HK1222808B - Novel combination treatment for acute myeloid leukemia (aml) - Google Patents

Description

Novel combination therapy for acute myeloid leukemia (AML)

Technical Field

The present invention relates to combination therapies for treating proliferative disorders, such as cancer, particularly acute myeloid leukemia (AML). More specifically, the present invention discloses a combination of cytarabine (Ara-C), a current mainstay of AML therapy, and a compound that acts as an inhibitor of the MDM2-p53 interaction. Surprisingly, this combination has been found to exhibit a greater-than-additive effect (synergistic).

Background Art

p53 is a tumor suppressor protein that plays a major role in preventing the development of cancer. It protects cell integrity and prevents the propagation of permanently damaged clones of cells by inducing growth arrest or apoptosis. At the molecular level, p53 is a transcription factor that activates a group of genes involved in regulating the cell cycle and apoptosis. p53 is a potent cell cycle inhibitor precisely regulated at the cellular level by MDM2. MDM2 and p53 form a feedback control loop. MDM2 binds to p53 and inhibits its ability to transactivate p53-regulated genes. In addition, MDM2 mediates the ubiquitin-dependent degradation of p53. p53 activates expression of the MDM2 gene, thereby increasing cellular levels of MDM2 protein. This feedback control loop ensures that MDM2 and p53 are maintained at low concentrations in normally proliferating cells. MDM2 is also a cofactor for E2F, which plays a major role in cell cycle regulation.

The ratio of MDM2 to p53 is dysregulated in many cancers. For example, molecular defects that commonly occur in the p16INK4/p19ARF locus have been shown to affect the degradation of MDM2 protein. Using functional p53 to inhibit the MDM2-p53 interaction in tumor cells should cause p53 accumulation, cell cycle arrest and/or apoptosis. Therefore, MDM2 antagonists can provide a new way to treat cancer in the form of a single agent or in combination with a wide range of other anti-tumor therapies. The possibility of this strategy has been demonstrated by using different macromolecular tools (such as antibodies, antisense oligonucleotides, peptides) for inhibiting the MDM2-p53 interaction. MDM2 also binds to E2F through a conserved binding region like p53 and activates E2F-dependent transcription of cyclin A, indicating that MDM2 antagonists can have an effect in p53 mutant cells with functional p53 signals.

Inhibitors of the MDM2-p53 interaction have been shown to induce apoptosis in the established human AML cell line MOLM-13 that overexpresses MDM2 (K. Kojima et al., Blood 2005, 106(9):3150-9). It has now been found that the combination of the compound of formula (I) and Ara-C provides an effect greater than the additive effect in the diffuse MOLM-13 AML model of mice without an immune response. The compound of formula (I) and its preparation are disclosed in WO2013/135648. These compounds are used as prodrugs of the following compounds:

4-{[(2R,3S,4R,5S)-4-(4-chloro-2-fluoro-phenyl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoic acid (Compound A of the present application).

Compound A is disclosed, for example, in US Pat. No. 8,354,444 and WO 2011/098398.

Summary of the Invention

The present invention relates to a pharmaceutical product comprising a) as a first component an inhibitor of the MDM2-p53 interaction (ie, an "MDM2 inhibitor"); and b) as a second component cytarabine; the pharmaceutical product being administered sequentially or simultaneously as a combined preparation for the treatment of cancer.

The present invention further relates to a method of treating a patient suffering from cancer, comprising administering to the patient a combination as mentioned above.

The present invention further relates to a method of treating a patient suffering from cancer, comprising administering to the patient a combination as described above.

The present invention also relates to a kit comprising a) a first component comprising as an active agent an inhibitor of the MDM2-p53 interaction; and b) a second component comprising as an active agent the compound cytarabine.

Furthermore, the present invention relates to the use of an MDM2 inhibitor and cytarabine for treating cancer.

Another aspect of the present invention is the use of an MDM2 inhibitor and cytarabine in the preparation of a medicament for treating cancer.

In one embodiment, the inhibitor of MDM2-p53 interaction is selected from compounds of formula (I):

Compounds of formula (I) are further described in this application.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the antitumor efficacy of the combination of Compound I-B and cytarabine against MOLM-13-luc.c4 (AML) tumor burden in SCID beige mice by quantifying bioluminescence.

