WO2006082448A1

WO2006082448A1 - Assays for agents with selective cytotoxicty to hdac resistant cell lines

Info

Publication number: WO2006082448A1
Application number: PCT/GB2006/000425
Authority: WO
Inventors: Torsten Nielsen; Nicholas Edwards; Maxwell Sehested
Original assignee: Topotarget Uk Limited
Priority date: 2005-02-07
Filing date: 2006-02-06
Publication date: 2006-08-10
Also published as: CA2596405A1; AU2006210994A1; EP1856522A1; JP2008529487A

Abstract

This invention relates to a method of identifying and/or obtaining an agent which is selectively cytotoxic to histone deacetylase inhibitor (HDACi) resistant cells, said method comprising the steps of: a) incubating HDACi resistant cells and HDACi sensitive cells with a test agent, b) determining the cytotoxicity of the test agent for said HDACi resistant cells and the cytotoxicity of the test agent for said HDACi sensitive cells, wherein an increase in cytoxicity of the test agent for said HDACi resistant cells relative to said HDACi sensitive cells is indicative that the test agent is an agent which is selectively cytotoxic to HDACi resistant cells. The invention is based on the recognition that cells which are resistant to inhibitors of histone deacetylases (HDACis) such as PXD101 are hypersensitized to certain anti-cancer agents.

Description

ASSAYS FOR AGENTS WITH SELECTIVE CYTOTOXICTY TO HDAC RESISTANT CELL

LINES

TECHNICAL FIELD This invention pertains generally to the field of drug resistance, more particularly to cells resistant to inhibitors of histone deacetylases (HDACi), and specifically to cell lines resistant to the histone deacetylase inhibitor PXD101.

BACKGROUND DNA in eukaryotic cells is tightly complexed with proteins (histones) to form chromatin.

Histones are small, positively charged proteins which are rich in basic amino acids (positively charged at physiological pH), which contact the phosphate groups (negatively charged at physiological pH) of DNA. There are five main classes of histones, H1 , H2A, H2B, H3, and H4. The amino acid sequences of histones H2A, H2B, H3, and H4 show remarkable conservation between species, whereas H1 varies somewhat, and in some cases is replaced by another histone, e.g., H5. Four pairs of each of H2A, H2B, H3, and H4 together form a disk-shaped octomeric protein core, around which DNA (about 140 base pairs) is wound to form a nucleosome. Individual nucleosomes are connected by short stretches of linker DNA associated with another histone molecule (e.g., H1 , or in certain cases, H5) to form a structure resembling a beaded string, which is itself arranged in a helical stack, known as a solenoid.

The majority of histones are synthesised during the S phase of the cell cycle, and newly synthesised histones quickly enter the nucleus to become associated with DNA. Within minutes of its synthesis, new DNA becomes associated with histones in nucleosomal structures.

A small fraction of histones, more specifically, the amino side chains thereof, are enzymatically modified by post-translational addition of methyl, acetyl, or phosphate groups, neutralising the positive charge of the side chain, or converting it to a negative charge. For example, lysine and arginine groups may be methylated, lysine groups may be acetylated, and serine groups may be phosphorylated. For lysine, the -(CH₂)₄-NH₂ sidechain may be acetylated, for example by an acetyltransferase enzyme, to give the amide -(CH₂)₄-NHC(=O)CH₃. Methylation, acetylation, and phosphorylation of amino termini of histones which extend from the nucleosomal core affects chromatin structure and gene expression. (See, for example, Spencer and Davie, 1999). Acetylation and deacetylation of histones is associated with transcriptional events leading to cell proliferation and/or differentiation. Regulation of the function of transcription factors is also mediated through acetylation. Recent reviews of histone deacetylation include Kouzarides, 1999 and Pazin et al., 1997.

The correlation between the acetylation status of histones and the transcription of genes has been known for over 30 years (see, for example, Howe et al., 1999). Certain enzymes, specifically acetylases (e.g., histone acetyltransferase, HAT) and deacetylases (e.g., histone deacetylase, HDAC), which regulate the acetylation state of histones have been identified in many organisms and have been implicated in the regulation of numerous genes, confirming the link between acetylation and transcription. See, for example, Davie, 1998. In general, histone acetylation correlates with transcriptional activation, whereas histone deacetylation is associated with gene repression.

A growing number of histone deacetylases (HDACs) have been identified (see, for example, Ng and Bird, 2000). The first deacetylase, HDAC1 , was identified in 1996 (see, for example, Tauton et al., 1996). Subsequently, two other nuclear mammalian deacetylases has been found, HDAC2 and HDAC3 (see, for example, Yang et al., 1996, 1997, and Emiliani et al., 1998). See also, Grozinger et al., 1999; Kao et al., 2000; and Van den Wyngaert et al., 2000.

