CA1307466C - Inhibition of protein kinase c by long-chain bases - Google Patents

Abstract

Inhibition of Protein Kinase C by Long-Chain Bases Abstract Compositions for inhibiting protein kinase C, comprising an inhibitory amount of a compound having the formula:

wherein Q is a hydrophobic group;

wherein X is -CH2-CH2- Or -CH=CH-, which may be substituted by one or more halogens or C1-C3 alkyl groups, wherein Y is , , , -CH2-, or , wherein W is a halogen;

wherein R1 and R2 may be the same or different and are selected from hydrogen, lower alkyl groups having from 1 to 7 carbon atoms, aralkyl, and aryl groups, and wherein Z is a phosphate or an organic group, and a pharmaceutically acceptable carrier material; and a method for inhibiting protein kinase C using such compositions.

Description

1 30746h , . .
~Description ?`Inhibition of Protein Kinase C by Long-chain Bases ;1 Technical Field The present invention relates to a method for inhibiting the enzyme protein kinase C by amphipathic long-chain bases such as sphingosine and sphinganine.
The invention further relates to compositions capable of producing inhibition of protein kinase C.
Inhibition of protein kinase C is useful in a variety of ways. Such inhibition can lead to inhibitio~ of the oxidative burst in neutrophils, whereby an anti-inflammatory effect is achieved. Inhibition of protein kinase C can also lead to inhibition of differentiation and growth of cells and can thereby produce an anti-tumor effect.

Background Art .~ .~'-.
Phospholipid/Ca+2-dependent protein kinase (protein kinase C) is a major protein phosphorylation ~;system which was first found ln brain (Takai et al, J.
Biol. Chem. 252, 3692, 1977) and subsequently shown to occur widely in tissues and phyla of the animal kingdom (Kuo et al, Proc. Natl. Acad. Sci. U.S.A. 77, 7039, . ~
1980). Protein kinase C plays an important role in signal transduction, cellular differentiation, tumor promotion and neurotransmission. The central unction of protein kinase C in transducing intracellularly extracellular signals has recently been recognized (Nishizuka, Nature 308, 693, 1984). Extracellular agents including neueotransmitters, hormones, and ,~ ,.~
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growth factors are bound by specific cell surface receptors and elicit, by transmembrane signalling, the generation of two second messengers by stimulating the degradation of phosphatidylinositols (Nishizuka, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 302, 101, 1983; Michel, Trends siochem. Sci. 4, 128, 1979; and -Berridge, siochem. J. 220, 345, 1984). In particular, the inositol phospholipid phosphatidylinositol-4-5-bisphosphate (PIP2) is hydrolyzed to inositol triphosphate (IP3) and sn-1,2-diacylglycerol (DAG) (Nishizuka, Science, 225, 1365, 1984). DAG activates protein kinase C. ~horbol-diesters, which are potent tumor promoters, also activate protein kinase C
(Castagna et al, J. 8iol. Chem. 257, 7847, 1982). In fact, it appears that protein kinase C is the phorbol-diester receptor (Niedel et al, Proc. Natl. Acad. Sci.
U.S.A. 80, 36, 1983) and mediates most, if not all, o the biological effects of the phorbol-diesters.
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Recentl~, protein kinase C has been shown to be inhibited by a lipoidal amine, 4-aminomethyl-1-[2,3(di-n-decyloxy)n-propyl]-4-phenylpiperidine dihydrochloride (CP-46, 665-1) in human leukemic cells (Shoji et al, Biochem. B_ophys. Research Comm., 590, 1985). This amine has also been shown to have antimetastatic properties in rodent tumor models (Wolff et al, Cancer Immunol. Immunother., 12, 97, 1982). This study supports the connection between inhibition of proteih -kinase C and anti-cancer activity, such as in human chronic myelogenous leukemia and other similar types of cancer.
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Dyson and Montano have reported an anti-tumor -agent which is a sphingosine derivative (J. Am. Chem.
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Soc., 100, 7441, 1978), but protein kinase C was not implicated.

Several other types of inhibitors of protein kinase C have also been reported. These include calmodulin antagonists (Mori et al, J. Biol. Chem. 255 8378, 1980; Wise et al, J. Biol. Chem. 257, 8489, 1982;
and Robinson, J. Cell. Biol. 101 1052, 1985), H-7 (Hidaka, Biochemistry 23, 5036, 1984), adriamycin (Wise, J. Biol. Chem. 257, 8489, 1982);
alkyllysosphospholipid (Helfman, Cancer Res. 43, 2955, 1983), a non-steroidal`anti-iestrogen, tamoxifen (O'Brien, Cancer Res. 45, 2462, 1985), amiloride (Besterman, J. Biol. Chem. 260, 1155, 1985), verapamil (Mori et al, J. Biol. Chem. 255, 8378, 1980), bilirubin (Sano, Ped. Res. 19, 587, 1985), palmitoylcarnitine (Nakadate, Cancer Res. 46, 1589, 1986), gangliosides GMl GDla GDlb and GTlb (Kim, J. Neurosci. Res. 15, 159, 1986), and retinal (Patarroyo, Immunobiology (Stuttgart) 170, 305, 1985). These various inhibitors of protein kinase C have widely varying potencies, and some are specific whereas others are non-specific.

In order to fully appreciate the present invention relating to inhibition of protein kinase C, it is :
necessary to understand other related work relating to the activation of protein kinase C. Such activation has been associated with tumor growth, and it is also believed that oncogenes may somehow affect the degree of activation of protein kinase C.

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ACTIVATION O~ PROTEIN KINASE C
Y' -. The mechanism of protein kinase C activation by ~ DAG, Ca+2, and phospholipid is of interest because ;~ activation of this en~yme is fundamental to tumor promotion, cellular transpormation and to understanding ;l the inhibition by anti-tumor agents. Protein kinase C
purified from rat brain cytosol consists of a single 80 kDa polypeptide which can be cleaved with trypsin into a 51 kDa catalytic fragment which is fully active in the absence of phosphalipid, Ca2+ and DAG, and a 32 kDa fragment which is fully capable of high affinity [3H]phorbol-dibutyrate binding. Phosphatidylserine `~ (PS) is the most effective phospholipid in activating -protein kinase C when sonicated with DAG. DAG
(phorbol-diesters) greatly reduced the concentration of Ca2+ required for activation. Translocation of the -enzyme from the cytosol to the membranes upon activation may be an important event of signal transduction. Alternatively, the enzyme may always be ~:
on the membrane "primed" to transduce DAG signals. ;
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The present inventors developed mixed micellar ~-methods to investigate the specificity, stoichiometry and mechanism of protein kinase C regulation by PS, DAG
and Ca2+ (see Experimental section herein). When the lipid cofactors are dispersed into detergent micelles of Triton X-100 or ~- octylglucoside at low mole fractions, the number of molecules present per micelle -can be systematically varied. Such analysis led to the conclusion that a single molecule of DAG is sufficient for protein kinase C activation and that 4 molecules of PS are required. Monomeric protein kinase C is the active species. Studies by the inventors employed over 20 DAG analogues to establish a precise DAG binding . .' ; ~ : .
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~ ~5~ 1 307~66 ., site (Ganong et al, Proc. Natl. Acad. Sci. U.S.A., 83, 1184, 1986) The 3-hydroxyl group and both oxygen esters of sn--1,2-DAG are essential. The structure function and stoichiometry studies are in full accord with DAG second messenger functions. A model in which the 4-carboxyl groups of PS bind Ca2+, and create a surface with which protein kinase C binds ~ut is inactive (see Figure 1) was developed. DAG activation occurs by three bonds to the protein kinase C-4PS~Ca2+
complex. A direct bond from DAG/phorbol-diester to Ca2+ was suggested to account for the increased affinity of Ca2+ in the compiex. The mixed micellar methods and mechanistic model have also been useful in analyzing protein kinase C inhibitors.
DAG Second Messengers '.' Direct evidence for the surprising function of DAG
as intracellular second messengers in a variety of cells exists from studies employing cell permeable DAGs. The limited solubility of DAG containing long ., , .
chain fatty acids, like those produced in response to extracellular signals, makes their addition to cells difficult. However, shorter chain length molecules have sufficient solubility to enter cells and activate protein kinase C. l-Oleoyl-2-acetylglycerol (OAG) was the first DAG of this type described. Dioctanoyl-glycerol (diC8) proved most effective among a series of -. DAGs containing fatty acids 3-10 carbons in length in activating protein kinase C when added to cells. DiC8 completely disclosed [3H]phorbol-dibutyrate rom its receptor, protein kinase C, in HL60 cells whereas OAG
would not. These cell permeable DAG's mimicked .
biological responses of phorbol-diesters whereas diC8 analogues modified at the 3-hydroxyl position would not :
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-6- 1 307~66 ..

activate protein kinase C, displace [3H]phorboldibutyrate or elicit biological responses.
Cell permeable DAG's are valuable tools to investigate DAG second messenger functions and the function of protein kinase C.
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Oncogenes and Protein Kinase C Activation The field of oncogene research has progressed to the point where more than 40 distinct oncogenes of viral and cellular origin ~ave been identified and their structures determined. The vast majority of oncogenes encode for altered forms of normal cellular proteins; thus, many of the protooncogene products are ~-thought to participate normally in regulation of growth and differentiation. Clues on the unction of the oncogenes whose products exist in the cytoplasm have emerged from their structures. Several of the gene -products appear related to growth factors or other -~
eiements involved in transmembrane signalling. These oncogene products may function by altering the level of -~
critical second messenger molecules, such as diacylglycerol (DAG). The oncogene products of nuclear location may modulate transcriptional activity. The oncogenes can be grouped into a small number of functional classes suggesting that only a small number of mechanisms of action underlie the functions oE the encoded proteins. -~

Transmembrane signalling, the molecular basis by which growth factors, neurotransmitters, and hormones regulate a variety of cellular functions including proliferation and differentiation merged with the metabolism of membrane phospholipids when protein kinase C was discovered. As pointed out above, ' ~:

~ ~7~ 1 307466 diacylglycerol was shown to be the molecular link between receptor occupancy, phosphatidylinositol (PI) turnover and cellular response when it was shown to activate protein kinase C. Protein kinase C is the intracellular receptor of phorbol-diesters and other tumor promoters; tumor promoters activate protein kinase C by interacting at the DAG site. As shown in Figure 1, the mechanism of transmembrane signalling is now thought to involve receptor mediated turnover of PIP2 (or other PI's) coupled through a GTP (G protein) dependent activation of phospholipase C. This mechanism is analogous to that established for transmembrane signalling involving c-AMP formation. In the case of transmembrane signalling linked to PIP2' two second messengers are produced. IP3 is believed to function as a second messenger in the mobilization of intracellular calcium. The role of Ca+2 as a second messenger is well known.
Oncogene Products Elevate DAG Levels ....
Since several of the oncogene products resembled elements involved in transmembrane signalling, the idea that these elements perpetually function to activate transmembrane signalling and elevate critical second messengers arose. ~As shown in Figure 1, cells containing sls become transformed by autocrine stimulation by a PDGF like molecule; the erbB gene product is a stump of the EGF receptor (neu and fms gene products are also receptors) and ras may be a G
protein activating phospholipase C. The inventors developed a sensi~ive assay, 20 pmol to 25 nmol, for DAG second messengers. The assay employed E. coli DAG
kinase whose structural gene was cloned and sequenced, to quantitatively convert the sn-1,2-DAG present in ~:
., :., -8- 1 307~6h crude lipid extracts to [32P]phosphatidic acid. K-ras transformed NRK cells had 2.5 times as much DAG as nontransformed cells at 34 or 38. This increase in DAG was similar to that observed in platelets in response to thrombin and in hepatocytes in response to vasopressin. When a temperature sensitive k-ras mutant was employed, DAG levels were elevated at the permissive but not at the restrictive temperature. Sis transformed cells had elevated levels as well.
Fleishman et al (Science 231, 407, 1986) have also reported elevated levels of DAG in ras transformed cells. Altered DAG levels ~ay be the (a) molecular mechanism explaining how certain oncogenes function. -. . .
Description of the Invention The novel hypothesis that sphingolipid metabolites may function as negative effectors, second messengers, of protein kinase C emerged from a serendipitous observation made during studies of the specificity of DAG activation of protein kinase C. Ceramide, a building block of sphingolipids, was tested because it resembled DAG. Ceramide did not activate or inhibit protein kinase C. Surprisingly, sphingosine, a building block of ceramide, prov~d to be as potent an inhibitor of protein kinase C activation as DAG was as an activator. This was intriguing because sphingosine is a normal component of cells; detailed mechanistic analysis had to await development of the mixed-micellar methods of protein kinase C activation and phorbol binding. In brief, sphingosine inhibited [3H~phorboldibut~rate binding to protein kinase C ln vitro, in human platelets and neutrophils, and in HL60 cells. Inhibition occurs wi~hout displacement of protein kinase C from the membrane/micelle surface.
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1 307~66 Moreover, sphingosine inhibited cellular responses known to be stimulated by cell permeable DAGs of phorboldiesters. Sphingosine inhibited 40 kDa protein phosphorylation in platelets, blocked the PMA dependent differentiation of HL60 cells to macrophages and inhibited the oxidative burst of human neutrophils in response to a number of different agents.

Based on the above observations, the present inventors have discovered that a variety of amphipathic ; amines, such as metaboliteg of sphingolipids which are major components of the cell surface, function as negative effectors of protein kinase C. Naturally -occuring sphingolipid metabolites thus seem to comprise a novel set of 'rsecond messengers" with vital functions in regulation of cell growth, differentiation, and development. These negative lipid effectors appear to underlie the action of negative growth factors, : biological activities of the gangliosides including growth inhibition, contact inhibition, differentiation ~i3 and oncogenesis, immiunosuppression, the pathobiology of ~:~ the sphingolipidoses, the action of tumor necrosis factor, and the cytopathic effects of HTLV-3 on T4+
lymphocytes.

These observations have created and shaped a ~ unifying hypothesis. The global implications stemming ;~ from this observation are striking.
Sphingosine/lysosphingolipids may function as second messengers to inhibit protein kinase C. Protein kinase ~i C activity would, therefore, be a function of positive effectors (DAG and Ca2+) and negative effectors (sphingosine/lysosphingolipids). These negative ~ii effectors could establish a "set point" for protein kinase C activation; negative effectors may explain ~hy ~1 ~

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' kinase C is not active at resting cellular DAG
levels. Such second messengers could be produced in response to extracellular signals and could define, in part, the functional significance of the sphingolipids. The origin of this putative set of second messengers, membrane sphingolipids, would be analogous to the origin of DAG second messengers, membrane glycerolipids (see Figure 2).

activation Protein inhibition sphingosine/
DAG > Kinase C < - - lysosphingolipids cellular response This hypothesis stems from an attempt to integrate and extend knowledge of glycosphingolipids which are present on cell surfaces and within cells. The patterns of cell surface glycosphlngolipids change during cell differentiation and development and in ~ :
transformed cells. Glycosphingolipids have been implicated in the cell's density sensing mechanisms (contact inhibition) through specific interactions with cell surface proteins. Importantly, glycosphingolipids can inhibit responses to growth factors by interacting with receptors present in fibroblasts, T-cells line, and epidermoid cells. Glycosphingolipids are also known to modulate the activities of membrane bound enzymes. Thus, the hypothesis provides a molecular model relevant to understanding the recurrent immunological observation that glycosphingolipids comprise prominent tumor associated antigens. It is possible to piece together a metabolic cycle as shown in Figure 4.
The inventors believe that lysosphingolipids 1 30746~

represent the "missing molecular link" between sphingolipids, signal transduction, differentiation, development, and cellular transformation. This hypothesis has the potential to make sense at a molecular level out of~diverse areas of biochemistry, neurobiology, cell biology, immunology and tumor biology.

