Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• the present invention relates to the treatment of mesothelioma patients through the use of a low molecular weight ATP-competitive CDK (Cyclin- Dependent Kinase) inhibitor.
• CDK Cyclin- Dependent Kinase
• Malignant mesothelioma is a locally invasive and rapidly fatal neoplastic disease, linked to asbestos exposure.
• the p16 protein leads to cell cycle arrest in the G1 phase by inhibiting cyclin dependent kinase.
• the G1 to S checkpoint control is lost, resulting in a hyperphosphorylation of the tumor suppressor Retinoblastoma (pRb) and cell progression to S phase.
• pRb tumor suppressor Retinoblastoma
• TRKA Thropomyosin Receptor Kinase A
• P-TRKA activated TRKA
• Chemotherapy options are limited and include gemcitabine, vinorelbine and other antifolate compounds [Zucali PA, Ceresoli GL, Garassino I, De Vincenzo F, Cavina R, Campagnoli E Cancer 2008;112(7):1555-1561].
• the invention provides a low molecular weight compound able to inhibit two of the main pathways involved in mesothelioma pathogenesis through inhibition of CDKs and of tyrosine kinase growth factor receptor-mediated signalling pathways and efficacious in inhibiting mesothelioma proliferation.
• the compound of the present invention showing the desired activity is a pyrazoloquinazoline designed to target the ATP pocket of protein kinases. The compound has revealed to be a potent ATP-competitive inhibitor of CDKs. The compound has been found to display a significant inhibitory potency towards TRKA.
• the compound of the invention offers a new path for the development of a treatment for the patient population suffering from mesothelioma.
• Figure 1 illustrates the cytofluorimetric profile of untreated cells (C) and of cells treated with two doses (1 and 3 ⁇ M) of the compound of formula (I) (T), to evaluate the effect on cell cycle progression and induction of apoptosis on MSTO-211 H cell line.
• Figure 2 illustrates protein expression in untreated cells (C) and in cells treated with two doses (1 and 3 ⁇ M) of the compound of formula (I) (T).
• the amount of phosphorylated Rb protein (the direct substrate of CDK2), the amount of Cyclin A and cdc ⁇ (proteins involved in the control of cell cycle progression) and the amount of phosphorylated AKT ; phosphorylated S6; phosphorylated p44/42MAPK and total p44/42MAPK was evaluated and shown.
• the present invention relates to a compound of formula (I)
• mesothelioma includes pleural, peritoneal and pericardial mesotheliomas and all the histological categories: epithelioid, sarcomatoid and mixed/biphasic.
• the compound of formula (I) has the chemical name 8-[4-(4-methyl- piperazin-1 -yl)-phenylamino]-1 ,4,4-trimethyl-4,5-dihydro-1 H-pyrazolo[4,3- h]quinazoline-3-carboxylic acid methylamide. It can be prepared as described in WO2004104007, is endowed with protein kinase inhibitory activity and is thus useful in therapy as antitumor agent. In particular, the preferred preparation of the compound of formula (I) is that described in example 58 of the above mentioned International Patent Application.
• Pharmaceutically acceptable salts of the compound of formula (I) include the acid addition salts with inorganic or organic acids, e.g., nitric, hydrochloric, hydrobromic, sulphuric, perchloric, phosphoric, acetic, trifluoroacetic, propionic, glycolic, lactic, oxalic, malonic, malic, maleic, tartaric, citric, benzoic, cinnamic, mandelic, methanesulphonic, isethionic and salicylic acid and the like.
• inorganic or organic acids e.g., nitric, hydrochloric, hydrobromic, sulphuric, perchloric, phosphoric, acetic, trifluoroacetic, propionic, glycolic, lactic, oxalic, malonic, malic, maleic, tartaric, citric, benzoic, cinnamic, mandelic, methanesulphonic, isethionic and sal
• Prodrugs are any covalently bonded compounds, which release the active parent drug, according to formula (I), in vivo.
