WO1998002153A1

WO1998002153A1 - Ceramide promotors of haematopoiesis

Info

Publication number: WO1998002153A1
Application number: PCT/CA1997/000509
Authority: WO
Inventors: Atsuo Ochi
Original assignee: The John P. Robarts Research Institute
Priority date: 1996-07-16
Filing date: 1997-07-16
Publication date: 1998-01-22
Also published as: GB9614889D0; AU3533997A

Abstract

The invention provides methods and pharmaceutical compositions employing cell permeable ceramides to increase haematopoiesis in mammals.

Description

CERAM1DE PROMOTERS OF HAEMATOPOIESIS

Field of the Invention This invention relates to promotion of haematopoiesis. More particularly, the present invention relates to the use of ceramides to stimulate haematopoiesis.

Background of the Invention The best available treatments for a number of diseases are accompanied by serious, undesirable side-effects. For example, chemotherapy and radiation therapy for cancer frequently result in reduced haematopoiesis. Similar side-effects are also seen as a result of the immunosuppressive therapies required for organ transplantation. For these therapies to be successful, it is frequently necessary to treat the patient also with promoters of haematopoiesis.

Currently, the drugs available for promotion of haematopoiesis are the naturally occurring haematopoietic growth factors. Those principally used clinically are granulocyte-colony stimulating factor (G-CSF), granulocyte- macrophage-colony stimulating factor (GM-CSF), thrombopoietin (Tpo) and erythropoietin. Of these, only thrombopoietin is an efficient stimulator of platelet production.

All of the group of naturally occurring growth factors are proteins, which are expensive to produce in vitro for clinical use. They are also subject to rapid breakdown in the body, particularly in the gastro-intestinal tract, so that they cannot conveniently be administered orally.

Sphingomyelin-ceramide turnover has been implicated in the signalling pathways of cell differentiation, but its role in these complex processes is poorly understood (Gill et al., (1994), Immunological Review, No. 142, p. 114). It has now been shown that ceramides are effective agents for increasing haematopoiesis in mammals. Summary of the Invention

In accordance with one embodiment, the invention provides a method for increasing haematopoiesis in a mammal comprising administering to the mammal an amount of a cell permeable ceramide or a derivative thereof effective to increase haematopoisis.

In accordance with a further embodiment, the invention provides a method for treating a disorder in a mammal associated with a reduced level of blood cells of a least one hematopoitic lineage comprising administering to the mammal an amount of a cell permeable cermide or a derivative thereof effective to increase the level of said blood cells.

In accordance with a further embodiment, the invention provides a method for increasing production of cells of at least one hematopoietic lineage in a mammal comprising administering to the mammal an amount of a cell permeable ceramide or a derivative thereof effective to increase production of cells of said at least one haematopoietic lineage.

In accordance with a further embodiment, the invention provides a method for increasing haemoglobin production in a mammal comprising administering to the mammal an amount of a cell permeable ceramide or a derivative thereof effective to increase haemoglobin production.

In accordance with a further embodiment, the invention provides a pharmaceutical composition comprising an effective amount of a cell permeable ceramide or a derivative thereof and a pharmaceutically acceptable carrier.

Brief Description of the Drawings

Certain embodiments of the invention are described, reference being made to the accompanying drawings, wherein:

Figure 1 shows the chemical structures of some of the compounds referred to herein.

Figure 2 shows a photograph of a Northern blot probed with (upper panel) a probe specific for the transcription factor GATA-1 mRNA and (lower panel) a probe specific for glyceraldehyde 3 ' phosphate dehydrogenase

(GAPDH) mRNA.

Figure 3 shows platelet number (Y axis) at indicated days after treatment (X axis) in mice treated with 1 mg C₂-ceramide (•) , C₂- dihydroceramide (O) or vehicle (■) .

Figure 4 shows individual platelet number values for two mice treated intra-peritoneally with 1 mg C₂-ceramide.

Figure 5 shows an autoradiograph of p21 -activated kinase stimulated by C₂ -ceramide or C₂-dihydroceramide. Figure 6 shows individual platelet number values for mice treated orally with C₂-ceramide (hatched bars) or vehicle (open bars).

Figure 7 shows a Western blot of cell proteins probed with a probe specific for p21^CIPl.

Detailed Description of the Invention

The present invention provides methods and pharmaceutical compositions employing ceramides for increasing haematopoiesis in vivo in mammals.

