# Patent

## Title
Combination therapy for proliferative disorders

## Abstract

The present invention relates to a combination therapy of propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]-amide}, or a pharmaceutically acceptable salt thereof, and an interferon for treating a patient suffering from a proliferative disorder, in particular a solid tumor, for example, colorectal cancer, melanoma, and thyroid cancer. In particular, the present invention relates to such a therapy wherein the interferon is peginterferon alfa-2a and the disorder is melanoma containing the V600E b-Raf mutation.

## Background
PRIORITY TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 61/422,690, filed Dec. 14, 2010, which is hereby incorporated by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates to a combination therapy for treating a patient suffering from a proliferative disorder, in particular a solid tumor, for example, colorectal cancer, melanoma, and thyroid cancer, comprising administering to the patient propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl}-amide and an interferon.
BACKGROUND OF THE INVENTION
Normally functioning b-Raf is a kinase which is involved in the relay of signals from the cell membrane to the nucleus and is active only when it is needed to relay such signals. Mutant b-Raf containing a V600E mutation, however, is constantly active and thus plays a role in tumor development. Such mutant b-Raf has been implicated in various tumors, for example, colorectal cancer, melanoma, and thyroid cancer.
Propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl}-amide (hereafter also referred to as “Compound I”) is a b-raf kinase inhibitor that specifically targets mutant b-Raf containing the V600E mutation. This compound is described in WO 2007/002325. Accordingly, such an inhibitor is used in the inhibition of tumors, particularly solid tumors, for example, colorectal cancer, melanoma, and thyroid cancer, which comprise b-Raf having the V600E mutation.
Interferons (IFNs) are naturally occurring proteins that have antiviral, antiproliferative, and immunoregulatory activity. The IFNα family represents the predominant class of IFNs produced by stimulated peripheral blood leukocytes and lymphoblastoid and myeloblastoid cell lines. Interferons downregulate the expression of bFGF. These drugs can be self administered by patients via subcutaneous injection with resultant good pharmacokinetics. For the purpose of the present specification, the term “interferon” shall refer also to modified interferons and/or recombinantly produced interferons such as peginterferon alfa-2a.
Peginterferon alfa-2a (sold as Pegasys® by Genentech, South San Francisco, USA) is a covalent conjugate of recombinant alfa-2a interferon (having an approximate molecular weight of 20,000 daltons) with a single branched bis-monomethoxy polyethylene glycol (PEG) chain (having an approximate molecular weight of 40,000 daltons). The PEG moiety is linked at a single site to the interferon alfa moiety via a stable amide bond to lysine. Peginterferon alfa-2a has an approximate molecular weight of 60,000 daltons. The advantage of peginterferon alfa-2a over interferons that do not contain the PEG moiety is that peginterferon alfa-2a exhibits a longer half-life, requiring less frequent dosing.
SUMMARY OF THE INVENTION
The present invention relates to a method of treating a patient suffering from a proliferative disorder, comprising administering to the patient: (A) a first component which comprises, as an active agent, Compound I, or a pharmaceutically-acceptable salt thereof; and (B) a second component which comprises, as an active agent, an interferon; the amounts of said active agents being such that the combination thereof is therapeutically-effective in the treatment of said proliferative disorder.
The present invention also relates to a kit comprising: (A) a first component which comprises, as an active agent, Compound I, or a pharmaceutically-acceptable salt thereof; and (B) a second component which comprises, as an active agent, an interferon.
The present invention further relates to a composition comprising: (A) a first component which comprises, as an active agent, Compound I, or a pharmaceutically-acceptable salt thereof; and (B) a second component which comprises, as an active agent, an interferon.
In addition, the present invention relates to the use of Compound I, or a pharmaceutically-acceptable salt thereof, and an interferon for the treatment of a proliferative disorder.
A yet further aspect of the present invention is the use of Compound I, or a pharmaceutically-acceptable salt thereof, and an interferon for the preparation of a medicament for the treatment of a proliferative disorder.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the tolerability, as demonstrated by % body weight change, of Compound I 75 mg/kg bid monotherapy, peginterferon alfa-2a 900 μg 1×/wk monotherapy, and Compound I 75 mg/kg bid/peginterferon alfa-2a 900 μg 1×/wk combination therapy.
FIG. 2 illustrates the antitumor activity, as demonstrated by tumor volume, of Compound I 75 mg/kg bid monotherapy, peginterferon alfa-2a 900 μg 1×/wk monotherapy, and Compound I 75 mg/kg bid/peginterferon alfa-2a 900 μg 1×/wk combination therapy.
FIG. 3 illustrates the effect on survival, as demonstrated by percentage of surviving mice over time, of Compound I 75 mg/kg bid monotherapy, peginterferon alfa-2a 900 μg 1×/wk monotherapy, and Compound I 75 mg/kg bid/peginterferon alfa-2a 900 μg 1×/wk combination therapy.
FIG. 4 illustrates the tolerability, as demonstrated by % body weight change, of Compound I 25 mg/kg bid monotherapy, peginterferon alfa-2a 450 μg 1×/wk monotherapy, and Compound I 25 mg/kg bid/peginterferon alfa-2a 450 μg 1×/wk combination therapy.
FIG. 5 illustrates the antitumor activity, as demonstrated by tumor volume, of Compound I 25 mg/kg bid monotherapy, peginterferon alfa-2a 450 μg 1×/wk monotherapy, and Compound I 25 mg/kg bid/peginterferon alfa-2a 450 μg 1×/wk combination therapy.
FIG. 6 illustrates the effect on survival, as demonstrated by percentage of surviving mice over time, of Compound I 25 mg/kg bid monotherapy, peginterferon alfa-2a 450 μg 1×/wk monotherapy, and Compound I 25 mg/kg bid/peginterferon alfa-2a 450 μg 1×/wk combination therapy.