• a therapeutically effective amount of the compound according to formula (I) may be administered to a subject upon determination of the subject as having a disease or unwanted condition that would benefit by treatment with said compound. Medical or clinical personnel may make the determination as part of a diagnosis of a disease or condition in a subject.
• the compound may also be used in the prevention of such conditions, which may be viewed as reducing the probability of a subject having one or more of the conditions.
• a "therapeutically effective amount" of a compound refers to an amount sufficient to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art based upon the achievement of a desired effect. An effective amount will depend on factors including, but not limited to, the size of a subject and/or the degree to which the disease or unwanted condition from which a subject suffers has progressed. The effective amount will also depend on whether the compound is administered to the subject in a single dosage or periodically over time.
• the compound of formula (I) of the present invention is intended for the treatment of subjects.
• the term "subject” encompasses mammals and non-mammals.
• mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
• non-mammals include, but are not limited to, birds, fish and the like.
• therapeutic benefit includes achieving a therapeutic benefit.
• therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
• therapeutic benefit includes eradication or amelioration of the underlying cancer.
• a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding the fact that the patient may still be afflicted with the underlying disorder.
• Another object of the present invention is a therapeutic combination comprising (a) the compound of formula (I) as defined above and (b) one or more cytotoxic or cytostatic chemical agents, for use in a method for treating a malignant mesothelioma.
• Exemplary cytostatic or cytotoxic chemical agents includes alkylating agents, alkylating-like agents (i.e. Platinum derivatives, such as Cisplatin and Carboplatin), antimetabolite agents (e.g. pemetrexed), antimicrotubules agents, hormonal agents, immunological agents, interferon-type agents, cyclooxygenase inhibitors (e.g. COX-2 inhibitors), matrixmetalloprotease inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor agents, anti-HER agents, anti-EGFR agents, anti-angiogenesis 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 Il inhibitors, and the like.
• the present invention provides a therapeutic combination comprising the compound of formula (I) as defined above, a Platinum derivative and pemetrexed, for use in a method for treating a malignant mesothelioma.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising a compound of formula (I) as defined above admixed with a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment of malignant mesotheliomas.
• composition according to the invention further comprises one or more cytotoxic or cytostatic chemical agents.
• 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 for treating a malignant mesothelioma.
• compositions containing the compounds of the invention are usually prepared following conventional methods and are administered in a suitable pharmaceutical form.
• the solid oral forms may contain, together with the active compound, diluents, e.g., lactose, dextrose, saccharose, sucrose, cellulose, corn starch or potato starch; lubricants, e.g., silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents, e.g., starches, arabic gum, gelatine, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disintegrating agents, e.g., starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations.
• diluents e.g., lactose, dextrose, saccha
• liquid dispersions for oral administration may be, e.g., syrups, emulsions or suspensions.
• the syrups may contain, as carrier, saccharose or saccharose with glycerine and/or mannitol and sorbitol.
• the suspensions and the emulsions may contain, as examples of carriers, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.
• the compound of formula (I) is administered to a subject at dosage levels of from about 10 mg/m 2 to about 400 mg/m 2 of body surface per day.
• a dosage level of from about 20 mg/m 2 to 200 mg/m 2 constitutes a particularly suitable range.
• a dosage of from about 20 mg to about 800 mg per dose, more preferably from about 40 mg to about 400 mg per dose, from 1 to 28 consecutive days may be used as a non-limiting example.
• a preferred schedule of treatment consists of a dose of 150 mg/day for seven days of treatment followed by seven days of rest in a two-week cycle. Cycles can be repeated as long as requested to control the diseases.
• This assay allows measurement of the inhibition of the kinase activity of a specific enzyme obtained with test compound. Different kinases can be tested in parallel.
• a biotinylated substrate is trans-phosphorylated by a specific kinase in the presence of ATP including a ⁇ 33-ATP tracer. At the end of the reaction the phosphorylated substrate is then captured using Streptavidin-coated SPA beads. A dense 5M CsCI solution is added and the mixture is incubated for four hours. This causes the SPA beads to float to the top of the CsCI solution containing the unincorporated radiolabeled ATP.
