HUT65932A - Methods for inducing cell differentiation using ceramides - Google Patents

Abstract

The present invention provides methods and compositions for inducing differentiation of cells. Compositions having formula (I) wherein R1 is C1 to about C20 alkyl or alkenyl; R2 is hydroxyl, alkoxy or H, R3 is H or lower alkyl; R4 is COR5, SO2R5, or CSR5, where R5 is C1 to C20 alkyl, alkenyl, or alkynyl, which may be substituted by one or more of the following functional groups: OH, SH, OR6, SR6, NR7R8, COOR9, and CONR10R8, where R6, R7, R8, R9, and R10 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 by hyperproliferation of cells.

Description

The present invention relates to compounds and methods for inducing cell differentiation. More particularly, the present invention relates to the use of ceramide and derivatives thereof for the induction of cell differentiation and for the treatment of conditions characterized by abnormal cell division.

Hyperproliferation (cell proliferation) of cells is one of the hallmarks of many human malignant and non-malignant diseases. In this disease, cells proliferate abnormally at high rates. In cancerous tumors and leukemias, the cells grow and divide indefinitely, which partly explains their rapid spread within the body. Similarly, in some non-malignant diseases such as in psoriasis, cells grow and divide at abnormally high rates. In these diseases, rapidly dividing cells are present in a relatively undifferentiated state. Undifferentiated cells are able to grow and divide. However, as a cell differentiates, it loses its ability to proliferate. Many treatments are proposed to stop cell proliferation by inducing cellular differentiation and thus controlling disease.

Recently, sphingolipids have been found to play an important role in cell growth, ontogeny and differentiation (Hannun, Y. A. and Bell, R. M. (1989) Science 243: 500507). Sphingolipid degradation products are considered a new class of cell regulatory molecules. Sphingolipid degradation products, sphingosine and lysophospholipids, inhibit protein kinase C, which is considered to be a key enzyme in cell regulation and signaling (Hannun, Y. A. et al. (1986) J. Biol. Chem. 261: 12604-12609). Sphingolipids and lysosphingolipids influence important cellular responses and exhibit antitumor activities in various mammalian cells (Hannun, Y. A. and Bell, R. M. (1987)).

Science 235: 670-674, Hannun, Y. A. et al. (1987) J. Bioi. Chem. 262: 13620-13626; Wilson, E. et al. (1987) Arch. Biochem. Biophys. 259: 204214).

U.S. Patent No. 4,710,490, issued December 1, 1987, discloses a composition comprising lipid-containing molecules having angiogenic activity. The lipids are derived from mammals and are predominantly of droplet origin. Known lipids have also been shown to have angiogenic activity in a mixture of gangliosides. The compositions stimulate the growth of blood vessels in vitro and in vivo. Gangliosides have the highest angiogenic activity, while glycolipids have, for example, the highest activity. ceramide derivatives have little or no such activity.

U.S. Patent No. 4,673,667, issued June 16, 1987, discloses plasmid inhibitors derived from mammalian spleen extracts. The materials contain lipid components. Gangliosides show the highest plasma-inhibiting activity. Samples containing different ceramide or ceramide derivatives have no or only minimal plasma inhibitory activity.

Japanese Patent Application H1-93562, published April 12, 1989, discloses sphingosine derivatives which are effective in the treatment of tumors.

U.S. Patent No. 4,816,450, issued March 18, 1989, to Bell, discloses long chain bases, generally sphingosine and sphingosine derivatives, which are effective inhibitors of protein kinase C. Activation of protein kinase C is considered essential for tumor promotion, cellular transformation, and understanding of inhibition by antitumor compounds.

The ability of cellular differentiation-inducing interferon to treat tumors has also been studied. Similarly, vitamin D3, which induces differentiation of the human myelocyte leukemia cell line HL-60, has been tested for its efficacy in the treatment of tumors. Although vitamin D3 induces cellular differentiation, it is not possible to use this compound in the treatment of tumors, since the high levels of vitamin D3 required for this cause harmful metabolism of calcium in the body.

Despite efforts to treat diseases characterized by cellular hyperproliferation, there is still a need to develop methods for treating these diseases. Accordingly, it is an object of the present invention to provide methods and compositions for inducing cell differentiation. The present invention also relates to methods and compositions that alter the phenotype of cells. Furthermore, the present invention provides methods and compositions for treating diseases characterized by cellular hyperproliferation. Still another object of the present invention is to provide compositions for the prevention and symptomatic treatment of diseases or abnormal conditions characterized by abnormal cell differentiation in mammals. Other objects of the invention will become apparent from the review of this specification and the claims.

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 for treating diseases characterized by cellular hyperproliferation. The methods of the present invention provide a therapeutically effective amount of a compound of formula (I) wherein the compound is administered to a mammal, generally a human patient.

R 1 has a maximum value of approx. An alkyl or alkenyl group containing 20 carbon atoms,

R ₂ is hydroxy or alkoxy or hydrogen,

R3 is hydrogen or lower alkyl,

R4 is COR5, SO2R5 or Csrs, wherein R ₅ is

Alkyl of 1 to 20 carbon atoms, alkenyl or alkynyl group optionally substituted by one or more OH, SH, ORg, SR $, NRyRg, COOR ₁₀ or CONR substituted with R8 - wherein the Rg, R7, Rg, Rg and R O is independently hydrogen or alkyl, aryl, alkaryl and arylalkyl having up to 10 carbon atoms.

The compounds of the invention are effective in the treatment of conditions in which hyperproliferation of cells or significant disruption of cell differentiation occurs. Accordingly, the compounds and pharmaceutical compositions of the invention are effective for the preparation and / or production of therapeutic agents that induce cell differentiation.

Figure 1 shows the dose dependence of ceramide formed by 1,25- (OH) ₂ D 3.

Figure 2 shows the increase in weight of ceramide over time as a result of 1,25- (OH) ₂ D ₃ .

Figure 3 shows the change in total phospholipid (A), phosphatidylcholine (B) and sphingomyelin (C) mass in HL-60 cells following treatment with 1,25- (OH) ₂ D ₃ .

Figure 4 shows the ability of C-g / C ₂ ceramide and 1 nM / L 1,25- (OH) ₂ D _{3 to} induce cell differentiation in HL-60 cells.

Figure 5 shows the effect of Cig / C ₂ ceramide on HL-60 cell growth.

Figure 6 shows the effect of Cig / C ₂ ceramide on HL-60 cell differentiation in the absence of 1,25- (OH) ₂ D 3.

Figure 7 shows the time dependence of HL-60 cell differentiation induced by Cig / C ₂ ceramide.

Figure 8 illustrates the effect of C-g / C ₂ ceramide on sphingomyelin mass growth in HL-60 cells.

Figures 9 and 10 show the effects of transient growth of C-fg / C ₂ ceramide on HL-60 cell differentiation.

Figure 11 shows an autoradiogram of a thin-layer chromatography plate after 24 hours of [ ³ H] -Cig / C ₂ ceramide uptake and metabolism by HL-60 cells.

Figure 12 shows lipids extracted from HL-60 cells showing [ ³ H] -Cig / C ₂ ceramide uptake and metabolized by HL-60 cells.

Figure 13 shows the effect of sphingosine on the differentiation of HL-60 cells induced by 1,25- (OH) ₂ D 3.

Applicants of the present invention find that delivery of ceramide or ceramide derivatives to HL-60 cells, a human myelocytic leukemia cell line, induces its differentiation. Treatment with ceramide or ceramide derivatives slows down the pro-Iteration of the cells and induces differentiation leading to the development of a phenotype characteristic of normal monocyte cells. This effect is also expected to occur in other cell types.

The HL-60 human cell line, originally isolated from a patient with acute myelocytic leukemia, is often used to study myeloid cell differentiation. These cells can become granulocytes when treated with compounds such as dimethyl sulfoxide or retinoic acid, or differentiated into monocyte / macrophage-like cells from phosphorus esters, e.g. 1α, 25-dihydroxy-vitamin D3 or G3β3 ganglioside. The mechanism of cellular maturation induced by most of these compounds is unknown. The characteristics of the HL60 promyelocytic leukemia cell line and its use as an assay model for cell differentiation are reviewed by Collins, S. J. (1987) Blood 70: 1233-1244. This well-known cell model is used to demonstrate the efficacy of the compounds of the present invention in the treatment of diseases characterized by cellular hyperproliferation.

