KR20100072253A - Formulation containing cyclin-dependent kinase inhibiting compound and method of treating tumors using the same - Google Patents

Abstract

This application discloses a novel formulation containing a 3-amino-4-sub stituted pyrazole derivative which has cyclin-dependent kinase inhibiting properties, and a method of treating tumors using the novel formulation.

Description

Formulation containing Cyclin-dependent Kinase Inhibiting Compound and Method of Treating Tumors Using the Same}

This application is issued to United States Provisional Patent Application 60 / 994,185, filed September 17, 2007; 60 / 999,469, filed October 17, 2007; And 35 USC 119 (e) of 61 / 053,784, filed May 16, 2008, the disclosures of which are incorporated herein by reference in their entirety.

The present application describes novel formulations containing 3-amino-4-substituted pyrazole derivatives having cyclin-dependent kinase inhibitory properties, and methods of treating tumors using the novel formulations.

The identification of any publication in this or any section of this application is not an admission of this publication as a prior art to the invention.

The mammalian cell cycle is a non-redundant process that includes extracellular signaling, DNA synthesis, and mitosis. Unregulation of cell cycle regulation is a hallmark of all human cancers and is also frequently associated with abnormal activity / regulation of cyclin-dependent kinases, designated herein as CDKs, for convenience. Lack of cell cycle regulation in tumor cells is believed to increase the characteristics of apoptosis in tumor cells compared to normal tissue. Thus, specific CDK inhibitors can selectively kill tumor cells while only inducing the reversible cell cycle to stop in the normally proliferating compartment. Tumor cell apoptosis can be achieved without mutagenic effects on normal tissues. Thus, the identification of compounds that inhibit the intrinsic, rate-limiting activity of CDK2 and CDK1 represents an attractive therapeutic strategy for the treatment and management of oncological prognosis.

U.S. Pat.No. 7,119,200, issued October 10, 2006, which discloses compounds having a cyclin-dependent kinase inhibitory property (CDK inhibitor) and containing a 3-amino-4-substituted pyrazole derivative structure. Guzi et al. . ("200 patents", herein incorporated by reference in their entirety). Effective synthesis of such CDK-inhibiting compounds, such as those of Formula I, is described in published US application 2006/0281756 (the '756 publication, which is incorporated herein by reference in its entirety):

[Formula I]

In formula (I) above, R ¹ , R ² , R ³ and R ⁴ are as defined in the '756 publication, which is incorporated herein by reference in its entirety.

Compounds of formula (I) are useful for the treatment of proliferative diseases such as tumors such as cancer.

As described in this' 200 patent, which is incorporated by reference in its entirety, the 2- (quinolin-5-yl) -4,5-disubstituted-azole derivatives of Formula (I) have been tested for in vitro CDK2 inhibition and curve-fitting calculations. It was shown to have CDK inhibitory activity with the desired IC ₅₀ value. For example, referring to formula (I) wherein R ¹ is pyridin-3-yl-methylamine, R ² is hydrogen, R ³ is 1-methyl-piperazine, and R ⁴ is hydrogen, the compound of formula (Ia) It was shown to have a calculated IC ₅₀ value of 5.6 Michaelomol for the CDK receptor site (see Example 1 of this' 200 patent, Table 87 in column 706).

Formula Ia

Other compounds having the structure of formula I, for example, R ¹ is 3-methyl-pyridine N-oxide, R ² is ethyl, R ³ is 2-piperidin-2-yl-ethanol, R ⁴ With reference to formula (I), wherein is hydrogen, the compounds of formula (II) also have useful CDK inhibitory activity. Compounds of formula (II) are useful in inhibiting tumors, including but not limited to proliferation, angiogenesis and / or metastasis.

[Formula II]

For example, the compound of formula (II) may comprise: bladder, breast, eg, breast cancer, colon, eg, colorectal cancer, kidney, bile, liver, lung, eg small cell lung cancer and non-small cell lung cancer, esophagus Gallbladder, pancreas, stomach, cervix, thyroid gland, prostate, ovarian cancer, vulvar cancer, peritoneal cancer, peritoneal pseudomyxoma, and skin, such as melanoma and squamous cell carcinoma; Leukemia, Acute Lymphocytic Leukemia, Acute Lymphocytic Leukemia, Chronic Lymphocytic Leukemia (CLL), B-Cell Lymphoma, T-Cell Lymphoma, Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma [eg, Mantle Cell Lymphoma (MCL)] Hematopoietic tumors of the lymphatic system, including hair cell lymphoma and Burkett's lymphoma; ; Hematopoietic tumors of myeloid lineage, including acute and chronic myeloid leukemia, myelodysplastic syndromes and promyelocytic leukemia; Tumors of mesenchymal cell origin, including fibrosarcoma and rhabdomyosarcoma; Tumors of the central and peripheral nervous systems, including astrocytoma, neuroblastoma, glioma and neuromyoma; And various cancers, including but not limited to testicular, teratocarcinoma, osteosarcoma, pigmentary dry skin, keratinocytes, thyroid vesicle cancer and Kaposi's sarcoma.

Summary and purpose of the invention

A pharmaceutical formulation capable of administering a compound of Formula (I) having useful CDK inhibitory activity to a patient in need of a therapy that is useful for the management or treatment of a disease state, eg, for the management or treatment of a tumor. Required. These and other objects and / or advantages are provided by the present invention.

One preferred aspect of the present invention provides a concentrated pharmaceutical formulation containing a compound of formula II, which may be suitable for administration to a mammal in need thereof having a tumor responsive to the compound of formula II, The formulation comprises a compound of Formula (II), citric acid, sodium citrate and water at about pH 2.5 to about pH 7.0. In one preferred embodiment, the buffer has a pH of about 3.4 to about 5. In some embodiments, it is preferable to use a mole ratio of 1 mole of the compound of formula II: 3.66 moles of sodium citrate: 8.8 mole of citric acid: sodium citrate: citric acid mole ratio. In some embodiments, the formulation provides wt./vol., Sufficient to adjust the pH of the concentrate to a pH of about pH 2.5 to about pH 7.0, preferably about pH 3.0 to about pH 4.0, more preferably about pH 3.5. On the basis of (a) 5 mg / ml of a compound of Formula II; (b) 21.4 mg / ml citric acid; And (c) an aqueous solution containing 11.4 mg / ml of sodium citrate dehydrate, optionally mixed with hydrochloric acid and / or sodium hydroxide.

Formula II

Another preferred aspect of the invention is an IV-insoluble formulation comprising a compound of formula II in a buffer having a pH between about pH 2.5 and about pH 7.0, wherein the formulation is injected into a human at the indicated dosage level, FIG. 2A And / or provide one or more PK behavior at a designated dose, depending on the dosage as shown in FIG. 2B and / or Table V. In one preferred embodiment, the buffer has a pH of about 3.4 to about 5. Preferably, the formulation provides one or more of the AUC / dose curves shown in FIGS. 2A and / or 3 when infused at the dose levels shown for each curve in a two-hour infusion.

Another preferred aspect of the invention is an IV-insoluble formulation comprising a compound of formula II in a buffer having a pH between about pH 2.5 and about pH 7.0, wherein the formulation is injected into a human at the indicated dosage level, FIG. 2C And / or provide one or more PK behavior at a designated dose, depending on the dosage as indicated in Table VI. Preferably, the formulation provides one or more of the AUC / dose curves shown in FIG. 2C when infused at the dosage levels shown for each curve in a 2-hour infusion. In one preferred embodiment, the buffer has a pH of about 3.4 to about 5.