The ceramides are a family of sphingolipids in which the amino alcohol sphinogosine is linked via an amide bond to a fatty acid. The fatty acid may be satured or unsaturated. The structure of a typical ceramide is shown in Figure 1 , (C_n-ceramide) where the fatty acid is represented by -CO(CH₂)_N- CH₃.

Although the term "ceramide" is sometimes reserved for the C14 compound, the most common fatty acid chain length in mammals, the term "ceramide" as used herein includes C14 ceramide and other analogues of the formula C_n-ceramide of Figure 1 having different fatty acid chain lengths.

Ceramides with shorter chain fatty acids penetrate freely through the cell membrane of mammalian cells; ceramides which can penetrate the membrane of mammalian cells are referred to herein as "cell permeable ceramides". Cell permeable ceramides include ceramides from C2 ceramide at least up to C18-ceramide.

The present invention also includes derivatives of cell permeable ceramides which are effective to stimulate haematopoiesis. Ceramide derivatives include ceramide phosphates, glucosyl ceramides and dihydroceramides (Figure 1 ).

In accordance with one embodiment, the present invention provides a method for increasing haematopoiesis in a mammal by administering to the mammal a cell permeable ceramide or a derivative of a cell permeable ceramide in an amount effective to increase haematopoiesis in the mammal.

Haematopoiesis is responsible for the production in mammals of circulating blood cells of various types, including lymphocytes, granulocytes, monocytes, macrophages, eosinophils, mast cells and erythrocytes and of platelets. Each of these cell types is derived from a separate haematopoietic cell lineage. These lineages are believed to be derived from a common progenitor or stem cell.

In accordance with further embodiments, the invention provides methods for increasing production of platelets and leukocytes, and for increasing the platelet count and leukocyte count in a mammal by administering a cell permeable ceramide.

In accordance with a further embodiment, the invention provides a method for increasing haemoglobin production and haemoglobin content of erythrocytes in a mammal by administering a cell permeable ceramide.

The invention provides methods of treatment for disorders in which a subject suffering from the disorder has a reduced level of blood cells of one or more haematopoietic cell lineages by administration to the subject of a cell permeable ceramide. Such disorders include anemias, for example aplastic anemia and autoimmune anemia, and bone marrow suppression resulting, for example, from immunosuppressive therapy or from radiation therapy or chemotherapy for cancer. In accordance with a further embodiment, the invention provides pharmaceutical compositions comprising a cell permeable ceramide or an effective derivative thereof and a pharmaceutically acceptable carrier.

Cell permeable ceramides for use in the methods and pharmaceutical compositions of the invention include C2 to C18 ceramides (Cn-ceramides where n is 2 to 18). Cn-ceramides where n is 2 to 8 are preferred. Cn- ceramides where n is 2, 4, 6, or 8 are especially preferred.

Effective derivatives of cell permeable ceramides may also be used in the methods and compositions of the invention, including C2 to C18 dihydroceramide, C2 to C18 ceramide-1 -phosphate, C2 to C18 glucosyl ceramide, C2 to C18 dihydroceramide-1 -phosphate and C2 to C18 dihydroglucosyl ceramide.

Ceramide derivatives may be screened for effectiveness, for example, by examining their effect on the platelet count of mice, as described herein. Cell permeable ceramides are of low molecular weight and can be very conveniently synthesised by acylation of neutral sphingosine with the appropriate aliphatic carboxylic acid anhydride. For example, caproic anhydride is used to prepare C6 ceramide. For the corresponding dihydroceramides, dihydro-sphingosine (sphinganine) is acylated with the appropriate anhydride. Suitable synthetic methods are described in the chemical literature, for example, in Okazaki et al. (1990), J. Biol. Chem., v. 265, pp. 15823 - 15831 and Gaver et al., (1996), J. Amer. Chem. Soc, v. 88, pp. 3643 - 3647, the contents of which are incorporated herein by reference. Many ceramides are also available commercially. Ceramide derivatives may also be prepared by methods described in the chemical literature. For example, ceramide-1 -phosphates may be prepared by phosphorylation of C_n-ceramide using diacylglycerol kinase as described in Olivera et al., (1992), J. Biol. Chem., v. 276, pp. 26121 - 26127 and glucosyl ceramides may be prepared by deoxyglucosylation of C_n- ceramides using glucosyltranferase, as described in Suzuki et al., (1982), Biochem. Biophys. Acta, v. 710, pp. 221 - 229. The chemical structure of ceramides is such that they are more resistant to degradation in the body than the protein growth factors previously available for haematopoiesis promotion. Ceramides are effective to promote haematopoiesis when administered orally or parenterally. 5 The pharmaceutical compositions of the invention may include a variety of pharmaceutically acceptable carriers, depending on the method of administration to be used for the composition.