Previous work (Okazaki, T. et al. (November 15, 1989) J. Bioi. Chem. 264: 19076-19080) reported that 1,25- (OH) 2D3-induced differentiation in HL-60 cells resulted in sphingomyelin degradation. together. Neutral sphingomyelinase activity detected in HL-60 cell extract has been shown to be induced by treatment with 1,25- (OH) 2D3 and associated with the formation of ceramide and phosphorylcholine. Sphingomyelin, ceramide, and phosphorylcholine levels return to baseline levels within four hours, suggesting a re-synthesis of sphingomyelin, thus completing the sphingomyelin cycle. These observations are thought to indicate the function of a sphingomyelin ft · · 4 · * «cycle in which inactive parental sphingolipids are converted to an active product upon cell activation. In contrast to the phosphatidylinositol cycle, sphingomyelin is degraded over time and may be involved in long-term changes in the cell.

At least 300 types of sphingolipids are synthesized in different mammalian cell types. Structurally, the sphingolipids consist of a long chain sphingoid base, an amide bonded fatty acid and a 1-position polar group. With the exception of ceramide, which has a hydroxyl group at position 1, and sphingomyelin, which has a polar group of phosphorylcholine, all sphingolipids contain carbohydrates as polar groups and are therefore called glycosphingolipids. These neutral lipids contain from 1 (cerebrosides) 20 or more glucose units, while acidic glycosphingolipids contain 1 or more sialic acid derivatives (gangliosides) or sulfate monoester groups (sulfatides). Most gangliosides and complex glycolipids are believed to be located in the outer layer of the cell membrane. However, sphingomyelin is also present in the outer membrane of cells.

The compounds useful in the present invention may be naturally occurring or synthetically produced. For use in the pharmaceutical compositions and methods of the present invention, the compounds of formula I wherein:

R! meaning max. An alkyl or alkenyl group containing 20 carbon atoms,

R ₂ is hydroxy or alkoxy or hydrogen, R ₃ is hydrogen or lower alkyl, R 4 is COR ₅ , SO 2 R ₅ or CSR ₅ where R ₅ is alkyl, alkenyl or alkynyl containing up to 1 to 20 carbon atoms, optionally one or more OH, SH, ORq, SRg, NRyRg, COORg or · * «

CONR _{10 is} substituted with R ₈ - wherein R ₈ , R ₇ , R ₈ , R ₈ and R ₁₀ are each independently hydrogen or alkyl, aryl, alkaryl and arylalkyl having up to 10 carbon atoms.

Preferably, the compounds are cell soluble, i.e., they can penetrate the cell wall and enter the cell's interior space. More preferred compounds are those in which R 1 and R 4 taken together are ca. They contain from 10 to 28 carbon atoms. The total length of the carbon chains of Rf and R4 can be divided in any proportion between Rf and R4, provided that both contain at least one carbon atom. For example, R 1 can be 1 and R _{4 is C} 12, or R 1, can be 10 and R _{4 is C} 7. Compounds of this size have been found to more readily pass through the cell membrane and enter the cell.

R1 is preferably an alkyl or alkenyl group containing from 1 to 20 carbon atoms, more preferably an alkyl having up to 1 to 20 carbon atoms or an alkyl or alkenyl group containing from 1 to 12 carbon atoms.

R2 is preferably hydrogen, hydroxy or alkoxy, more preferably hydroxy or alkoxy. In a preferred embodiment of the invention, R _{2 is} a methoxy group.

Preferably R 3 is hydrogen or lower alkyl (i.e., C 1-6 alkyl), more preferably hydrogen.

Preferably _R4 COR5, SO2R5, or CSR5, where alkyl, alkenyl or alkynyl group having 1 to 20 carbon atoms in _R5, which are optionally substituted by one or more OH, SH, ORg, SRG, NR ₇ R _8, COOR ₈ group or CONRiqR They substituted. In the formulas, R ₈ , R ₇ , R ₈ , R ₈ and R 10 are each independently hydrogen or lower alkyl (i.e., C 1-6 alkyl) and C 1-12 aryl, alkaryl and arylalkyl. More preferably, R ₄ is COR ₅ , wherein R ₅ is alkyl or alkenyl of 1 to 20 carbon atoms. Suitable aryl groups include phenyl, substituted phenyl and pyridine. Suitable arylalkyl groups may be benzyl or phenethyl.

Preferred compounds include ceramide, C18 / C2 ceramide, C3 / C8 ceramide and 3-O-methyl sphingosine. The nomenclature of these compounds is described in the experimental section of this specification.

The compounds of the present invention are effective in inducing cellular differentiation. They are delivered to mammalian cells in amounts sufficient to induce cell differentiation. Cellular differentiation, cell differentiation, and the like are used to refer to the biological process of cell maturation and acquisition of the characteristics of a functional cell. During differentiation, the cell, e.g. it may acquire or lose its morphological form or characteristics and may become or lose its ability to bind to certain substances or undergo chemical reactions. Induction of differentiation is used to describe the process of influencing cells capable of differentiation to obtain a differentiated phenotype. In general, mammalian cells are initially immature, undifferentiated cells, which then undergo differentiation and acquire the characteristics of mature, differentiated cells.

The compounds of formula I are also effective in altering the phenotype of cells. The shape, behavior and other characteristics of a cell, including its biochemical activities, are generally known as the phenotype of a cell. In this embodiment of the invention, the compounds of the invention are introduced into cells that have an altered phenotype in a mammal, generally a human patient, in an amount effective to produce the same type of normal cell phenotype of the cell phenotype.

. By a normal phenotype of a cell is meant a cell which, e.g. shape, cell markers, growth, response to changes in the environment, and regulation seem normal. Transformed cells are cells that are derived from normal cells by spontaneous or artificial intervention and have the characteristics of cancer cells, e.g. immature / less differentiated phenotype, faster growth, lack of responsiveness to environmental effects or control of cell growth, poor response and ability to develop tumors in experimental animal models. Changing the phenotype of a cell thus means changing at least one property of the cell, e.g. the ability to bind certain compounds, its enzymatic activity, its environmental response and other cellular characteristics.

Because the compounds of the present invention are capable of inducing cellular differentiation and altering the phenotype of cells, these compounds are believed to be useful in the treatment of diseases characterized by cellular hyperproliferation or in cases of significant disruption of cellular differentiation. Diseases characterized by cellular hyperproliferation are those in which the disease results in or is an abnormal proliferation of a given cell.

Abnormal cell proliferation is usually manifested by an increase in the number of cells compared to the number of cells present in the absence of disease. Hyperproliferation of cells can occur in normal, abnormal or malignant cells. Diseases that can be characterized by cellular hyperproliferation include cancerous tumors, leukemias, non-malignant tumors, psoriasis, atherosclerosis, and other diseases. The above list is intended as an example, but is not intended to be exhaustive

4 4 all such diseases. These diseases are consistent with being caused primarily by increased and abnormal proliferation of malignant (eg cancer, leukemia and lymphoma), premalignant (eg myelodysplasia) or benign (eg lymphoproliferative, benign tumors and psoriasis) cells. Inhibition of cell proliferation by the compounds of the present invention may slow down the growth of the cells attacked by the disease, resulting in significant therapeutic and possibly curative effects.

Many of these disorders are also characterized by non-differentiated cells. Undifferentiated cells, or undifferentiated phenotype, refers to immature cells that are generally unable to function like mature cells due to the lack of the necessary biochemical and physiological mechanism required for mature cells. During the process of cell differentiation, immature cells begin to show the biochemical and physiological properties characteristic of mature cells. For example, stem cells become granulocytes or cells in vivo. differentiate into monocytes. The lack of non-differentiated cells to transform into cells with a more differentiated, healthier phenotype contributes to the lack of normal function. The ability of the compounds of the present invention to induce differentiation also helps to attenuate the increased proliferation of these cells and allows them to obtain important biochemical and phenotypic characteristics that ensure that they function as normal cells. For example, the compounds of the present invention are believed to induce differentiation of cells to healthy skin by inducing differentiation of abnormally proliferating cells in psoriasis. Similarly, in myelodysplasia, these compounds are thought to induce differentiation of early and undifferentiated myeloid cells, which may play an important role in counteracting the most deleterious effects of these diseases, i.e., the reduction of normal, ··· well differentiated blood cells. Leukemia, lymphoma, and other cancers can also be treated by increasing the differentiation of malignant cells. This helps in treating the above diseases, since differentiated cells are generally unable to divide, so that individual cells are no longer able to populate the malignant clone and generate metastases.

