WO2004064823A1

WO2004064823A1 - Ceramide derivatives for the treatment of inflammation

Info

Publication number: WO2004064823A1
Application number: PCT/GB2004/000173
Authority: WO
Inventors: Yvonne Perrie; Helen Rosemary Griffiths; Darren Charles Phillips
Original assignee: Aston University
Current assignee: Aston University
Priority date: 2003-01-21
Filing date: 2004-01-21
Publication date: 2004-08-05
Anticipated expiration: 2005-07-21
Also published as: GB0301395D0

Abstract

The present invention relates to the use of: (i) a ceramide, a ceramide analogue, a phytoceramide; or (ii) a compound which is metabolisable into or otherwise capable of releasing a ceramide, a ceramide analogue or a phytoceramide; or (iii) a compound which modulates the level of endogenous ceramide as a pharmaceutically active agent in the production of a medicament for the treatment of an inflammatory disorder. The invention also relates to a method of inhibiting monocyte recruitment, and/or adhesion and/or transmigration to sites of inflammation and/or of inducing growth arrest in proliferating monocytes, comprising exposing the monocytes to a therapeutically effective amount of a ceramide, a ceramide analogue, a phytoceramide or a compound which is metabolised into or breaks down to release a ceramide, a ceramide analogue, a phytoceramide in situ, or a compound which modulates the level of ceramide as a pharmaceutically active agent.

Description

CERAMIDE DERIVATIVES FOR THE TREATMENT OP INFLAMMATION

This invention relates to a novel medicament for the treatment of inflammatory disorders.

The rapid transition between adherent and non-adherent states is of key importance to the immunological function of cells of the circulatory and immune system. In the presence of foreign bodies, pathogens, antigens, or at sites of injury, cells of the immune system congregate in the lymphoid organs, or cross endothelium and basement membranes to accumulate at the site of infection. This process requires the up-regulation of cell adhesion, but becomes deregulated under conditions of vascular and chronic inflammatory disease.

The vascular endothelium represents a primary site in the pathophysiology of several vascular diseases and disorders of inflammatory origins including atherosclerosis, rheumatoid arthritis (RA) and acute and chronic respiratory lung disease. Monocyte adhesion and transmigration across resting endothelial cells occurs at very low rates, however, an early observation in the development of atherosclerotic lesions is the enhanced recruitment of monocytes into subendothelial segments of arteries which ultimately leads to foam cell formation.

An elevation in adhesion molecules expressed on the endothelium in RA is associated with the augmented homing of leukocytes to inflamed synovial tissue compartments. Further, the changes in the expression and function of adhesion molecules involved in cell-cell interaction are involved in a variety of pulmonary diseases. Additionally, monocytes isolated from the whole blood of patients with RA possess elevated adherence to fibronectin and resting or activated endothelial cells. The appearance of monocytes within the synovium of rheumatoid patients is followed by their differentiation into tissue macrophages and type A synoviocytes, which contribute to joint destruction through secretion of matrix metalloproteinases.

In the past, many successful anti-inflammatory drugs have been discovered fortuitously: these include non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit prostoglandins, disease modifying anti- rheumatic drugs and cytotoxic agents which inhibit cellular replication. None of these classes of anti-inflammatory drugs is specifically targeted to inflammatory cells and therefore may result in undesirable side effects. More recently work has been focussed on growth factors, such as Tumour Necrosis Factor (TNF) which is a potent inflammatory cytokine, and its neutralisation or blocking using antibodies. This treatment can lead to severe side effects and also can be prohibitively expensive.

It is an object of the present invention to provide an alternative medicament for the treatment of inflammatory disorders which obviates or mitigates one or more of the above disadvantages.

According to a first aspect, the present invention resides in the use of: (i) a ceramide, a ceramide analogue, a phytoceramide; or (ii) a compound which is metabolisable into or otherwise capable of releasing a ceramide, a ceramide analogue or a phytoceramide; or (iii) a compound which modulates the level of endogenous ceramide, as a pharmaceutically active agent in the production of a medicament for the treatment of an inflammatory disorder.

The invention resides in the surprising discoveries that ceramides, ceramide analogues and phytoceramides inhibit monocyte recruitment, adhesion, proliferation and transmigration to sites of inflammation.

