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WO2008073304A2 - Procédés de traitement du cancer - Google Patents

Procédés de traitement du cancer Download PDF

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Publication number
WO2008073304A2
WO2008073304A2 PCT/US2007/025095 US2007025095W WO2008073304A2 WO 2008073304 A2 WO2008073304 A2 WO 2008073304A2 US 2007025095 W US2007025095 W US 2007025095W WO 2008073304 A2 WO2008073304 A2 WO 2008073304A2
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compound
dose
administered
cells
hours
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WO2008073304A3 (fr
WO2008073304A9 (fr
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Daniel C. Adelman
Ute Hoch
Duncan Walker
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Viracta Therapeutics Inc
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Sunesis Pharmaceuticals Inc
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Publication of WO2008073304A3 publication Critical patent/WO2008073304A3/fr
Publication of WO2008073304A9 publication Critical patent/WO2008073304A9/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles

Definitions

  • the present invention relates to N-[5-[[[5-(l,l-dimethylethyl)-2- oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide, compositions thereof, and methods for its use to treat cancer.
  • Compound I is suitable as an inhibitor of protein kinases such as the cyclin dependent kinases (cdks), for example, cdc2 (cdkl), cdk2, cdk3, cdk4, cdk5, cdk6, cdk7, cdk8 and cdk9.
  • cdks protein kinases
  • Other proliferative diseases that Compound I may be useful to treat include inflammation, arthritis, Alzheimer's disease and cardiovascular disease. Two of the principal hallmarks of cancer are uncontrolled cell proliferation and the avoidance of cell death.
  • Molecules that directly control cell-cycle progression and apoptosis can be altered during tumorigenesis enabling tumor cells to survive and proliferate uncontrollably, independent of normal mitogenic and antigrowth signals.
  • the CDK family of protein serine/threonine kinases includes key drivers of cell-cycle progression and transcriptional control.
  • the inappropriate activation of both cell- cycle and transcriptional-regulatory CDKs in cancers can lead to unregulated proliferation and avoidance of apoptosis, suggesting that these kinases may be important targets for cancer therapeutics.
  • Flavopiridol the first nonselective CDK inhibitor to enter clinical trials, has had disappointing activity against solid tumors in part due to unacceptable toxicity and poor PK properties.
  • CDK2 is a cell cycle-regulated kinase that controls the entry into and normal progression through DNA synthesis. Inappropriate activation of CDK2 occurs in many human cancers and is associated with a poorer prognosis for patients. Activation of CDK2 in several cancer types including breast, NSCLC, and ovarian cancers is mediated through either overexpression of the associated activating subunit (cyclin E) or underexpression of the CDK2-inhibitor, p27. hi melanoma, CDK2 protein is directly upregulated, and its overexpression correlates with sensitivity to CDK inhibitors.
  • cyclin E activating subunit
  • CDK7 and CDK9 are involved in transcriptional control, particularly of short half- life mRNAs, many of which encode antiapoptotic and growth regulatory genes.
  • the deregulation of CDK7 or 9 in cancers has not been widely studied to date.
  • CDK9 may play a key role in cell survival in B-cell malignancies; CDK9 and its cyclin partner, cyclin Tl, are both highly expressed in some lymphomas and other B-cell malignancies, hi particular, it has been shown that CDK9 mediates transcriptional expression of myeloid cell leukemia sequence- 1 (McI- 1), an antiapoptotic protein related to Bcl-2 that is associated with survival and resistance to chemotherapeutic intervention.
  • McI-1 myeloid cell leukemia sequence- 1
  • An inhibitor of CDKs 2, 7, and 9 in tumor cells may be expected to cause cell cycle arrest and induce apoptosis in tumors both as a single agent and in combination with chemotherapy.
  • the invention provides a method of treating cancer comprising administering to a human having cancer a first dose of Compound I and a second dose of Compound I, wherein the first dose and the second dose are administered in the same 24-hour period.
  • the first dose and the second dose are administered parenterally, preferably intravenously.
  • the first dose and the second dose are administered orally.
  • the first dose and the second dose are administered essentially contiguously.