DETAILED DESCRIPTION OF THE INVENTION
As stated above, “Compound I” shall herein refer to propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]-amide}. This is a compound having the following structure.




Compound I is a b-Raf kinase inhibitor that specifically targets the V600E mutation of b-Raf.
The “V600E” mutation of b-Raf, as used herein, refers to a mutation in the b-Raf protein wherein the valine residue at residue position 600 of b-Raf is replaced by glutamic acid.
As used herein, the term “pharmaceutically acceptable carrier” indicates that the indicated carrier does not have properties that would cause a reasonably prudent medical practitioner to avoid administration thereof to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration.
As used herein, the term “pharmaceutically acceptable salt” of a compound refers to any conventional salt or base addition salt that retains the biological effectiveness and properties of the compound and which is formed from a suitable non-toxic organic or inorganic acid or organic or inorganic base.
As used herein, the term “therapeutically effective” means an amount of drug, or combination or composition, which is effective for producing a desired therapeutic effect upon administration to a patient, for example, to stem the growth, or result in the shrinkage, of a cancerous tumor or to increase the patient's life span.
The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, colorectal cancer, melanoma, and thyroid cancer.
The term “colorectal tumor” or “colorectal cancer” refers to any tumor or cancer of the large bowel, which includes the colon (the large intestine from the cecum to the rectum) and the rectum, including, e.g., adenocarcinomas and less prevalent forms, such as lymphomas and squamous cell carcinomas.
“Inhibiting cell growth or proliferation” means decreasing a cell's growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.
The phrase “substantially reduced” or “substantially different,” as used herein, refers to a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values.
The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as referred to herein.
“Regression” of a tumor is said to occur following treatment when the volume of said tumor is reduced. If the tumor remains present (tumor volume >0 mm3) but its volume is reduced from what it was at the initiation of treatment, “partial regression” (PR) is said to have occurred. If the tumor is palpably absent following treatment, “complete regression” (CR) is said to have occurred.
The present invention relates to a method of treating a patient suffering from a proliferative disorder, comprising administering to the patient: (A) a first component which comprises, as an active agent, Compound I, or a pharmaceutically-acceptable salt thereof; and (B) a second component which comprises, as an active agent, an interferon; the amount of said active agents being such that the combination thereof is therapeutically-effective in the treatment of said proliferative disorder.
Treatment of a proliferative disorder shall be understood to include maintaining or decreasing tumor size, inducing tumor regression (either partial or complete), inhibiting tumor growth, and/or increasing the life span of a patient suffering from said disorder.
The present invention also relates to a kit or a composition comprising: (A) a first component which comprises, as an active agent, Compound I, or a pharmaceutically-acceptable salt thereof; and (B) a second component which comprises, as an active agent, an interferon. The kit or composition may be used, for example, in the treatment of a proliferative disorder.
In addition, the present invention provides the use of Compound I, or a pharmaceutically-acceptable salt thereof, and an interferon for the treatment of a proliferative disorder.
The invention further provides the use of Compound I, or a pharmaceutically-acceptable salt thereof, and an interferon for the preparation of a medicament for the treatment of a proliferative disorder.
In an embodiment of the present invention, the patient is a human.
In an embodiment of the invention, the proliferative disorder is a solid tumor.
In another embodiment of the invention, the proliferative disorder is a tumor containing the V600E b-Raf mutation.
In a further embodiment of the invention, the tumor is a solid tumor is selected from the group consisting of: colorectal cancer, melanoma, and thyroid cancer and the cancer involves a tumor comprising b-Raf having the V600E mutation.
In yet a further embodiment of the invention, the proliferative disorder is a solid tumor comprising b-Raf having the V600E mutation.
In yet a further embodiment of the invention, the tumor is a solid tumor containing the V600E b-Raf mutation and said tumor is selected from the group consisting of: colorectal cancer, melanoma, and thyroid cancer.
In yet a further embodiment of the invention, the tumor is melanoma.
In yet a further embodiment of the invention, the tumor is melanoma containing the V600E b-Raf mutation.
In yet a further embodiment of the invention, the interferon is selected from the group consisting of: peginterferon alfa-2a, interferon alfa-2a, peginterferon alfa-2b, and interferon alfa-2b.
In yet a further embodiment of the invention, the interferon is peginterferon alfa-2a.
In yet a further embodiment of the invention, the interferon is interferon alfa-2b.
In yet a further embodiment of the invention, the present invention relates to a method of treating a patient suffering from melanoma containing the V600E b-Raf mutation, comprising administering to the patient: (A) a first component which comprises, as an active agent, Compound I, or a pharmaceutically-acceptable salt thereof; and (B) a second component which comprises, as an active agent, peginterferon alfa-2a; the amount of said active agents being such that the combination thereof is therapeutically-effective in the treatment of said melanoma.
The amount of each component administered according to the present method may, but does not have to be therapeutically effective by itself. That is, this invention specifically contemplates combinations wherein the amount of Compound I, or a pharmaceutically-acceptable salt thereof, and/or the amount of interferon, in the combination may be less than the amount that is therapeutically-effective for each active agent when said agent is administered in monotherapy.
Compound I, or a pharmaceutically-acceptable salt thereof, may, for example, be administered orally. Peginterferon alfa-2a may, for example, be administered subcutaneously. The first component and the second component of the present invention are administered in any amount and for any duration that the combined amounts thereof are therapeutically effective in treating a proliferative disorder.
In embodiments of the present invention, Compound I is administered daily at a dosage amount of from about 200 mg/day to about 3000 mg/day, from about 800 mg/day to about 2500 mg/day, from about 1400 mg/day to about 2100 mg/day, about 960 mg/day, about 1440 mg/day, or about 1920 mg/day.
In an embodiment of the present invention, the foregoing amounts of Compound I may be administered as a single dose daily or divided, for example into equal doses (though this is not required), and administered twice daily (bid). For example, Compound I may be administered daily in a dosage amount of from about 100 mg to about 1500 mg bid, from about 400 mg to about 1250 mg bid, from about 700 mg to about 1050 mg bid, about 480 mg bid, about 720 mg bid, or about 960 mg bid.
In an embodiment of the present invention, the administration of Compound I, or a pharmaceutically acceptable salt thereof, occurs until disease progression or unacceptable toxicity.
In an embodiment of the present invention, peginterferon alfa-2a is administered at a dosage of from about 1 μg/week to about 1,000 μg/week, from about 50 μg/week to about 800 μg/week, or from about 90 μg/week to about 630 μg/week. In yet another embodiment, the dosage amount is about 180 μg/week.