Eight human HDACs have been cloned so far:

HDAC1 (Genbank Accession No. NP_004955)

HDAC2 (Genbank Accession No. NP_001518) HDAC3 (Genbank Accession No. 015739)

HDAC4 (Genbank Accession No. AAD29046)

HDAC5 (Genbank Accession No. NP_005465)

HDAC6 (Genbank Accession No. NP_006035)

HDAC7 (Genbank Accession No. AAF63491) HDAC8 (Genbank Accession No. AAF73428)

These eight human HDACs fall in two distinct classes: HDACs 1 ,2,3 and 8 are in class I, and HDACs 4,5,6 and 7 are in class II.

There are a number of histone deacetylases in yeast, including the following: RPD3 (Genbank Accession No. NP_014069) HDA1 (Genbank Accession No. P53973) H0S1 (Genbank Accession No. Q12214) HOS2 (Genbank Accession No. P53096) HOS3 (Genbank Accession No. Q02959)

There are also numerous plant deacetylases, for example, HD2, in Zea mays (Genbank Accession No. AF254073_1).

HDACs function as part of large multiprotein complexes, which are tethered to the promoter and repress transcription. Well characterised transcriptional repressors such as Mad (Laherty et al., 1997), pRb (Brehm et al., 1998), nuclear receptors (Wong et al., 1998) and YY1 (Yang et al., 1997) associate with HDAC complexes to exert their repressor function.

The study of inhibitors of histone deacetylases indicates that these enzymes play an important role in cell proliferation and differentiation. The inhibitor Trichostatin A (TSA) (Yoshida et al., 1990a) causes cell cycle arrest at both G1 and G2 phases (Yoshida and Beppu, 1988), reverts the transformed phenotype of different cell lines, and induces differentiation of Friend leukaemia cells and others (Yoshida et al., 1990b). TSA (and SAHA) have been reported to inhibit cell growth, induce terminal differentiation, and prevent the formation of tumours in mice (Finnin et al., 1999).

Trichostatin A (TSA)

Suberoylanilide Hydroxamic Acid (SAHA)

Cell cycle arrest by TSA correlates with an increased expression of gelsolin (Hoshikawa et al., 1994), an actin regulatory protein that is down regulated in malignant breast cancer (Mielnicki et al., 1999). Similar effects on cell cycle and differentiation have been observed with a number of deacetylase inhibitors (Kim et al., 1999). Trichostatin A has also been reported to be useful in the treatment of fibrosis, e.g., liver fibrosis and liver cirrhosis. See, e.g., Geerts et al., 1998.

Recently, certain compounds that induce differentiation have been reported to inhibit histone deacetylases. Several experimental anti-tumour compounds, such as trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid (SAHA), and phenylbutyrate have been reported to act, at least in part, by inhibiting histone deacetylase (see, e.g., Yoshida et al., 1990; Richon et al., 1998; Kijima et al., 1993). Additionally, diallyl sulfide and related molecules (see, e.g., Lea et al., 1999), oxamflatin (see, e.g., Kim et al., 1999), MS-27-275, a synthetic benzamide derivative (see, e.g., Saito et al., 1999; Suzuki et al., 1999; note that MS-27-275 was later renamed as MS-275), butyrate derivatives (see, e.g., Lea and Tulsyan, 1995), FR901228 (see, e.g., Nokajima et al., 1998), depudecin (see, e.g., Kwon et al., 1998), and m-carboxycinnamic acid bishydroxamide (see, e.g., Richon et al., 1998) have been reported to inhibit histone deacetylases. In vitro, some of these compounds are reported to inhibit the growth of fibroblast cells by causing cell cycle arrest in the G1 and G2 phases, and can lead to the terminal differentiation and loss of transforming potential of a variety of transformed cell lines (see, e.g., Richon et al, 1996; Kim et al., 1999; Yoshida et al., 1995; Yoshida & Beppu, 1988). In vivo, phenybutyrate is reported to be effective in the treatment of acute promyelocytic leukemia in conjunction with retinoic acid (see, e.g., Warrell et al., 1998). SAHA is reported to be effective in preventing the formation of mammary tumours in rats, and lung tumours in mice (see, e.g., Desai et al., 1999).

The clear involvement of HDACs in the control of cell proliferation and differentiation suggest that aberrant HDAC activity may play a role in cancer. The most direct demonstration that deacetylases contribute to cancer development comes from the analysis of different acute promyelocytic leukaemias (APL). In most APL patients, a translocation of chromosomes 15 and 17 (t(15;17)) results in the expression of a fusion protein containing the N-terminal portion of PML gene product linked to most of RARα (retinoic acid receptor). In some cases, a different translocation (t(11 ; 17)) causes the fusion between the zinc finger protein PLZF and RARα. In the absence of ligand, the wild type RARα represses target genes by tethering HDAC repressor complexes to the promoter DNA. During normal hematopoiesis, retinoic acid (RA) binds RARα and displaces the repressor complex, allowing expression of genes implicated in myeloid differentiation. The RARα fusion proteins occurring in APL patients are no longer responsive to physiological levels of RA and they interfere with the expression of the RA-inducible genes that promote myeloid differentiation. This results in a clonal expansion of promyelocytic cells and development of leukaemia. In vitro experiments have shown that TSA is capable of restoring RA-responsiveness to the fusion RARα proteins and of allowing myeloid differentiation. These results establish a link between HDACs and oncogenesis and suggest that HDACs are potential targets for pharmaceutical intervention in APL patients. (See, for example, Kitamura et al., 2000; David et al., 1998; Lin et al., 1998).