  • the treatment cycle is repeated.
  • the dose of Compound I is administered once per treatment cycle. In certain embodiments, the dose is administered once per week for two or more weeks. In certain embodiments, the method comprises administering the dose once per week for three weeks. In embodiments in which two or more doses are given per treatment cycle, a waiting period may be interposed between treatment r cycles. For example, in a regimen comprising two or more treatment cycles, each comprising three doses of Compound I per week, i.e., doses on days 1, 8, and 15 of the cycle, subsequent treatment cycles may begin on day 29 following the first dose, thereby defining a 28-day cycle.
  • the waiting period between the last dose of one treatment cycle and the first dose of a subsequent treatment cycle may be more than two weeks, optionally three weeks (5 -week treatment cycle), four weeks (6-week treatment cycle), or longer if appropriate as determined by a physician.
  • the first dose is administered over a period of about 2 to 20 minutes
  • the second dose is administered over a period of about 2 to 24 hours.
  • the first dose is about 1 mg/m 2 to about 20 mg/m 2 .
  • the second dose is about 1 mg/m 2 to about 110 mg/m 2 .
  • the application provides a method of treating cancer comprising administering to a human having cancer an amount of Compound I sufficient to achieve and maintain a plasma concentration of at least about 100 ng/mL for at least about 4-24 hours.
  • the treatment further comprises administration of a second active agent.
  • the second active agent is an agent to prevent or manage tumor lysis syndrome (TLS).
  • TLS tumor lysis syndrome
  • the second active agent is an anticancer cytotoxic agent
  • the second active agent is a DNA-damaging agent.
  • Compound I is administered as a solution comprising a hemitartrate salt of Compound I.
  • the cancer to be treated by the above methods is a hematologic malignancy.
  • the hematologic malignancy is acute myelogenous leukemia (AML).
  • the cancer to be treated is a B-cell malignancy.
  • the cancer to be treated is multiple myeloma (MM).
  • the cancer to be treated is chronic lymphocytic leukemia (CLL).
  • the cancer to be treated is multiple myeloma.
  • the cancer to be treated is mantle cell lymphoma (MCL).
  • the application provides a method of determining whether a patient is responding to treatment with Compound I comprising measuring a difference in the PBMC or cancer cell level before and after treatment with the compound of at least one RNA transcript selected from the group consisting of: Cyclin Dl, Cyclin D2, XIAP, BCI-X L , BAG-I, MCI-I , c-myc, and VEGF, in each case as normalized to a transcript that is not modulated by inhibition of RNA polymerase II.
  • RNA transcript selected from the group consisting of: Cyclin Dl, Cyclin D2, XIAP, BCI-X L , BAG-I, MCI-I , c-myc, and VEGF
  • the application provides a method of determining whether a patient is responding to treatment with Compound I comprising measuring a difference in the PBMC level before and after treatment with the compound of at least one protein selected from the group consisting of: Cyclin Dl, Cyclin D2, XIAP, BCI-X L , BAG-I, McI-I, c-myc, and VEGF, in each case as normalized to a protein not modulated by inhibition of RNA polymerase II.
  • Figure 1 shows target modulation in tumor-bearing mice that were dosed intraperitoneally (IP) with a single injection of Compound I.
  • Figure 2 A shows the dose-dependent effects of Compound I on mean tumor growth volume in HL-60 tumor-bearing mice.
  • Figure 2B shows the effect of Compound I on mean body weights of the xenograft mice.
  • the vehicle control group is depicted in diamonds; the group treated with 30 mg/kg IP every four days for three administrations is shown in squares, the group treated with 15 mg/kg IP every four days for three administrations is depicted in circles and the group treated with 7.5 mg/kg IP every four days for three administrations is depicted in x's.
  • Figure 2C shows the dose-dependent effect of Compound I on mean tumor growth volume in RPMI 8226 (multiple myeloma) tumor-bearing mice.
  • the effect of Compound I on mean body weights of the xenograft mice is depicted in Figure 2D.