The use of the compounds of the present invention against a wide range of neoplastic diseases is strongly supported by the observation that tumor necrosis factor (TNF) and interferon gamma increase ceramide levels, and the resulting ceramide may mediate the effects of these reagents on HL-60 cell differentiation. Therefore, these compounds are believed to be effective in the treatment of tumors by inducing tumor death and tumor regression.

Because ceramide and its derivatives are capable of slowing lymphocyte growth, the compounds of the present invention are also found to be effective in inducing immunosuppression in mammals, particularly humans. Other lymphocyte growth inhibitors are e.g. steroids, anti-lymphocyte antibodies and others play an important role in inducing immunosuppression. Immunosuppression is very important in the case of organ rejection, which can occur e.g. kidney, heart, liver and other organ transplants. Because steroids also increase levels of ceramide, ceramide may mimic the immune system's inhibitory effects on steroids. By slowing the growth of cells, particularly lymphocytes, is meant to suppress or prevent the normal rate of cell growth and cell division. Thus, compounds that slow cell growth affect the growth rate and normal functioning of these cells.

Autoimmune disorders are characterized by increased activity and proliferation of lymphocytes responsive to the body's own structures. Ceramide's potential to inhibit the growth of lymphocytes is expected to significantly contribute to the inhibition of the incidence of autoimmune disorders. Because corticosteroids play a therapeutic role in autoimmune disorders, it is now believed that ceramide and other compounds of the present invention may mediate the effects of steroids in these disorders.

Obesity can also be characterized by increased proliferation and metabolism of fat cells in the body. The compounds of the present invention can slow the growth of these cells and thus contribute to the reduction of obesity. The main relationship arose from the fact that tumor necrosis factor increased ceramide levels. TNF is thought to play a role in the induction of cachexia and its potential therapeutic use for obesity. Nowadays, ceramid is believed to be effective in treating obesity.

Atherosclerosis is another disorder characterized by increased and possibly abnormal proliferation of smooth muscle cells and endothelial cells. It is believed that slowing the growth of these cells by treatment with compounds of the present invention may contribute to the treatment of atherosclerosis.

The compounds of the present invention are also believed to be effective in anti-aging treatment. Retinoic acid, which is widely used in anti-aging treatments, increases ceramide levels in skin cells. Because retinoic acid increases ceramide levels, ceramide may mediate the function of retinoic acid and may be effective in anti-aging treatments.

:

The present invention may also be useful in chemical prevention, that is, in the treatment of cells by preventing or slowing the transformation of a cell of normal phenotype into a transformed malignant phenotype. Ceramide is capable of inducing the differentiation of malignant and other non-differentiated cells, and the compounds of the present invention are believed to be effective in inducing differentiation and slowing down proliferation of early (clinically undetectable) malignant cells. This makes it possible to develop a strong chemical preventive reagent. Similarly, retinoic acid can be effective as a chemical preventive reagent, and since retinoic acid increases ceramide levels, the compounds of the invention may similarly be useful for chemical prevention.

The compounds of the present invention and / or their pharmaceutically acceptable active salts may be formulated in the form of pharmaceutical compositions or medicaments which may be administered to mammals, e.g. can be used to treat human beings suffering from various diseases or conditions caused by other defective differentiation processes described herein. Preferably, the pharmaceutical compositions or medicaments comprise a therapeutically effective amount of a compound of the present invention and / or pharmaceutically acceptable salts thereof. The compounds of the invention may be administered to a mammal suffering from or suspected of suffering from a disease, or in combination with other compounds of the invention or other therapeutic or therapeutic agents. The compositions of the present invention may be administered by any route, i.e., orally, parenterally, intradermally, intramuscularly, intravenously, subcutaneously or topically. The method can be quickly determined by analogy with known methods and depends on the condition of the disease being treated, its severity, the age and condition of the patient. For oral administration in the form of tablets, capsules or solutions or as a solution for injection. parenterally in the form of a solution or a suspension. For injection, a sterile liquid may be used as a solvent. Water for injection may be used which contains the stabilizing reagents, solubilizing reagents and / or buffers customary for injection solutions. The desired additives may be e.g. tartrate and borate buffer, ethanol, dimethylsulfoxide, complexing agents (e.g., ethylene diamine tetraacetic acid), high molecular weight polymers (e.g., polyethylene oxide) for appropriate viscosity, or polyethylene derivatives of sorbitic anhydrides.

The total routine dose (i.e. daily, weekly, monthly, etc.) of the compounds of the present invention is such as to be effective in differentiating the respective cells, reducing cell proliferation, or improving the condition to be treated. stabilize. Those skilled in the art can readily determine the optimal therapeutically effective dose for the particular case using the range outlined above as a starting point.

In the manufacture or manufacture of a composition for the treatment of a disease, a compound of the present invention, or a physiologically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a mixture of these with a physiologically acceptable carrier, carrier, paste, binder, preservative, stabilizer, flavoring and / or other excipient, in unit dosage form. Typical examples of additives for use in tablets and capsules include tragacanth, gum arabic, corn starch and gelatin as binder, microcrystalline cellulose as binder, corn starch, pregelatinized starch and alginic acid, magnesium stearate as a lubricant, and magnesium stearate as a lubricant. Other additives for capsules include liquid carriers for cooking oil, shellac, sugar, and combinations thereof to form tablets.

In the case of parenteral injection, the following can be used as a carrier for dissolving or suspending the active ingredient: water, natural vegetable oils, e.g. sesame seed oil, coconut oil, peanut oil or cotton seed oil, synthetic oils e.g. ethyl oleate and may contain buffer solutions, preservatives and antioxidants as required.

The method of the present invention may be carried out by directly adding an effective amount of the composition of the invention to cells. However, it is also within the scope of the invention to provide the process indirectly, i.e., by administering a compound or composition which in vivo induces the production of one of the compounds of the present invention. Furthermore, it is within the scope of the present invention to convert prodrugs of drugs into in vivo compounds of the invention.

Many of the compounds used herein are abbreviated to denote the total number of carbon atoms of the compounds of formula (I). For example CI8 / _C2 ceramide is a (I) a compound of structural formula wherein R | represents an alkyl group having 15 carbon atoms, _R2 is hydroxy, R3 is hydrogen, _R4 is COR5 and R5 is methyl. In the Rf portion of Cfg / Cz ceramide, the double bond is in the same position as in ceramide. Thus, 18 carbon atoms are present in the first-mentioned chain, which contains the carbon atoms in Rf, the carbon to which R _{2 is} attached, the carbon to which nitrogen is attached, and the CH ₂ OH group at the end of the chain. There are two carbon atoms in the second chain, namely the carbon atom in R ₄ amide bond and the carbon atom in R 5 methyl group. In the compounds so named, the first carbon chain contains R 1 and the carbon atoms to which R ₂ , N and OH are attached, the second carbon chain represents the carbon atoms in R ₄ . Similarly the Cfg / Cg is a ceramide of formula (I) compound of the structural formula wherein represents an alkyl group having 15 carbon atoms, _R2 is hydroxy, R3 is hydrogen, R4 is COR5 and _R5 pentanilcsoport. C ^ / / Cg ceramide means a compound wherein R- is C 8 alkenyl, R ₂ is hydroxy, R 3 is hydrogen, R 4 is COR 5 and R 5 is heptanyl. In each of the examples herein, the double bond in Rj is in the same position in the ceramide, although unsaturation at other sites may be used. 3-O-Methyl-sphingosine is a compound of general formula (I) wherein represents an alkyl group having 15 carbon atoms, _R2 is methoxy, R3 is hydrogen, R4 is COR5 and R5 is methyl.

16,25-Dihydroxy Vitamin D 3 (1,25- (OH) ₂ D 3) was obtained from Hoffman-LaRoche, Nutley, New Jersey. We purchased the SM and PC from Avanti Polar Lipids, ceramide from Supelco. Insulin, transferrin, NBT and α-naphthyl acetate are from Sigma Chemical Co., St. Louis, Missouri.