Detailed Description of the Invention

The compounds of formula (I) and particularly I-A and I-B as disclosed herein are polyethylene glycol (PEG) prodrugs of compound (A) that have been synthesized to provide the desired solubility for intravenous (iv) formulations of the active parent molecule, compound (A). IV formulations are contemplated to improve dose-limiting gastrointestinal intolerance and exposure variability, as well as to provide an acceptable route of administration for the treatment of hematological malignancies and for pediatric use.

In MOLM-13 model, the antitumor activity of compound I-B and oral (po) administration of compound (A) was compared with monotherapy and in combination with AML standard of care, cytarabine (Ara-C) once a week.Compound I-B and (A) both induced significant lifespan increase (ILS) with monotherapy, wherein up to 37% ILS was observed compared with vehicle control animals.Although Ara-C lacks monotherapy activity, it significantly prolongs survival when combined with compound I-B or (A), wherein 54% or 68% maximum ILS was observed respectively.The synergistic effect demonstrated by the data of the present invention shows that the combination of using MDM2 inhibitor targeting MDM2-p53 and using cytarabine (Ara-C) to induce S phase arrest is an effective therapeutic strategy for the treatment of AML.These data also show that the efficacy of compound (A) can be maintained using formula (I) compound and especially I-A and/or I-B by this prodrug mode.

Thus, in one embodiment, the present invention relates to a pharmaceutical product comprising a) an inhibitor of MDM2-p53 interaction as a first component; and b) cytarabine as a second component; the pharmaceutical product being administered sequentially or simultaneously as a combined preparation for the treatment of AML.

In another embodiment, the inhibitor of MDM2-p53 interaction is selected from the group consisting of compounds of formula (I):

wherein n is 3 to 70.

In one embodiment, n is from 30 to 60.

In another embodiment, n is 40 to 50.

In yet another embodiment, n is 41, 42, 43, 44, 46, 47, 48 or 49.

In another embodiment, the compound of formula (I) is: 4-{[(2R,3S,4R,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoic acid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW of ˜2000). This compound is referred to herein as Compound I-A.

In another embodiment, the compound of formula (I) is: 4-{[(2R,3S,4R,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoic acid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW of ˜2200). This compound is referred to herein as Compound I-B.

In another embodiment, the MDM2 inhibitor of the present invention may be compound (A). In this embodiment, compound (A) is preferably provided in the form of an oral pre-administration formulation comprising an amorphous solid dispersion, preferably a microprecipitated bulk powder (MBP), comprising compound (A) and a polymer (preferably HPMCAS) that stabilizes compound (A) in its amorphous form. The oral pre-administration formulation is reconstituted before administration as a suspension in Klucel/Tween. Compound (A) can be prepared according to the method disclosed in, for example, U.S. Patent No. 8,354,444 or WO2011/098398. The dosage selection in the current study is based on the optimal dose for compound (A) previously determined in preclinical (animal) and clinical (Phase 1) trials.

The pharmaceutical products or methods of the present invention are particularly useful for treating or controlling hematological malignancies (e.g., leukemias), and are particularly useful for treating acute myeloid leukemia (AML). They may also be used to treat other cell proliferative disorders caused by dysregulated MDM2-p53 interactions, such as cancers, more particularly, solid tumors, such as breast cancer, colon cancer, lung cancer, melanoma, prostate cancer, kidney cancer, head and neck cancer, or sarcoma.

In one embodiment, the present invention provides a pharmaceutical product and/or method of the present invention for use in the treatment of acute myeloid leukemia (AML).

In another embodiment of the invention, pharmaceutical products and/or methods of the invention are provided for the treatment of cell proliferative disorders caused by dysregulation of the MDM2-p53 interaction, such as cancer, more particularly solid tumors, such as breast cancer, colon cancer, lung cancer, melanoma, prostate cancer, kidney cancer, head and neck cancer or sarcoma.

Formulations of the compounds of formula (I) include those suitable for oral, nasal and/or parenteral or intravenous administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

In one embodiment, the compound of formula (I) is provided in a stable lyophilized formulation for intravenous administration comprising about 0.1 mg to about 100 mg of compound (I), about 10 mM to about 100 mM buffer, about 25 mg to about 125 mg of a lyophilized bulking agent, and an isotonicity building agent. The resulting formulation should have a pH of about 5-7 via adjustment with HCl or NaOH.