Specifically, sphingosine/lysosphingoglycolipid inhibition of protein kinase C appear to underlie the role of glycolipids in cellular transformation and the biological activities of gly'colipids when added to cells (i.e. inhibition of mitogenesis by PDGF and EGF ~-and immunosuppression by blocking IL-2 stimulation of T
cells). Furthee, negative growth factors, "chalones"
including transforming growth factor -~, fibroblast -growth regulator (FGR-s (13K) from 3T3 Cells) and others might work through sphingosine based negative effectors.

The concept of negative effectors is attractive for reciprocal regulation is the rule in biology.
These effectors could be the "signals" of contact inhibition. Glycolipids have been implicated in processes where cells become non-responsive to growth factors. Finally, it is possible that modulation of these effectors might underlie the dramatic effects of tumor necrosis factor, and the effects of the AIDS
virus, HTLV-III, on T4+ helper cells. Production of sphingosine signals could be highly cytotoxic.
Sphingosine is highly cytotoxic; 2-3 ~g/ml inhibits growth of CHO cells. Alteration of the level of these negative effectors by oncogene products seems to eesult ;
in developmental trapping and underlie promyelocytic :~
leukemias. ~

-,, ,:. ' -12- 1 3074h6 Protein kinase C inhibition might be involved in the pathobiology of the sphingolipidoses where glycolipids accumulate because of genetic defects of lysosomal enzymes of glycolipid catabolism. The inventors prepared deacylated (lyso-forms) of many of the s~hingolipids. All were found to inhibit protein kinase C (see Figure 4), whereas the parental sphingolipids did not. Conversion of the accumulated glycolipids to lysosphingolipids or sphingosine occurs in these disorders. The pathobiology which has remained a mystery is likely caused by these agents which interfere with signal transduction.

Detailed understanding of the mechanism and structure of protein kinase C is of utmost importance because this target is the site of tumor promoter action, and an essential element of signal transduction which responds to elevated levels of DAG second messengers produced by growth factors and certain oncogene products. Molecular understanding of this target is invaluable for drug design and/or discovery projects. Specifically, protein kinase C may be considered a target for anti-tumor agents. If DAG
signals are elevated by specific oncogenes, ras, sls, erbB, fms, etc., then inhibition of protein kinase C
should block the action of the oncogene product. The same logic extends to other enzymes of second messenger generation and signal attenuation.

Because protein kinase C has also been implicated in at least some neutrophil oxidative activation mechanisms, it has also been of interest to study the effect of protein kinase C inhibitors on these mechanisms. Compounds which inhibit protein kinase C
are not automatically capable of inhibiting human ., neutrophil activation. For example, the protein kinase C inhibitors H-7 and H-9 (Wright, Biochem. BiophYs.
Res. Commun. 135, 749, 1986) are incapable of inhibiting the respiratory burst in human neutrophils. Inhibition of the oxidative burst can reduce inflammation in affected tissue. Neutrophil oxidative metabolism also plays an important role in tumor generation (Schwarz, Carcinogenesis 5, 1663, 1984), so inhibition of the oxidative burst can inhibit tumor formation.

Disclosure of the Invention Accordingly, it is an object of the present invention to provide new and effective compositions for the inhibition of protein kinase C.

It is also an object of the present invention to provide a method of inhibitin~ protein kinase C which involves contacting this enzyme either ln vitro or in vivo with an inhibitory concentration of a compound or composition according to the present invention.

It is yet another object of the present invention to provide a method of treating inflammation which involves treating a mammal afflicted with inflammation with a protein kinase C inhibitory concentration of a protein kinase C inhibitor according to this invention. ~;

It is yet another object of the present invention to provide a method for inhibiting the oxidative burst in neutrophils which involves contacting neutrophils either in vitro or in vivo with a protein kinase C
inhibitory concentration of a compound capable of inhibiting this enzyme.

These and other objects as will hereinafter become more apparent have been accomplished by discovering that certain cornpounds, amphipathic amines such as notably sphingosine, sphinganine and related compounds, are capable of potently inhibiting protein kinase C
both in vitro and in vivo, and further that these compounds are capable of preventing inflammation by inhibiting the neutrophil oxidative burst activation mechanisms. The compounds according to the present invention have the following general structure:
Q-X-Y,CH-~H2-0-z I
NRlR2 wherein Q is a hydrophobic group;

wherein X is -CH2-CH2- or -CH=CH-, which may be substituted by one or more halogens or Cl-C3 alkyl groups, wherein Y is -CH-, -C-, -CH2-, -CH-, or -CH-wherein W is a halogen;

wherein R1 and R2 may be the same or different and -are selected from hydrogen and lower alkyl groups having from 1 to 7 carbon atoms, aryl groups having from 6 to 12 carbon atoms, and aralkyl groups having ;
from 6 to 15 carbon atoms, and wherein Z is any organic group or a phosphate, and :.
is not necessarily furtiler limited. More specifically, -15- l 307466 Z may be any alkyl or aralkyl group, which is cyclic, branched or straight chain, and which may be substituted by conventional pharmaceutically acceptable substituents such as halogens, nitro groups, hydroxyl groups, etc.; nucleotides or nuc]eosides, polynucleotides or polynucleosides, amino acids, peptides, saccharides, polysaccharides, acetyl groups, etc. When Z is an alkyl or aralkyl group, it may preferably have from 1 to 12 carbon atoms, more preferably from l to lO carbon atoms, and most preferably 3 to 8 carbon a~oms. Z may preferably be selected from the group consisting of H, glycyl, arginyl, lysyl, galactosyl, sulfogalactosyl, glucosyl, lactosyl, trihexosyl, phosphorylcholine, GalNAc-Gal-Glc, Gal-Gal-Glc, Sia-Gal-Glc, Gal-GalNAc and GalNAc Sia-Gal-Glc- Sia-Gal-Glc.

By GalNAc is meant N-acetyl galactosamine; by Glc is meant glucose, by Gal is meant galactose, and by Sia is meant sialic acid. By trihexosyl is meant polysaccharides composed of three hexoses such as galactose~ glucose, mannose, gulose, etc. Both D and L
isomers are contemplated.
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Q may be any hydrophobic group, such as a long chain alkyl group having from 2 to 30 carbon atoms, I which may be singly or multiply unsaturated, straight ! chain, branched, or may contain cycloalkyl groups.
'.. ': ' ¦ The compounds of the invention can also be used in ¦ the orm of pharmaceutically acceptable salts, such as acid addition salts formed from inorganic or organic ~, .

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acids. Included among such salts are the following:
acetate, adipater alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulEonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methansulfonate, 2-naphthalanesulfonate, nicotinate, oxalate, palmitate, pectinate, persulfate, 3-phenylpropionate, picraté, pivalate, propionate, succinate, sulfate, tartraté, thiocyanate, tosylate, and undecanoate. Of these salts, simple inorganic salts, such as salts of the hydrogen halides are preferred. The compounds of the general formula which are not salts are particularly preferred.

8rief Description of the Drawings FIGURE 1: Mechanism of transmembrane signalling and protein kinase C activation by DAG second messengers. The figure illustrates oncogene products which resemble elements of transmembrane signalling.

FIGURE 2: Mechanism of transmembrane signalling and protein kinase C inhibition by sphingosine/
lysosphingolipids putative second messengers. The figure depicts a model for the generation of sphingosine/lysosphingolipids and their inhibition of protein kinase C.

FIGURE 3: Positive and negative effectors of Protein kinase C. An overall view of the complex metabolism of glycerolipids producing diacylglycerol "second messengers" and sphingosine "negative protein kinase C eE~ectors" is shown. PIP's stand for the phosphatidylinositol phosphates. The inventors recognize that numerous routes could underlie the arrows and that more than one enzyme may lie under a given arro~. The question of whether sphingosine arises directly from palmitoyl-CoA and serine or via dihydroceramide is not illustrated. Sphingomyelin synthesis occurs by the reaction, phosphatidylcholine +
ceramide --> sphingomyelin ~ DAG. Evidence that this enzyme resides in the plasma membrane provides another potential route for DAG for~ation. The figure also points out how the negative effector (and positive effector) functions of sphingosine may be involved in biochemistry, cell biology and pathology.
Consideration of sphingosine providing a functional regulatory link for sphingolipids should not be -ignored.
' ' .:-' FIGURE 4: Lysosphingolipids/sphingosine inhibition of Protein Kinase C. A. This figure illustrates the relevance of lysosphingolipids/
sphingosine inhibition of protein kinase C/signal transduction to the sphingolipidosis.

FIGURE 5: Effect of Sphinganine on HL-60 Cell :
Growth. Sphinganine at varying concentrations was added to 1 x 105 cells and incubated for the times shown. The closed circles are viable cells (all groups had >90~ viability, except where otherwise indicated) and the open circles reflect both viable and non-viable cells.

FIGURE 6: Competition of Sphinganine for Phorbol Dibutyrate-Binding by H~-60 Cells. Sphinganine at the indicated cncentrations or fatty acid-free bovine serum ~ :
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-18- l 307~6 albumin at 20 nM was added to the cells with 12 nM
[3H]phorbol dibutyrate and binding (including correction for nonspecific binding] was determined as described in the Experimental section.

FIGURE 7: Effect of Different Long-chain Bases on HL-60 Cell Attachment. The different compounds were added to the cells with 8 nM PMA and the % attachment determined as described in the Experimental section.
The compounds used were sphingosine (So), sphinganine (Sa), stearylamine (St), octylamine (Oct), and ceramide (similar res~lts were obtained with bovine brain ceramides and N-palmitoylsphinganine).

FIGURE 8: Kinetics of Sphinganine Uptake bX HL-60 cells. Approximately l x 106 cells were suspended in medium containing 1 ~M [3-3~I]sphinganine and after different intervals of incubation at 37 aliquots were removed for measurement of the radiolabel in the medium ~
and associated with the cells. ;

FIGURE 9: Levels of Different [3H]sphingolipids in HL-60 Cells at Various Time-points. The liqids from the experiment described in Figure 8 were separated by thin-layer chromatography and visualized by fluorography. The radiolabel was coincident with ceramide, sphinganine, and polar sphingolipid standards and was quantitated by liquid scintillation counting.

FIGURE 10: Inhibition of Protein Kinase C
Activity by Sphingosine. Mixed micelles were formed with 3% (w/v) Triton X 100 containing PS at 6 mol6, diCl8 1 at 2 mol6 and sphingosine at 10 fold the indicated concentrations. The mixed micelles were then diluted 1:10 into the assay mixture. 1 mol6 of * Trademark , .
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sphingosine is equivalent to 43 ~M . Effect of sphingosine on protein kinase C (~) and on protein kinase M (0). Identical results were obtained with protein kinase M when sphingosine was added in 0.3%
Triton X-100 solution without PS and diC18 1.

FIGURE 11: Effect of Mixed Micelle Concentration on Potency of Sphingosine Inhibition. A. Mixed micelles were formed with Triton X-100 at 12% (w/v, (O) 6% (~), 3% (~) and 1.5% ( ~ ) containing 7 mol% PS, 1 mol% diC18 1' and sphingosine at 10 fold the indicated concentrations. The mixed micelles were then diluted 1:10 into the assay mixture. B. Mixed micelles were formed with 3% Triton X-100, 7 mol% PS, 1 mol% diC18 1' and 2-5 mol% sphingosine. These were then diluted 1:20 ( ~ ) 1:10 (~), 1:5 (~), and 2:5 ( ~ ) into the assay mixture. In these experiments, 1 mol% sphingosine corresponds to 21.5 ~M ; 43 ~M ; 86 ~M ; and 172 -~M respectively.
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FIGURE 12: Interaction of Sphingosine with Dioleoylglycerol. Mixed micelles were formed at 3% -Triton X-100, 6 mol% PS, 0-10 mol% diC18 1' and sphingosine at 0 mol% (~), 2 mol% (~), 3 mol% ( ~ ), and 4 mol% (~ ). A. Protein kinase C activity assayed in presence of ~0 ~M CaC12. B. Double reciprocal plots with diC18 1 concentration in mol%.

FIGUR~ 13: Interaction of Sphingosine with Phorbol Dibutyrate. Mixed micelles were formed with 10 mol~ PS and 0 molO (a), 6 mol~ (~), and 8 mol% ( ~ ) sphingosine. PDBu was added as an aqueous solution.
A. Protein kinase C activity assayed in presence of ;
10 ~M CaC12. B. Double reciprocal plots of data in A. -~' : . -:
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-20- 1 307~66 FIGURE 14: Potency o~ Sphingosine Inhibition as a Function of Phosphatidylserine. Mixed micelles contained 2 mol% diC18 1 and 5 mol~ (C~ ), 6 mol-O (~), and 7 mol% (~) PS, and variable mold of sphingosine.

FIGURE 15: Interaction of Sphingosine with Phosphatidylserine. Mixed micelles were formed with diC18 1 at 1 mol% and sphingosine at 0 mol-O (~), 2 mol-O
(~), and 4 mol-O ( ~ ), activity was measured in presence of 50 ~M CaC12.

FIGURE 16: Interaction of Sphingosine.with Ca2 .
Mixed micelles contained 6 mol-O PS, 1 mol% diC18 1 and O molO (~), 3 MolO ~) and 4 mol-O (O ) sphingosine.
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FIGURE 17: Sphin~osine Inhibition of Phorbol Dibutyrate Binding. Mixed micelles contained PS at 16 mol~ ( a, and 20 mol~ (a ); or PA at 20 mol~ (~) and 0-20 mol% of sphingosine. Binding studies were performed as described in the Experimental section. The data are plotted as -O of maximal binding under each condition.
With 16 mol% PS there was 82~ of the [3H]PDBu binding seen with 20 mol% PS. At 20 mol% PA there was ony 51%
of the binding seen with 20 mol% PS.
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FIGURE 18: Sphingosine Inhibition of Platelet Protein Phosphorylation. A. The effect of increasing sphingosine concentration on phosphorylation of the 40kDa protein induced by thrombin. Lane 1: control platelets; Lane 2: thrombin (1 unit/ml); Lane 3:
10 ~M sphingosine; Lane 4: 25 ~M sphingosine; Lane 5:
50 ~M ; Lane 6:100 ~M ; Lane 7: 200 ~M . B. The 40k band was cut out and 32P was counted in Aquasol II.
The -0 of maximal phosphorylation induced by thrombin is plotted as a function of sphingosine concentration.
' l -21- 1 307466 .

FIGURE 19: Sphingosine Inhibition of PDBu binding `;i to Platelets. [3H]PDBu was at 25 nM and platelets at `` 2.5x108 platelet/ml.

FIGURE 20: Effects of Stearylamine and Octylamine on Protein Kinase C. Mixed micelles contained 5.5 mol%
PS, 1.0 mol% diC18 1' and sphingosine ( a,, octylamine or stearylamine (a).
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FIGURE 21: Structure of Lysosphingolipids.
Lysosphingolipids are derivatives of the long-chain base sphingosine where the l-hydroxyl is substituted by different head groups. With the exception of lysosphingomyelin (sphingosylphosphorylcholine) where the linkage is through a phosphodiesteric bond, lysosphingolipids have a glycosidic bond at C-l.