PREPARATION OF CERAMIDE AND SPINGINGOSIN ANALOGES

N-acetyl and [ ³ H] -N-acetyl sphingosine are described by Gaver, RC and Sweely, C.

C., J. Amer. Chem. Soc. 88: 3643-3647 (1966). Briefly summarizing the N-acetyl sphingosine (Cig / _C2 ceramide) and N-hexanoyl-sphingosine (C ^ g / Cg ceramide) or neutral sphingosine with acetic anhydride. by acylation with caproic anhydride (90% yield). The ^[3 H] -N-acetyl-sphingosine (specific activity 2.5 x 10 ⁴ cpm / nmol / L)

It is prepared by reduction of 3-oxo-1-hydroxy-2-acetamide-4-octadecene with pHJNaBfy (40% yield after TLC). 3-Oxo-1-hydroxy-2-acetamide-4-octadecene is obtained by oxidation of cold N-acetyl sphingosine with dry chromic anhydride (yield after 80% TLC). N-ethyl sphingosine was prepared from N-acetyl sphingosine by reduction with LiBH ₄ and purified by preparative TLC (30% yield). The Cn / Cg ceramide is prepared according to the procedure described by Liotta et al., Tetrahedrom Letters 29: 3037 (1988). The structure of each compound was confirmed by NMR and the purity of the preparations was assayed by TLC, which is estimated to be greater than 97%. These compounds are dissolved in ethanol and transferred to the culture medium (in which the final concentration of ethanol is less than 0.1%).

PREPARATION OF 3-O-METHYL-SPINGOSIN

3-O-Methyl sphingosine was synthesized according to the method described by Carter et al., Journal of Biochemistry 192: 197-207 (1951). 100 mg of bovine cerebrosides (Cat. No. A-46, Serdary Research Labs, London, Ontario, Canada) were dissolved in a round-bottom flask containing 112 μΙ of concentrated sulfuric acid and 2.3 mL of methanol. The mixture was heated and refluxed for 6 hours with stirring. After heating the reaction mixture on ice for approx. Cool for 15 minutes. The precipitate from the fatty acids and methyl esters was removed by filtration using Whatman filter paper 1 and a Brücner funnel. The fatty acids and esters remaining in the filtrate were extracted with 4 x 1 mL of petroleum ether. The ether was removed in vacuo for ca. In 15 minutes. The solution was neutralized with 4M KOH in methanol (approximately 6.5 mL required pH checked paper) and the precipitated potassium sulfate was filtered through Whatman filter paper 1 using a Brüchner funnel. The pelleted solution was stored at 4 ° C (refrigerator) overnight, and any additional potassium sulfate precipitate formed upon standing was removed. The solution was adjusted to pH 10 with 6 M NaOH (checked with pH paper). The solution was then extracted twice with diethyl ether. The extracts were combined, washed once with water and dried over sodium sulfate in vacuo. The dry material was dissolved in chloroform / methanol (1: 1).

The sample was purified by thin layer chromatography (TLC) using the procedure 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: 2 M ammonium hydroxide (40: 10: 1) as solvent and sphingosine as standard. A small portion of the plate is visualized with a ninhydrin spray. 3-O-Methyl Sphingosine runs farthest on the plate, the fastest migrating component. The silica strip corresponding to 3-O-methyl sphingosine is scraped off and the 3-O-methyl sphingosine is eluted with chloroform: methanol (1: 1). Elute the eluate in an IEC centrifuge at 2000 rpm for approx. Centrifuge for 5 minutes and pour the supernatant into a clean tube. Elution of 3-O-methyl sphingosine from silica with chloroform: methanol (1: 1) was repeated and the supernatants were combined. The combined supernatants were dried under vacuum and the total yield of 11 mg of 3-O-methyl sphingosine was suspended in ethanol.

Cell culture

HL-60 human myelocytic leukemia cells (after 45 passages) were grown in RPMI1640 medium containing 10% fetal calf serum (FCS) (Sigma Chemical Co., St. Louis, Missouri) at 37 ° C in a 5% CO ₂ incubator. . The cells were washed twice in phosphate buffered saline (PBS) and resuspended in serum-free medium containing insulin (5 mg / L) and transferrin (5 mg / L) prior to treatment with the various compounds. DETERMINATION OF THE QUANTITY OF LIPIDS

At the indicated times, the cells were harvested and the lipids were harvested by Bligh and Dyer, Can. J. Biochem. Physiol. 37: 911-917 (1959). The samples were dried under nitrogen and then dissolved in 0.1 ml of chloroform: 40 μ visz was applied to a thin layer chromatography (TLC) plate (Merek) and 40 μΙ was used to measure phospholipid phosphate (duplicate). The phosphate is · Van · Veldhoven, P. and Mammaerts, G, Ann. Biochem. 161: 45-48 (1987). Of sphingomyelin (SM) and the phosphorylcholine (PC), the purpose of identifying the TLC plates with chloroform / methanol / acetic acid / H ₂ O (50: 30: 8: 5) (solution A) and chloroform / methanol / 2 M / l NH ₄ OH (60: 35: 5) (Solution B). For two-dimensional TLC plates, use a combination of solution A and solution B. After staining the plates with iodine vapor, the spots corresponding to SM and PC were scraped off, extracted with chloroform / methanol (1: 1), dried under nitrogen and the phospholipid phosphate was measured.

DETERMINATION OF CERAMIDE

The amount of ceramide was measured enzymatically using sn-1,2-diacylglycerol (DAG) kinase as described by Preiss et al., J. Bioi. Chem. 261: 86978700 (1986) and Van Veldhoven, Anal. Biochem. 183: 177-189 (1989). To check for ceramide conversion to ceramide-1-phosphate in HL-60 cells, TLC plate was run with ceramide phosphate and phosphatidic acid in various solvent systems, e.g. chloroform / methanol / acetic acid (65: 15: 5), chloroform / pyridine / formalic acid (60: 30: 8) and chloroform / acetone / methanol / acetic acid / H ₂ O (6: 2: 2). Rf values are compared with those obtained for the ceramide standard. Also ceramide-1-phosphate (70 C, 20 h, 1 M / l NaOH) is converted to sphingosine-1-phosphate by alkaline hydrolysis, and the Rf values of the sphingosine-1-phosphate standard one, butanol / acetic acid / H ₂ O ( 6: 2: 2) or chloroform / acetone / methanol / acetic acid / H ₂ O (10: 1 compared with the value obtained) solvent, and found to be identical values of 0.45 or 0.42: 4: 2: 2nd

TESTING FOR GROWTH AND DIFFERENCE OF HL-60 CELLS

Cell growth was measured with a hemocytometer. Live cells were determined by their displacement of trypan blue dye. The percentage of live cells is always greater than 80%, otherwise it is specifically labeled. Monocyte / macrophage and

Nitrogen tetrazolium blue (NBT) reduction was used to identify granulocyte cell line, and non-specific esterase was used to identify monocyte / macrophage cell line. Both markers are described in Okazaki, T. et al., J. Cell

Physiol. 131: 50-57 (1987).

Uptake and metabolism of [ ³ H] -C ₁₈ C ₂ ceramide

After resuspension of the HL-60 cells in serum free medium labeled ^[3 H] -C18 / C2 ceramide (1 x 10 ⁵ cpm / ml), harvested at the indicated time and were washed three times with PBS. The lipids were extracted according to the procedure of Bligh and Dyer, supra. Dried samples were dissolved in 100 μΙ chloroform: 20 μΙ was transferred to a TLC plate and 40 μΙ was used to measure phospholipid phosphate. The plates were developed in chloroform / methanol / 2M / NH4OH (40: 10: 1) in C- | | |. ₈ / C2 for the separation of ceramide, SM and sphingosine (SPH). The resulting spots were scraped off and weighed in a Safety Solve solvent (Research Products International Corp.) in an LKB scintillation counter (LKB).