Another preferred aspect of the invention provides a formulation comprising an aqueous organic acid, a conjugated salt thereof, and a compound of formula (II) that provides a PK profile shown in FIGS. 2A, 2B, and / or 2C upon IV infusion to a human. Preferably, the formula comprises a molar ratio of 1 mol of the compound: 8.8 mol of the organic acid: 3.7 mol of the conjugate salt of the compound of formula II: organic acid: its conjugate salt. Preferably, the art formulations 2 hour infusion of a dose containing 1.85 mg / M ² having about 2.15 times of t _1/2, from about 77 ng / ml of C _{up to} and / or AUC of about 181 ng hr / mL Provide a PK for. Preferably 2 hour infusion of the capacity to the art formulation comprises from about 2.75 time of t _1/2, from about 136 ng / ml of C _{up to,} and / or 3.7 mg / M ² having an AUC of about 336 ng hr / mL Provide a PK for. Preferably 2 hour infusion of the capacity to the art formulation comprises from about 2.86 time of t _1/2, from about 353 ng / ml of C _{up to,} and / or 7.4 mg / M ² having an AUC of about 994 ng hr / mL Provide a PK for. Preferably, the art formulations 2 hours of capacity containing about 2.78 times of t _1/2, from about 396 ng / ml of C _{up to,} and / or about 1053 ng hr / mL 14.8 mg / M 2 having an AUC of Provide PK for injection. Preferably, the art formulations 2 hours of capacity containing about 2.79 times of t _1/2, about 1020 ng / ml of C _{up to,} and / or 29.6 mg / M ² having an AUC of about 2531 ng hr / mL Provide PK for injection. Preferably, the art formulations 2 hours of capacity containing 41.4 mg / M ² has a C _maximum, and / or AUC of about 3041 ng hr / mL of t _1/2, about 1343 ng / ml for about 2.8 hours Provide PK for injection. More preferably, the art formulations for about 2.9 hours t _1/2, about 1820 ng / in ml C _maximum, and / or two of the dose containing about 4050 ng hr / mL 50 mg / M ^2, with the AUC of Provide PK for time injection. Preferably, the formulation is 2 hours at a dose comprising 58 mg / M ² having a t _1/2 of about 3.2 hours, a C _peak of about 1460 ng / ml, and / or an AUC of about 3290 ng hr / mL. Provide PK for injection.

Table VIII shows the accumulation of data in Tables V, VI and VII with increasing number of patients at certain doses as indicated in Table VIII. Another aspect relates to formulations comprising an aqueous organic acid, its conjugate salt, a compound of Formula (II), which, upon IV injection into a human, provide the PK profile shown in FIGS. 2A, 2B, and / or 2C. Preferably, the formula comprises a molar ratio of 1 mole of the compound: 8.8 moles of the organic acid: 3.7 moles of the conjugate salt of the compound of formula II: organic acid: the conjugate salt. Preferably two of the capacity of the art formulations comprises from about 2,29 times t _1/2, from about 91 ng / ml of C _{up to,} and / or 1.85 mg / M ² has a AUC of about 211 ng hr / mL Provide PK for time injection. Preferably 2 hour infusion of the capacity to the art formulation comprises from about 2.75 time of t _1/2, from about 136 ng / ml of C _{up to,} and / or 3.7 mg / M ² having an AUC of about 336 ng hr / mL Provide a PK for. Preferably 2 hour infusion of the capacity to the art formulation comprises from about 2.86 time of t _1/2, from about 353 ng / ml of C _{up to,} and / or 7.4 mg / M ² having an AUC of about 972 ng hr / mL Provide a PK for. Preferably 2 hour infusion of the capacity to the art formulation comprises from about 2.78 time of t _1/2, from about 396 ng / ml of C _{up to,} and / or about 1050 ng hr / mL 14.8 mg / M 2 having an AUC of Provide a PK for. Preferably 2 hour infusion of the capacity to the art formulation comprises from about 2.98 time of t _1/2, from about 890 ng / ml of C _{up to,} and / or about 2430 ng hr / mL 29.6 mg / M 2 having an AUC of Provide a PK for. Preferably 2 hour infusion of the capacity to the art formulation comprises 41.4 mg / M ² has a C _maximum, and / or AUC of about 2890 ng hr / mL of t _1/2, about 1300 ng / ml for about 2.8 hours Provide a PK for. More preferably about 2 hour infusion of a dose containing 50 mg / M ² has a C _maximum, and / or AUC of about 4090 ng hr / mL of t _1/2, about 1820 ng / ml for about 2.9 hours Provide PK. Preferably the art formulations 2 hour infusion of a dose containing about of 3.2 hours t _1/2, about 1460 ng / ml of C _{up to,} and / or about 3300 ng hr / mL 58 mg / M 2 having an AUC of Provide a PK for.

In some embodiments, the solution containing the compound of formula (II) contains an organic acid and its conjugate salt, until the desired amount of the compound of formula (II) is dissolved and the resulting solution is watered until the desired pH is obtained. It is preferable to prepare the concentrate by titrating with carboxylic acid and an aqueous base and then diluting the buffer with water or an aqueous solution such as saline to achieve the desired concentration of the compound of formula II. In one preferred embodiment, the buffer has a pH of about 3 to about 5. In some embodiments, it is preferred to use the solution to prepare concentrates from organic acids and conjugate salts selected from citric acid / sodium citrate and lactic acid / sodium lactate.

In one aspect, the present invention is directed to administering a concentrate, a diluted form of the concentrate, to a mammal in need of treating or managing a disease state, and thus preferably a Normal Saline USP IV bag, preferably a standard. By mixing the contents of a 250 ml saline IV bag with a concentrate, the treatment or management of a disease state that provides a medicament suitable for intravenous infusion. In some embodiments, it is desirable to mix sufficient amounts of the concentrate to provide about 0.92 micrograms of saline solution / compound of formula II to about 372 micrograms / salt solution of formula II.

In some embodiments, the amount of the diluted concentrate is about 0.33 mg / m ² to about 58 mg / m ² , preferably about 0.33 mg / m ² to about 58 mg / m ² , more preferably a compound of Formula II. Preferably, the compound of formula (II) is administered to a mammal in need of treatment equivalent to about 50 mg / m ² to about 58 mg / m ² . In some embodiments, it is preferred to administer about 50 mg / m ² of the compound of Formula II. In some embodiments, it is desirable to administer the diluted concentrate by IV infusion.

In some embodiments, it is preferred to administer by IV infusion the amount of diluted concentrate necessary to provide the desired amount of the compound of Formula II over a period of about 1 hour to about 24 hours. In some embodiments, it is preferred to administer by IV infusion comprising a dose of about 0.33 mg / m ² of a compound of Formula II to about 58 mg / m ² of a compound of Formula II. It is preferred to administer the infusion over a period of about 2 hours. In some embodiments, provide a dose of 14.8 mg / m ² of compound of Formula II at an infusion rate that yields a steady state plasma concentration of about 58 ng / ml, preferably at a rate that delivers the diluted concentrate over 24 hours It is desirable to administer the amount of diluted concentrate necessary to administer to the mammal in need of treatment.

In certain preferred embodiments, the compound of formula II is administered in one or more cycles at the desired dose density. In one preferred embodiment, the compound of formula (II) is administered once a week for three weeks and is administered in a 28-day cycle comprising one week off. In another preferred embodiment, the compound of formula II is administered in a 21-day cycle which comprises administering from 1 day to 20 days.

1 shows a graph measuring the concentration (ng / ml) vs. hours profile for a 24-hour infusion of a solution comprising 14.8 mg / m ² of a compound of Formula II.
FIG. 2A shows a human patient who obtained one of nine different 2 hour IV infusion-administered doses of the formulation of the present invention over an amount of 12. 3 mg / m ² to 41.4 mg / m ² A graph showing the volunteer's C _highest and average concentration / time profile is shown.
FIG. 2B shows a human patient who obtained one of nine different 2 hour IV infusion-administered doses of the formulation of the present invention over an amount ranging from 0.33 mg / m ² to 41.4 mg / m ² of the compound of Formula II over 24 hours. A graph showing the average concentration / time profile of volunteers is shown.
FIG. 2C shows a human patient who obtained one of eight different 2 hour IV infusion-administered doses of the formulation of the present invention in an amount ranging from 1.85 mg / m ² to 58 mg / m ² of the compound of Formula II over 12 hours A graph showing the average concentration / time profile of volunteers is shown.
FIG. 3 shows exposure in healthy human volunteers for a series of doses of the formulation of the invention containing an amount of a compound of Formula II in the range of 0.33 mg / m ² to 41.4 mg / m ² administered with a 2 hour IV infusion. Show the graph.
4 shows a graph showing the effect of various dose regimens of compounds of Formula II on tumor volume inhibition in A549 NSCLC mouse xenograft model.
FIG. 5 shows a graph showing the binding affinity of compounds of Formula II to cyclin A / CDK2 complexes.
FIG. 6 shows a graph showing the inhibitory effects of compounds of Formula II in A2780 mouse xenograft egg cancer model.
FIG. 7 shows a microscope of typical hair follicles recovered from nude mice exposed to doses of the compound of Formula II, showing the suppression of phospho-Rb in proliferated epithelial cells of basal epithelium and hair follicles.
FIG. 8 is a graph showing concentration / time profiles for patients administered with a 2 hour IV infusion of a compound of Formula II in an amount of 1.85 mg / m ² as observed on days 1 and 15 of the 28 day dose cycle. Indicates.
9A and 9B show individual patient PK profiles over 24 hours after obtaining IV infusion doses of compounds of Formula II in amounts of Formulas 29.8 mg / m ² and 41.4 mg / m ² , respectively.
10 shows a graph showing the effect of various dose levels of a compound of Formula II on BrdU incorporation in an ex vivo blood sample.
FIG. 11 shows a chart showing decreasing levels of biomarkers after obtaining 6 cycles of IV infusion doses of compounds of Formula II in patients diagnosed with pseudomyxoma peritonei.
12A and 12B show graphs showing the effect of various dose levels of a compound of Formula II on BrdU incorporation in ex vivo blood samples.
FIG. 13 is a graph showing the inhibition of BrdU incorporation 8 hours after exposure to various dose levels of a compound of Formula II in an ex vivo blood sample.

