WO2014000027A1

WO2014000027A1 - Prevention and treatment of haematological conditions

Info

Publication number: WO2014000027A1
Application number: PCT/AU2013/000682
Authority: WO
Inventors: Andrew Hutchinson
Original assignee: University Of Technology, Sydney
Priority date: 2012-06-26
Filing date: 2013-06-25
Publication date: 2014-01-03

Abstract

The present invention relates generally to methods for the prevention and treatment of haematological conditions that exhibits elevated levels of free immunoglobulin light chains (FLCs), including multiple myeloma, B-cell lymphomas, and associated diseases and conditions.

Description

TITLE OF THE INVENTION

PREVENTION AND TREATMENT OF HAEMATOLOGICAL CONDITIONS

FIELD OF THE INVENTION

[0001] This invention relates generally to methods for preventing or treating haematological conditions, including haematological malignancies and pre-malignancies, that exhibit elevated levels of free immunoglobulin light chains (FLCs), including multiple myeloma, B-cell lymphomas, and their associated diseases and conditions.

BACKGROUND OF THE INVENTION

[0002 ] Multiple myeloma (MM) is a plasma cell malignancy characterised by the uncontrolled proliferation of plasma cells and the accumulation of their secreted free immunoglobulin light chains (FLCs).

[0003] A complication frequently observed in patients suffering from MM is the deposition of FLC aggregates in organs. These can be as ordered amyloid fibrils (referred to as LC amyloidosis), or amorphous deposits (referred to as LC deposition disease).

[0004] In work leading up to the present invention, the inventors determined that FLCs interact with sphingomyelin, a major component of cellular membranes, and that this interaction appears to promote aggregation and stable association of the FLC with the lipid bilayer of myeloma plasma cells (Hutchinson et ah 2010).

[0005] During some of the experiments performed in researching these FLC aggregates, the inventors employed GW4869, which is a highly specific small molecule neutral sphingomyelinase inhibitor. Neutral sphingomyelinase is responsible for the breakdown of sphingomyelin into ceramide and phosphocholine. Ceramide plays a key role in apoptosis, and thus GW4869 (which inhibits sphingomyelinase and thus the production of ceramide) is used in many assays because of its anti-apoptotic properties.

[0006] Surprisingly, the inventors discovered that GW4869 induces cell death in multiple myeloma cells, and that this cytotoxic activity is not observed in other cell lines tested.

[0007] Without wishing to be bound by theory or mode of operation, the present inventors propose that while the interaction between FLCs and sphingomyelin promotes FLC aggregation and stable association within the lipid bilayer, these membrane bound FLC aggregates are in fact cytotoxic as they lead to disruption of cell membrane integrity, leakage of extracellular components such as calcium ions across the membrane, and thus cell death. The present inventors have also found that inhibition of neutral sphingomyelinase leads to an accumulation of FLC aggregates in the endoplasmic reticulum (ER), which is proposed to cause ER stress. Based on these proposals, the present inventors consider that by increasing the sphingomyelin levels (e.g. , by addition neutral sphingomyelinase inhibitors, illustrative examples of which include GW4869 and sphingolactone 24), the number of FLC aggregates accumulating in the ER and bound to sphingomyelin within the lipid bi layer of the multiple myeloma cells is increased, leading to death of these cells. As such, it is believed that this discovery can be applied therapeutically to induce selective toxicity in multiple myeloma cells in an individual suffering from multiple myeloma.

[0008] Levels of FLCs are also elevated in some other haematologicai malignancies, including B-cell lymphomas, in some pre-malignancies, including monoclonal gammopathy of undetermined significance (MGUS), and in associated disease or conditions, including AL amyloidosis and LC deposition disease. As such, the present inventors also propose that increasing sphingomyelin levels in these other haematologicai conditions that exhibit elevated levels of FLCs will reduce or inhibit the proliferation of the malignant or undesirable cells, and/or to stimulate their death. SUMMARY OF THE INVENTION

[0009] Accordingly, the present inventors consider that an increase in sphingomyelin levels in an individual suffering from a haematologicai condition (e.g., haematologicai malignancy, pre-malignancy, or associated disease or condition) that exhibits elevated levels of FLCs will lead to an increase in the number of FLC aggregates accumulating in the ER and bound to sphingomyelin within the lipid bilayer of malignant or undesirable cells and thus to a reduction or inhibition in the proliferation or to the death of malignant or undesirable cells in the haematologicai condition. The

haematologicai condition may be multiple myeloma, a B-cell lymphoma, or an associated disease or condition.

[0010] Accordingly, the present inventors propose that increasing levels of sphingomyelin in an individual will be beneficial in the prophylaxis or treatment of a haematologicai condition that exhibits elevated levels of FLCs. [0011] Thus, in a first aspect the present invention provides an agent that increases the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of FLCs in the individual.

Suitably, the haematological condition is multiple myeloma, B-cell lymphoma, or an associated disease or condition. Suitably, the agent comprises an inhibitor of neutral sphingomyelinase.

[0012] In another aspect, the present invention provides a method of preventing or treating a haematological condition that exhibits elevated levels of FLCs in an individual in need thereof, the method comprising administering to the individual an agent that increases the level of sphingomyelin in the individual. Suitably, the haematological condition is multiple myeloma, B-cell lymphoma, or an associated disease or condition. Suitably, the agent comprises an inhibitor of neutral

sphingomyelinase.

[0013] The present invention also provides a use of an agent that increases the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of FLCs in the individual. Suitably, the haematological condition is multiple myeloma, B-cell lymphoma, or an associated disease or condition. Suitably, the agent comprises an inhibitor of neutral

sphingomyelinase.

[0014] The present invention also provides a use of an agent that increases the level of sphingomyelin in an individual in the manufacture of a composition for preventing or treating a haematological condition that exhibits elevated levels of FLCs in the individual. Suitably, the haematological condition is multiple myeloma, B-cell lymphoma, or an associated disease or condition. Suitably, the agent comprises an inhibitor of neutral sphingomyelinase.

[0015] In illustrative embodiments of aspects of the present invention, the agent that increases the level of sphingomyelin comprises a compound having the structure:

[0016J or a pharmaceutically acceptable salt thereof. Specifically contemplated is the dihydrochloride salt thereof, namely the compound 3,3'-(l ,4- phenylene)bis[N-[4-(4,5-dihydro-lH-imidazol-2-yl)phenyl]-hydrochloride-2- propenamide (also termed "GW4869" and having CAS number: 6823-69-4).

[0017] In yet another aspect, the invention provides methods for identifying agents that increase the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of FLCs.

[0018] One suitable method comprises contacting a sample of cells expressing sphingomyelin with a test agent, wherein a detected increase in sphingomyelin relative to a reference or control level in the absence of the test agent, indicates that the agent increases sphingomyelin levels and that it is useful for preventing or treating a haematological condition that exhibits elevated levels of FLCs.

[0019] Another suitable method comprises contacting a sample containing sphingomyelin (or an analogue thereof) and sphingomyelinase (e.g., neutral

sphingomyelinase, illustrative examples of which include nSMasel (encoded by the sphingomyelinase (SM) phosphodiesterase 2 (Smpd2) gene), nSMase2 (encoded by the Smpd3 gene) and nSMaseS (encoded by the Smpd4 gene)) with a test agent, wherein a detected decrease in the level of sphingomyelin degradation (e.g. , a higher level of sphingomyelin or a lower level of ceramide and/or phosphocholine produced) relative to a reference or control level in the absence of the test agent, indicates that the agent inhibits or otherwise antagonises sphingomyelinase activity and is therefore an agent that increases sphingomyelin levels useful for preventing or treating a haematological condition that exhibits elevated levels of FLCs. [0020] In accordance with the present invention, the agents identified using the methods broadly described above are useful for preventing or treating a

haematological condition that exhibits elevated levels of FLCs.

[0021] Still another aspect of the present invention provides methods of producing an agent that increases the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of FLCs in the individual. These methods generally comprise: testing a test agent as broadly described above; and synthesising the agent on the basis that it tests positive for increasing sphingomyelin levels. Suitably, the method further comprises derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing a haematological condition that exhibits elevated levels of FLCs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a graphical representation showing the results of incubating MM cell lines (NCI-H929, JJN-3, ARH-77, and RPMI-8226) and non- MM cell lines (RAW264.7, K562, HEK293 and Toledo) with various concentrations of GW4869 for 3 days and assessing cell death by propidium iodide or TO-PRO-3 uptake. GW4869 was found to be cytotoxic for each MM cell line but not for the non-MM cell lines. Percentage viable cells normalised to DMSO vehicle control.

[0023] Figure 2 is a graphical representation showing the results of incubating MM cell lines (NCI-H929, OPM-2, RPMI-8226 and JJN-3) and a non-MM cell line ( 562) with various concentrations of sphingolactone 24 for 2 days and assessing cell death by propidium iodide or TO-PRO-3 uptake. Sphingolactone 24 was found to be cytotoxic for each MM cell line but not for the non-MM cell line. Percentage viable cells normalised to DMSO vehicle control.

[0024] Figure 3 is a graphical representation showing the results of the Example 3 in vivo experiment performed using a MM murine model. Mice were treated with GW4869 or a vehicle control for 2 weeks and serum FLC levels and the number of engrafted MM cells assessed. As can be seen in Figure 3A, the serum FLC levels of GW4869-treated mice were significantly lower than the control group. Figure 3B shows the percentage of MM cells (as a percentage of total cells) in the femur/tibia in both groups of mice. Figure 3C shows the survival of GW4869 and vehicle control treated mice (9-10 mice per group) over 94 days after commencement of the treatment (p value < 0.05).

[0025] Figure 4 is a graphical representation showing the effect of GW4869 on primary myeloma cells. Ficoll purified bone marrow cells (IgG kappa myeloma) cultured for 24h in medium (RPMI8226/10%FCS/PSG/Hepes), medium supplemented with vehicle or GW4869. Proportion of myeloma cells (defined as CD138+ or CD38+ events) relative to other mononuclear cells in the bone marrow were determined by flow cytometry. (A) Representative dot plots of CD 138 vs. SSC show proportions of myeloma cells (CD 138+ events) recovered following no treatment, GW4869 and vehicle treatment. (B) Proportions of myeloma cells (defined as CD138+ or CD38+ events) recovered following no treatment, increasing concentration of vehicle (0.06-0.62% DMSO) or GW4869 (2.2-22 μΜ).