In another embodiment, the compound of formula (I) is dissolved in 0.9% sodium chloride in sterile water by vortexing and then filtered through a filter into a septum-sealed vial for intravenous administration.

The term "buffer" as used herein refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and are described in the literature. Preferred pharmaceutically acceptable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and phosphate buffer, especially succinic acid (20-50 mM) and phosphoric acid (10-50 mM). The most preferred buffers include citrate, L-histidine, or a mixture of L-histidine and L-histidine hydrochloride. Other preferred buffers are acetate buffers. Independent of the buffer used, acid or alkali known in the art (such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, citric acid, sodium hydroxide, and potassium hydroxide) can be used to adjust the pH.

A preferred "bulking agent" is trehalose dihydrate, but lactose, sucrose, sorbitol, glucose, raffinose, mannitol, dextran, and lower molecular weight amino acids (e.g., glycine, valine, and arginine) and others described in the scientific literature may also be used.

As diluents for the formulated solution or reconstituted solution from the lyophilized powder, the following diluents may also be used, such as sodium chloride (0.9%), 5% dextrose, water for injection, lactated Ringers solution or semi-normal saline. It will be appreciated that fillers may also serve as isotonicity building agents.

The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of Formula I that produces a therapeutic effect. Typically, out of 100%, the amount of active ingredient will be from about 1% to about 99%, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%. Typical formulations are prepared by mixing a compound of the invention with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, for example, Ansel, Howard C. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems . Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al., Remington: The Science and Practice of Pharmacy . Philadelphia: Lippincott, Williams & Wilkins, 2000 and Rowe, Raymond C. Handbook of Pharmaceutical Excipients . Chicago, Pharmaceutical Press, 2005.

Cytarabine was purchased from Hospira, Inc. Lake Forest, IL 60045 USA in the form of a sterile solution for intravenous (iv) injection (100 mg/ml).

Formula (I) compound and cytarabine are administered in a therapeutically effective amount. More specifically, the compound of formula (I), especially formula I-A and I-B, is administered to deliver the therapeutically active amount of compound A to the patient. The therapeutically effective amount of the compound of the present invention refers to the amount of the compound that effectively prevents, alleviates or improves the symptoms of the disease or prolongs the survival of the treated individual. Those skilled in the art are familiar with measuring the amount of prodrug (e.g., formula (I) compound such as I-A or I-B) to deliver the desired amount of active ingredient (e.g., compound A) to the patient. In one embodiment of the invention, a dose of 437 mg/kg of formula I-B prodrug is equivalent to the parent MDM2 inhibitor of 100 mg/kg of compound (A), due to a loading of 22.88% of the active compound in the prodrug.

The therapeutically effective amount or dosage of the compounds of the present invention may vary within wide limits and may be determined in a manner known in the art. The dosage is adjusted to individual needs according to each specific situation, including the specific compound administered, the route of administration, the condition being treated, and the patient being treated. In general, in the case of an adult weighing about 70 kg for oral or parenteral administration, a daily dose of about 10 mg to about 3,000 mg, preferably about 80 mg to about 1600 mg of compound (A) should be appropriate, but the upper limit may be exceeded when indicated. The daily dose may be administered in a single dose or in divided doses, or for parenteral administration; it may be given as a continuous infusion.

In one embodiment, the pharmaceutical product of the present invention comprises a compound of formula (I), characterized in that it is administered in the following manner: Compound (A) is delivered in an amount of about 50 mg/day to about 3000 mg/day, or about 80 mg/day to about 2500 mg/day, or about 80 mg/day to about 1600 mg/day, or about 200 mg/day to about 1600 mg/day, or about 400 mg/day to about 1600 mg/day, or about 400 mg/day to about 1200 mg/day, or about 400 mg/day to about 1000 mg/day, or about 400 mg/day to about 800 mg/day, or about 400 mg/day to about 600 mg/day on days 1-7, or preferably days 1-5, of a 28-day treatment cycle over an administration period of up to about 7 days, preferably up to about 5 days, followed by a drug-free period of about 21 days to about 23 days, preferably up to about 23 days. The daily dose (ie the amount of Compound (A) expressed in mg/day) may be administered in a single dose (qd) or in two doses (BID). When two doses are given, they are preferably administered in equal amounts, once in the morning and once in the afternoon.