In an embodiment of the present invention, the administration of peginterferon alfa-2a occurs until disease relapse, disease progression or unacceptable toxicity. In another embodiment, the peginterferon alfa-2a is administered at the dosage amounts described above for a period of up to 12 months, up to 24 months, up to 36 months, or up to 60 months.
The present invention also provides a method of treating a patient suffering from a proliferative disorder, comprising administering to the patient: (A) a first component which comprises, as an active agent, Compound I in an amount of from about 200 mg/day to about 3000 mg/day, from about 800 mg/day to about 2500 mg/day, from about 1400 mg/day to about 2100 mg/day, about 960 mg/day, about 1440 mg/day, or about 1920 mg/day; and (B) a second component which comprises, as an active agent, peginterferon alfa-2a in an amount of from about 1 μg/week to about 1,000 μg/week, from about 50 μg/week to about 800 μg/week, from about 90 μg/week to about 630 μg/week, or about 180 μg/week. In an embodiment of this invention, the proliferative disorder is a solid tumor, in particular the disorder is selected from the group consisting of: colorectal cancer, melanoma, and thyroid cancer. In another embodiment of this invention, the proliferative disorder involves a tumor comprising b-Raf having the V600E mutation. In a particular embodiment of this invention, the proliferative disorder is melanoma comprising b-Raf having the V600E mutation.
In another aspect of this invention, the components herein described above are administered in conjunction with radiotherapy and/or in conjunction with another active agent.
Compound I exists in its natural state in a crystalline form. However, the amorphous form of the compound has greater solubility in water as compared with the crystalline form and thus has an improved dissolution rate and, therefore, improved bioavailability as compared to the crystalline form. As such, the amorphous form of the compound is preferred. Accordingly, in preferred embodiments of the present invention, Compound I is in substantially amorphous form and, more preferably, in amorphous form. As used herein, the term “substantially amorphous” material embraces material which has no more than about 10% crystallinity; and “amorphous” material embraces material which has no more than about 2% crystallinity.
In an embodiment of the present invention, Compound I is contained in a solid molecular complex formed with hydroxypropyl methyl cellulose acetate succinate (HPMC-AS). As used herein, the term “solid molecular complex” means a composition wherein Compound I is randomly distributed (“molecularly dispersed”) within a matrix formed by HPMC-AS. In certain embodiments Compound I is present in the polymer in a final state of subdivision. In certain embodiments, Compound I is molecularly dispersed within the HPMC-AS matrix such that it is immobilized in its amorphous form. By “immobilized”, it is meant that the molecules of Compound I interact with molecules of HPMC-AS in such a way that they are held in the aforementioned matrix and prevented from crystal nucleation due to lack of mobility. In some embodiments the polymer may prevent intramolecular hydrogen bonding or weak dispersion forces between two or more molecules of Compound I.
In some embodiments the ratio of the amount by weight of Compound I within the solid molecular complex to the amount by weight of HPMC-AS therein is from about 1:9 to about 5:5. In an embodiment, said ratio is from about 2:8 to about 4:6. In another embodiment, said ratio is about 3:7.
In certain embodiments of the method and kit of the present invention, the first component comprises the aforementioned solid molecular complex of Compound I and HPMC-AS blended with colloidal silicon dioxide. In certain embodiments, the blend is at least 0.5% by weight silicon dioxide. In an embodiment of the present invention, the blend is about 97% complex and about 3% silicon dioxide.
In another embodiment, the first component includes a composition comprising the aforementioned solid molecular complex, either blended or not blended with silicon dioxide as described above, and a pharmaceutically acceptable carrier. In certain embodiments, the aforementioned complex or blend comprising the same is suspended in the carrier. An example of a carrier is hydroxypropylcellulose (HPC). In an embodiment, the vehicle contains about 2% by weight HPC.
Each component may also contain additional agents such as preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents and antioxidants.
In certain embodiments, the first component may comprise a solid molecular complex of Compound I and HPMC-AS blended with colloidal silicon dioxide, hydroxypropylcellulose, Crospovidone (a disintegrating agent), magnesium stearate (a lubricant that may be used in tablet and capsulation operations), and/or croscarmellose sodium (a disintegrating agent).
In an embodiment, the first component is a hard gelatin capsule comprising a solid molecular complex of Compound I and HPMC-AS blended with colloidal silicon dioxide, hydroxypropylcellulose, magnesium stearate, and croscarmellose sodium.
In an embodiment, the first component is a tablet comprising Compound I, or a pharmaceutically acceptable salt thereof. In an embodiment, the tablet comprises a solid molecular complex of Compound I, or a pharmaceutically acceptable salt thereof, and HPMC-AS. The complex may, for example, be blended with colloidal silicon dioxide, hydroxypropylcellulose, magnesium stearate, and croscarmellose sodium. The tablet may, for example, be coated with a film coating. The film coating may, for example, comprise polyvinyl alcohol, titanium dioxide, polyethylene glycol 3350, talc, and iron oxide red.
In certain embodiments, the second component may comprise peginterferon alfa-2a as an injectable solution.
Pegasys® is available as an injectable solution in vials and prefilled syringes. Each 180 μg/1.0 ml vial contains approximately 1.2 ml of solution to deliver 1.0 ml of drug product. Subcutaneous (sc) administration of 1.0 ml delivers 180 μg of drug product (expressed as the amount of interferon alfa-2a), 8.0 mg sodium chloride, 0.05 mg polysorbate 80, 10.0 mg benzyl alcohol, 2.62 mg sodium acetate trihydrate, and 0.0462 mg acetic acid. The solution is colorless to light yellow and the pH is 6.0±0.5. Each 180 μg/0.5 ml prefilled syringe contains 0.6 ml of solution to deliver 0.5 ml of drug product. Subcutaneous (sc) administration of 0.5 ml delivers 180 μg of drug product (expressed as the amount of interferon alfa-2a), 4.0 mg sodium chloride, 0.025 mg polysorbate 80, 5.0 mg benzyl alcohol, 1.3085 mg sodium acetate trihydrate, and 0.0231 mg acetic acid. The solution is colorless to light yellow and the pH is 6.0±0.5.
Applicants have conducted studies using mice containing a human melanoma xenograft.
Applicants found that, while the combination of Compound I at 75 mg/kg bid and peginterferon alfa-2a at 900 μg 1×/wk produced significantly increased life span (ILS) in mice in comparison to what was achieved with 900 μg 1×/wk peginterferon alfa-2a monotherapy, the ILS results were statistically equivalent to what was achieved with Compound I 75 mg/kg bid monotherapy.
In order to unmask the effect of combination therapy, applicants conducted studies in which Compound I was administered at 25 mg/kg bid and peginterferon alfa-2a was administered at 450 μg 1×/wk. In mice, 25 mg/kg bid Compound I achieved 321% increased life span (ILS) and 450 μg 1×/wk peginterferon alfa-2a achieved 114% ILS. By contrast, when a combined therapy of 25 mg/kg bid Compound I and 450 μg 1×/wk peginterferon alfa-2a was administered to mice, 2843% ILS was achieved. As such, ILS achieved by the combination therapy is significantly better than correlative monotherapy results at p<0.05.
It is important to note that no increased toxicity was seen in the combination groups and there was no antagonism between the two agents.
These studies indicate that treating patients with a combination of Compound I and peginterferon alfa-2a is superior to treatment with either agent alone, and that combining the two agents allows for reduction in the dose of either agent needed to obtain equivalent or better results.
EXAMPLES
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.
Abbreviations used herein are as follows:
















