WO2005044274A1

WO2005044274A1 - Roscovitine treatment of mantle cell lymphoma

Info

Publication number: WO2005044274A1
Application number: PCT/GB2004/004700
Authority: WO
Inventors: Simon Green; Athos Giannella-Borradori; Francesco Bertoni
Original assignee: Cyclacel Limited
Priority date: 2003-11-06
Filing date: 2004-11-08
Publication date: 2005-05-19

Abstract

The present invention relates to the use of roscovitine, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating mantle cell lymphoma.

Description

ROSCOVITINE TREATMENT OF MANTLE CELL LYMPHOMA

The present invention relates to the therapeutic uses of the compound 2-[(l-ethyl-2- hydroxyethyl)amino]-6-benzylamine-9-isopropylpurine and pharmaceutically acceptable salts thereof.

BACKGROUND TO THE INVENTION

The prior art has described several compounds that are capable of regulating the cell cycle by virtue of inhibiting cyclin dependent kinases. These compounds include butyrolactone, flavopiridol and 2-(2-hydroxyethylamino)-6-benzylamino-9- methylpurine (olomoucine). Oloumucine and related compounds have been shown to be inhibitors of cdc2. cdc2 (also known as cdkl) is a catalytic sub-unit of a family of cyclin dependent kinases that are involved in cell cycle regulation.

These kinases comprise at least two sub-units, namely a catalytic sub-unit (of which cdc2 is the prototype) and a regulatory sub-unit (cyclin). The cdks are regulated by transitory association with a member of the cyclin family: cyclin A (cdc2, CDK2), cyclin B1-B3 (cdc2), cyclin C (CDK8), cycline D1-D3 (CDK2-CDK4- CDK5-CDK6), cyclin E (CDK2), cyclin H (CDK7).

Each of these complexes is involved in a phase of the cellular cycle. CDK activity is regulated by post-translatory modification, by transitory associations with other proteins and by modifications of their intra-cellular localization. The CDK regulators comprise activators (cyclins, CDK7/cyclin H, cdc25 phosphateses), the p9.sup.CKS and pl5.sup.CDK-BP sub-units, and the inhibiting proteins (pl6.sup.INK4A, pl5.sup.LNK4B, p21.sup.Cipl, pl8, p27.sup.Kipl).

There is now considerable support in the literature for the hypothesis that CDKs and their regulatory proteins play a significant role in the development of human tumours. Thus, in numerous tumours a temporal abnormal expression of cyclin-dependent kinases, and a major de-regulation of protein inhibitors (mutations, deletions) has been observed. Roscovitine is the compound 6-benzylamino-2-[(R)-l-ethyl-2-hydroxyethylamino]-9- isopropylpurine. It induces apoptosis from all phases of the cell cycle in tumour cell lines and reduces tumour growth in human tumour xenongrafts in nude mice. The compound is currently in development as an anti-cancer agent.

Roscovitine has been demonstrated to be a potent inhibitor of cyclin dependent kinase enzymes, particularly CDK2/cyclin E and CDK2/cyclin A, CDK7/cyclin H and CDK9/cyclin T and it has been shown to have cytotoxic activity against a broad range of tumours in vitro and in vivo.

CDK inhibitors are understood to block passage of cells from the Gl/S and the G2/M phase of the cell cycle. Roscovitine has also been shown to be an inhibitor of retinoblastoma phosphorylation and therefore implicated as acting more potently on Rb positive tumours.

It has now been observed that roscovitine has therapeutic applications in the treatment of certain proliferative disorders that have to date been particularly difficult to treat.

STATEMENT OF INVENTION A first aspect of the invention relates to the use of roscovitine, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating mature B-cell malignancies, for example, mantle cell lymphoma.

A second aspect of the invention relates to a method of treating a patient suffering from mantle cell lymphoma comprising administering a therapeutically effective amount of roscovitine or a pharmaceutically effective salt thereof.

A third aspect of the invention relates to a pharmaceutical composition comprising (i) roscovitine, or a pharmaceutically acceptable salt thereof; and optionally (ii) a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment of mantle cell lymphoma. A fourth aspect of the invention relates to a method of down regulating expression of an anti-apoptotic gene in mantle cell lymphoma cells, the method comprising contacting the cells with roscovitine, or a pharmaceutically acceptable salt thereof.

A fifth aspect of the invention relates to a method of treating mantle cell lymphoma in a subject, the method comprising administering roscovitine, or a pharmaceutically acceptable salt thereof, to the subject in an amount sufficient to down regulate the expression of an anti-apoptotic gene in the subject.