In another embodiment, the pharmaceutical product of the present invention comprises a compound of formula (I), I-A or I-B, characterized in that it is administered in the following manner: Compound A is delivered in an amount of about 120 mg/day to about 1200 mg/day over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period. The dosage is administered as a single dose (qd) or as two doses (BID). Within this embodiment, Compound I-B is preferred.

In another embodiment, the pharmaceutical product of the present invention comprises a compound of formula (I), I-A or I-B, characterized in that it is administered in the following manner: Compound A is delivered in an amount of about 400 mg/day to about 1200 mg/day over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period. The dosage is administered as a single dose (qd) or as two doses (BID). Within this embodiment, Compound I-B is preferred.

In another embodiment, the pharmaceutical product of the present invention comprises a compound of formula (I), I-A or I-B, characterized in that it is administered in the following manner: Compound A is delivered in an amount of about 400 mg/day to about 800 mg/day over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period. The dosage is administered as a single dose (qd) or as two doses (BID). Within this embodiment, Compound I-B is preferred.

In another embodiment, the pharmaceutical product of the present invention comprises a compound of formula (I), I-A or I-B, characterized in that it is administered in the following manner: Compound A is delivered in an amount of about 400 mg/day to about 600 mg/day over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period. The dosage is administered as a single dose (qd) or as two doses (BID). Within this embodiment, Compound I-B is preferred.

In another embodiment, the pharmaceutical product of the present invention comprises a compound of formula (I), I-A or I-B, characterized in that it is administered in the following manner: Compound A is delivered in an amount of about 120 mg/day to about 800 mg/day over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period. The dosage is administered as a single dose (qd) or as two doses (BID). Within this embodiment, Compound I-B is preferred.

In another embodiment, the pharmaceutical product of the present invention comprises a compound of formula (I), I-A or I-B, characterized in that it is administered in the following manner: Compound A is delivered in an amount of about 120 mg/day to about 600 mg/day over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period. The dosage is administered as a single dose (qd) or as two doses (BID). Within this embodiment, Compound I-B is preferred.

In another embodiment, the pharmaceutical product of the present invention comprises a compound of formula (I), I-A or I-B, characterized in that it is administered in the following manner: Compound A is delivered in an amount of about 120 mg/day to about 400 mg/day over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period. The dosage is administered as a single dose (qd) or as two doses (BID). Within this embodiment, Compound I-B is preferred.

In another embodiment, the pharmaceutical product of the present invention comprises a compound of formula (I), I-A or I-B, characterized in that it is administered in the following manner: Compound A is delivered in an amount of about 120 mg/day over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period. The dosage is administered as a single dose (qd) or in two doses (BID). Within this embodiment, Compound I-B is preferred.

The therapeutically effective amount (or "effective amount") of cytarabine of the present invention refers to an amount that can effectively achieve a synergistic (i.e., greater than an additive) effect, as shown by the data disclosed in this application (e.g., see Figure 1). Since cytarabine has been used as a primary therapy for AML for many years, a person skilled in the art (e.g., a clinician) can obtain a large amount of information about effective and tolerable doses in humans. For example, it has been found that cytarabine can be administered over 2 hours every 12 hours on days 1 to 6 as a single agent in higher amounts (e.g., an amount of up to 3 g/ ^m2 (intravenously)) to treat AML (induction regimen). A review of the use of cytarabine to treat leukemia is provided, for example, in "Nicholas D. Reese, Gary J. Schiller; Curr Hematol Malig Rep, 2013, 8: 141-148". In certain combination therapies (e.g., induction therapy for acute nonlymphocytic leukemia), a typical dose of cytarabine in combination with other anticancer drugs is 100 mg/ ^m2 /day by continuous intravenous infusion (Days 1-7) or 100 mg/ ^m2 intravenously every 12 hours (Days 1-7). (See, e.g., www.hospira.com).

