OA16774A - Multiple myeloma treatment. - Google Patents

Abstract

A method of treating a subject presenting with multiple myeloma at a stage characterized by an increase in the prevalence of MM cells that (1) are IL-6 non-responsive and/or (2) have a CD45phenotype, comprising administering to the subject an amount of a compound of formula Ib.

Description

This invention relates to the enzyme Janus kinase 2, or JAK2, More particularly, the invention relates to the use of JAK2 inhibitors in the treatment of multiple myeloma and related myel oproli ferat î ve neop lasms.

Background to the Invention

Multiple myeloma (MM) is an incurable drug résistant clonal B cell malignant neoplasm localized to the bone marrow that is associated ultimately with overproduction of monoclonal antibody by plasma cells leading to destructive lytic bone lestons and end organ damage in the form of rénal and cardiac dysfonction. Current research is focused on agents that reduce activity of interlcukin-6 (IL-6) which is believed to play a central rôle in the pathogenesis of MM through its stimulation ofthe JAK/STAT pathway responsible for plasma cell growth and prolifération.

Some human myeloma cell lines (HMCL) cannot proliferate or survive without exogenous IL-6 [9, 10] and some conventional drugs are ineffective in the presence of 1L-6 [l I13]. The bone marrow niicroenvironmenl (BMME), known to provide supportive signais to MM cells, produces IL-6 [14] hence reducing the pro-survival effect of IL-6 may abrogate the drug résistant phenotype of MM.

JanLis-activated Kinases (JAKs) are well characterized signalling kinases comprising four family members JAKl, JAK2, JAK3 and TYK2 thaï are important in haematological malignancy as JAK mutations hâve been shown to contribute to the pathogenesis of both myeloproliferative disorders [l-3] and leukaemias [4]. JAKs hâve an established rôle in signalling for many cells [reviewed by 5], In MM, JAKs are activated by a variety of cytokines including interlukin-6 (IL-6) [6, 7], inlerferon-a [6, 8] and cpidermal growth factor [6]. Many pathways downstreain of JAKs are exploited by malignant cells.

A variety of JAK inhibitors hâve been developcd reccntly, and their utility as treatments ol’MM is being investigated. CYT387 is a novel JAK inhibitor that can inhibil JAKl, JAK2, JAK3 and TYK2 kinase activity [15, 16]. The structure and development ofthe compound has recently been described [17], Other JAK inhibitors currently in various stages of development and investigation include INCB000020 [18], 1NCB16562 [19], AG490 [20, 21], AZDI480 [22] and Pyrîdone 6 [23], as well as WP1066 [24]. Given lhe putative rôle of IL-6 in MM drug résistance JAK inhibitors are being investigated for their potential use as single agent or in combination therapy for MM. Furthermore, preliminary in vitro data has demonstrated the potential of JAK/STAT inhibition to sensitize MM cells to conventional thérapies [21],

-2Despite these efforts, MM remains an incurable disease, resulting annually in 11,000 deaths and affecting 16,000 additional sufferers each year. It would be useful to provide additional agents and methods for the treatment of subjects afflicted with this disease.

Summaryofthe Invention

It has now been found that CYT 387 is active in the treatment of multiple myeloma, and particularly in treating forms of MM in which the target MM cells are CD45- and/or are IL6 non-responsive. This effect of CYT 387 thus expands on the types of multiple myeloma that can be treated, relative to other agents that also exhibit JAK inhibition activity.

More particularly, and in one of its aspects, lhe présent invention provides for the use of CYT 387 to inhibit growth and/or prolifération, i.e., the viability, of MM cells having a CD45phcnotype. In addition, and in another of its aspects, the présent invention provides for the use of CYT387 to inhibit the growtli and/or prolifération, i.e., the viability, of MM cells that are considered IL-6 non-responsive. Compounds having a JAK kinase inhibition profile like that of CYT 387 are also useful in lhe présent method.

The activity ofCYT 387 allows for the treatment of MM at a later stage ofthe disease, when the MM cells shifl phenotypically from a CD45+ phenotype to a predominantly CD45plienotype, thereby allowing for prolongée! survival in patients desperate for treatment.

In a related aspect, the présent method comprises the step of assessing the subject or a biological sample obtained thcrelrom, identifying multiple myeloma subjects meeting at least one ofthe criteria noted above, and then treating tlie identified subjects with CYT387 or a related compound.

In another aspect ofthe présent invention, there is provided an article of manufacture, comprising CYT 387 or a related compound in combination with a label indicating treatment of a subject presenting with al least one of the noted criteria.

In a related aspect ofthe présent invention, there is provided a kit comprising CYT 387 or a related compound in combination with printed instruction teaching a method of selecting a subject for CYT387 or a related compound therapy based on the sélection criteria herein described.

These and other aspects and embodiments ofthe présent invention are now described in greater detail with reference to the accompanying drawings in which:

Reference to the Figures

Figure 1 : CYT387 prevents signalling downstream of IL-6 or coculture stimulation. HMCL were incubated with or without CYT387 (0,5 - 2 μΜ) for 1 hour before stimulation with 10 ng/nil IL-6 for 15 minutes. Cells were then harvested and p-STAT3 (pY705) was measured. (A) By intraccllular FACS with the géométrie mean fluorescence intensily measured and graphed (n = 3, mean ± SE, Slimulated cells ± CYT387 were analyzed )

-3 using a One-way ANOVA with Tukey post-test * = p <0.05, ** = p < 0.01,*** = p < 0.001). (B) By western blot for p-STAT3 (pY705), total STAT3 and α-tubulin as a loading control.(C) p-STAT3 was also induced in IIMCL using a direct cocullure (CC) with IIS5 immortalized bone marrow stromal cells or primary bone marrow stromal cells or a transwell (TW) “soluble only” CC with HS5. I IMCL were fluorescently labelled with CD38 or CD 138 and stimulated for 15 minutes with or without co-trealment with 2 μΜ CYT387. Représentative plots of NCIH929, (n = 3, for NCI-H929, OCI-MYl and U266). (D) NC1-H929, OCI-MYl and U266 cells were starved overnight and stimulated with 5 ng/ml IL-6 and lOOng/ml IGF-l for 15 minutes with or without co-lreatment with 2 μΜ CYT387. p-AKT (pS473) and p-ERKl/2 (pT202/pY204) were measured by intracellular FACS with the géométrie mean fluorescence intensity normalized to the untreated (UT) control and averaged (n = 4, mean ± SE. Stimulated cells ± CYT387 were analyzed using a One-way ANOVA with Tukey post-test * = p <0.05, ** = p < 0.01 ).

Figure 2: CYT387 inhibits I IMCL prolifération. (A) CYT387 inhibits IIMCL in a time and dose dépendent manner. IL-6 phenotype HMCL (ANBL-6, OCIMY1,U266 and XGI ) and non-IL-6 phenotype HMCL (LP-I, NCI-I1929, OPM2 and RPM1-8226) were cultured for 24, 48 and 72 hours UT, with CYT387 (0.1,0.5, 1,2.5 or 5 μΜ) or with vehicle (DMSO). Cell prolifération was then detennined by M I S Assay (72 liour data shown, n = 3, mean ± SE) (B) Treatment with CYT387 inhibits myeloma cell prolifération even in the presence of IL-6. Absolute cell numbers of viable cells were detennined by haemocylomcler counts of HMCL cultured alone (Untreated) with IL-6 (10 ng/ml) with CYT387 (0.5 - 1 μΜ) or with IL-6 and CYT387. Culture with CYT387 greally decreased the prolifération of lhe IIMCL over 72 hours (treatment at time 0 only). Results represent the mean of 3 independent experiments ± SE. (C) CYT387 prevents cell cycling. HMCL were treated with CYT387 (1 μΜ or 5 μΜ) for 24 and 72 hours then they were harvested and fixed and cell cycle analysed by FACS. Représentative cell cycle plots of Cil 1929 UT or 5 μΜ CYT387 for 24 or 72 hours, with mean of 4 independent experiments ± SE of cycling cells in G2/M phase of the cell cycle.

Figure 3: CYT387 induces apoplosis in IIMCL. (A) Représentative Annexin-V and Propidium Iodide (PI) plots of NCI-H929. UT, Vehicle (DMSO) treated, 24 hours 5 μΜ CYT387 treatment and 72 hours 5 μΜ CYT387 treatment. (B) Proportion of viable (AnnexinV- and PI-) cells after CYT387 treatment compared to UT. Data shown is the mean of 4 independent experiments ± SE.