Furthermore, different lines of evidence suggest that HDACs may be important therapeutic targets in other types of cancer. Cell lines derived from many different cancers (prostate, colorectal, breast, neuronal, hepatic) are induced to differentiate by HDAC inhibitors (Yoshida and Horinouchi, 1999). A number of HDAC inhibitors have been studied in animal models of cancer. They reduce tumour growth and prolong the lifespan of mice bearing different types of transplanted tumours, including melanoma, leukaemia, colon, lung and gastric carcinomas, etc. (Ueda et al., 1994; Kim et al., 1999).

SUMMARY OF THE INVENTION

One aspect of the invention provides a method to identify and/or obtain an agent which displays increased cytotoxicity to HDACi-resistant cells in comparison to the HDACi, said method comprising the steps of: incubating said HDACi resistant cells and sensitive cells with an agent to be tested; determining the cytotoxicity of said agent for HDACi resistant cells and sensitive cells; and, identifying agents which display a reduced resistance factor (IC50 value for resistant cells divided by IC50 value for parental cells) compared to the HDACi.

A further aspect of the invention provides a method to identify agents which display selective cytotoxicity to HDACi resistant cells in comparison to the HDACi, said method comprising the steps of: incubating said HDACi resistant cells and sensitive cells with an agent to be tested; determining the cytotoxicity of said agent for HDACi resistant cells and sensitive cells; and, identifying agents which display a resistance factor (IC50 value for resistant cells divided by IC50 value for parental cells) < 1.

In yet another aspect, the invention provides the agents identified according to the methods disclosed herein which are selectively cytotoxic to HDACi resistant cells.

Another aspect of the invention is a method to selectively inhibit the growth of HDACi resistant cells in vitro or in vivo, said method comprising contacting said resistant cells with one or more of said cytotoxic agents identified according to the methods disclosed herein. Another aspect of the invention is a method to identify agents which are chemosensitizers of HDACi, said method comprising the steps of incubating HDACi resistant cells with the HDACi to which the cells are resistant, in the presence and absence of an agent to be tested and determining the cytotoxicity of the HDACi for the cells, wherein increased cytotoxicity in cultures incubated in the presence of the agent compared to cultures incubated in the absence of the agent indicates that the agent is a chemosensitizer.

In yet another aspect, the invention provides the chemosensitizers of HDACi's identified according to the methods disclosed herein.

In a still further aspect, the invention provides a method to inhibit the growth of HDACi resistant cells in vitro or in vivo, said method comprising contacting said HDACi resistant cells with one or more HDACi's and one or more of the chemosensitizers identified according to the method disclosed herein.

In another aspect, the invention provides a method to identify HDACi analogs, for example compounds with a mechanism of action similar to that of HDACi's, said method comprising the steps of contacting an HDACi resistant cell line and the corresponding HDACi sensitive parental cell line with increasing concentrations of a test substance; and, determining resistance of the HDACi resistant cell line to the test substance compared to resistance of the parental cell line to the test substance wherein greater resistance to the test substance of the HDACi resistant cell line compared to the parental cell line suggests that the test substance may act on the same signalling pathway as the HDACi. The thus identified agents could include, but do not have to be restricted to, HDACi and their analogs.

In still another aspect, the invention provides agents, including HDACi analogs, identified according to the method disclosed above herein are also herein included as an aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention generally relates to HDACi resistant cell lines, such as HDACi resistant sublines of the P388 mouse leukemia cell line.

An HDACi resistant cell may be resistant to one or more HDACi, including, for example, PXD101 , trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid (SAHA), phenylbutyrate, diallyl sulfide, oxamflatin, MS-27-275, butyrate, FR901228, depudecin, and m-carboxycinnamic acid bishydroxamide. In some embodiments, an HDACi resistant cell line may be resistant to PXD101 and may include the WT/PXD101-2C cell line described herein.