Thus, in one embodiment, the present invention provides a pharmaceutical product comprising a) as an MDM2 inhibitor, a compound of formula (I), I-A or I-B, wherein the compound is administered intravenously (iv) once or twice daily on days 1 to 5 of a 28-day treatment cycle, followed by a 23-day rest period; and b) as a second component an effective amount of the compound cytarabine; the pharmaceutical product being administered simultaneously or sequentially as a combined preparation for the treatment of cancer, preferably AML. In this embodiment, compounds I-A and I-B are preferably administered in the following manner to deliver compound (A) in an amount of about 50 mg/day to about 3000 mg/day, or about 80 mg/day to about 2500 mg/day, or about 80 mg/day to about 1600 mg/day, or about 200 mg/day to about 1600 mg/day, or about 400 mg/day to about 1600 mg/day, or about 400 mg/day to about 1200 mg/day, or about 400 mg/day to about 1000 mg/day, or about 400 mg/day to about 800 mg/day, or about 400 mg/day to about 600 mg/day. The dosage is administered in a single dose (qd) or in two doses (BID). In this embodiment, compound I-B is preferred. Furthermore, within this embodiment, the compound of Formula (I), IA or IB is administered in an amount of about 120 mg/day to about 1200 mg/day to deliver Compound A over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period; or the compound of Formula (I), IA or IB is administered in an amount of about 400 mg/day to about 1200 mg/day to deliver Compound A over a period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period; or The compound of formula (I), I-A or I-B is administered in the following manner: Compound A is delivered in an amount of about 400 mg/day to about 800 mg/day on days 1-5 of a 28-day treatment cycle over a period of up to 5 days, followed by a 23-day rest period; or The compound of formula (I), I-A or I-B is administered in the following manner: Compound A is delivered in an amount of about 400 mg/day to about 600 mg/day on days 1-5 of a 28-day treatment cycle over a period of up to 5 days, followed by a 23-day rest period; or The compound of formula (I), I-A or I-B is administered in the following manner: Compound A is delivered in an amount of about 400 mg/day to about 600 mg/day on days 1-5 of a 28-day treatment cycle over a period of up to 5 days, followed by a 23-day rest period The invention provides a method for administering Compound A in an amount of about 120 mg/day to about 800 mg/day on days 1-5 of a 28-day treatment cycle over a period of up to 5 days, followed by a 23-day rest period; or a compound of Formula (I), IA or I-B is administered in an amount of about 120 mg/day to about 600 mg/day on days 1-5 of a 28-day treatment cycle over a period of up to 5 days, followed by a 23-day rest period; or a compound of Formula (I), IA or I-B is administered in an amount of about 120 mg/day to about 600 mg/day on days 1-5 of a 28-day treatment cycle over a period of up to 5 days, followed by a 23-day rest period. -5 days to deliver Compound A in an amount of about 120 mg/day to about 400 mg/day over an administration period of up to 5 days, followed by a 23-day rest period; or the compound of Formula (I), I-A or I-B is administered in the following manner: Compound A is delivered in an amount of about 120 mg/day over an administration period of up to 5 days on days 1-5 of a 28-day treatment cycle, followed by a 23-day rest period; or the compound of Formula (I), I-A or I-B is administered at a dose of 600 mg twice daily (BID) to deliver a total daily dose of about 1200 mg of Compound (A) to the patient.

Furthermore, within these embodiments and if not explicitly indicated, the dosage is administered as a single dose (qd) or in two doses (BID), and Compound I-A or I-B is the preferred compound.

In another embodiment, cytarabine is combined with a compound of Formula (I), I-A or I-B according to the following regimen as needed by two specific patient populations, namely:

1) Patients with relapsed/refractory AML or de novo AML with adverse features according to the European Leukemia Net guidelines; or patients with a previous blood disorder that has transformed into AML. In this embodiment, cytarabine is administered as a single dose (qd) of 1 g/m ² over 6 days as an intravenous (iv) infusion over 1-3 hours.

2) Patients with unfavorable features according to the European Leukemia Network guidelines may be considered candidates for intensive chemotherapy treatment using a standard dose of cytarabine and an anthracycline (daunorubicin or idarubicin) regimen ("7+3 induction regimen"). Within this regimen, cytarabine is administered at 100-200 mg/m ² daily as a continuous intravenous infusion over 7 days + 45 mg/m ² to 60 mg/m ² of daunorubicin or + idarubicin (12 mg/m ² intravenously daily for 3 days).

In any of the above embodiments 1) or 2), the compound of formula (I), I-A or I-B is administered as defined above. In addition, in any of the above embodiments 1) or 2), the compound of formula (I), I-A or I-B is administered in the following manner: Compound A is delivered in an amount of about 120 mg/day to about 1200 mg/day or about 400 mg/day to about 1200 mg/day on days 1-5 of a 28-day treatment cycle over a period of up to 5 days, followed by a 23-day rest period. In this embodiment, compound I-B is preferred.