Figure 4: CYT387 synergizes with melphalan and bortezomib in IIMCL. (A)

Dose effect curves of NCI-H929, OC’l-MYl and U266 after 24 and 48 hours treatment CYT387 treatment (0.5 - 10 μΜ) as determined by the proportion of PI+ cells minus background death (untreated). Meanof'4 independent experiments ± SE. (B) CYT387 in combination with melphalan or bortezomib show synergism. Synergy was measured using a combination index calculated by Calcusyn software, where values less than l represent synergism, plotted against the fraction of cells killed with various dose/ratios of the drugs. Syncrgy is seen between melphalan and CYT387 at a range of doses/ratios/cell lines and lime points. Bortezomib and

CYT387 demonstrated synergistic or nearly additive in 18/24 combinations. Synergism was calculated from dose effect curves of the mean of 4 independent experiments.

Figure 5: CYT387 induces apoplosis in primary samples as a single agent or in combination with melphalan and bortezomib. (A) Proportion of apoptotic (Apo 2.7+) CD38+CD45- primary patient myeloma cells after 48 hours CYT387 treatment (n = 6). (B) Synergy between CYT387 and melphalan or bortezomib after 24 hours treatment as determined by calcusyn software in primary patient CD38+CD45- cells.

Detailed Description of the Invention

CYT 387 is a phenylaminopyrimidine compound having CAS registration number CAS 1056634-68-4, the chemical name N-(cyaiioiiiethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-4pyrimidinyl]-benzamide, and the structure shown below:

NH

CN

Synthesis, formulation and therapeutic use of CYT 387 is described in WO 2008/109943 published 18 September 2008; and in Blood, 2010, 115(25):5232-40. Of course, CYT387 can be used in the form of a sait, solvaté or prodrug if desired.

“Related compounds” are compounds related to CYT 387 by their sélective J AK inhibition signature, in which a préférence is shown for binding lo and inhibition of JAK2 and JAK.1, relative to JAK3 and other members of the kinase family, and by their structural conformance to thc formula:

Ib wherein

Z is independently selected from N and CH;

R¹ is independently selected from 1 1, halogen, OH, CONFIR², CON(R²)₂, CF3, R²OR²,

CN, morpholino, thiomorpholinyl, thiomorpholino-l, I-dioxide, substituted or

-5unsubstitutcdpiperidinyl, substituted or unsubstitutedpiperazinyl, imidazolyl, substituted or unsubstitutedpyrrolidinyl and C|.₄alkylene wherein the carbon atoms arc optionally replaced with NR^Y and/or O substituted with morpholino, thiomorpholinyl, thiomorpholino-l,l-dioxide, substituted or unsubstitutedpiperidinyl, substituted or unsubstitutedpiperazinyl, imidazolyl or substituted or unsubstitutedpyrrolidinyl;

R² is substituted or unsubstituted C^alkyl;

R^Y is II or substituted or unsubstituted Cj.₄alkyl;

R^K is R^XCN;

R^x is substituted or unsubstituted C^alkylene wherein up to 2 carbon atoms can be optionally replaced with CO, NSO2R¹, NR^Y, CONR^y, SO, SO₂ or O;

R is l-I or C_Malkyl, or an enantiomer thereof, a prodrug thereof or a phannaceutically acceptable sait thereof.

The term “Ci.₄alkyl” refers to straight chain or branched chain hydrocarbon groups having from l to 4 carbon atoms. Examples include methyl, ethyl, propyl, ïsopropyl, butyl, isobutyl, sec-butyl, and tert-butyl.

The term “halogen” refers to fluorine, chlorine, bromine and iodine.

The term “substituted” refers to a group that is substituted with one or more groups selected from C|.₄ alkyl, Cj._(lcycloalkyl, C₂.(,alkenyl, C₂.6alkynyl, C|.(,alkylaryl, aryl, heterocycylyl, halo, haloC|.₆alkyl, haloCi.6cycioalkyl, haloC₂.6alkenyl, haloC₂.(,alkynyl, haloaryl, haloheterocycylyl, hydroxy, C|.₆alkoxy, C₂.(,alkenyloxy, C₂.6alkynyloxy, aryloxy, heterocyciyloxy, carboxy, haloC|.₆alkoxy, haloC₂.₆alkenyloxy, haloC₂.₆alkynyloxy, haloaryloxy, nitro, nitroCi_₆,alkyl, nitroC₂.₆alkenyl, nitroaryl, nitroheterocyclyl, azido, amino, Ci.fcalkylamino, C₂.ftaikenylamino, C₂.₆alkynylainino, arylamino, heterocyclamino acyl, C|. ₆alkylacyl, C₂.₆alkcnylacyl, C₂.(,alkynylacy], arylacyl, heterocycylylacyl, acylatnino, acyloxy, aklehydo, C|._f)alkylsulphonyl, arylsulphonyl, C[.₆alkylsulphonylamino, arylsulphonylamino, C|. _f,alkylsulphonyloxy, arylsulphonyloxy, Ci.₍,aikylsulphenyl, C₂.₍,alklysulphenyl,arylsulphonyl, carboalkoxy, carboaryloxy, mercapto, C|._f,alkylthio, arylthio, acylthio, cyano and the like. Preferred substituents are selected from the group consisting of Cm alkyl, C^cycloalkyl, C₂. (.alkenyl, C’₂.₍,alkynyl, Ct-oalkylaryl, aryl, heterocycylyl, halo, haloaryl, haloheterocycylyl, hydroxy. C|„ialkoxy, aryloxy, carboxy, amino, C|.₍,alkyiacyl, arylacyl, heterocycylylacyl, acylamino, acyloxy, C|.₍,alkylsulphenyl, arylsulphonyl and cyano.

The term “aryl refers to single, polynuclear, conjugated or fused residues of aromatic hydrocarbons. Examples include phenyl, biphenyl, terphenyl, quaterphcnyl, naphlhyl, tetrahydronaphtliyl, anthracenyl, dihydroanthracenyl, benzanlhracenyl, dibenxantliracenyl and phenanlhrcnyl.

The term “unsaturaled N-containing 5 or 6-membered hcterocyclyl” refers to unsaturaled, cyclic hydrocarbon groups containing al least one nitrogen. )

-6Suitable N-containing heterocyclîc groups include unsaturated 5 to 6-æembered heteromonocyclic groups containing l to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyi, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl; unsaturated 5 or 6-membered heteromonocyclic group containing l to 2 oxygen atoms and l to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl or oxadiazolyl; and unsaturated 5 or 6-membered heteromonocyclic group containing l to 2 sulphur atoms and l to 3 nitrogen atoms, such as, thiazolyl or thiadiazolyl.

In preferred embodiments, compounds related to CYT 387 include those in which K¹ is substituted in the para position by morpholinyl and in the orlho position by H, Z is carbon, and R¹¹ is H, methyl or methoxy.

In particularly preferred embodiments, R⁸ is -C(O)-NH-CH2-CH^N; -C(O)-N11-C(C1 hhCHzN; or -NH-C(O)-CT1₃-CHzN.

Spécifie compounds related to CYT 387 useful in accordance with the présent method include:

/V-(cyanomethyl)-4-(2-(4-morpholinophenylammo)pyrimidin-4-yl)benzamide; /V-(cyanomcthyl)-3-(2-(4-morpholinophenyianiino)pyrimidin-4-yl)benzamide; ₍V-(cyanomethyl)-3-melhyl-4-(2-(4-mori)holinophenylamino)pyrimidin-4-yl)benzamide; /V-(cyanomethyl)-2-methyl-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide; 2-cyano-/V-(3-(2-(4-inorpholinophenylamino)pyrimidin-4-yl)benzyl)acetamide; 2-cyano4V-(3-(2-(4-morpholinophenylamino)pyrimidin-4-yl)phenyl)acetamide;

JV-(cyanomethyl )-4-(2-(3-morphol inophenylamino)pyri midi n-4-yl)benzamide; /V-(cyanomethyl)-4-(2-(4-thiomorpholinophenylamino)pyrimidm-4-yl)benzamide; and jV-(cyanomethyl)-4-(2-(4-(morpholinomethyl)phenylamino)pyrimidin-4-yl)benzamide.

In lhe présent invention, CYT 387 and related compounds are used to ireat multiple myeloma (MM) cells that hâve a CD45 négative (CD45-) phenotype, and/or MM cells that are considered IL-6 non-responsive. MM cells are the disease cells that form plasmacytomatumours that are the hallmark of multiple myeloma. “CD45- phenotype” refers to a MM cell that tests négative or dim, as distinct from intermediate to bright, for surface expression of the protein marker known as CD45, which is a well-known marker of ail hematopoietic cells. The CD45phenotype is also ascribed herein with reference to a population of MM cells in which the prevalence of CD45- cells within that population exceeds at least about 10% of that population, such as at least about 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45% or al least about 50% of that population. Détection of CD45 on the cellular surface is readily achieved using fluorescence-labeled CD45 monoclonal antibody and established techniques of fluorescenceactivation cell sorting (FACS) or any related means for identifying cells that bind the CD45 antibody. Reference can be made for instance to the articles published by Moreau et al, q/'

-7Haematologica, 2004, 89(5):547, and by Kumar et al, Leukemia, 2005, 19:1466, the disclosures of which are incorporated herein by reference.