q.s.
as much as needed



x
times



po
orally



sc
subcutaneously



bid
twice daily



wk
week



BWL
body weight loss










Example 1
This example describes the formation of a suspension comprising Compound I.
A solid molecular complex comprising Compound I and hydroxypropyl methyl cellulose acetate succinate (HPMC-AS) was first formed.
Compound I and HPMC-AS in a ratio of approximately 3:7, respectively, were dissolved in dimethylacetamide (DMA). The resulting solution was then added with stirring to very cold dilute hydrochloric acid resulting in the co-precipitation of Compound I and HPMC-AS as a solid molecular complex wherein Compound I was present in a nanoparticulate size range. The ratio of DMA to acid was in the range of 1:5 to 1:10.
The co-precipitate was then washed with water to remove DMA, filtered, dried to <2% moisture content and passed through a #30 mesh screen prior to evaluation. The resulting solid molecular complex was 30% by weight Compound I and 70% by weight HPMC.
The complex was then blended with colloidal silicon dioxide (available as Aerosil® 200 from Evonik Industries AG, Essen, Germany) such that, per 100 g of the blend, 97 g was the complex and 3 g was colloidal silicon dioxide.
An aqueous vehicle containing 2% hydroxypropylcellulose (available as Klucel® LF from Aqualon, Wilmington, Del., USA) and 1N HCL at Qs to pH4 for the purpose of pH adjustment was then prepared. 23.2 mL of the vehicle was equilibrated to room temperature and slowly transferred into 773.2 mg of the aforementioned blend. The resulting preparation was then slowly mixed until a homogenous suspension was obtained. The suspension was stored at 2-8° C. and protected from light.
The suspension contained 9.375 mg/mL of Compound I.
Example 2
This example describes an injectable solution of peginterferon alfa-2a.