[0026] Figure 5 is a graphical representation showing that nSMase2 is upregulated in MM primary cells and cell lines. qRT-PCR quantification of nSMase2 is shown in non-MM cells (blue histograms) and MM cells (red histograms). All data are normalised to qRT-PCR quantification of nSMase2 in PBMC (peripheral blood mononuclear cells).

[0027] Figure 6 is a photographic representation showing the results of treating RPMI8226 MM cells with GW4869 or DMSO vehicle control for 18h, fixing the treated cells with 4% paraformaldehyde, permeabilising the cells with 0.01 % digitonin and staining the cells intracellularly with anti-FLC mAb AlexaFluor 488 (mAb clone #4G7, Abeam). Stained cells were than assessed by confocal microscopy. GW4869 was found to cause accumulation of intracellular λΡΙ in treated MM cells.

[0028] Figure 7 is a graphical representation showing the results of treating RPMI8226 and OPM2 MM cells with GW4869 or DMSO vehicle control for 18h, fixing the treated cells with 4% paraformaldehyde, permeabilising the cells with 0.01 % digitonin and staining the cells intracellularly with anti-FLC mAb AlexaFluor 488 (mAb clone #4G7, Abeam). Stained cells were than assessed by flow cytometry. GW4869 was found to cause accumulation of intracellular XFLC in treated MM cells.

[0029] Figure 8 is a graphical representation showing the results of treating

RPMI8226 and OPM2 MM cells with sphingolactone 24 or DMSO vehicle control for 18h, fixing the treated cells with 4% paraformaldehyde, permeabilising the cells with 0.01% digitonin and staining the cells intracellularly with anti-FLC mAb AlexaFluor 488 (mAb clone #4G7, Abeam). Stained cells were than assessed by flow cytometry.

Sphingolactone 24 was found to cause accumulation of intracellular λΡΙ in treated MM cells.

[0030] Figure 9 is a photographic representation showing the results of treating RPM18226 MM cells with 5 μΜ GW4869, fixing the cells with 4%

paraformaldehyde, permeabilising the cells with 0.01% digitonin, and staining the cells intracellularly with anti- FLC mAb AlexaFluor 488 (mAb clone #4G7, Abeam), anti- calnexin (Abeam) + anti-rabbit AlexaFluor 568 and DAPI. Confocal microscopy of the cells (Blue - DAPI (nucleus), red = calnexin (ER marker), green = FLC) revealed that GW4869 causes MM cells to accumulate intracellular λΡΙ in the ER of treated MM cells.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0032] The articles V and "an" are used herein to refer to one or to more than one {i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0033] The term "about" is used herein to refer to conditions {e.g. , amounts, concentrations, times etc) that vary by as much as 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%>, 7%, 6%, 5%, 4%, 3%, 2%, or 1% to a specified condition.

[0034] By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to

immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. [0035] "Antigenic or immunogenic activity^'" refers to the ability of a polypeptide, peptide, fragment, variant or derivative to produce an antigenic or immunogenic response in an animal, suitably a mammal, to which it is administered, wherein the response includes the production of elements which specifically bind the polypeptide or fragment thereof.

[0036] By "biologically active fragment is meant a fragment of a full-length parent polypeptide which fragment retains an activity of the parent polypeptide. As used herein, the term "biologically active fragment'^'' includes deletion variants and small peptides, for example of at least 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 contiguous amino acid residues, which comprise an activity of the parent polypeptide. Peptides of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard which is included in a publication entitled "Synthetic Vaccines" edited by Nicholson and published by

Blackwell Scientific Publications. Alternatively, peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu- C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.

[0037] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises'^'' and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements,

[0038] By "corresponds to" or "corresponding to" (and the like) is meant (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein; and (b) a peptide Or polypeptide having an amino acid sequence that displays substantial similarity and/or identity to a sequence of amino acids in a reference peptide or protein.

[0039] By "derivative" (and the like) is meant an agent that has been derived from a basic or parent compound of agent, including by chemical or sequence

modification, by conjugation or complexing with other chemical moieties or by post- translational modification techniques as would be understood in the art. Methods to produce derivatives of agents, including derivatives of agents that increase levels of sphingomyelin, which are themselves also agents that increase levels of sphingomyelin, are well known in the art, including methods for derivatizing the agent and methods for testing the activity of the derivative.

[0040] By "effective amount" or "effective dose" in the context of preventing or treating a haematological condition that exhibits elevated levels of FLCs in an individual is meant the administration of that amount to an individual in need of such prophylaxis or treatment, either in a single dose or as part of a series, that is effective for the intended purpose, for example the prevention or treatment of a haematological condition that exhibits elevated levels of FLCs as herein defined. The effective amount or effective dose will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the age, sex, and weight of the individual to be treated, the formulation of the composition including the bioavailability and pharmacokinetic parameters of the agent that increases sphingomyelin levels, the route of administration, the assessment of the medical situation including the progression of the haematological condition (if any) over time, and other relevant factors. In this regard, precise amounts of the agent that increases

sphingomyelin levels in the individual for administration will depend on the judgement of the practitioner. In any event, it is expected that the amount or dose will fall in a relatively broad range that can be determined through routine trials.

[0041] The term "expression product" refers to production of mRNA, translation of R A message into proteins or polypeptides, or processed forms of those proteins or polypeptides.

[0042] The term "gene" as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions. The term "gene" is also intended to mean the open reading frame encoding specific polypeptides, introns and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control signals such as promoters, enhancers, termination, and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals. The DNA sequences may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.

[0043] Reference herein to "immuno-inter active" includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.

[0044] The terms "individual", "patient" and "subject" are used

interchangeably herein to refer to individuals of human or other animal origin and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that these terms do not imply that symptoms are present. Suitable animals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals {e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes, avians, reptiles).

[0045] By "isolated'^'' is meant material that is substantially or essentially free from components that normally accompany it in its native state.

[0046] By "modulating" is meant increasing or decreasing, either directly or indirectly, the level or functional activity of a target molecule. For example, an agent may indirectly modulate the level/activity by interacting with a molecule other than the target molecule. In this regard, indirect modulation of a gene encoding a target polypeptide includes within its scope modulation of the expression of a first nucleic acid molecule, wherein an expression product of the first nucleic acid molecule modulates the expression of a nucleic acid molecule encoding the target polypeptide.

[0047] The term "oligonucleotide" as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or

ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotide residues, but the term can refer to molecules of an length, although the term "polynucleotide" or "nucleic acid" is typically used for large oligonucleotides.

[0048] By "operably ' linked" is meant that transcriptional and translational regulatory polynucleotides are positioned relative to a polypeptide-encoding

polynucleotide in such a manner that the polynucleotide is transcribed and the polypeptide is translated.

[0049] By "pharmaceutically acceptable carrier or diluent" is meant a solid or liquid filler, diluent or encapsulating substance that can be safely used in topical or systemic administration to a mammal.

[0050] The term "pharmaceutically acceptable salt" or "pharmaceutically compatible salt" as used herein refers to a salt which is toxicologically safe for administration to an individual. The salt may be formed with acids (including organic acids), including those selected from hydrochloric acids, phosphoric acids, sulphuric acid, acetic acid, oxalic acid, lactic acid, tartaric acid, malic acid, maleic acid, succinic acid, and the like. The salt may be formed with bases (including inorganic bases), including those selected from sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases isopropylamine, trimethylamine, 2-ehtyamino ethanol, histidine, procaine, and the like. In exemplary embodiments, the salt may be selected from a group that including hydrochlorides, hydrobromides, sulphates, bisulphates, nitrates, citrates, lactates, tartrates, bitartrates, phosphates, malates, maleates, napsylates, fumarates, succinates, acetates, terephthalates, pamoates, and pectinates.

[0051]_. The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA, or DNA. The term typically refers to oligonucleotides greater than 30 nucleotide residues in length.

[0052] The terms "polynucleotide variant" and "variant" refer to

polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions as known in the art (see for example Sambrook et al. , Molecular Cloning. A

Laboratory Manual", Cold Spring Harbor Press, 1989). These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide. The terms "polynucleotide variant" and "variant" also include naturally-occurring allelic variants.

[0053] "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0054) The term "polypeptide variant" refers to polypeptides in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter. These terms also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acids.

[0055[ By "preventing or treating", "prevent", "preventing", "prophylaxis", "treatment", "treat", and the like is meant to include both prophylactic and therapeutic treatment, including but not limited to preventing, relieving, altering, reversing, affecting, inhibiting the development or progression of, ameliorating, or curing (1) a haematological condition (including a haematological malignancy, pre-malignancy or associated disease or condition, e.g., multiple myeloma, B-cell lymphoma, AL amyloidosis, LCDD), or (2) a symptom of the haematological condition. In some embodiments, these terms include reducing the number of malignant or undesirable cells, reducing the number of viable malignant or undesirable cells, inhibiting the growth of a malignant or undesirable cell, inhibiting the proliferation of a malignant or undesirable cell, or killing a malignant or undesirable cell. The malignant or undesirable cell may be a cancerous or pre-cancerous cell. The terms also include prolonging or extended the survi val of an individual suffering from the haematological condition. In illustrative embodiments, the

haematological condition may be an early stage cancer or a late stage cancer.

[0056] "Probe" refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term "probe^''^'' typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridisation conditions. Probes can be labelled directly or indirectly.

[0057) The term "recombinant polynucleotide" as used herein refers to a _t polynucleotide formed in vitro by the manipulation of a polynucleotide into a form not normally found in nature. For example, the recombinant polynucleotide can be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory polynucleotide operably linked to the polynucleotide.

[0058] By "recombinant polypeptide^'1'' is meant a polypeptide made using recombinant techniques, i.e. , through the expression of a recombinant or synthetic polynucleotide.

[0059] The term "reference or control level" as used herein refers to any suitable reference or control level, including, but not limited to, a normal healthy individual, an individual without a haematological condition that exhibits elevated FLC levels, a level measured in the same individual at a different time, and a level measured in a biological sample taken from a different tissue in the same individual or control.

[0060] By "reporter molecule" (and the like) as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that allows the detection of a desired compound or complex, including a gene or an expression product of a gene.