As mentioned above, compounds of formula II (as defined herein) have activity as useful pharmaceutical compounds with cyclin-dependent kinase inhibitory properties. In particular, the inventors have surprisingly found that compounds of formula (II) provide therapeutic levels when administered as IV formulations. Vitro the CDK2, and the drug affinity (IC ₅₀₎ calculated for 1nM having a drug affinity (IC ₅₀₎ calculated for CDK1, 1nM having a drug affinity (IC ₅₀₎ calculated for the compound of formula II 4nM in The calculation shows inhibition of cyclin-dependent kinases with CDK9 having a calculated drug affinity (IC ₅₀ ) of CDK5, 4 nM. In addition, the inventors have surprisingly found the inhibition of CDK2 / CDK1 in tumor cells causing cell cycle arrest and apoptosis in more than 100 tumor cell lines comprising standard NCI panels. In addition, the inventors have found exposure of xenograft models to plasma concentrations below 25 nM of compounds of Formula II which surprisingly cause tumor growth inhibition and regression for less than 2 hours. The inventors surprisingly find that formulations containing a compound of formula II suitable for IV administration to a human can be prepared comprising a buffered aqueous solution of a compound of formula II to achieve advantageous plasma levels for the treatment or management of a proliferative disorder. Revealed. The formulations comprising the compounds of formula II, the methods of treating benign and proliferative disease states using the formulations of the invention containing the compounds of formula II, and examples are described in more detail below.

Chemical formula II Containing compounds of IV  Preparation of the Formulation

One aspect of the invention is a formulation comprising a compound of formula II suitable for intravenous (IV) administration. Compounds of formula (II) are soluble in acidic media but poorly soluble in neutral and basic media. The inventors surprisingly dissolve the compound of formula (II) in an acidic solution prepared by dissolving an organic acid in water suitable for insertion into a buffer system, and then titrating a solution of the compound of formula (II) and the dissolved organic acid to adjust the pH. It has been found that as long as the appropriate base and the appropriate acid can be used to produce the desired final pH.

Suitable acids can provide aqueous solutions having a pH more acidic than about pH 7.0, preferably at least pH 3.5, and when titrated with a suitable base, a buffer having a pH ranging from about pH 7 to about pH 2.5 Any organic acid that can provide, preferably a buffer of about pH 3.5. Preferably, the selected organic acid is freely water soluble, for example lactic acid or citric acid, and more preferably the selected organic acid has more than one acidic proton, for example citric acid.

Compounds of formula (II) that are poorly soluble in neutral water are readily soluble in aqueous acid solutions. Thus, in some embodiments, in the preparation of the formulations of the invention, an acid solution comprising an aqueous acid solution, preferably at least about 0.111 M of the selected acid, is initially prepared, wherein the conjugated salts, such as sodium citrate and It is preferable to dissolve the sodium lactate and then dissolve an aliquot of the compound of formula II in the resulting buffer. After dissolution is complete, the resulting solution is titrated to prepare the solution using an amount of acid, such as hydrochloric acid, and a base, such as sodium hydroxide, in an amount necessary to prepare the desired pH or within the desired pH range. to achieve pH. Preferably, the solution is more basic than about pH 2.5, more preferably a solution having a pH of about pH 3 to about pH 5. In one preferred embodiment, the solution has about pH 3.0 to about pH 4.2, more preferably about pH 3.4 to about 3.6. Once the desired pH is reached, the volume of the buffered acid solution containing the API is adjusted to one or more aliquots of water to prepare the solution to yield a solution that provides about 5 mg / ml of API. It will be appreciated that suitable solutions may be prepared under sterile preparation conditions or may be sterilized after preparation.

Formulations of the present invention may be prepared without departing from the formulations of the present invention, with a desired concentration of API, a pH of about pH 2.5 to about pH 7.0, preferably about pH 3 to about pH 5, more preferably about pH 3.0 to about It will be appreciated that other procedures may be used to produce a buffered acidic aqueous solution having a pH of 4.2.

How to use

In one embodiment, the formulations of the present invention are concentrated to be diluted as needed by adding the concentrate into a standard IV bag containing 250 ml of 0.9% saline in an amount that provides milligrams of the desired number of APIs resulting in treatment. Formulation. It is within the scope of the present invention to adjust the concentration of the formulation as necessary to provide a sufficiently concentrated formulation in the API to accommodate the amount of concentrate providing 250 mg of the desired number of API in the free space of the 250 mL saline bag. Preferably, however, if the concentrate contains about 5 mg / ml and an amount of excess is acceptable in the free space of the 250 mL saline bag, the amount of concentrate required is divided into aliquots and various saline solution for administration to the patient. Is applied to the bag. It will be appreciated that the pH will change as the amount of concentrated solution introduced into the saline bag is small and large. Preferably, the pH of the injectable solution is between pH 3.4 and pH 7, more preferably between about pH 3.4 and about pH 5. More acidic solutions can be obtained at high dilution levels of the concentrate due to the lowest buffering capacity of the concentrate with increasing dilution as will be appreciated. It will be appreciated that any pH value appropriate for the physiology of the patient can be used to control the infusion time as long as possible to prevent or minimize damage to the vein into which the composition is infused.

In another embodiment, a formulation of the present invention provides an IV injectable solution containing a sufficient amount of a compound of Formula II, when infused into a patient, to provide a therapeutic level of a compound of Formula II in a patient. A sufficient amount aliquot of the concentrate described above diluted in amounts.

Without being bound by theory, but 58 mg / m may be administered as desired in an amount of greater than ² and less than, believed to typically administered compound of about 58 mg / m ² or less of the formula II to a patient is need of treatment. Preferably, about 50 mg / m ² to about 58 mg / m ² of the compound of Formula II are administered to a patient in need of treatment. In one preferred embodiment, about 50 mg / m ² is administered to the patient in need of treatment.

The total amount of concentrate diluted in saline solution is injected into the patient over a period of time, typically from about 2 hours to about 24 hours. In some embodiments, it is desirable to infuse the entire amount of the formulation to the patient over two hours. In another embodiment, it is preferred to infuse the entire amount of the formulation to the patient over 8 hours. In another embodiment, to enable longer steady-state plasma levels of the API, it is desirable to infuse the patient with the full amount of the formulation over 24 hours. In some embodiments, it is desirable to administer from about 50 mg / m ^2/3 Note capacity density. In some embodiments, it is desirable to administer the dose density for a 28-day cycle, ie, a 16.7 mg / m ² dose for 3 weeks on a weekly basis and for a week off. In some embodiments, it is desirable to administer the dose density for a 21-day cycle, ie 50 mg / m ² single dose every three weeks (ie, up to 20 days after a daily dose). In another aspect, it is preferred that the administration of the art capacitance density usually about 58 mg / m ^2/3. In some embodiments, it is desirable to administer the dose density for a 28-day cycle, ie, 19.3 mg / m ² doses for 1 week to 1 week for 3 weeks and one week off. In some embodiments, it is desirable to administer the dose density as a 21-day cycle, i.e., a 58 mg / m ² single dose every three weeks (ie, one day followed by 20 days). More or less amount can be used. Dosing cycles that provide a controlled dose, for example, a 28-day cycle comprising 3 weeks once a week and taking 1 week off, or a 21-day cycle comprising administering from 1 day to 20 days Can be used to provide the desired capacity density.