DOSE AND TIME DETERMINATION OF CERAMIDE FORMATION 1,254OH) ₂ D ₃

The amount of ceramide is measured using the diacylglycerol kinase method previously developed for diacylglycerol (see Van Veldhove et al., Supra). DAG kinase from E. coli is capable of converting ceramide to ceramide-1-phosphate. After converting ceramide into ceramide1-phosphate, the cells in the cells are identified by thin-layer chromatography (TLC) plates by co-migration of ceramide phosphate with the standard. Cellular and standard ceramide phosphate give the same Rf value after developing TLC plates in 4 different solvent systems. Further identification was carried out by alkaline hydrolysis of ceramide phosphate. This leads to the formation of sphingosine-1-phosphate, which in the TLC plate is butanol / acetic acid /

Η ₂ 0 (2: 2: 2) when developed in a solvent system, it interacts (Rf 0.45) with the standard.

As shown in Figure 1, treatment of HL-60 cells with 1,25- (OH) ₂ D 3 results in a dose-dependent increase in ceramide levels in the second hour after treatment. Data are expressed as% of control (without C g / C ₂ ceramide). Vertical sections represent standard deviation values from two parallel measurements. The base ceramide level is 26.1 * 0.82 nM / nM phospholipid. The results represent three different experiments.

Maximum increase in ceramide level occurs at 100 nM / Ι 1,25- (OH) ₂ D3. At this concentration, there was a 41% increase in the level of ceramide compared to the control level. The dose dependence of ceramide formation by 1,25- (OH) ₂ D3 is very similar to that of HL-60 cell differentiation by 1,25- (OH) ₂ D3, which is maximal at 100-300 nM / l. Thus, these results suggest a quantitative relationship between ceramide formation and cell differentiation.

We also investigate the time dependence of the change in ceramide levels by 1,25- (OH) ₂ D3 on HL-60 cells. As shown in Figure 2, HL-60 cells are harvested at the indicated times after treatment with 100 nM / Ι Cig / C ₂ ceramide. The amount of ceramide is measured as described in the Experimental section. The results are derived from two different definitions. The vertical sections represent the standard deviation value from a single measurement. The results represent three experiments where HL-60 cells are treated with 100 nM / l 1,25- (OH) ₂ D3 (which is an optimal concentration based on the dose-dependency assay shown in Figure 1), with a gradual increase in ceramide levels over the first 2 hours. , and then return to baseline. The fastest growth (7% of baseline) was observed after 30 minutes after treatment with 1,25- (OH) ₂ D3, reaching a maximum of 41% after 2 hours (Figure 2).

• «·

These studies show that 1,25- (OH) ₂ D ₃ induces a time- and dose-dependent transient increase in ceramide levels, which introduces HL-60 cell differentiation (maximal ceramide formation at 2 h and phenotypic changes in differentiation). in hours 2-4). As ceramide formation appears to be the earliest biochemical change following treatment with 1,25- (OH) ₂ D3, these results point to the role of ceramide as a lipid mediator.

CERAMIDE FORMING FROM SPINGOMIELINE

Previous studies (Okazaki, T., et al., Supra) have already shown that 1,25- (OH) ₂ D3 induces hydrolysis of sphingomyelin with simultaneous changes in phosphorylcholine and ceramide levels, followed by sphingomyelin re-synthesis and return to baseline. To verify that ceramide is indeed formed from sphingomyelin, the amount of sphingomyelin hydrolyzed is measured and compared with the amount of ceramide formed. As shown in Figure 2, the maximum level of ceramide occurs at hour 2 and a total of 13+ 2 pM ceramide / nM phospholipid is produced. The ceramide level then returns to baseline at 4 o'clock. Figure 3 shows no significant change in total phospholipid levels (Figure 3A) and phosphatidylcholine levels (Figure 3B) during the first 4 hours after 1,25- (OH) ₂ D ₃ treatment. In contrast, sphingomyelin levels decrease from 51 + 6 pM / nM phospholipid to 34+ 2 pM / nM phospholipid at hour 2 (Figure 3C). The total change in sphingomyelin weight is 17 + 4 pM / nM phospholipid, which is very similar to the total amount of ceramide in the total 13 pM / nM phospholipid. These results strongly suggest that ceramide is derived from sphingomyelin degradation by treatment with 1,25- (OH) ₂ D _{3 in} HL-60 cells. The labeling of sphingosine precursors with [ ³ H] -palmitate shows that during this period no significant change occurs in other sphingolipids upon treatment with 1,25 (OH) ₂ D ₃ , and that of the various sphingolipids, the · ·· most of the signal. Therefore, it is unlikely that ceramide could be derived from hydrolysis of a sphingolipid other than sphingomyelin. However, these assays do not preclude de novo synthesis of ceramide by treatment with 1,25- (OH) ₂ D ₃ , although this is unlikely given the significant and proportional hydrolysis of sphingomyelin.

These results also show that the total phospholipid level in HL-60 cells is 15.6+ 1.0 µM / cell, of which 53.3+ 2.3% PC and 5.1 ± 0.6% SM. This is in good agreement with previous data that using the labeling of two lipids with [ ³ H] choline, the SM / PC ratio is 0.05 to 0.1 (Okazaki, T. et al. (1989) J. Biol. Chem. 264 : 19076-19080). These results also explain why no significant change in total phospholipid levels can be observed, since a total decrease in SM levels corresponds only to a change of 1.5+ to 2.0% in total phospholipid levels.

C ₁₈ / C ₂ CERAMIDE PROMOTES THE EFFECTS OF 1,25- (OH) ₂ D ₃ HL-60 ON CELL DIFFERENCE

Exogenous use of bacterial sphingomyelinase, which induces membrane sphingomyelin hydrolysis and ceramide formation, has been shown to allow 1,25- (OH) ₂ D ₃ cell differentiation in HL-60 cells (Okazaki, T. et al. supra). However, the use of bacterial sphingomyelinase is complicated by the fact that at higher concentrations of sphingomyelinase the membrane is also hydrolyzed by phosphatidylcholine, thus limiting its use.

To overcome this problem and to investigate whether ceramide can replace the action of bacterial sphingomyelinase, a synthetic cell-permeable ceramide, C-g / C ₂ , is prepared containing acetate in an amide bond. In comparison to the wild ceramidokkal the Cig / _C2 ceramide contains less than from 14 to 16 carbon atoms, and thus more soluble in water. Under the same conditions, naturally occurring ceramide with long N-acyl chains at 50 μΜ does not affect cell growth or cell differentiation, which can be explained by poor uptake of ceramides with long N-acyl chains.

This phenomenon holds true for cell-permeable DAG analogs e.g. and dioctanoylglycerol and oleoylacetylglycerol, which have acyl chains shorter than the naturally occurring DAG. Increased cell differentiation was observed when HL-60 cells were treated with sub-optimal concentrations of 1,25- (OH) 2D3 (1 nM / l, which is 100% of the optimal concentration) and different concentrations of ceramide.

HL-60 cells (2.5 x 10 ⁵ cells / mL) was treated simultaneously with various concentrations 0ιβ / θ2 ceramide and 1 nM / l 1,25- (OH) ₂ D3 ~ mal. Cellular differentiation is assessed by NBT reduction (bars) and NSE activity (bars). The effect of Cig / C ₂ ceramide on cell growth is shown in the graph. The results are from three different experiments. The vertical sections represent the standard deviation calculated from one definition.

As shown in Figure 4, ceramide C18 / C2 increases dose-dependent cellular differentiation induced by 1,25- (OH) ₂ D 3 and exerts its maximum effect at 1 μΜ. On the 4th day after treatment, 1 μf Cfg / C ^ ceramide increases the NBT reducing and / or NBT from 11.9+ to 2.9% to 57.8 to 1.4%. Increases non-specific esterase activity (NSE) from 2.2 + 0.9% to 39.2 ± 7.2%. At the same concentration range, ceramide slightly inhibits cell growth without significantly affecting cell viability. In all cases, cell viability is greater than 80%. Although one μΜ Οιβ / Ο ceramide ₂ slightly inhibited the absolute number of cellular growth, the NBT- and NSE-positive cells 0.9 x 10 ⁵ cells / ml to cells / ml and 2.54 x 10 ⁵ respectively. Increases from 0.16 x 10 ⁵ cells / ml to 1.72 x 10 ⁵ cells / ml on day 4 compared to controls.

