HK1160779B - Cdk inhibitor for the treatment of mesothelioma - Google Patents

Description

CDK inhibitors for the treatment of mesothelioma

Technical Field

The present invention relates to the treatment of mesothelioma patients by using low molecular weight ATP-competitive CDK (cyclin dependent kinase) inhibitors.

Background

Malignant mesothelioma is a locally invasive and rapidly fatal neoplastic disease associated with asbestos exposure.

There are many genetic defects that contribute to the outcome of mesothelioma. Syntypic deletion of p16/CDKN2A (cyclin-dependent kinase inhibitor 2A), the most common genetic alteration in this cancer, was found in about 75% of mesotheliomas [ Hirao T, Bueno R, Chen CJ, Gordon GJ, Heilig E, Kelsey kt. carcinogenesis 2002; 23(7): 1127-; whitson BA, Kratzke ra. cancer Lett 2006; 239(2): 183-189]. In terms of prognosis, the p16/CDKN2A deletion was associated with a more aggressive clinical behavior of mesothelioma [ Kobayashi N, Toyooka S, Yanai H, Soh J, Fujimoto N, Yamamoto H et al, Lung Cancer 200862 (1): 120-5; davidson B, Reich R, Lazarovici P, Florenes VA, Ri sberg B, Nielsen S, Lung Cancer 2004; 44(2): 159-165].

The p16 protein causes the cell cycle to stop in the G1 phase by inhibiting cyclin-dependent kinases. As a result of the lack of p16/CDKN2A, control of G1 to the S checkpoint was lost, resulting in hyperphosphorylation of the tumor suppressor retinoblastoma (pRb) and cell progression to S phase.

In addition, the Thropomyosin Receptor Kinase A (TRKA) also plays a significant role in the biology of the disease. Indeed, frequent expression of activated TRKA (P-TRKA) is often found in malignant mesotheliomas and is mainly found in effusion and peritoneal lesions, in tumors present in young patients [ Whitson BA, Kratzke ra. molecular pathways in macromolecular pleurant pleuralmeastern theta. cancer Lett 2006; 239(2): 183-189].

Mesothelioma is increasingly present in high incidence worldwide, and only a small proportion of patients benefit from surgical resection. For patients who are not amenable to therapeutic resection, the median total survival time is about 6-7 months. The choice of treatment is limited. Most patients, either treated or untreated, die of complications of local disease. None of the commercially available chemotherapeutic agents, either as single agents or in combination, have proven to significantly affect survival.

Current treatment options include the use of pemetrexed (under the trademark pemetrexed) as a single drugSales), has demonstrated a modest response rate of 14.1% with a median total survival time of 10.7 months [ Scagliotti GV, Shin DM, Kindler HL, Vasconcelles MJ, KepplerrU, Manegold C et al, J Clin Oncol 2003; 21(8): 1556-1561]Or a combination of a platinum derivative (cisplatin or carboplatin) and pemetrexed [ Castagneto B, Botta M, Aitini E, Spigno F, Degiovanni D, Alabiso O et al, Ann Oncol 2008；19：370-373]。

There are no approved drugs for second line treatment of mesothelioma. The choice of chemotherapeutic drugs is limited, including gemcitabine, vinorelbine, and other antifolate compounds [ zucai PA, cerenoli GL, garrasino I, De Vincenzo F, Cavina R, campagonoli ecanecer 2008; 112(7): 1555-1561].

Indeed, there is a very high unmet medical need for new effective drugs for the treatment of mesothelioma. The present invention solves this problem.

Disclosure of Invention

The present invention provides a low molecular weight compound that inhibits two major pathways involved in the pathogenesis of mesothelioma and effectively inhibits mesothelioma proliferation by inhibiting CDK and tyrosine kinase growth factor receptor-mediated signaling pathways.

It was shown that the desired compound of the invention is a pyrazoloquinazoline designed to target the ATP pocket of a protein kinase. The compound has been shown to be a potent ATP-competitive CDK inhibitor. We have found that this compound shows a significant inhibitory effect on TRKA.

Given its biological activity, the compounds of the invention provide a new avenue for developing treatments for mesothelioma-bearing patient populations.

Drawings

FIG. 1 shows the cytofluorescence plots of untreated cells (C) and cells (T) treated with two doses (1 and 3 μ M) of a compound of formula (I) to evaluate the effect on cell cycle progression and induction of apoptosis in the MSTO-211H cell line.