Cell permeable ceramides may be administered by intravenous, intramuscular or subcutaneous injection or by oral, nasal, buccal, rectal, l o vaginal, transdermal or ocular routes in a variety of formulations, as is known to those skilled in the art.

For intravenous infusion, ceramides may be prepared in a water- based, physiologic buffer. More concentrated ceramide preparations, for administration for example by intra-muscular injection, may be prepared as 15 lipid vesicles or micelles by techniques known to those of ordinary skill in the art.

The pharmaceutical compositions of the present invention also provide the convenience of oral administration. Ceramides may be dissolved in any suitable edible oil, for example olive oil, for oral administration. 20 Micelle preparations of ceramides may also be used for oral or nasal administration and creams containing ceramides may be used for topical mucosal administration.

The dosage level and frequency of administration of the pharmaceutical composition of the invention will be the level and frequency 25 required to provide a desired level of haematopoiesis or other outcome in the treated subject. A dosage in the range of about 10 mg to about 50 mg / kg body weight of the treated subject is likely to be a suitable initial treatment dosage and the dosage may then be optimised by reference to the response of the treated subject, as can be determined by those of ordinary skill in the 30 art.

In accordance with a further series of embodiments, the invention provides methods for increasing haematopoiesis in a mammal by administration of a combination of a cell permeable ceramide and one or more haematopoietic growth factors. The administration of a cell permeable ceramide in combination with a haemtopoietic growth factor enhances the haematopoietic effect of a given dose of growth factor. Such combination therapy may involve simultaneous administration of a cell permeable ceramide and a haematopoietic growth factor, administration of a cell permeable ceramide after pretreatment with a growth factor or administration of a growth factor after pretreatment with a cell permeable ceramide. The combination therapy may be repeated at selected intervals to produce a desired level of haematopoiesis.

A cell permeable ceramide and a haematopoietic growth factor may be prepared as a single pharmaceutical composition for combination therapy. For example, a micelle preparation may include a ceramide and a growth factor. Suitable haematopoitic growth factors for administration in combination with a cell permeable ceramide include G-CSF, GM-CSF, Tpo and EPO.

EXAMPLES The examples are provided for illustration and are not intended to limit the invention. Materials and methods

Balb/c mice (H-2^d female, 5-7 wks of age) were purchased from Jackson Laboratory (Bar Harbor, ME). The human erythroid leukemia cell line, K562, and mouse thymoma cell line, EL4, were obtained from American Type Culture Collection, Rockville, MD. C2-, C6- and C8- ceramides and dihydroceramides were purchased from BioMol or Metrea, dissolved in absolute alcohol at a concentration of 10 mM and used as the stock reagent. For in vivo injections, ceramides were further diluted with polyethylene glycol, PEG600 (Fluka), to provide the desired dose of ceramide in a volume of 0.1 ml. For cell culture studies, ceramides were further diluted in growth medium. 0.1 ml ceramide suspension was injected into the peritoneal cavity by

23 gauge needle. 120 μl blood samples were obtained from the eye venous plexus using 100 μl capillary pipettes (Fisher Scientific). 5 μl of 250 mM EDTA was immediately added to 100 μl blood and was diluted into 200 μl preservation buffer (10 mM NaCl/150 mM KCI/10 mM glucose/1 mM MgCl^.δ mM potassium phosphate) and platelet number and haemoglobin concentration were determined using standard techniques on a Coulter

Center Model STKR (Coulter Science) in accordance with the manufacturer's instructions. 10 μl of blood was used for the assay of haematocrit in 10 μl glass capillary. Example 1 : K562 human erythroid leukemia cells (3x10⁶/ml) were cultured in medium (RPMI1640, 5% FCS, 2ME 5 x10^"5) containing 10 μM C6-ceramide and cells were harvested after 3 or 5 hours culture. Control cells were cultured with vehicle (medium containing 3-5 μl/ml ethanol) alone. Total cellular RNA was extracted by the Guanidine-phenol method of Chomczyns et al., (1987), Anal. Biochem, v. 162, p. 156, and fractionated by electrophoresis on 1% agarose gel and transferred to nylon membrane by vacuum blotting method. The membrane was probed with a ³²P-dCTP- labeled GATA-1 -specific DNA fragment, prepared by reverse PCR and representing approximately 0.63kb DNA sequence from 5 ' initiation codon. A GAPDH (glyceraldehyde 3' phosphate dehydrogenase) - specific probe was used as positive control probe to indicate the amounts of RNA loaded. Both C6-ceramide and control groups had a similar level of RNA. The results are shown in Figure 2. GATA-1 mRNA increased during incubation with C6-ceramide. Example 2:

7 Balb/c mice were treated with 1 mg C2-ceramide in 0.1 ml PEG600 by intraperitoneal injection and blood samples were collected at intervals for determination of platelet number. A control group of 7 mice was treated with vehicle alone. The results are shown in Figure 3. At 8 days after treatment, C2- ceramide-treated animals showed a 20% increase in platelet number compared to control animals (p = 0.003, < 0.005). By 15 days after treatment, the platelet level had begun to decline towards basal level.

Figure 4 shows the individual platelet number values for two of the mice treated with 1 mg C2-ceramide. Platelet number peaked around 8 days after treatment (data not shown).

C2-dihydroceramide was used in a similar study and gave the same stimulation of platelet number as that seen with C2-ceramide (Figure 3). Example 3:

6 Balb/c mice were treated with 0.25 mg C2-ceramide in 0.1 ml PEG600 by intraperitoneal injection. Control mice were treated with vehicle.

Haematocrit values and haemoglobin concentrations were measured at various time intervals after treatment. The haematocrit value showed a maximum reduction of about 20% at day 15 after treatment (P = 0.003) and by day 22 had returned to normal, although haemoglobin concentration was not changed. The results at Day 15 are shown in Table 1.

In order to evaluate the haemoglobin content relative to RBC volume, haemoglobin concentration values were divided by haematocrit values to give a haemoglobin/total RBC volume index. This index was increased significantly, compared to baseline value, at day 15. The haematocrit samples at day 8 and day 15 after treatment showed a 30 to 50% increase in the volume of buffy coat, indicating that this treatment increases the proportion of white blood cells.

C2-dihydroceram.de was used in a similar study and gave the same effect on haemoglobin/total RBC volume index as that seen with C2- ceramide. Example 4:

3 Balb/c mice were injected with 50 μg staphylococcal enterotoxin B (SEB, Sigma), which is known to activate about a third of T cells in mice. A similar group of mice were injected with 50 μg SEB plus 1 mg C6-ceramide. Two days after treatment, mice were sacrificed and spleen size measured. Both treatment groups had similar spleen enlargement (about three times larger by weight than normal) and both had a large number of T cell blasts in spleen and lymph node, indicating that the treatment with ceramide does not impair SEB stimulation of T cells in vivo.

Example 5: There have been previous reports of stimulation of differentiation of HL

60 cells (Okazaki et al., (1990), J. Biol. Chem., v. 265, p. 15823; Bielawska et al., (1992), J. Biol. Chem., v. 287, p. 18493) and of apoptosis (Obeid et al.,

(1993), Science, v. 259, p. 1769), by ceramides but not by dihydroceramides.

This is in contrast to the effect of C2-ceramide and C2-dihydroceramide on haematopoiesis, where the dihydro compound was as active as the reduced compounds, indicating that the observed effects are not dependent on the presence of the 4-5 trans double bond.

The effect of C2-ceramide and C2-dihydroceramide on activity of a serine/threonine-specific, p21 -activated kinase (PAK) in a haematopoietic cell line, the T lymphocyte line EL4, was examined. PAK's have been implicated in the mechanism of stimulation of cell differentiation by ceramides. PAK assay

The mouse thymoma cell line EL4 was maintained in RPMI 1640 with 10% fetal calf serum and 40μg/ml of gentamycine. Cells were resuspended to 1 x 10⁷ per ml of serum-free medium and stimulated with 100nM C2 ceramide, 100nM C2-dihydroceramide, or vehicle (ethanol) for 2 min at 37°C. The cells were then lysed in 1ml of RIPA buffer (20mM Tris (pH7.4), 1% Triton X-100, 0.1% SDS, 150mM NaCI, 0.5% sodium deoxycholate, 2mM EGTA, 1 mM sodium molybdate, 1mM orthovanadate, and 20 μg/ml aprotinin) followed by centrifugation at 17000g for 20 min at 4°C. After preclearing the lysates with protein A/G agarose, immunoprecipitates were prepared by the addition of 1 μg of affinity purified antibody to PAK (santa Crux Biotechnology) to the lysates for 1 hr at 4°C followed by 25μl of protein A/g agarose. Immune complexes were washed twice in RIPA buffer, once in RIPA buffer containing 0.5M LiCI instead of NaCI, twice in RIPA buffer, and twice in kinase buffer (50mM HEPES (pH7.5), 10mM MnCI₂, 10mM MgCI₂ and 1mM DTT). The kinase reactions were performed in 18μl of kinase buffer containing 10μg of myelin basic protein (MBP, Sigma), 5μM ATP, and 20μCi [γ-³²P] ATP. After