MM cells that are “IL-6 non-responsive” are identified as cells that do not rely for survival on the presence of interleukin-6 (IL-6). Thus, a MM cell that is IL-6 non-responsive shows insubstantial response, in ternis such as IL-6 receptor stimulation or downstream signalling events, when incubated with an otherwise stimuiatory amount of IL-6. Such MM cells can particularly include those MM cells that are résident in the bone marrow environment, and which thus grow in the same environment as bone marrow stromal cells, but they also include MM cells in circulation that are not exposed lo the marrow environment.

Within the realm of MM and its progression and development, CD45 represents an early marker of the disease MM cells. As the disease progresses, a shift occurs in CD45 phenotype of those cells, in which the prédominance of CD45+ cells wanes, and the population of disease plasma cells becomes predominantly CD45- (see Kumar et al, Leukemia, 2005, 19(8): 1466). A shift also occurs in the number of IL-6 non-responsive cells, with this cell form becoming prédominant in the later stages of disease.

In the présent method, the use of JAK inhibitors is proposed for the treatment of MM cells, and plasmacytomatumours that anse therefrom, that hâve acquired the CD45- and/or IL-6 non-responsive phenotype. The effect of JAK inhibition on this particular cell population is surprising, given that the JAK2 response to stimulation of the IL-6 receptor is believed to hâve a central and important rôle in progression of MM. Even when this IL-6 pathway is not involved in MM disease progression, CYT 387 fonctions to inhibit the growth and/or prolifération of these cells.

“Related disorders” are disorders related to MM as plasma cell disorders characterized by a clonal population of hematopoietic B cells that produce a monoclonal protein (M protein, or paraprotein). The clinical manifestations of these disorders resuit from lhe uncontrolled and progressive prolifération of a plasma cell clone, the effect of normal bone marrow replacement, and the overproduction of monoclonal proteins. MM is a plasma cell dyscrasia and includes newly diagnosed as well as relapsed MM.

Subjects, most notably human patients, who présent with MM are identifiable using any of the established diagnostic criteria and staging parameters. These include criteria established by the International Myeloma Workshop which distinguishes symptomatie MM subjects from those having asymptoinalic MM or gammopathy of undetermined significance (MGUS), by lhe presence of M protein in sérum or urine, clonal bone marrow plasmacytosis or plasmacytoma and related organ and tissue impairment. For staging of MM, the guidelines proposed by the Soulhwesl Oncology Group (SWOG) can be used, which rely essentially on measurements of B2-miciOglobulin and lhe relative presence of sérum albumin, with >5.5mg/L β2-Μ and <3.0g/dL indicaling stage IV disease. Ollier useful guidelines bave been established using the c-Z

-8Duire and Salmon staging system (using hemoglobin, sérum calcium, radiography and M protein).

In the présent method, subjects selected for treatment are those presenting with MM that also display an increase in the presence of CD45- cells within the population of MM cells. The increase in the presence of the CD45- cells is seen in subjects having newly diagnosed or relapsed MM, relative to subjects afflicted with smoldering MM or MGUS. Subjects having a relatively dramatic increase in the prevalence of CD45- MM cells particularly include those MM subjects diagnosed with stage III or stage IV ofthe disease. In a preferred embodiment, the number of CD45- cells within the population is at least 10% ofthe total population, such as 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more ofthe total MM cell population. Thus, in a représentative sample of 100 MM cells extracted from the subject, the patient population targeted for treatment by the présent method includes those presenting with a MM cell population in which 10-50% or more of the MM cells test négative for the CD45 marker.

In the présent method, subjects diagnosed with MM can first be screened lo select those subjects presenting with a MM cell population in which the CD45- phenotype is increased relative lo subjects afflicted with early stages of the disease, such as the smoldering MM stage. Screening is achieved using a MM cell population extracted from the subject, and then assaying the population such as by CD45 MAb-based flow cytometry to identify subjects in which there is an increase in CD45- MM cells. The MM cell population can also be assessed to reveal the prevalence of IL-6 non-responsive cells, the presence of which indicates the subject is a candidate for treatment by the présent method.

For use in the présent method, CYT 387 or a related compound is formulated according to standard pharmaceutical practice.

The compounds may be prepared as salts which are pharmaceutically acceptable, such as salts of pharmaceutically acceptable calions such as sodium, potassium, lithium, calcium, magnésium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, cilric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacctic, methancsulfonic, trihalomethanesulfonic, toluenesulfonic, benzenesulIonic, iscthionic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic, valeric and orotic acids. Salts of amine groups may also comprise quatemary ammonium salis in which the amino nitrogen atom carries a suitable organic group such as an alkyl, alkenyl, alkynyl oraralkyl moiety.

Where a compound possesses a chiral center Lhe compound can be used as a purified enantiomer or diastereomer, or as a mixture of any ratio of stereoisomers. It is however -D preferred that the mixture comprises at least 70%, 80%, 90%, 95%, 97.5% or 99% of the preferred isomer, where the preferred isomer gives the desired level of potency and selectivity.

Prodrugs of the compounds of formula Ib can also be administered. For example, compounds of formula Ib having free amino, amido, hydroxy or carboxylic acid groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy and carboxylic acid groups of compounds of the invention. The amino acid residues include the 20 naturally occurring amino acids coinmonly designated by three letter symbole and also include, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-mcthylhistidine, norvlin, beta-alanine, gammaaminobutyric acid, citrulline, homocystéine, hoinoserine, omithine and methioinesulfone. Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of compounds of the présent invention through the carbonyl carbon prodrugsidechain. Prodrugs also include phosphate dérivatives of compounds (such as acids, salts of acids, or esters) joined through a phosphorus-oxygen bond to a free hydroxyl of compounds of formula Ib, Prodrugs may also include N-oxides, and S-oxides of appropriate nitrogen and sulfur atoms in formula Ib.

The compound may be administered as a pharmaceutical composition comprising at least one of lhe compounds of the formula Ib and a pharmaceutically acceptable carrier. The carrier must be pharmaceutically acceptable means that it is compatible with lhe other ingrédients of the composition and is not dclelerious to a subject. The compositions may contain other therapeutic agents as described below, and may be formulated, for examplc, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavours, etc.) according to techniques such as those well known in the art of pharmaceutical formulation (See, for example, Remington: The Science and Practice of Pharmacy, 2lst Ed., 2005, Lippincott Williams & Wilkins).

The compound may be administered by any suitable means, for examplc, orally, such as in the form of tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subeutaneous, intravenous, intramuscular, intra(trans)dermal, or intracisternal injection or infusion techniques (e.g., as stérile injectable aqueous or non-aqueous solutions or suspensions); nasally such as by inhalation spray or insufflation; topically, such as in the form of a cream or ointment ocularly in the form of a solution or suspension; vaginally in the form of pessaries, tampons or creams; or rectally such as in the form of suppositories; in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents. The compounds may, for example, be administered in a form suitable for immédiate release or extended release. Immédiate release or extended release may be achieved by the use of suitable pharmaceutical c*

- lOcompositions comprising lhe compounds, or, particularly in lhe case ol'extended release, by lhe use of devices such as subcutaneous implants or osmolic pumps.

The pharmaceutical compositions for the administration may conveniently be presented in dosage unit form and may be prepared by any of lhe methods well known in (lie art of pharmacy. These methods generally include the step of bringing lhe compound of formula lb into association with the carrier which conslitutes one or more accessory ingrédients. In general, the pharmaceutical compositions are prepared by uiiiformly and intimately bringing lhe compound into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In lhe pharmaceutical composition the active object compound is included in an amount sufficient lo produce the desired effect upon the process or condition of diseases. As used herein, the term composition is intended to encompass a product comprising the specified ingrédients in (lie specified amounts, as well as any product which results, directly or indirectly, from combination ofthe specified ingrédients in the specified amounts.

The pharmaceutical compositions may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, émulsions, hard or soil capsules, or syrups or élixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents such as sweetcning agents, flavouring agents, colouring agents and preserving agents, e.g. lo provide pharmaceutically stable and palatable préparations. Tablets contain the compound of formula lb in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulaling and disinlegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnésium stéarate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in lhe gastrointestinal tract and thereby provide a suslained action over a longer period. For example, a lime delay material such as glycerylmonoslearate or glyceryldistearate may bc employed. They may also be coated to form osmolic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsules wherein lhe compound of formula lb is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the compound of formula lb is mixed with water or an oil medium, for cxample peanul oil, liquid paraffin, or olive oil,

Aqueous suspensions contain lhe active materials in admixture with excipients suitable for lhe manufacture of aqueous suspensions. Such excipients are sitspending agents, for J

- Il example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for cxample poiyoxyethylene stéarate, or condensation products of ethylene oxide with long chain aliphalic alcohols, for example heptadecaethyleneoxycelanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as poiyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitanmonooleate. The aqueous suspensions may also contain one or more préservât ives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the compound of formula lb in a vegetable oil, for example arachîs oil, olive oil, sesame oil or coconut oil, or in a minerai oil such as liquid paraffin. The oily suspensions may contain a thickcning agent, for example beeswax, hard paraffin orcetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral préparation. These compositions may be preserved by the addition ofan anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for préparation ofan aqueous suspension by the addition of water provide the compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be présent.

l he pharmaceutical compositions may also be in the form of oil-in-water émulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a minerai oil, l'or example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturallyoccurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy beau, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitanmonooleate, and condensation products ofthe said partial esters with ethylene oxide, for example polyoxyethylenesorbitanmonooleate. The émulsions may also contain sweetening and flavoring agents.