CELL PERMABIC CERAMIDES INDUCE HL-60 CELL DIFFERENCE INDEPENDENT FROM 1,25- (OH) 2D3

The strong synergy subthreshold concentrations of 1,25- (OH) ₂ D3 and low concentration (100 nM / l -1 μΜ / l) CI8 / C between _two ceramide suggests that the _C18 / _C2 ceramide concentrations higher than 1 , Can induce cell differentiation independent of 25- (OH) 2D3. The effect of synthetic Cig / C ₂ ceramide on cell growth and cell differentiation was investigated. Treatment of HL60 cells with increasing concentrations of _C18 / _C2 ceramide results in dose-dependent inhibition of cell growth, as shown in Figure 5. Various _C18 / _C2 ceramide concentrations are shown as follows: p - control, set the black rectangle corner μΜ -1 / I, black square - 3 μΜ / I, black circle - 6 μΜ / I and the black triangle -10 μΜ / l . The results are derived from three different definitions. The vertical sections represent the standard deviation value from a single measurement. The C- | Concentration of ₈ / C ₂ ceramide at 10 μΜ / L on day 7 leads to severe cell death. Cell death is due, at least in part, to the cell differentiation inducing effect of Cj e / C ₂ ceramide and not to toxicity, as more than 30% of cells are differentiated by day 2 after treatment.

As increasing concentrations of ₈ Ci / _C2 ceramide is shown in Figure 6 would result in a progressive increase in reducing power according to the NBT and NSE activity in the induction of Day 4, the HL-60 cell differentiation. NBT-reducing capacity is indicated by bar columns and NSE activity is indicated by blank columns. The results are from three different experiments. The vertical sections represent the standard deviation calculated from one definition. The cells for 4 days with various concentrations of C- | 8 treated with ceramide / C _2nd NBT-positive cells from 0+ 1.0% to 53.2 + 1.6%, and NSE • · · positive cells from 1.0+ 1.9% to 46.4 ± 6.0% increase after treatment with 6 μΜ / Ι Cig / C ₂ ceramide. Significant growth can be observed in differentiated cells even at 1 μΜ / Ι Cj ₈ / C ₂ ceramide concentration.

Morphological examination of the phenotype of ceramide-treated HL-60 cells shows that the morphological changes are consistent with the monocyte phenotype induced by 1,25- (OH) ₂ D3. These cells are characterized by a high cytoplasm: nuclear ratio, lobe and eccentric nucleus, and nuclear bodies. azurophilic granules do not appear. Cells acquire NBT-reducing ability and NSE activity, a specific marker of monocyte differentiation.

This differentiation követeöen assayed in the C ^ ₈ / _C2 ceramide 6 as a result of time dependency of μΜ / Ι ₈ Ci / _C2 ceramide as an optimal concentration, which results in maximal differentiation and cell toxicity is minimal. Cell differentiation is judged by NBT-reducing ability and NSE activity. At six μΜ / Ι Cj ₈ / C ₂ ceramide doses, there was a progressive increase in the number of NBT- and NSE-positive HL-60 cells, up to 61% and 7 days after treatment. To 56% as shown in Figure 7. Figure 7 ₁₈ / _C2 ceramide-treated HL-60 cells were labeled empty symbols 6 μΜ / C Ι black symbols, untreated cells. NBT-reducing ability is indicated by circles. NSE activity is represented by rectangles.

These results show that C- Ceramide ₈ / C ₂ alone can induce the differentiation of HL-60 cells into cells with a monocyte phenotype, with 6 μΙ / Ι Cfg / C _{2 having} similar efficacy to 1,25- (OH) ₂ D ₃ . At an optimum concentration of 100 nM / L, ceramide Οΐβ / 74 ₂ was found to reduce NBT-reducing ability or 74 + 6% of cells. 51 + 4% induces NSE activity compared to 53.2+ 1.6% and 46.4+ 6% for C 1 -C ₂ ceramide on day 4.

As shown in Figure 8, addition of C18 / C2 ceramide to HL-60 cells does not alter the cellular level of sphingomyelin. At the times indicated in the HL-60 cells treated with 5 μΜ / Ι ₈ Ci / _C2 ceramide. Sphingomyelin is extracted and measured as described in the Experimental section. Data are expressed as% of control (without addition of C18 / C2 ceramide). The vertical sections represent the standard deviation calculated from one definition. The results are from two different experiments. The results strongly suggest that the difference in ceramide and bacterial sphingomyelinase (SMase) HL-60 cells is mediated by ceramide and not by changes in SM levels.

To further clarify the role of ceramide as a second messenger, we investigate whether brief incubation of HL-60 cells with ceramide is sufficient to induce differentiation. C18 / C2 ceramide repeated washings added to cells may be applied again to the medium, so that after 3 washes less than 20% of the original Cig / _C2 ceramide quantity remains sejtpellettel associated.

Since 1,25- (OH) 2D3 results in an increase in endogenous ceramide levels of approx. After 2 hours, HL-60 cells were incubated with C18 / C2 ceramide (0.5-2 μΜ / Ι) for 2 hours. ₁₈ C / C2 ceramide is extracted and evaluated differentiation. Cells were treated with C18 / C2 (0.5.1 or 2 μΜ / Ι) without ceramide for 2 hours (Figs. 9 and 10) or 4 hours (Fig. 10), washed three times in RPM11640 medium and then serum free. Suspended in RPM11640 medium. On the indicated day (Figure 9) or on the fourth day (Figure 10), differentiation is measured by the NBT reduction capacity as described in the Experimental section. The vertical sections represent the standard deviation calculated from one definition. In Figure 9, the empty circles represent 0 μΜ / Ι C18 / C2. The black triangles represent 0.5 μΜ / Ι 0 ^ 8 ^ 2 ceramide. Black rectangles represent 1 μΜ / Ι C18 / C2 ceramide. The black circles represent 2 μΜ / Ι C18 / C2 ceramide. In Figure 10, ceramide 10 and 0 μΜ / Ι C / Cg are indicated by blank columns. Ceramide at 0.5 μ ig / Ι C is labeled with vertically shaded columns. The 1 μΜ / Ι C18 / C2 ceramide is labeled with oblique shaded columns. Ceramic 2μΜ / Ι C-18 / C2 is marked with oblique columns. The results are from two different experiments. Cig / Cz ceramide (1 or 2 μΜ / Ι) results in significant differentiation of HL-60 cells under these conditions (Fig. 9), indicating that 2 h treatment of HL-60 cells with C18 / C2 is sufficient for differentiation. To induce. Four hours of treatment do not result in further significant differentiation (Figure 10). These studies indicate that short term treatment of HL-60 cells with elevated ceramide levels is sufficient for cellular commitment to differentiation.

EFFECT OF CERAMIDE AND SPINGINGOSIN ANALOGIES ON D3-INDUCED HL-60 CELL DIFFERENCE

Sphingosine is a pharmacological inhibitor of protein kinase C in vitro and in various cellular systems. Since ceramide can be metabolized to sphingosine by acidic and / or neutral ceramidases, it is examined whether the effect of ceramide on HL-60 cells can be attributed to sphingosine formation. No sphingosine was detected during treatment of HL-60 cells with 1,25- (OH) 2D3. Furthermore, addition of C18 / C2 ceramide to HL-60 cells does not result in detectable sphingosine formation. For these experiments, C18 / C2 ceramide is labeled with [ ³ H] on the third carbon atom of the sphingosine base. Cells (5x10® cells / ml) labeled 4 μΜ / Ι ^[3 Η] -Οΐβ / Ο2 ceramide (1 x 10 ⁵ cpm / ml). C18 / C2 ceramide is used in ethanol. Addition of [ ³ Η] -0ΐ8 / θ2 ceramide to HL-60 cells results in immediate uptake of labeled C18 / C2 ceramide, but does not convert to sphingosine as shown in Figures 11 and 12. The C-tg / Cz ceramide was ca. 20% is taken up within 0.5 hours of treatment.

Ceramide residue (80%) remains unchanged in the medium. Figure 11 shows an autoradiographic image of a TLC plate (24 hour exposure). THE

In Figure 12, the lipids are extracted, separated on a TLC plate and the radioactivity is measured as described in the Experimental section. Ceramide is indicated by blank circles. Sphingomyelin is indicated by blank rectangles. Sphingosine is indicated by black circles.