FIG~RE 22: Inhibition of Protein Kinase C by Lysosphingoli~lds. A. Psychosine (~), sulfogalactosylsphingosine (O), and lyso-GM2 (~) were potent inhibitors of protein kinase C when activity was measured at 6 mol~ phosphatidylserine, 2 mol ~ sn-1,2-dioleoylglycerol, and 100 ~M Ca2+. These data are representative of the inhibition observed with the rest of the related lysosphingolipids. The parental sphingolipids were also evaluated for their effects on protein kinase C activity. They were dried down from chloroform/methanol solutions with the lipid cofactors of protein kinase C and solubilized in Triton X-100.
None of the parent compounds inhibited protein kinase C, as shown for cerebrosides, sphingomyelin, and GM2.
To further investigate the specificity of inhibition of protein kinase C by lysosphingolipids, the N-acetyl derivatives of galactosylsphingosine, sphingosylphosphorylcholine, and lyso GM2 were ~ ~ .
. .......

-22- 1 307~66 prepared as described by Gaver and Sweeley for the N-acetyl derivative of sphingosine. The compounds were purified on a CN HPLC column and then tested for their effects on protein kinase C activity. None of these N-acetyl derivatives inhibited protein kinase C. ~. The lysosphingolipids displayed surface dilution kinetics ( the data shown for galactosylsphingosine is representative of the group). Galactosylsphingosine was less potent when protein kinase C was assayed at higher concentrations of Triton X-100 mixed micelles containing ~ixed amounts of phosphatidylserine (6 mol~) and dioleoylglycerol (2 mol %). When the data are plotted as mol~ psychosine:Triton X-100 (inset), the inhibition profiles at the different concentrations of Triton X-100 (0;15% w/v,~ O.3%, ~ , O.6%, ~ , and 1.8%, ~ ) appeared identical. These results indicate that the lysosphingolipids preferentially partition into the mixed micelles; therefore, ffective concentrations should be expressed as mol~ of the lipid compounds of Triton X-100 and not as bulk concentrations.

FIGURE 23: Inhibitlon of 40 kDa phosphorylation in Platelets. The phosphorylation of 40 kDa polypetpide in human platelets in response to thrombin -was carried out. Lysosphingolipids inhibited the phosphorylation of this polypeptide which is known to ~-~
be effected by protein kinase C. Lane l; unstimulated platelets, lanes 2-15; thrombin 1 ~m/ml, lanes 3-7;
galactosylsphingosine 5, 10, 25, 50 and 100 ~M , lanes 8-11; lysosulfatide 5, 15, 50, and 100 ~M , lanes 12-15; lysoGM2 25, 50, 100, and 200 ~M .
Galactosylsphingosine (25 uM ) did not affect the generation of diacylglycerol second messengers in response to thrombin. Also, the N-acetyl derivatives -23- 1 307~66 of galactosylsphingosine and sphingosylphosphorylcholine did not affect 40 kDa phosphorylation when tested at concentrations less than 100 ~M, indicating that the inhibition by lysosphingolipids was specific and not due to non-specific detergent actions.

FIGURE 24: Effect of Sphinganine on the Oxidative Burst. Panel A shows tracings of oxygraph recordings. Tracing 1 shows the effect of 1 ~M PMA on oxygen consumption. In Tracing 2 cells were preincubated for 3 minutes with sphinganine (50 lJM ?
prior to addition of PMA. Tracing 3 shows the addition of 40 ~M sphinganine after activation with PMA. Panel B shows recordings of reduction of cytochrome c -(monitored at 550 nm). Tracing 1 shows PMA (1 ~M ) induction of cytochrome c reduction. In control experiments the cytochrome reduction was shown to be -completely inhibited by superoxide dismutase. Tracing 2 shows effects of preincubation of cells with 10 ~M sphinganine prior to addition of PMA.
`:
FIGURE 25: Concentration Dependence for Inhibition of Oxidative Events by Sphinganine with Several Types of Activators. Panel Ao Cells (7.5 x 106 cells/ml) were preincubated with the indicated concentration of sphinganine for 3 min. prior to activation with 100 ~M dioctanoyl glycerol (open `
squares), PMA (filled squares), or opsonized zymosan (open triangles). Oxygen consumption was measured as described in Experimental section using a Clark oxygen electrode. Panel 8. Cells (6.5 x 105 cells/ml) were incubated 3 minutes with the indicated concentrations of sphinganine prior to activation with PMA (open squares), FMLP (open triangles), or arachidonate :
.
,.

(filled triangles). Superoxide production was monitored as in Figure 24.

FIGURE 26: Dose Dependence for Inhibition of O~ygen Consumption b~_Structural Analogs of Spinganine. Cells (7.5 x 106 cells/ml) were incubated wiht sphinganine (open squares), stearylamine tfilled squares), or octylamine (open triangles) at the indicated concentration' prior to activation with 1 ~M phorbol myristate acetate. Oxy~en consumption was measured using a Clark oxygen electrode.

FIGURE 27: Inhibition by Sphin~anine of Cellular Phorbol Dibutyrate Binding. Cells (1 x 106 cells/ml) were preincubated with the indicated concentrations of sphinganine for five minutes prior to addition of 50 nM
(final concentration) of 3[H]-phorbol dibutyrate. The cells were then incubated for 15 minutes in a shaking water bath, 37C. The incubation mixtures were filtered with ice cold PBS-glucose. The filters were then counted to quantitate bound phorbol dibutyrate.
100~ represents 47,000 cpm.

Best Mode for CarrYing Out the Invention Protein kinase C according to the present invention includes the enzyme discussed by Nishizuka Science, 233, 305, 1986. Functional equivalents of this enzyme (i.e., homologous proteins which have essentially the same activity as protein kinase C) which are known or are discovered are also included under the meaning of protein kinase C. For example, isozymes of protein kinase C, three of which are known, are included in the meaning of protein kinase C.

. ~ ,.

-25- 1 307~66 A compound is an inhibitor of protein kinase C if it is capable oE blocking cell adherence or preventing phorbol ester-dependent differentiation of human promyelocytic leukemic (HL-60) cells in vitro at concentrations up to approximately 10 mmolar. The precise method by which inhibition was determined for sphinganine, sphingosine and related compounds is i described ln detail in the experimental section herein. For example, using the assays described in the experimental section, inhibitors which decrease `
adherence by 50% at a~concentration on the order of 10 millimolar or lo~er are preferred. More preferred are compounds exhibiting such an effect at concentrations ~ on the order of up to about 1 mmolar. Particularly ¦ preferred are those compounds exerting such activity at - -concentrations up to 100 ~molar . This invention is not limited, however, by the particular methods of measuring inhibition of protein kinase C described herein. One of skill in the art may formulate other possible assay methods for determining whether a compound can inhibit protein kinase C. In general, in assays in which a Ki (inhibition constant) is determined for a particular compound with protein kinase C, a Ki lower than the millimolar range is desired, preferably a Ki in the micromolar range or submicromolar range (e.g., the nanomolar range).
, ' .

The critical structural features of sphingosine and related compounds required for inhibition of protein kinase C are the amine moiety and a hydrophobic character. Substitution at the l-hydroxyl group does not abolish inhibition. Accordingly, lysosphingolipids l containing a variety of l-hydroxyl substituents have i also been found to be inhibitors of protein kinase C.
In the lysosphingolipids, the preferred head groups at ..~', ,-, ..

-26- 1 3 07 ~ 66 the 1-hydroxyl position are phosphorylcholine, amino acids (such as glycine, arginine, lysine, alanine, serine, etc.) glucose, galactose, lactose, sulfogalactose, trihexoses, and other more eomplex sugar residues with sialic acid substituents that oeeur in the gangliosides. The 3-keto and threo- isomer of sphingosine, and sphinganine (no double bond) will inhibit protein kinase C. Further, the lysoderivatives of all sphingolipids tested whieh are modified at 1-hydroxyl are active. Stearylamine is active and has been reported to be eytotoxie.

The speeificity of protein kinase C inhibition by sphingosine was investigated (see the Experimental section). As a result, it was diseovered that swainsonine, a moleeule strueturally related to sphingosine (Colegate, Am. J. Chem. 32, 2257, 2979) was not an inhibitor. Moreover, the following eompounds were also diseovered not to be inhibitors of protein kinase C: N-acetylsphingosine, ceramide, 1,3-dihydroxy-2-amino-3-phenylpropane~ fatty aeids, and eetyl triethylammonium bromide. On the other hand, 3-ketosphinganine, erythro- and threo- sphinganine were inhibitors.

In the general formula, Q may be any hydrophobie group. So long as Q is a hydrophobic group, it ls ineonsequential what group it is as long as Q does not interfere with inhibition. For example, Q may be an alkyl group eontaining from 2 to 30 carbon atoms, whieh may eontain one or more (preferably 1 to 5) double bonds, and may be straight chain or branched and may contain substituent groups (e.g. halogen such as Cl, Br, F; or aryl such as phenyl).

:~
.~ .' .. ..

, Preferred Q's are:
CH3-(CH2)n n = 1 - 29 and CH3-(CH2)m-CH=cH (CH2)p p = 1 - 15 . .:
In the above formulas, n is preferably 2-20, more preferably 10-16, and most preferably 11-13; m and p are each preferably 2-10.
- `:. '"
~ X in the general formula is preferably -CH2-CH- or ¦ -CH = CH-.
3 ` ` ~ -~ Y in the general formula is preferably -CH- or -C, . I 11 OH O
and more preferably -CH-.

OH
:. : .

Rl and R2 are both preferably hydrogen atoms, but may also be Cl-C7 alkyl groups, such as methyl, ethyl, propyl, butyl, etc., or may be aryl, such as phenyl, ~-methoxy-phenyl, _-chloro-phenyl; or aralkyl~ such as benzyl, phenethyl, etc. Aryl and aralkyl preferably have 5 to 10 carbon atoms.

The compounds falling within the scope of this invention may be naturally occurring or synthetically produced. Preferred naturally occurring compounds are sphingosine, sphinganine and lysosphingolipids. ~
. ~
The structural requirements for inhibition of the oxidative burst of neutrophils includes a hydrophobic `
group such as a long aliphatic chain and an ~`~
aminocontaining head group. There is modest ...

specificity for native (erythro) isomer of sphinganine.
.--The sphingolipids such as sphingosine and sphinganine are well known compounds and are available commercially or are readily synthesized by known methods (see Experimental section).

The ways in which the above compounds have been tested to show their ability to inhibit protein kinase C, and other features relative to the mode of inhibition of these co~mpounds are described in the Experimental Examples.

The compounds of the present invention and their pharmaceutically active salts, may be formulated into pharmaceutical compositions which may be used to treat mammals such as man, which are afflicted with various conditions in which the activity of protein kinase C is accelerated, or conditions which are treatable by inhibiting protein kinase C. For example, the present compounds can be used to treat asthma, inflammation, psoriasis or tumor metastases.

The compositions of the present invention may be administered in any mode such as orally, parenterally, 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 will be a sterile liquid. As an injection medium, it is preferred to use water which contains the stabilizing agents, solubilizing agents and/or buffers , . ~ ' ' ~ -29-conventional in the case oE injection solutions.
Desirable additives include, or example, tartrate and borate buffers, ethanol, dimethylsulfoxide, complex forming agents (for example, ethylenediaminetetraacetic acid), high molecular weight polymers (for example liquid polyethylene oxide) for viscosity regulation or polyethylene derivatives of sorbitan anhydrides.

The total routine (e.g., daily, weekly, monthly, etc.) dose of the compounds according to the present invention will be that; sufficient to result in an ln vivo protein kinase C inhibitory concentration. The inhibitory concentration will generally comprise a range, the mid-point of which is the experimentally measured concentration for 50~ inhibition of e.g., PMA-induced attachment or cell growth of the compound, and the limits of the range being concentrations which are a factor of 10 above and below the 50~ inhibitory concentration. For example, if the experimentally measured inhibitory concentration is 5 ~molar, the desired inhibitory concentration to be achieved will be approximately 5 to 500 ~molar. One of skill in the art can readily ascertain the optimum dosage to use for a particular case, using as a starting point the range delineated above.
:' When a composition for the treatment of a disease is formulated, a compound or a physiologically acceptable salt of a compound according to this invention or a mixture thereof is 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 :

~30- 1 307466 :'.
tablets and capsules are binders such as tragacanth gum, gum arabic, corn starch and gelatin; excipients such as microcrystalline cellulose, swelling agents such as corn starch, pre-gelatinized starch and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose and aspartame; and flavorings such as peppermint. Other additives include edible oil as a liquid carrier (in capsules); and shellac, sugar and a combination thereof (tablet coating).
.~
Parenteral inject1on may employ, as a vehicle to dissolve or suspend the active ingredient, water, 3 natural vegetable oils such as sesame oil, coconut oil, peanut oil and cottonseed oil, and synthetic oils such ethyl oleate, and may also contain buffering agents, preservatives and anti-oxidants as required.

The present compounds are also capable of inhibiting the neutrophil oxidative burst. This effect is believed to be due to inhibition of protein kinase C
mediated processes involved in the activation of neutrophils. The concentrations and modes of administratibn may be any of those mentioned above. To reduce inflammation, a topical route (e.g. cream or ~1 lotion) is generally desirable, formulated to achieve an in vivo concentration of from about 0.1 to 1000 ~m, preferably 1 to 100 ~m of the inhibitory compound.
.. ~
`,' .
~, The method of this invention may be carried out by directly contacting an inhibitory compound or I composition according to the invention with protein ,~ kinase C. However, it is also possible, and within the scope of the invention, to carry out the above method indirectly, e.g., by administering a compound or composition which has an in vivo activity of inducing ~1 .
~' .
,~ , ........ ..

~ -31-production of one of the inhibitory compounds of this invention and thereby causing inhibition of protein kinase C. Precursors of sphingosine or sphinganine which are converted into sphingosine (see Figure 3) or sphinganine in vivo may be administered to livihg cells to result in protein kinase C inhibition.
:
Further, pro-drug precursors which are converted in vivo into a protein kinase C inhibitor are within the scope of this invention. For example, compounds of this invention having àn N-lower acyl (Cl to C8) group can be cleaved i~ vivo by enzymes such as amidases or esterases to produce inhibiting compounds according to this invention. The nitrogen may be acylated with acetyl, propionyi, etc., or with an amino acid (i.e., one oE the 20 naturally occurring amino acids such as glycine, lysine, etc.) or a peptide, which can be cleaved in vivo. Other precursors produced by analogy to conventional pro-drug methodology are also within the scope of this invention. Such precursors of protein kinase C inhibitors may be administered in the same manner to achieve similar concentrations as in the -case of the direct inhibitors as described above.
Compounds which act as cellular messengers and which elicit an increased intracellular concentration of a naturally occurring inhibitor of protein kinase C also fall within the scope of the invention. Examples of such compounds are chalones such as transforming growth factor -3 and fibroblast growth regulator.
.`~ ,.
The concentration of such precursors or pro-drugs required to result in an in vivo inhibitory concentration of the direct inhibitor, may be ; determined experimentally by way of the assays ~ described herein or by other standard assays. Undue .
,~

~ -32-experimentation would not be required.

The invention now being generally described, the same will be better understood by reference to certain specific examples which are ~included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof, unless specified.

