

1

/C

2 ceramide caused a progressive increase in differentiation of HL-60 cells by 4 days as quantitated by NBTreducing ability and induction of NSE activity. NBT activity is represented by shaded bars, and NSE activity is represented WO 912/03129 PCIJ/US9I/0543 24 by open bars. The results are averages of three determinationl. Bars represent one standard deviation. The cells were treated with various concentrations of C/C 2 ceramide for 4 days. NBT positive cells increased from 0+1.0 to 53.2+1.6%, and NSE-positive cells increased from 1.0+1.9% to 46.4+6.0% following treatment with 6 /M of Cz 1

/C

2 ceramide.

Significant increases in differentiated cells were also observed with concentration of Cz 1

/C

2 ceramide as low as 1 AM.

Examination of the morphologic phenotype of cells treated with ceramide showed morphological changes consistent with the monocytic phenotype induced by 1,25- (OH) D3, These cells here characterized by a larger cytoplasmic to nuclear ratio, lobulated and eccentric nucleus, and disappearance of nuclear bodies and azurophilic granules.

The cells also acquired NBT-reducing ability, and NSE activity, the latter being a specific marker of monocytic differentiation.

The time-dependence of differentiation in response to Cz 1

/C

2 ceramide was next examined using 6 AM C 1 8

/C

2 ceramide as an optimal concentration that produces maximal differentiation with minimal cytotoxicity. Cell differentiation was judged by NBT reducing ability and NSE activity. The addition of 6gM Cz 1 /Cz ceramide caused a progressive increase in NBT and NSE positive HL-60 cells with up to 61% and 56% respectively, by day 7 of treatment, as shown in Figure 7.

In Figure 7, HL-60 cells treated with 6 m ceramide are represented by closed figures and cells not treated with ceramide are represented by open figures. NBT reducing ability is represented by and circles. NSE activity is represented by squares.

These results show that the addition of C 18

/C

2 ceramide alone can induce HL-60 cell differentiation into a monocytic phenotype with 6,M C 1 z/C 2 ceramide displaying similar effectiveness as 1,25-(OH) 2

D

3 At an optimal concentration of (I Z5' -CO.H) 2 ._3 100 nM,A Cn/C, cramidc induces NBT reducing ability in 74±6% of cells and NSE in 51+4% of cells compared to 53.2+1.6% and 46.4+6% for Cz 8

/C

2 ceramide on day 4.

WO 92/03129 PCT/US91/05743 25 As shown in Figure 8, the addition of C 18

/C

2 ceramide to HL-60 cells did not modulate cellular levels of sphingomyelin. HL-60 cells were treated with 5 pM C 18

/C

2 ceramide for the indicated times. Sphingomyelin was extracted and measured as described in "Experimental Procedure". The data are shown as of control (in the absence of C 18

/C

2 ceramide). Bars show one standard deviation. The results were obtained from two different experiments. These results strongly suggest that the effects of C18/Cz ceramide and bacterial sphingomyelinase (SMase) on HL-60 cell differentiation are mediated by ceramide and not by the changes in SM levels per se.

To further evaluate the role of ceramide as a second messenger, experiments were performed to examine whether a short duration of exposure of HL-60 cells to ceramide is sufficient for induction of differentiation. C 18

/C

2 ceramide, added to cells, could be back-extracted into media following repeated washing of cells, so that after 3 washes, less than of original C 18

/C

2 ceramide remained in association with the cell pellet.

Since 1,25-(OH) 2

D

3 caused elevation of endogenous ceramide over approximately 2 hrs, HL-60 cells were exposed to CI 8

/C

2 ceramide (0.5 2pM) for 2 hr. C 18

/C

2 ceramide was back-extracted, ar,d differentiation was evaluated. The cells were treated without or with Cj 8

/C

2 ceramide 1 or 2 pM) for two (Figures 9 and 10) or four hours (Figure 10), washed with RPMI 1640 media three times and then resuspended in serum-free RPMI 1640 media. At the indicated day (Figure 9) or four days (Figure 10) after washing out treatment, the differentiation was measured by NBT reducing ability as described in "Experimental Procedure". Bars mean one standard deviation. In Figure 9, open circles represent 0 IM CIO/C 2 ceramide. Closed triangle represent 0.5 pM C 16

/C

2 ceramide.