The addition of 1,25- (OH) ₂ D 3 to cells labeled with Cig / C ₂ ceramide does not result in sphingosine formation either. A small percentage of the mark is converted to sphingomyelin (2.8% after 12 hour mark). These studies show that 1,25 (0H) ₂ D3 does not lead to sphingosine formation and that when exogenous ceramide analogs are added to cells (with or without 1,25- (OH) ₂ D3), they do not convert to sphingosine .

Sphingosine is slowly metabolized in HL-60 cells and is mainly incorporated in the form of ceramides and other sphingolipids (Merrill, A.H. et al. (1986) J. Biol. Chem. 261: 12610-12615). However, the above assays do not exclude the rapid metabolism of ceramide-derived sphingosine, which cannot be detected by the above procedures. To determine the role of sphingosine in mediating the effects of ceramide, we investigate whether sphingosine can mimic the function of ceramide.

As shown in Figure 13, when HL-60 cells were simultaneously treated with various concentrations of sphingosine and suboptimal amounts of 1,25- (OH) ₂ D3 (1 nM / Ι) for 4 days, NBT-reducing ability and NSE activity does not change compared to control. The HL-60 cells (2.5 x 10 ⁵ cells / ml) were treated with various concentrations of sphingosine for 4 days at 1 nM / l 1,25 (OH) ₂ D3 in the presence of. Cell differentiation is assessed by NBT-reducing ability (bared columns) and NSE activity (empty columns). The effect of sphingosine on the growth of HL-60 cells is shown in the graph. The results are from three different experiments. The vertical sections represent the standard deviation calculated from one definition.

These studies show that sphingosine does not increase the ability of 1,25 (OH) ₂ D ₃ HL-60 to induce cell differentiation, which is a sharp distinction from the effect of ceramide (compare Figures 4 and 13). [ ³ H] Sphingosine is efficiently uptake by HL-60 cells, indicating that ineffectiveness of sphingosine is not due to poor uptake. Furthermore, sphingosine actually slows the growth of HL-60 cells (graph plotted at the top of Figure 13) to a level comparable to that achieved with Cie / C ₂ ceramide, indicating that the cellular effect of sphingosine is different from cellular differentiation. The ability of sphingosine to slow the growth of HL-60 cells without promoting cell differentiation supports the notion that ceramide induces cell differentiation independently of the cell growth rate of HL-60 cells. To further demonstrate the role of ceramide in cell differentiation, sphingosine-independent syntheses, synthetic ceramide and sphingosine analogs were prepared and tested for their ability to increase HL-60 differentiation in the presence of suboptimal 1,25- (OH) ₂ D ₃ . Table 1 shows that both C 1 -C 4 ceramide and C 8 / C ₂ ceramide result in significant increases in NBT-reducing ability and NSE activity observed with 100 nM / l 1,25- (OH) ₂ D ₃ value. Cj f / Cg ceramide also significantly increases NBT-reducing ability and NSE activity. Studies with C ^ / Cg ceramide play a significant role in excluding the role of sphingosine. The Cn / Cg ceramide deacylation may result in a CN ^{W ^, n} 9 ^{oz 'N} analog, already has been shown to not have the in vitro and cellular effects of sphingosine (Norjiri, H. et al, supra).

· · ·

i co Table Effect of various sphingolipids 1 nM / l 1,25- (011) D (C

Ul 'β ω' 0 <—i '{C • H o c φ Lí

V

U-i ς-ι • H

Λ3 Φ

4J • m

Φ

CD o o

4J r-i

Φ

N Φ v

ni Ξ> • H 4 = T dP

N

C Λ4 a φ

I 4J K * m CS Φ z ω> • H

4-> • H

N o a i C-i dφ

4-1 • m e tt

N tt 4J

• r->

e κ u. - • C '0 Ή Ή z o • h -rí <—I' -J -O 44 r-H ~ c c φ z • H 0 2 <44 C - N 0 CC Aí ω Ό i — 1

Φ

N Φ: <

cl cl tendon CS m C c She « • She <N r * She - * 1 l * 1 +1 * 1 -rl • H -1 +1

cs tendon TENDON ΓΊ 10 n c <N 10 cl the ci · cw ΠΊ • M ci

in in • · <N CN

« « « « cl CD m m tendon tendon r * • (N She She "C. c m • H H -I +1 -I -rl r | - | un CS CN ΓΊ cs m tendon r *. « • • • • • • • • m r * She CS r * She ΙΛ She un · r i r-

N & c «Μ CS Ό 04 10 Shoot c »-» - She -1 -1 -1 d 1 * 1 -rl -1 -I rl IC T · 04 C9 SHE 04 CS «0 r * r * r * tendon

HL-60 cells (2.5 x 10 ^b cells / ml) with the labeled lipid and below the threshold concentration of 1,25- (OH)? D ₃ (1 nM / L) -inal was treated simultaneously for 4 days. The results are derived from three determinations. The asterisks indicate that the deviation from the control (1 nM / l 1,25- (OH) ₂ O ₃ ) is significant p <0.01.

* · ·· ·. · ···;

: ·%. · ·· *

On the other hand, sphingosine does not induce a significant change in the two parameters of cell differentiation. Similar results have already been reported for sphingosine (Stevens, VL et al. (1989) Cancer Rev. 49: 3229-3234). Furthermore, N-ethyl sphingosine, a potent inhibitor of protein kinase C, does not significantly increase NBT-reducing ability and NSE activity from baseline. These studies show that ceramide derivatives increase HL-60 cellular differentiation to sphingosine derivatives by 1,25 (OH) ₂ D3.

Previous studies have found that in HL-60 cells, 1,25 (OH) ₂ D3 results in hydrolysis of sphingomyelin with the simultaneous formation of ceramide and phosphorylcholine, which is regulated by the sphingomyelin cycle (Okazaki, T. et al. (1989) J. Bioi. Chem. 254: 19076-19080). It has been suggested that hydrolysis of sphingomyelin plays a role in the differentiation of HL-60 cells since administration of exogenous bacterial sphingomyelinase promotes HL-60 cell differentiation upon exposure to a concentration below 1,25- (OH) ₂ D3.

Applicants have discovered that ceramide acts as a lipid mediator to mediate HL-60 cell differentiation by 1,25- (OH) ₂ D3. Low concentrations of ceramide (100 nM / l - 3 μΜ / Ι) increase HL-60 cell differentiation induced by the concentration of 1,25- (OH) ₂ below the D3 threshold. More importantly, ceramide is able to induce HL-60 cell differentiation at higher concentrations (1-6 μΜ / nélkül) in the absence of 1,25- (OH) ₂ D3. The phenotype of differentiated HL-60 cells closely resembles the monocyte phenotype produced by 1,25- (OH) ₂ D3. These results suggest that ceramide plays an essential role in mediating cellular differentiation by 1,25- (OH) ₂ D3. Furthermore, the C ₁₈ / C ₂ ceramide prove effective in inducing cell differentiation even if cells are treated for only 2 hours. This indicates that the ceramide formed by 1,25- (OH) 2D3 is sufficient to induce cell differentiation. Since the ceramide added to the cells is ca. 20% of the cells are taken up, these results also indicate that the effective concentration of C18 / C2 ceramide is in the nM range (20-1000 nM / l).

Applicants are not yet aware of the mechanism by which ceramide mediates cellular differentiation by 1,25- (OH) 2D3. A target for ceramide function has not yet been identified. Since ceramide may serve as a precursor of sphingolipids and sphingosine, its function may also be mediated by metabolites. Ganglioside GM3 has been reported to increase cellular differentiation of HL-60 by phorbol esters and cell differentiation on the monocyte line (Norjiri, H. et al. (1986) Proc. Natl. Acad. Sci. 83: 782-786). Ceramides are gangliosides, e.g. It can serve as a precursor to GM3. However, 1,25- (OH) ₂ D3 does not modify GM3 (Norjiri, H. et al., Proc. Natl. Acad. Sci. USA (1988) 83: 782-786), and according to the applicants an amount of ceramide is converted to ganglioside. Furthermore, the dose response of HL-60 cells to ceramide is much lower than that observed for GM3 (increasing the likelihood of GM3 being degraded to ceramide).