¦ EXPERIMENT~L EX~MPLES
I. INHIBITION OF PHORBOL ESTER-DEPENDENT
DIFFER~NTIATION OF HUMAN PROMYELOCYTIC
LEUKEMIC (HL-60) CELLS BY SPHINGANINE AND
OTHER LONG-CHAIN BASES
Materials :,~
RMPI 1640 medium was purchased from Gibco ~Grand Island, NY) and defined bovine calf serum was from HyClone Laboratories (Logan, UT). Fatty acidfree bovine serum albumin and all other tissue culture reagents were obtained from Sigma (St. Louis, MO). The sphingolipid standards: erythro-dihydrosphingosine (sphinganine), sphingosine, N-palmitoyldihydro-sphingosine, and ceramides (from bovine brain sphingomyelin) were purchased from Sigma; N-acetylsphinganine and 3-ketosphinganine were synthesized according to Gaver and Sweeley, J. Am.
Chem. Soc. 88, 3643-3647, 1966. The other homologs .,~
were provided by Dr. Dennis Liotta at Emory University. Phorbol 12-myristate 13-acetate (PMA) was purchased from LC Services Corp. (Woburn, MA) and [3H]phorbol dibutyrate (8.3 Ci/mmol) was from Amersham;
1,2-dioctanoylglycerol was obtained from Avanti Polar Lipids (Birmingham, AL).

~':

.~
.~ , ~` -33~ 1 307466 ..~
i The [3-3H]sphinganine was synthesized by the s reduction of N-acetyl-3-ketosphinganine (Gaver and Sweeley) by NaB3H4 (Amersham, Arlington Heights, IL) ~3 followed by acid hydrolysis, and purified by silica gel column chromatography (Unasil, Clarkson Chemical Co., Williamsport, PA). The product yielded a single spot coincident with sphinganine when examined by TLC with silica gel H plates developed in CHC13:methanol:2 N
NH40H (40:10:1). The specific activity was adjusted to 17,000 cpm/nmol by quantitating the amount of sphinganine as the TNBS derlvative Yamamoto and Rouser, ~ Lipids 5, 442-444, 1970.
.. ~ - ,.-~ Cell Culture !~ The HL-60 cells (obtained from the American Type -~ Culture Collection, ATCC CCL240) were grown at 37 as a suspension culture in 175 cm2 Nunc tissue culture flasks (Vangard International, Neptune, NJ). The cells were subcultured at a density of 0.25 x 106 cells/ml and used between passage numbers 30 and 40.
:,~ ~' Incubation of HL-60 Cells Cells were centrifuged at 600 ~ for 3 min, and added at 1 x 105 cells per well in 12-well culture dishes. Medium, PMA, and long-chain bases (prepared as the 1:1 molar comple~ with fatty acid-Eree bovine serum albumin) at the indicated concentrations were added for a total volume of 2.0 ml. After the desired times, the cells in suspension ~ere counted and checked for viability (trypan blue exclusion) with a hemacytometer. The attached cells were quantitated by measuring DNA content according to the method of West et al (Anal. Biochern., 147, 289-295, 1985) and by '~s ~'i - -3~- 1 307466 ;, .

assaying for acid phosphatase according to the method of Schnyder (J. Ex~. Med., 148, 434-445, 1978).

~`i When 1,2-dioctanoylglycerol was used, the cells were treated essentially as described by Ebling et al i Proc. Natl. Acad. Sci. U.S.A. 82, 815-819, 1985.
. Sphinganine was added to the cells, then . dioctanoylglycerol in 1 ~1 of ethanol to yield 100 uM, followed by additional maintenance additions of 20 ~m diacylglycerol every 2 h for 12 h.
. . .
Displacement of Phorbol by Sphinganine ;: Competitive binding assays were conducted as described by Goodwin and Weinberg (J. Clin. Invest, 70, 699-706, 1982 and Ebling et al (ibid.). Approximately 1 x 106 cells/ml were incubated with 12 nM ~3H]phorbol c dibutyrate (8.3 Ci/mmol) and varying concentrations of sphinganine ~1:1 with BSA) for 1 h at 37. The cells were recovered on Millipore filters, washed, and counted. The data were corrected for nonspecific binding by subtracting the cpm obtained in the presence of 300 nM PMA.
... .
Kinetics of [3-3H]sphinganine Uptake by HL-60 Cells Approximately 1 x 106 HL-60 cells in 1 ml of medium were mixed with an equal volume of medium containing 2.5 ~m [3-3H]sphinganine (equimolar with BSA). After varying time intervals, an aliquot of the cells was removed and counted and a portion was e~tracted as described below. The extracts were applied to Silica gel H plates and developed in CHC13:methanol: 2N NH40H (40:10:1, v/v/v), air dried, sprayed with Amplify (Amersham), and subjected to ~,''~-' ' '?`,~, ~, * Trademark ~35~ 1 3 074 66 fluorography. Radiolabel was observed in only three regions of the chromatogram, coincident with: ceramides near the solvent front, sphinganine with an Rf of approximately 0.45, and in a region near the origin that encompassed sphingomyelïn and other more polar complex sphingolipids (Rf of 0.1 to 0.2).
Analysis of Long-chain Bases ' From 1 to 4 x 107 cells were recovered by ¦ centrifugation, washed thrice with phophate buffered ! saline, and extracted immediately by a minor modification of ,the procedure of Bligh and Dyer (Canad.
J. Biochem., 37, 911-917, 1959): 1.5 ml of chloroform:methanol (1:2) were added and mixed thoroughly; 1 ml each of chloroform and water were added and the two phases were separated by centrifugation; the upper phase was discarded, and the chloroform phase was washed twice with water and dried by passage through a small column containing Na2SO4.
The extracts were saponified in methanolic KOH (0.1. M, and incubated at 37 for 1 h) to remove estercontaining ¦ glycerolipids. The DNP-derivatives were prepared according to Braun and Snell (J. ~iol. Chem., 243, 3775, 3783, 1968) as follows: the lipids were dissolved in 50 ~1 of methanol-ether (1:1), then 0.5 ml of 0.2% fluorodinitrobenzene (Sigma) in methanol-ether (1:1) and 0.5 ml of 2 M K3BO3 (pH 9.6) were added.
After incubating for 1 h at 37, 2 ml of ether and 2 ml of water were added. The ether was collected and the aqueous layer was reextracted with an additional 1 ml of ether. The combined ether extracts were washed with 2 ml of water and dried over Na2SO4, and the solvent was removed under a stream of N2. The recovery of [3H]sphinganine (60~) was used to correct for losses during extraction. For each experiment, the DNP ~-,., '.' ,, :

derivatives of standard sphingosine, sphinganine (dihydrosphingosine), and phytosphingosine (Sigma) were also prepared.

The derivatives were dissolved in 50 ~1 of methanol-5 mM potassium phosphate (pH 7.0) (90:10) and 10 ~1 was injected onto a 0.5 x 25 cm C18-column (ISCO) and eluted isocratically with this same solvent. The derivatives were detected at 360 nm with an ISCO V4 Detector. The standard sphinganine eluted at 11.2 min and sphingosine at 9 min, and both were well resolved from other speciçs.
, Statistical Methods Data given in tables and figures are results typical for several different experiments. The results are expressed as means + SD and the significance of differences between groups was evaluated with the Student's t-test for unpaired data.
.
RESULTS
Effect of Sphinganine on Cell Growth ~--The effect of sphinganine on HL-60 cell viability and growth was investigated because sphinganine has been reported to alter growth and to be cytotoxic for Chinese hamster ovary cells (Merrill, Biochem. Biophys.
Acta., 754, 284-291, 1983). Sphinganine was chosen over sphingosine because the former is available commerically as a homogeneous compound whereas the latter is a mixture of various homologs. -:, Untreated cells doubled during the first 24 h (Fig. 5), and this was not changed by 1 ~M sphinganine, .:
. ..

but both 2.5 and 5 ~M limited growth. None of the cells exhibited a loss of cell viability for the first 24 h. By the second day, all concentrations of sphinganine were still somewhat inhibitory; 1 and 2.s ~M inhibited growth without cytotoxicity whereas 5 ~M resulted in significant cell death. The change in total cell numbers between day 2 and 3 indicated that growth inhibition had ceased for the cells in 1 and 2.5 ~M sphinganine. This may have been due to removal of sphinganine by metabolism (see below).

These effec~s depended on both the cell number, which probably reflected surface dilution, and the sphinganine to albumin ratio; therefore, these parameters were kept constant except where noted.

Effects on PMA-Induced Adherence and Growth Inhibition Upon adding 8 nM PMA, growth of the HL-60 cells was inhibited by 70% within 24 h and the majority of the cells (61~) attached to the petri dish (Table I), which is typical for this cell line. When 1 ~M
sphinganine was also added, the cells continued to grow and only 26% of the total adhered. These data establish that sphinganine prevented PMA-induced growth inhibition at a concentration where sphinganine itself did not affect growth (cf., Table I and Fig. 5).

Because the cells in sphinganine continued to gro~, the number available for adherence in response to PMA was higher. This results in similar numbers of adherent cells (i.e., 0.51 x 105 for PMA plus sphinganine versus 0.76 x 105 for PMA alone, or a 33%
difference) while the adherent cells as a O of the total was much lower (i.e., a 7~% dlEference).

. :

-38- I 307~66 Adherence has been expressed as the % of the total viable cells to normalize for difference in growth.

Adherence was further limited to 17.6 + 6.4~ and 15.0 + 2.1% by 2.5 and 5 ~M sphinganine, respectively, without decreasing cell viabilities. Bovine serum albumin added alone at equivalent concentrations had no effect on growth or adherence.

Acid Phosphatase Activlties of Treated Cells For a more ~uantitative inde~ of differentiation, the acid phosphatase activities of suspended and attached cells were compared (Table II). Essentially, all of the acid phosphatase activity was associated with the cells in suspension until PMA treatment, when varying percentages were transferred from the media to the dish. Sphinganine caused a concentration-dependent increase in the activity remaining in suspension and a decrease in the adherent activities, which reflects inhibition of cell adherence.
': . '.
Inhibition of Phorbol Dibutyrate Binding by Sphinganine -.
The effect of sphinganine on phorbol dibutyrate binding was investigated because the blockage o~
attachment may be due to inhibition of protein Itinace C, which, which is thought to be the phorbol ester receptor of HL-60 cells. Sphinganine blocked [3H]phorbol dibutyrate binding (Fig. 6), with 50%
inhibition at appro.Yimately 15 ~M. Addition of 20 ~M bovine serum albumin alone did not alter binding significantly (8%). This concentration cf sphinganine as higher than that resulting in 50~ inhibition of attachment, but a higher cell number and shorter , ~ ' -39- l 307~66 ':
..
incubation time was used for binding. Therefore, less ` sphinganine would have been taken up by the cells in ~ the binding experiment, and the effective concentration 3 in the membrane would probably also be lower.

Inhibition of Diacylglycerol-Induced Cell Attachment by Sphinganine !, Dioctanoylglycerol, a cell-permeant activator of protein kinase C, also induces HL-60 cell l~ differentiation. Dioctanoylglycerol was added at j 100 ~M with or without sphinganine in an initial :3 loading dose, and additional dioctanoylglycerol was given to the cells in maintenance doses (20 ~M ) every 2 h for 16 h total. Sphinganine reduced adherence by ~, 50~ at approximately 5 ~M (Table III). This trend was observed in three separate experiments; however, more precise comparisons were thwarted by variability in the response of the cells to dioctanoylglycerol, which must be added as described above to elicit differentiation.

Structural Specificity of the Inhibition ,~ The concentration dependence of sphinganine inhibition of PMA-induced attachment is shown in Fig.
3; results of similar experiments using other longchain bases are also shown. Sphinganine at 3 ~M caused 50~
~.~ inhibition and sphingosine, the predominant longchain `~ base found in mammalian sphingolipids, caused 50 -~ inhibition at 1 ~M. Stearylamine, which is structurally related but lacks the 1,3-dihydroxy .~ groups, effected similar inhibition at 10 ~M. Other a evidence for the minimal involvement of the 3-hydroxyl was similar inhibition by 3-ketosphinganine (not shown).
: .

.~ .
.~ .

~40- 1 3 07 4 66 , Both the free amino and the long alkyl chain were important. Ceramides from bovine brain and Npalmitoyldihydrosphingosine were not inhibitory, nor was N-acetylsphinganine at up to 500 ~M. Octylamine ~:' did not inhibit, nor did another short-chain analog of sphinganine, 1,3-dihydroxy-2-amino-3-phenylpropane.

. Effects of Sphinganine on Cell Morphology and ;~ Histochemical Parameters ~ .
Expression of most othér signs of HL-60 cell differentiation requires longer time periods after ~
treatment with PMA. For these experiments, the cells were examined on day 3 for adherence and acid phosphatase activity (Table IV) and morphology and the marker enzymes alpha-naptholacetate (ANA) esterase and acid phosphatase (Table V).
.. ~ ' .
By day 3, most of the viable cells and acid phosphatase activities were adherent (Table IV). The majority of the cells had lost promyelocyte morphology, :
and resembled macrophages with visibly higher alphanaptholacetate (ANA) esterase activity (Table V). Acid phosphatase activities were higher for adherent cells when expressed as activity per 105 cells, increasing 3fold upon addition of PMA with and without sphinganine. This is a typical response of HL-60 cells tO P~A.
'7'.`
None of the few viable cells in suspension had clear signs of differentiation.

~`:1 : .
,~.

. ;Y' ~' l ,~, 1 307~66 , J, Kinetics of Sphinganine Uptake and Metabolism , The rate of disappearance of [3H]sphinganine from 'q the culture medium and its appearance in the cells is ;~ shown in Figure 4. Approximately half of the sphinganine was taken up from the medium by the cells ~"~ within 6 hr. Free sphinganine accounted for most of ,~ the cellular radiolabel (Figure 5). A large fraction ,~, was rapidly incorporated into ceramides but little was ~, found in more polar sphingolipids (sphingomyelin and glycolipids). By days`2 and 3, only 0.07 and 0.06 nmol ~;~,r'' of free [3H]sphi,nganinè was associated with the '.~ cells. The radioactivity that could not be accounted Eor in lipids was in the aqueous phase and may reflect :~c~' degradation, which produces 3H2O.
;~.'', .
The removal of sphinganine by metabolism probably accounts for the absence of inhibition by day 3.
~.`''.' ` .
~' Free Long-chain Base Content of HL-60 Cells ~:' The endogenous level of long-chain bases in HL-60 cells was quantitated by HPLC. Sphingosine was the major free long-chain base detected (i.e., >80% of the total) and was present at 12.3 + 1.2 pmoles/106 .~ cells. Since leukocytes contain approximately 5 nmol ." of sphingolipids/106 cells, this corresponds to about ,~ 0.2% of the total long-chain bases present in the ,' cells. Previous studies have found that free long-~''' chain bases are not artifacts of the isolation or ,'' derivatization procedures.

~'"' ~`''~'' ~r ; ., .~iC~

1 307~66 ' -42-i~
.'~
j Table I
.~Effects of Sphinganine on the Response of HL-60 Cells to PMA
.~ .
Cells were incubated for 24 h with 1 ~M sphinganine, 8 nM PMA, or both, and then the total number of viable cells in suspension and attached to the petri dish was determined.
.'.'::
.. :
-~ Treatment , None .Sa PMA ` PMA + Sa ' ' ".~ '' . Period of Incubation Cells/dish (x 10 5) :
.
..
0 h 1.0 + 0.1 - - -24 h 1.78 + 0.18 1.91 + 0.14 1.25 + 0.05a 1.95 + 0.15 . . % Adherence 24 h 4.5 + 1.53.7 _ 0-7 61.3 + 1.0 25.5 + 7.7 - % Viability ~ ~ .
24 h 97 95 80 93 p<0.05 compared to all other groups.

`:-' ~:~

:

, .~
, ~ ~.

,'.