Component
Amount




















Peginterferon alfa-2a
4.5
mg



Benzyl alcohol
10.0
mg



Sodium chloride
8.00
mg



Sodium acetate trihydrate
2.617
mg



Acetic acid, glacial
0.0462
mg



Polysorbate 80
0.05
mg










Sodium acetate trihydrate, 10% w/v
q.s. pH 6.0



Acetic acid, 10% w/v
q.s., pH 6.0











Water for injection
q.s. to 1.0
ml







The solution was stored at 2 to 8° C.






Example 3
Mice were implanted with human LOX-IMVI melanoma cell xenografts. The mice, cell line used, and implantation are described below.
Female athymic Crl:NU-Foxn1nu mice were used for efficacy testing (Charles River, Wilmington, Mass., USA). Mice were 10-12 weeks of age and weighed 23-25 grams. The health of the mice was assessed daily by observation and analysis of blood samples taken from sentinel animals on shared shelf racks. All animals were allowed to acclimate and recover from shipping-related stress for one week. Autoclaved water and irradiated food (5058-ms Pico Lab mouse chow, Purina Mills, Richmond, Ind., USA) were provided ad libitum, and the animals were kept in a 12 hour light and dark cycle. Cages, bedding and water bottles were autoclaved before use and changed weekly. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, local regulations, and protocols approved by the Roche Animal Care and Use Committee in our AAALAC accredited facility.
LOX-IMVI cells (aka LOX, National Cancer Institute—Bethesda, Md.) were grown in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% of 200 nM L-glutamine, scaled up, harvested, and prepared so that each mouse received 2×106 cells/0.2 ml calcium and magnesium free phosphate-buffered saline (PBS). Cells were implanted in the subcutaneous right flank of each mouse.
Mice implanted with human xenografts were randomized into eight groups of 10 mice each according to tumor volume so that all groups had similar starting mean tumor volumes. The approximate starting mean tumor volume for this study was 130 mm3.
Example 4
Compound I was formulated as a suspension as described in example 1. Peginterferon alfa-2a was formulated as an injectable solution as described in example 2.
Treatment began on day 5 post-cell implant and ended at day 18 post-cell implant. Four groups of mice developed in example 3 were used. Each group was subjected to a different therapy as follows:

    (1) mice receiving Compound I vehicle bid po and peginterferon alfa-2a vehicle 1×/wk sc;
    (2) mice receiving Compound I at 75 mg/kg bid bid po;
    (3) mice receiving peginterferon alfa-2a at 900 μg 1×/wk sc; and
    (4) mice receiving Compound I at 75 mg/kg bid bid po and peginterferon alfa-2a at 900 μg 1×/wk sc.


The Compound I suspension and its corresponding vehicle were dosed using a sterile 1 cc syringe and 18-gauge gavage needle (0.2 ml/animal) twice daily. The peginterferon alfa-2a solution and its corresponding vehicle were dosed using a sterile 1 cc syringe and 26-gauge needle (0.2 ml/animal) once weekly on days 5 and 12 post-cell implant for a total of two injections.
Tumor measurements were taken once or twice per week. All animals were individually followed throughout the experiment.
Weight loss was graphically represented as percent change in mean group body weight, using the formula: ((W−W0)/W0)×100, where ‘W’ represents mean body weight of the treated group at a particular day, and ‘W0’ represents mean body weight of the same treated group at initiation of treatment. Maximum weight loss was also represented using the above formula, and indicated the maximum percent body weight loss that was observed at any time during the entire experiment for a particular group.
Efficacy data was graphically represented as the mean tumor volume±standard error of the mean (SEM). In addition, tumor volumes of treated groups were presented as percentages of tumor volumes of the control groups (% T/C), using the formula: 100×((T−T0)/(C−C0)), where T represented mean tumor volume of a treated group on a specific day during the experiment, T0 represented mean tumor volume of the same treated group on the first day of treatment; C represented mean tumor volume of a control group on the specific day during the experiment, and Co represented mean tumor volume of the same treated group on the first day of treatment.
Tumor volume (in cubic millimeters) was calculated using the ellipsoid formula: (D×(d2))/2, where “D” represents the large diameter of the tumor and “d” represents the small diameter.
Also, tumor regression and/or percent change in tumor volume was calculated using the formula: ((T−T0)/T0)×100, where ‘T’ represents mean tumor volume of the treated group at a particular day, and ‘T0’ represents mean tumor volume of the same treated group at initiation of treatment.
Statistical analysis was determined by the rank sum test and One Way Anova and a post-hoc Bonferroni t-test (SigmaStat, version 2.0, Jandel Scientific, San Francisco, Calif., USA). Differences between groups were considered to be significant when the probability value (p) was ≦0.05.
For survival assessment, the percent of increased life space (ILS) was calculated as: 100×[(median survival day of treated group−median survival day of control group)/median survival day of control group]. Median survival was determined utilizing Kaplan Meier survival analysis. Survival in treated groups was statistically compared with the vehicle group and survival comparisons were done between groups using the log-rank test (Graph Pad Prism, La Jolla, Calif., USA). Differences between groups were considered significant when the probability value (p) was ≦0.05.
Toxicity
No signs of toxicity were noted in any dose group in any of the studies described as assessed by measuring changes in body weight and gross observation of individual animals. These results are depicted in Table 1 and FIG. 1.