In general, due to the important role of CDK in the regulation of cell proliferation, new formulation (s) containing a CDK inhibitor of formula (II) are for example solid tumors such as skin, such as melanoma , Breast, eg breast cancer, brain, colon and colorectal cancer, prostate cancer, eg prostate cancer, gallbladder, thyroid, cervical cancer, testicular cancer, vulva cancer, peritoneal pseudomyxoma, peritoneal cancer, ovarian cancer, non-small cell It is believed to act as a reversible cell proliferative agent useful in treating any disease process characterized by abnormal cell proliferation, eg, tumor growth, in treating cancers including lung cancer (NSCLC) and the like. More particularly, cancers that can be treated with the compounds, compositions, and methods of the present invention include, but are not limited to, heart : sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyosarcoma, fibroma, lipoma and teratoma; Lung : tracheal support carcinoma (squamous cell, undifferentiated cell, undifferentiated large cell, adenocarcinoma), alveolar (tribronchial) carcinoma, bronchial adenocarcinoma, sarcoma, lymphoma, chondroma, mesothelioma; Gastrointestinal tract : Esophagus (squamous cell carcinoma, adenocarcinoma, leiomysarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyoma), pancreas (tubular adenocarcinoma, insulinoma, glucagon, gastrin, carcinoid, vip) Small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroid), large intestine (adenocarcinoma, thyroidoma, chorionic adenocarcinoma, leiomyoma, leiomyoma); Urogenital tract : Kidney (adenocarcinoma, Wilm's tumor) (renal blastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testicle (normal Carcinoma, teratoma, germ carcinoma, teratoma, chorionic carcinoma, sarcoma, stromal cell sarcoma, fibrosarcoma, fibrosarcoma, glandular tumor, lipoma); Liver : Liver tumor (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, hemangiosarcoma, hepatocellular adenoma, hemangioma; Bone : Osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (net cell sarcoma), multiple myeloma, malignant giant cell tumor stromal sarcoma, osteochondroma (osteochondral cartilage) Sex exosteoma), benign chondroma, chondroma, chondrocyte-like fibroblasts, osteomyomas and giant cell tumors; Nervous system : skull (osteomas, hemangiomas, granulomas, yellowomas, degenerative osteoarthritis), meninges (pathological meningiomas, meningiosarcoma, glioma), brain (astrogliomas, medulloblastoma, glioma, ventricular cell tumor, seed cell carcinoma) Glioblastoma multiforme, oligodendrocyte glioma, glioma, retinoblastoma, congenital tumor), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecology : Uterine (endometrial carcinoma), Cervical (cervical carcinoma, pre-tumor cervical dysplasia), Ovarian (ovarian carcinoma (serum cystic carcinoma, myxedema cyst, unclassified carcinoma), granulosa-follicular cell tumor, Sertoli lei Dih's tumor, undifferentiated cell carcinoma, malignant teratoma, vulva (squamous cell carcinoma, epithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (transparent cell carcinoma, squamous cell carcinoma, staphylosarcoma), fallopian tube ( Carcinoma); Blood : blood (myeloid leukemia (acute and chronic), acute lymphocytic leukemia, acute and chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (Coat cell lymphoma), B-cell lymphoma, T-cell lymphoma, hairy cell lymphoma, Burkett's lymphoma, promyelocytic leukemia; skin : malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma , Moles dysplastic nevus, lipoma, hemangioma, Cutaneous fibroids, keloids, psoriasis, adrenal glands , neuroblastomas, and other cancers , such as, for example, pigmentary dry skin, keratinous blastoma and thyroid vesicle cancer.

In some embodiments, formulations of the invention comprising a CDK inhibitor of Formula (II) can be used for non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, acute leukemia (eg, acute myeloid leukemia (AML) or acute lymphocytic leukemia (ALL) )], Chronic leukemia (eg CLL), non-Hodgkin's lymphoma (eg MCL), melanoma, and multiple myeloma.

In one embodiment, a formulation of the invention comprising a CDK inhibitor of Formula (II) is administered to a patient over 8 hours. Without limitation, for example, the dosage range of the formulation of the invention comprising a CDK inhibitor of Formula II during an 8 hour infusion period can range from about 1.85 mg / m ² to about 70 mg / m ² and about 15 mg / m ² To about 30 mg / m ² . In some embodiments, the dosage is administered as a 21 day cycle (administered from 1 day to 20 days).

In another embodiment, a formulation of the invention comprising a CDK inhibitor of Formula (II) is administered to a patient over 24 hours. Without limitation, for example, the dosage range for 24 hours can range from about 1.85 mg / m ² to about 100 mg / m ² , about 50 mg / m ² to about 80 mg / m ² , and about 30 mg / m ² To about 70.0 mg / m ² . In some embodiments, the dose is administered as a 21 day cycle (administered from day 1 to day 20).

In other embodiments, the solid tumor (eg, breast, melanoma, ovary) is treated by the methods described herein. Without limitation, for example, in this embodiment, a dosage of about 1.85 mg / m ² to about 58 mg / m ² (eg, 20 mg / m ² , of a formulation of the invention comprising a CDK inhibitor of Formula II) 30 mg / m ² , 40 mg / m ² , 50 mg / m ² ) are administered on a 21 day cycle (ie, administered from 1 day to 20 days). Dosages may be infused over 2 hours, 8 hours or 24 hours.

In alternative embodiments, solid tumors (eg, breasts, melanoma, ovaries) are treated by the methods described herein in combination with growth factors (eg, GCSF and GMCSF). Without limitation, for example, the dosage range of a formulation of the invention comprising a CDK inhibitor of Formula II can range from about 0.33 mg / m ² , and from about 100 mg / m ² to a 21 day schedule (1 day followed by 20 days). Administered up to). Dosages may be infused over 2 hours, 8 hours or 24 hours.

In another embodiment, from about 1.85 mg / m ² to about 100 mg / m ² , and about 40 mg / m ² to about 70 mg / m ² of the dosage form of the formulation comprising a CDK inhibitor of Formula II is 21 days Cycle (ie, administered from 1 day to 20 days). Dosages may be infused over 2 hours, 8 hours or 24 hours.

In another embodiment, a dosage of about 0.33 mg / m ² to about 30 mg / m ² , about 0.33 mg / m ² to about 14 mg / m ² or about 7 in a dosage form of the formulation comprising a CDK inhibitor of Formula II mg / m ² to about 12 mg / m ² are administered on a 28 day cycle (ie, a dose of 1 week off after 3 weeks of dosing once a week). Dosages may be infused over 2 hours, 8 hours or 24 hours. In some embodiments, the hematological cancer is treated. In other embodiments, the solid tumor is treated.

As used herein, cancer treatment includes the treatment of cells, such as cancer, including cells corresponding to any one of the conditions indicated above.

In addition, the formulations of the present invention may be useful for chemoprevention of cancer. Chemoprevention is defined as inhibiting the progression of invasive cancer by blocking the onset of mutagenicity, or by blocking the progression of pre-malignant cells that have already occurred or by inhibiting tumor recurrence. The formulations of the present invention may also be useful for inhibiting tumor angiogenesis and metastasis.

Example

A number of examples describing the formulations and methods of treatment of the present invention are described below. All animal experiments were performed according to pharmaceutical industry standards and good laboratory practice. All reagents used in the preparation of the formulation were USP or Food Grade.