A sixth aspect of the invention relates to a method of down-regulating Mcl-1 expression in mantle cell lymphoma cells, said method comprising contacting said cells with roscovitine, or a pharmaceutically acceptable salt thereof.

A seventh aspect of the invention relates to a method of treating mantle cell lymphoma in a subject, said method comprising administering roscovitine, or a pharmaceutically acceptable salt thereof, to the subject in an amount sufficient to down-regulate the expression of Mcl-1 in said subject.

An eighth aspect of the invention relates to the use of roscovitine, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating mantle cell lymphoma, wherein the roscovitine or a pharmaceutically acceptable salt thereof, is in an amount sufficient to down-regulate the expression of Mcl-1.

DETAILED DESCRIPTION As mentioned above, the present invention relates to the use of roscovitine in the treatment of mantle cell lymphoma.

Roscovitine or 2-[(l-ethyl-2-hydroxyethyl)amino]-6-benzylamine-9-isopropylpurine, is also described as 2-(l-D,L-hydroxymethylproρylamino)-6-benzylamine-9- isopropylpurine. As used herein, the term "roscovitine" encompasses the resolved R and S enantiomers, mixtures thereof, and the racemate thereof. The in vitro activity of roscovitine is as follows:

Although the use of roscovitine as an antiproliferative agent is known in the art, to date, there has been no suggestion that it would be effective in the treatment of mantle cell lymphoma, which is known to be particularly difficult to treat and is often resistant to conventional treatments.

THERAPEUTIC ACTIVITY Mantle cell lymphoma (MCL) is a type of non-Hodgkin's lymphoma (NHL). In the US, NHL represents approximately 4% of all cancer diagnoses and is the seventh most common cancer. In 1999, more than 55,000 new cases of NHL were diagnosed and the incidence of NHL of all types has increased by about 40% over the last 20 years. Internationally, NHLs are five times more common than Hodgkin's disease, representing about 4% of all cancers diagnosed internationally.

There are over twenty different types of non-Hodgkin's lymphoma and MCL is a relatively uncommon type accounting for about 5-8% of all cases. MCL is now recognised as a distinct clinicopathologic entity, but was previously often characterised as diffuse small cleaved cell type or centrocytic type. Despite being originally considered a low-grade and indolent lymphoma, MCL appears to have the worst characteristics of both low and high grade lymphomas, i.e. poor response to conventional therapy and rapid growth. Exact incidence of MCL is difficult to estimate internationally because of lack of uniform classification and procedures used for diagnosis.

More specifically, MCL is a lymphoproliferative disorder derived from a subset of naive pregerminal center cells localized in primary follicles or in the mantle region of secondary follicles. Most cases of MCL are associated with chromosome translocation t(ll;14)(ql3;q32). This translocation involves the immunoglobulin heavy-chain gene on chromosome 14 and the BCL1 locus on chromosome 11. The molecular consequence of translocation is overexpression of the protein cyclin Dl (coded by PRAD1 gene located close to the breakpoint). Cyclin Dl plays a key role in cell cycle regulation and progression of cells from Gl phase to S phase by activation of cyclin- dependent kinases.

To date, MCL has been treated by conventional chemotherapy using single alkylating agents such as chlorambucil. Alternatively, combination chemotherapy may be used; for example, cyclophosphoramide, vincristine, prednisone ["CVP"], or cyclophosphoramide, hydroxydaunomycin, oncovin, prednisone ["CHOP"]. Newer regimens such as moderately high dose cyclophosphoramide, vincristine, Adriamycin® and decadron, alternating with methotrexate and cytarabine, have been reported to yield higher response rates, but the efficacy of these regimens has not been tested in randomized studies. More recently, the use of nucleoside analogues (fludarabine, cladribine), interferon and monoclonal antibodies (rituximab) have been investigated, together with high dose chemotherapy with autologous bone marrow or stem cell transplantation.

However, many of these prior art treatments are of limited efficacy and are associated with adverse side effects such as infection, neutropenia, hypersensitivity, anemia, thrombocytopenia, fatigue, neuropathy, dehydration from diarrhea or vomiting, and cardiac toxicity. MCL remains a difficult problem because of the lack of reliable curative treatments and paucity of prospective clinical trials. Although 50-90% of patients respond to combination chemotherapy, relatively few (30%) have a complete response and the disorder is rarely curable with currently available treatments. Generally, the prognosis for patients is poor, with a median survival of 2 to 5 years, with only 5-10% of patients surviving 10 years.