Methods for the selection of drug resistant cell lines are known in the art and generally involve culturing and subculturing cells in the presence of increasing concentrations of a drug. Surviving colonies of cells are further expanded in the presence of higher concentrations of drug which eventually results in individual resistant cell lines and sub-lines of cells (see, for example Akiyama et al., Somat. Cell. MoI. Genetics 11 :117-126 (1985)). As used herein "resistance" of a cell to an agent refers to the ability of the cell to tolerate higher concentrations of a drug than a sensitive cell. Thus, the resistance of a cell that has been continually exposed to the PXD101 can be determined relative to the parental sensitive cell from which the drug resistant cell was derived. Resistance of a cell to an agent (e.g. PXD101) is typically quantitated as the increase in IC50 (concentration of the agent needed to inhibit cell growth by 50%) relative to a control sensitive cell,

As used herein, "test cells" refers to cells that may or may not be PXD101 resistant. One HDACi resistant cell line disclosed herein was obtained from culturing and subculturing P388 cell lines in the presence of increasing concentrations of PXD101. The P388 cell line may be obtained from the ATCC (Manassas, VA, USA). This cell line may be grown in monolayer using RPMI supplemented with 10% fetal calf serum, 1 % L- glutamine, penicillin (50 Units/ml) and streptomycin (50 μg/ml) and maintained at 37°C in 5.0% CO2. Media and supplements are all available from Gibco Life Technologies, Rockville, MD.

The HDACi resistant P388/PXD101-C sub-line disclosed herein is derived from incubating the parental P388 breast adenocarcinoma cell line as above in PXD-101. As used herein, the term "subline" (or "subclone") refers to a cell line that is derived from a parental cell line by virtue of its resistance to one or more HDACis. It is intended that the invention encompasses other PXD101 resistant cell lines that may be selected by culturing cells of the P388 cell line in the presence of other concentrations of PXD101. The increase in fold resistance of the cell to PXD101 can be assessed relative to the parental cell line from which the resistant cell line was derived (e.g., IC50 of PXD101 for the resistant cell line versus IC50 of PXD101 for the parental cell line).

Some aspects of the invention provide methods for identifying a substance that is a chemosensitizer of HDACis. As used herein, the term "chemosensitizer of HDACis" refers to a substance that can increase the efficacy of a therapeutic agent against a resistant cell and/or decrease the resistance of a cell for a therapeutic agent. For example, verapamil is a chemosensitizer of P-gp-mediated multidrug resistance; in the presence of verapamil, a multidrug resistant cell is more susceptible to the cytotoxic effect of anthracyclines. The cell lines described herein are particularly useful in a method to identify substances that are chemosensitizers of HDACis. This could comprise the steps of incubating the HDACi resistant cells with an HDACi to which the cells are resistant in the presence and absence of an agent to be tested; and determining the cytotoxicity of the HDACi for the cells, wherein increased cytotoxicity in cultures incubated with the agent compared to cultures incubated in the absence of the agent indicates that the agent is a chemosensitizer. Cytotoxicity can be measured using methods known to one of skill in the art, for example, a colorimetric assay system such as CELL TITER 96@ AQUEOUS assay (PROMEGA, Madison, Wl).

Aspects of the invention also provide a method for identifying HDACi analogues. As used herein, the term "HDACi analog" refers to a synthetic or natural compound that inhibits histone deacetylase activity. As contemplated herein, the cell lines described herein can be used to identify HDACi analogues that may partially overcome the resistance of cell lines for HDACis. For example, one could incubate an HDACi resistant cell line and corresponding parental cell line with increasing concentrations of a test substance and then determine the relative resistance factor by dividing the IC50 determined for the resistant cell line by that determined for the parental cell line. A test compound with a decreased resistance factor (compared to the HDACi included in the same assay as a reference) would constitute a potential analogue of interest

Other aspects of the invention provide methods for identifying substances that are selectively cytotoxic to HDACi resistant cells. As used herein, the term "cytotoxic agent" refers to compounds, including anticancer drugs, that inhibit cell proliferation and induce cell death. The method could comprise incubating the resistant cell lines disclosed herein with the substance to be tested and, using standard methods, determining the cytotoxicity of the substance for the resistant cell line and comparing it to that of the parental cell line. Compounds that preferentially kill HDACi-resistant cell lines, i.e. compounds where the IC50 for growth inhibition is lower on the resistant cells than on the corresponding parental cells, would be a hit. Examples of selectively cytotoxic agents include protein kinase C inhibitors such as staurosporine and DNA methylation inhibitors such as 5-azacytidine.