In another embodiment, the present invention provides a method for treating cancer comprising administering a pharmaceutical product as defined above to a patient in need of treatment. In this embodiment, the MDM2 inhibitor is preferably selected from Compound I-A or I-B. The dosage form, dosage, and treatment schedule for Compound I-A or I-B and cytarabine are preferably as described above. Additionally, in this embodiment, the cancer is a solid or non-solid tumor, preferably acute myeloid leukemia (AML).

In another embodiment, the present invention provides the use of a compound of formula (I), preferably I-A or I-B, and cytarabine in the preparation of a medicament for the treatment of cancer, especially acute myeloid leukemia (AML).

In another embodiment, the present invention provides a pharmaceutical product comprising a) a compound of formula (I) as a first component; and b) a compound cytarabine as a second component, both administered intravenously once or twice a day, which is administered simultaneously or sequentially as a combined preparation for the treatment of cancer; characterized in that the dosage of the compound of formula (I) corresponds to the dosage of compound (A) in the following range: about 50 mg/day to about 3000 mg/day or about 80 mg/day to about 2500 mg/day or about 80 mg/day to about 1600 mg/day or about 200 mg/day to about 1600 mg/day or about 400 mg/day to about 1600 mg/day or about 400 mg/day to about 1200 mg/day or about 400 mg/day to about 1000 mg/day or about 400 mg/day to about 800 mg/day or about 400 mg/day to about 600 mg/day. In this embodiment, the compound of formula (I) is preferably compound I-A or I-B, the cancer is AML, the daily dose is about 200 to about 1600 mg and is administered once or twice daily, and the dosage regimen for compound I-A or I-B is administration on days 1 to 5 of a 28-day treatment cycle, followed by a 23-day rest period. More preferably, in this embodiment, the compound of formula (I) is compound I-B, the cancer is AML, the daily dose is about 1200 mg and is administered once or twice daily (BID, 600 mg), administered on days 1 to 5 of a 28-day treatment cycle, followed by a 23-day rest period.

The present invention will now be further illustrated by the following additional working examples.

Example

Materials and methods

animal

Female SCID light brown mice (10/group) obtained from Charles River Laboratories (Wilmington, DE) were used, which were approximately 8-12 weeks old and weighed approximately 20-25 grams. The health of the mice was assessed daily by visual observation and analysis of blood samples obtained from sentinel animals on a shared storage rack. All animals were allowed to adapt and recover from any transport-related stress for at least 72 hours before experimental use. Autoclaved water and irradiated food (5058-ms Pico Lab mouse food, Purina Mills, Richmond, IN) were provided ad libitum, and the animals were maintained on a 12-hour day and night cycle. Cages, bedding, and water bottles were autoclaved and replaced weekly before use. All animal experiments were performed in AAALAC-certified facilities according to the Guide for the Care and Use of Laboratory Animals, local regulations, and protocols approved by the Roche Animal Care and Use Committee.

tumor

Use Luc2 lentiviral particle in the presence of polybrene (8ug/ml) by parental generation MOLM-13 human AML cell stable transfection 24hr and then select 3 weeks in the presence of blasticidin (Blasticidin).Subsequently, single cell tiling is carried out in the presence of 0.1mg/mL G418 to select a kind of clone and be called MOLM-13.luc.c4.Lentiviral Luc2 expression plasmid is built by incorporating Luc2 gene (Promega) into pLOC lentiviral plasmid backbone (Thermo Fisher Scientific).By as recommended using trans-lentiviral packaging system (Trans-Lentiviral Packaging System, Thermo Fisher Scientific) prepare Luc2 lentiviral particle.

MOLM-13.luc.c4 was maintained using RPMI 1640 with L-glutamine (2 mM) culture medium (GIBCO/Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; GIBCO/Invitrogen, Carlsbad, CA) and 1% 100 mM sodium pyruvate. The newly dissociated MOLM13-Luc.c4 cells (1 × ¹⁰ or 5 × ¹⁰ ) suspended in phosphate-buffered saline (PBS) were then inoculated intravenously into female SCID beige mice via the tail vein.