[0061] By "vector" is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector may contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.

Accordingly, the vector can be an autonomously replication vector, i. e. , a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated with the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system can comprise a single vector or plasmid, two or more vectors or plamids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In the present case, the vector is suitably a viral or viral-derived vector, which is operably functional in animal and suitably mammalian cells. Such vector may be derived from a poxvirus, an adenovirus, or yeast. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transforrnants. Examples of such resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.

Prevention and treatment methods

[0062] The present invention is based, at least in part, on the surprising discovery that increasing sphingomyelin levels leads to the death of multiple myeloma cells, but not to the other cell lines that were tested. Multiple myeloma cells are known to have elevated levels of FLCs, and the inventors have demonstrated that at least some of those FLCs form protein aggregates that accumulate in the ER and are bound to sphingomyelin in the lipid bilayer of myeloma plasma cells.

[0063] Without wishing to be bound by theory, the inventors postulate that the interaction of FLC aggregates with sphingomyelin in the lipid bilayer of the cell that expresses FLC destabilises the cell membrane and thus decreases cell viability and/or stimulates cell death. It is also postulated that the accumulation of FLC aggregates in the ER lead to binding of the aggregates to the ER membrane, resulting in ER stress and stimulation of cell death. Accordingly, it is believed that by increasing the

sphingomyelin levels in an individual with a haematological condition that exhibits elevated FLC levels, the number of FLC aggregates accumulating within the ER and/or bound to sphingomyelin within the lipid bilayer of the malignant or undesirable cells will increase, and thus proliferation of the malignant or undesirable cells will reduce or be inhibited or the malignant or undesirable cells will be killed. The malignant or undesirable cells may be cancerous cells or pre-cancerous cells. Suitably, reduction or inhibition of the proliferation of the malignant or undesirable cells or the death of the malignant or undesirable cells occurs without substantially reducing or inhibiting proliferation of, or causing death of non-malignant cells.

[0064] Accordingly, the present invention provides an agent that increases the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of PLCs in the individual. The present invention also provides a method of preventing or treating a haematological condition that exhibits elevated levels of FLCs in an individual in need thereof, the method comprising administering to the individual a composition comprising an agent that increases the level of sphingomyelin in the individual. Also contemplated by the present invention is the use of an agent that increases the level of sphingomyelin in the individual for preventing or treating a haematological condition that exhibits elevated levels of FLCs. The present invention also provides the use of an agent that increases the level of sphingomyelin in the individual in the manufacture of a composition for preventing or treating a haematological condition that exhibits elevated levels of FLCs.

[0065] As used herein, an individual with a haematological condition that exhibits elevated levels of FLCs means that the individual has a higher level of FLCs than a reference or control level.

[0066] Suitably, the individual has a higher level of serum FLCs (sFLCs) than a reference or control level of sFLCs. Normal range data for FLCs (including sFLCs) has been published many times, the most reliable being those published in Abraham et al. 1974, Soiling 1975, Brouwer et al. 1985, Axiak et al. 1987, Wakasugi et al. 1991 , Nelson et al. 1 92, Wakasugi et al. 1995, Abe et al. 1998, Bradwell et al. 2001, Katzmann et al. 2002, Nakano et al, 2004, and Nakano et al. 2006. These studies are reviewed in Bradman AR, 2010, see especially Chapter 5. The most detailed of those studies is that published by atzman et al. 2002 which examined samples from 127 healthy blood donors aged 21-62 years, and from 155 health blood donors aged older than 62. The median values and reference ranges from Katzman et al. 2002 are shown in Table 1 below:

[0067] Table 1 : Median values and ranges for free and total light chain concentrations and κ/λ ratios in the sera of 282 normal individuals as determined by Katzman et al. 2002. Free light chains Total light chains

Kappa (95% range) 7.3 mg/L (3.3-19.4) 2.520 mg/L

Lambda (95% range) 12.7 mg/L (5.7-26.3) 1.430 mg/L κ/λ ratio ( 100% range) 0.6 (0.26-1.65) 1.78 (mean) κ/λ ratio (95% range) 0.6 (0.31-1.2) N/A

[0068] Suitably, the individual has a higher level of renal FLCs than a reference or control level of renal FLC levels. Normal range data for renal FLCs is known in the art, including Katzman et al. 2002 which describes a reference level for normal renal κ/λ ratio of FLCs of 0.37-3.1.

[0069] Katzman et al. 2002 also noted that FLC levels in an individual appear to be affected by the age of the individual, with higher sFLC values seen in older people. It is believed these higher levels can be explained by small reductions in glomerular filtration rate (Deinum et al. 2000). Suitably, the individual has a higher level of serum FLCs (sFLCs) than a reference or control level of sFLCs, wherein the reference or control level takes into account the age of the individual (see, especially Bradman AR, 2010, Table 5.3).

[0070] Suitably, the individual with a haematological condition with elevated levels of FLC has a higher level of serum FLCs (sFLCs) than the reference level of Katzman et al. 2002 in Table 1 above.

[0071] In some embodiments, the individual with a haematological condition with elevated levels of FLC is an individual with free κ light chains greater than 8.0 mg/L, greater than 10.0 mg/L greater than 12.0 mg/L, greater than 14.0 mg/L, greater than 16.0 mg/L, greater than 18.0 mg/L, greater than 20.0 mg/L, greater than 22.0 mg/L, greater than 24.0 mg/L, greater than 26.0 mg/L, greater than 28.0 mg/L, greater than 30.0 mg/L, greater than 35.0 mg/L, greater than 40.0 mg/L, greater than 50.0 mg/L, or greater than 75 mg/L. Suitably the level of KFLC is measured in the serum of the individual (e.g., sFLC measurement). [0072] . In some other embodiments, the individual with a haematological condition with elevated levels of FLC is an individual with free λ light chains greater than 13.0 mg/L, greater than 15.0 mg/L, greater than 17.0 mg/L, greater than 19 mg/L, greater than 21.0 mg/L, greater than 23.0 mg L, greater than 25.0 mg/L, greater than 26.0 mg/L, greater than 27.0 mg/L, greater than 28.0 mg/L, greater than 29.0 mg/L, greater than 30.0 mg L, greater than 35.0 mg/L, greater than 40.0 mg/L, greater than 50.0 mg/L, greater than 75.0 mg/L. Suitably the level of FLC is measured in the serum of the individual (e.g. , sFLC measurement).

[0073] In some other embodiments, the individual with a haematological condition with elevated levels of FLC is an individual with a κ/λ ratio of FLCs of greater than 0.65 mg/L, greater than 0.75 mg/L, greater than 1.0 mg/L, greater than 1.25 mg/L, greater than 1.35 mg/L, greater than 1.45 mg/L, greater than 1.55 mg/L, greater than 1.65 mg/L, greater than 1.75 mg/L, greater than 1.85 mg/L, greater than 2,0 mg/L, greater than 2.5 mg/L, greater than 3.0 mg/L. Suitably, the individual with a haematological condition with elevated levels of FLC is an individual with a κ/λ ratio of FLCs of greater than 1.65 mg/L. Suitably the FLC κ/λ ratio is measured in the serum of the individual (e.g. , sFLC measurement).

[0074] The level of FLC in an individual may be measured using any suitable method known in the art, including those reviewed in Bradman AR, 2010, see especially Chapter 4 which discusses suitable immunoassays that can be used and Chapter 17 which reviews suitable instrumentation currently available to measure FLC levels.

[0075] In some embodiments, the haematological condition is multiple myeloma (MM) or an associated disease or condition. MM is a cancer of the plasma cells. Although MM is not the most common cancer, there is no known curative therapy for this cancer. Therefore, the identification of new therapeutic agents with anti-MM activity remains an urgent priority.

[0076] Also contemplated within the scope of the present invention is the prevention or treatment of diseases or conditions associated with MM. These include monoclonal gammopathy of undetermined significance (MGUS), AL amyloidosis (also referred to as LC amyloidosis or amyloid fibrils), and LC deposition disease (or LCDD).

MGUS is a condition in which abnormal levels of a monoclonal immunogloblulin (also called an M-protein) are observed in the blood. In -80% of individuals, MGUS does not appear to lead to any malignant condition, but in the remaining ~20%, MGUS is either associated with a malignant disease or condition, or can progress over years to a malignant disease or condition, including some haerhatological malignancies, including MM. In contrast to MGUS which may precede MM, AL amyloidosis and LC deposition disease are both conditions that often result from MM or are associated with MM after an individual has presented with MM. AL amyloidosis (or primary systemic amyloidosis) is a condition characterised by the accumulation of misfolded monoclonal FLCs, or their fragments, as amyloid deposits. In LCDD, monoclonal serum free light chains (sFLCs) are precipitated on the basement membranes of cells in the kidneys and other organs.

[0077] Other haematological conditions with elevated levels of FLCs include B-cell lymphoma (including B-cell, non-Hodgkin Lymphomas), solitary plasmacytoma of bone, extramedullary plasmacytoma, multiple solitary plasmacytoma (+/- recurrent), plasma cell leukaemia, Waldenstrom's macroglobulinaemia, non-Hodgkin lymphomas complicated by AL amyloidosis, B-cell, chronic lymphocytic leukaemia, POEMS syndrome, and cryoglobulinaemia (see, for example, Bradwell AR, 2010, Chapter 18). It is thus proposed that the methods of the present invention can be used to prevent or treat these haematological conditions, including haematological malignancies, pre- malignancies, and their associated diseases or conditions.

[0078] As discussed herein, the inventors therefore propose that increasing levels of sphingomyelin in an individual will be beneficial in the prophylaxis or treatment of a haematological condition that exhibits elevated levels of FLCs, as they believe that an increase in sphingomyelin levels in the individual will lead to an increase in the number of FLC aggregates bound to sphingomyelin within the lipid bilayer of malignant or undesirable cells and thus to a reduction or inhibition in the proliferation or to the death of malignant or undesirable cells in the haematological condition. The malignant or undesirable cells may be malignant cells of the haematological condition, for example, the abnormal plasma cells (myeloma cells) an individual suffering from multiple myeloma, or may be undesirable cells of the haematological condition, for example, benign or pre-malignant cells of a condition, including benign or pre-malignant B cells or plasma cells (e.g., MGUS) where the cells exhibit elevated levels of FLC.