15 min incubation at 30°C, the reactions were stopped by addition of denaturing SDS-PAGE buffer. [32P] - labeled proteins were resolved by

SDS-PAGE and visualised using a Molecular Imager (BioRad).

5 The results are shown in Figure 5. C2-ceramide and C2- dihydroceramide stimulated PAK activity to a similar extent.

Example 6:

3 Balb/c mice were orally administered with 1mg C2 ceramide in 0.1 ml

PEG600 and blood samples were collected on day 9 for determination of l o platelet number. A control group of 3 mice was treated with vehicle alone (0.1 ml PEG600).

The results are shown in Figure 6. Each bar indicates the platelet number of an individual mouse. C2-ceramide-treated animals showed -15% increase in mean platelet number compared to control animals.

15 Example 7:

Induction of cyclin-dependent kinase inhibitor p21 ^CIP in response to C2 ceramide was observed using human histiocytoma U937 cells. 2 x 10⁶ U937 cells were incubated for 24 hrs. with 5μM C2-ceramide, 40 nM phorbol, 12-myristate, 13-acetate (PMA) or a combination of C2 ceramide and PMA.

20 Control cells were incubated with vehicle (DMSO). Cells were pelleted, resuspended in residual media and lysed in 100μl of lysis buffer (25mM Tris, pH 7.4 50mM NaCI, 0.5% sodium deoxycholate, 2% Nonidet p-40, 0.2% SDS, 50μg/ml aprotinin, 20μg/ml leupeptin, 50mM NaF). Lysates were immediately clarified by centrifugation at 17000g for 15 min at 4°C. Whole cell lysates

25 were analyzed by 14% SDS-PAGE and proteins were transfered to nitrocellulose membrane and the blots blocked overnight in 5% nonfat dried skimmilk in PBS + 0.1% Tween 20. Blots were incubated overnight 4°C with anti-p21 ^C1P1 at 0.5μg/ml. Results are shown in Figure 7.

P21 ^CIP1 was induced by C2-ceramide treatment. This induction was

30 inhibited by PMA. Example 8: A number of groups of mice are treated weekly for 2 to 6 weeks with GM-CSF (0.05μg/mouse — sub-optimal dosage for granulocyte and macrophage stimulation by GM-CSF) by intravenous injection and either orally or intraperitoneally with a different dose of C2-ceramide per group (eg. 5 0.1 , 0.2, 0.4 and 0.8 mg/mouse). A control group is treated with GM-CSF (0.05 mg/mouse) and vehicle instead of C2-ceramide.

Blood samples are collected weekly and analysed for numbers of B lymphocytes, T lymphocytes, natural killer cells, neutrophilic granulocytes, monocytes/macrophages, eosinophils and megakaryocytes. l o This experiment is repeated with different doses of GM-CSF to determine the optimal combination or combinations of GM-CSF and ceramide doses for synergistic enhancement of granulocyte and macrophage proliferation. Example 9:

15 Groups of mice are treated as described in Example 8 but employing

G-CSF, Epo or Tpo instead of GM-CSF, at doses sub-optimal for hematopoietic stimulation by the particular growth factor. The optimal combination or combinations of growth factor and ceramide doses for synergistic enhancement of haematopoiesis is determined as described.

20 Example 10:

Groups of mice are treated with various doses of C2-ceramide at various time intervals (1 , 2, 4 or 8 days) before treatment with a sub-optimal dose of a hematopoietic growth factor, as described in Example 8. Similar treatments may be repeated bi-weekly. Analysis of blood samples and

25 determination of optimal growth factor and ceramide dose combinations are carried out as described in Example 8. Example 11:

New Zealand Black (NZB) mice, which spontaneously develop autoimmune disease, usually characterised by an autoimmune haemolytic

30 anemia, are treated with doses of 0.2 to 1.5 mg/mouse of C2-ceram.de in 0.1 ml PEG 600 intraperitoneally at weekly intervals for 2 to 6 weeks. Blood samples are collected weekly for determination of erythrocyte count and haemoglobin level. A control group of mice are treated with vehicle alone.