Lipids, which include ceramides, represent a useful class of pharmaceutical compounds as they can evade the immune system due to their flexible structures and their ability to rapidly partition into membranes.

Ceramide comprises a sphingosine molecule linked to a second fatty acid by an amide bond (formula 1).

CH₂OH

H — C NH

H — C— -OH C=0

CH C_nH_2n+i

C₁₄H₂8

Formula 1

Preferably, said ceramide, ceramide analogue or phytoceramide has the structure of formula 1 where n is no more than 18, preferably no more than 8, and more preferably from 2 to 6.

Preferably, said compound which modulates the level of endogenous ceramide is one which increases levels of endogenous ceramide or reduces the degradation of endogenous ceramide. For example said compound may be an inhibitor of ceramidase, which normally acts to degrade ceramide, such as DMAPP which blocks alkaline ceramidase, or B13 which blocks acid ceramidase.

According to a second aspect of the present invention there is provided a method of inhibiting monocyte recruitment, and/or adhesion and/or transmigration to sites of inflammation and/or of inducing growth arrest in proliferating monocytes, comprising exposing said monocytes to a therapeutical ly effective amount of a ceramide, a ceramide analogue, a phytoceramide or a compound which is metabolised into or breaks down to release a ceramide, a ceramide analogue, a phytoceramide in situ, or a compound which modulates the level of ceramide as a pharmaceutically active agent.

According to a third aspect of the present invention there is provided a method of treating a patient having an inflammatory disorder comprising, administering to said patient as a pharmaceutically active agent a therapeutical ly effective amount of a ceramide, a ceramide analogue, a phytoceramide or a compound which is metabolised into or breaks down to release a ceramide, a ceramide analogue, a phytoceramide in situ, or a compound which modulates the level of endogenous ceramide.

Preferably said treatment reduces recruitment, adhesion and/or transmigration of monocytes to sites of inflammation and/or induces growth arrest in proliferating monocytes. Preferably, said medicament according to the first aspect is characterised in that it acts by inhibiting at least one of and preferably all of monocyte recruitment, adhesion, transmigration to an inflammatory site and growth of proliferating monocytes.

Preferably, said medicament comprises means for delivery of the pharmaceutically active agent to an inflammatory site.

Preferably, said delivery means allows selective delivery to the inflammatory sites.

Said delivery means may be any known delivery means e.g. a surfactant- based vesicle delivery system, for example liposomes, a viral vector delivery system, an antibody associated delivery system or any other ligand-receptor associated targeted delivery system.

Administration of said medicament may be by any convenient route e.g. by intravenous, intramuscular, or subcutaneous injection, topical administration as an ointment, salve, cream or tincture, oral administration as a tablet, capsule, suspension or liquid and nasally as a spray (e.g. aerosol).

In the methods or medicament of the present invention, said pharmaceutically active agent may be in admixture with one or more excipients, carriers, pH regulators, flavourings, colourings, preservatives, or other commonly used additives in the field of pharmaceuticals as appropriate for the mode of administration. Examples of inflammatory disorders which may be suitable for treatment by the medicament according to the first aspect include, rheumatoid arthritis, atherosclerosis, acute or chronic inflammatory lung disease and inflammatory bowel disease.

Preferably, particularly when said pharmaceutically active agent is administered systemically, it is administered at a level to obtain a steady state blood concentration of ceramide, ceramide analogue, or phytoceramide of 1 to 20mg/l, more preferably 2 to 10mg l, most preferably 3 to 8mg/l.

Preferably, when said pharmaceutically active agent is delivered non- systemically sufficient agent is delivered to achieve a concentration of ceramide, ceramide analogue, or phytoceramide in the inflammatory cells of greater than 100 amol, more preferably from 500 amol to 5 fmol, most preferably from 750 amol to 3 fmol.

When said pharmaceutically active agent is a ceramide, a ceramide analogue, a phytoceramide or a compound which is metabolisable into or otherwise capable of releasing a ceramide, a ceramide analogue or a phytoceramide, said blood and cellular concentrations may be achieved by administering a dosage of pharmaceutically active agent of 10 to 100 mg/day, or preferably, 20 to 80 mg/day.