The present invention provides an alternative treatment for mantle cell lymphoma which comprises the use of roscovitine. To date, there has been no teaching or suggestion in the prior art that roscovitine would be suitable for treating this particular disorder.

Experiments have demonstrated that roscovitine can inhibit the transcription of a wide range of RNA polymerase II transcripts (Lam et al., 2001, Genome Biology 2(10) RESEARCH0041). Furthermore, roscovitine can disrupt nucleolar structure also indicative of a transcription inhibitor. Consistent with these results, roscovitine inhibits CDK7/CyclinH/Mat 1 and CDK 9/Cyclin Tl in vitro at low μM concentrations (McClue et al, 2002, Int. J. Cancer. 102(5):463-8). These kinases are required for phosphorylation of the heptapeptide repeats in the carboxy-terminal domain (CTD) of RNA polymerase II resulting in the activation of transcriptional elongation. Inhibition of transcription exerts greatest effect on gene products where both the mRNA and protein have short half lives resulting in rapid decline of the protein levels. A number of key proteins that regulate apoptosis fit into this category, most notably Mcl-1, with a half-life of 30 minutes.

Mcl-1 is a protein with homology to Bcl-2 that can inhibit apoptosis by blocking the activity of pro-apoptotic members of the Bcl-2 family such as Bax. Cell survival is regulated by this delicate balance between pro- and anti-apoptotic factors. Mcl-1 has been shown to play a critical role in the survival of a number of cancer cell types including multiple myeloma and B cell chronic lymphocytic leukaemia (Bannerman et al., 2001, J Biol Chem. 276 (18): 14924-32; Derenne et al., 2002, Blood. 100(l):194-9; Liu et al., 2002, J. Exp. Med. 194(2):113-26; Pederson et al., 2002, Blood. 100(5): 1795-801; Zhang et al., 2002, Blood. 99(6): 1885-93). Specific reduction in Mcl-

1 levels induce apoptosis in these cell types.

In vitro studies on a number of MCL cell lines have demonstrated that roscovitine exhibits a significant anti-proliferative effect. In particular, an antiprohferative effect was observed after 24h of exposure to roscovitine in Granta-519 and NCEB-1 cells and after 48h in REC and JeKo-1 cells. Flow-cytometry analysis showed a block in G2M after 24h of treatment in REC, Granta-519 and NCEB-1; the block persisted up to 72h of continuous drug exposure. JeKo-1 cells showed an induction of apoptosis after 48h of treatment. These results suggest that roscovitine induces apoptosis by down- regulating transcription of key genes required for survival of malignant B-cells.

In one preferred embodiment of the invention, the roscovitine is administered in an amount sufficient to inhibit at least one CDK enzyme.

Preferably, the CDK enzyme is selected from CDK1, CDK2, CDK4, CDK7 and CDK9.

In one particularly preferred embodiment, the CDK enzyme is CDK2.

In another particularly preferred embodiment, the CDK enzyme is selected from CDK7 and CDK9.

In another preferred embodiment, the roscovitine down regulates expression of an anti- apoptotic gene. Preferably, the anti-apoptotic gene is Mcl-1.

Thus, in another preferred embodiment, the roscovitine is administered in an amount sufficient to cause a down-regulation of Mcl-1.

In another preferred embodiment, the roscovitine is administered in an amount sufficient to cause a down-regulation of cyclin Dl protein expression. In another preferred embodiment, the roscovitine is administered in an amount sufficient to cause a down-regulation of cyclin H protein expression.

In another preferred embodiment, the roscovitine is administered in an amount sufficient to induce apoptosis.

In one particularly preferred embodiment, the roscovitine or pharmaceutically acceptable salt thereof is administered in the absence of another active agent, i.e. in a monotherapeutic regimen. For this embodiment, the roscovitine or pharmaceutically acceptable salt thereof is administered optionally in combination with one or more pharmaceutically acceptable diluents, excipients and/or carriers, but in the absence of one or more other active agents.

Another embodiment of the invention relates to a method of treating a patient suffering from mantle cell lymphoma, wherein said method consists essentially of administering a therapeutically effective amount of roscovitine or a pharmaceutically effective salt thereof.

Yet another embodiment of the invention relates to a method of treating a patient suffering from mantle cell lymphoma, wherein said method consists of administering a therapeutically effective amount of roscovitine or a pharmaceutically effective salt thereof.