Example  1: chemical formula II Of formulations containing compounds of

400 kg water was placed at ambient temperature (between 20 ° C. and 30 ° C.) into a 500 L stainless steel vessel equipped with a stirring paddle. Citric acid (10,682 g) was dissolved therein with stirring. The vessel was stirred for 10 minutes while maintaining the ambient temperature, then the addition apparatus was washed with 0.2 kg additional water that had been added to the solution. When citric acid dissolution was completed, stirring was continued and 5,710 g of sodium citrate was added to the solution and stirred until the solution dissolved while maintaining the ambient temperature. The addition apparatus was washed by adding 0.2 kg water added to the citric acid / sodium citrate solution. The temperature of the solution was adjusted at a temperature between 20 ° C. and 30 ° C. and then maintained. 2,585 g of compound of formula II (96.7% activity, API, compound of formula II prepared according to the method described in the above mentioned '756 publication) were added into the solution with stirring. Stirring was continued until all added API dissolved in the citric acid / sodium citrate solution. The addition apparatus was washed in solution by adding 1.2 kg of water. Upon completion of dissolution, the volume of the resulting solution was adjusted to 490 kg by addition of water, then the pH was adjusted by titration with 1 M sodium hydroxide solution, and 0.1 M HCl solution to give a final pH 3.5. The volume of the buffer solution was adjusted to 507 kg by adding water.

Example  2: Study of xenograft cancer model tumors in nude mice

The efficacy of the formulations comprising the compound of Formula II prepared in Example 1 was investigated in A2780 xenograft ovarian cancer model. For all experimental models, the maximum inhibited dose was defined as the dose over a given period of time, which resulted in a 20% weight loss in the subject mouse.

In carrying out this study, A2780 cells were seeded subcutaneously into nude mice and tumors were grown until the mice had a volume of about 100 mm ³ (about 7 days). Ten groups of these test animals were 8 mg / kg (13% maximum inhibited dose (MTD)); 16 mg / kg (27% MTD); 32 mg / kg (53% MTD); And IP injections daily for 10 days with the formulations prepared in Example 1 at a dosage level of 48 mg / kg (80% MTD). A solution of paclitaxel was IP injected into the control group at 20 mg / kg dose level (50% MTD) twice a week with the control group injected in a formulation that did not include API. The results of the inhibition of tumor growth are shown in FIG. 6. FIG. 6 shows that the control group had tumor growth in a volume of about 800 mm ³ over a 10 day period, whereas animals that were dosed with paclitaxel had tumor growth inhibited to 63%. Groups of mice that obtained formulations comprising the compound of Formula II were 70% (both 8 mg / kg and 16 mg / kg dose levels), 90% (32 mg / kg dose levels), and 96% (48 mg / kg) kg dose level) tumor growth inhibition. These data show that the 50% inhibition level (which defines the minimum effective amount in that model) is less than about 8 mg / kg. In addition, animals exposed to the maximum dosage level of the compound of formula II exhibited weight loss over the experimental route of about 5%, demonstrating that the formulation is well inhibited.

The formulation is administered to BALB / c mice for 5 days at a dosage level of 40 mg / kg once per day. Blood samples were obtained on day 1 and 7 followed by final doses and then analyzed on an Advia 120 hematology analyzer immediately after dilution to 1: 5 in PBS. All different blood counts were performed, including erythrocyte analysis (including reticulocytes, parameter calculations, and hemoglobin analysis), leukocyte analysis (including different lineage counts and peroxidase staining), and plateletogenesis analysis. No effect on platelets or erythrocytes was observed.

In a related study, an escalating dose of a formulation prepared in Example 1 and containing a compound of Formula III (flavopyridol) was administered once daily for 7 days to nude mice containing A2780 ovarian carcinoma xenografts. Was administered. For this study, the maximum inhibited dose considered the dose level inducing 20% weight loss over the duration of the experiment, and the minimum effective amount (MED) was defined as the dose level inducing at least 50% inhibition of tumor growth. The results are shown in Table I, which reports various treatment indices for each compound. The data in Table I show that compounds of Formula III are insufficiently inhibited by obtaining a nominal therapeutic index (MTD / MED) of less than 1, while compounds of Formula II are well inhibited and are effective at significantly lower doses below MTD, and greater than 10 To obtain a nominal therapeutic index of.

[Formula III]

TABLE I

Referring to FIG. 4, nude mice containing A549 NSCLC xenografts and established in volumes greater than 100 mm ³ were (a) 20 mg / kg once daily for 13 days; (b) 29 mg / kg for 3 days followed by 3 days of 2 days off; (c) 52 mg / kg on days 1, 4, 7, 10, and 13; (d) 17 mg / kg twice a day (equivalent to 52 mg / kg in 24 hours) on days 1, 4, 7, 10, and 13; And (e) infusion at a dose equivalent to 260 mg / kg of compound of Formula II using the formulation prepared in Example 1 over five different schedules representing 87 mg / kg on Days 1, 7, and 13 It was. As shown in FIG. 4, degeneration in tumor volume and mass was observed on all dosing schedules. Similar studies were performed using mice containing xenografts on tumor type models of RPMI 8226, A549 NSCLC, NCI H460, PC3 (prostate), H82 (SCLC), SW527 (breast), and MDA MB231 (breast). The results are presented in Table II below in comparison to the reference cytotoxic agents shown.

[Table II]

Nude mice were injected intraperitoneally with a single dose of the formulation prepared in Example 1 equivalent to 40 mg / kg. Skin samples were collected at various intervals after dosing and compared to biopsies collected prior to dosing (T0). Biopsied samples were fixed overnight in 10% formalin. The samples were then placed in 70% alcohol for storage, embedded in paraffin blocks, sectioned and stained with phospho-Rb Ser 807 / 811-specific antiserum.

As shown in FIG. 7, which shows a typical sample (dose after T2) and (dose after T4) obtained over several hours, the sample obtained at post-administration of the formulation contains the epithelial cells in which the formulation has grown in the basal epithelium and hair follicles. Induction of rapid and sustained inhibition of phospho-Rb.

Referring to FIG. 4, nude mice containing xenografts also had a single daily dose (20 mg / kg) at a rate of 87 mg / kg once every 6 days, single dosing followed by two days of dosing for three days. Dose (29 mg / kg single dose), single dose every three days (52 mg / kg, single dose), 17 mg / kg dose three times per day, repeated every three days, and one given every six days The dosing schedule was divided into 87 mg / kg doses. Tumor degeneration was observed in animals treated with the compound of formula II compared to the control. The extent of tumor degeneration was similar to all dosing schedules investigated.

Example  3: chemical formula II After a short exposure to the compound, many human tumor cell lines Thymidine  Insert or Alama blue Of alamar blue viability  Inhibition and cell Self-defeating  Confirm

Referring to Table 4, many human tumor cell lines were exposed to increasing concentrations of the compound of formula II for 24 hours, followed by inhibition of thymidine insertion (adhesive cells) or Alamar Blue viability assay (suspension cell lines). Was evaluated. In addition, the activity level of apoptosis was assessed by determining the level of caspase activated in each cell line. In each experiment, the cells prepared various batches of media containing different amounts of the compound of formula II and then exposing different batches of cells to the media containing the compound of formula II at each particular concentration. The cell medium was exposed to increasing concentrations of the compound of formula II for 2 hours. After removal of the medium, cells were re-cultured in complete medium without the compound of formula II. Cells were evaluated by fluorescence for levels of activated caspase 6 hours after washing. Samples of each cell line were also subjected to SDS-PAGE, followed by immunoblotting against the p85 PARP caspase cleavage product. The detectable activity of caspase and the accumulation of p85 PARP were judged as positive apoptosis results when observed at concentrations of less than 0.1 micromole of the compound of formula II after 2 hours of cell line exposure.

These data are shown in Table III. IC ₅₀ values were calculated using the procedure described in the above-referenced '200 patent (Table 87 herein). The data in Table III shows that all tumor types that have experienced the cell cycle have stopped at least a portion of the different cell lines tested. In addition, the tumor types tested showed no specific sensitivity or resistance to the compounds of Formula II. In addition, these data show that caspase activity and apoptosis were detected in at least 85% of the cell lines tested after a single exposure to the compound of Formula II. It should be mentioned that about 50% of the NCI-60 screening set is positive for MDR1 (multiple drug resistance gene 1) gene expression.

[Table III]

The activity of apoptosis after a short exposure time of A2780 cells to the compound of formula II was determined by exposing the A2780 ovarian cancer cell culture to increased concentrations of the compound of formula II dissolved in growth medium for 2 hours, after which the cells were removed. These were then identified by re-incubation (wash period) for 6 hours in complete medium without the compound of formula II. After a 6 hour wash period, cell extracts were prepared and evaluated by fluorescence analysis for activated caspase (RFLU). In this study, caspase activity was reproducibly detected after 2 hours exposure to compounds of formula (II) which were present at less than 0.05 micromolar concentration. It was found that there was no increase in caspase activity when compared to cells treated with higher concentrations (up to 5.0 micromoles) of the compound of formula II. These data show that caspase activity is maximal at an exposure level of 0.05 micromolar.