  • Figures 2C and 2D the vehicle control group is depicted in diamonds; the group treated with 30 mg/kg IP per day for 5 days is depicted in squares, and the group treated with 15 mg/kg IP per day for 5 days is depicted in triangles.
  • Figure 3 shows the effect of Compound I on: (A-B) a RPMI 8226 mouse xenograft model, and (C) RPMI 8226 cells.
  • FIG 3 A The dose-dependent effect of Compound I on mean tumor growth volume is shown in Figure 3 A.
  • the effect of Compound I on mean body weights of the xenograft mice is depicted in Figure 3B.
  • Figures 3 A and 3B the vehicle control group is depicted in diamonds; the group treated with 30 mg/kg IP per day for 5 days is depicted in squares and the group treated with 15 mg/kg IP per day for 5 days is depicted in triangles.
  • Figure 3C the time-dependent effect of Compound I on colony growth is shown. Cells were treated for 4 hours (squares), 8 hours (triangles), 16 hours (inverted triangles), and 24 hours (diamonds).
  • Figures 4A-B show TUNEL assays of RPMI 8226 cells: Ml corresponds to normal cells; M2 corresponds to apoptotic cells.
  • Figure 4A cells were exposed for 8 hours to (top) 300 nM Compound I or (bottom) DMSO.
  • Figure 4B (left panels) cells were exposed for 6 hours to Compound I or DMSO as indicated.
  • Figure 4B (right panels) cells were exposed for 6 hours to Compound I or DMSO as indicated, followed by a 2 hour washout.
  • Figure 4C illustrates the effect of vehicle (left) and Compound I (right) on viability of RPMI 8226 cells after a 6 hour exposure, followed by staining with propidium iodide and Annexin V.
  • FIG. 4D shows the effect of Compound I (first and third rows) and DMSO vehicle (second and fourth rows) on viability of RPMI 8226 cells over time. Quadrants are as described for Figure 4C.
  • the first row shows exposure to 300 nM Compound I for (left to right) 2 hours, 4 hours, and 6 hours, respectively.
  • the second row shows exposure to DMSO vehicle for (left to right) 2 hours, 4 hours, and 6 hours, respectively.
  • the third row shows exposure to 300 nM Compound I for 6 hours, followed by (left to right) 2 hours of washout, 18 hours of washout, and 48 hours of washout, respectively.
  • the fourth row shows exposure to DMSO vehicle for 6 hours followed by (left to right) 2 hours of washout, 18 hours of washout, and 48 hours of washout, respectively.
  • Figure 5 depicts dose-dependent inhibition of A) Cdk9 and B) Cdk7 in RPMI 8226 cells by incubation with Compound I for 16 hours. Phosphorylation of Ser2 in the CTD of RNA polymerase II was used to measure Cdk9 activity, and phosphorylation of Ser5 in the CTD of RNA polymerase II was used to measure Cdk7 activity. Phosphorylation levels in both figures are represented as a percentage of phosphorylation relative to vehicle-treated cells.
  • Figure 6 A illustrates the effect of fetal bovine serum (FBS) and human serum on the activity of flavopiridol and Compound I in a MTT cytotoxicity assay using RPMI 8226 cells. Shown is the percent activity as a function of compound concentration: Compound I in the presence of FBS (squares); Compound I in the presence of human serum (triangles); flavopiridol in the presence of FBS (inverted triangles); and flavopiridol in the presence of human serum (diamonds).
  • FBS fetal bovine serum
  • human serum Shown is the percent activity as a function of compound concentration: Compound I in the presence of FBS (squares); Compound I in the presence of human serum (triangles); flavopiridol in the presence of FBS (inverted triangles); and flavopiridol in the presence of human serum (diamonds).
  • Figure 6B-C illustrates the effect of human serum and fetal bovine serum (FBS) on inhibition of cdk9 activity by (B) Compound I and (C) flavopiridol, as measured by extent of phosphorylation of Ser2 of CTD of RNA polymerase II in RPMI 8226 cells.
  • Cdk9 activity is shown as a percent of DMSO.