A further aspect of the invention relates to a method of down regulating expression of an anti-apoptotic gene in mantle cell lymphoma cells, the method consisting essentially of contacting the cells with roscovitine, or a pharmaceutically acceptable salt thereof.

A further aspect of the invention relates to a method of down regulating expression of an anti-apoptotic gene in mantle cell lymphoma cells, the method consisting of contacting the cells with roscovitine, or a pharmaceutically acceptable salt thereof. A further aspect of the invention relates to a method of treating mantle cell lymphoma in a subject, the method consisting essentially of administering roscovitine, or a pharmaceutically acceptable salt thereof, to the subject in an amount sufficient to down regulate the expression of an anti-apoptotic gene in the subject. A further aspect of the invention relates to a method of treating mantle cell lymphoma in a subject, the method consisting of administering roscovitine, or a pharmaceutically acceptable salt thereof, to the subject in an amount sufficient to down regulate the expression of an anti-apoptotic gene in the subject.

Yet another aspect of the invention relates to a method of down-regulating Mcl-1 expression in mantle cell lymphoma cells, said method consisting essentially of contacting said cells with roscovitine, or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to a method of down-regulating Mcl-1 expression in mantle cell lymphoma cells, said method consisting of contacting said cells with roscovitine, or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to a method of treating mantle cell lymphoma in a subject, said method consisting essentially of administering roscovitine, or a pharmaceutically acceptable salt thereof, to the subject in an amount sufficient to down-regulate the expression of Mcl-1 in said subject.

Another aspect of the invention relates to a method of treating mantle cell lymphoma in a subject, said method consisting of administering roscovitine, or a pharmaceutically acceptable salt thereof, to the subject in an amount sufficient to down-regulate the expression of Mcl-1 in said subject.

Another aspect of the invention relates to the use of roscovitine, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating mantle cell lymphoma, wherein the roscovitine is administered in a monotherapeutic regimen. A further aspect of the invention relates to the use of roscovitine alone, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating mantle cell lymphoma.

Another aspect of the invention relates to the use of roscovitine, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating mantle cell lymphoma, wherein the roscovitine or a pharmaceutically acceptable salt thereof, is in an amount sufficient to down-regulate the expression of Mcl-1, and wherein the roscovitine is administered in a monotherapeutic regimen.

PHARMACEUTICAL COMPOSITIONS

Although roscovitine, (or a pharmaceutically acceptable salt, ester or pharmaceutically acceptable solvate thereof) can be administered alone, for human therapy it will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent.

Thus, one aspect of the invention relates to a pharmaceutical composition comprising (i) roscovitine, or a pharmaceutically acceptable salt thereof; and optionally (ii) a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment of mantle cell lymphoma.

Another aspect relates to a pharmaceutical composition consisting essentially of (i) roscovitine, or a pharmaceutically acceptable salt thereof; and optionally (ii) a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment of mantle cell lymphoma.

Yet another aspect relates to a pharmaceutical composition consisting of (i) roscovitine, or a pharmaceutically acceptable salt thereof; and optionally (ii) a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment of mantle cell lymphoma. One preferred embodiment of the invention relates to the administration of roscovitine in combination with a pharmaceutically acceptable excipient, diluent or carrier.

Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients, 2^nd Edition, (1994), Edited by A Wade and PJ Weller.

Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.

The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.

Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. SALTS/ESTERS

The active agent of the present invention can be present in the form of a salt or an ester, in particular a pharmaceutically acceptable salt or ester.

Pharmaceutically acceptable salts of the active agent of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. sulphuric acid, phosphoric acid or hydrohalic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Ci-G -alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.

Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Cι-C )-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen). ENANTIOMERS/TAUTOMERS

The invention also includes where appropriate all enantiomers and tautomers of the active agent. The man skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolatedprepared by methods known in the art.

STEREO AND GEOMETRIC ISOMERS

The active agent of the invention may exist in the form of different stereoisomers and/or geometric isomers, e.g. it may possess one or more asymmetric and or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of the agent, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).

The present invention also includes all suitable isotopic variations of the active agent or pharmaceutically acceptable salts thereof. An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷0, ¹⁸0, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶C1, respectively. Certain isotopic variations of the agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as ³H or ¹⁴C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferced in some circumstances. Isotopic variations of the agents of the present invention and pharmaceutically acceptable salts thereof can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

SOLVATES

The present invention also includes solvate forms of the active agent of the present invention. The terms used in the claims encompass these forms.