Syrups and élixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcenl, a preservative and flavoring and coloring agents.

The pharmaceutical compositions may be in the form of a stérile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which hâve been mentioned p/

- 12 above. The stérile injectable préparation may also be a stérile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in l ,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonie sodium chloride solution. In addition, stérile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the préparation of injectable formulations.

For administration to the respiratory tract, including intranasal administration, the active compound may be administered by any ofthe methods and formulations employed in the art for administration to the respiratory tract.

Thus in general the active compound may be administered in the form of a solution or a suspension or as a dry powder.

Solutions and suspensions will generally be aqueous, for example prepared from water alone (for example stérile or pyrogen-free water) or water and a physiologically acceptable cosolvent (for example éthanol, propylene glycol or polyethylene glycols such as PEG 400).

Such solutions or suspensions may additionally contain other excipients for example preservatives (such as benzalkonium chloride), solubilizing agents/surfaclants such as polysorbates (eg. Tween 80, Span 80, benzalkonium chloride), bnffering agents, isotonicityadjusting agents (for example sodium chloride), absorption enhancers and viseosity enhancers. Suspensions may additionally contain suspending agents (for example microcrystalline cellulose and carboxymethyl cellulose sodium).

Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in single or multidose form. In the latter case a means of dose metering is desirably provided. In lhe case of a dropper or pipette this may be achieved by the subject administering an appropriate, predetennined volume ofthe solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomising spray pump.

Administration to the respiratory tract may also be achieved by means of an aérosol formulation in which the compound is provided in a pressurized pack with a suitable propcllant, such as a chlorofluorocarbon (CFC), for example dichlorodifluororncthane, trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aérosol may conveniently also contain a surfactant such as lecilhin. The dose of active compound may be controlled by provision of a metered valve.

Alternatively lhe active compound may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch dérivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine ( P VP). Conveniently lhe powder carrier will form a gel in the nasal cavity. The powder composition j

- 13may be presented in unit dose form, for example in capsules or cartridges of eg. gelatin, or blister packs from which the powder may be administered by means of an inhaler.

In formulations intended for administration to the respiratory iract, including intranasal formulations, the active compound will generally have a small particle size, for example ofthe order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronisation.

When desired, formulations adapted to give sustained release ofthe active compound may be employed.

The active compound may be administered by oral inhalation as a free-flow powder via 10 a “Diskhaler” (trade mark of Glaxo Ciroup Ltd) or a mcter dose aérosol inhaler.

The compound may also be administered in the form of suppositories for rectal administration ofthe drug. These compositions can be prepared by mixing lhe drug with a suitable non-irritating excipient which is solid at ordinary températures but liquid at the rectal température and will therefore melt in lhe rectum to release the drug. Such materials are cocoa 15 butter and polyethylene glycols.

Compositions suitable for vaginal administration may be presented as pessarîes, tampons, crearns, gels, pastes, foams or sprays containing in addition to the active ingrédient such carriers as are known in the art to be appropriate.

For topical use, crearns, ointments, jellies, solutions or suspensions, etc., containing the 20 compound are employed. For purposes of this application, topical application can include mouthwashes and gargles.

For application to the eye, the active compound may be in the form of a solution or suspension in a suitable stérile aqueous or non-aqueous vehicle. Additives, for instance buffers, preservatives including bactericidal and fungicidal agents, such as phenyl mercurîc acetate or

5 nitrate, benzalkonium chloride, or chlorohexidine and thickening agents such as hypromellose may also be included.

Thecompound can also be administered in the form ofliposonies. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous 30 medium. Any non-toxic, physiologically acceptable and mclabolisable lipid capable of forming liposomes can be used. The présent compositions in liposome form can contain, in addition to a compound ofthe présent invention, stabilizers, preservatives, excipients and the like. The preferred lipids are the phospholipids and pliosphatidylcholines, both natural and synthetic. Methods to form liposomes are known in the art.

5 The compound may also be presented for use in the form of velerinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for: d (a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); ta blets or bol uses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the longue;

(b) parentéral administration for example by subculaneous, intramuscular or intravenous injection, e.g. as a stérile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced in the udder via the teat;

(c) topical applications, e.g. as a cream, ointment or spray applied lo lhe skin; or (d) reclally or intravaginally, e.g. as a pessary, cream or foam.

The pharmaceutical composition may further comprise other therapcutically active compounds as noted herein which are usually applied in the treatment of the above mentioned pathological conditions. Sélection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prévention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Examples of other therapeutic agents include the following: endothelin receptor antagonists (egambrisentan, bosentan, sitaxsentan), PDE-V inhibitors (eg sildenafil, tadalafil, vardcnafil), Calcium channel blockers (eg ainlodipine, felodipine, varepamil, diltiazem, menthol), prostacyclin, treprostinil, iloprost, beraprost. nitric oxide, oxygen, heparin, warfarin, diuretics, digoxin, cyclosporins (e.g., cyclosporin A), CTLA4 lg. antîbodies such as ICAM 3, anti-IL 2 receptor (Anti Tac), anti CD45RB, anti CD2, anti CD3 (OKT 3), anti CD4, anti CDSO, anti CD86, agents blocking the interaction between C’D40 and gp39, such as antîbodies spécifie forCD40 and/or gp39 (i.e., CDI54), fusion proteins construcled fromCIMO and gp39(CD40lg and CD8gp39), inhibitors, such as nuclear translocation inhibitors, of NE kappa B function, such as deoxyspergualin (DSG), cholestérol biosynthesis inhibitors such as ÎIMG CoA reductase inhibitors (lovastalin and simvastatin), non-steroidal anti-inflammatory drugs (NSAlDs) such as ibuproten, aspirin, acetaminophcn, leflunomidc, deoxyspergualin, cyclooxygenase inhibitors such as celecoxib, steroids such as prednisoione or dexamethasone. gold compounds, beta-agonists such as salbutamol, LABA's such as salmeterol, leukotriene antagonists such as montelukast, antiproliférative agents such as methotrexate, EK.506 (tacrolimus, Prograf), inycophenolatemofetil, cylotoxic drugs such as azathioprine, VP-16, etoposide, fludarabine, doxorubin, adriamycin, amsacrinc, camptothecin, cytarabine, gemcitabine, fiuorodeoxyuridine, melphalan and cyclophosphamide, antimetabolites such as methotrexate, topoisomerase inhibitors such as camptothecin, DNA alkylators such as cisplatin, kinase inhibitors such as sorafenib, microtubule poisons such as paclitaxel, TNF-a inhibitors

- 15 such as tenidap, anti-TNF antibodies or soluble TNF receptor, hydroxy urea and rapamycin (sirolimus or Rapamune) or dérivatives thereof.

In one embodiment, MM subjects are treated with a combination of CYT 387 or a related compound, and melphalan.

In another embodiment, MM subjects are treated with a combination of CYT 387 or a related compound, and borlezomib.

When other therapeutic agents are employed in combination with the compounds ofthe présent invention they may be used for example in amounts as noted in the Physician Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

In a preferred embodiment, the présent method utilizes CYT 387 in combination with a compound selected from melphalan and borlezomib.

The présent invention also provides both an article of manu facture and a kit, comprising a container comprising CYT387 or a related compound in an amount effective to treat MM. The container may be simply a bottle comprising the compound in oral dosage form. each dosage form comprising a unît dose ofthe compound, in an amount for instance from about 50mg to 400mg, such as 200mg or 3()0mg. The kit will further comprise prînted instructions teaching the présent method of selecting subjects for treatment. The article of manufacture will comprise a label or the like, indicating treatment of a subject according to the présent method of patient sélection.

Generally, the term treatment means affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiologiesI effect and include: (a) preventing the disease from occurring in a subject that may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresling its development; or (c) relieving or ameliorating the effects of the disease, i.e., cause régression of tlie effects of lhe disease. In one embodiment, treatment achieves the resuit of reducing the number of CD45- and/or IL-6 non-responsive MM cells in the récipient subject.