Ceramide can also serve as a precursor of sphingosine, which is possible by one-step hydrolysis with neutral or acidic ceramidases. Applicants' findings contradict that sphingosine mediates the effects of ceramide. This can be demonstrated by: (1) sphingosine not being detected by 1,25- (OH) 2D3 in HL-60 cells, (2) sphingosine not increasing or causing HL-60 cell differentiation by 1,25- (OH) 2D3 the differentiation of HL-60 cells into monocytes alone, 3) unlike other ceramide derivatives, sphingosine and its related analogue, N-ethyl sphingosine, do not increase cellular differentiation by 1,2536 (OH) ₂ D3, and 4) C-ι j / Cg ceramide, which hydrolysis yields a lower sphingosine, which is not an inhibitor of protein kinase C (Merril, AH et al, (1989) Biochemistry 28: 31383145) is as effective as 0 ₁₈ / θ2 ceramide cell differentiation induction.

Studies with Cn / C8 ceramide play a very important role in excluding the important role of sphingosine. Deacylation of C? / Cg ceramide may result in a Cn-sphingosine analog that has been shown to have no in vitro and cellular effects of sphingosine (Norjiri, H. et al., Supra).

Since these two pathways are unlikely to exist, other targets of ceramide may mediate their function. Sphingomyelin serves as a cellular repository for sphingomyelinases to produce ceramide, a potential lipid mediator (second messenger), just as glycerolipids serve as a cellular repository for phospholipase C to produce diacylglycerol as a second messenger.

Claims (30)

PATENT CLAIMS CLAIMS 1. A method of inducing cell differentiation, comprising contacting cells capable of differentiation with a compound of formula I wherein: R-1 is alkyl or alkenyl having up to kh20 carbon atoms, R ₂ is hydroxy or alkoxy or hydrogen, R ₃ is hydrogen or lower alkyl, R ₄ represents COR 5, SO ₂ R, or CSR5, where alkyl, alkenyl or alkynyl group having 1 to 20 carbon atoms in _R5, which are optionally substituted by one or more OH, SH, ORg, SRG, NRyRg, COOR or CONR ₁₀ R g They substituted - wherein the Rg, R7, Rg, Rg and _R10 are independently hydrogen or alkyl of a maximum of 10 carbon atoms, aryl, alkaryl, and arylalkyl. A variety of the above compound is used that is effective in inducing differentiation of a given cell. The process according to claim 2, wherein R- | and the number of carbon atoms of R ₄ taken together is approx. 10-28. A process according to claim 1, wherein R 1 is alkyl of 1-20 carbon atoms or alkyl or alkenyl of 1-12 carbon atoms. The process of claim 2, wherein the number of carbon atoms of R ₁ and R ₄ taken together is approx. 12-26. The process of claim 2, wherein R 1 is and the number of carbon atoms of R ₄ taken together is approx. 14-24. • · · The process of claim 1, wherein R f is C 15 alkenyl, R ₂ is hydroxy-purified, R ₃ is hydrogen, R ₄ is COR 5 and R 5 is methyl. 7. The process of claim 1, wherein R 1 is is C 15 alkenyl, R ₂ is hydroxy, R ₃ is hydrogen, R ₄ is COR 5 and R 5 is pentanyl. 8. A method of Claim 1, wherein R f is an alkyl group having 8 carbon atoms, _R2 is hydroxy, _R3 is hydrogen, _R4 is COR5 and R5 is heptanyl. The process of claim 1, wherein R ₁ is C 15 alkenyl, R ₂ is hydroxy, R ₃ is hydrogen, R ₄ is COR 5 and R ₅ is C 17 alkyl. 10. The process of claim 1, wherein R; represents an alkyl group having 15 carbon atoms, _R2 is methoxy, _R3 is hydrogen, _R4 is COR5 and R5 is methyl. 11. The method of claim 1, wherein said cell is a leukemic lymphocyte. 12. A pharmaceutical composition for inducing cellular differentiation comprising a pharmaceutically acceptable carrier and a compound of formula I wherein R 1 has a maximum value of approx. Alkyl having 20 carbon atoms Alkenyl sweep containing 1 to 12 carbon atoms, R ₂ is hydroxy or alkoxy radical, or hydrogen, R ₃ is hydrogen or lower alkyl, R ₄ is COR 5, SO ₂ R 5 or CSR 5 where R 5 is C 1-20 alkyl, alkenyl or alkynyl optionally substituted with one or more OH, SH, ORg, SRg, NRyRg, COORg or CONR ₁₀ substituted with R ₈ - wherein each of R ₈ , R 7, R 8, R 8 and R ₁₀ is independently hydrogen, or alkyl, aryl, alkaryl, and arylalkyl having up to 10 carbon atoms and wherein the total number of R 1 and R 4 carbon atoms is about . 10-28. 13. The composition of claim 12, wherein the number of carbon atoms of R1 and R4 taken together is about. 10-28. 14. A13. A composition according to claim 1, wherein the number of carbon atoms of Rf and R4 taken together is about. 12-26. 15. The composition of claim 13, wherein the total number of carbon atoms of R1 and R4 is in the range of about 1 to about 10. 14-24. 16. A composition according A12.igénypont, wherein Rf is an alkyl group having 15 carbon atoms, _R2 is hydroxy, R3 is hydrogen, R4 is COR5 and R5 is methyl. 17. The composition of claim 12, wherein R ₁ is C 15 alkenyl, R ₂ is hydroxy, R ₃ is hydrogen, R 4 is COR 5 and R 5 is pentanyl. 18. A composition according to claim 12, wherein R f is C 8 alkenyl, R ₂ is hydroxy, R 3 is hydrogen, R 4 is COR and R 5 is heptanyl. 19. The composition of claim 12, wherein R 1 is C 15 alkenyl, R ₂ is hydroxy, R ₃ is hydrogen, R 4 is COR ₅ and R ₅ is C 17 alkyl. 20. A composition according to claim 12, characterized in that R f is an alkyl group having 15 carbon atoms, _R2 is methoxy, _R3 is hydrogen, _R4 is COR5 and R5 is methyl. 21. A process for the preparation of a pharmaceutical composition for inducing cell differentiation, comprising the step of: providing a compound of formula I wherein: R-ι has a maximum value of approx. Alkyl having 20 carbon atoms Alkenyl containing 1 to 12 carbon atoms, R ₂ is hydroxy or alkoxy or hydrogen, R3 is hydrogen or lower alkyl, R ₄ is COR ₅ , SO ₂ R ₅ or CSR ₅ , where R ₅ is a maximum of approx. Alkyl of 20 carbon atoms, alkenyl or alkynyl groups optionally substituted with one or more OH, SH, ORQ, SRQ, NRyRg, COOR and CONRfoRg group - the formulas, Rg, R7, Rs, Rg and _R10 are each independently hydrogen, or alkyl, aryl, alkaryl and arylalkyl containing up to 10 carbon atoms and wherein the total number of carbon atoms of R ₁ and R ₄ is about 1 to about ₁₀ carbon atoms. 10-28 - formulated in a pharmaceutical composition in admixture with excipients and / or carriers conventionally used in the preparation of pharmaceutical compositions. 22. The method of claim 21, wherein the therapeutically effective amount of the compound is a therapeutically effective amount. 23. The process of claim 21, wherein the total number of carbon atoms of R ₁ and R ₄ is about 1 to about 10. 10-28. 24. The process of claim 23, wherein the total number of carbon atoms of R ₁ and R ₄ is about. 12-26. 25. The method of claim 23, wherein the number of carbon atoms in R ₁ and R 4 together with approx. 14-24. The process of claim 21, wherein R ₁ is C 15 alkenyl, R ₂ is hydroxy, R ₃ is hydrogen, R 4 is COR 5 and R 8 is methyl. The process of claim 21, wherein R f is C 15 alkenyl, R ₂ is hydroxy, R ₃ is hydrogen, R 4 is COR 5 and R 5 is pentanyl. The process of claim 21, wherein R 1 is C 8 alkenyl, R ₂ is hydroxy, R ₃ is hydrogen, R 4 is COR 5 and R 5 is heptanyl. 29. The process of claim 21, wherein R 1 is C 15 alkenyl, R ₂ is hydroxy, R ₃ is hydrogen, R 4 is COR 5 and R 5 is C 17 alkyl. 30. The method of claim 21, characterized in that R represents an alkyl group having 15 carbon atoms, _R2 is methoxy, _R3 is hydrogen, R4 is COR5 and R5 is methyl. Id oIcIclLv ΙΈ oldocL ·