, -43- 1 307~66 ~ Table II
,., Acid PhosDhatase ~ctivities of HL-60 Cells .4fter Treatment with Pl1A and Sphin~anine Cells were incubated ~or 24 h with 8 nM PMA and varying concentrations of sphinganine and the acid phosphatase activities of the ~ cells in suspension and attache~ to the petri dish were determined. .-:,~`. ' .,' :
! ~,. .
'!~" Treatment ~cid phosphatase activitya Adherence (nmol/h/dish) (~0 of total) ~,: Suspended Adherent ~: No PMA
0~M Sphinganine 1.57 + 0.08 o.o6 + 0.02 4 9 6 % ~ b 1~M Sphinganine 1.53 + 0.01 - o.o6 + 0.03 4 ~, ( 9096 1 . 2.5~M Sphinganine 1.25 + o. 16 o.o6 + 0.02 5 (98~) 5~M Sphinganine 0.91 + 0.16 0.04 + 0.01 4 (87%) 8 nM PMA
0~M Sphinganine 0.11 + 0.05 0.52 + 0.01 83 ~i (60%) M Sphinganine 0.22 + 0.07 0.44 + o.11 67 2.5~M Sphinganine 0.49 + 0.28 0.30 + 0.04 38 .~ (94~) 5~M Sphinganine 0.52 + 9.23 0.l4 ~ 0.04 21 ;
~;" (80%) ~' _ .~ a The ~ctivity o~ the susperlded cells on day 0 was 0.84 ~ 0.05 .molih/d ish .
?encent viabilit~ o~` SU~pt':~2'1 cells.

~:.'';, ' 1 307~66 ~ ~ -~!Table III

~iInh.~_icn of Dioctano~ lycerol-lnducPd Gif~eren~iatlon o~ ~L-60 Cells by ~phinganir.e Ceils r,7ere incubated ~ith dioctanoylg1~-corol ard ~arying concentrations of sphinganine.

[Spninganine] ~ ~iable cells at~ached~
~,~ ( y~
~.,. o `,10 0 + 10 -; ~ 5 63 + 21b 5.0 50 - 25b Ccmpared .i~ cells treated ~7ith diacylglycerol only.
~sbSi~ificancly di ferent from control (P < 0.05),".
,~':. :.:
'.:.

~''''' ,".'.

.; ~
.
: ~ .
i~

..

.~,~."

~`:

': ~45- 1 307~66 .
, Table I~

Adherence and Acid Phosphatase Activities of HL-60 Cells ~,l After Treatment with PMA and Sphinganir.e ~or 72 h . .
Cells were incubated for 72 h with 8 nM PMA and varying concentrations of sphlnganine and the acid phosphatase activities of the cells in suspension and ;~ at'ached to the petri dish were determined.
.,j; .
.~. T~eatment activity Adherent cellsAcid phosphatase . Suspended Adherent s, ~: No PMA (~) (nmol/hidish) (7,) 0~M Sphinganine 8 2.31 + 0.40 0.08 + 0.03 (3) M Sphinganine 9 2.77 + 0.25 0.05 + 0.03 (2) 2.5~M Sphinganine 11 - 2.35 + 0.11 . 0.05 + 0.02 (2)-5~M Sphinganine 6 0.24 ~ 0.01 0.04 + 0.01 (14) ;~.-8 nM PMA
. ~ 0uM Sphinganine 96 0.05 + 0.01 2.20 + 0.09 (98) . 1~M Sphinganine 85 0.05 + 0.01 2.95 + 0.62 (98) ~;
.~ 2.5 ~M Sphinganine 74 0.06 + 0.01 3.10 ~ 0.30 (98) ' 5~M Sphinganine 70 0.12 + 0.08 0.82 + 0.38 (87) .~
,.~

~'''~' , ' .~, ..

.:~ .
:~`

, .
?i Table V

`,Markers of ~IL-60 Cell Differentiation After Treatment IWith PMA and Sphinganine -Cells were treated with 8 nM PMA and sphinganine for 3 days and examined for morphology, alpha-naptholacetate esterase (ANA), and acid phosphatase (AP) activity.
, ,:r` _ . .
~ [Sphinganine] (~M) - 0 1 2.5 5 -Parameter :~ - ,. . .
.; , .
ir No PMA--Suspended cells Promyelocytic +++ +++ +++ +++
morphology ~`~ Macrophage - - - +
morphology ANA activity + + ++ NAa ~, Acid phosphatase 0.20 0.25 0.22 0.16b ~, 8 nM PMA---Suspended cells ~` ANA activity NA + + +
~ Acid phosphatase 0.30 0.19 0.12 o.o6 3i . 8 nM PMA---Adherent cells ~'~' Promyelocytic + + + +
morphology ~,~ Macrophage +++ +++ +++ +-~+
'i morphology ANA activity ++ ++ ++ +~+
~` Acid phosphatase 0.60 0.64 o.60 0.64 ~', aToo few viable cells to score.
, '- bActivity in nmol/h/105 cells.

.,.:; j ,.
,; ~.~ ' ~ -47- 1 307~66 , . --. .
..
~x II. SPHINGOSINE INHIBITION OF PROTEIN KINASE C
ACTIVITY AND OF PHORBOL-DIBUTYRATE BINDING
,,i IN VITRO AND IN HUMAN PLATELETS

Materialsl ~
.~

Charles River CD female rats were used for the source of protein kinase C. Ultrogel* AcA 44 and Ultrogel AcA 202 were from LKB. 32P-orthophosphate, Aquasol*II, [-32P]ATP, and [3~]PDBu (12.5 Ci/nmol) were from New England Nuclear. Calf thymus histone type III-S, phospholipase C, thrombin, phenylmethylsulfonyl-fluoride, stearylamine, bovine serum albumin, phorbol dibutyrate, 4-sphingenine, threo-sphingosine and dihydrosphingosine were from Sigma. Leupeptin was from the Peptide Institute (Osaka, Japan). 1,2-Dioleoyl-sn-glycerol-3phosphoserine, dioctanoylglycerol and 1,2-dioleoyl-sn- glycero-3-phosphocholine were from Avanti Polar Lipids, Inc. sn--1,2-Dioleoylglycerol was synthesized from dioleoylphosphatidylcholine as previously described. Triton X-100 was from Research Products International Corp. Octylamine was from Aldrich Chemical Company, Inc. Swainsonine was a gift --from Harry Broquist (Department of Biochemistry, Vanderbilt University); ceramide was a gift from Jim Walsh (Department of Biochemistry, Duke University); N-acetylsphingosine was a gift from Barry Ganong (Department of Biochemistry, Duke University); 1,3-Dihydroxy-2-amino-3-phenylpropane was a gift from Dennis Liotta (Department of Chemistry, Emory -University). Phorbol 12-myristate-13-acetate was from PL Biochemicals.
., ~:
* Trademark . ".:
. .

-48- ~ 307~66 METHODS
j Partial Purification of Protein Kinase C

Protein kinase C was partially purified from rat I brain as described by Hanrun et~al (J. Biol. Chem., '~! 260, 10043, 1985), which is hereby incorporated by ~,! reference.

:.; Mixed Micellar Assay for Protein Kinase C Activity Protein kinase C`!was assayed with Triton X-100 ~ mixed micelles as described by Hanrun et al, (ibid.), -:~; Sphingosine was dried down with the lipid cofactors.
:1~' ....................................................................... ..
[3H]PDBu binding was performed as previously .~ described (26). j;""
Preparation of Human Platelets ~- Human platelets were prepared from freshly drawn ~i blood essentially as described by Siess et al (_. Biol.
Chem, 258, 11236, 11242, 1983). They were then suspended in modified Tyrode's buffer to a concentration of 2.5x103 platelets/ml.
,~`.'.: .
[3H]PDBu Binding to Human Platelets Human platelets, prepared as described above, were suspended at a concentration of 2.5x103 platelet/ml.
50 ~1 of the platelets were then incubated for ~ive minutes with the indicated concentration of sphingosine in Eppendorf microfuge tubes. Sphingosine was prepared in 50~ ethanol at a concentration 100-fold the final concentration so that ethanol was kept at 0.5%.
~3H~PDBu was added tb 10 nM and incubated with the platelets at 37 for 10 minutes. The samples were then 1 307~6h , -49-:, .

1 filtered on Whatman GF/C filters pre-washed with 5 ml . of modified Tyrode's buffer containing 0.1% bovine ' serum albumin, washed with 10 ml of the same buffer, ' dried, and counted in 10 ml of Aquasol II in an LKB
', beta counter. Non-specific binding was determined in .'. the presence of 1 ~M unlabelled PDBu and subtracted from the total counts to yield the specific binding.
.~, ~' 40K Phosphorylation in Human Platelets ~. :
" 32p at 0.2 mCi/ml was added to the platelet suspension and labelling was allowed to proceed for,75 ' minutes at 37C, after which the platelets were ~ pelleted at 600,xg for 10 minutes and resuspended in r~ Tyrode's buffer to the same concentration. They were ~, then aliquoted in Eppendorf microfuge tubes and pre-~ incubated at 37C for 5 minutes with the varying '~, concentrations of sphingosine. Platelets were then stimulated with either 5 ~M dioctanoylglycerol (added as an ethanol solution with the final concentration of ' ethanol 0.5%), 10 ~M TPA, or 1 unit/ml thrombin. The '` reactions were stopped after 30 seconds by the addition of an equal volume of 2x sample buffer, and the samples ~,, were then boiled for 3 minutes. 0.1 ml were then ~', loaded on 10% sodium dodecylsulfate polyacrylamide gels , and electrophoresis was performed according to the method of Laemmli (Nature, 227, 680-685, 1970). Gels "~ were subsequently fixed in water/methanol/acetic acid (60:30:10), dried, and autoradiographed.
,~
','. Phosphollpld Quantltatlon .' Phospholipids were extracted from whole platelets ~, by the method of Bligh and Dyer (Can. J. Biochem.
~- Physiol., 37, 911-917, 1959). Phosphatidylserine was ~', purified by two dimensional thin layer chromatography ."',.'' '..
":
~"~, , -50- 1 307466 .1 .
on Silica gel H plates developed in chloroform:methanol:acetic acid (65:25:10 v/v) in the first dimension and in chloroform:methanol:88% ~ormic acid (65:Z5:10 v/v) in the second dimension.
Phospholipids were quantitated by measuring phosphates according to the method of Ames (J. Biol. Chem., 235, 769-775, 1960).
The data shown is representative of at least 3 sets of experiments.

RESULTS AND DISCUSSION
' When the effect of sphingosine on protein kinase C
activity was tested using the Triton X-100 mixed micelle assay containing 6 mol% PS and 2 mol% diC18 1' sphingosine proved to be a potent inhibitor (Figure 10). Under these conditions, the bulk concentration of PS was 260 uM and diC18 1 was 86 ~M. Therefore, 50%
inhibition (100 ~M ) occurred on a molar basis equivalent to [diC18 1] or to 0.4 of [PS]. The potency of sphingosine inhibition was markedly affected by the number of Triton X-100 mixed micelles containing 7 mol~ PS and 1 mol% diC18 1 present in the assay (~igure llA). Thus, the effect of sphingosine was subject to surface dilution. When the data are expressed as mol%, (sphingosine:Triton X-100), sphingosine inhibition at four different levels of Triton X-100 mixed micelles (containing PS and diC18 1) was identical (Figure llB) implying that it is the number of sphingosine molecules present in each mixed micelle that determines the potency and not the absolute concentration. ~or surface active amphipathic molecules, the expression of inhibitor potencies must be relative to the amount of surface (micelles in this case) as bulk concentrations are misleading. These results also imply that . '';
'''' ' ~. ~
,' '''';

-51- 1 30746b sphingosine interacts with the surface-bound protein kinase C probably by interfering with the function of its regulatory domain. To test this hypothesis, the catalytic domain (protein kinase M) was generated by proteolysis of protein kinase C2. The activity of this catalytic domain which is independent of Ca2+, phospholipid and DAG/phorbol esters was not inhibited by sphingosine (Fig. 10). Thus, sphingosine does not appear to inhibit by interaction with the active site.

To further inves~igaté the mechanism of sphingosine inhibition, the sn-1,2-diC18 1 dependency of protein kinase C activation was investigated at fixed PS (6 molO) and Ca2+ (50 ~M ) and at several levels of sphingosine. Importantly, the inhibition by sphingosine was modulated strongly by diC18 1 (Figure 12A). Double reciprocal plots (Figure 12B) indicated essentially a competitive type of inhibition with respect to diC18 1. Similarly, inhibition by sphingosine was overcome by increasing concentration of phorbol dibutyrate (Fig. 13A), and double reciprocal plots (Figure 13B) showed competitive inhibition.

When the concentration of sphingosine was varied at 5, 6 and 7 mol~ PS and fixed diC18 1 (2 mol~), the potency was modulated markedly (Figure 14). This was especially true when PS was not saturating at 5 mol%.
Shifting from 6 to 7 mol% PS, a level at which PS
becomes saturating (Figure 15), revealed that the curves were simply not displaced on a mole for mole basis. When PS was in excess, higher concentrations of sphingosine were required for inhibition (Figure 14).
i;.:.
5",-'`- Next, the effect of 2 and 4 mol-O sphingosine On the PS dependence of protein kinase C activation was investigated ~Figure 15). Interestingly, sphingosine .:T ' ~, .~-,, .
~,:~ ;'.;
r i ~ . . :
~, r :;
., ~ .
~i: i;` :~, ....

`~ caused a displacement of the PS dependence to higher levels which remained strongly cooperative in the 3 presence or absence of inhibitor. When the PS
i dependencies were then plotted according to Hill, Hill l numbers of 5.4, 6.9 and 8.8 were obtained for the curves generated at 0, 2 and 4 molz sphingosine, respectively. Double reciprocal plots were not constructed because of the cooperativity observed with PS and the sphingosine-dependent change in Hill numbers. In that additional PS completely overcame the inhibition by sphingosine, a competitive form of ~ inhibition appe~rs likely.

i~; To further explore the mechanism of sphingosine ~!; inhibition, the efEect of the level of Ca2+ employed ,!~ was examined at fixed PS and diC18 1 and at 0, 3, and 4 mol% sphingosine. Double reciprocal plots of calcium ~, dependencies were linear and appeared competitive (Figure 16).

The effect of sphingosine on phorbol ester binding was examined to further substantiate the sphingosine ~' inhibition occurs by interfering with the regulatory domain of protein kinase C. Sphingosine inhibited phorbol-ester binding to Triton X-100 mixed micelles containing 16 and 20 molz PS (Figure 17). The ~; concentration dependence of sphingosine displacement of phorbol-dibutyrate, paralleled its ability to inhibit protein kinase C activation by PDBu (Figure 13A).
Sphingosine also inhibited phorbol binding when ~;~ phosphatidic acid was used as the lipid cofactor (PA
supports only 50z of the binding measured with PS).
.' i..;'~' ,; Next, experiments were conducted tested in human platelets to determine whether sphingosine would , inhibit protein kinase C activation by thrombin. This ~. .

~53~ 1 307~66 was determined by monitoring the protein kinase C
induced phosphorylation of the 40 KDa polypeptide (Figure 18A). As shown in Figure 18B, sphingosine caused nearly complete inhibition of 40 K
phosphorylation at a concentration of 25 ~M. Partial inhibition was seen with concentrations as low as 10 ~M. Sphingosine also inhibited TPA and diC8-induced 40 K phosphorylation (data not shown).

To further analyze the mechanism by which sphingosine inhibits protein kinase C activation, PDBu binding to wholç human platélets was studied ~Figure 19). Saturable and displaceable binding of ~3H]PDBu to platelets was demonstrated (data not shown).
Sphingosine was able to inhibit this binding in concentrations identical to those required to inhibit 30 K phosphorylation.