The term subject refers to any animal having a disease which requires treatment by lhe présent method. In addition to primates, such as humans. a variety of other mammals can be treated using lhe methods of lhe présent invention. For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated. Dogs in particular are known to expérience multiple myeloma.

The terni “administering should be underslood to mean providing a compound ofthe invention to a subject in need of treatment.

The term therapeutically effective amount refers to lhe amount ofthe compound that will elicit lhe biological or medical response of a tissue, system, animal or human that is being soughl by the researcher, veterinarian, medical doctor or other clinician. _/

- 16 In the treatment or prévention of multiple myeloma, an appropriate compound dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably aboui 0.5 to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing l .0 to 1000 milligrams ofthe active ingrédient, particularly l .0,5,0, 10.0, I5.0. 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0. 250.0, 300.0,400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams ofthe active ingrédient. The dosage may bc selected, for example to any dose within any ofthese ranges, for therapeutic efficacy and/or symptomatic adjustment ofthe dosage to the patient to be treated. The compound will preferably be administered on a regimen of l to 4 times per day. preferably once or twice per day.

Il will be understood that the spécifie dose level and frequency of dosage for any particular patient may be variée! and will dépend upon a variety of factors including the activity of the spécifie compound employed. the metabolîc stability and length of action of that compound, the âge, body weight, general health, sex, diet, mode and time of administration, rate ofexcrétion, drug combination, the severity ofthe particular condition, and the host undergoing therapy.

In order to exemplify the nature ofthe présent invention such that it may be more clearly understood, the following non-limiting examples are provided.

Ail publications mentioned in this spécification are herein incorporated by reference. Il will be appreciated by persons skilled in the art thaï numerous variations and/or modifications may be made to the invention as shown in the spécifie embodiments without departing from the spirit or scope ofthe invention as broadly described. The présent embodiments are, therefore, to be considered in ail respects as illustrative and not restrictive.

Examples

CYT387 is an inhibitor of the kinase enzymes JAKl and JAK2, which have been implicated in a family of hcmatological conditions known as myeloproliferative neoplasms, including myclofibrosis. and as well in numerous disorders including indications in hcmalology, oncology and inflammatory discases. Myclofibrosis is a chronic debilitating disease in which a patient's bone marrow is replaçai by scar tissue and for which treatment options arc limited or unsatisfactory.

Synthesis ofCYT 387

Λ mixture of 4-cthoxycarbonylphenyl boronic acid (23.1 I g, 119 mmol), 2,4dichloropyrimidine (16.90 g, I I3 mmol), toluene (230 mL) and aqueous sodium carbonate (2

- 17M, 56 mL) was stirred vigorously and nitrogen was bubbled through the suspension for 15 minutes. Tetrakis(lriphenylphosphine)palladium[0] (2.61 g, 2.26 mmol) was added. Nitrogen was bubbled through for another 10 min., the mixture was heated to !00.°C, then at 75°C overnight. The mixture was cooled, diluted with ethyl acetate (200 mL), water ( 100 mL) was added and lhe layers were separated. The aqueous layer was extracted with ethyl acetate (100 ml) and the two organic extracts were combined. The organics were washed with brine, filtered through sodium sulfate, concentrated, and the résultant solid was triturated with methanol (10Ü mL) and filtered. The solids were washed with methanol (2x30 mL) and air dried. This material was dissolved in acetonitrile (150 mL) and dichloromethane (200 inL), stirred with MP.TMT Pd-scavenging resin (Agronaut part number 800471) (7.5 g) over 2 days. The solution was filtered, the solids were washed with dichloromethane (2x100 mL), and lhe filtrate concentrated to give ethyl 4-(2-chloropyrimidin-4-yl)benzoate as an off-white solid (17.73 g, 60%)— additional washing with dichloromethane yielded a further 1.38 g and 0.5 g of product.

Λ mixture of ethyl 4-(2-chloropyrimidin-4-yl)benzoate (26.15 g, 99.7 mmol) and 4inorpholinoaniline (23.10 g, 129.6 mmol) was suspended in 1,4-dioxane (250 mL). pToluenesulfonic acid monohydrate ( I 7.07 g, 89.73 mmol) was added. The mixture was heated at reilux for 40 h., cooled to ambienl température, concentrated then the residue was partitioned between ethyl acetate and 1:1 saturated sodium bicarbonale/water ( I L total). The organic phase was washed with water (2x100 mL) and concentrated. The aqueous phase was extracted with dichloromethane (3x200 mL). The material which precipitated during this workup was collected by filtration and set aside. The liquid organics were combined, concentrated, triturated with methanol (200 mL) and filtered to yield additional yellow solid. The solids were combined, suspended in methanol (500 mL), allowed to stand overnight then sonicatcd and filtered. The solids were washed with methanol (2x50 mL) lo give, after drying, ethyl 4-(2-(4niorphonlinophenylamino)pyrimidin-4-yl)benzoate (35.39 g, 88%).

A solution of ethyl 4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzoate (35.39 g, 87.6 mmol) in 3:1 methanol/leirahydrofuran (350 mL) was treated with lithium hydroxide (4.41 g, 183.9 mmol) in water (90 mL). l he mixture was heated ai reflux for 2 h., cooled, concentrated and acidified with hydrochloric acid (2M, 92.5 ntL, 185 mmol). The dark precipitate was filtered, washed with water, and dried under vacuum, l he solid was ground to a powder with a mortar and pestle, triturated with methanol (500 mL) then filtered again lo yield 4-(2-(4-morpholinophenylamino)pyrirnidin-4-yl)benzoic acid as a muddy solid. This material was washed with ether, air dried overnight, and ground to a fine powder with mortar and pestle. On lhe basis of mass recovery (34.49 g) the yield was assumed to be quantitative.

To a suspension of 4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)bcnzoic acid (lheorelically 32.59 g, 86.6 mmol) in DMF (400 mL) was added triethylamine (72.4 mL, 519.6 mmol, 6 eq.) The mixture was sonicaled to ensure dissolution. Ammoacetonitrile hydrochloride af

-18 (16.02 g, 173.2 mmol) was added followed by N-hydroxybenzotriazole (anhydrous, 14.04 g, 103.8 mmol) and 1 -ethyl-3-(dimelhylaminopropyl)carbodiÎmide hydrochloride (19.92 g, 103.8 mmol). The suspension was stirred vigorously overnight. The solvent was evaporated under reduced pressure, lhe residue was diluted with 5% sodium bicarbonate (400 mL) and water (300 mL), giving a yellow solid, which was broken up and filtered. The solids were washed several times with 100 mL portions of water, triturated with bot melhanol/dichloromethane (500 mL, 1:1), concentrated to a volume of approximately 300 mL), cooled and filtered. The solids were washed with cold methanol (3x100 mL), ether (200 mL) and hexane (200 mL) prior to drying to afford CYT 387 (31.69 g, 88%). M.p. 238-243°C.

Synthesis of related compounds is described by Bunis et al, in Bioorganic & Médicinal Chemistry Letters, 2009, 19:5887 and in W02008/109943, both disclosures being incorporated herein by reference.

MATERIALS AND METHODS

Rcagenls

The JAK.1/2 inhibitor CYT387 was dissolved in DMSO. The protéasome inhibitor bortezomib (Jansscn-Cilag) was reconslituted in saline. The alkylating agent melphalan (Sigma) was dissolved in 0.5% HCl.EtOH. Ail stock drug solutions were diluted in complété RI’MI1640 culture medium to various concentrations for expérimentation.

Cell Lines and Culture conditions

HMCL LP-1, NCI-11929, OPM2, RPMI-8226 and U266 and the human stromal cell line HS5 were obtained from the American Type Culture Collection, USA. ANBL6, 0CI-MY1 and XG-1 were a ktnd gift from the Winthrop P Rockefeller Cancer Institute, Arkansas. HMCL were grown and treated at densifies between 2.0 and 5.0 x 105 cell/ml in RPMI-1640 media (Gibco, Invitrogen) supplemented with 10% heat inaclivaled foetal bovine sérum (FBS, Lonza) and 2 mMLglutaminc (Gibco, Invitrogen). IL-6 dépendent cells lines were cultured with 2-5 ng/tnl IL-6 as required. Ail cells were cultured in a humidified ïncubator at 37°C with 5% CO2. Al! HMCL were passaged 24 hours prior to experimental set-up to ensure high viability and cycling.