Closed squares represent 1 AM C 18

/C

2 ceramide. Closed circles represent 2 iM C18/C 2 ceramide. In Figure 10, 0 iM C 18 /Cz ceramide is represented by unshaded bars. 0.5 M C 18

/C

2 ceramide is represented by light cross-hatched bars. 1 AM WO 92/03129 PCT/US91/05743 26

C

1

I/C

2 ceramide is represented by dark cross hatched bars. 2 AM C 18

/C

2 ceramide is represented by shaded bars. The results were obtained from two different experiments. The r-sulte- SC,8/C z (1 or 2 .wer obtained from two different cmper-imcnts. Cj./C. (1 or 2 gM) caused significant differentiation of HL-60 cells under these conditions (Figure 9) indicating that a 2-hr exposure of HL-60 cells to CI8/Cz ceramide was sufficient for induction of differentiation. A 4-hr exposure did not result in significantly more differentiation (Figure 10). These studies suggest that a short exposure of HL-60 cells to elevated ceramide levels is sufficient for commitment to differentiation.

EFFECTS OF CERAMIDE AND SPINGOSINE ANALOGS ON Da-INDUCED UL- CELL DIFFERENTIATION.

Sphingosine is a pharmacologic inhibitor of protein kinase C activity in vitro and in different cell systems. Because ceramide can potentially be metabolized to sphingosine by the action of acid and/or neutral ceramidases, we investigated whether the actions of ceramide on HL-60 cells could be attributed to the formation of sphingosine. No sphingosine could be detected following treatment of HL-60 cells with 1,25-(OH) 2

D

3 Moreover, the addition of C 18

/C

2 ceraride to HLcells did not result in any measurable sphingosine formation. For these experiments, C 16

/C

a was labeled with [3H] on the third carbon of the sphingosina base. Cells x 10 6 cells/ml) were labeled with 4 AM H]Cei/C ceramide (1 x 10 5 cpm/ml). ceramide was delivered in ethanol. The additional L"H]Cli/Cz ceramide to HL-60 cells resulted in prompt uptake of labeled C18/C 2 ceramide but no conversion to sphingosine as shown in Figures 11 and 12. The uptake of

C

18

/C

2 ceramide was about 20% .t 0.5 hr after treatment. The rest of the ceramide (80%)remained unchanged in the medium.

Figure 11 shows an autoradiography of a TLC plate (24 h exposure). In Figure 12 lipids were extracted, separated by TLC plates and radioactivity was counted as described in "Experimental Procedures". Ceramide is represented by open circles. Sphingomyeline is represented by open squares.

i *.Er WO 92/03129 PCT/US91/05743 27 Sphingosine is represented by closed circles.

Also, the addition of 1,25-(OH) 2

D

a to C 18

/C

2 ceramidelabeled cells did not result in the formation of sphingosine.

A small percentage of label was converted to sphingomyelin at 12h after labeling). These studies show that 1,25-

(OH)

2

D

3 does not lead to the formation of sphingosine and that exogenous ceramide analogs, when added to cells (in the presence or absence of 1,25-(OH),D 3 are not metabolized to sphingosine.

Sphingosine is slowly metabolized in HL-60 cells by primarily becoming incorporated into ceramide and other sphingolipids. Merrill, et al., J. Biol. Chem.

261:12610-12615 (1986). However, the above studies do not rule out rapid metabolism of sphingosine generated from ceramide, thus escaping detection by the above methods. Since the objective was to evaluate the role of sphingosine in mediating the effects of ceramide, the ability of sphingosine to mimic the action of ce-amide was therefore tested.

As shown in Vigure 13, when HL-60 cells were treated simultaneously with various concentrations of sphingosine and suboptimal concentrations of 1,25-(OH) 2

D

3 (InM) for 4 days, NBT reducing ability and NSE activity did not change compared to control. HL-60 cells (2.5 x 10 5 cells/ml) were treated with various concentrations of sphingosine for 4 days in the presence cf InM 1,25-(OH) 2

D

3 Cell differentiation was judged by NBT reducing ability (shaded bar) and NSE activity (open bar). The effects of sphingosine on HL-60 cell growth are shown in the inset. The results were obtained from three determinations. Bars represent one standard deviation.