To confirm that the 2 hour exposure profile was sufficient to induce a sustainable effect on cell cycle distribution, asynchronous growth A2780 cells were 0.001 in 16 batches, each of which approximately doubled the concentration of the compound of formula II over the previous batch concentration. It was exposed to the concentration of compound of formula II dissolved in growth medium for 2 hours in the range of Michaelomol to 30 micromol. In each experiment, after exposure, the cells were washed and then re-incubated for 24 hours in growth medium without the compound of formula II, followed by a pulse of BrdU for 30 minutes to indicate the percentage of cells that experienced active DNA replication. Was established. Cells were then fixed and stained with FITC-conjugated, anti-BrdU-monoclonal antibodies and propidium iodide. Cells thus prepared were analyzed by doublet-discrimination using a two-color flow cytometer. The study showed that at exposures greater than 0.5 micromolar, the compound of Formula II completely inhibited DNA synthesis 24 hours after exposure. In addition, samples exposed to micromolar concentrations greater than 0.5 showed accumulation of subG1 (associated with apoptotic cells). Elevated exposures of concentrations greater than 0.5 micromolar, up to 30 micromolar did not increase the apoptotic phenotype.

Evaluation of the mechanical effect of short exposure of the compound of formula II was carried out by exposing the asynchronous growth A2780 cells to growth medium containing 0.1 mM of the compound of formula II for 2 hours, washing the cells, and then treating the cells with the compound of formula II It was performed by re-culture with a medium that does not contain. After washing, lysates were prepared from the cells to assess the duration of the exposure effect. Samples were separated on SDS-PAGE and then immunoblotted with anti-serum specific for hypo-phosphorylated retinoblastoma protein and p85 PARP to assess the activity of CDK inhibition and trigemination. Two hours exposure of cells to 0.1 mM of compound of formula II was sufficient to induce RB phosphorylation and inhibition of caspase activity for a period of six hours after the wash period.

Asynchronous growth A2780 cells were exposed to increasing concentrations of the compound of formula II or compound of formula III for 16 hours. Cell lysates were isolated on SDS-Page and then immunoblotted with rabbit polyclonal antiserum specific for the CDK phosphorylation site (Ser 07/811) on retinoblastoma tumor-inhibitory protein (RB, known CDK substrate). It was. In addition, the accumulation of p85 PARP product was monitored to assess the correlation between caspase activity and the beginning of apoptosis and inhibition of CDK. In connection with the studies described above in the cellular data evaluating the inhibition of thymidine insertion (IC _{50 of} 4 nM), the formulation comprising the compound of formula II inhibited phospho-Ser 807/811 after exposure. Phospho-Ser 807/811 was undetectable in lysates exposed to concentrations greater than 6.25 nM in compounds of Formula II, and complete inhibition of detectable RB phosphorylation was required at the beginning of apoptosis known as detectable p85 PARP. Was observed. In comparison, exposure to a compound of formula III (flavopyridol) requires a concentration of about 1 micromole to induce a detectable effect on phospho-Ser 807/811 levels, while significantly inhibiting RB phosphorylation Did not induce. At the concentration levels below that required to observe inhibition of RB phosphorylation, apoptosis activity was observed. In addition, there was a poor correlation between IC ₅₀ / EC ₅₀ for the compounds of Formula III in a number of intracellular assays of mechanisms based on thymidine insertion and biomarkers such as phospho-RB. Was observed.

Table IV shows that compounds of Formula II are selective for the CDK system of Ser / Thr kinase, as determined by Upstate kinase Profiler counter-screening, while compounds of Formula III are c -Src has more general activity, including inhibition of tyrosine kinase.

TABLE IV

5 also shows that the compounds of formula II bind with high affinity for the cyclin A / CDK2 complex.

Example  4: man At the applicant  Research

The formulations prepared in Example 1 were diluted in 250 ml standard 0.9% saline IV bags and then injected into volunteer human cancer patients. The contents of the IV bag were injected over 2 hours. The dosage is 0.33 mg / m ² , 0.66 mg / m ² , 1.32 mg / m ² , 1.85 mg / m ² , 2.59 mg / m ² , 3.63 mg / m ² , 5.08 of the compound of formula II for the infused patient. It was prepared using an amount of formulation providing a dose of mg / m ² , 7.11 mg / m ² , 10.00 mg / m ² , 12.00 mg / m ² and 14.00 mg / m ² . Each patient was dosed on a 28-day cycle, including once weekly infusions during the first three weeks of the cycle and no doses after the third dose on days 1, 8, and 15 of each cycle. Plasma samples were collected before initiation of infusion and then at 1 hour, 2 hours (completion of infusion), at 2.25, 2.5, 3.0, 3.5, 4.0, 5.4, 6.0, 8.0, and 24 hours after initiation of infusion. In addition, volunteer human cancer patients received 1.85 mg / m ² , 3.7 mg / m ² , 7.4 mg / m ² , 14.8 mg / m ² via 2 hour IV infusion once every 3 weeks (ie, 21 day cycle). , 29.6 mg / m ² , 41.4 mg / m ² , 50 mg / m ² , and 58 mg / m ² . The PK and AUC results obtained were similar to the 21-day dosing schedule and the comparable 28-day dosing schedule described above.

Referring to Figures 2A, 2B, and 2C, the plasma concentrations in ng / ml of the compound of Formula II after the start of the 2 hour infusion for the various dose levels shown for each infusion, the C _highest is after the start of the infusion. It is achieved between 1 hour and 2 hours. When the injection is completed, it is possible to observe the range of from about 1.4 to quickly removed from the plasma by T _1/2 of about 3.3 hours to the compounds of formula II depends on the dose in each case.

Referring to Figure 3, one day of the 28 days dosing period (diamond), and 15 days shows the ^{AUC (ng 'hr / mL)} / dose (mg / m ²⁾ for the various dose administered to the (square), which is exposed A dose-distribution increase in is observed for doses between 0.33 mg / m ² and 41.4 mg / m ² , with clearance appearing to be dose independent. In addition, FIG. 2B shows that a plasma level of 25 nM (obtained at a plasma concentration of 10 ng / mL) is available from infusion for 2 hours at a dose greater than 1.32 mg / m ² .

Referring to FIG. 8, mean AUC data is shown for patients administered at 1.85 mg / m ² on days 1 and 15 of the 28 day dosing schedule, and the AUC data for each dose during the cycle is similar. The AUC ratio expressed as AUC-15 days / AUC-1 days was 1.09. In addition, it was found that at day 1, 8, and 15 of cycle 1, and day 1 of cycle 2, plasma concentrations were similar, indicating constant exposure to dose regimen. In addition, it appears that drug accumulation is not certain using this regimen.

1 shows the calculation of the concentration / time profile for a 24 hour infusion of a 14.8 mg / m ² dose of a compound of Formula II, which extends the infusion time up to 24 hours or less, whereby steady state plasma concentrations begin infusion. It is expected to be established within 2 hours and extend over the infusion period. Thus, by changing the IV infusion time, the PK profile of the compound of formula II in the formulation of the present invention can be altered. In this case, the therapeutically effective plasma levels are maintained for an extended period of time.

The PK parameters observed in this study are summarized in Tables V, VI, and VII. Specifically, the data shown in FIGS. 2A and 2B correspond to Table V and the data shown in FIG. 2C corresponds to Tables VI and VII. These data alleviate PK robust internal-object diversity (C _highest and AUC) and relatively low internal-object diversity (about 30%) at doses from about 0.33 mg / m ² to about 58 mg / m ² . It has been shown to mitigate. The observed t _1/2 was between about 1.4 hours and about 3.3 hours.

Table VIII shows the accumulation of data in Tables V, VI and VII with an increase in the number of patients at certain doses as shown in Table VIII.