Primary Samples

Primary MM samples were obtained from Bone Marrow Aspirâtes from relapsed and refractory MM patients following written informer! consent with approval from lhe Alfred Hospital Research and Elhics Committee and isolated and treated as previously described [25], Briefly, bouc marrow mononuclear cells (BMMC) were isolated with Ficoll-Paque Plus (Amersham Biosciences), washed in PBS and red blood cells were lysed with NH4CI solution (8.29 g/L ammonium chloride, 0.037 g/L EDTA, 1 g/L potassium bicarbonate). Cells were then washed again in PBS and quanlilated by haemocylometer. BMMC samples were then cultured

- 19in complété RPMI-l 640 media (as above for HMCL) for 24 hours. Subsequently the BMMC were plated at 5 x 105 cells/ml and were treated with CYT387 (5 - 50 pM) alone or (dépendant on cell numbers) in combination with bortezomib (5 - 40 nM) or melphalan (50 - 200 pM) for 24 and/or 48 hours. Drug-induced MM spécifie cell apoptosis was then compared to untreated and vehicle controls by staining for CD45 FITC (BD), CD38 PerCP-Cy5.5 (BD) and Apo 2.7 PE (Immunotech) to détermine apoptosis in CD45-CD38+ MM cells. Samples were subsequently analyzed by FACS. Primary bone marrow stromal cells (BMSC) were also collected from patient BMMC, cells that adhered to the flask after an initial 24 hour culture were cullured with conlinued sélection for adhèrent cells over several passages. Once cells had expandcd in culture they were used in coculture (CC) to stimulatc MM cells in parallel to experiments utilising the HS5 stromal cell line.

Western Blots (WB)

HMCL were treated with CYT387 (1 or 2 pM) for 60 minutes and then stimulated with ng/inl IL-6 for 15 minutes. Protein lysâtes of CYT387 treated and untreated HMCL were made with RIPA Buffer (50 mMTrîs.HCI pH 7.4. 150 mMNaCl, ! mM PMSF, 1 mM EDTA, 5 pg/ml Aprotiuin, 5 pg/ml Leupeptin, 1% Triton X-100, 1% Sodium deoxycholate and 0.1% SDS). Brielly cells were incubated in RIPA buffer on ice for 30-60 minutes before being cenlrifugcd ai 16,100 x g for 20 minutes at 4°C and the supematant collected. Protein concentration was quanti lied using DC Protein Assay (Bio- Rad) as per manufacturées instructions. Subsequently, 100 pg of each protein lysate was separated by 10% SDS-PAGE and blotled onto nitrocellulose (Hybond ECL, Amersham) using the Bio-Rad semi-dry transfer system. Membranes wereblocked with 5% skim milk powder 0.1% Tween-20/PBS for 60 minutes then incubated with mouse monoclonal antiphospho-STAT3 (pY705, Santa Cruz), mouse monoclonal anti-STAT3 (Santa Cruz) or mouse monoclonal anti-a-tubulin (Sigma25 Aldrich) for 1-2 hours at room température or overnight at 4°C. The blots were washed three times for 15 minutes in 0.1% Twecn-20/PBS, then incubated with secondary HRP tagged antibody (swinc anli-rabbit Ig MRP (Dako) or rabbit anti-mouse lg HRP (Dako)) for 1-2 hours at room température before washing as above. Blots were visitaiized with Supersignal west pico ECL reagents (Pierce).

Intracellular FACS

Activation of the JAK/STAT, P13K/AKT and Ras/MAPK pathways was investigated using intracellular flow cytometry to measure the phosphorylation of STAT3 at tyrosine 705 (pSTAT3), AKT at serine 473 (p-AKT) and ERK.1/2 at threonine 202 and tyrosine 204 (p-ERK). HMCL were stimulated alone with 10 ng/ml IL-6 ± 200 ng/ml IGF-I or stimulated in CC with

HS5 stromal cells or primary BMSC with or without CYT387 treatment. For CC HS5 and primary BMSC were seeded into a 24 well plate at 2 x 105 cells/ml and allowed lo establish for J

-204 hours, aller which HMCL prestained with CD38 or CD138 F1TC (BD) were added. MM cells were stimulated alone ( 10 ng/ml IL-6 or 5 ng/ml lL-6 and 100 ng/ml IGF-1 ), or in CC (direct CC with stroma or transwell (TW) CC with stroma) with or without either 60 minutes of CYT387 prelreatmcnt or 15 minutes CYT387 co-treatment. After stimulation ± treatment, MM cells were harvested and fixed with 2% paraformaldéhyde for 10 - 30 minutes, washed then permeabilised with methanol overnight. Methanol was washed off and the cells were resuspended in p-STAT3 PE (BD), p-AKT PE (BD) or p-ERK (BD) and stained for 45 - 60 minutes at room température. Unbound antibody was washed off and the cells resuspended in 2% FBS PBS and acquired by FACS.

Prolifération and Viability Assays

The viability and prolifération of CYT387 treated HMCL and untreated/vehicle controls were determined using various methods as described previously [26]. Prolifération was measured first using Cellliter 96 AQeous one solution cell prolifération assay MTS reagent (Proniega) on a panel of 8 HMCL. Cells were cultured at 2.0 * 105 cell/ml in 100 μΐ fresh media in 96 well plates for 24, 48 and 72 hours with CYT387 (0.1 - 5 μΜ). 20 μ! of MTS reagent was added for the final 4 hours of treatment and the plates were read at 490 nm using a Fluostar Optima plate reader (BMG Labtech). The viable cell numbers of a panel of 5 HMCL that were treated with CYT387 with and without IL-6 co-treatment was also measured by trypan blue staining and haemocytomcter count. The HMCL NCI-H929, OC1-MY1 and U266 were then selected for further analysis. Apoptosis of CYT387 treated cells was assessed by FACS with Annexin-V and propidium iodide (PI) staining. HMCLs were treated for 24 or 72 hours with 1 or 5 μΜ CYT387 then harvested and washed in Annexin Buffer (0.01 HEPES, 0.14 M NaCI, 2.5 mM CaC12, pl i 7.4) and stained with Annexin-V FITC (Biosource) made up in Annexin Buffer for 30 minutes at room température. Unbound antibody was then washed off with Annexin Buffer and cells were resuspended in Annexin Bufler with 62.5 ng/ml PI (SigmaAldrich) and analyzed by FACS.

For the synergy experiments HMCL were treated with CYT3S7 in combination with bortezomib or melphalan for 24 and 48 hours before being harvested and resuspended in FACS Buffer (0.5% III FBS in PBS) supplemented with 62.5 ng/ml PI (Sigma-Aldrich). Cells were iinmediately analyscd by FACS. The proportion of PI positive cells was quantitated by subtracting lhe background death of unlreated cells. Single drug treated cells were compared to combination treated cells and synergism was calculated using Calcusyn software (Biosoft).

Cell Cvcling

The effect of CYT387 treatment on HMCL cell cycling was measured aller 24 and 72 hours. CYT387 (I or 5 μΜ) treated and untreated HMCL were harvested, washed in PBS and resuspended in 100 μΙ PBS. Cells were fixed with I ml of cold 70% éthanol while being o/

-2l vortexed. Tubes were stored at -20°C until analysis. Once all samples were collected tubes were centrifuged at 500 x g for 10 minutes, the supematant was carefully removed and the cells washed in 5 ntl PBS. After Lhe final wash cells were resuspended 250 - 500 pl of Pl/RNase staining buffer (BD) and incubated in the dark at room température for 15 minutes before being analyzed by FACS.

Data Analysis

All FACS data was acquired on a BD FACScalibur and data analysis done using Flowjo 7.6 Software (Treestar, USA). Ail statistical analysis was done using GraphPad Prism 5.03 software (USA).

RESULTS

JAK/STAT signal]mg is inhibited by CYT387 IL-6 signalling through the JAK/STAT palhway is well characterized in MM cells with binding of IL-6 to its receptor inducing JAK2 to phospliorylate STAT3. The ability of CYT387 to inhibit JAK2 was first confirmed by measuring the level of STAT3 phosphorylation by western blolting and FACS. FIMCL (NClH929, OCl-MYl and U266) were incubated with CYT387 for I hour before being stimulaled with IL-6 for 15 minutes to induce p-STAT3. CYT387 (0.5 - 2 pM) inhibited the phosphorylation of STAT3 in IL-6 stimulated samples as demonstraled by FACS (Figure l A) and confirmed by western blolting (Figure l B). Total STAT3 protein was unaffected.

Given the importance ofthe BMME in MM growth and survival it was important to establish if CYT387 could similarly modulate signalling in MM cells in CC with BMSC. This was done with both immortalized BMSC (IIS5) and primary patient BMSC. In each case 15 minutes of CC with BMSC (with or without contact) was able to induce phosphorylation of STAT3 in the MM cells, whereas conlemporaneous treatment with CYT387 dramatically reduced the amount of p-STAT3 in the HMCL (Figure IC). Thus, demonstrating that CYT387 is able to prevent STAT3 activation in MM cells induced by the soluble within lhe MM-BMSC microenvironment and also the contact mediated signalling provided to MM by BMSC.