POLYMORPHS The invention furthermore relates to various crystalline forms, polymorphic forms and (an)hydrous forms of the active agent. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.

PRODRUGS

The invention further includes the active agent of the present invention in prodrug form. Such prodrugs are generally compounds wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include esters (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.

ADMINISTRATION

The pharmaceutical compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration. For oral administration, particular use is made . of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 2000 mg and more preferably from 50-1000 mg, of active ingredient per dose.

Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredients can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredients can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.

Injectable forms may contain between 10 - 1000 mg, preferably between 10 - 500 mg, of active ingredient per dose.

Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.

In a particularly preferred embodiment, the combination or pharmaceutical composition of the invention is administered intravenously.

DOSAGE

A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation.

Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the active agent, the metabolic stability and length of action of the agent, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. Dosages and frequency of application are typically adapted to the general medical condition of the patient and to the severity of the adverse effects caused, in particular to those caused to the hematopoietic, hepatic and to the renal system. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Depending upon the need, the agent may be administered at a dose of from 0.1 to 30 mg/kg body weight, or from 2 to 20 mg/kg body weight. More preferably the agent may be administered at a dose of from 0.1 to 1 mg/kg body weight.

As described above, roscovitine is preferably administered in a therapeutically effective amount, preferably in the form of a pharmaceutically acceptable amount. This amount will be familiar to those skilled in the art. By way of guidance, roscovitine is typically administered orally or intravenously at a dosage of from about 0.05 to about 5g/day, preferably from about 0.5 to about 5 g/day or 1 to about 5g/day, and even more preferably from about 1 to about 3 g/day. Roscovitine is preferably administered orally in tablets or capsules. The total daily dose of roscovitine can be administered as a single dose or divided into separate dosages administered two, three or four times a day.

COMBINATIONS

In one preferred embodiment of the invention, roscovitine is administered in combination with one or more other antiproliferative agents. In such cases, the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other antiproliferative agents.

It is known in the art that many drugs are more effective when used in combination. In particular, combination therapy is desirable in order to avoid an overlap of major toxicities, mechanism of action and resistance mechanism(s). Furthermore, it is also desirable to administer most drugs at their maximum tolerated doses with minimum time intervals between such doses. The major advantages of combining drugs are that it may promote additive or possible synergistic effects through biochemical interactions and also may decrease the emergence of drug resistance which would have been otherwise responsive to initial treatment with a single agent.

Beneficial combinations may be suggested by studying the activity of the test compounds with agents known or suspected of being valuable in the treatment of a particular disorder. This procedure can also be used to determine the order of administration of the agents, i.e. before, simultaneously, or after delivery. The present invention is further illustrated by way of example, and with reference to the following figures. Further details of the figures may be found in the Examples section.

Figure 1 shows an evaluation of the cytotoxic effect of CYC202 after 72 hours of exposure (IC50 dose evaluation) as measured by an MTT assay.

Figure 2 shows the growth curve of Granta-519, NCEB-1, REC and Jeko-1 mantle lymphoma cells continuously exposed to the conesponding IC₃₀, IC50 and IC₇₀ doses of CYC202.

Figure 3 shows (A) representative fluorescence-activated cell sorter analysis of DNA content (histogram of PI fluorescence); cell cycle analysed after 24 and 48 hours of treatment with the IC50 dose of CYC202; and (B) contour plot of DNA content in Jeko- 1 cells. RI represents the sub Gl peak (percentage of apoptotic cells).

Figure 4 shows TUNEL analysis of apoptosis in mantle lymphoma cells carried out by flow cytometry, after 72 hours of exposure to the ICso dose of the compound. Filled lines represent the negative control incubated in the absence of terminal transferase; Empty lines represent the samples incubated with TUNEL reaction mixture. The percentage of TUNEL-positive cells in each sample is reported. Figure 5 shows a representative Western blotting experiment illustrating the expression of the different cell cycle related proteins in mantle lymphoma cells, after 48 hours of exposure to the IC₅₀ dose of CYC202. Tubulin was used as a control for loading.

Figure 6 shows representative Western blotting experiments illustrating the expression of the different apoptosis related proteins in mantle lymphoma cells, after 48 hours of exposure to the IC₅₀ dose of CYC202. Tubulin was used as a control for loading.

EXAMPLES Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods. See, generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.; as well as Guthrie et al., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif), McPherson et al, PCR Volume 1, Oxford University Press, (1991), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, NJ.). These documents are incorporated herein by reference.