  • Figure 7 A shows a Western blot of phosphorylated serine 2 (pSer2) of the CTD of RNA pol II in comparison to total RNA Pol II levels, cleaved PARP (poly ADP ribose polymerase) levels, and actin levels in RPMI 8226 cells treated with various concentrations of Compound I.
  • DMSO vehicle-treated control (C) is in the left lane.
  • Figure 7B shows a Western blot of XIAP, three different iso forms of BAG, pSer5 of the CTD of RNA Pol II, and b- Actin in RPMI 8226 cells treated with 1 micromolar Compound I over time.
  • DMSO vehicle-treated control (C) is in the left lane.
  • Figures 7C-D show Western blots from RPMI 8226 cells treated with vehicle or with 300 nM Compound I over time. The same cells were used for both blots: C) shows levels of total RNA pol II, RNA pol II phosphorylated on Ser2 of the CTD, full length (FL) PARP, cleaved PARP, and actin; D) shows levels of XIAP, Bcl-X L , McI-I, BcI- 2, and Actin.
  • Figures 8A-C show the effect of Compound I on RNA transcript levels.
  • Figure 8 A shows microarray data for RPMI 8226 cells treated with 300 nM Compound I.
  • Figure 8B shows changes in levels (as compared with DMSO) of Cyclin Dl, Cyclin D2, McI-I, XIAP, and VEGF transcripts after a 6-hour treatment with Compound I.
  • Figure 8C shows time-dependent changes in levels (as compared with DMSO) of Cyclin D2, XIAP, and VEGF transcripts upon exposure to Compound I.
  • Figure 9 illustrates modulation of protein levels by Compound I in the peripheral blood mononuclear cells of patients having advanced solid tumors treated with the compound.
  • Figure 1 OA depicts predicted plasma-concentration-time profiles of a 15-minute intravenous loading infusion of Compound I followed by a 6-hour intravenous maintenance infusion.
  • Figure 10 shows pharmacokinetic data from patients treated with a fifteen minute infusion of 10 mg/m 2 Compound I followed by a 6 hour infusion of 12 mg/m 2 Compound I, each in comparison to the corresponding simulated pharmacokinetic data for Cohort 2 shown in Figure 1OA. Patient data is shown in symbols connected by a solid line, whereas the modeled data is shown as a plain solid line.
  • Figure 1OD shows predicted plasma concentration-time profiles of a five minute infusion followed by a 6 hour infusion of Compound I (dashed line), as compared with a 15 minute loading infusion followed by a 6 hour maintenance infusion (solid line). In each model, the loading infusion is 10 mg/m 2 and the maintenance infusion is 40 mg/m 2 .
  • the inset shows the difference predicted plasma concentration-time profiles within the first hour of administration.
  • Figure 11 shows the effect on cell death in CLL cells of varied concentrations of, and times of exposure to, Compound I and flavopiridol;
  • Figure 1 IB shows the selective apoptotic effect of Compound I on CLL cells as opposed to human PBMC.
  • Figure 12 shows the comparative effects of concentration of, and time of exposure to, Compound I and flavopiridol on RNA synthesis in CLL cells.
  • Figures 13 (A-top) and 13 (B-bottom) show the effects of concentration of, and time of exposure to, Compound I on Cdk9/7 mediated phosphorylation of Ser2 and Ser5, respectively, on the C-terminal domain of RNA polymerase II in CLL cells. Results for cells exposed to Compound I for 6 hours are depicted in triangles; results for cells exposed to Compound I for 24 hours are depicted in squares.
  • Figure 14 shows the effect of concentration of, and time of exposure to, Compound I on mRNA levels of short-lived anti-apoptotic proteins McI-I, XIAP, and Bcl-2 in CLL cells.
  • Figure 15 shows representative dose-dependent inhibition of McI-I (Fig. 15 -left) and XIAP (Fig. 15-right) protein levels (as a percentage of actin expression levels in the tested cells) in CLL cells. Results for cells exposed to Compound I for 6 hours are depicted in squares; results for cells exposed to Compound I for 24 hours are depicted in triangles.
  • Figure 16 shows an experimental protocol under which the persistence of Compound I in cell culture medium was tested, along with experimental data obtained.