These studies show that sphingosine does not enhance the ability of 1,25-(OH) D 3 to induce HL-60 cell differentiation in clear distinction from the effects of ceramide (compare Figs. 4 and 13). 3 H]sphingosine was taken up efficiently by HL-60 cells demonstrating that the lack of effects of sphingosine were not due to poor uptake. Moreover, sphingosine did slow the growth of HL-60 cells (Fig. 13 inset) to a level comparable to that induced by Cs/C 2 ceramide WO 92/03129 PCT/US91/05743 28 indicating a cellular effect of sphingosine other than the induction of differentiation. The ability of sphingosine to slow HL-60 growth without enhancing differentiation also supports the notion that ceramide is primarily acting to induce differentiation independent of growth rate of cells. To further support a role for ceramide in cell differentiation, independent of sphingosine, synthetic ceramide and sphingosine analogs were prepared and tested for their ability to enhance HL-60 cell differentiation by suboptimal concentrations of 1,25-(OH) 2

D

3 As shown in Table 1, both C18/C 6 ceramide and C 18

/C

2 ceramide caused significant enhancement of NBT reducing ability and NSE activity comparable to that observed with 100 nM 1,25-(OH),D 3

C

1

/C,

also caused significant enhancement of NBT reducing ability and NSE activity. Studies with C 1

/C

8 ceramide are particularly relevant in ruling out an important role for sphingosine. Deacylation of C 1

/C

8 ceramide would result in the formation of a C 1 -sphingosine analog which has been shown toArt- the in vitro and cellular effects of sphingosine.

Norjiri, et al., supra.

TABLE 1 Effects of various sphingolipids on HL-60 cell differentiation treated with 1 nM4 1,25-(OH),D 3 Treatment Concentration Cell 5Number NBT positive cells NSE positive cells of sphingolipid (xlO cells/ml)

(!LM)

1 nM 1,25-(OH),D 3 0 7.6 1.2 11.9 2.9 2.2 0.9 C18/C2 ceramide 0.1 7.6 1.0 35.4 +1 4.8* 26.5 2.4* I nM 1,25-(OH) 2

D

3 1.0 4.4 0.1 57.8 1.4* 39.2 7.9* C18/C6 ceramide 0.1 7.2 1.8 33.5 0.5* 18.3 1 nM 1,25-(OH),D, 1.0 6-8 1.6 58.1 4+ 1.5* 29.6 4+ 1.8* H-ethylsphingosine 0.1 5.0 1.4 16.5 0.5 1.3 0.3 1 nM 1,25- (QH) 2

D

3 1.0 4.2 1.2 17.8 4.5 1.0 sphingosine 0.1 7.8 1.6 11.3 0.5 2.5 1 nM 1,25-(011) 2

D

3 1.0 5.6 0.6 10.7 1.7 2.5

C

1 1 ceraxuide 0.1 25* 18* mnM 1,25-(OH),D 3 1.0 38.5* 34.5* cells (2.5 x 10 5 cells./mi) were treated simultaneously with the indicated lipid and subthreshold 1, 25- (OH) ZD 3 for four days. The results were obtained from three determinations. Asterisks show that the difference from the control (lnM 1,25-(OH),D 3 is significant at a p value <0.01.

WO 92/03129 W 2CT/tS91/05743 30 On the other hand, sphingosine failed to induce any significant changes in those two parameters of cell differentiation. Similar results with sphingosine have also been noted. Stevens, et al. Cancer Res. 49:3229-3234 (1989). Moreover, N-ethyl sphingosine, which is a potent inhibitor of protein kinase C, failed to increase NBT and NSE activity significantly over baseline. These studies show that ceramide derivatives, but not sphingosine and its analog, enhance 1,25-(OH) 2

D

3 HL-60 cell differentiation.

In a previous study, it was discovered that 1,25-

(OH)

2

D

3 caused hydrolysis of sphingomyelin in HL-60 cells with the concomitant generation of ceramide and phosphorylcholine in what appeared to be a regulated "sphingomyelin cycle".

Okazaki, et al., J. Biol. Chem. 254:19076-19080 (1989).

Sphingomyelin hydrolysis was suggested to play a role in HLcell differentiation since the addition of exogenous bacterial sphingomyelinase potentiated the ability of subthreshold concentrations of 1,25-(OH) 2

D

3 to induce cell differentiation.

Applicants have now discovered that ceramide functions as a lipid mediator transducing the effects of 1,25-

(OH)

2

D

3 on HL-60 cell differentiation. Low concentrations of ceramide (100 nM 3 AM) enhanced the ability of subthreshold concentrations of 1,25-(OH) 2

D

3 to induce cell differentiation.