Preparation of Roscovitine

Roscovitine was prepared in accordance with the method disclosed in EP0874847B (CNRS). As used herein, the term "CYC202" refers to a single enantiomer of roscovitine, namely, 2-(l -R-hydroxymethylpropylamino)-6-benzylamino-9-isopurine. In vitro activity of CYC202 against mantle cell lymphoma cells

Studies were carried out to investigate the effect of CYC202 on 4 human MCL cell lines: REC, Granta-519, JeKo-1 and NCEB-1. Granta-519 were obtained from the

Deutsche Sammlung von Mikroorganismen und Zellkulturen DSMZ. JeKo cells were obtained from Dr T Akagi, Dept of Pathology, Chosun University Medical School,

Kwangju, Korea (Jeon et al, British Journal of Haematology, 1998, 102, 1323-6).

NCEB-1 were obtained from Dr C M Steel, MRC Clinical and Population Cyto genetics

Unit, Western General Hospital, Edinburgh, UK (Saltman et al, Blood 1988, 72, 2026-

30).

REC, Granta-519, JeKo-1 and NCEB-1 cells were exposed to CYC202 doses ranging from 2.5 to lOOμM to assess, by MTT assay, the IC50 at 72h. REC, Granta-519 and Jeko-1 presented an IC50 dose of 25μM, whilst NCEB-1 of 50μM. Growth inhibition was assessed after treatment with roscovitine at the IC50 doses specific of each cell line.

MTT Assay

The MTT assay was used to evaluate the effects of roscovitine on the various cell lines.

Standard 72-h MTT (thiazolyl blue; 3-[4,5-dimethylthiazol-2-yl]-2,5-diρhenyl tetrazolium bromide) assays were performed (Haselsberger, K.; Peterson, D. C;

Thomas, D. G.; Darling, J. L. Anti Cancer Drugs 1996, 7, 331-8; Loveland, B. E.;

Johns, T. G.; Mackay, I. R.; Vaillant, F.; Wang, Z. X.; Hertzog, P. J. Biochemistry

International 1992, 27, 501-10). In short: cells were seeded into 96-well plates according to doubling time and incubated overnight at 37 °C. Test compound (CYC202, Cyclacel Ltd, UK) was made up in DMSO and a 1/3 dilution series prepared in 100 μL cell media, added to cells (in triplicates) and incubated for 72 hours at 37 °C.

MTT (Sigma) was made up as a stock of 5 mg/mL in cell media and filter-sterilised.

Media was removed from cells followed by a wash with 200 μL PBS. MTT solution was then added at 20 μL per well and incubated in the dark at 37 °C for 4 h. MTT solution was removed and cells again washed with 200 μL PBS. MTT dye was solubilised with 200 μL per well of DMSO with agitation. Absorbance was read at 540 nm and data analysed using curve-fitting software (GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego California USA) to determine IC₅0 values

(concentration of test compound which inhibits cell growth by 50%).

The results of the MTT assay evaluation of the cytotoxic activity of CYC202 (IC₅₀ dose evaluation) after 72 hours of exposure are shown in Figure 1.

Cell Growth inhibition

For cell growth, cells were individually seeded in the 24-well culture plates at the density of 3xl0⁵ cells per well. After 24hr, CYC202 was added at the concentration corresponding to IC30, IC50 and IC70. Cell number and cell viability were determined daily using a Coulter Counter (Beckman Coulter-Z2).

Table 1 below shows the percentage of cell growth inhibition after treatment with CYC202.

Table 1:

The growth curves of Granta-519, NCEB-1, REC and Jeko-1 mantle lymphoma cells continuously exposed to the conesponding IC₃₀, IC₅₀ and IC₇₀ doses of CYC202 are shown in Figure 2.

Flow Cytometry Analysis (Cell Cycle)

Cells were seeded onto 90 mm diameter plates. After 24 h, cells were treated with CYC202 or the equivalent amount of vehicle (DMSO). Cells were harvested at various time points after addition of the drug. After washing once in PBS, cells were fixed in ice-cold 70% v/v ethanol and stored for up to two weeks at -20 °C. Cells were washed twice in PBS + 1% w/v BSA to remove fixative and resuspended in PBS containing 50 μg/ml propidium iodide (PI, Sigma) and 50 μg/ml RnaseA (Sigma). After incubation at room temperature for 20 minutes, cells were analysed using flow cytometry. The analysis of cell cycle and apoptosis was performed using a FACScan flow cytometer

(Becton Dickinson, USA) and the Cell Quest software package (Becton Dickinson).