FIG. 2 shows protein expression of untreated cells (C) and cells (T) treated with two doses (1 and 3 μ M) of a compound of formula (I). The amount of phosphorylated Rb protein (a direct substrate for CDK 2), cyclin a and cdc6 (a protein involved in controlling cell cycle progression), and the amount of phosphorylated AKT, phosphorylated S6, phosphorylated p44/42MAPK, and total p44/42MAPK were evaluated and shown.

Detailed Description

In a first aspect, the invention relates to compounds of formula (I)

Or a pharmaceutically acceptable salt thereof, for use in a method of treating mesothelioma.

The term "mesothelioma" as used herein includes pleural, peritoneal and pericardial mesotheliomas and all histological classifications: epithelioid, sarcoma-like, and mixed/biphasic.

The chemical name of the compound of formula (I) is 8- [4- (4-methyl-piperazin-1-yl) -phenylamino ] -1, 4, 4-trimethyl-4, 5-dihydro-1H-pyrazolo [4, 3-H ] quinazoline-3-carboxylic acid methylamide. It can be prepared as described in WO2004104007, has protein kinase inhibitory activity, and therefore can be used as an antitumor agent for treatment. In particular, a preferred process for the preparation of the compound of formula (I) is described in example 58 of the above-mentioned International patent application.

Pharmaceutically acceptable salts of the compounds of formula (I) include acid addition salts with inorganic or organic acids such as nitric acid, hydrochloric acid, hydrobromic acid, sulfuric acid, perchloric acid, phosphoric acid, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, lactic acid, oxalic acid, malonic acid, malic acid, maleic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, isethionic acid, salicylic acid and the like.

Within the scope of the claimed invention are all possible isomers of the compounds of formula (I) and their mixtures, as well as metabolites and pharmaceutically acceptable biological precursors (also called prodrugs) thereof. Prodrugs are any covalently bonded compounds which release the active parent compound of formula (I) in vivo.

When a patient is determined to have a disease or unwanted condition that would benefit from treatment with the compound, a therapeutically effective amount of the compound of formula (I) may be administered to the patient. As part of diagnosing a disease or condition in a patient, medical or clinical personnel may make such a decision. The compounds may also be used to prevent these disorders, which may be viewed as reducing the chance of a patient suffering from one or more of the disorders.

As used herein, a "therapeutically effective amount" of a compound refers to an amount sufficient to achieve its intended purpose. Determination of an effective amount is within the ability of those skilled in the art, depending on the achievement of the desired effect. An effective amount depends on various factors including, but not limited to, the size of the patient and/or the extent of the disease or unwanted condition from which the patient is suffering. An effective amount will also depend on whether the compound is administered to the patient in a single dose or periodically over time.

The compounds of formula (I) of the present invention are intended for use in the treatment of patients. The term "patient" as used herein includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: human, non-human primates such as orangutans, and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals include rodents, such as rats, mice and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like.

The term "treating" as used herein includes achieving a therapeutic benefit. Therapeutic benefit refers to eradication or amelioration of the underlying (underlying) disease being treated. For example, in cancer patients, therapeutic benefit includes eradication or amelioration of the underlying cancer. At the same time, therapeutic benefit may also be achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disease, such that an improvement is observed in the patient despite the fact that the patient may still be afflicted with the underlying disease.

Another object of the present invention is a therapeutic combination comprising (a) a compound of formula (I) as defined above and (b) one or more cytotoxic or cytostatic chemical agents, for use in a method of treating malignant mesothelioma.

Exemplary cytotoxic or cytostatic chemical agents include alkylating agents, alkylating-like agents (i.e., platinum derivatives such as cisplatin and carboplatin), antimetabolites (e.g., pemetrexed), antimicrotubule agents, hormonal agents, immunological agents, interferon-type agents, cyclooxygenase inhibitors (e.g., COX-2 inhibitors), matrix metalloproteinase inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor agents, anti-HER agents, anti-EGFR agents, anti-angiogenic agents (e.g., angiogenesis inhibitors), farnesyl transferase inhibitors, ras-raf signal transduction pathway inhibitors, cell cycle inhibitors, other cdks inhibitors, tubulin binding agents, topoisomerase I inhibitors, topoisomerase II inhibitors, and the like.