CYT387 inhibits P13K/AKT and Ras/MAPK signalling JAK signalling kinases are involved in many cellular palhways, which lcd us to investigate the effect of CYT387 on IL-6 and IGF-l induced P13K/AKT and Ras/MAPK signalling in NCI11929, OCI-MYI and U266cells (Figure ID). OCl-MYl showed distinct p-AKT activation after IL-6 and IGF-l stimulation that was signifîcantly reduced by CYT387 cotreatment. IL-6 and IGF-l stimulation induced p-ERK in U266 cells which was significantly inhibited by CYT387. l'he levels of p-AKT anti p-ERK showed only a small increase tn response to IL-6 and IGF-l stimulation in NCI-H929.

-22CYT387 inhibits prolifération in HMCL

The effect of CYT387 (O.l - 5 μΜ) on cell prolifération was measured by MTS assay at 24, 48 and 72 hours (Figure 2A) on a panel of 8 HMCL (IL-6 non-responsive phenotype - LP-l, NCI-H929, OPM2, RPMI-8226 and IL-6 responsive phenotype - ANBL-6, OCI-MY l, U266 and XG-l). Six of 8 HMCL had a time and dose dépendent response to CYT387 with inhibition in some HMCL within 24 hours. At 72 hours NCI-H929 and XG-l were the most sensitive to CYT387treatment.

Prolifération of HMCL cultured with CYT387 over 72 hours was also assessed by quantitation of viable cell number by haemocytomelcr (Figure 2B). Because ofthe obvious relationship between IL-6 signalling and the inhibition of JAK2 by CYT387 the prolifération of 3 HMCL (NCI-H929, OCI-MYl and U266) was measured with the addition of IL-6 and/or CYT387. The 3 HMCL proliferated well in complété media with or without supplémentation with 10 ng/ml IL-6 and CYT387 (0.5 - l μΜ) was able to rcduce the prolifération of HMCL in culture even in tlie presence of IL-6. CYT387 inhibited cell prolifération by 50% NCI-H929 (l μΜ). 50% OCl-M Y l (0.5 μΜ) and 44% U266 ( l μΜ) after 72 hours.

Treatment of HMCL with CYT387 results in an accumulation of cells in G2/M phase of the cell cycle

The anti-proliferative effects of CYT387 were further characterised by evaluating the cell cycle of HMCL treated with CYT387. HMCL (NCI-H929, OCI-MYl and Ό266) treated with CYT387 (l - 5 μΜ) showed a marked accumulation of cells in the G2/M phase ofthe cell cycle. This was most pronounced in NCI-H929 cells (Figure 2C), where there was more than a two fold increase in cells in G2/M in the drug treated sample compared to untreated or vehicle treated control after24 and 72 hours treatment. An additional polyploid population was found in the CYT387 treated samples - suggesting thaï CYT387 treatment causes further aberration from the normal cell cycle of MM cells.

CYT387 induces apoptosis in HMCL and primary MM cells

Apoptosis of CYT387 treated cells was investigated using Annexin-V/Propidium Iodide FACS slaining in 3 HMCL (NCIII929, OCI-MYl and U266). An increased proportion of apoptotic cells was detected in ail 3 HMCL which was most évident in NCI-H929 (Figure 3A), with a 2I and 52% réduction in viable cells detected at 24 and 72 hours, respectively, after treatment with 5 μΜ CYT387 (Figure 3B). To assess CYT387 as pari of a combination therapy NCI-H929, OCI-MYl and U266 were treated with a range of doses of CYT387, bortezomib, melphatan to establish dose effect curves for each drug before combining with CYT387 and ineasurîng the synergy using CalcusynTM software. Dose effect curves generaied for each compound (melphalan and bortezomib data not shown) show CYT387 (0.5 - 10 μΜ) induced apoptosis in 3 HMCL in a time and dose dépendent manner (Figure 4A). CYT387 displayed o(

-23 synergism with both bortezomib and melphalan but with variations in the level of synergy seen at differing drug dosages and analysis time-points (Figure 4B).

The effect of CYT387 on ex-νίνο primary MM cells was also investigated, both as a single agent and as part of combination therapy. MM Patient BMA were cultured with various of doses of CYT387 (5 - 50 μΜ) for 24 and 48 hours after wliich the CD38+CD45- MM cell populations were asscssed for apoptosis by flow cytometry. Six patients were treated and apoptosis was seen in between 5 and 59% of MM cells treated with 20 μΜ CYT387 after 48 hours (Figure 5A). The eflect of CYT387 in combination with melphalan and bortezomib was also investigated. CYT387 was seen to synergize with melphalan in 2/3 patients, and synergy was also observed with bortezomib in some patients/doses (Figure 5B).

DISCUSSION

Multiple lines of evidence bave confinned the rôle oflL-6/JAK/STAT signalling in MM including experiments demonstrating lhe IL-6 dependence of some MM cells, the upregulalion of prolifération of MM cells with IL-6, the inhibition of drug induced apoptosis by IL-6, and most important to this investigation, the direct induction of apoptosis in MM cells by inhibition ofthe IL-6/JAK/STAT pathway. These data and the abundance of IL-6 in the BMME makes JAK/STAT signalling a rational target for inhibition with new chemotherapeutics and is supportée! by preliminary in vitro studies that hâve demonstrated MM cell apoptosis induction via siRNA targeting ofthe JAK/STAT pathway [27]. Furthermore, the signal transduction rôle of JAKs în other important pathways [reviewed by 5] and the pro-survival effects of JAK mutations in other haemalologicai malignancies [l-4] hâve already demonstrated the therapeutic potential of JAK inhibition. The therapeutic challenge is then inhibiting MM cells in the presence of IL-6 or BMSC. Here we hâve expanded upon previous work evaluating JAK inhibition in MM by studying a broader range of IIMCL, by demonstrating that CYT387 can inhibit JAK-STAT activation in the context of coculture tnodels and by demonstrating the impact of CYT387 on primary MM tumour cells. It has been hypothesized that in the earlier stages of MM that the malignant cells are predominantly CD45+, whereas in more advanced, drug-resistant disease, CD45- MM cells predominate [28], Moreover, CD45- MM cells are considered less IL-6 responsive and express fewer IL-6 receptors than CD45+ MM [29], Other studies of JAK inhibition bave focuscd predominantly on CD45+ IL-6 responsive I-IMCL and while this is a logical locus for preliminary investigation il must be stressed that such target cell populations represents only a subset of MM cells. Furthermore, patients may demonstrate mixed populations of both C’D45-and CD45+ MM cells [29], Importantly, we bave demonstrated lhe effectiveness of CYT387 against NCI-H929, a IIMCL considered to bave an IL-6 non-responsive phenotype suggesting thaï CYT387 will be effective against a range of MM phenotypes, whereas others [19] bave reported only limited success against CD45- MM

-24using alternative JAK inhibitors. Our data demonslrating synergy between CYT387 and bortezomib or melphalan against several HMCL and primary MM tumour cells confirais previously published work [l 9).

Available data suggests that the inhibition of JAK in MM cells may hâve downstream effects other than the direct inhibition ofthe JAK/STAT pathway. The importance ofIL-6 in MM cell survival has been well characterized in ternis of the JAK/STAT pathway but there is now increasing evidence of lL-6 induced P13K/AKT activation [l 8, 30] and Ras/MAPK activation [20, 30-33] in various IIMCL. Given the established importance of both the PBK/AKT andRas/MAPK pathways in addition to the JAK/STAT pathway in MM, small molécule inhibitors that could modulate ail three could hâve enormous clinical potential.

hivestigating the effects of JAK inhibition on the P13K/AKT and Ras/MAPK pathways has yielded contrasting results. AZD1480 and AG490 bave beeti shown to reduce IL-6 induced activation of Ras/MAPK [20, 22], but AG490 in lhe hands of others showed no decrease in IL-6 induced Ras/MAPK activation [31 ]. The JAK inhibîtor 1NCB20 could inhibit IL-6 induced Ras/MAPK activation in MM.l S cells, but did not affect constitutive Ras/MAPK activation iti lNA-6 cells [l 8]. The inhibition of JAK with AG490 could also abrogate AKT activation in PTEN mutated OPM2 MM cells [30]. Similarly 1NCB20 could also inhibit IL-6 induced pΛΚΤ, but had no effect on IGF-l induced p-AKT in INA-6 cells [l8]. Variability in the available data may be the resuit of différences in inhibitors and heterogeneity amongst HMCLs that are commonly studied. In our study there was a signilicant réduction in IL-6 and IGF-l induced ρ-ERK in U266 cells, as well as a dramatic réduction of IL-6 and IGF-l induced pAKT in OCIMYl cells supporting the data of others that suggests JAK inhibition may hâve broader anti-MM activity than would be initially expected. The inhibition of IL-6 induced JAK/STAT and PI3K/AKT signalling may also resull in a réduction in IL-6R expression on the surface of MM cells [30], which could also lead to a réduction in the pro-survival effects of IL-

6.