  • Figure 17 shows the effect of concentration of, and time of exposure to, Compound I on mantle cell lymphoma cell lines.
  • Figure 18A shows the effect of concentration of, and time of exposure to, Compound
  • Figures 19, 20, and 21 show Western blots of pSer2 and pSer5 on CTD of RNA pol
  • RNA Pol II in comparison to total RNA Pol II levels, as well as levels of cyclin Dl, McI-I, BcI- 2, c-myc, PARP (full length - top; cleaved - bottom), CDK2 (pCDK2 [phosphorylated at Thrl ⁇ O]- green/top; total CDK2 red/bottom), and actin in Jeko, Mino, and SP53 MCL cells, respectively, treated with various concentrations of Compound I (vehicle-treated control is in the left lane).
  • Figure 22 shows the effects of concentration of, and time of exposure to, Compound I on the induction of SubGl DNA in four MCL cell lines. DESCRIPTION OF THE INVENTION
  • methods for treating a human or other mammal having a cancer by using administering a dose of about 1-150 mg/m 2 of Compound I to the human or other mammal.
  • methods for using Compound I comprise daily administration of Compound I for at least 2 days. In other embodiments, Compound I is administered daily for at least 3 days. In other embodiments, Compound I is administered daily for at least 4 days. In other embodiments, Compound I is administered daily for at least 5 days. In still other embodiments,
  • Compound I is administered daily for 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, and 14 days.
  • methods for using Compound I comprise a weekly administration of Compound I.
  • the weekly dose is administered over 2 to 12 hours, hi another embodiment, the weekly dose is administered over 5 to 8 hours. In another embodiment, the weekly dose is administered over 6 to 7 hours.
  • Compound I is administered weekly for three weeks. Li another embodiment, Compound I is administered weekly for three weeks (days 1, 8, and 15), followed by two weeks with no treatment with Compound I, in a 28-day schedule.
  • Compound I is administered to a patient so as to achieve a given plasma concentration and maintain the plasma concentration for a given time period.
  • Compound I is administered to a patient so as to achieve a plasma concentration of at least about 100 ng/mL for approximately 4- 24 hours, hi one embodiment, Compound I is administered to a patient so as to achieve a plasma concentration of at least about 115 ng/mL for at least about 4 hours, hi one embodiment, Compound I is administered to a patient so as to achieve a plasma concentration of at least about 115 ng/mL for at least about 6 hours, hi another embodiment, Compound I is administered to a patient so as to achieve a plasma concentration of at least about 115 ng/mL for at least about 8 hours, hi another embodiment, Compound I is administered to a patient so as to achieve a plasma concentration of at least about 115 ng/mL for at least about 10 hours, hi another embodiment, Compound I is administered to a patient so as to achieve a plasma concentration of at least about 115 ng/mL for
  • Compound I is administered to a patient so as to achieve a plasma concentration of at least about 115 ng/mL for at least about 18 hours, hi another embodiment, Compound I is administered to a patient so as to achieve a plasma concentration of at least about 115 ng/mL for at least about 24 hours, hi another embodiment, Compound I is administered to a patient so as to achieve a plasma concentration of about 115-300 ng/mL for at least about 4-24 hours, hi one embodiment, the dose of Compound I is held constant throughout the administration period. In another embodiment, dose of Compound I is varied during the administration period. For example a dose given over a shorter period may be followed by a different dose given over a longer period.
  • the first dose and second dose can be adjusted to achieve a desirable drug exposure over a given time period. Either the dose or the dose rate, or indeed, both of these parameters can be varied between doses. For example, in one embodiment, the first dose is lower than the second dose, hi another embodiment, the first dose is administered over a shorter period than the second dose. In another embodiment the first dose is administered in a period of about 5 to 30 minutes.
  • the first dose is administered in about 10 to 25 minutes, hi another embodiment, the first dose is administered in about 10 to 15 minutes, hi another embodiment, the first dose is administered in about 15 to 20 minutes, hi another embodiment, the first dose is administered in about 3 minutes, hi another embodiment, the first dose is administered in about 5 minutes, hi another embodiment, the first dose is administered in about 10 minutes, hi another embodiment, the first dose is administered in about 15 minutes, hi another embodiment, the first dose is administered in about 20 minutes, hi another embodiment, the second dose is administered in a period of about 2 to 10 hours, hi another embodiment, the second dose is administered in a period of about 4 to 24 hours, hi another embodiment, the second dose is administered in a period of about 5 to 7 hours.