TABLE 1








% Change in









Body Weight at
Max %
Max %





end of Study
Weight
Weight
# animals ≧ 20%


Group
Frequency
Route
Day 18
Loss
Gain
BWL
Mortality






















Combo
bid, 1x/wk
po, sc
13.5
5.3
13.5
0
0


Vehicle


Compound I
bid
po
−7.0
−7.0
−3.6
0
0


75 mg/kg


Peginterferon
1x/wk
sc
2.7
0.7
2.7
0
0


alfa-2a 900 μg


Compound I
bid, 1x/wk
po, sc
−1.1
−1.1
0.5
0
0


75 mg/kg +


Peginterferon


alfa-2a 900 μg









Tumor Growth Inhibition (TGI)

The group receiving Compound I monotherapy at 75 mg/kg bid exhibited greater than 100% TGI with 10 out of 10 complete regressions (CRs). The group receiving peginterferon alfa-2a monotherapy at 900 μg 1×/wk exhibited 98% TGI, 2 partial regressions (PRs) and 3 CRs out of 10. The group receiving combination therapy of Compound I at 75 mg/kg bid and peginterferon alfa-2a at 900 μg 1×/wk exhibited greater than 100% TGI with 10 out of 10 CRs. See Tables 2 and 3 and FIG. 2.















TABLE 2








Mean Tumor


Mean Tumor







Volume (mm3)


Volume (mm3)





Start Study


End Study


Group
Frequency
Route
DAY: 5
SEM
SD
DAY: 18
SD
SEM























Combo Vehicle
bid, 1x/wk
po, sc
132.83
±2.65
±8.38
3918.69
±914.15
±289.08


Compound I
bid
po
134.36
±2.52
±7.98
0.00
±0.00
±0.00


75 mg/kg


Peginterferon
1x/wk
sc
131.77
±2.84
±8.98
193.98
±220.41
±69.70


alfa-2a 900 μg


Compound I
bid, 1x/wk
po, sc
131.79
±1.68
±5.32
0.00
±0.00
±0.00


75 mg/kg +


Peginterferon


alfa-2a 900 μg
























TABLE 3






% T/C
% Inhibition end
P value
Average







end of study
of study
End of study
% Regression per
Partial
Complete
Animal per
% Tumor Growth


Group
Day: 18
Day: 18
Day: 18
Group
Regression
Regression
Group
Inhibition























Combo Vehicle
—
—
—
—
0
0
10
—


Compound I
−4
regression
<0.001
100
0
10
10
>100


75 mg/kg bid


Peginterferon alfa-
2
98
<0.001
—
2
3
10
98


2a 900 μg 1x/wk


Compound I
−3
regression
<0.001
100
0
10
10
>100


75 mg/kg bid +


Peginterferon alfa-


2a 900 μg 1x/wk









Assessment of Survival

The group receiving Compound I monotherapy at 75 mg/kg bid exhibited 3500% increased life span (ILS). The group receiving peginterferon alfa-2a monotherapy at 900 μg 1×/wk exhibited 143% ILS. The group receiving combination therapy of Compound I at 75 mg/kg bid and peginterferon alfa-2a at 900 μg 1×/wk exhibited 3400% ILS. See Table 4 and FIG. 3.










TABLE 4









ILS Calculations













50%
50%




Group
Treatment Days
Vehicle Days
% ILS
P value





Combo Vehicle
—
—
—
—


Compound I
504
14
3500
<0.0001


75 mg/kg bid


Peginterferon
 34
14
 143
<0.0001


alfa-2a


900 μg 1x/wk


Compound I
490
14
3400
<0.0001


75 mg/kg bid +


Peginterferon


alfa-2a


900 μg 1x/wk









Statistical Analysis

The % TGI in the Compound I 75 mg/kg bid/peginterferon alfa-2a 900 μg 1×/wk combination therapy group was statistically superior to that of the peginterferon alfa-2a 900 μg 1×/wk monotherapy group but equivalent to that of the Compound I 75 mg/kg bid monotherapy group. The % ILS in the Compound I 75 mg/kg bid/peginterferon alfa-2a 900 μg 1×/wk combination therapy group was statistically superior to that of the peginterferon alfa-2a 900 μg 1×/wk monotherapy group but equivalent to that of the Compound I 75 mg/kg bid monotherapy group. See Table 5.