Test reagent

4-{[(2R,3S,4R,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoic acid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW is ～2200) (i.e., compound I-B) is dissolved in 0.9% sodium chloride in sterile water by vortexing. It is then filtered through a 0.22 micron filter into a septum-sealed vial for intravenous administration. A drug dose of 437 mg/kg is equivalent to 100 mg/kg of the parent MDM2 inhibitor, since the prodrug has an active compound load of 22.88%. Stock cytarabine (Ara-C injection, 100 mg/ml) was diluted to 44 mg/ml in sterile 0.9% sodium chloride according to the manufacturer's instructions and administered intravenously at 200 mg/kg twice weekly.

Compound I-B and cytarabine were administered intravenously using a 1cc syringe and a 26-gauge needle at 437 mg/kg (9 ml/kg) weekly (q7d) and 200 mg/kg (4.5 ml/kg) twice weekly (2x/week), respectively. On concurrent administration days, Compound I-B was administered in the morning and cytarabine was administered 6 hours later according to IACUC regulations for intravenous volume administration. The duration of treatment was 3 weeks.

monitor

For increased lifespan (ILS) assessment, animal body weights were measured two to three times per week, and animals were monitored daily for any clinical signs of toxicity or excessive tumor burden (i.e., hind limb paralysis or morbidity). In addition, disease progression was monitored using a spectral system by in vivo bioluminescence imaging (BLI). For each BLI session, mice received 100 mg/kg D-luciferin (Caliper Life Sciences/Perkin-Elmer) via intraperitoneal (ip) injection and were imaged 20 minutes after luciferin injection with either a 5s or 10s exposure time. Images were captured by a spectral system and data were collected and analyzed using Living Image 4.2.0 software (Caliper Life Sciences, Hopkinton, MA). The total photon flux (ph/s) representing luciferase activity within each fixed region of interest (ROI) covering the entire tumor of a single mouse was determined. Actual images of the mice are not disclosed in this application. Quantitative data from the monitored bioluminescence are provided in Figure 1.

Computational and statistical analysis

Weight loss is expressed graphically as the percent change in mean group body weight using the following formula: ((W ₀ )/W ₀ )×100, where "W" represents the mean body weight of the treatment group on a particular day, and "W ₀ " represents the mean body weight of the same treatment group at the start of treatment. Maximum weight loss is also expressed using the above formula and indicates the maximum percent body weight loss observed for a particular group at any one time throughout the experiment. Toxicity is defined as ≥20% of the mice in a given group showing ≥20% body weight loss and/or death.

Bioluminescence quantification allows direct longitudinal comparison of tumor burden between treatment groups before reaching the surrogate death endpoint. Tumor burden is graphically represented as mean BLI photon flux + standard error of the mean (SEM), and median survival is determined using Kaplan-Meier survival analysis. Statistical analysis of group comparisons was performed by two-way ANOVA and post-hoc Bonferroni test (GraphPadPrism, Version 4.3). Differences between groups were considered significant at probability values (p) ≤ 0.05.

For survival evaluation, endpoint monitoring of morbidity or hind limb paralysis was used and the results were plotted as percent survival versus days after tumor implantation (GraphPad Prism, Version 4.3). Hind limb paralysis, morbidity, or ≥20% body weight loss were used as surrogates for death. ILS% was calculated as 100 × [(median survival days in the treatment group - median survival days in the control group) / median survival days in the control group]. Median survival was determined using Kaplan Meier survival analysis. The treatment group and vehicle group survival were compared by Log-rank (Mantel-Cox) test (GraphPad Prism, Version 4.3). The difference between the groups was considered significant when the probability value (p) ≤ 0.05.

result

toxicity

No toxicity, as assessed by animal weight loss or gross clinical signs, was observed in the current study. However, sporadic deaths of uncertain etiology (possibly technical, but not confirmed, see Table 1) occurred immediately after intravenous administration of Compound I-B.

Table 1: Summary of toxicity data

Antitumor efficacy and evaluation of survival/lifespan increase (ILS)

5 million cells were inoculated into mice and drug treatment was started on day 3. BLI showed that the photon counts of mice receiving compound I-B monotherapy were significantly reduced, while cytarabine (Ara-C) alone showed no difference compared to vehicle-treated control mice (see Figure 1). These significant reductions in tumor burden (as evaluated by BLI) can be converted into a significant increase in lifespan, wherein 37% ILS was observed in the group treated with q7d 437mg/kg compound I-B. Cytarabine (Ara-C) showed a lack of anti-tumor activity, as evaluated by tumor burden (BLI) or ILS. On the other hand, the combination of Ara-C and compound I-B induced a statistically significant ILS% compared to the vehicle control or monotherapy arm, indicating a significant enhancement of the combination's anti-tumor activity. These data are summarized in Table 2 below, also including a comparison with orally administered compound (A).