Embodiments of the invention will now be described by way of example only, with reference to the accompanying drawings in which: Fig. 1 is a graph showing the effect of ceramide on in vitro monocyte recruitment to LPS activated endothelial cells,

Fig. 2 is a graph showing the effect of exposure to ceramide for 16 hours on the membrane expression of a number of cell surface adhesion molecules.

Fig. 3 is a graph showing the effects of exposure to ceramide on the numbers of monocytes becoming growth arrested.

Materials and Methods

Ceramide stock solution production

C2-ceramide (N-acetyl-sphingosine) and Cβ-ceramide (N-hexanoyl- sphingosine) (obtained from Biomol Research Laboratories, Plymouth Meeting, PA, USA) were separately dissolved in anhydrous DMSO to a stock solution of 20mM. Subsequent dilutions were made in 1 mM fatty acid free BSA.

Cell growth conditions

The acute human monocytic cell line, U937 was maintained in RPMI 1640 media, supplemented with 10% heat inactivated foetal calf serum and 1 % penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Determination of cell numbers

The number of viable cells per ml was determined by trypan blue exclusion using an improved Neubauer haemocytometer (Weber Scientific International Ltd., Teddington, UK).

Treatment of cells with ceramide solutions

Cells at a concentration of 2x107ml were serum starved for 4 hours prior to treatment with ceramide. Cells were treated with C_- or Cβ-ceramide for the times and at the concentrations noted in the figures. Incubations were performed at 37°C in a humidified 5% CU2/95% air incubator.

Cell adhesion assay

The cell adhesion assay was performed as described by Woollard (Woollard KJ, Phillips DC, Griffiths HR. Direct modulatory effect of CRP on primary human monocyte adhesion to human endothelial cells. Clin. Exp. Immunol. 2002; 130, 256-262.). Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from umbilical cords by digestion with collagenase and cultured in Endothelial Growth Medium (EGM; BioWhittaker) at 37°C, 5% CO2, 95% air humidity, as described previously Gaffe EA, Nachman RL, Becker GC et a/. Culture of human endothelial cells derived from umbilical veins. J. Clin. Invest. 1973; 52: 2745-2756. Holland JA, Pritchard KA, Rogers NJ et al. Perturbation of cultured human endothelial cells by atherogenic levels of LDL. Am. J. Pathol. 1988; 132: 474-478.). HUVEC were grown to confluence in 24 well plates (Orange Scientific) up to passage 3 in EGM media and were used 24 hours after confluence. HUVEC were washed and LPS (1μg/ml) or HBSS as control added for 0, 5 or 24 hours (37°C, 5% CO2, 95% air humidity). Each well was then washed twice with 1 ml of M199 (Sigma), before addition of monocytes. Monocytes were resuspended to 5x107ml and labelled with 2', 7'-bis-2-carboxyethyl-5-(6)-carboxyfluorescein- acetoxymethylester (BCECF-AM; Sigma; 10μg/ml) for 30 minutes at RT in the dark. Dye loading was quenched by adding 10mls of PBS (0.1 % BSA) and centrifuging at 235g for 8 minutes. Monocytes were washed and resuspended in M199. Monocytes (0.5x107ml) were added to HUVEC and incubated for 30 minutes, under the above described culture conditions., Non-adhered cells were removed by centrifugation of inverted plates (Weber C, Wolfgang E, Weber K et al. Increased adhesiveness of isolated monocytes to endothelium is prevented by vitamin C intake in smokers. Circulation 1996; 93: 1488-1492.). Lysis buffer (1 ml; 0.1 % Triton-X; Sigma) was added to each well and incubated in the dark at RT for 30 minutes. Lysed cells were pipetted into 96 well plates (Nalge Nunc (Europe) Ltd, Hereford, U K) in replicates of 9 and fluorescence was measured at an excitation of 485 nm and emission of 535 nm on a Wallace S pectroflu or i meter.