A Becton Dickinson LSR flow cytometer was used for these studies, in accordance with the manufacturers recommendations. The argon ion laser set at 488nm was used as an excitation source. Red fluorescence (575±26nm) was acquired on a linear scale and pulse width analysis was used to exclude cell doublets and aggregates from the analysis. Cells with a DNA content of between 2n and 4n were designated as being in Gl, S or G2/M phases of the cell cycle, as defined by the level of red fluorescence. Cells showing less than 2n DNA content were designated as sub-Gl cells. The number of cells in each cell cycle compartment was expressed as a percentage of the total number of cells present.

Table 2 below shows the percentage of G2/M phase of untreated and CYC202 treated cells (IC₅₀). The data indicate that CYC202 causes perturbation of the cell cycle resulting in G2/M phase accumulation.

Table 2:

REC NCEB-1 Granta-519

Figure 3 shows (A) representative fluorescence-activated cell sorter analysis of DNA content (histogram of PI fluorescence); cell cycle analysed after 24 and 48 hours of treatment with the IC₅₀ dose of CYC202; and (B) contour plot of DNA content in Jeko- 1 cells. Ri represents the sub Gl peak (percentage of apoptotic cells). Tunel assay

Cells were harvested and fixed in 4% paraformaldeyde for 45 minutes at room temperature, after 72hr of exposure to the CYC202 (IC50 dose),. After rinsing with

PBS, the cells were permeabilized in a solution of 0.1% Triton X-100 in sodium 0.1% citrate for 2 minutes on ice. Samples, washed with PBS IX, were incubated in -the

TUNEL reaction mixture (Boehringer Mannheim-Roche) for lhr at 37°C in the dark, and after rinsing with PBS IX and analysed by FACScan cytofluorimeter (Becton

Dickinson, USA). The results were expressed as the percentage of TUNEL-positive cells in the overall cell population.

The results of the TUNEL analysis of apoptosis in mantle lymphoma cells carried out by flow cytometry, after 72 hours of exposure to the IC5₀ dose of the compound are shown in Figure 4. Filled lines represent the negative control incubated in the absence of terminal transferase; empty lines represent the samples incubated with TUNΕL reaction mixture. The percentage of TUNEL-positive cells in each sample is reported.

Western blot analysis

Human MCL cells were solubilised in lysis buffer (0.01 M Tris-HCl [pH 7.5], 0.144- M

NaCl, 0.5% Nonidet P-40, 0.5% sodium dodecyl sulfate [SDS], 0.1% aprotinin, 10 mg/ml leupeptin, and 2 mM phenyhnethylsulfonyl fluoride).The protein content in the different samples was quantified using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL, USA). The proteins resolved on 10% or 12% SDS-polyacrylamide gel electrophoresis, were blotted to a nitrocellulose membrane, blocked in TBS buffer containing 5% blotting-grade blocker non fat milk for lhr. Blots were incubated with primary antibodies: anti cyclin Dl(cloneG124-326, PharMingen, San Diego, CA, USA), anti cdk4 (clone H-22, Santa Cruz Biotechnology, CA, USA), anti cyclin H (clone G301-1, PharMingen, San Diego,CA), anti cdk7 (clone 17, PharMingen, San Diego, CA), anti cyclin Bl (SC-245, Santa Cruz Biotechnology, CA, USA), anti Bcl-2 (clone N-19, Santa Cruz Biotechnology,CA, USA) anti Bax (Cell Signaling Technology), anti MCL-1 (clone-22, PharMingen, San Diego,CA) and anti Tubulin (Ab-1 Oncogene). Peroxidase labeled anti-mouse and anti-rabbit antibodies (Amerstαam Life Science, Arlington Heights, IL, USA) were used as secondary antibodies. The immunoblots were processed for enhanced chemiluminescence detection (Amersham

Life Science). The relative amount of transfened proteins in a given sample was quantified and estimating the relative arbitrary density units, normalized to the correspective Tubulin content in each sample.

The results of the above Western blotting experiments are shown in Figures 5 and 6.

Figure 5 illustrates the expression of the different cell cycle related proteins in mantle lymphoma cells, after 48 hours of exposure to the IC50 dose of CYC202. Tubulin was used as a control for loading. Figure 6 shows representative Western blotting experiments illustrating the expression of the different apoptosis related proteins in mantle lymphoma cells, after 48 hours of exposure to the IC₅₀ dose of CYC202. Again,

Tubulin was used as a control for loading.