In a particularly preferred embodiment, the present invention provides a therapeutic combination comprising a compound of formula (I) as defined above, a platinum derivative and pemetrexed for use in a method of treatment of malignant mesothelioma.

The invention also relates to a pharmaceutical composition comprising a compound of formula (I) as defined above, together with a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment of malignant mesothelioma.

In another embodiment, the pharmaceutical composition of the invention further comprises one or more cytotoxic or cytostatic chemical agents.

In a particularly preferred embodiment, the present invention provides a pharmaceutical composition comprising a compound of formula (I) as defined above, a platinum derivative and pemetrexed for use in a method of treating malignant mesothelioma.

The pharmaceutical compositions comprising the compounds of the invention are generally prepared according to conventional methods and administered in the appropriate pharmaceutical form.

For example, solid oral dosage forms may contain, together with the active compound, diluents, such as lactose, dextrose, saccharose, sucrose, cellulose, corn starch or potato starch; lubricants, for example silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycol; binders, for example, starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disintegrating agents, such as starch, alginic acid, alginates or sodium starch glycolate; boiling complexing agent; a dye; a sweetener; wetting agents, such as lecithin, polysorbate, lauryl sulfate; and non-toxic and pharmaceutically inactive substances commonly used in pharmaceutical formulations. These pharmaceutical preparations can be prepared in a known manner, for example, by mixing, granulating, tabletting, sugar-coating or film-coating processes.

Oral liquid dispersions may be, for example, syrups, emulsions or suspensions.

For example, the syrup may contain sucrose or sucrose and glycerin, and/or mannitol and sorbitol as carriers.

As examples of carriers, suspensions and emulsions may contain natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.

In therapeutic applications, the compound of formula (I) is present at about 10mg/m per day²-about 400mg/m²A dosage level on the body surface is administered to the patient. About 20mg/m²-200mg/m²The dosage level of (a) constitutes a particularly suitable range. For adult patients, a dosage of about 20mg to about 800mg per dose, more preferably about 40mg to about 400mg per dose, for a period of 1 to 28 days, may be used as a non-limiting example. A preferred treatment regimen comprises treatment at a dose of 150 mg/day for 7 days over a 2 week period, followed by 7 days off. The treatment cycle may be repeated as long as disease control is desired.

Lower or higher doses than those described herein can be used if desired. However, these dosages may be varied depending upon a number of variables not limited to the activity of the compound employed, the condition being treated, the mode of administration, the treatment regimen, the individual requirements of the patient, the severity of the condition being treated and the judgment of the practitioner. The above ranges are only suggestive, as the number of variables associated with an individual treatment regimen is large, and large deviations from these recommended values are not uncommon.

For the purpose of better illustrating the invention (and not of limiting it), the following examples are now given.

Examples

Example 1 Scintillation Proximity Assay (SPA) mode of kinases

This assay allows the determination of the inhibition of the kinase activity of a particular enzyme obtained with a test compound. Different kinases can be tested in parallel.

The biotinylated substrate is transphosphorylated by a specific enzyme in the presence of ATP (including a gamma 33-ATP tracer). At the end of the reaction, streptavidin-coated SPA beads were then used to capture the phosphorylated substrate. A dense 5M CsCl solution was added and the mixture was incubated for 4 hours. This resulted in SPA beads being suspended on top of CsCl solution containing unincorporated radiolabeled ATP.

The extent of phosphorylation was determined using a beta-counter. In these assays, the compounds of formula (I) showed potent inhibitory activity (IC) against the CDK 2/cyclin A complex₅₀45nM), the activities shown were against closely related CDKs, CDK1, CDK4 and CDK5 (IC, respectively)₅₀398, 160 and 265nM), but also against the thrombomycin receptor kinase a (trka) (IC)₅₀＝53nM)。

Example 2 Effect of Compounds of formula (I) in vitro mesothelioma cells

MSTO-211H, NCl-H2052 and NCl-H28 mesothelioma cell lines were cultured in RPMI 1640 supplemented with 10% FCS, 2mM glutamine, 1mM sodium pyruvate and 10mM HEPES. For all experiments, 1X 10 on day 2 after the prescribed period of treatment with the compounds of the invention⁴/cm²The cells were seeded at the density of (1) and then harvested at the time reported.