Agents-that increase sphingomyelin levels

[0079] As used herein, the phrase "agent that increases sphingomyelin levels" and the like refers to any agent which increases the levels of sphingomyelin in an individual, including those agents which interact with the sphingomyelin degradation pathway so as to antagonise a component of the degradation pathway (e.g. ,

sphingomyelinase) and thus increase sphingomyelin levels, and those agents which interact with the sphingomyelin production pathway so as to agonise a component of the production pathway (e.g. , sphingomyelin synthase) and thus increase sphingomyelin levels. .1 Antagonists of sphingomyelin degradation pathway

[0080] In some embodiments, the agent that increases sphingomyelin levels modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes a component involved in the degradation of sphingomyelin. Representative genes encoding a component involved in the degradation of sphingomyelin include a gene encoding a sphingomyelinase, especially a neutral sphingomyelinase, including those with GenBank accession nos. AL 109947.19 and CH471051.2 and those described in Horinouchi et al. 1995. Representative expression products of genes encoding components involved in the degradation of sphingomyelin include a sphingomyelinase, especially neutral sphingomyelinase.

[0081] In other embodiments, the agent that increases sphingomyelin levels modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression of a gene encoding a component involved in the degradation of sphingomyelin.

[0082] In other embodiments, the agent that increases sphingomyelin levels modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression product of a gene encoding a component involved in the degradation of sphingomyelin.

[0083] In some embodiments, the agent that increases sphingomyelin levels increases the expression of a gene or the level or functional activity of an expression product of that gene. In other embodiments, the agent that increases sphingomyelin levels reduces the expression of a gene (e.g. , a gene encoding a sphingomyelinase, especially a gene encoding neutral sphingomyelinase) or the level or functional activity of an expression product of that gene (e.g. , a sphingomyelinase, especially neutral sphingomyelinase).

[0084J Suitably, the agent that increases sphingomyelin levels increases or reduces the expression of the gene or the level of functional activity of an expression product of that gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to the expression, level or functional activity in the absence of the agent that increases sphingomyelin levels.

[0085] Suitable agents for reducing or abrogating gene expression include, but are not restricted to, oligoribonucleotide sequences, including anti-sense RNA, DNA molecules and ribozymes, that function to inhibit the translation of mRNA. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regards to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions may be used.

[0086) Ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridisation of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyse endonucleolytic cleavage of target sequences. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridisation with complementary oligonucleotides, using ribonuclease protection assays.

[0087J Both anti-sense RNA and DNA molecules and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesising oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.

Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of

DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesise antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

[0088] Various modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

[0089] Alternatively, RNA molecules that mediate RNA interference (RNAi) of a target gene or gene transcript can be used to reduce or abrogate gene expression. RNAi refers to interference with or destruction of the product of a target gene by introducing a single stranded, and typically a double stranded RNA (dsRNA) that is homologous to the transcript of a target gene. Thus, in some embodiments, dsRNA per se and especially dsRNA-producing constructs corresponding to at least a portion of a target gene may be used to reduce or abrogate its expression. RNAi-mediated inhibition of gene expression may be accomplished using any of the techniques reported in the art, for instance by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfected nucleic acid construct having homology for a target gene from between convergent promoters, or as a head to head or tail to tail duplication from behind a single promoter. Any similar construct may be used so long as it produces a single RNA having the ability to fold back on itself and produce a dsRNA, or so long as it produces two separate RNA transcripts which then anneal to form a dsRNA having homology to a target gene.

[0090] Absolute homology is not required for RNAi, with a lower threshold being described at about 85% homology for a dsRNA of about 200 base pairs (Plasterk RH, et al. 2000). Therefore, depending on the length of the dsRNA, the RNAi-encoding nucleic acids can vary in the level of homology they contain toward the target gene transcript, i.e., with dsRNAs of 100 to 200 base pairs having at least about 85% homology with the target gene, and longer dsRNAs, i.e., 300 to 100 base pairs, having at least about 75% homology to the target gene. RNA-encoding constructs that express a single RNA transcript designed to anneal to a separately expressed RNA, or single constructs expressing separate transcripts from convergent promoters, are suitably at least about 100 nucleotides in length. RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding are suitably at least about 200 nucleotides in length.

[0091] The promoter used to express the dsRNA-forming construct may be any type of promoter if the resulting dsRNA is specific for a gene product in the cell lineage targeted for destruction. Alternatively, the promoter may be lineage specific in that it is only expressed in cells of a particular development lineage. This might be advantageous where some overlap in homology is observed with a gene that is expressed in a non-targeted cell lineage. The promoter may also be inducible by externally controlled factors, or by intracellular environmental factors.

[0092] In other embodiments, RNA molecules of about 21 to about 23 nucleotides, which direct cleavage of specific mRNA to which they correspond, as for example described in US 2002/0086356 (the entire contents of which is incorporated herein by reference), can be utilised for mediating RNAi. Such 21-23 ht RNA molecules can comprise a 3' hydroxyl group, can be single-stranded or double stranded (as two 21 -

23 nt RNAs) wherein the dsRNA molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3').

[0093] Exemplary RNAi methods suitable for use in accordance with the present invention include those known in the art, including delivery by way of liposome or virus, and including lipofectamine 2000 (Invitrogen) and the delivery systems described in Nguyen T et al. 2008. Based on sequences known in the art a person of skill in the art would readily know how to prepare suitable agents for reducing or abrogating gene expression as described herein.

[0094] Suitable agents for modulating the level or functional activity of an expression product of a gene include, but are not restricted to small organic molecules, nucleic acids, aptamers, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, as further described herein.

[0095] Suitably, sphingomyelinase is the subject of the targeting, especially neutral sphingomyelinase. For example, the agent that increases sphingomyelin levels may inhibit or otherwise reduce sphingomyelinase activity. [0096] In some embodiments, the agent that increases sphingomyelin levels may inhibit or otherwise reduce sphingomyelinase activity and modulate (increase or decrease) the activity of at least one other component (e.g., a component of the sphingomyelin degradation pathway or another component). In other embodiments, the agent that increases sphingomyelin levels selectively inhibits or otherwise reduces sphingomyelinase inhibitor activity, i.e., inhibits or otherwise reduces sphingomyelinase activity without modulating (increasing or decreasing) the activity of another component.

[0097] Inhibitors of neutral sphingomyelinase suitable for use in the present invention include those described in Wascholowski el al. 2006, in which the authors reviewed a number of neutral sphingomyelinase inhibitors and drew a distinction between those that are compounds containing epoxy groups and "epoxy-free" compounds developed by the authors which they term "sphingolactones".

[0098J Illustrative examples of inhibitors of neutral sphingomyelinase (and thus agents that increases sphingomyelin levels) comprising compounds containing epoxy groups suitable for use in the present invention include the following:

[0099] (A) The compound termed "scyphostatin" having the formula:

[0100] and analogues thereof (including the compound termed

"spiroepoxide" discussed below, including "spiroepoxide 1"). Scyphostatin was originally isolated from Trichopeziza mollissima and is a non-competitive inhibitor of neutral sphingomyelinase. Scyphostatin and analogues thereof, methods for their preparation and their biological activity are disclosed in Nara et al. 1999 (1), Inoue et al. 2004, Nara et al. 1999 (2), and US 6,790,992 which is incorporated herein by reference.

[0101] (B) The compound termed "spiroepoxide" having the formula:

[0102] and analogues thereof. Spiroepoxide is an analogue of scyphostatin. These compounds, methods for their preparation and their biological activity are disclosed in Arenz et al. 2000 (1), Arenz et al. 2000 (2), and Arenz et al. 2001 (1).

[0103J (C) The compound termed "manumycin A" (also referred to as Chlorogentisylquinone Manumycin A) having the formula:

(0104J and analogues thereof including manumycin B, manumycin C, and manumycin D. These compounds, methods for their preparation and their biological activity are disclosed in Arenz et al. 2001 (2).

[0105] Illustrative examples of inhibitors of neutral sphingomyelinase (and thus agents that increases sphingomyelin levels) comprising compounds not containing epoxy groups ("epoxy-free") suitable for use in the present invention include the class of compounds (D) termed "sphingolactones" described in Wascholowski et al. 2006. This class of compounds is based on the authors determination of the desired structural feature of a epoxy-free neutral sphingomyelinase inhibitor to be as follows:

[0106} this structure/formula being based on the authors recognition that: (1) both the epoxy-containing neutral sphingomyelinase inhibitors scyphostatin and manumycin (each being described above) contain a polyunsaturated fatty acid residue as a common structural element; (2) synthetic manumycin analogues with unsaturated carboxylic acids in the amide moiety possess a higher affinity to neutral

sphingomyelinase relative to those with saturated fatty acid chains; (3) the primary hydroxyl group of spiroepoxide (also described above) is essential for the inhibitory activity of that compound; and also based on (4) the selection of the γ-butyrolactone scaffold as a replacement for the reactive epoxy groups of scyphostatin, manumycin and spiroepoxide.

[0107] The sphingolactones class of compounds with this shared

structure/formula are described as being potent, selective, and stable inhibitors of neutral sphingomyelinase, and are thus agents that increases sphingomyelin levels suitable for use in the present invention. Specific compounds with the shared structure/formula, methods for their preparation, their biological activity, and also identification of the essential features of the structure/formula of these compounds are described in

Wascholowski et al. 2006.

[0108] The present invention thus contemplates the use of any candidate compound comprising a sphingolactone having the shared structure/formula identified in Wascholowski et al. 2006 as an agent that increases sphingomyelin levels for use in the present invention. The compound may be further designed having regard to the important structural features that enhance neutral sphingomyelinase inhibitory activity that were also identified in Wascholowski et al. 2006. Methods for evaluating the neutral sphingomyelinase inhibitory effect of a compound can be employed using the methods described in Wascholowski et al. 2006 to determine whether the candidate compound exhibits neutral sphingomyelinase inhibitory activity.