By way of summary, an anti-proliferative effect was already observed after 24h of exposure to CYC202 in Granta-519 and NCEB-1 cells and after 48h in REC and JeKo- 1 cells. Flow-cytometry analysis showed a block in G2M after 24h of treatment in REC, Granta-519 and NCEB-1; the block persisted up to 72h of continuous drug exposure. JeKo-1 cells showed an induction of apoptosis after 48h of treatment.

The results suggest that CYC202 induces apoptosis by down-regulating transcription of key genes required for survival of malignant B-cells. More specifically, the results suggest that the mechanism of action of CYC202 involves the down regulation of cyclin Dl and cyclin H protein expression, and the down regulation of Mcl-1 protein expression.

Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

Claims

1. Use of roscovitine, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating mantle cell lymphoma.

2. Use according to claim 1 wherein the roscovitine is administered in combination with a pharmaceutically acceptable carrier, diluent or excipient.

3. Use according to claim 1 or claim 2 wherein the roscovitine is administered in an amount sufficient to inhibit at least one CDK enzyme.

4. Use according to claim 3 wherein the CDK enzyme is selected from CDK1, CDK2, CDK4, CDK7 and CDK9.

5. Use according to claim 3 wherein the CDK enzyme is selected from CDK1 and CDK2.

6. Use according to claim 3 wherein the CDK enzyme is selected from CDK7 and CDK9.

7. Use according to any preceding claim wherein the roscovitine is administered in combination with one or more other antiproliferative agents.

8. Use according to any preceding claim wherein the roscovitine down regulates expression of an anti-apoptotic gene.

9. Use according to any preceding claim wherein the roscovitine is administered in an amount sufficient to cause a down-regulation of Mcl-1.

10. Use according to any preceding claim wherein the roscovitine is administered in an amount sufficient to cause a down-regulation of cyclin D.

11. A method of treating a patient suffering from mantle cell lymphoma comprising administering a therapeutically effective amount of roscovitine or a pharmaceutically effective salt thereof.

12. A method according to claim 11 wherein the roscovitine down regulates expression of an anti-apoptotic gene.

13. A method according to claim 11 wherein the roscovitine is administered in an amount sufficient to cause a down-regulation of Mcl-1.

14. A method according to claim 11 wherein the roscovitine is administered in an amount sufficient to cause a down-regulation of cyclin D.

15. A method according to claim 11 wherein the roscovitine is administered in an amount sufficient to inhibit at least one CDK enzyme.

16. A method according to claim 15 wherein the CDK enzyme is selected from CDK1, CDK2, CDK4, CDK7 and CDK9.

17. A method according to claim 15 wherein the CDK enzyme is selected from CDK1 and CDK2.

18. A method according to claim 11 wherein the CDK enzyme is selected from CDK7 and CDK9.

19. A method according to any one of claims 11 to 18 wherein the roscovitine is administered in combination with a pharmaceutically acceptable carrier, diluent or excipient.

20. A method according to any one of claims 11 to 19 wherein the roscovitine is administered in combination with one or more other antiproliferative agents.

21. A pharmaceutical composition comprising (i) roscovitine, or a pharmaceutically acceptable salt thereof; and optionally (ii) a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment of mantle cell lymphoma.

22. A method of down regulating expression of an anti-apoptotic gene in mantle cell lymphoma cells, the method comprising contacting the cells with roscovitine, or a pharmaceutically acceptable salt thereof.

23. A method of treating mantle cell lymphoma in a subject, the method comprising administering roscovitine, or a pharmaceutically acceptable salt thereof, to the subject in an amount sufficient to down regulate the expression of an anti-apoptotic gene in the subject.

24. The method of claim 22 or 23 wherein the anti-apoptotic gene is Mcl-1.

25. A method of down-regulating Mcl-1 and/or cyclin D expression in mantle cell lymphoma cells, said method comprising contacting said cells with roscovitine, or a pharmaceutically acceptable salt thereof.

26. A method of treating mantle cell lymphoma in a subject, said method comprising administering roscovitine, or a pharmaceutically acceptable salt thereof, to the subject in an amount sufficient to down-regulate the expression of Mcl-1 and/or cyclin D in said subject.

27. Use of roscovitine, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating mantle cell lymphoma, wherein the roscovitine or a pharmaceutically acceptable salt thereof, is in an amount sufficient to down-regulate the expression of Mcl-1 and/or cyclin D.

28. Use or a method substantially as described herein, with reference to the accompanying figures.