C2-ceramide treatments are similarly administered to groups of WCB6F/J, Mof^/Mαf^ or WBB6F1/J - Kit^w/Kit -^U mice, all of which have inherited defects leading to anemia and blood samples are analysed as described.

The present invention is not limited to the features of the embodiments described herein, but includes all variations and modifications within the scope of the claims.

volume of whole blood.

Claims

Claims:

1. A method for increasing haematopoiesis in a mammal comprising administering to the mammal an amount of a cell permeable ceramide or a derivative thereof effective to increase haematopoiesis.

2. The method of claim 1 wherein the cell permeable ceramide or derivative thereof is selected from the group consisting of

(a) C2 to C18 ceramide; (b) C2 to C18 dihydroceramide;

(c) C2 to C18 ceramide-1 -phosphate;

(d) C2 to C18 glucosyl ceramide;

(e) C2 to C18 dihydroceramide- 1 -phosphate; and

(f) C2 to C18 dihydroglucosyl ceramide.

3. The method of claim 1 wherein the cell permeable ceramide or derivative thereof is selected from the group consisting of C2 ceramide to C8 ceramide.

4. The method of claim 1 wherein the cell permeable ceramide or derivative thereof is selected from the group consisting of C2 ceramide, C4 ceramide, C6 ceramide and C8 ceramide.

5. A method for increasing haematopoiesis in a mammal comprising administering to the mammal a combination of a cell permeable ceramide or an effective derivative thereof and at least one haematopoietic growth factor in a combined amount effective to increase haematopoiesis.

6. The method of claim 5 wherein the hematopoietic growth factor is selected from the group consisting of granulocyte-colony stimulating factor, granulocyte-macrophage-colony stimulating factor, erythropoietin and thrombopoietin.

7. A method for treating a disorder in a mammal associated with a reduced level of blood cells of at least one hematopoietic lineage comprising administering to the mammal an amount of a cell permeable ceramide or a

5 derivative thereof effective to increase the level of said blood cells.

8. The method of claim 7 wherein the disorder is selected from the group consisting of anemia or bone marrow suppression.

0 9. A method for increasing production of cells of at least one hematopoietic lineage in a mammal comprising administering to the mammal an amount of a cell permeable ceramide or a derivative thereof effective to increase production of cells of said at least one haematopoietic lineage.

5 10. The method of claim 9 wherein the cells of at least one hematopoietic lineage are selected from the group consisting of platelets and leukocytes.

11. A method for increasing haemoglobin production in a mammal comprising administering to the mammal an amount of a cell permeable o ceramide or a derivative thereof effective to increase haemoglobin production.

12. A pharmaceutical composition comprising an effective amount of a cell permeable ceramide or a derivative thereof and a pharmaceutically acceptable carrier. 5

13. The pharmaceutical composition of claim 12 further comprising an effective amount of at least one haematopoietic growth factor.

14. The pharmaceutical composition of claim 12 or 13 wherein the cell 0 permeable ceramide or derivative thereof is selected from the group consisting of (a) C2 to C18 ceramide;

(b) C2 to C18 dihydroceramide;

(c) C2 to C18 ceramide-1 -phosphate;

(d) C2 to C18 glucosyl ceramide; (e) C2 to C18 dihydroceramide- 1 -phosphate; and

(f) C2 to C18 dihydroglucosyl ceramide.

15. The pharmaceutical composition of claim 12 or 13 wherein the cell permeable ceramide or derivative thereof is selected from the group consisting of C2 ceramide to C8 ceramide.

16. The pharmaceutical composition of claim 12 or 13 wherein the cell permeable ceramide or derivative thereof is selected from the group consisting of C2 ceramide, C4 ceramide, C6 ceramide and C8 ceramide.

17. The pharmaceutical composition of any claims 13 to 17 wherein the haematopoietic growth factor is selected from the group consisting of granulocyte-colony stimulating factor, granulocyte-macrophage-colony stimulating factor, erythropoietin and thrombopoietin.

18. The method of any claims 1 to 11 wherein the ceramide or derivative is administered orally.

19. The method of any claims 1 to 11 wherein the ceramide or derivative is administered parenterally.

20. The method of any claims 1 to 11 , 18 or 19 wherein the mammal is a human.