[01091 Exemplary sphingolactones suitable for use in the present invention include the following:

[0110] (D. l) Compounds 18, 19, 20 and 21 as described in Wascholowski et al. 2006 having the following formulae: O

H A R

[0111] wherein:

[0112] (i) for Compound 18, R is CH=CHCH=CHCH₃;

[0113] (ii) for Compound 19, R is CH=CH(CH₂)₂CH₃;

[0114] (iii) for Compound 20, R is (CH₂)₄CH₃;

[0115] (iv) for Compound 21, R is Ph; and

[0116] (D.2) Compounds 24 and 25 as described in Wascholowski et al. 2006 having the following formulae

o

[0117] wherein:

[0118] (i) for Compound 24, R is CH=CHCH=CHCH₃; and

[0119] (ii) for Compound 25, R is (CH₂)₄CH₃.

[0120] In other illustrative embodiments, the agent that increases

sphingomyelin levels comprises a compound E) having the formula:

[0121] or a pharmaceutically acceptable salt thereof. Specifically

contemplated is the dihydrochloride salt thereof, namely the compound 3,3 '-(1,4- phenylene)bis[N-[4-(4,5-dihydro-lH-imidazol-2-yl)phenyl]-hydrochloride-2- propenamide (also termed "GW4869" and having CAS number: 6823-69-4) and having the following structure:

[0122] wherein these compounds, methods for their preparation and their biological activity are disclosed in Luberto et al. 2002, Marchesini et al. 2003 and Walton et al. 2006.

[0123] Also contemplated are sphingomyelinase inhibitors prepared based on the structures of scyphostatin and manumycin, including those described by Arenz et al. 2001 (3), Arenz et al. 2001 (1), Arenz et al. 2000 (1), Tanaka et al. 1997, Saito et al. 2000, Hoye et al. 2000, Izuhara et al. 2001, Runcie et al. 2001, Chau et al. 2001, Arenz et al. 2001 (2), US 5,079,263 (the entire contents of which is incorporated herein by reference), and US 5,444,087 (the entire contents of which is incorporated herein by reference).

[0124] Other sphingomyelinase inhibitors suitable for use in the present invention as agents that increase sphingomyelin levels include:

[0125] (F) Glutathione, which has been shown to inhibit neutral

sphingomyelinase at physiological concentrations (>95% inhibition at 5mM) as described in WO 2008-122037;

[0126] (G) Desipramine as described in WO 2008-122037;

[0127] (H) Imipramine as described in WO 2008- 122037;

[0128] (I) The compound termed "NB6" with the formula: (3-carbazol-9-yl- propyl)-[2-(3,4-dimethoxy-phenyl)-ehtyl)-methyl-aminej this compound is an inhibitor of sphingomyelinase gene transcription, as described in WO 2008-122037; [0129] (J) Hexanoic acid (2-cyclo-pent-l-enyl-2 -hydroxy- 1-hydroxy-methyl- ethyl)-amide, also termed "NB12" as described in WO 2008-122037;

(0130) (K) The compound termed "C 1 1 AG" as described in WO 2008- 122037 and in Amtmann et al. 2003;

[0131] (L) Macquarimicin A, a non-competitive inhibitor of neutral sphingomyelinase, as described in WO 2008-122037;

[0132] (M) Alutenusin, a non-competitive inhibitor of neutral

sphingomyelinase, as described in WO 2008-122037;

[0133] (N) Ubiquinol and homologues thereof including those described in Martin et al. 2001 and Martin SF et al. 2002;

[0134] (O) The serine protease inhibitors N-tosyl-L-phenylalanyl chloromethyl ketone and dichloroisocoumarin as described in Mansat et al. 1997;

[0135] (P) L-carnitine and related compounds as described in Andriue- Abadie et al. 1999, Katircioglu et al. 1999 and Gunther et al. 2000, and US 6,284,798 (the entire contents of which is incorporated by reference);

[0136] (Q) Gentamicin and gentamicin derivatives and other aminoglycosides as described in Ghosh et al. 1987 and US 2007/0212348 (the entire contents of which is incorporated herein by reference); and

[0137] (R) The water-soluble polysaccharide fraction termed "CME-1 " having a molecular mass of 27.6 kDA, derived from Cordyceps mycelia, and described in Wang et al. 201 1.

[0138] An additional sphingomyelinase inhibitor suitable for use in the present invention is the compound terms "RY221B-a" having the formula:

Neutral Sphingomyelinase Inhibitor (IC₅₀ = 1.2 μΜ)

[0139] ^" as described in Imagawa et al. 2010. RW21 ΙΒ-a contains a bipyridyl moiety as a metal coordination site designed based upon the mechanism of phosphate ester hydrolysis. The synthesis and biological activity of this compound is also disclosed in Imagawa et al. 2010.

[0140] Further sphingomyelinase inhibitors suitable for use in the present invention include those described in WO 2000/072833, including:

[0141] (i) [3(10,1 l -Dihydro-dibenzo[b,f]azepin-5-yl)-N-propyl]-[2(3,4- dimethoxyphenyl)-ethyl]methylamin,

[0142] (ii) [3(10,1 l -Dihydro-dibenzo[b,fJazepin-5-yl)-N-propyl]-[2(4- methoxyphenyl)-ethyl]methylamin,

[0143] (iii) [2(3,4-Dίmethoxyphenyl)-ethyl]-[3(2-chlo henothiazin-10-yl)- N-propyl]-methylamin,

[0144] (iv) [2(4-Methoxyphenyl)-eth l]-[3(2-chlo henothiazin-10-yl)-N- propy 1 ] -methy lam in,

[0145] (v) [3(Carbazol-9-yl)-N-propyl]-[2(3,4-dimethoxyphenyl)- ethyljmethylamin,

[0146] (vi) [3(Carbazol-9-yl)-N-propyl]-[2(4-methoxyphenyl)- ethyl]methylamin,

[0147] (vii) [2(3,4-Dimethoxyphenyl)-ethyl]-[2(phenothiazin-l 0-yl)-N- ethyl]-methylamin,

[0148] (viii) [2(4-Methoxyphenyl)-ethyl]-[2(phenothiazin-10-yl)-N-ethyl]- methylamin,

[0149] (ix) [(3 ,4-Dimethoxyphenyl)-acetyl] - [3 (2-chlorphenothiazin- 10-yl)-N- propylj-methylamin,

]0150] (x) n(l-naphthyl)-N'[2(3,4-dimethoxyphenyl)-ethyl]-ethyl diamine,

[0151] (xi) n(l-naphthyl)-N[2(4-methoxyphenyl)-ethyl]-ethyl diamine,

[0152] (xii) n[2(3,4-Dimethoxyphenyl)-ethyl]-n[l-naphthylmethyl]amine,

[0153| (xiii) n[2(4-Methoxyphenyl)-ethyl]-n[l -naphthylmethyljamine,

[0154] (xiv) [3(10,1 1 -Dihydro-dibenzo[b,f]azepin-5-yl)-N-propyl]-[(4- methoxyphenyl)-acetyl]-methylamin, [0155] (xv) [2(10,1 l -Dihydro-dibenzo[b,f]azepin-5-yl)-N-ethyl]-[2(3 ,4- dimethoxyphenyl)-ethyl]methylamin,

[0156] (xvi) [2(10,1 1 -Dihydro-dibenzo[b,f]azepin-5-yl)-N-ethyl]-[2(4- methoxyphenyl)-ethyl]-methylamin,

[0157] (xvii) [2(10,1 1 -Dihydro-dibenzo[b,f]azepin-5-yl)-N-ethyl]-[(4- methoxyphenyl)-acety-l]-methylamin,

[0158] (xviii) n[2(Carbazol-9-yl)-N-ethyl]-N'[2(4-methoxyphenyl)- ethyl]piperazin,

[0159] (xix) 1 [2(Carbazol-9-yl)-N-ethyl]-4[2(4-methoxyphenyl)-ethyl]-3,5- dimethylpiperazin,

[0160] (xx) [2(4-Methoxyphenyl)-ethyl]-[3(phenoxazin-10-yl)-N-propyl]- methylamin,

[0161] (xxi) [3(5,6,1 l,12-Tetrahydrodibenzo[b,f]azocin)-N-propyl]-[3(4- methoxyphenyl)-propyl]methylamin,

[0162] (xxii) n(5H-Dibenzo[A,D]cycloheptan-5-yl)-N'[2(4-methoxyphenyl)- ethyl] -propylene diamine, and

[0163] (xxiii) [2(Carbazol-9-yl)-N-ethyl]-[2(4-methoxyphenyl)- ethyl]methylamine,

[0164] where these compounds, methods for their preparation and their biological activity are disclosed in WO 2000/072833.

[0165] Additional sphingomyelinase inhibitors suitable for use in the present invention include the sphingosine compounds with the formula:

[0166] wherein (i) one of R¹ and R² is hydrogen, and the other is a group represented by the formula:

[0167] n is 0 or 1, and R³ is hydrogen, C|_-23 alkyl, C₃-8 cycloalkyl, C₂.₆ alkenyl, Ci_-6 alkoxy, Cj.g cycloalkyloxy, phenyl, or furil;

[0168] (ii) wherein R¹ is a group represented by the formula:

[0170] (iii) wherein R¹ is hydrogen, and R² is a group represented by the formula:

[0171] (iv) salts thereof,

[0172] wherein these compounds, methods for their preparation and their biological activity are disclosed in US 2010/0099881, the entire contents of which is incorporated herein by reference.

[0173] The neutral sphingomyelinase inhibitors described above and other additional neutral sphingomyelinase inhibitors suitable for use in the present invention include those reviewed in Delgado et al. 2006 and those discussed in PCT publication WO 2008/122037 (see, in particular, paragraphs [0028 to 0031] on pages 1 1-13), Uchida et al. 1999, Tanaka et al. 1999 (1), Tanaka et al. 1999 (2), Uchida et al. 2001, Martin al. 2002, Zhou et al. 2004, Arenz et al. 2001 (3), Pitsinos et al. 2003, Claus et al. 2005, Hakogi et al. 2000, Yokomatsu et al. 2001, Hakogi et al. 2002, Lindsey et al. 2002, Yokomatsu et al. 2003, Taguchi et al. 2003 ( 1 ), Taguchi et al. 2003 (2), US 6,613,322 (the entire contents of which is incorporated by reference), US 2003/0026796 (the entire contents of which is incorporated by reference), PCT publication WO 99/11283, US 2007/0212348 (the entire contents of which is incorporated by reference), US

2004/0047851 (the entire contents of which is incorporated by reference), and US

2003/0162191 (the entire contents of which is incorporated by reference). .2 Agonists of sphingomyelin production pathway

[0174} In some embodiments, the agent that increases sphingomyelin levels modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes a component involved in the production of sphingomyelin. Representative genes encoding a component involved in the production of sphingomyelin include a gene encoding a sphingomyelin synthase, including those with GenBank accession nos. AC069547.7, AL1 17341.26, AL596137.7, and

CH471 142.2. Representative expression products of genes encoding components involved in the production of sphingomyelin include a sphingomyelin synthase.