TABLE V

Table VI

TABLE VII

TABLE VIII

Blood samples, 0.33 mg / m ² ; 0.66 mg / m ² ; And initially 2, 3, 4, 6, and 8 hours post infusion from patients infused (2 hours) at a dose level of a compound of Formula II in an amount equivalent to 1.85 mg / m ² . Samples were collected in tubes containing heparin and then sent to a central laboratory for evaluation. Whole blood obtained from each sample was diluted 1: 5 in complete RPMI medium, and then 200 aliquots of aliquots were added to the wells of a 96-well plate. From each time point the sample was stimulated or not stimulated with PHA (0.01 mg / ml final concentration). Sample well plates were incubated for 48 hours at 37 ° C. under 5% CO ₂ , followed by BrdU (10 micromol) was added to each well. Plates were incubated for an additional 24 hours. Samples were stained with FITC conjugated anti-CD45 antibodies. After lysing RBCs, the remaining cells were fixed, permeabilized and treated with DNase. Thereafter, the samples were incubated with anti-BrdU antibodies. The washed cells were resuspended and then CD45 and BrdU-positive and BrdU-negative populations were detected by FACS. 10, 12a, and 12b show the percentage of BrdU insertion for samples taken at various doses.

Referring to FIG. 10, inhibition of BrdU insertion was not observed at dose 0.33 mg / m ² , but inhibition of BrdU insertion at dose 0.66 mg / m ² was observed up to 8 hours after infusion. Inhibition of BrdU incorporation was observed after 2 hours infusion at a dose of 1.85 mg / m ² . These data indicate that the compound of formula II interacts with the mechanical pathways of the cell in the same way it degrades tumor cells.

Referring to FIG. 12A, good inhibition of BrdU incorporation is observed after 2 hours infusion at a dose of 1.85 mg / m ² . In contrast, as shown in FIGS. 12A and 12B, inhibition of BrdU incorporation was observed after up to 3 hours of infusion at doses of 3.7 mg / m ² and 7.4 mg / m ² , and 29.6 mg / m ² , 41.4 mg / m Observed after 8 hours or less injection at doses of ² , 50 mg / m ² , and 58 mg / m ² . The effect of the compound of formula (II) on inhibition of BrdU insertion observed at a 1.85 mg / m ² dose (see FIG. 12A) may be due to internal-subject variability compared to that shown in FIG. 10.

Referring to FIG. 13 and Table IXI, the inhibition rate of BrdU incorporation after 8 h infusion was generally increased over the range of 1.85 to 58 mg / m ² . Indeed, the inhibition rate of BrdU insertion has already risen above 75% at a dose of 7.4 mg / m ² . These data reflect that inhibition of lymphocyte proliferation, as measured by BrdU incorporation, correlated with the dose level of the compound of formula II to which the blood sample is exposed.

TABLE IX

NSCLC, colorectal cancer, prostate cancer, ovarian cancer, breast cancer, melanoma, vulvar cancer, peritoneal pseudomyxoma, peritoneal cancer, bile duct cancer, pancreatic cancer, soft tissue sarcoma, gastric cancer, gastrointestinal stromal tumor (GIST), esophageal cancer, adenocarcinoma Patients diagnosed with adenoidcystic carcinoma, neuroendocrine tumor or hepatocellular carcinoma were administered various doses of the compound of formula II by 2-hour IV infusion on a 28-day dosing cycle. Each 28-day dosing cycle was administered once weekly for three weeks and included one week off. No complete or partial response was observed by RECIST criteria in the population (see, eg, Therasse et al., J Natl Cancer Inst, 92 (3): 205-216 (2000)). Nevertheless, one patient diagnosed with NSCLC had stable disease after 6 cycles of 28 days of treatment at 0.66 mg / m ² , and one patient diagnosed with peritoneal pseudomyxoma was 2.59 mg / m, as shown in FIG. 11. ^They had tumor markers that decreased after 6 cycles of ² to 28 days of treatment and continued for a total of 9 cycles. For accuracy, tumor markers are denoted as "CA" as CA125. In addition, one patient with prostate cancer obtained four cycles of a 28-day dosing regimen dosed at 3.63 mg / m ² . One patient with melanoma was treated for 9 cycles at 3.63 mg / m ² until disease progressed. 1 patients with soft tissue sarcoma obtained for 6 cycles at 7.11 mg / m ² was maintained to have a capacity of one Ahn disease as patients with GIST obtained for 5 cycles at 7.11 mg / m ^2. One patient with esophageal cancer who obtained 5 cycles at 7.11 mg / m ² has a stable disease. One patient with adenoid cystic carcinoma who obtained 5 cycles at 7.11 mg / m ² has a stable disease until the disease progresses.

Similarly, carcinoid, thyroid cancer, leiomyarcoma, NSCLC, colorectal cancer, squamous cell carcinoma of the head and neck (HNSCC), unknown primary adenocarcinoma, melanoma, ovarian cancer, non-Hodgkin's lymphoma, neuroendocrine, testicular cancer Various types of compounds of formula II by 2-hour IV infusion in 21-day dosing cycles in patients diagnosed with breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, thyroid cancer, liposarcoma, cholangiocarcinoma, peritoneal pseudomyxoma, chordoma or sarcoma Doses were administered. Patients diagnosed with liposarcoma, colorectal cancer, neuroendocrine tumor, NSCLC, and esophageal cancer were administered various doses of the compound of formula II by 2 hour IV infusion on a 21-day dosing cycle. Each 21-day dosing cycle included the administration of a dose of 1 day during the 21-day period. Although no complete or partial response was observed by RECIST criteria in the population, stable disease was achieved in some of the aforementioned tumor types after 4 or 21-day or more dosing cycles. Specifically, two carcinoid patients achieved stable disease states for four cycles at doses of 29.6 mg / m ² and 41.4 mg / m ² . Similarly, thymic cancer patients achieved stable disease states for 4 cycles at a dose of 50 mg / m ² . Patients with anal cancer achieved stable disease for 6 cycles at a dose of 50 mg / m ² . In addition, patients with leiomyosarcoma achieved a stable disease state for 8 cycles at a dose of 41.4 mg / m ² . NSCLC patients achieved a stable disease state for 12 cycles at a dose of 50 mg / m ² . Chordoma patients achieved stable disease for 8 cycles at a dose of 58 mg / m ² , and prostate cancer patients achieved stable disease for 7 cycles at a dose of 50 mg / m ² .

The above description of the invention is illustrative and is not intended to be limiting. Many variations or modifications of the embodiments described herein may occur to those skilled in the art. These changes can be made without departing from the scope or spirit of the invention.

TABLE X

TABLE XI

The above description of the invention is illustrative and is not intended to be limiting. Many variations or modifications of the embodiments described herein may occur to those skilled in the art. These changes can be made without departing from the scope or spirit of the invention.

Claims (52)