Protein Kinase C (PKC) is composed of a family of phospholipid-dependent kinases which phosphorylate proteins at serine and threonine residues. Phorbol esters bind and activate PKC and PKC has been implicated in phorbol ester-mediated responses such as the induction of differentiation markers in primary keratinocytes. One of the most potent PKC inhibitors presently available is staurosporine (Tamaoki, T. et al, "Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase," Biochem. Biophys. Res. Commun., 135: 397- 402, 1986), which inhibits PKC at nanomolar doses in vitro by interacting with its catalytic domain (Nakadate, T. et al, "Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action," Biochem. Pharmacol., 37: 1541-1545, 1988; and Gross, J. L. et al, "Characterization of specific [3H]dimethylstaurosporine binding to protein kinase C," Biochem. Pharmacol., 40: 343-350, 1990). Other PKC inhibitors include bryostatin I , tamoxifen, safingol, UCN-01 , CGP-41251 , bisindolylmaleimides such as GF109203X, calphostins, and antisense to PKC, such as ISIS3521 (reviewed in Da Rocha AB et al, "Targetting Protein Kinase C: New Therapeutic Opportunities against Malignant Glioma." The Oncologist, 7(1): 17-33, 2002)

The DNA methylation inhibitors 5-azacytidine and decitabine are activated to a triphosphate and are degraded by cytidine deaminase. 5-Azacytidine incorporates into RNA and, to a lesser extent, DNA. Decitabine incorporates primarily into DNA. Incorporation into RNA produces disassembly of polyribosomes, defective methylation and acceptor function of transfer RNA, and marked inhibition of protein production. Insertion into DNA results in covalent linking with methyltransferase and blocking of DNA synthesis that ultimately results in direct cytotoxicity. 5-Azacytidine is highly cytotoxic to cells in S phase and exerts its action mainly on rapidly dividing cells. (Reviewed in Santini et al "Changes in DNA Methylation in Neoplasia: Pathophysiology and Therapeutic Implications" Ann. Int. Med 134:537-586, 2001)

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, cell culture, immunology and the like which are in the skill of one in the art. These techniques are fully disclosed in the current literature and reference is made specifically to Sambrook, Fritsch and Maniatis eds., "Molecular Cloning A Laboratory Manual, 2nd edition, (Cold Spring Harbor Laboratory Press, 1989; the series Methods of Enzymology (Academic Press, Inc); and Antibodies: A Laboratory Manual, Harlowet al., eds., Cold Spring Harbor Laboratory Press (1987).

The cytotoxic agents and chemosensitizers of HDACis identified according to the methods of the present invention are particularly useful in a method to inhibit the growth of HDACi resistant cell lines. A method to inhibit growth of HDACi resistant cell lines using cytotoxic agents and chemosensitizers of HDACis would comprise contacting said HDACi resistant cells with one or more HDACis and one or more of the chemosensitizers. A method to inhibit growth of HDACi resistant cell lines using cytotoxic agents identified according to the methods of the present invention would comprise contacting said resistant cells with one or more cytotoxic agents. The term "contacting" is intended to include in vitro, ex vivo or in vivo administration. The cytotoxic agents and chemosensitizers identified according to the methods of the present invention may also be of therapeutic use in subjects bearing an HDACi resistant tumor (i.e., in situations where HDACis are not or no longer effective therapeutic agents due to natural or acquired resistance). Suitable pharmaceutical compositions of such cytotoxic agents and chemosensitizers, methods of administration and dosages would be apparent to one of skill in the art and could be performed according to conventional methodologies, including those discussed below.

Aspects of the invention provide the use of a PKC inhibitor and/or a DNA methylation inhibitor in the manufacture of a medicament for use in the treatment of an HDACi resistant cancer condition and a method of treating an HDACi resistant cancer condition in an individual comprising administering a PKC inhibitor and/or a DNA methylation inhibitor to the individual.

An HDACi resistant cancer condition may be resistant to PXD101.

A suitable PKC inhibitor may be selected from the group consisting of staurosporine, bryostatin I, tamoxifen, safingol, UCN-01 , CGP-41251 , bisindolylmaleimides, calphostins, PKC antisense molecules and analogues, derivatives or salts of any of these compounds. In some preferred embodiments, the PKC inhibitor may be staurosporine or an analogue, derivative or salt thereof.

A suitable DNA methylation inhibitor may include 5-azacytidine, decitabine or an analogue, derivative or salt of either of these compounds.

A method may comprise the step of identifying the cancer condition as an HDACi resistant cancer condition.

A cancer condition may be identified as an HDACi resistant cancer condition using standard techniques. For example, HDACi resistant cells may be detected in a tumor sample in vitro or in vivo using conventional methods. In some embodiments, tumor tissue removed from a patient can be used as the tumor sample. A sample can be used immediately or frozen and stored at temperature below -20°C for later use. A tumor section on a microscope slide can be reacted with antibodies using standard immunohistochemistry techniques or with nucleic acids by standard in situ hybridization techniques. Additionally, if a single cell suspension of tumor cells is achievable, tumor cells can be reacted with antibody and analyzed by flow cytometry. Alternatively, an HDACi resistant tumor cell can be detected in vivo in a subject bearing a tumor. Labelled antibodies can be introduced into the subject and antibodies bound to the tumor can be detected. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Reagents useful for identifying an HDACi resistant tumor, for example, antibodies specific for HDACi resistant cell antigens, can be incorporated into a diagnostic kit. The kit can contain standards to which a sample is compared. The various reagents can be included in the kit in suitable containers and the kit can include a holder for the containers as well as an instruction manual for the use of the kit. In other embodiments, the sensitivity of cancer cells from the individual to HDACi such as PXD101 , trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid (SAHA), phenylbutyrate, diallyl sulfide, oxamflatin, MS-27-275, butyrate, FR901228, depudecin, or m-carboxycinnamic acid bishydroxamide may be determined in vitro.