[0175] In other embodiments, the agent that increases sphingomyelin levels modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression of a gene encoding a component involved in the production of sphingomyelin.

[0176] In other embodiments, the agent that increases sphingomyelin levels modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression product of a gene encoding a component involved in the production of sphingomyelin.

[0177] In some embodiments, the agent that increases sphingomyelin levels increases the expression of a gene (e.g., a gene encoding a sphingomyelin synthase) or the level or functional activity f an expression product of that gene (e.g., a

sphingomyelin synthase). In other embodiments, the agent that increases sphingomyelin levels reduces the expression of a gene or the level or functional activity of an expression product of that gene. [0178] Suitably, the agent that increases sphingomyelin levels increases or reduces the expression of the gene or the level of functional activity of an expression product of that gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to the expression, level or functional activity in the absence of the agent that increases sphingomyelin levels.

[0179] Suitable agents for reducing or abrogating gene expression include, but are not restricted to, oligoribonucleotide sequences, including anti-sense RNA, DNA molecules and ribozymes, that function to inhibit the translation of mRNA, and RNAi, as described above.

[0180] Suitable agents for modulating the level or functional activity of an expression product of a gene include, but are not restricted to small organic molecules, nucleic acids, aptamers, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, as further described herein.

[0181] Suitably, sphingomyelin synthase is the subject of the targeting. For example, the agent that increases sphingomyelin levels may increase sphingomyelin synthase activity.

[0182] In some embodiments, the agent that increases sphingomyelin levels may increase sphingomyelin synthase activity and modulate (increase or decrease) the activity of at least one other component (e.g., component of the sphingomyelin production pathway or another component). In other embodiments, the agent that increases sphingomyelin levels selectively increase sphingomyelin synthase activity, i.e., increases sphingomyelin synthase activity without modulating (increasing or decreasing) the activity of another component.

[0183] In some embodiments, the agent that increases sphingomyelin levels comprises sphingomyelin or an analogue thereof. Illustrative examples of sphingomyelin analogues suitable for use in the present invention include 3-O-methylsphingomyelin and 3-O-ethylsphingomyelin, each of which are described in WO 2008/122037.

[0184] In some other embodiments, the agent that increases sphingomyelin levels is not a sphingomyelin precursor or sphingomyelin prodrug. Identification of agents that increase levels of sphingomyelin

[0185] The present invention also provides methods for identifying agents that increase the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of FLCs.

[0186] The method may comprise screening for an agent that modulates the expression of a gene or the level and/or functional activity of an expression product of that gene, wherein the gene is selected from a gene encoding a component of the sphingomyelin production or degradation pathway a gene whose expression product modulates directly or indirectly the expression of a gene encoding a component of the sphingomyelin production or degradation pathway, and a gene whose expression product modulates directly or indirectly the expression product of a gene encoding a component of the sphingomyelin production or degradation pathway. Components of the sphingomyelin production or degradation pathway include sphingomyelin,

sphingomyelin synthase, and sphingomyelinase.

[0187] One suitable method comprises contacting a sample of cells expressing sphingomyelin with a test agent. A detected increase in sphingomyelin relative to a reference or control level in the absence of the test agent, indicates that the agent increases sphingomyelin levels and that it is useful for preventing or treating a haematological condition that exhibits elevated levels of FLCs. Any suitable method to detect an increase in level of sphingomyelin may be used.

[0188] Another suitable method comprises contacting a sample containing sphingomyelin (or an analogue thereof) and sphingomyelinase {e.g., neutral

sphingomyelinase) with a test agent. A detected decrease in the level of sphingomyelin degradation (e.g., a higher level of sphingomyelin or a lower level of ceramide and/or phosphocholine produced) relative to a reference or control level in the absence of the test agent, indicates that the agent inhibitors or otherwise antagonises sphingomyelinase activity and is therefore an agent that increases sphingomyelin levels useful for preventing or treating a haematological condition that exhibits elevated levels of FLCs. In some embodiments, the sphingomyelin analogue is a sphingomyelin conjugate where cleavage of the conjugate by sphingomyelinase releases a ceramide analogue with a detectable moiety (e.g., free thiol) that is detectable (e.g., by use of a thiol detector that analyses fluorescence wavelength). Suitable methods include the Sphingomyelinase Inhibitor Screening Assay sold by Cayman (Cayman Chemical Item Number 700330) or the Amplex® Red Sphingomyelinase Assay Kit sold by Invitrogen.

[0189] Yet another suitable method comprises contacting a preparation with a test agent, wherein the preparation comprises (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of a polypeptide component of the sphingomyelin production or degradation pathway, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence (e.g. a transcriptional control element such as a promoter or a cw-acting sequence) that regulates a nucleotide sequence that encodes at least a biologically active fragment of a polypeptide component of the sphingomyelin production or degradation pathway, or a variant or derivative thereof, which is operably linked to a reporter gene; or (iii) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide according to (i). A detected change in the level and/or functional activity of the polypeptide component, or an expression product of the reporter gene, relative to a reference and/or control level or functional activity in the absence of the test agent, indicates that the agent modulates sphingomyelin levels and that it is useful for preventing or treating a haematological condition that exhibits elevated levels of FLCs. In some embodiments, the method comprises identifying agents that agonise a component of the sphingomyelin production pathway, wherein an increase in the level and/or functional activity of a component in the sphingomyelin production pathway (e.g., sphingomyelin, sphingomyelin synthase), or an expression product of the reporter gene, relative to a reference and/or control level or functional activity in the absence of the test agent, indicates that the agent agonises the sphingomyelin production pathway and thus is an agent that increases sphingomyelin levels and is therefore useful for preventing or treating a haematological condition that exhibits elevated levels of FLCs. In some other embodiments, the method comprises identifying agents that antagonise a component of the sphingomyelin degradation pathway, wherein a decrease in the level and/or functional activity of a component in the sphingomyelin degradation pathway (e.g. ,

sphingomyelinase, including neutral sphingomyelinase), or an expression product of the reporter gene, relative to a reference and/or control level or functional activity in the absence of the test agent, indicates that the agent inhibits or otherwise antagonises the sphingomyelin degradation pathway and thus is an agent that increases sphingomyelin levels and is therefore useful for preventing or treating a haematological condition that exhibits elevated levels of FLCs. [0190] Methods of identifying suitable agents that increase sphingomyelin levels, including inhibitors of sphingomyelinase, are known in the art and include those described in Arenz C et al. 2001 (3) and Kornhuber et al. 2008. In some embodiments, the methods further comprise identifying agents that increase sphingomyelin levels that are inhibitors of sphingomyelinase, wherein the agents are capable of crossing the cell membrane. Suitable such methods are well known in the art.

[0191] Candidate test agents encompass numerous chemical classes.

Candidate organic molecules comprise functional groups necessary for structural interaction with components of the sphingomyelin production or degradation pathways, including sphingomyelin, sphingomyelin synthase and sphingomyelinase {e.g., neutral sphingomyelinase), including proteins, particularly by way of hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, suitably at least two of the functional chemical groups. The candidate test agent often comprises cyclical carbon or heterocyclic structures or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate test agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof.

[0192] In some embodiments, small molecules are used as candidate test agents because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals. Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene {e.g., by interacting with the regulatory region or transcription factors involved in gene expression); or affect the activity of a gene by inhibiting or enhancing the binding of accessory molecules.

[0193] Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification,

amidification, etc to produce structural analogues. [0194] Screening may also be directed to known pharmacologically active compounds and chemical analogues thereof.

[0195] Suitable methods contemplated include high throughput screening of putative agents such as proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries

[0196] In accordance with the present invention, the agents identified using the methods broadly described above are useful for preventing or treating a

haematological condition that exhibits elevated levels of FLCs in an individual. Methods of producing agents that increase levels of sphingomyelin

[0197] The present invention also provides methods of producing an agent that increases levels of sphingomyelin for preventing or treating a haematological condition that exhibits elevated levels of FLCs in an individual. These methods generally comprise: testing a test agent as broadly described above; and synthesising the agent on the basis that it tests positive for increasing sphingomyelin levels.

[0198] Suitably, the method further comprises derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing a haematological condition that exhibits elevated levels of FLCs. Suitable methods are known in the art. Formulations and administration routes

[0199] In accordance with the present invention, it is proposed that agents that increase sphingomyelin levels are useful for the prevention or treatment of haematological conditions that exhibits elevated levels of FLCs.

[0200] Such agents can be administered to an individual either by themselves, or in pharmaceutical compositions where they are mixed with a suitable pharmaceutically acceptable carrier or diluent.

[0201] The agents of the present invention may be conjugated with biological targeting agents which enable their activity to be restricted to particular cell types. Such biological-targeting agents include substances which are immuno-interactive with cell- specific surface antigens.

[0202] The agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the drugs of the invention may be formulated in aqueous solutions, suitably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal

administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Intra-muscular and subcutaneous injection is also contemplated.

[0203] The agents can be formulated readily using pharmaceutically acceptable carriers or diluents well known in the art into dosages suitable for oral administration. Such carriers or diluents enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers or diluents may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

[0204] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. The dose of agent administered to an individual should be sufficient to effect a beneficial response in the individual over time such reducing the number of malignant or undesirable cells, reducing the number of viable malignant or undesirable cells, inhibiting the growth of a malignant or undesirable cell, inhibiting the proliferation of a malignant or undesirable cell, or killing a malignant or undesirable cell.

The quantity of the agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the agent(s) for administration will depend on the judgement of the practitioner. In determining the effective amount of the drug to be administered, the physician may evaluate the characteristics of the patient, their response to the drug and the safety profile of the drug. In any event, those of skill in the art may readily determine suitable dosages of the agents. [0205] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilisers or other components which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0206] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium

carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more drugs as described above with the carrier or diluent which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0207] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterise different combinations of active compound doses.

[0208] Pharmaceuticals which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilisers may be added.