When injecting one or more of the indicated dosage levels into a human, from about pH 3.4 to about pH 5, providing one or more PK behavior and / or AUC as shown in one of FIGS. 2A, 2B or 2C. IV-infusible formulations comprising a compound of Formula II in a buffer solution having a pH. The formulation of claim 1, which provides the AUC / dose curves shown in FIG. 3 when infused for 2 hours in an amount providing one or more dose levels shown. The formulation of claim 1, wherein the buffer solution is an aqueous buffer solution comprising an acid and its conjugated sodium salt, wherein the acid is citric acid or lactic acid. The formulation of claim 1, wherein the buffer solution comprises citric acid and sodium citrate. The formulation of claim 4, wherein the ratio of citric acid: sodium citrate in the buffer solution is about 1 mole sodium citrate for about 2.4 moles citric acid. The formulation of claim 5, wherein the sodium further comprises a dilution of sodium chloride solution. Injecting a solution comprising a compound of formula (II) into a patient in need of treatment of a tumor over a period of 1 hour to about 24 hours, each of days 1, 8, and 15 of the 28 day cycle in an amount effective to treat the tumor condition A method of treating a tumor condition treatable by administration of a compound of Formula (II). 8. The method of claim 7, wherein the cycle is repeated until tumor suppression or complete regression is observed. The method of claim 7 or 8, wherein the amount of solution injected provides a plasma level of at least about 10 ng / mL. The method of claim 7, wherein the solution administered comprises the solution of claim 5. 11. A process for preparing a concentrate comprising preparing a buffer solution of an organic acid and its conjugate salt, dissolving the compound of formula II in a buffer solution, and then adjusting the pH of the resulting solution to about pH 3 to about pH 5.1. The method of claim 11, further comprising providing an injectable formulation by mixing the concentrate with the contents of a standard saline IV bag. The process according to claim 11 or 12, wherein the organic acid is citric acid and the conjugate salt is sodium citrate. The process according to any one of claims 11 to 13, wherein the ratio of organic acid, conjugate salt and compound of formula II is 1 mole of compound of formula II: 3.66 moles of conjugate salt: 8.8 moles of organic acid. The method of claim 11, wherein the compound of Formula II is present in the concentrate at a level of 5 mg / mL. Formulations comprising an aqueous organic acid, its conjugate salt, and a compound of Formula (II) which, upon IV injection into a human, provide the PK profile shown in FIG. 2A. A formulation comprising an aqueous organic acid, a conjugate salt thereof, and a compound of Formula II, which provides one or more of the PK profiles shown in Table V upon IV infusion into a human in an amount that provides one or more of the indicated dosages. The formulation of claim 17 comprising a molar ratio of 1 mole: 8.8 mole: 3.7 mole of the compound of Formula II: organic acid: conjugate salt. Of about 2.15 time t _1/2, from about 77 ng / ml C _maximum, and / or to provide about 181 ng hr / mL of PK for a two hour infusion of a dose containing 1.85 mg / M ² having an AUC , Wherein about 1 mole of compound of formula II: about 8.8 mole of organic acid: conjugate salt is about 3.7 moles of the compound of formula II: molar ratio of organic acid: conjugate salt. Of about 2.75 time t _1/2, from about 136 ng / ml C _maximum, and / or to provide about 336 ng hr / mL of PK for a two hour infusion of a dose containing 3.7 mg / M ² having an AUC , Wherein about 1 mole of compound of formula II: about 8.8 mole of organic acid: conjugate salt is about 3.7 moles of the compound of formula II: molar ratio of organic acid: conjugate salt. Of about 2.86 time t _1/2, from about 353 ng / ml C _maximum, and / or to provide about 994 ng hr / mL of PK for a two hour infusion of a dose containing 7.4 mg / M ² having an AUC , Wherein about 1 mole of compound of formula II: about 8.8 mole of organic acid: conjugate salt is about 3.7 moles of the compound of formula II: molar ratio of organic acid: conjugate salt. Of about 2.78 time t _1/2, from about 396 ng / ml C _maximum, and / or about 1053 ng hr / mL, which provides a PK for a two hour infusion of a dose containing 14.8 mg / M ² having an AUC , Wherein about 1 mole of compound of formula II: about 8.8 mole of organic acid: conjugate salt is about 3.7 moles of the compound of formula II: molar ratio of organic acid: conjugate salt. About 2.79 times of t _1/2, to provide about 1020 ng / ml of C _{up to,} and / or PK for a two hour infusion of a dose containing 29.6 mg / M ² having an AUC of about 2531 ng hr / mL , Wherein about 1 mole of compound of formula II: about 8.8 mole of organic acid: conjugate salt is about 3.7 moles of the compound of formula II: molar ratio of organic acid: conjugate salt. About 2 to about 2.8 hours t _1/2, about 1020 ng / ml to about 1343 ng / ml of C _{up to,} and / or about 2531 ng hr / mL to about 3041 ng hr / mL of 41.4 mg / having an AUC About 1 mole: organic acid about 8.8 mole: conjugate salt about 3.7 moles of compound of formula II: molar ratio of organic acid: conjugate salt, providing PK for a two hour injection of a dose comprising M ² Formulations comprising. Of about 1.5 hours t _1/2, about 1820 ng / ml of C _{up to,} and / or about 3290 ng hr / mL, which provides a PK for a two hour infusion of a dose containing 50 mg / M ² having an AUC , Wherein about 1 mole of compound of formula II: about 8.8 mole of organic acid: conjugate salt is about 3.7 moles of the compound of formula II: molar ratio of organic acid: conjugate salt. For about 1 hour t _1/2, to provide about 1460 ng / ml of C _{up to,} and / or about 3290 ng hr / mL of PK for a two hour infusion of a dose containing 58 mg / M ² having an AUC , Wherein about 1 mole of compound of formula II: about 8.8 mole of organic acid: conjugate salt is about 3.7 moles of the compound of formula II: molar ratio of organic acid: conjugate salt. A formulation comprising a compound of formula (II) exhibiting the PK values and C _highest values shown in Table V when administered by IV infusion in an amount providing the corresponding doses indicated in Table V. A formulation comprising a compound of Formula (II) exhibiting the PK values and C _highest values shown in Table V when administered by IV infusion in an amount providing the corresponding doses indicated in Table VI. Injecting a solution comprising a compound of Formula II administered between 1 day and 20 days (21 day cycle) in an amount effective to treat a tumor condition over a period of from 1 hour to about 24 hours in a patient in need of treatment of the tumor condition A method of treating a tumor condition treatable by administration of a compound of Formula (II). The method of claim 29, wherein the cycle is repeated until tumor suppression or complete degeneration is observed. The method of claim 29 or 30, wherein the amount of solution injected provides a plasma level of at least about 10 ng / mL. 32. The method of any one of claims 29-31, wherein the solution administered comprises the solution of claim 5. 33. The method of any one of claims 7-10 and 29-32, wherein the amount of solution injected is provided at about 58 mg / m ² or less of the compound of formula II. 33. The method of any one of claims 7-10 and 29-32, wherein the amount of solution injected is provided at about 50 mg / m ² of the compound of formula II. The method of claim 29, wherein the tumor is non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, acute leukemia (eg, acute myeloid leukemia (AML) or acute lymphocytic leukemia (ALL)), chronic leukemia (eg , CLL), non-Hodgkin's lymphoma (eg, MCL), melanoma, and multiple myeloma. The method of claim 29, wherein the time for infusion is selected from the group consisting of 2 hours, 8 hours and 24 hours. The method of claim 29, wherein the amount of the compound of Formula II administered is from about 0.33 mg / m ² to about 20 mg / m ² , about 0.33 mg / m ² to about 14 mg / m ² or about 7 mg / m ² to Administered at about 12 mg / m ² . A solution comprising a compound of Formula (II) administered at 1, 8 and 15 days of a 28-day cycle, respectively, in an amount effective to treat a tumor condition into a patient in need of treatment of the tumor condition over a period of 1 hour to about 24 hours. A method of treating a tumor condition treatable by administration of a compound of Formula (II), comprising injecting a compound. The method of claim 29, wherein the tumor is non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, acute leukemia (eg, acute myeloid leukemia (AML) or acute lymphocytic leukemia (ALL)), chronic leukemia (eg , CLL), non-Hodgkin's lymphoma (eg, MCL), melanoma, and multiple myeloma. The method of claim 38, wherein the time for infusion is selected from the group consisting of 2 hours, 8 hours and 24 hours. The method of claim 40, wherein the time for infusion is 8 hours. The method of claim 41, wherein the amount of compound of Formula II administered is about 1.85 mg / m ² to about 70 mg / m ² or about 15 mg / m ² to 30 mg / m ² . The method of claim 40, wherein the time for infusion is 24 hours. The compound of claim 43, wherein the amount of the compound of Formula II administered is from about 1.85 mg / m ² to about 100 mg / m ² , about 50 mg / m ² to about 80 mg / m ² or about 30 mg / m ² to about 70.0 mg / m ² . The method of claim 38, wherein the solid tumor is treated. 46. The method of claim 45, wherein the solid tumor is selected from the group consisting of breast cancer, melanoma and ovarian cancer. The method of claim 45, wherein the amount of solution injected is provided from about 1.8 mg / m ² to about 58 mg / m ² . 47. The method of claim 45 or 46, further comprising administration of a growth factor. 49. The method of claim 48, wherein the growth factor is selected from the group consisting of GCSF and GMCSF. The method of claim 48 or 49, wherein the amount of solution injected is provided between about 0.33 mg / m ² and about 100 mg / m ^{2 on a} 21 day schedule. A formulation comprising an aqueous organic acid, its conjugate salt, and a compound of Formula (II), which provides the PK profile shown in FIG. 2B upon IV injection into a human. A formulation comprising an aqueous organic acid, a conjugate salt thereof, and a compound of Formula II, which, upon IV injection into a human, provide the PK profile shown in FIG. 2C.

Images