Inhibition of cell proliferation

At 72 hours post-treatment, cells were washed and counted. Cell proliferation was determined by a cellular adenosine triphosphate detection system. Cell proliferation was compared to control cells. The concentration inhibiting 50% of cell proliferation (IC) was calculated₅₀)。

Analysis of cell cycle progression and Induction of apoptosis

Cells were washed 24 hours after treatment, fixed with 70% glacial methanol, and stored at-20 ℃. The fixed cells were washed with PBS to remove methanol and stained with 25. mu.g/mL propidium iodide, 5. mu.g/mL RNA se, and 0.125. mu.g/mL Noni det P40. Cells were kept in the dark at room temperature for 60 minutes and passed through a cell line with BD CellQuest^TM3.3 MacintoshG4 computer-connected BD FACSCalibur for software^TMAnd (5) analyzing the system. During the analysis, all doublets or aggregates were removed by appropriate gating on FL3-A/FL3-W dot, and DNA content analysis was performed in gate cells ≧ 10,000. With ModFit LT^TMTo analyze the DNA histogram (as shown in FIG. 1).

For example, as shown in figure 1, influencing cell cycle progression by compounds of formula (I) resulted in a clear G1 blockade in cells treated with 1 μ M and an increase in the sub G1 peak (induction of apoptosis) in cells treated with 3 μ M.

Table 1 also reports the results of these analyses. The compound of formula (I) was active on all mesothelioma cells tested (first column): it can inhibit proliferation, its IC₅₀In the range of 0.23-1.56. mu.M (th)Two columns), it induces apoptosis as determined by cytofluorimetric analysis on the percentage of cells with sub G1DNA content (third column).

TABLE 1

Mesothelioma cell line	IC50(μM)	Sub G1％
			MSTO-211H	0.23±0.10	24
NCl-H2052	0.74±0.37	14
			NCl-H28	1.56±0.52	5

Example 3 evaluation of the mode of action of the Compounds of formula (I) by Western blot analysis

The treated cells were lysed by adding SDS sample buffer (0.125M Tris-HCl pH6.8, 5% SDS). The sample was heated to 95 ℃ for 5 minutes and then sonicated with Ultrasonic 2000 ARTEK. The lysed cells were centrifuged at 13,000 RPM for 10 minutes. The amount of protein was determined using BCA buffer (Pierce) and BSA standard curve. The wells were loaded with 20. mu.g of protein extract and separated by SDS-PAGE gel 7.5-10% (PAGE-PLUS 40% concentrate AMRESCO). The gel was coated onto nitrocellulose filter paper (Hybond Amersham) in a buffer containing 25mM Tris HCl pH 8.3, 192mM glycine and 20% methanol. The filter paper was saturated in 5% low-fat milk for 2 hours at room temperature in TBS containing 0.1% Tween 20(TBS-T), then incubated with primary monoclonal antibodies overnight at 4 ℃, then washed in TBS-T, and incubated with secondary anti-mouse antibodies. These bands were observed with "Super Signal West Pico" Pierce.

The results obtained in all the cell lines tested indicate that the compounds of formula (I) are capable of interfering with two major pathways involved in mesothelioma pathogenesis.

A reduction in cell cycle-associated markers was observed in all the experimental mesothelioma cell lines. For example, FIG. 2 shows the results obtained in NCl-H28 cells (mutated in p16/CDKN 2A) treated with two doses of the compound of formula (I) for 24 hours. Strong inhibition of Rb phosphorylation and cyclin a and cdc6 expression was evident.

The ability of compounds of formula (I) to inhibit tyrosine kinase growth factor receptor mediated channels was also evaluated. Inhibition of phosphorylation of AKT, S6 and MAPK in NCl-H28 cells was also reported in FIG. 2.

Claims (4)

1. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of malignant mesothelioma,

2. use of a therapeutic composition comprising (a) a compound of formula (I) as defined in claim 1 and (b) one or more cytotoxic or cytostatic chemical agents selected from pemetrexed and platinum derivatives in the manufacture of a medicament for the treatment of malignant mesothelioma.

3. Use of a pharmaceutical composition comprising a compound of formula (I) as defined in claim 1 together with a pharmaceutically acceptable carrier, diluent or excipient in the manufacture of a medicament for the treatment of malignant mesothelioma.

4. Use according to claim 3, wherein the pharmaceutical composition further comprises one or more cytotoxic or cytostatic chemical agents selected from pemetrexed and platinum derivatives.