[0209] Dosage forms of the drugs of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of an agent of the invention may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as

hydroxypropylmethyl cellulose. In addition, controlled release may be effected by using other polymer matrices, liposomes or microspheres.

[0210] The agents that increase sphingomyelin levels may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

[0211] For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating

concentration range that includes the IC50 as determined in cell culture (e.g. , the concentration of an agent, which achieves a half-maximal inhibition in activity of a neutral sphingomyelinase inhibitor). Such information can be used to more accurately determine useful doses in humans.

[0212] Toxicity and therapeutic efficacy of such drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio

LD50/ED50. Compounds that exhibit large therapeutic indices may be employed. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. In some embodiments the dosage of such compounds lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See for example Fingl et al. , 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi ).

[0213] Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to increase sphingomyelin levels to the desired level. Usual patient dosages for systemic administration range from 1-2000 mg/day, commonly from 1-250 mg/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02-25 mg kg/day, commonly from 0.02-3 mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5-1200 mg/m2/day, commonly from 0.5-150 mg/m2/day, typically from 5-100 mg/m2/day.

[0214] Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a tissue, which may be subcutaneous or omental tissue, often in a depot or sustained release formulation.

[0215] Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tissue-specific antigen-binding molecule (e.g., antibody). The liposomes will be targeted to and taken up selectively by the tissue.

Ancillary therapy

[0216] The agents that increase sphingomyelin levels may be administered concurrently with at least one ancillary therapy that treats or ameliorates the symptoms or reverses or inhibits the development or progression of the hematologic condition in the subject. The agent that increases sphingomyelin levels may be used therapeutically after the ancillary therapy or may be used before the therapy is administered or together with the therapy. Accordingly, the present invention contemplates combination therapies, which employ both an agent that increases sphingomyelin levels and concurrent administration of an ancillary (e.g., medical treatment), non-limiting examples of which include radiotherapy, surgery, chemotherapy, hormone abalation therapy,

immunotherapy, and other therapies used for haematological conditions. .1 Radiotherapy

[0217] Radiotherapies include radiation and waves that induce DNA damage for example, γ-irradiation, X rays, UV irradiation, microwaves, electronic emissions, radioisotopes, and the like. Therapy may be achieved by irradiating the localized tumor site (e.g. tumor deposits, bone lesions) with the above described forms of radiations. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and

maintenance of chromosomes.

[0218] Dosage ranges for X rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0219J Non-limiting examples of radiotherapies include conformal external beam radiotherapy (50-100 Grey given as fractions over 4-8 weeks), either single shot or fractionated, high dose rate brachytherapy, permanent interstitial brachytherapy, systemic radio-isotopes (e.g., Strontium 89). In some embodiments the radiotherapy may be administered in combination with a radiosensitizing agent. Illustrative examples of radiosensitizing agents include but are not limited to efaproxiral, etanidazole, fluosol, misonidazole, nimorazole, temoporfin and tirapazamine. .2 Chemotherapy

[0220] Chemotherapeutic agents may be selected from any one or more of the following categories:

[0221 J (i) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyridines like 5- fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea; anti-tumor antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like paclitaxel and docetaxel; and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin);

[0222] (ii) cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), UH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5a-reductase such as finasteride;

[0223] (iii) agents which inhibit cancer cell invasion (for example

metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function);

[0224] (iv) inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti- erbb2 antibody trastuzumab [Herceptin™] and the anti-erbbl antibody cetuximab

[C225]), farnesyl transferase inhibitors, MEK inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example other inhibitors of the epidermal growth factor family (for example other EGFR family tyrosine kinase inhibitors such as N-(3- chloro-4-fluorophenyl)-7-methoxy-6-(3-mo holino ropoxy)quinazolin-4- -amine (gefitinib, AZD 1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3- morphoIinopropoxy)quinazoli- n-4-amine (CI 1033)), for example inhibitors of the platelet-derived growth factor family and for example inhibitors of the hepatocyte growth factor family;

[0225] (v) anti-angiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [Avastin™], compounds such as those disclosed in

International Patent Applications WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin .alpha.v.beta.3 function and angiostatin); [0226] (vi) vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO00/40529, WO 00/41669, WO01/92224, WO02/04434 and WO02/08213; ·

[0227] (vii) antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense; and

[0228] (viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy. .3 Immunotherapy

[0229] Immunotherapy approaches, include for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokirie- transfected tumor cell lines and approaches using anti-idiotypic antibodies. These approaches generally rely on the use of immune effector cells and molecules to target and destroy malignant cells. The immune effector may be, for example, an antigen-binding molecule (e.g., antibody) specific for some marker on the surface of a malignant cell. The antigen-binding molecule alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antigen-binding molecule also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a malignant cell target. Various effector cells include cytotoxic T cells and NK cells. .4 Other Therapies

[0230] Examples of other ancillary therapies include phototherapy, cryotherapy, or toxin therapy. One of skill in the art would know that this list is not exhaustive of the types of treatment modalities available for cancer and other hyperplastic lesions. Exemplary therapies for multiple myeloma include administration of steroids, administration of thalidomide, administration of lenalidomide, administration of bortezomib, and stem cell transplants.

[0231] It is well known that chemotherapy and radiation therapy target rapidly dividing cells and/or disrupt the cell cycle or cell division. These treatments are offered as part of the treating several forms of cancerous haematological malignancies, aiming either at slowing their progression or reversing the symptoms of disease by means of a curative treatment. However, these ancillary treatments may lead to an

immunocompromised state and ensuing pathogenic infections and thus the present invention also extends to combination therapies, which employ both an agent that increases sphingomyelin levels and an anti-infective agent that is effective against an infection that develops or that has an increased risk of developing from an

immunocompromised condition resulting from a medical treatment. The anti-infective drug is suitably selected from antimicrobials, which include without limitation compounds that kill or inhibit the growth of microorganisms such asviruses, bacteria, yeast, fungi, protozoa, etc. and thus include antibiotics, amebicides, antifungals, antiprotozoals, antimalarials, antituberculotics and antivirals. Anti-infective drugs also include within their scope anthelmintics and nematocides. Illustrative antibiotics include quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin; gemifloxacin; and garenoxacin), tetracyclines, glycylcyclines and oxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline; linezolide, eperozolid), glycopeptides, aminoglycosides (e.g., amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin, spectinomyein, streptomycin, tobramycin), β-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam, carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, F -037, DQ- 2556, FK-518, cefozopran, ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000, LY206763), rifamycins, rnacrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramicin, roxithromycin, troleandomycin), ketolides (e.g., telithromycin, cethromycin), coumermycins, lincosamides (e.g., clindamycin, lincomycin) and chloramphenicol.

[0232] Illustrative antivirals include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, and zidovudine. [0233] Non-limiting examples of amebicides or antiprotozoals include atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride, and pentamidine isethionate. Anthelmintics can be at least one selected from mebendazole, pyrantel pamoate, albendazole, ivermectin and thiabendazole. Illustrative antifungals can be selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, fluconazole, flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, and terbinafine hydrochloride. Non-limiting examples of antimalarials include chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and pyrimethamine with sulfadoxine. Antituberculotics include but are not restricted to clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate.

[0234] As noted above, the present invention encompasses co-administration of an agent that increases sphingomyelin levels in concert with an additional agent. It will be understood that, in embodiments comprising administration of the agent that increases sphingomyelin levels with other agents, the dosages of the actives in the combination may on their own comprise an effective amount and the additional agent(s) may further augment the therapeutic or prophylactic benefit to the patient. Alternatively, the agent that increases sphingomyelin levels and the additional agent(s) may together comprise an effective amount for preventing or treating the haematological condition. It will also be understood that effective amounts may be defined in the context of particular treatment regimens, including, e.g., timing and number of administrations, modes of

administrations, formulations, etc. In some embodiments, the agent that increases sphingomyelin levels and optionally the ancillary treatment are administered on a routine schedule. Alternatively, the ancillary treatment may be administered as symptoms arise. A "routine schedule" as used herein, refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined. For instance, the routine schedule may involve administration of agent that increases sphingomyelin levels on a daily basis, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc. Alternatively, the predetermined routine schedule may involve concurrent administration of the agent that increases sphingomyelin levels and the ancillary therapy on a daily basis for the first week, followed by a monthly basis for several months, and then every three months after that. Any particular combination would be covered by the routine schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.

[0235] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples. EXAMPLES

EXAMPLE 1

GW4869 INDUCES SELECTIVE CELL DEATH IN MM CELL LINES

[0236] Multiple myeloma (MM) cell lines (NCI-H929, JJN-3, ARH-77, and RPMI-8226) and non- MM cell lines (RAW264.7, 562, HEK293 and Toledo) were incubated with various concentrations of GW4869. GW4869 is a highly specific small molecule neutral sphingomyelinase inhibitor (3,3'-(l ,4-phenylene)bis[N-[4-(4,5-dihydro- lH-imidazol-2-yl)phenyl]-hydrochloride-2-propenamide; CAS number: 6823-69-4). [0237] After 3 days in culture, cells were harvested and cell death was assessed by flow cytometry measuring the uptake of the DNA strain, TO-PRO-3.

[0238] It was found that GW4869 was cytotoxic to all MM cell lines,tested with an EC50 of approximately Ι μΜ. GW4869 had no effect on non-MM cell lines that were tested.

[0239] These results, which are presented in Figure 1 A, show a significant decrease in cell viability of the MM cell lines (NCI-H929, JJN-3, ARH-77, and RPMI- 8226) as a function of GW4869 concentration, while comparatively no (or very small) change in cell viability is seen for the non-MM cell lines (RAW 264.7, K562, and

HEK293).

[0240] These findings were confirmed in a separate experiment in which MM cell lines (NCI-H929, OPM2, RPMI8226, JJN3 and RPMI8226-TGL) as well as peripheral blood mononuclear cells (PBMC) and non-MM cell lines (Jurkat, K562 and Daudi) were treated with GW4869 or DMSO vehicle control for 2 days. Cells were then stained with propidium iodide (dead cell stain) and assessed by flow cytometry to facilitate calculation of percentage viable cells.

[0241] The results from this experiment clearly demonstrate that GW4869 is cytotoxic to MM cell lines (Figures lB-1 F) but is not cytotoxic to PBMC or non-MM cell lines (Figure 1 G-1J).