Since sphingosine is an amphiphilic molecule and would be expected to partition into a bilayer or micelle, a direct comparison of its biologic effects to its in vitro potency in inhibiting protein kinase C
requires that its bulk concentration be expressed as mol% sphingosine to phospholipids. To accomplish this, the amount of PS and total phospholipids in platelet membranes were quantitated. Human platelets were found to have 20% of their total phospholipids as PS and the absolute PS concentration under the assay conditions was 200 ~M. Therefore, the sphingosine concentrations (expressed as mol% sphingosine:phospholipids) required to inhibit platelet ~0 K phosphorylation were similar to those required for in vitro inhibition of protein kinase C; 3-5 mol% sphingosine required for complete inhibition (or 15-25~ of PS). In addition, this inhibition demonstrated the same reversibility observed in the in vitro system in that increasing the :, -'.'.':

'.' concentration of diC8 or of platelets (i.e. PS) overcomes the inhibition.

The speciEicity of protein kinase C inhibition by sphingosine was investigated using a number of related molecules. As seen in Figure 20, octylamine did not inhibit at the concentrations tested whereas stearylamine was nearly as effective as sphingosine.
Swainsonine, structurally related to sphingosine, was not an inhibitor. N-acetylsphingosine, ceramide and 1,3-dihydroxy-2-amino~3 phenylpropane derivative were without effect. Fatty acids, and cetyl triethylammonium bromide were also inactive. However, 3-ketosphinganine, erythro- and threo-sphinganine were all inhibitors. The results presented show that sphingosine is a potent and reversible inhibitor of protein kinase C. This suggests that sphingosine may be a useful inhibitor of protein kinase C in different cell systems. Results with platelets, in HL-60 cells and in neutrophils further attest to the usefulness of sphingosine as a protein kinase C inhibitor.

Sphingosine differs from the other known inhibitors of protein kinase C in that it is a natural component of cells comprising a critical component of ceramide, the building block of sphingomyelin and the glycosphingolipids. Sphingosine and other naturally occuring long-chain (sphingoid) bases are synthesized by serine palmitoyltransferase. Sphingosine could also be generated by the action of ceramidases (N-acyl sphingosine amidohydrolases). These metabolic pathways raise the possibility that the generation of sphingosine intracellularly may serve as a regulatory negative effector of protein kinase C activity.
Sphingosine levels may be regulated in response to either intra- or extracellular signals. In fact, -'' .

sphingomyelin was observed to undergo rapid deacylation and N-acylation when lymphocytes were stimulated to undergo blastogenesis. The deacylation of sphingomyelin leads to the generation of sphingosylphosphorylcholine (lysosphingomyelin). This molecule may lead to the generation of sphingosine through hydrolysis oE the phosphorylcholine head group. Therefore, these catabolic pathways for the ` generation of sphingosine or one of its analogues may ', play a physiologic role in modulating the activity of ¦ protein kinase C. The activity of protein kinase C is expected to be a function of the concentration of PS
I (phospholipid), DAG, Ca2+ and the negative effector, I sphingosine3.
:1 FooTNoTEs lThe abbreviations used are: PS, phosphatidylserine;
DAG, sn-1,2-diacylglycerol; diC18 1' sn-1,2dioleoylglycerol; diC8' sn-1'2-dioctanoylglycerol;
¦ EGTA, ethylene glycol bis ( ~- aminoethyl)N,N,Nl,Nl-tetraacetic acid; PA, phosphatidic acid; PDBu, phorbol `I dibutyrate, TPA, phorbol 12-myristate, 13-acetate ~ ( PMA ) .
'I .: .
2The catalytic domain was generated by trypsin -treatment of purified protein kinase C; and purified by Ultrogel AcA-44 molecular sieve chromatography. The fractions showing protein kinase activity independent of Ca2+, PS, and DAG were pooled and used for the above experiments.
'~
3This suggests an explanation for the different -concentration dependencies of protein kinase C
activation by the cell permeable diacylglycerol, diC8' observed in different cell types. In platelets, neutrophils, and A~31 cells, ~M diC8 was effective;
whereas, in tracheal 2C5 cells, pituitary cells, and :
:'-..

-56- l 307466 HL60 cells, 10-lOO ~M amounts were required. Perhaps the higher diC8 concentrations required reflects the presence of an anti-signal (negative effector) in these cell types, such as sphingosine.

III. LYSOSPHINOGOLIPID INHIBITION 0~ PROTEIN KINASE C
TABLE VI: Inhibition of protein kinase C activity and phorbol-dibutyrate binding by lysosphingolipids. The first column lists different disease entities that constitute the sphingolipidosis, and the second column shows the lysosphingolipids that are expected to accumulate in each of these diseases. Accumulation has been demonstrated in the case of galactosylsphingosine, glucosylsphingosine, and lyso GM2. The remaining two columns sho~ the observed effects of the lysosphingo-lipids of protein kinase C activity and phorbol diester binding in vitro and in human platelets.
Lysogangliosides were prepared from their parental gangliosides by hydrolysis in methanolic KOH esentially as described by Neuenhofer et al. in Bioch. 24, 525, 1985. During this preparation, with approximately 90-95% conversion, the N-acetate of N-acetylneuraminic acid is partially hydrolyzed. Fatty acids were partitioned into heptane after acidification of the methanolic reaction mixture. Galactosylsphingosine, glucosylsphingosine, and lactosylsphingosine were prepared as previously described for -galactosylsphingosine (Radin, Lipids 9, 358, 1974) Sphingosylphosphorylcholine was prepared by hydrolysis in HCl-butanol (Kaller, Biochemische Zeitschrift 334, 451, 1961). The purity of the lyso derivatives (greater than 90%) was checked by thin layer chromatography (TLC) on silica gel a plates developed in chloroform/methanol/2N NH40H (65/35/9) and visualized by ninhydrin reaction for N-lyso compounds, by ~- naph~hol/sulfuric acid for both gangliosides and ~' lysogangliosides, and by a phospholipid spray for sphingosylphosphorylcholine. Lysosphingolipids were quantitated by reaction of the amine group with trinitrobenzenesulfonate. Gangliosides and lysosphingolipids were also quanti~tated by reaction with anthrone. The lysosphingolipids were then studied for their effect on protein kinase C activity. Protein kinase C was purified to near homogeneity by the method of Wolf et al. (J. Biol. Chem. 260, 15718, 1985), and activity was measured using a mixed micellar assay (described herein). Lysosphingolipids were added directly in aqueous solution, and allowed to equilibrate with the Triton X-100 (0.3% w/v) mixed micelles containing 6 mol~i phosphatidylserine and 2 mol~ dioleoylglycerol. All the lysosphingolipids proved to be potent inhibitors of protein kinase C (1 mol% = 43 ~M ). The complex acidic lysogangliosides were more potent than the neutral glycosphingolipid derivatives, possibly due to partial hydrolysis of the N-acetate groups leading to the generation of more than one amine per molecule. Phorbol-dibutyrate binding was measured in a mixed micellar assay. Lysosphingolipids inhibited phorbol-dibutyrate binding to protein kinase -~
C in the presence of Triton X-100 mixed micelles containing 20 mol~ phosphatidylserine.
Lysosphingolipids appeared less potent at inhibiting -~
phorbol dibutyrate binding than protein kinase C
activity. The difference is related to the higher concentration of phosphatidylserine used in the binding assay, as both activity and binding show a high cooperative dependence on phosphatidylserine, which modulates the inhibition by lysosphingolipids.
Phorbol-dibutyrate binding to human platelets and 40 kDa phosphorylation in platelets ~7ere carried out as described above. Lysosphingolipids inhibited phorbol dibutyrate binding to human platelets at concentrations -:~
.: . ' ', ' -58- 1 307~66 '~ in the 10-50 ~M range. Inhibition of 40 kDa phosphorylation occurred over the same concentration range.

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1 307~66 ;
IV. INHIBITION OF THE OXIDATIVE BURST IN HUMiAN
NEUTROPHILS BY SPHING~ID LONG BASIS AND THE
:
ROLE OF PROTEIN KINASE C THEREIN
Materials HESPAN (6.0~ hetastarch in 0.9% NaCl) was obtained from American Critical Care Division of American Hospital Supply Corporation (McGraw Park, IL).
Lymphocyte Separation Medium (LSM) (6.2~ Ficoll, 9.4 sodium diatrizoate) was obtained from Bionetics Laboratory Products (Kensington, MD).
Formylmethionylleucylphenylalanine (FMLP), Phorbol 12myristate 13-acetate (PMA), cytochalasin B, A23187, fatty acid ~ree-bovine serum albumin, (BSA), ceramides (from bovine brain cerebrosides), cytochrome c (horse heart, type III), NADH, NADPH, Trypan Blue, superoxide dismutase, latex particles, palmitic acid, psychosine, erythro-dihydrosphingosine (sphinganine), and sphingosine were obtained from Sigma (St. Louis, MO).
Na2H[32P]04 was obtained from New England Nuclear (NEN) -(Boston, MA) (1000 mCi/nmol) and ICN Radiochemicals (Irvine, CA) (285 Ci/mg P). [3H]-Phorbol dibutyrate (8.3 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). Electrophoresis reagents were obtained from Bio-Rad (Rockville Center, NY); 1,2-dioctanoylglycerol was obtained from Avanti Polar Lipids (Birmingham, AL). Phenyl analogs of sphinganine were synthesized by Dr. Dennis Liotta (Emory University Department of Chemistry). N-acetyl sphinganine was synthesized by the method of Gaver and Sweely.
.
The [3H]-sphinganine was prepared by the reduction . of N-acetyl-3-ketosphinganine by NaB3H~ (Amersham) followed by hydrolysis, and purified by silica gel column chromatography (Unasil, Clarkson Chemical Co., Williamsport, PA). The product yielded a single spot ~
., . .
. ;', coincident with sphinganine when examined by TLC with silica gel H plates developed in CHC13:methanol: 2 N
NH40H (40:10:1). The specific activity was adjusted to 17,000 cpm/nmol by quantitating sphinganine as the TNBS
derivative.

METHODS
Isolation of Human Neutrophils Human neutrophils were obtained by continuous flow leukapheresis from normal adults. Residual erythrocytes were removed by hypotonic lysis with a resulting purity of > 95~ neutrophils. Alternatively peripheral blood was obtained by phlebotomy and neutrophils were isolated by HESPAN (6% Hetastarch, 0.9~ NaCl) sedimentaion of erythrocytes, centrifugation through lymphocyte separation medium (9.4 ~ sodium diatrizoate, 6.2~ Ficoll), and hypotonic lysis of residual erythrocytes. Isolated cells were resuspended -in phosphate buffered saline (PBS)-glucose, containing O.6 m~ CaC12' 2.6 mM KCl, 1.5 mM KH2PO4' 0.5 mM MgC12' 136 mM NaCl, 8 mM Na2HPO4' 5.5 mM glucose.
.~ .
Preparation of Stock Solutions of EEfectors Effectors of the oxidative burst include activators, inhibitors, and modulators. Stock solutions of FMLP, PMA, Cytochalasin B, were prepared at 5 mg per ml in dimethylsulfoxide (DMSO). The calcium ionophore A23187 was prepared as a 1 mM stock ~- in DMSO. Long-chain bases, fatty acids, and other ~^
i.~ inhibitor analogs were prepared as equimolar concentrations of the effector and fatty acid-free BSA.

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-62- 1 307~66 ., ~ Measurement of Oxygen Consumption .e Oxygen consumption was measured using a Clark-type electrode with YSI model 53 oxygen monitor. Assays were conducted at 37C with 6.5 x 1o6 cells per ml with a total volume of either 2.5 ml or 4 ml. Effectors were injected into the electrode chamber using a Hamilton syringe. The conditions for each measurement are given in figure legends. Initial slopes after the addition of the stimuli were used for rate calculations. For activators with a lag prior to the onset of the oxidative burst, maximal rates following the lag were used.
.
Assay for Superoxide Production Superoxide production by cells was quantitated from superoxide-mediated reduction of cytochrome c, monitored as an increase in absorbance at 550 nm and an extinction coefficient at 21,000 M 1 cm 1. Measurements were carried out at 37C using approximately 5 x 105 neutrophils per ml, stirred continuously. The cells were allowed to equilibrate several minutes prior to the addition of effectors. Conditions for individual experiments are described in figure legends and tables. Controls utilizing added superoxide dismutase verified that the cytochrome c reduction was mediated by superoxide.
..
Superoxide production was also measured spectrophotometrically in isolated membranes (see below) by the method of Curnutte et al iN. Eng. J. Med.
293, 628-632) also using superoxide dismutase inhibitable reduction of cytochrome c. Assays utilized paired cuvettes with the reference cuvette containing 30 ~g/ml superoxide dismutase. Both cuvettes also ~s:'-' .:
~'''''. .
~"`'`"':` .

i -, -63- 1 307466 ,l, contained 65 mM potassium phosphate pH 7.0, 125 mM
sucrose, 81 ~M cytochrome c, and 0.1 mg of membrane protein. The reaction was initiated with 200 ~M (final) NADPH in both cuvettes. The treatments are as described in Table I. All spectrophotometric measurements were carried out using a Cary 219 UT-visible spectrophotometer.
. -.

Cell Viability : ' Cell viability was assessed by two criteria:
Trypan Blue exclusion and release oE the cytosolic enzyme, lactate dehydrogenase (LDH). LDH activity was monitored by followihg the oxidation of NADH at 340 nm. Activity was determined in three groups of cells: control (no additions), neutrophils plus 50 ~M sphinganine, and cells permeabilized with 0.5%
. .
Triton X-100 (final concentration) for complete release of cellular LDH. Trypan Blue exclusion was determined microscopically. ~ ^
. - ::,., Isolation of Activated Membranes -,, .......
. ;:~

Neutrophil membranes activated for superoxide production were prepared as described previously (Hohn, -J. Clin Invest. 55, 707-713, 1975). Neutrophils (108) --were suspended in PBS-glucose and incubated in a shaking waterbath at 37C. Cells were activated by the ~-addition of 10 ~g PMA/ml and allowed to incubate 5 min. The reaction was stopped by adding an equal volume of ice-cold PBS. The cells were centrifuged at 400 x g for 4 min at 4~C and resuspended in PBS-glucose containing 120 ~M FAD. The cells were lysed by homogenization with a teflon-glass homogenizer. The homgenate was centrifuged at 250 x g for 10 min. to remove unlysed cells. The supernatant was centrifuged ..

,,.

. ::

` -6~- 1 307~66 . , , at 27,000 x g for 30 min. to yield the membrane/granule-rich ~raction. The pellet was resuspended in Tris-HCl, pH 8.6, containing 15%
glycerol and 120 ~M FAD.

Phagocytos1s Latex particles were opsonized by incubating at 37C 1 ml of particles with 0.2 ml of human serum plus 1 ml Tris buffer, pH 8.5. Neutrophils (2 x 106 cells) in 0.1 ml of PBS-gluco,se (either containing 40 ~M sphinganine or with no addition) were incubated with 108 particles at 37C for 5 min. The reaction was terminated by the addition of 0.4 ml of ice-cold buffer containing 1 mM EDTA. The cells were then diluted by 80~ with 0.15 M NaCl and layered onto a ficoll/sodium metrizoate solution (density = 1.077) and centrifuged 20 minutes at 400 x g at 4C. Cells are sedimented, while excess beads remain at the ficoll-saline interface. The number of latex beads internalized per cell was determined by microscopic examination.