Additional embodiments of the invention relate to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed herein. Such pharmaceutical compositions may include, but are not limited to cytotoxic agents and chemosensitizers identified using methods described above. In some embodiments, pharmaceutical compositions may further comprise one or more HDACi.

The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.

The compositions may be administered to a patient alone, or in combination with other agents, including other chemotherapeutic agents, drugs or hormones. As used herein, the term "other chemotherapeutic agent" refers especially to any chemotherapeutic agent that is or can be used in the treatment of tumor diseases, such as chemotherapeutics derived from the following classes: (A) Alkylating agents, preferably cross-linking chemotherapeutics, preferably bis-alkylating agents;

(B) antitumor antibiotics, preferably doxorubicin (ADRIAMYCIN™, RUBEX™);

(C) antimetabolites; (D) plant alkaloids;

(E) hormonal agents and antagonists;

(F) biological response modifiers, preferably lymphokines or interferons;

(G) inhibitors of protein tyrosine kinases and/or serine/threonine kinases; (H) antisense oligonucleotides or oligonucleotide derivatives; (I) microtubule stablizers/destabilizers; or

(J) miscellaneous agents or agents with other or unknown mechanisms of action.

The pharmaceutical compositions encompassed by the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-articular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means. In addition to the active ingredients, these pharmaceutical compositions may contain pharmaceutically acceptable carriers or diluents comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The term "pharmaceutically acceptable carrier or diluent" includes, but is not limited to, fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and binders, such as starch pastes using for example com, wheat, rice or potato starch, gelatin, tragacanth, methylcellulose, hydroxylpropylmethyl- cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate. Excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable, optionally enteric, coatings, there being used, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as ethylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Capsules are dry- filled capsules made of gelatin and soft sealed capsules made of gelatin and a plastiziser, such as glycerol or sorbitol. The dry-filled capsules may comprise the active ingredient in the form of granules, for example with fillers, such as lactose, binders, such as starches, and/or glidants, such as talc or magnesium stearate, and if desired with stabilizers. In soft capsules the active ingredient is preferably dissolved or suspended in suitable oily excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols, and also stabilizers and/or antibacterial agents may be added. Dyes or pigments may be added to the tablets or dragee coatings or the capsule casings, for example for identification purposes or to indicate different doses of active ingredient.

As used herein, "therapeutically effective amount" refers to that amount of active ingredient, for example an amount of antibodies raised against the HDACi resistant cell lines disclosed herein which when coupled to a substance having toxic or therapeutic activity, can ameliorate a symptom or condition e.g. cause the death of a tumor cell. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

For any compound, the therapeutically effective amount can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks, or once every three weeks, depending on half-life and clearance rate of the particular formulation.

Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. Pharmaceutical formulations suitable for oral administration of proteins are described, e.g., in U.S. Patents 5,008,114; 5,505,962; 5,641 ,515; 5,681 ,811 ; 5,700,486; 5,766,633; 5,792,451 ; 5,853,748; 5,972,387; 5,976,569; and 6,051 ,561. In the case of combinations with other chemotherapeutic agent(s), the other chemotherapeutic agents(s) is/ are used in standard formulations that are marketed and known to the person of skill in the art.

Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.).

All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. The invention generally described above will be more readily understood by reference to the following examples, which are hereby included merely for the purpose of illustration of certain embodiments of the present invention and are not intended to limit the invention in any way.

METHODS AND EXPERIMENTAL

Assay Method Cells were cultured, exposed to candidate compounds, and incubated for a time, and the number of viable cells was then assessed using the Cell Proliferation Reagent WST-1 from Boehringer Mannheim (Cat. No. 1 644 807), described below.

Cells were plated in 96-well plates at 3-10x103 cells/well in 100 μl_ of culture medium. The following day, different concentrations of candidate compounds were added and the cells incubated at 37⁰C for 48 h. Subsequently, 10 μL/well of WST-1 reagent was added and the cells reincubated for 1 hour. After the incubation time, absorbance was measured.

WST-1 is a tetrazolium salt which is cleaved to formazan dye by cellular enzymes. An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. This augmentation in the enzyme activity leads to an increase in the amount of formazan dye formed, which directly correlates to the number of metabolically active cells in the culture. The formazan dye produced is quantified by a scanning multiwell spectrophotometer by measuring the absorbance of the dye solution at 450 nm wavelength (reference wavelength 690 nm).