Flow cytometry

Monocytes were incubated with 0-20μM C2- or Cβ- ceramide in serum free medium for 16 hours and then washed three times in PBS. Subsequently samples were analysed for CD1 1 b, CD31 , CD18, CD1 1 a and CD62L expression by flow cytometry (Beckman Coulter, Miami, Florida, USA), using appropriate three-way colour compensation and isotype negative controls for each sample. Following treatment, cell samples (monocytes) were incubated with appropriate primary (anti-human) Mab (CD1 1 b-RPE [ICRF44]), CD31-RPE [WM59], CD1 1 a-FITC [AT10], CD62L-RPE-C/5 [TUK-4]; Serotec Ltd, Kidlington, UK; 10μl/10⁶ cells or 10Oμl PWB) on ice in the dark for 30 minutes. Optilyse (Beckman Coulter) was added to lyse RBC and fix the samples. Samples were vortexed and incubated in the dark at RT for 10 minutes. Each sample was diluted 1 :2 with Isoton (Beckman Coulter), vortexed and left at RT in the dark for no longer than 4 hours, until analysis by flow cytometry.

Examples

Example 1, Effect of ceramide on the recruitment of monocytes to the endothelium.

Figures 1 A, B and C show the effect of ceramide on U937 monocyte recruitment to confluent HUVEC endothelial monolayers which have been activated with lipopolysaccharide (LPS) for varying times. LPS is a bacterial antigen which stimulates the immune response. It is used as a model of inflammation and activates integrins and adhesion molecules on endothelial cells and monocytes. After LPS treatment, the HUVEC cells were exposed for 30 min to monocytes which had previously been incubated in media containing ceramide for 16 hours. Figure 1 A is a negative control (no LPS), Figure 1 B 5 hours exposure to media containing 1 μg/m! LPS, Figure 1 C 24 hours exposure to media containing 1 μg/ml LPS. In all figures, column 1 is a negative control (no ceramide), column 2 20μM C2-ceramide, column 3 20μM Cβ-ceramide, column 4 10μM Ce- ceramide. Figure 1 A shows that ceramide has no significant effect on the level of monocyte recruitment to non LPS activated HUVEC monolayers.

Figure 1 B shows that after 5 hours exposure to LPS, cells then exposed to 20μM C2- or Cβ-ceramide show a significant reduction in the recruitment of monocytes to about 50% of the control level of recruitment. There is less reduction in recruitment when the cells are exposed to 10μM Ce- ceramide.

Figure 1 C shows that in all cases, there is a further reduction in the recruitment of monocytes in response to ceramide after 24 hours induction of HUVEC monolayers with LPS. Monocyte recruitment in the presence of ceramide drops to about 40% of that seen in the control.

It can be seen from these figures that ceramide has a significant effect on the recruitment of monocytes to sites of inflammation, whilst having no significant effect on recruitment of monocytes to other sites.

Example 2, Effect of ceramide on the expression of adhesion molecules on the surface of U937 monocytes.

Figure 2 shows the effect of exposure of monocytes to ceramide for 16 hours on the expression of various cell surface adhesion molecules. Fig. 2 shows the effect on the expression of CD31 (Panel A), CD1 1 a (panel B), CD26L (panel C), CD18 (panel D) and CD1 1 B (panel E). In each case column 1 is a negative control (no ceramide), column 2 is 20μM C2- ceramide and column 3 is 20 μM C6-ceramide.

Figure 2 shows that in all cases expression of the cell surface adhesion molecules is significantly reduced by addition of ceramide for 16 hours. Reduction in cell surface adhesion molecule expression results in fewer monocytes binding to the endothelial tissue at sites of inflammation.

Example 3, Effect of ceramide on the cell cycle of U937 monocytes.

Figure 3 shows the effect of ceramide on the percentage of U937 monocytes in a population entering the G0/G1 (growth arrested) phase of the cell cycle. Panel A shows the effect of C2-ceramide on cell cycle stage after 8, 16, 20 and 24 hours exposure to media containing ceramide, and panel B shows the effect of Cβ-ceramide. In all cases column 1 is a negative control, column 2 is 1 μM ceramide, column 3 is 10μM and column 4 is 20μM.

2 x 10⁶ U 937 monocyte cells were serum starved for 4 hours before the addition of media containing ceramide. The proportion of cells in the GO/ G1 stage of the cell cycle was measured at 8, 16, 20 and 24 hours using flow cytometry (Nicoletti et al, A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry, 1991 , J. Immunol. Methods, 139: 271-279).