TABLE 5






TGI
ILS


Treatment versus Treatment
p value*
p value**


















Compound I
Peginterferon alfa-2a
<0.05
<0.0001


75 mg/kg bid
900 μg 1x/wk


Compound I 
Compound I 75 mg/kg bid +
>0.05
0.1343


5 mg/kg bid
Peginterferon alfa-2a 900 μg



1x/wk


Peginterferon
Compound I 75 mg/kg bid +
<0.05
<0.0001


alfa-2a
Peginterferon alfa-2a 900 μg


900 μg 1x/wk
1x/wk





*One-Way ANOVA, post-hoc Bonferroni


**Breslow-Gehan-Wilcoxon






Example 5
Compound I was formulated as a suspension as described in example 1. The Compound I vehicle was 2.0 grams of Klucel LF in Peginterferon alfa-2a was formulated as an injectable solution as described in example 2.
Treatment began on day 6 post-cell implant and ended at day 19 post-cell implant. Four groups of mice developed in example 3 were used. The treatment groups were as follows:

    (1) mice receiving Compound I vehicle bid po and peginterferon alfa-2a vehicle 1×/wk sc;
    (2) mice receiving Compound I at 25 mg/kg bid po;
    (3) mice receiving peginterferon alfa-2a at 450 μg 1×/wk sc; and
    (4) mice receiving Compound I at 25 mg/kg bid and peginterferon alfa-2a at 450 μg 1×/wk.


The Compound I suspension and its corresponding vehicle were dosed using a sterile 1 cc syringe and 18-gauge gavage needle (0.2 ml/animal) twice daily. The peginterferon alfa-2a solution and its corresponding vehicle were dosed using a sterile 1 cc syringe and 26-gauge needle (0.2 ml/animal) once weekly on days 6 and 13 post-cell implant for a total of two injections.
Tumor measurements were taken once or twice per week. All animals were individually followed throughout the experiment.
Weight loss was graphically represented as percent change in mean group body weight, using the formula: ((W−W0)/W0)×100, where ‘W’ represents mean body weight of the treated group at a particular day, and ‘W0’ represents mean body weight of the same treated group at initiation of treatment. Maximum weight loss was also represented using the above formula, and indicated the maximum percent body weight loss that was observed at any time during the entire experiment for a particular group.
Efficacy data was graphically represented as the mean tumor volume±standard error of the mean (SEM). In addition, tumor volumes of treated groups were presented as percentages of tumor volumes of the control groups (% T/C), using the formula: 100×((T−T0)/(C−C0)), where T represented mean tumor volume of a treated group on a specific day during the experiment, T0 represented mean tumor volume of the same treated group on the first day of treatment; C represented mean tumor volume of a control group on the specific day during the experiment, and C0 represented mean tumor volume of the same treated group on the first day of treatment.
Tumor volume (in cubic millimeters) was calculated using the ellipsoid formula: (D×(d2))/2, where “D” represents the large diameter of the tumor and “d” represents the small diameter.
Also, tumor regression and/or percent change in tumor volume was calculated using the formula: ((T−T0)/T0)×100, where ‘T’ represents mean tumor volume of the treated group at a particular day, and ‘T0’ represents mean tumor volume of the same treated group at initiation of treatment.
Statistical analysis was determined by the rank sum test and One Way Anova and a post-hoc Bonferroni t-test (SigmaStat, version 2.0, Jandel Scientific, San Francisco, Calif., USA). Differences between groups were considered to be significant when the probability value (p) was <0.05.
For survival assessment, the percent of increased life space (ILS) was calculated as: 100×[(median survival day of treated group−median survival day of control group)/median survival day of control group]. Median survival was determined utilizing Kaplan Meier survival analysis. Survival in treated groups was statistically compared with the vehicle group and survival comparisons were done between groups using the log-rank test (Graph Pad Prism, La Jolla, Calif., USA). Differences between groups were considered significant when the probability value (p) was ≦0.05.
Toxicity
No signs of toxicity were noted in any dose group in any of the studies described as assessed by measuring changes in body weight and gross observation of individual animals. These results are depicted in Table 6 and FIG. 4.














TABLE 6








% Change in









Body Weight at
Max %
Max %





end of Study
Weight
Weight
# animals ≧ 20%


Group
Frequency
Route
Day 19
Loss
Gain
BWL
Mortality






















Combo
bid, 1x/wk
po, sc
15.1
3.3
15.1
0
0


Vehicle


Compound I
bid
po
1.3
−1.0
1.3
0
0


25 mg/kg


Peginterferon
1x/wk
sc
8.1
3.7
8.1
0
0


alfa-2a 450 μg


Compound I
bid, 1x/wk
po, sc
2.1
0.1
2.1
0
0


25 mg/kg +


Peginterferon


alfa-2a 450 μg









Tumor Growth Inhibition (TGI)

The group receiving Compound I monotherapy at 25 mg/kg bid exhibited greater than 100% TGI with 10 out of 10 CRs. The group receiving peginterferon alfa-2a monotherapy at 450 μg 1×/wk exhibited greater than 100% TGI, 6 PRs and 1 CR out of 10. The group receiving combination therapy of Compound I at 25 mg/kg bid and peginterferon alfa-2a at 450 μg 1×/wk exhibited greater than 100% TGI with 10 out of 10 complete regressions (CRs). See Tables 7 and 8 and FIG. 5.