• the extent of phosphorylation is measured using a ⁇ -counter.
• Example 2 Effect of compound of formula (I) in mesothelioma cells in vitro
• MSTO-211 H, NCI-H2052 and NCI-H28 mesothelioma cell lines were cultured in RPMI 1640 added with 10% FCS, 2mM glutamine, 1 mM sodium pyruvate and 10 mM HEPES.
• cells were seeded at the density of 1x10 4 /cm 2 , the day after treatment with the compound for the prescribed duration and then collected at the reported times.
• Cells were washed and counted 72 hours after the treatment. Cell proliferation was determined by a cellular adenosine triphosphate monitoring system. Cell proliferation was compared to control cells. The concentration inhibiting cell proliferation by 50% (ICso) was calculated. Analysis of cell cycle progression and apoptosis induction [0043] Cells were washed 24 hours after the treatment, fixed with cold methanol 70% and stored at -20°C. Fixed cells were washed with PBS to remove methanol and were stained with 25 ⁇ g/mL of propidium iodide, 5 ⁇ g/mL of RNase, and 0.125 ⁇ g/mL of Nonidet P 40.
• Cells were kept at room temperature for 60 minutes in the dark and were analyzed by BD FACSCaliburTM system interfaced to a Macintosh G4 computer with BD CellQuestTM 3.3 software. All doublets or aggregates were removed during analysis by an appropriate gate on an FL3-A/FL3-W dot plot and DNA content analysis was performed in >10,000 gated cells. DNA histograms (shown in Figure 1) were analyzed using ModFit LTTM .
• cell cycle progression is affected by the compound of formula (I) resulting in a G1 block evident in cells treated with 1 ⁇ M and increase of the sub G1 peak (induction of apoptosis) in cells treated with 3 ⁇ M.
• the results of these assays are also reported in Table 1.
• the compound of formula (I) is active on all the tested mesothelioma cells (first column): it is able to inhibit proliferation with an ICso in the range 0.23-1.56 ⁇ M (second column) and it is able to induce apoptosis as measured by cytofluorimetric analysis of percentage of cells with a sub G1 DNA content (third column).
• Treated cells were lysed by adding SDS sample buffer (0.125M Tris-HCI pH6.8, 5% SDS). Samples were heated to 95°C for 5 minutes and then sonicated using an Ultrasonic 2000 ARTEK. Lysed cells were centrifuged at 13,000 RPM for 10 minutes. Protein quantification was determined using BCA buffer (Pierce) and a BSA standard curve. 20 ⁇ g protein extract per well were loaded and separated by SDS-PAGE gel 7.5-10% (PAGE- PLUS 40% concentrate AMRESCO). The gel was blotted onto nitrocellulose filters (Hybond Amersham) in a buffer containing 25 mM Tris HCI pH 8.3, 192 mM Glycine and 20% methanol.
• the filters were saturated in 5% low-fat milk in TBS containing 0.1 % Tween 20 (TBS-T) for 2 hours at room temperature and than incubated overnight at 4°C with the primary monoclonal followed by washes in TBS-T and incubation using a secondary anti mouse antibody.
• TBS-T Tween 20
• FIG. 2 shows the results obtained with two doses of compound of formula (I) in NCI-H28 cells (mutated in p16/CDKN2A) treated for 24 hours. A strong inhibition of Rb phosphorylation as well as of cyclin A and cdc ⁇ expression is evident.

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Cited By (32)

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• 2009
  • 2009-11-23 EP EP09760831.9A patent/EP2376084B1/en active Active
  • 2009-11-23 CN CN2009801464496A patent/CN102223885B/zh active Active
  • 2009-11-23 JP JP2011536889A patent/JP2012509859A/ja active Pending
  • 2009-11-23 US US13/130,903 patent/US8912194B2/en active Active
  • 2009-11-23 WO PCT/EP2009/065643 patent/WO2010058006A1/en active Application Filing
• 2012
  • 2012-02-08 HK HK12101219.9A patent/HK1160779A1/xx unknown

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