Panels A and B show an increase with time in the percentage of cells becoming growth arrested both in the presence and absence of ceramide. The most dramatic increase in cells entering G0/G1 being seen between 8 and 16 hours,. and levelling off by 20 hours. It can be seen from both panels that there is a clear trend toward cell stasis with increasing concentrations (10μM, 20μM) of both C2- and Gs-ceramide.

This trend toward cell stasis reduces the potential for differentiation of monocytes at sites of inflammation which can contribute to joint destruction through the secretion of matrix metal loproteinases.

The data show that ceramide can reduce the recruitment of monocytes to sites of inflammation and also reduce their adhesion to these sites, it also shows that monocytes can be growth arrested by treatment with ceramide. Thus, making this naturally occurring lipid, which has no known long term side effects, a useful agent for the treatment of inflammatory disorders.

Claims

1. The use of:

(i) a ceramide, a ceramide analogue, a phytoceramide; or

(ii) a compound which is metabolisable into or otherwise capable of releasing a ceramide, a ceramide analogue or a phytoceramide; or

(iii) a compound which modulates the level of endogenous ceramide as a pharmaceutically active agent in the production of a medicament for the treatment of an inflammatory disorder.

2. The use according to claim 1 , wherein said ceramide, ceramide analogue or the phytoceramide has the structure of formula 1 where n is no more than 18.

Formula 1

3. The use according to claim 2, wherein n is from 2 to 6.

4. The use according to claim 1 , wherein said compound which modulates the level of endogenous ceramide is one which increases levels of endogenous ceramide or reduces the degradation of endogenous ceramide.

5. The use according to claim 4, wherein said compound is an inhibitor of alkaline or acid ceramidase.

6. The use according to any preceding claim, wherein said medicament acts by inhibiting at least one of monocyte recruitment, adhesion, transmigration to an inflammatory site and growth of proliferating monocytes.

7. The use according to any preceding claim, wherein said medicament comprises means for delivery of the pharmaceutically active agent to an inflammatory site.

8. The use according to claim 7, wherein said delivery means is a surfactant-based vesicle delivery system, a viral vector delivery system, an antibody associated delivery system or another ligand-receptor associated targeted delivery system.

9. A method of inhibiting monocyte recruitment, and/or adhesion and/or transmigration to sites of inflammation and/or of inducing growth arrest in proliferating monocytes, comprising exposing said monocytes to a therapeutical ly effective amount of a ceramide, a ceramide analogue, a phytoceramide or a compound which is metabolised into or breaks down to release a ceramide, a ceramide analogue, a phytoceramide in situ, or a compound which modulates the level of ceramide as a pharmaceutically active agent.

10. A method of treating a patient having an inflammatory disorder comprising, administering to said patient as a pharmaceutically active agent a therapeutical ly effective amount of a ceramide, a ceramide analogue, a phytoceramide or a compound which is metabolised into or breaks down to release a ceramide, a ceramide analogue, a phytoceramide in situ, or a compound which modulates the level of endogenous ceramide.

1 1. The method of claim 10, wherein said medicament according to the first aspect is characterised in that it acts by inhibiting at least one of and preferably all of monocyte recruitment, adhesion, transmigration to an inflammatory site and growth of proliferating monocytes.

12. The method of claim 1 1 , wherein said inflammatory disorder is rheumatoid arthritis, artherosclerosis, acute or chronic inflammatory lung disease or inflammatory bowel disease.

13. The method of any one of claims 10 to 12, wherein said pharmaceutically active agent is administered systemically, at a level to obtain a steady state blood concentration of ceramide, ceramide analogue, or phytoceramide of 1 to 20mg/l.

14. The method of any one of claims 10 to 12, wherein said pharmaceutically active agent is delivered non-system ically to achieve a concentration of ceramide, ceramide analogue, or phytoceramide in the inflammatory cells of from 100 amol to 5 fmol.

15. The method of any one of claims 12 to 14, wherein said pharmaceutically active agent is a ceramide, a ceramide analogue, a phytoceramide or a compound which is metabolisable into or otherwise capable of releasing a ceramide, a ceramide analogue or a phytoceramide, and is administered at by administering a dosage of 10 to 100 mg/day.