Percent activity (% activity) in reducing the number of viable cells was calculated for each test compound as:

% activity = { (SC - B) / (S⁰ - B) } x 100 wherein SC denotes signal measured in the presence of the compound being tested, S° denotes signal measured in the absence of the compound being tested, and B denotes the background signal measured in blank wells containing medium only. The IC50 corresponds to the concentration which achieves 50% activity. IC50 values were calculated using the software package Prism 3.0 (GraphPad Software Inc., San Diego, CA) , setting top value at 100 and bottom value at 0.

RESULTS

The ratios of the IC50 of the parental P388 cell line (WT) to the HDACi resistant cell line (PXD-101-C) are as shown in the table below:

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Claims

1. A method of identifying and/or obtaining an agent which is selectively cytotoxic to histone deacetylase inhibitor (HDACi) resistant cells, said method comprising the steps of: a) incubating HDACi resistant cells and HDACi sensitive cells with a test agent, b) determining the cytotoxicity of the test agent for said HDACi resistant cells and the cytotoxicity of the test agent for said HDACi sensitive cells, wherein an increase in cytoxicity of the test agent for said HDACi resistant cells relative to said HDACi sensitive cells is indicative that the test agent is an agent which is selectively cytotoxic to HDACi resistant cells.

2. A method according to claim 1 wherein the cytotoxicity of the test agent for said HDACi resistant cells and the cytotoxicity of the test agent for said HDACi sensitive cells are determined as IC50 values.

3. A method according to claim 2 wherein a resistance factor (IC50 value for resistant cells divided by IC50 value for sensitive cells) for the test agent which is less than the resistance factor for the HDACi is indicative that the test agent is an agent which is selectively cytotoxic to HDACi resistant cells.

4. A method according to claim 2 wherein a resistance factor (IC50 value for resistant cells divided by IC50 value for sensitive cells) < 1 is indicative that the test agent is an agent which is selectively cytotoxic to HDACi resistant cells.

5. A method according to any one of claims 1 to 4 wherein the agent is useful in the treatment of HDACi resistant cancer conditions.

6. A method according to any one of claims 1 to 5 wherein said HDACi is PXD101.

7. A method according to any one of claims 1 to 6 wherein the test agent is a protein kinase C (PKC) inhibitor.

8. A method according to claim 7 wherein the test agent is selected from the group consisting of staurosporine, bryostatin I₁ tamoxifen, safingol, UCN-01 , CGP-41251 , bisindolylmaleimides, calphostins, PKC antisense molecules and analogues, derivatives or salts of any of these compounds.

9. A method according to claim 8 wherein the test agent is staurosporine or an analogue, derivative or salt thereof.

10. A method according to any one of claims 1 to 6 wherein the test agent is a DNA methylation inhibitor.

11. A method according to claim 10 wherein the DNA methylation inhibitor is 5-azacytidine, or an analogue, derivative or salt thereof.

12. A method according to claim 10 wherein the DNA methylation inhibitor is decitabine, or an analogue, derivative or salt thereof.

13. A method according to any one of claims 1 to 12 comprising identifying the test agent as an agent that is selectively cytotoxic to HDACi resistant cells.

14. A method according to claim 13 comprising formulating the agent with a pharmaceutically acceptable excipient.

15. A method of inhibiting the growth of HDACi resistant cells in vitro or in vivo, said method comprising contacting said resistant cells with one or more cytotoxic agents identified and/or obtained by a method according to claim 13.

16. A method according to claim 15 wherein the one or more cytotoxic agents comprise a PKC inhibitor.

17. A method according to claim 16 wherein the PKC inhibitor is selected from the group consisting of staurosporine, bryostatin I , tamoxifen, safingol, UCN-01 , CGP-41251 , bisindolylmaleimides, calphostins, PKC antisense molecules and analogues, derivatives or salts of any of these compounds.

18. A method according to claim 17 wherein the PKC inhibitor is staurosporine or an analogue, derivative or salt thereof.

19. A method according to claim 15 wherein the one or more cytotoxic agents comprise a DNA methylation inhibitor.

20. A method according to claim 19 wherein the DNA methylation inhibitor is 5-azacytidine, or an analogue, derivative or salt thereof.

21. A method according to claim 19 wherein the DNA methylation inhibitor is decitabine, or an analogue, derivative or salt thereof.

22. A method of identifying and/or obtaining a chemosensitizer of HDACi, said method comprising the steps of: a) incubating an HDACi with a cell which is resistant to said HDACi in the presence and absence of a test agent; and, b) determining the cytotoxicity of the HDACi for the cell, wherein an increase in cytotoxicity in the presence relative to the absence of the test agent is indicative that the agent is a chemosensitizer of HDACi.

23. A method according to claim 22 wherein said HDACi is PXD101.

24 A method according to claim 22 or claim 23 wherein the test agent is a PKC inhibitor.

25. A method according to claim 24 wherein the PKC inhibitor is selected from the group consisting of staurosporine, bryostatin I , tamoxifen, safingol, UCN-01 , CGP-41251 , bisindolylmaleimides, calphostins, PKC antisense molecules and analogues, derivatives or salts of any of these compounds.