Sphinganine Inhibition of Cellular Phorbol Dibutyrate Binding Neutrophils (1 x 108 cells/ml) were preincubated with the indicated concentration of sphinganine, palmitic acid, or ceramide for five minutes prior to the addition of 50 nM (final concentration) of [3H]phorbol dibutyrate (specific activity of 8.3 Ci/mMole). The cells were incubated an additional 15 min. in a shaking waterbath at 37C. The incubation mixtures were filtered using an Amicon filtration manifold model VFM-~III and washed 5 times with 5 m1s of ice-cold P~S-glucose. The filters were counted using a Beckman LS7000 scintillation counter.
~:
. .

- -65- 1 307~66 .
Phosphorylation Studies Neutrophils (1.4 x 108 cells) were preincubated in with 0.5 mCi Na2H32PO4 in buffer containing 136 nM
NaCl, 5.56 mM glucose, 10 ml~ Hepes, and 0.33 mM CaC1 for a total of 1 hr at 37C in a shaking waterbath.
Excess unbound counts were removed by centrifugation and resuspension, repeated five times. The cells were then divided into 4 treatment groups (see RESULTS
below) containing approximately 3.5 x 107 cells each.
All groups were incubated at 37C in a shaking waterbath for the same total time. Some samples were incubated 10 min. with sphinganine (40 ~M ) prior to addition of 1 ~M PMA ~final concentration), with which ;l they were incubated for an additional 15 min. The reaction was stopped by addition of an equal volume of -boiling SDS dissociation buffer (9% SDS, 1%
mercaptoethanol, 15% glycerol, 30 mM Tris, pH 7.8). The samples were run on a 12~ SDS-polyacrylamide gel using the method of Rudolph and Krueger (Adv. Cyclic Nucleotide Res. 101, 107-133, 1979). The gels were _ .
dried using a Bio-Rad*model ~400 Gel Slab Dryer.
Autoradiography was performed on the dried gels using Kodak*X-omat X-ray film and Dupont Cronex*intensifying screens. The gels were exposed for 12 hrs at -70C.
.i .,' Alternatively, neutrophils were preincubated as ~; described above using 0.1 mCi Of 32p labeled phosphate. The cells were broken by 5 min. of sonication using an Ultramet III waterbath sonicator, and 5% (final concentration) trichloroacetic acid was added to each sample. The precipitate was filtered and trichloroacetic acid was added to each sample. The precipitate was filtered and washed extensively using an Amicon*filtration system and counted using a Beckman*
~,' *Trademarks :~ ~ -.!
LS7000 scintillation counter.

Measurement of Cytoplasmic Calcium Concentration Intracellular calcium was measured by the Quin2 method. Briefly, Quin2/acetoxymethylester (50 ~M ) was preincubated with 1 x 108 cell/ml for 20 min. at 37, prior to 10-fold dilution and continuation of incubation for an additional 40-60 minutes. Cells were reisolated by centrifugation to remove extracellular fluorophore, and fluorescence measurement and calibration were carried out as described by Tsien et al. in J. Cell. Biol. 94, 325-334, 1982, using a Perkin-Elmer MPF-44B fluorescence spectrophotometer.
Buffer consisted of 140 mM NaCl, 5 mM KCl, 1 mM Na2PO4' 5.5 mM glucose, 0.5 ~M MgS04' 20 mM Hepes, and 1 mM
CaC12.

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'I TABLE VII

EFFECT OF SPHINGANIME ON NADPH-OXIDASE ACTIVITY
j IN ISOLATED NEUTROPHIL MEMBRANES :

I ADDITIONCytochrome c Reduction : :
¦ (nmols/min/mg/protein) .:

NONE 18 .~.
BSA (50 ~M) 18 ;
: SPHINGANINE (18 ~M) , 27 :
Cytochrome c reduction was monitored as described in the Experimental section.
, ,.'..', :
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L~

-68- 1 307~66 , ! TABLE VIII

, EFFECT OF SPHINGANINE ON OXYGEN CONSUMPTION
USING VARIOUS ACTIVATORS
RATE~
(nmols 2 consumed/min/106 cells) ,~ Phorbol Myristate 1 Acetate 4.3 0.2 M) Dioctanoyl Glycerol 6.1 0.3 (100 ~M) FMLP (0.5 ~M) +
Cytochalasin B 5.3 0.3 (5 ~g/ml) Opsonized zymosan (30 ~g/ml) 5.3 0 3 Arachidonate (83 ~M) 4.2 0.2 ~Oxygen consumption was measured using a Clark oxygen electrode, as described in the Experimental Section.
For incubations in the presence of sphinganine, ~ 50 ~M sphinganine (final) plus 50 ~M fatty acid-Eree - BSA were added to the cells 3 minutes prior to addition of the activator. In control experiments, 50 ~M BSA
alone prior to activation did not affect the activated rate.
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: TABLE IX
~ SPHINGANINE ANALOGS
j STRUCTURE/FUNCTION EFFECTS

,~ .
-~ ANALOGConcentration required '~ for 50% inhibition - .
uM
erythro-Sphinganine 7 threo-Sphinganine 1 24 Sphingosine Stearylamine 25 Octylamine 300 3-amino-3-hydro;Yy-3-ph-enyl-propanol R,R-S n.i. *
R,R-R n.i.
N-acetylsphinganine n.i.
Ceramide n.i.
Palmitic acid n.i.
~CTAB n.l.
~ .
n.i. = no inhibition at 100 ~iM analog ~cetyl trimethylammonium bromide .:
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TABLE X

INHIBITION BY SPHINGANINE OF [32P]-PHoSPHATE
INCORPORATION INTO TCA-PRECIPITABLE MATERIAL
! - $
~ ADDITION COUNTS PER MINUTE

¦ NONE 2932 . PMA (1 ~M) 4912 : PMA (1 ~M) plus Sa (25 ~M) 2845 . Sa (25 ~M) 2506 .' . ~see Experimental section for methodology.
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.~ ., -71- l 307466 -, TABLE XI -:
EFFECTS OF SPHINGANINE ON INTRACELLULAR
CALCIUM LEVELS AND ON FMLP-INDUCED
CALCIUM TRANSIENTS IN HUMAN NEUTROPHILS
-.' Calcium~
Pretreatment Resting FMLP-Peak ~M ~M
None 0.16 +/-0.02 (4) 0.59 +/-0.13 (3) Sphinganine (30 ~M) 0.20 +/-0.02 (3) 0.66 ~/-0.14 (3) .: _ ~Intracellular calcium was measured using Quin2, as described in the Experimental Section. Each measurement utilized 7.5 x 106 cells in 2 mls buffer.
The "FMLP-Peak" value refers to the maximum calcium Concentration achieved following 1 ~M FMLP addition, but prior to the gradual return of calcium towards resting levels. Numbers in parenthesis refer to the number of experiments averaged to obtain the reported values. Fluorescence was corrected in the sphinganine experiments for a small amount of fluorescence of the ,.
I albumin carrier.
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,~
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72 l 307466 RESULTS
'~ Inhibition by Sphinganine of 0~-consumption of ¦ Superoxide-Mediated Reduction of Cytochrome c -~ Sphinganine was tested for its ability to inhibit the "oxidative burst" of normal human neutrophils. This i lipid rather than sphingosine was used initially as the ~, prototypical long-chain base, since it is commercially ,~ available in synthetic chemically wellcharacterized form. When neutrophils were preincubated with ~; sphinganine, activatio,n of 02-consumption of PMA was abolished (See Figure 24, Panel A). The increased ;~ oxygen consumption is though to reflect reduction of 2 to superoxide anion, which can be detected by monitoring superoxide dismutase-inhibitable cytochrome c reduction. Figure 24, panel B, shows the ~ preincubation with sphinganine also blocks PMA
.. ~ activation of superoxide generation.
.. ~;, .
Figure 24 (Panel A, Tracing 3) also illustrates that the addition of sphinganine several minutes after inhibition of the oxidative burst causes a gradual return to the basal rate of oxygen consumption. (The apparent loss of activity at later times in the control tracing (P~A stimulated, no sphinganine addition) was due to oxygen depletion, in contrast to the true inactivation seen following sphinganine addition.) The decay to basal rates followed first order kinetics as determined by plotting the natural 109 of the rate of oxygen consumption after addition of sphinganine as a function of time (not shown). The half-time for inactivation varied from 30 sec to 6 minutes with the individual donors examined. Since sphinganine added 2-3 minutes prior to activation with PMA completely abolished the oxidative burst ~or all donors, the inactivation rate does not appear to reflect the rate ~5 '');', ,: '~
.. ~,`,.',`;;' .

~l ~73- 1 307466 i at which sphinganine gains access to the cell. Rather, we suggest that the variable rate of this decay represents individual variation in a normal inactivation process which is revealed when activation mechanisms are blocked.
.
Evidence that Sphinganine acts Reversibly and without 3 Cytotoxicity on the NADPH-oxidase Activation Mechanisms :~ :
The inhibitory effects of sphinganine were not due . '~7~ to cytotoxic efects of~long-chain bases. When neutrophils (1.2 x 107 cells/ml) were incubated with r,~ 50 ~M sphinganine, a completely inhibitory concentration, there was no effect on cell viability.
Trypan Blue exclusion showed greater than 95~
viability, and LDH relese showed greater than 96~ of cellular LDH was retained in both the presence and absence of sphinganine.

To determine whether the sphinganine inhibition was reversible, neutrophils (1.2 x 107 cells/ml) were first incubated for 5-10 minutes with an inhibitory concentration (40 ~M) of sphinganine. Rates of oxygen consumption in the freshly isolated cells were about 4 nmols/min/106 cells in the absence and 0.2 to 0.3 in the presence of sphinganine. Cells were then washed twice by centrifugation and resuspension in PBS-glucose containing 50 uM fatty acid-free BSA. After such procedures, restoration of PMA-induced oxygen consumption comparable to that in untreated cells was seen (3.7 and 3.8 nmols/min/106 cells respectively), ~ indicating that inhibition of the oxidative burst by ;~i sphinganine is reversible.

To determine whether the observed effects of ~:~d, sphinganine were on the activation process or on the ."~J, !

~7~~ 1 307466 NADPH-dependent oxidase enzyme itself, membranes were prepared from PMA-pretreated cells, and examined for sphinganine effects on superoxide generation.
Membranes isolated from PMA-pretreated cells have previously been shown to exhibit an approximately 10-fold increase in NADPH-dependent 2 generation. No inhibition was observed when these membranes were incubated with concentrations of sphinganine which inhibited the cellular response (See Table VII). In fact, a slight stimulation was seen, as is also seen with some detergents., Thus, sphinganine does not inhibit the oxidase enzyme directly but must act at some site(s) involved in activation.
.~
I Lack of Effects of Sphinganine on Phagocytosis ¦ Neutrophils were incubated with and without sphinganine in the presence o opsonized latex beads. -~¦ These cells were reisolated, free of uningested beads : (see Experimental section) and examined microscopically for the presence of ingested beads. In 208 control cells, there were an average of 7.4 + 1.0 (S.E.M.) beads per cell, while in the presence of sphinganine, there were 7.2 + 0.8 beads/cell (178 cells counted).
Thus, there is no significant inhibition of phagocytosis by sphinganine. Therefore, not only is there no evident toxicity during the experimental time frame, but also this complex cellular Eunction remains unaffected.

Effect of Sphinganine on the Oxidative Burst Induced by A VarietY of Activators.

The inhibitory effect of sphinganine was tested using a variety of activators. Table VIII shows that with all activators-tested there was complete ,. . ,.
',`' . '.' _75- 1 307~66 inhibition of the oxidative burst by sphinganine. To test whether all stimulants utilized a common, sphinganine-inhibitable step, the concentration dependence for inhibition was determined for a variety of activators. In initial studies with -dioctanoylglycerol and PMA, oxygen consumption was monitored (Figure 25, Panel A). Using this assay, half maximal inhibition occurred at 7.4 and 6.7 and 5.6 ~M using diotanoylglycerol, PMA, and opsonized zymosan, respectively. These values are identical within experimental error and individual variation among donors. Thus, às has been suggested by a variety of earlier studies, phorbol esters and diacylglycerols appear to activate by a common mechanism. In addition, activation by opsonized zymosan appears to share this sphinganine-inhibitable mechanism.

To quantitate sphinganine inhibition using a . series of other activators, the superoxide assay was 1 used (Figure 25, Panel B). This assay is considerably '3, more sensitive and less time-consuming than the oxygen , consumption assay, therefore requiring fewer cells and facilitating data aquisition. Activation by both FMLP
and arachidonate was 50% inhibited at essentially the same concentration as that which effected PMA
~3 activation, again implicating a common site of inhibition. ~Particulate activators could not be assessed using this assay due to turbidity and baseline noise.]
s~
There is an apparent but readily explainable discrepancy in sphinganine concentration required for SO~ inhibition using the two assays (compare, for ~, example PMA activation in panel A versus Panel B of ~3 Figure 25). This is due to the diference in cell ~ number used in the two assays. The oxygen consumption ?,Jj -76- l 307466 assay requires large numbers of cells (approximately 7.5 x 106 cells/ml) and shows a concentration dependence for 50O inhibition of 6-8 ~Im sphinganine.
In separate e~periments (not shown) it was found that inhibition of oxygen consumption required a higher sphinganine concentration when the cell number was increased and lo~er when the cell number was decreased. The superoxide production measurements were obtained using about lO-fold less cells (approximately 5 x 105 cells/ml), and required about l ~M sphinganine for 50% inhibition for~ all activators. Thus the difference in required concéntration can be readily explained by a cell number effect. The concentration requirement appears to be approximately directly proportional to cell number as is also seen for many lipophilic compounds (e.g. diacylglycerols and arachidonate) whose activity requires partitioning into cellular membranes. This is in agreement with sphinganine inhibition being subject to surface dilution effects in an in vitro mixed micelle system and in platelets.

To determine how much inhibitor was actually becoming associated with the cells, radiolabeled sphinganine (3, lO, 30 ~M) plus albumin carrier was ;~
incubated with neutrophils (2.5 x 106 cells) for 5 min (i.e. a time longer than that needed to achieve inhibition). After washing by centriEugation, the cell-associated radioactivity was measured.
Approximately 20~ of the added sphinganine became cell-associated. Therefore, because of partitioning between the albumin and the cells, the cellular concentration -~
of sphinganine was considerably less than the total.
Thus, if generated intracellularly, long-chain bases are expected to be more effective than the concentration dependence for externally added inhibitor '''.
.~
'3~' ' ":

indicates.