More importantly, higher concentrations of ceramide (1-6 MM) were able to induce HL-60 cell differentiation in the absence of 1,25-(OH) 2

D

3 The phenotype of differentiated HL-60 cells closely resembles the monocytic phenotype induced by 1,25-

(OH)

2

D

3 These studies strongly suggest that ceramide may play an essential role in mediating the action of 1,25-

(OH)

2

D

3 on cell differentiation. Moreover, C 18 /C ceramide was effective in causing differentiation when cells were exposed to it for only 2 hrs. This strongly suggests that the ceramide response to 1,25-(OH) 2 D action is sufficient for the induction of differentiation. Also, since approximately of added ceramide was taken up by cells, the results indicate that the effective concentration of C 1 a/C 2 ceramide is the nM WO 92/03129 PCT/US91/05743 31 range (20-1000 nM).

At the present time, Applicants do not know the mechanism by which ceramide mediates the effects of 1,25-

(OH)

2

D

3 on cell differentiation. No immediate target for ceramide action could be identified. Because ceramide may serve as a precursor to sphingolipids and sphingosine, its action may be mediated by metabolites. The ganglioside GM 3 has been reported to increase in response to phorbol esterinduced HL-60 cell differentiation and also to induce cell differentiation along a monocytic lineage. Norjiri, H. et al., Proc. Natl. Acad. Sci. 8.:782-786 (1986). Ceramide may serve as a precursor to gangliosides such as GM 3 However, 1,25-

(OH)

2

D

3 was found not to modulate GMa (Nojiri, H. et al. (1988) Proc. Natl. Acad. Sci. USA 83: 782-786), and Applicants found very little ceramide converted to gangliosides. Moreover, the dose response of HL-60 cells to ceramide is much lower than that reported for GM 3 (raising the possibility that the action of GM 3 may be due to its further metabolism to ceramide).

Ceramide may also serve as a precursor to sphingosine which would be generated through a single hydrolysis step by the action of neutral or acid ceramidases.

Applicants' data strongly argue against a role for sphingosine for mediating the effects of ceramide. This is supported by: 1) no sphingosine could be detected in response to the action of 1,25-(OH) 2

D

3 on HL-60 cells; 2) sphingosine did not enhance the ability of 1,25-(OH) 2

D

3 to induce HL-60 cell differentiation nor did it cause monocytic differentiation of cells on its own; 3) other ceramide derivatives were able to induce HL-60 cell differentiation but sphingosine and its related analog N-ethyl sphingosine failed to enhance 1,25-

(OH)

2

D

3 induced differentiation, and 4) Cjn/C 8 ceramide, whose hydrolysis results in a short chain sphingosine that does not inhibit protein kinase C (Merril, A.H. et al. (1989) Biochemistry 28: 3138-3145) was as effective as C18/C ceramide in inducing cell differeptiation.

Studies withAn ceramide are particularly relevant in ruling out an important role for sphingosine. Deacylation I I^ B 9. ,V WO 92/03129 PCT/US91 /05743 32 ofA 4 C ceramide would result in the formation of a Cnsphingosine analog which has been shown to lack the in vitro and cellular effects of sphingosine. Norjiri, et al., supra.

Si'ice these two pathways appear unlikely, ceramide may have other targets mediating its actions. Sphingomyelin serving as cellular reservoir acted upon by sphingoinyelinase to produce ceramide, a potential lipid mediator (second messenger) is analogous to the glycerolipids serving as cellular reservoirs acted upon by phospholipase C to produce diacylglycerol second messengers.

Claims (30)

1. A method of inducing cell differentiation comprising adding to a mammalian cell capable of undergoing differentiation a compound having the formula H H I I R 1 C C CH 2 0H I I OH NH COR S I I a. ai I I a. t *i wherein R 1 is C 8 to about C 15 alkyl and R 5 is C 1 to C 7 alkyl; said addition being in an amount effective to induce differentiation of said cell,

2. The method of claim 1 wherein R 1 is C 14 or C 15 alkyl.

3. The method of claim 1 wherein R 1 is C 14 alkyl.

4. The method of claim 1 wherein R 5 is C 1 to C 5 alkyl,

5. The method of claim 1 wherein R 5 is methyl.

6. The method of claim 1 wherein R 1 is C 14 or C 15 alkyl and R 5 is C 1 to Cs alkyl.

7. The method of claim 1 wherein RI is C1 4 alkyl and R 5 is methyl. 25 8, The method of claim 1 wherein said cell is a leukemic lymphocyte.