26. A method according to claim 25 wherein the PKC inhibitor is staurosporine or an analogue, derivative or salt thereof.

27. A method according to claim 22 or claim 23 wherein the one or more cytotoxic agents comprise a DNA methylation inhibitor.

28. A method according to claim 27 wherein the DNA methylation inhibitor is 5-azacytidine, or an analogue, derivative or salt thereof.

29. A method according to claim 27 wherein the DNA methylation inhibitor is decitabine, or an analogue, derivative or salt thereof.

30. A method according to any one of claims 22 to 29 comprising identifying the test agent as a chemosensitizer of HDACi.

31. A method according to claim 30 comprising formulating the agent with a pharmaceutically acceptable excipient.

32. A method according to claim 30 comprising contacting an HDACi resistant cell with said test agent and an HDACi.

33. A method of inhibiting the growth of a HDACi resistant cell in vitro or in vivo, said method comprising contacting said HDACi resistant cell with one or more HDACi and one or more chemosensitizers identified by the method of claim 30.

34. A method of identifying and/or obtaining an inhibitor of the HDAC signalling pathway comprising the steps of: a) contacting an HDACi resistant cell line and the corresponding HDACi sensitive parental cell line with increasing concentrations of a test agent; and, b) determining resistance of the HDACi resistant cell line to the test substance compared to resistance of the parental cell line to the test agent, wherein increased resistance of the HDACi resistant cell line relative the HDACi sensitive parental cell line is indicative that the test agent is an inhibitor of the HDAC signalling pathway.

35. A method of identifying and/or obtaining an HDACi analogue comprising the steps of: a) contacting an HDACi resistant cell line and the corresponding HDACi sensitive parental cell line with increasing concentrations of a test agent; and, b) determining resistance of the HDACi resistant cell line to the test agent compared to resistance of the parental cell line to the test agent, wherein a decreased resistance factor for said test agent relative to said HDACi is indicative that the test agent is an HDACi analogue.

36. A method according to claim 35 wherein said HDACi resistant cell line is resistant to PXD101.

37 Use of a PKC inhibitor in the manufacture of a medicament for use in the treatment of an HDACi resistant cancer condition.

38. Use according to claim 37 wherein said HDACi resistant cancer condition is resistant to PXDIOl

39. Use according to claim 37 or claim 38 wherein the PKC inhibitor is selected from the group consisting of staurosporine, bryostatin I , tamoxifen, safingol, UCN-01 , CGP-41251 , bisindolylmaleimides, calphostins, PKC antisense molecules and analogues, derivatives or salts of any of these compounds.

40. Use according to claim 39 wherein the PKC inhibitor is staurosporine or an analogue, derivative or salt thereof.

41. Use of a DNA methylation inhibitor in the manufacture of a medicament for use in the treatment of an HDACi resistant cancer condition.

42. Use according to claim 41 wherein said HDACi resistant cancer condition is resistant to PXDIOl

43. Use according to claim 41 or claim 42 wherein the DNA methylation inhibitor is 5- azacytidine, or an analogue, derivative or salt thereof.

44. Use according to claim 41 or claim 42 wherein the DNA methylation inhibitor is decitabine, or an analogue, derivative or salt thereof.

45. A method of treating an HDACi resistant cancer condition in an individual comprising; administering a PKC inhibitor to the individual.

46. A method of treating a cancer condition in an individual comprising; identifying the cancer condition as an HDACi resistant cancer condition, and; administering a PKC inhibitor to the individual.

47. A method according to claim 45 or claim 46 wherein said HDACi resistant cancer condition is resistant to PXD101.

48. A method according to any one of claims 45 to 47 wherein the PKC inhibitor is selected from the group consisting of staurosporine, bryostatin I, tamoxifen, safingol, UCN-01 , CGP-41251 , bisindolylmaleimide, calphostin, PKC antisense molecules and analogues, derivatives or salts of any of these compounds.

49. A method according to claim 48 wherein the PKC inhibitor is staurosporine or an analogue, derivative or salt thereof.

50. A method of treating an HDACi resistant cancer condition in an individual comprising; administering a DNA methylation inhibitor to the individual.

51. A method of treating a cancer condition in an individual comprising; identifying the cancer condition as an HDACi resistant cancer condition, and; administering a DNA methylation inhibitor to the individual.

52. A method according to claim 50 or claim 51 wherein said HDACi resistant cancer condition is resistant to PXD101.

53. A method according to any one of claims 50 to 52 wherein the DNA methylation inhibitor is 5-azacytidine, or an analogue, derivative or salt thereof.

54. A method according to any one of claims 50 to 52 wherein the DNA methylation inhibitor is decitabine, or an analogue, derivative or salt thereof.