EXAMPLE 2

SPHINGOLACTONE 24 INDUCES SELECTIVE CELL DEATH IN MM CELL LINES

[0242] MM cell lines (NCI-H929, OPM-2, RPMI-8226 and JJN-3) and a non- MM cell line (K562) were incubated with various concentrations of sphingolactone 24. Sphingolactone 24 (i.e. , compound 24, Wascholowski et ai. 2006, supra) is a highly specific small molecule neutral sphingomyelinase inhibitor having the following structure:

[0243]

[0244] After 2 days in culture, cells were harvested and cell death was assessed by flow cytometry measuring the uptake of the DNA strain, TO-PRO-3.

[0245] It was found that sphingolactone 24 was cytotoxic to all MM cell lines tested with an EC50 of approximately l μΜ. GW4869 had no effect on non-MM cell lines that were tested.

[0246] These results, which are presented in Figure 2A, show a significant decrease in cell viability of the MM cell lines (NCI-H929, OPM-2, RPMI.8226 and JJN- 3) as a function of sphingolactone 24 concentration, while comparatively no (or very small) change in cell viability is seen for the non-MM cell line (K562).

[0247] A similar experiment was performed in which MM cell lines (JJN3,

OPM2 and RPMI8226) and PBMC were incubated with sphingolactone 24 or DMSO vehicle control for 2 days. Cells were then stained with propidium iodide (dead cell stain) and assessed by flow cytometry to facilitate calculation of percentage viable cells. The results from this experiment clearly demonstrate that sphingolactone 24 is cytotoxic to MM cell lines (Figures 2B-2D) but not to PBMC (Figure 2E).

EXAMPLE 3

GW4869 SUPPRESSES TUMOUR GROWTH IN A MURINE MM MODEL

[0248] To assess whether GW4869 shows cytotoxic effects on MM cells in vivo, a murine model of MM was obtained. The model used relies on the depletion of CD 122+ cells (NK and myeloid cells) from NOD/SCID mice, which allows for the engraftment of human MM cell lines and their establishment in bone marrow. This model can be contrasted with other models where MM cells are not established in the bone marrow. This model thus better replicates the human disease because the MM cells reside in the same place as the human disease.

[0249] Mice were injected intravenously with the MM cell line RPMI-8226 and their serum monitored for human FLC (a MM cell biomarker) to confirm successful engraftment of the tumour. Mice were then given daily intraperitoneal injections of either GW4869 (1.25 μΜ) or a vehicle control over two weeks.

[0250] At the end of the two week treatment period, mice were sacrificed and assessed for serum FLC levels (by ELISA) and the number of RPMI-8226 in various tissues (by flow cytometry). [0251] Success of the treatment was indicated by a significant decrease in serum FLC values as compared to controls (mean FLC levels; GW4869: 684.6 ng/mL versus control: 1908 ng/mL; pO.01). These results are shown in Figure 3A.

[0252] Similarly, the percentage of MM engrafted cells was reduced in the GW4869 treated group in both the femur/tibia (mean MM cell %; GW4869: 0.50 versus control: 3.42; p=0.07). These results are shown in Figure..3B. The percentage of engrafted MM cells was also reduced in the GW4869 treated group in the lumbur spine (mean MM cell %; GW4869: 2.04 versus control: 6.33; p=0.28). Significantly, mice treated with GW4869 showed increased survival rates as compared to vehicle control treated mice over 94 days after commencement of the treatment (Figure 3C).

EXAMPLE 4

EFFECT OF GW4869 ON PRIMARY MYELOMA CELLS

[0253] Experiments were also performed to assess the effect of GW4869 on primary myeloma cells. The results from these experiments, presented in Figure 4, suggest that GW4869 treatment reduces the proportion of myeloma cells (defined as

CD138⁺ or CD38⁺) as compared to other mononuclear cells in the bone marrow.

Discussion of the results in Examples 1-4

[0254] The sphingomyelinase inhibitors GW4869 and sphingolactone 24 were found to be cytotoxic to all MM cell lines tested. Furthermore, they had no effect on non-MM cell lines. Upon evaluating GW4869 in an in vivo (murine) model of MM, it was found that mice treated with GW4869 showed delayed tumour growth as measured by serum FLC levels and by the proportion of MM cells found in various tissues and showed increased survival of treated mice. Additionally, GW4869 was also shown to reduce the proportion of primary myeloma cells as compared to other mononuclear cells in the bone marrow.

[0255] Together, these results suggest that up-regulation of cell membrane sphingomyelin (for example, via inhibition of neutral sphingomyelinase) may constitute a novel therapeutic approach for the treatment of MM and other haematological Conditions known to express elevated levels of FLC as it is postulated by the inventors that the increase of sphingomyelin levels by GW4869 leads to more FLC aggregates and thus more cell instability. EXAMPLE 5

NSMASE2 IS UPREGULATED IN MM PRIMARY CELLS AND CELL LINES

[0256] Expression of nSMase2 in MM cells and in non-MM cells was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Cells were harvested and RNA was purified using an RNeasy mini kit (Qiagen). cDNA was synthesized using the Superscript III First-Strand Synthesis SuperMix kit (Invitrogen) according to the manufacturer's protocol. qRT-PCR was performed using an Eppendorf Mastercycler EP realplex using a SYBR GreenER qPCR SuperMix kit (Invitrogen).

Briefly, the PCR protocol was as follows: initial incubation for 2 min at 50 C, followed by heating to 95°C for 5 min, 40 cycles of 95°C for 15 sec, 60°C for 1 min and 72°C for 30 sec. All samples were amplified in triplicate, and quantification of gene copy number was calculated with reference to the house keeping gene, RPL36AL, using the 2-AACt method as described in Livak et al. (2001, Methods 25: 402-408). All data was normalized to PBMCs (peripheral blood mononuclear cells). Primers were designed using LaserGene software. Primer sequences were SMPD3 F:5'- ACTTTGATAACTGCTCCTCTGAC-3 ' R : 5 ' -TTCGTGTCC AGC AG AGTACC-3 ' , and for RPL36AL F:5'-GTTAGGCGAGAGCTGCGAAAGG-3' R:5'- GGTTCTTCGGGTTTTAGGTACGTT-3*.

[0257] The results presented in Figure 5 show that nSMase2 (SMPD3 gene) is mostly up regulated in MM cells, as compared to non-MM cells.

EXAMPLE 6

ACCUMULATION OF INTRACELLULAR FLC IN GW4869 TREATED MM CELLS

[0258] RPMI8226 and OPM2 MM cells were treated with 5 μΜ GW4869 or DMSO vehicle control for 18 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.01% digitonin, then stained intracellularly with anti FLC mAb AlexaFluor 488 (mAb clone #4G7, Abeam). Stained cells were assessed by confocal microscopy and flow cytometry.

[0259] The results presented in Figures 6 and 7 show accumulation of FLC in GW4869 treated MM cells. EXAMPLE 7

ACCUMULATION OF INTRACELLULAR FLC IN SPHINGOLACTONE 24 TREATED MM

CELLS

[0260] RPMI8226 and OPM2 MM cells were treated with 100 μΜ sphingolactone 24 or DMSO vehicle control for 18 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.01% digitonin, then stained intracellularly with anti- FLC mAb AlexaFluor 488 (mAb clone #4G7, Abeam). Stained cells were assessed by flow cytometry.

[0261] The results presented in Figures 8 show accumulation of XFLC in sphingolactone 24 treated cells.

EXAMPLE 8

ACCUMULATION OF INTRACELLULAR FLC ΓΝ THE ENDOPLASMIC RETICULUM

(ER) OF GW4869 TREATED MM CELLS

[0262] RPMI8226 MM cells were treated with 5 μΜ GW4869 for 18 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.01% digitonin, then stained intracellularly with anti-lFLC mAb AlexaFluor 488 (mAb clone #4G7, Abeam), anti-calnexin (Abeam) + anti-rabbit AlexaFluor 568 and DAPI. Stained cells were assessed by confocal microscopy.

[0263] The results presented in Figures 9 indicate that λFLC accumulates in the ER of GW4869 treated cells.

[0264] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0265) The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0266] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims. Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention.

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Claims

WHAT IS CLAIMED IS:

1. An agent that increases the level of sphingomyelin in an individual for use in preventing or treating a haematological condition that exhibits elevated levels of FLCs in the individual.

2. A method of preventing or treating a haematological condition that exhibits elevated levels of FLCs in an individual in need thereof, the method comprising administering to the individual an agent that increases the level o sphingomyelin in the individual.

3. A use of an agent that increases the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of

FLCs in the individual.

4. A use of an agent that increases the level of sphingomyelin in an individual in the manufacture of a composition for preventing or treating a haematological condition that exhibits elevated levels of FLCs in the individual.

5. The agent of claim 1 , the method of claim 2, or the use of claim 3 or claim

4, wherein the haematological condition is selected from the group consisting of multiple myeloma and B-cell lymphoma.

6. The agent of claim 1, the method of claim 2, or the use of claim 3 or claim 4, wherein the agent comprises an inhibitor of neutral sphingomyelinase.

7. A method for identifying an agent that increases the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of FLCs, the method comprising contacting a sample of cells expressing sphingomyelin with a test agent, wherein a detected increase in sphingomyelin relative to a reference or control level in the absence of the test agent, indicates that the agent increases sphingomyelin levels and that it is useful for preventing or treating a haematological condition that exhibits elevated levels of FLCs.

8. A method for identifying an agent that increases the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of FLCs, the method comprising contacting a sample containing sphingomyelin or a sphingomyelin analogue and sphingomyelinase with a test agent, wherein a detected decrease in the level of sphingomyelin degradation relative to a reference or control level in the absence of the test agent, indicates that the agent inhibits or otherwise antagonises sphingomyelinase activity and is therefore an agent that increases sphingomyelin levels useful for preventing or treating a haematological condition that exhibits elevated levels of PLCs.

9. A method of producing an agent that increases the level of sphingomyelin in an individual for preventing or treating a haematological condition that exhibits elevated levels of FLCs in the individual, the method comprising identifying an agent that increases the level of sphingomyelin as claimed in claim 7 or claim 8, synthesising the agent, optionally derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing a haematological condition that exhibits elevated levels of FLCs.