Table 2: Summary of efficacy data

*p<0.05 compared with vehicle control

Compared with the monotherapy group, p < 0.05

Cpd = compound.

Claims (25)

1. A pharmaceutical product comprising a) an inhibitor of MDM2-p53 interaction as a first component; and b) cytarabine as a second component, said pharmaceutical product being administered sequentially or simultaneously as a combination formulation for the treatment of cancer. The inhibitors of MDM2-p53 interaction mentioned above are selected from: Compounds of formula (I): Where n is between 3 and 70; and Compounds of formula A: 2. The pharmaceutical product of claim 1, wherein the inhibitor of MDM2-p53 interaction is selected from compounds of formula (I): Where n ranges from 3 to 70. 3. The pharmaceutical product of claim 2, wherein n is 30 to 60. 4. The pharmaceutical product of claim 2, wherein n is 40 to 50. 5. The pharmaceutical product of claim 2, wherein n is 41, 42, 43, 44, 46, 47, 48 or 49. 6. The pharmaceutical product of claim 1, wherein the inhibitor of the MDM2-p53 interaction is 4-{[(2R,3S,4R,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoic acid 1-mPEG-carbonyloxy-ethyl ester, wherein the average MW of mPEG is ~2000. 7. The pharmaceutical product of claim 1, wherein the inhibitor of the MDM2-p53 interaction is 4-{[(2R,3S,4R,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoic acid 1-mPEG-carbonyloxy-ethyl ester, wherein the average MW of mPEG is ~2200. 8. The pharmaceutical product of any one of claims 1 to 7, for the treatment of acute myeloid leukemia. 9. The pharmaceutical product of any one of claims 1 to 7, for the treatment of solid tumors. 10. Use of the compound of any one of claims 2 to 7 and cytarabine in the preparation of a medicament for treating cancer. 11. Use of the compound of any one of claims 2-7 and cytarabine in the preparation of a medicament for the treatment of acute myeloid leukemia. 12. A pharmaceutical product comprising a) a compound of formula (I) of any one of claims 2-7 as a first component; and b) a compound cytarabine as a second component, wherein both components are administered intravenously, and the pharmaceutical product is administered as a combination formulation simultaneously or sequentially for the treatment of cancer; characterized in that the dose of compound (I) corresponds to a dose of compound (A) in the range of 200 mg/day to 1600 mg/day, wherein compound (A) is: 13. The pharmaceutical product of claim 12, wherein the compound of formula (I) is compound 4-{[(2R,3S,4R,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoic acid 1-mPEG-carbonyloxy-ethyl ester, wherein the average MW of mPEG is ~2000. 14. The pharmaceutical product of claim 12, wherein the compound of formula (I) is compound 4-{[(2R,3S,4R,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoic acid 1-mPEG-carbonyloxy-ethyl ester, wherein the average MW of mPEG is ~2200. 15. The pharmaceutical product of any one of claims 12 to 14, wherein the compound of formula (I) is administered on days 1 to 5 of a 28-day treatment cycle, followed by a 23-day withdrawal period. 16. The pharmaceutical product of claim 15, wherein the compound of formula (I) is administered once or twice daily. 17. The pharmaceutical product of any one of claims 12 to 14, wherein the cancer is acute myeloid leukemia. 18. The pharmaceutical product of claim 15, wherein the cancer is acute myeloid leukemia. 19. The pharmaceutical product of claim 16, wherein the cancer is acute myeloid leukemia. 20. The pharmaceutical product of claim 1, wherein the inhibitor of MDM2-p53 interaction is a compound of formula (A): 21. The pharmaceutical product of claim 20, wherein the compound of formula (A) is administered on days 1 to 5 of a 28-day treatment cycle, followed by a 23-day withdrawal period. 22. The pharmaceutical product of claim 21, wherein the compound of formula (A) is administered once or twice daily. 23. The pharmaceutical product of claim 21 or 22, wherein the cancer is acute myeloid leukemia. 24. Use of the pharmaceutical product of any one of claims 20 to 22 in the preparation of a medicament for treating cancer. 25. The use of claim 24, wherein the cancer is acute myeloid leukemia.