Effect of Structural Analogs of Sphinganine on the Oxidative Burst Using P~A activation, analogs with structural I Eeatures similar to those of sphinganine were tested for their effects on the oxidative burst (see Figure 26 and Table IX). Compounds which also showed inhibitory effects include stearylamine, sphingosine, threo-sphinganine, and octylamine. Other compounds tested (See Table IX) did not show inhibition at 100 ~M concentrations.
!., It appears that the structural features necessary for inhibition include a free amino group and a hydrophobic group such as a long alkyl chain. There i5 also a modest selectivity for the native stereoisomer, erythro-sphinganine, since the threo isomer was 3-4 times less effective. The longer alkyl chain derivatives were more effective than shorter, and a hydrophobic benzene ring could not substitute for an alkyl chain. Thus, sphinganine and sphingosine were the most effective inhibitors tested, and inhibition appears to exhibit some degree of structural specificity.
~:
Sphinganine Inhibition oE Radiolabelled Phorbol Dibutyrate Binding Displacement of bound, radiolabeled phorbol dibutyrate by diacylglycerols has been used previously to provide evidence for a common cellular site of action (protein kinase C) for these two activators.
Here, the invelltors used the same technique to evaluate ~ whether, as indicated by phosphorylation studies, i'` l :~:,. }
.. . l sphinganine is acting by binding to protein kinase C.
Figure 27 shows that sphinganine displaces phorbol dibutyrate from its cellular binding site. Control studies were also carried out using 50 ~M ceramide and palmitic acid, two analogs which did not show significant inhibition (see Table I~). With these analogs, there ~as 18% and 3% displacement, respectively compared with 60% displacement with the same concentration of sphinganine. Residual apparent binding is non-specific, since an excess of unlabeled PMA produced the same`degree of displacement. These studies show that the decrease in binding of the phorbol ester does not appear to be due to detergent or other effects. Rather, there appears to be a specific inhibition oE the phorbol ester binding by sphinganine. Such behavior was also seen in the micelle-reconstituted system and in human platelets.
.
In the present studies, the lipids sphinganine and sphingosine are shown to be potent inhibitors of the neutrophil oxidative burst. Sphinganine, a sphingoid long-chain base, is the product of the enzymatic ~-condensation of palmitoyl-CoA and serine by serinepamitoyl transferase. Sphinganine can be -incorporated into a variety of more complex biomolecules, the sphingolipids (eg. sphingomyelin, ceramide, and glycosphingolipids). Sphingosine is also an intermediate in sphingolipid biosynthesis, can be generated from the breakdown of sphingolipids, and is the predominant long-chain base in vivo. The present studies provide support for the proposal that one or more of the long-chain bases is an intracellular regulatory molecule. Although it remains to be shown whether these molecules are synthesized or mobilized in response to physiologic stimuli, Eree long-chain bases are present in HL-60 cells and in mature neutrophils S~

~79~ 1 307~66 (A. Merrill, personal communcation) in quantities expected to affect the function of cellular protein kinase C. If, as has been proposed, neutrophil oxidative metabolism plays an important role in tumor generation, then long-chain bases might under some conditions function as "anti-tumor promoters".

The inhibition by long-chain bases of the oxidative burst appears to be a specific rather than a generalized metabolic effect. Incubation with sphinganine for the time required Eor the assays had no direct inhibitory effect on the NADPH-oxidase enzyme system. It may therefore be concluded that the inhibition of the oxidative burst involves the activation process rather than the oxidase itself.

That sphinganine does not have generalized effects on a variety of regulatory systems is indicated by at least three observations. ~irst, in a system known to be c~P-dependent, ACTH stimulation of steroidogenesis in the Y-1 tumor cell line, sphinganine did not affect the ACTH-stimulated steroidogenic rate (E. Wilson, unpublished studies). Second, sphinganine does not appear to affect Ca++/calmodulin-dependent protein kinase, as evidenced by lack of inhibition of phosphorylation of the 20K protein in platelets using concentrations which completely abolished phosphorylation of the 40K protein known to be phosphorylated by protein kinase C. Third, long-chain bases do not significantly affect resting or FMLP-stimulated calcium levels. FMLP is thought to influence calcium levels initially via receptor-mediated activation of a phopholipase which cleaves phophatidylinositol-4,5-bisphosphate into diacylglycerol plus inositol triphosphate. The latter promotes release of lntracellular calcium stores. Thus, ~' :

-80- 1 307~66 it appears unlikely that long-chain bases exert their effects on the phopholipase or on calcium levels.

Two lines of evidence from the present studies implicate protein kinase C as a specific site of inhibition by long-chain bases. ~irst, the inventors have shown that sphinganine can displace [3H]-phorbol dibutyrate from the phorbol ester binding site.
Phorbol esters have been shown to bind to and activate protein kinase C. According to data shown elsewhere in this section, sphinganine and phorbol esters compete for binding to protein kinase C. Secondly, PMA-stimulated phosphorylation is inhibited by sphinganine. Thus, long-chain bases appear i specifically to block activation of protein kinase C.
J, It is possible that sphinganine may have additional as ~ yet unknown modulatory effects on other enzymatic ;~/ systems. Nevertheless, the combination of data ~, presented herein strongly implicates protein kinase C
;i as the affected site.
.:
~I The mechanism(s) of activation of the oxidative tj burst by what appears to be a diverse array of stimuli is (are) poorly understood. Sphinganine has been used -~
~, in the present studies as a general probe of these r~ mechanism(s) in human neutrophils. More ~pecifically, the inventors wished to determine whether protein kinase C participated in some or all of the activation ~! processes. Probably the best understood of the chemical activators are the phorbol esters such as PMA, which are potent activators of the enzyme. Also, synthetic diacylglycerols have been shown to be direct activators of protein kinase C. These compounds appear to activate by binding to protein kinase C. In the above experiments, it has been shown that activation of the oxidative burst by these compounds is inhibited by ,",.

'.' ''.

1 307~66 identical concentrations of sphinganine.

More complex in its mechanism, the chemotactic peptide FMLP functions by first binding to a plasma membrane surface receptor, thus promoting the hydrolysis of phosphatidylinositol-4,5 bisphosphate in a pertussus toxin-inhibitable process. Furthermore, stimulation with FMLP causes an increase in the concentrations of both calcium and diacylglycerol.
Thus, a direct pathway for protein kinase C involvement in the oxidative burs~ can be postulated for FMLP
activation via the generated diacylglycerol, but an elevation in cytosolic calcium has also been proposed to mediate the effects. In the present studies, sphinganine inhibited the oxidative burst without affecting the cytoplasmic FMLP-dependent rise in calcium concentrations. Thus a direct mediator role for calcium in activation of the oxidative burst seems unlikely. In measurements of 2 production, the inventors have shown that the half maximal concentration of sphinganine required to inhibit superoxide production was virtually the same when either FMLP or PMA was used as the activator, implicating a common inhibition site (i.e. protein kinase C).
', Likewise, activation of the oxidative burst by ~-opsonized particles (e.g., zymosan) is inhibited by sphinganine in the same concentration range which inhibited PMA activation. The inventors conjecture that concurrent with phagocytosis, a protein kinase C
activator (e.g. diacylglycerol or arachidonate) is generated, resulting in the observed oxidative burst.
Thus, while phagocytosis itself is not inhibited by sphinganine, the concurrent activation of the oxidative burst is blocked, consistent with the involvement of r. .

!
; !

`^ -82- 1 3074h6 activators, the inventors propose that the activation mechanisms for not only diacylglycerol and phorbol esters, but also FMLP, arachidonate and opsonized zymosan converge to act through protein kinase C. The latter may act directly on the NADPH oxi~dase, or may phosphorylate another regulatory component, such as the cytosolic factor recently reported by Curnutte (Blood 66, 77a, 1985).

The invention now being fully described, it will be apparent to one of``ordinary skill in the art that .
many changes and modifications can be made thereto - .
without departing from the spirit or scope of the invention as set forth herein.

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Claims (41)

1. A method of inhibiting protein kinase C, which comprises contacting protein kinase C in vitro with an inhibitory amount of a compound having the formula:

wherein Q is a hydrophobic group;
wherein X is -CH2-CH2- or -CH=CH-, which may be substituted by one or more halogens or C1-C3 alkyl groups, wherein Y is , , , -CH2-' or , wherein W is a halogen, wherein R1 and R2 may be the same or different and are selected from hydrogen, lower alkyl groups having from 1 to 7 carbon atoms, aralkyl, and aryl groups, and wherein Z is a phosphate or an organic group.

2. The method of Claim 1, wherein Z is selected from the group consisting of phosphate, H, galactosyl, sulfogalactosyl, glucosyl, lactosyl, trihexosyl, phosphorylcholine, GalNAc-Gal-Glc, Gal-Gal-Glc-, Sia-Gal-Glc, and .

3. The method of Claim 1, wherein Q is CH3-(CH2)n- or CH3-(CH2)m-CH=CH-(CH2)n- wherein n is 2-30;
m is 1-15 and p is 1-15.

4. The method of Claim 1, wherein Y is or .

5. A method of inhibiting an oxidative burst in neutrophils, which comprises contacting a neutrophil in vitro with a proteinkinase C inhibitory concentration of a compound having the formula:

wherein Q is a hydrophobic group;
wherein X is CH2-CH2- or -CH=CH-, which may be substituted by one or more halogens or C1-C3 alkyl groups, wherein Y is , , , -CH2-, or , wherein W is a halogen;
wherein R1 and R2 may be the same or different and are selected from hydrogen, lower alkyl groups having from 1 to 7 carbon atoms, aralkyl, and aryl groups, and wherein Z is a phosphate or an organic group.

6. The method of Claim 3, wherein Z is selected from the group consisting of phosphate, H, galactosyl, sulfogalactosyl, glucosyl, lactosyl, trihexosyl, phosphorylcholine, GalNAc-Gal-Glc, Gal-Gal-Glc-, and Sia-Gal-Glc, and .

7. The method of Claim 6, wherein Q is CH3-(CH2)n- or CH3-(CH2)m-CH=CH-(CH2)p- wherein n is 2-30;
m is 1-15 and p is 1-15.

8. The method of Claim 6, wherein Y is or .

9. The method of Claim 1, wherein said compound is sphingosine.

10. The method of Claim 1, wherein said compound is sphinganine.

11. The method of Claim 1, wherein said compound is psychosine.

12. Use of an inhibitory concentration of a compound having the formula:

wherein Q is a hydrophobic group;
wherein X is -CH2-CH2- or -CH=CH-, which may be substituted by one or more halogens or C1-C3 alkyl groups, wherein Y is , , , -CH2-' or , wherein W is a halogen;
wherein 1 and R2 may be the same or different and are selected from hydrogen, lower alkyl groups having from 1 to 7 carbon atoms, aralkyl, and aryl groups, and wherein Z is a phosphate or an organic group, for treating inflammation in a mammal.

13. Use of Claim 12, wherein Q is CH3-(CH2)n- or CH3-(CH2)m-CH=CH-(CH2)n- wherein n is 2-30; m is 1-15 and p is 1-15.

14. Use of Claim 13, wherein Y is or .

15. A method for inhibiting protein kinase C, which comprises contacting a cell containing protein kinase C in vitro, with a metabolic precursor of sphingosine, sphinganine or a naturally occuring lysosphingolipid, said precursor having the ability of increasing the intracellular concentration of a protein kinase C inhibitory compound which produces inhibition of protein kinase C activity in vitro.

16. A method for inhibiting protein kinase C, which comprises contacting a cell containing protein kinase C in vitro, with a pro-drug analogue of a protein kinase C inhibitor according to Claim 1, wherein the nitrogen atom of said inhibitor is acylated with an in vitro enzyme cleavable C1-C7 acyl group or an amino acid.

17. A composition capable of inhibiting protein kinase C upon routine administration in vivo, which comprises a protein kinase C inhibitory concentration of a compound having the formula:

wherein Q is a hydrophobic group;
wherein X is -CH2-CH2- or -CH=CH-, which may be substituted by one or more halogens or C1-C3 alkyl groups, wherein Y is , , , -CH2-' or , wherein W is a halogen;
wherein R1 and R2 may be the same or different and are selected from hydrogen, lower alkyl groups having from 1 to 7 carbon atoms, aralkyl, and aryl groups, and wherein Z is a phosphate or an organic group, and a physiologically acceptable carrier.

18. The composition of Claim 17, wherein Z
is selected from the group consisting of H, galactosyl, sulfogalactosyl, glucosyl, lactosyl, trihexosyl, phosphorylcholine, GalNAc-Gal-Glc, Gal-Gal-Glc, Sia-Gal-Glc, and .

19. The composition of Claim 17, wherein Q
is CH3-(CH2)- or CH3-(CH2)m-CH=CH-(CH2)p- wherein n is 1 to 29; m is 1-15 and p is 1-15.

20. The composition of Claim 17, wherein Y
is or .

21. The composition of Claim 17, wherein said physiologically acceptable carrier is a sterile liquid or cream.

22. The composition of Claim 17, wherein said compound is sphingosine.

23. The composition of Claim 17, wherein said compound is sphinganine.

24. The composition of Claim 17, wherein said compound is psychosine.

25. Use of an inhibitory amount of a compound having the formula:

wherein Q is a hydrophobic group;
wherein X is -CH2-CH2- or -CH=CH-, which may be substituted by one or more halogens or C1-C3 alkyl groups, wherein Y is , , , -CH2-' or , wherein W is a halogen;
wherein R1 and R2 may be the same or different and are selected from hydrogen, lower alkyl groups having from 1 to 7 carbon atoms, aralkyl, and aryl groups, and wherein Z is a phosphate or an organic group, for inhibiting protein kinase C in vivo.

26. Use of Claim 25, wherein Z is selected from the group consisting of phosphate, H, galactosyl, sulfogalactosyl, glucosyl, lactosyl, trihexosyl, phosphorylcholine, GalNAc-Gal-Glc, Gal-Gal-Glc-, Sia-Gal-Glc, and .

27. Use of Claim 25, wherein Q is CH3-(CH2)n- or CH3-(CH2)m-CH=CH-(CH2)n- wherein n is 2-30 m is 1-15 and p is 1-15.

28. Use of Claim 25, wherein Y is or .

29. Use of a protein kinase C inhibitory concentration of a compound having the formula:

wherein Q is a hydrophobic group;
wherein X is -CH2-CH2- or -CH=CH-, which may be substituted by one or more halogens or C1-C3 alkyl groups, wherein Y is ,, , -CH2-, or , wherein W is a halogen;
wherein R1 and R2 may be the same or different and are selected from hydrogen, lower alkyl groups having from 1 to 7 carbon atoms, aralkyl, and aryl groups, and wherein Z is a phosphate or an organic group with a neutrophil, for inhibiting an oxidative burst in neutrophils.

30. Use of Claim 27, wherein Z is selected from the group consisting of phosphate, H, galactosyl, sulfogalactosyl, glucosyl, lactosyl, trihexosyl, phosphorylcholine, GalNAc-Gal-Glc, Gal-Gal-Glc-, Sia-Gal-Glc, and .

31. Use of Claim 27, wherein Q is CH3-(CH2)n- or CH3-(CH2)m-CH=CH-(CH2)p- wherein n is 2-30;
m is 1-15 and p is 1-15.

32. Use of Claim 27, wherein Y is or .

33. Use of Claim 25, for treatment in vivo in a human being.

34. Use of Claim 25, wherein said compound is sphingosine.

35. Use of Claim 25, wherein said compound is sphinganine.

36. Use of Claim 25, wherein said compound is psychosine.

37. Use of a protein kinase C inhibitory concentration of a compound having the formula:

wherein Q is a hydrophobic group;
wherein X is -CH2-CH2- or -CH=CH-, which may be substituted by one or more halogens or C1-C3 alkyl groups, wherein Y is , , , -CH2-' or , wherein W is a halogen;
wherein R1 and R2 may be the same or different and are selected from hydrogen, lower alkyl groups having from 1 to 7 carbon atoms, aralkyl, and aryl groups, and wherein Z is a phosphate or an organic group, for treating inflammation.

38. Use of Claim 37, wherein Q is CH3-(CH2)n- or CH3-(CH2)m-CH=CH-(CH2)n- wherein n is 2-30; m is 1-15 and p is 1-15.

39. Use of Claim 37, wherein Y is or .

40. Use of a metabolic precursor of sphingosine, sphinganine or a naturally occurring lysosphingolipid, with a cell containing protein kinase C, for increasing the intracellular concentration of a protein kinase C inhibitory compound thereby producing inhibition of protein kinase C activity.

41. Use of a pro-drug analogue of a protein kinase C inhibitor according to claim 1 with a cell containing protein kinase C, wherein the nitrogen atom of said inhibitor is acylated with an in vivo enzyme cleavable C1-C7 acyl group or an amino acid, for inhibiting protein kinase C.