9. The method of claim 1 wherein said cell is a hyperpro- liferative basal cell residing in the skin of a mammal and the adding is carried out by contacting the skin of the 30 mammal with the compound. A method of altering the phenotype of a mammalian cell, comprising adding to a mammalian cell capable of undergoing phenotypic alteration a compound having the formula H H I I R C C CH 2 0H I I OH NH COR\ wherein RI is C 8 to about Cs 15 alkyl and R 5 is C 1 to C 7 alkyl; said addition being in an amount effective to induce alteration of the phenotype of said cell. I1. The method of claim 10

12. The method of claim 10

13. The method of claim 10 14, The method of claim 10 The method of claim 10 and R 5 is C 1 to C 5 alkyl,

16. The method of claim 10 is methyl, 17, The method of claim 10 lymphocyte 18, The method of claim 10 wherein R 1 wherein R 1 wherein R 5 wherein R 5 wherein R 1 is C 1 4 or C 1 5 alkyl. is C14 alkyl. is C1 to C5 alkyl. is methyl. is C 1 4 or CI 5 alkyl w "e*ain Pl is C14 alkyl and wherein said cell is a leukemic wherein said cell is a hyperpro-liferative basal cell residing in the skin of a mammal and the adding is carried out by contacting the skin of the mammal with the compound. g

19. A method of treating a disease characterized by hyperproliferation of cells comprising administering to an animal suspected of having the disease a therapeutically effective amount of a compound having the formula H H I I R1 C C CH 2 0H I I OH NH COR wherein RI is Ci to about C15 alkyl and R5 is C 1 to C 7 alkyl. The method or claim 19 wherein said disease is manifested by the proliferation of malignant cells, 21, The method of claim 19 wherein said disease is a malignant tumor.

22. The method of claim 19 wherein said disease is leukemia.

23. The method of claim 19 wherein said disease is a lymphoma.

24. The method of claim 19 wherein said disease is a premalignant tumor. The method of claim 19 wherein said disease is myelodyspl&sia.

26. The method of claim 19 wherein said disease is characterized by the hyperproliferation of benign cells.

27. The method of claim 19 wherein said disease is a benign tumor,

28. The method of claim 19 wherein said disease is psoriasis. s 36

29. A method of slowing the growth of cells comprising administering to cells in an animal a compound having the formula H H I I R 1 -C CH 2 0H OH NH wherein R 1 is C 8 to about C 1 5 alkyl and R 5 is C1, to C 7 alkyl. The method of claim 29 wherein said cell is a fat cell.

31. rlhe method of claim 29 whereir, said cell a lymphoc-yte.

32. The method of claim 29 wherL-2.n said cell is a smooth muscle cell.

33. The method of clalta 29 wherein said cell is an

34. A pharmaceutical ptenarattion for inducing cellular differeri'ation comprising a pharmaceutically acceptable carrier and a compound having the formula U.H H R 1 C C CHiZOH 31OH NH1 COR whev:e R 1 is C 8 to about C 15 alkyl, R 5 is C 1 to carrier being pharmaceutically acceptable f'r in mammals. The pharmaceutical composition of claim is C1 4 or C 1 5 alkyl.

36. The pharmaceutical composition of claim is C14 alkyl.

37- The pharmaceutical composition of claim is C 1 to 0 5 alkyl.

38. The pharmaceutical composition of claim is methyl.

39. The pharmaceutical composition of claim is C 1 4 or C 1 5 alkyl and R 5 is C 1 to C 5 alkyl. The pharmaceutical composition of claim is C 1 4 alkyl and R5 is methyl.

41. A compound having the formula C 7 alkyl; said administration 34 wherein R 1 34 wherein R 1 34 wherein R 34 wherein R 34 wherein RI 34 wherein R 1 1, H H I I RI- C C CH 2 0H OH NH COR where R 1 is C 1 4 alkyl, and R 5 is C 1 to C 7 alkyl.

42. The compound of claim 41 in a carrier pharmaceutically acceptable for administration in mammals.

43. The compound of claim 41 wherein R 5 is methyl. DATED this 29th day of July 1994 DUKE UNIVERSITY Patent Attorneys for the Applicant: F.B. RICE CO.