TABLE 7








Mean Tumor


Mean Tumor







Volume (mm3)


Volume (mm3)





Start Study


End Study


Group
Frequency
Route
DAY: 6
SEM
SD
DAY: 19
SD
SEM























Combo Vehicle
bid, 1x/wk
po, sc
130.71
±1.46
±4.63
2906.48
±463.14
±146.46


Compound I
bid
po
131.02
±2.06
±6.52
0.00
±0.00
±0.00


25 mg/kg


Peginterferon
1x/wk
sc
131.12
±1.19
±3.77
122.93
±117.57
±37.18


alfa-2a 450 μg


Compound I
bid, 1x/wk
po, sc
132.29
±1.53
±4.84
0.00
±0.00
±0.00


25 mg/kg +


Peginterferon


alfa-2a 450 μg
























TABLE 8






% T/C
% Inhibition end
P value
Average







end of study
of study
End of study
% Regression per
Partial
Complete
Animals per
% Tumor Growth


Group
Day: 19
Day: 19
Day: 19
Group
Regression
Regression
Group
Inhibition























Combo Vehicle
—
—
—
—
0
0
10
—


Compound I
−5
regression
<0.001
100
0
10
10
>100


25 mg/kg bid


Peginterferon alfa-
0
regression
<0.001
6
6
1
10
>100


2a 450 μg 1x/wk


Compound I
−5
regression
<0.001
100
0
10
10
>100


25 mg/kg bid +


Peginterferon alfa-


2a 450 μg 1x/wk









Assessment of Survival

The group receiving Compound I monotherapy at 25 mg/kg bid exhibited 321% increased life span (ILS). The group receiving peginterferon alfa-2a monotherapy at 450 μg 1×/wk exhibited 114% ILS. The group receiving combination therapy of Compound I at 25 mg/kg bid and peginterferon alfa-2a at 450 μg 1×/wk exhibited 2843% ILS. See Table 9 and FIG. 6.










TABLE 9









ILS Calculations













50%
50%




Group
Treatment Days
Vehicle Days
% ILS
P value





Combo Vehicle
—
—
—
—


Compound I
59
14
321
<0.0001


25 mg/kg bid


Peginterferon
30
14
114
<0.0001


alfa-2a


450 μg 1x/wk


Compound I
412 
14
2843 
<0.0001


25 mg/kg bid +


Peginterferon


alfa-2a


450 μg 1x/wk









The % TGI in the Compound I 25 mg/kg bid/peginterferon alfa-2a 450 μg 1×/wk combination therapy group was statistically superior to that of the peginterferon alfa-2a 450 μg 1×/wk monotherapy group but equivalent to that of the Compound I 25 mg/kg bid monotherapy group. The % ILS in the Compound I 25 mg/kg bid/peginterferon alfa-2a 450 μg 1×/wk combination therapy group was statistically superior to that each monotherapy group. See Table 10.









TABLE 10






TGI
ILS


Treatment versus Treatment
p value*
p value**


















Compound I
Peginterferon alfa-2a
<0.05
<0.0001


25 mg/kg bid
450 μg 1x/wk


Compound I
Compound I 25 mg/kg bid +
>0.05
0.0464


25 mg/kg bid
Peginterferon alfa-2a 450 μg



1x/wk


Peginterferon
Compound I 25 mg/kg bid +
<0.05
<0.0001


alfa-2a
Peginterferon alfa-2a 450 μg


450 μg 1x/wk
1x/wk





*One-Way ANOVA, post-hoc Bonferroni


**Breslow-Gehan-Wilcoxon

## Claims

1. A method of treating a patient suffering from a proliferative disorder, comprising administering to the patient: (A) a first component which comprises, as an active agent, propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]amide}, or a pharmaceutically acceptable salt thereof; and (B) a second component which comprises, as an active agent, peginterferon alfa-2a;
wherein said propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]amide} is administered in an amount of from about 1700 mg/day to about 2100 mg/day, wherein said amount is about 25 mg/kg, and
said peginterferon alfa-2a is administered in an amount of from about 180 μg/week to about 630 μg/week, and
wherein said proliferative disorder is selected from the group consisting of: colorectal cancer, melanoma, and thyroid cancer, wherein said cancer involves a tumor comprising b-Raf having the V600E mutation.

2. A method according to claim 1 wherein said patient is a human.

3. A method according to claim 1 wherein said proliferative disorder is melanoma containing the V600E b-Raf mutation.

4. A method according to claim 1 wherein propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]amide}, or a pharmaceutically acceptable salt thereof, is substantially in amorphous form.

5. A method according to claim 1 wherein propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]amide}, or a pharmaceutically acceptable salt thereof, is in amorphous form.

6. A method according to claim 1 wherein propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]amide}, or a pharmaceutically acceptable salt thereof, is contained in a solid molecular complex formed with hydroxypropyl methyl cellulose acetate succinate such that it is immobilized in its amorphous form.

7. A method according to claim 6 wherein the amounts of propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]amide}, or a pharmaceutically acceptable salt thereof, are hydroxypropyl methyl cellulose acetate succinate in said complex are in a ratio of from about 1:9 to about 5:5, respectively.

8. A method according to claim 6 wherein the amounts of propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]amide}, or a pharmaceutically acceptable salt thereof, and hydroxypropyl methyl cellulose acetate succinate in said complex are in a ratio of about 3:7, respectively.

9. A method according to claim 6 wherein said first component comprises a blend wherein about 97% by weight of the blend is said complex and about 3% by weight of the blend is silicon dioxide.

10. A method according to claim 6 wherein said first component comprises a suspension of said complex in a pharmaceutically acceptable carrier.

11. A method according to claim 1 wherein said first component comprises a tablet comprising a solid molecular complex of propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]amide}, or a pharmaceutically acceptable salt thereof, and hydroxypropyl methyl cellulose acetate succinate.