The profound anti-proliferative effect of CYT387 on various HMCL after a single dose is an important démonstration of its elTectiveness. Furthermore, the ability of CYT387 lo inhibit HMCL growth even in the presence ofexogenous IL-6 which has been shown many limes as a medialor of drug résistance [l 1-13] ts noteworthy. This signilicant effect of JAK inhibition may be the result of HMCL having some dependence on JAK/STAT for proliférative signais, or more likely the involvement and subséquent inhibition of alterative signalling pathways mentioned above. The effect on prolifération is also seen in the cell cycling analysis which demonslrates that CYT387 can prevent cell cycling.

Also of interest was lhe additional polyploidy population (8n) induced by CYT387 treatment, which may suggest a rôle for JAK signalling leading to cell cycle régulation or may be the resuit of CYT387 inhibition of other kinases involved in cell cycle such as aurora B,

-25 aurora C, cyclin A or cyclin B as described by Pardanani et aL, 2009. A direct or downstream effect of JAK inhibitors on cell cycling proteins is supported by studies on other JAK inhibitors; Scuto et al. found AZD1480 inhibited Cyclin D2 in two HMCL [22]. The induction of apoptosis in MM cells in primary marrow cocultures is a critical démonstration of the potential of CYT387. In contrast, other studies inhibiting IL-6 signalling hâve failed to demonstrate any convincing evidence of apoptosis when MM cells were treated in the presence of BMSC [32]. The investigators suggested that this might represent MM cell independence from IL-6 in the presence of BMSC. Our findings with CYT387 could bc interpreted as either refuting the latter or alternatively being consistent with the capacity of JAK signalling inhibition to interrupt non1L6 mediated survival pathways. Consistent with the latter was that the primary MM samples treated with CYT387 were représentative of autologous whole marrow cocultures with lhe CD38+CD45- MM cells making up between 4 and 67% of treated cells. Therefore the démonstration that CYT387 was able to induce apoptosis of these heavily-treated MM cells was particularly encouraging.

Additional compound évaluation

The compounds of formula lb can tested in the mouse model of multiple myeloma as described in Dalton, W. and Anderson, K. C., Clinical Cancer Rcsearch, 2006; 12(22), 66036610. Mouse models 5T2MM and 5T33MM are reported in Dalton et al as having clinical characteristics similar to the human disease, including localisation of multiple myeloma cells to the bone marrow, measurable M-protein in sérum, induction of osteolytic bone disease, and increased angiogenesis in the marrow. Both models may therefore be used to test the compounds of formula lb and allow for lestîng whether the compounds affect multiple myeloma cell homing before disease onset or after onset ofthe disease. Using these models, the effect ofthe compounds on lhe levels of sérum M-protein may be determined and the effect of the compounds on inhibition of disease development such as réduction of osteolytic bone disease, réduction in tumor burden and improvement in survival, may also be determined.

In order to examine the effects ofthe compounds of formula lb against a human myeloma, the compounds may be tested against xenograft models of human myeloma in mice. For these tests, the xenograft models of human myeloma turmors or cell lines may be transplanted into, for example, SCID-Ilu or NOD/SCID mice. The SCID-lIu model may be used to study myeloma in a human microenvironmenl and the impact of lhe compounds of formula lb on the reproducible growth of primary myeloma cells may also be studied. The NOD/SCID model involves labelling multiple myeloma cells with green fluorescent protein. This model may be used to follow disease progression. j

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13. Perez, L.E., N. Parquet, K. Shain, et al., Bone marrow stroma confers résistance to Apo2 Ugand/TRAIL inmidtiple myeloma in part by regulating c-FLIP. J Immunol, 2008.180(3): p. 1545-55.

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15. Tyner, J.W., T.G. Bumm, J. Deîninger, et al., CYT387, a novel JAK2 inhibitor, induces hématologie responsesand normalizes inflammatory cytokines in murine myeloproliferative neoplasms. Blood, 2010.115(25): p. 5232-5240.

16. Pardanani, A., T. Lasho, G. Smith, C.J. Burns, E. Fantino, and A. Tefferi, CYT387, a sélective JAK17JAK2inhibitor: in vitro assessment of kinase selectivity andpreclinical studtes using cell lines andprimary cellsfrompolycythemia vera patients. Leukemia, 2009.23(8): p. 1441-5.

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31. Puthier, D., R. Bataille, and M. Amiot, IL-6 up-regulates md-l in human myeloma cells through J AK / STATrather than ras / MAP kinase pathway. Eur J Immunol, 1999. 29(12): p. 3945-50.

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33. Ferlin, Μ., N. Noraz, C. I-Iertogh, J. Brochier, N. Taylor, and B. Klein, Insulin-likegrowth factor induces thesurvival and prolifération of myeloma cells through an interleukin-6independent transductionpathway. Br JHaematol, 2000.111(2): p. 626-34. cf

-29l. The use of a compound in preparing a médicament for treating a subject presenting with multiple myeloma at a stage characterized by an increase in the prevalence of MM cells that (l) are IL-6 non-responsive and/or (2) hâve a CD45- phenotype, the compound having the formula Ib:

Claims (10)

1. WE CLAIM:

   wherein

   Z is independently selected from N and Cil;

   R¹ is independently selected from H, halogen, OH, CON11R*. CON(R²)₂, CF», R^{2 * *}OR^{2 *}, CN, morpholino, thiomorpholinyl, thiomorpholino-l, l-dioxide, substituted or unsubstitutedpiperidinyl, substituted or unsubstitutcdpipcrazinyl, imidazolyl, substituted or unsubstitutedpynOlidinyl and Cj^alkylene wherein the carbon atoms are optionally replaced with NR^V and/or O substituted with morpholino, thiomorpholinyl, thiomorpholino-l, l-dioxide, substituted or unsubstitutedpiperidinyl, substituted or unsubstitutedpiperazinyl, imidazolyl or substituted or unsubstitutedpynOlidinyl;

   R² is substituted or unsubstituted C|.qalkyl;

   R^Y is H or substituted or unsubstituted Cj^alkyl;

   R⁸ is R^XCN;

   R^x is substituted or unsubstituted Ci^alkylene wherein up to 2 carbon atoms can be optionally replaced with CO, NSO2R', NR^Y, CONR^Y, SO, SO2 or O;

   R¹¹ is II or Ci^alkyl, or an enantiomer thereof, a prodrug thereof or a pharmaceutically acceptable sait thereof effective to reduce the viability of said MM cells.
2. 2. The use of a compound of in preparing a médicament for treating a subject with multiple myeloma according to claim 19 including the steps of (1) selecting, for treatment, a subject diagnosed with an increase in the prevalence of MM cells that ( i ) are IL-6 non-responsive, and/or (2) hâve a CD45- phenotype; and (2) administering to the subject an amount the compound of formula 1b effective to reduce the viability of the MM cells.
3. 3. The use of a compound of in a method of preparing a médicament for treating a subject with multiple myeloma according to claim 20, including the steps of ( l ) obtaining a sample of MM cells front said subject; (2) analyzing said MM cells for the prevalence of cells that are IL-6 non-responsive and/or have a CD45- phenotype; (3) selecting for treatment a subject in which the prevalence of said MM cells is increased relative to subjects at an early stage of multiple myeloma, and (4) administering to the selected subject an amount of the compound of formula Ib effective to reduce the viability of said MM cells.
4. 4. The use of a compound in preparing a médicament for use in treating a subject presenting with multiple myeloma according to any one of daims 19 to 21, wherein the subject is treated with CYT 387 which is N-(cyanomethyl)-4-[2-[[4-(4morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide, having the structure shown below:

   CN , or an enantiomer thereof, a prodrug thereof or a pharmaceutically acceptable sait thereof
5. 5. The use of a compound in preparing a médicament for use in treating a subject presenting with multiple myeloma according to claim 22, wherein the subject is afflicted with newly diagnosed or relapsed multiple myeloma.
6. 6. The use of a compound in a method of preparing a médicament for use in treating a subject with multiple myeloma according to any one of daims 18 to 23 wherein the subject présents with a population of MM cells in which at least I0% of said cells are CD45-.
7. 7. The use of a compound in a method of preparing a médicament lor use in treating a subject with multiple myeloma according lo daim 24, wherein the subject is treated with CYT 387 and a second therapeutic agent selected from mclphalan and bortezomib.

   -3l -
8. 8. An article of manufacture, comprising a container comprising the compound of formula Ib defined in claim l, and, associated with the container, a label indicating treatment of a subject presenting with newly diagnosed or relapsed multiple myeloma in which MM cells are IL-6 non-responsive or hâve a CD45- phenotype.
9. 9. A kit comprising a container comprising the compound of formula Ib defined in claim l in an amount useful to trcat newly diagnosed or relapsed multiple myeloma in which MM cells are IL-6 non-responsive or bave a CIJ45- phenotype and, in association
10. 10 lherewith, printed instruction teaching a method according to any one of claims 1 -7. p/