  • the second dose is administered in a period of about 6 hours.
  • methods of using Compound I comprise administering a first dose of Compound I in a first dosing period and a second dose of Compound I in a second dosing period.
  • the first dosing period and the second dosing period are generally performed on the same day (i.e., within the same 24-hour period).
  • the second dose is administered within 2 hours after the first dose is administered.
  • the second dose is administered within 1 hour after the first dose is administered.
  • the duration and spacing of the dosing periods may be designed so as to maintain a desired plasma concentration of Compound I in the blood of the subject.
  • the second dosing period is essentially contiguous with the first dosing period.
  • the practitioner may determine that it would be useful to introduce an interval between the completion of the first dosing period and the start of the second dosing period.
  • Such an interdose interval may be determined by practical considerations, but it is preferred that the plasma concentration of Compound I not fall below the desired level.
  • the interval between doses is selected to prevent the plasma concentration of Compound I from falling below about 115 ng/mL at any time during the course of the administration of the first and second doses.
  • Doses, dosage rates and interdose intervals may be varied relative to one another to accommodate maintenance of the plasma concentration of Compound I above the desired level.
  • Compound I is administered once a week.
  • Compound I is administered weekly for three weeks. In another embodiment, Compound I is administered weekly for three weeks, followed by one week off, in a 28-day schedule, hi one embodiment, the first dosing period of Compound I is 15 minutes and the second dosing period of Compound I is 6 hours. In another embodiment, a first dose of 5 mg/m 2 of Compound I is administered in about 15 minutes and a second dose of 10 mg/m 2 of Compound I is administered in about 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 12 mg/m 2 is administered in 6 hours.
  • a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 20-30 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 30-40 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 40-50 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 50-60 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 60-70 mg/m 2 is administered in 6 hours.
  • a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 70-80 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 80-90 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 90-100 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in 15 minutes and a second dose of 100-110 mg/m 2 is administered in 6 hours.
  • a first dose of 10 mg/m 2 of Compound I is administered in 15 minutes and a second dose of 40-100 mg/m 2 of Compound I is administered in 6 hours.
  • a first dose of 10 mg/m 2 of Compound I is administered in 5 minutes and a second dose of 40-100 mg/m 2 of Compound I is administered in 6 hours.
  • the first dosing period is 15 minutes and the second dosing period is 6 hours.
  • a first dose of 5 mg/m 2 of Compound I is administered in about 5 minutes and a second dose of 10 mg/m 2 of Compound I is administered in about 6 hours.
  • a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 12 mg/m 2 is administered in 6 hours.
  • a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 20-30 mg/m 2 is administered in 6 hours.
  • a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 30-40 mg/m 2 is administered in 6 hours.
  • a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 40-50 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 50-60 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 60-70 mg/m 2 is administered in 6 hours. In another embodiment, a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 70-80 mg/m 2 is administered in 6 hours.
  • a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 80-90 mg/m 2 is administered in 6 hours.
  • a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 90-100 mg/m 2 is administered in 6 hours, hi another embodiment, a first dose of 10 mg/m 2 is administered in about 5 minutes and a second dose of 100-110 mg/m 2 is administered in 6 hours.
  • a first dose of 10 mg/m 2 of Compound I is administered in about 5 minutes and a second dose of 40-100 mg/m 2 of Compound I is administered in about 10 hours to 12 hours.
  • a first dose of 10 mg/m 2 of Compound I is administered in about 5 minutes and a second dose of 40-100 mg/m 2 of Compound I is administered in 10 hours
  • a first dose of 10 mg/m 2 of Compound I is administered in about 5 minutes and a second dose of 40- 100 mg/m 2 of Compound I is administered in 11 hours.
  • a first dose of 10 mg/m 2 of Compound I is administered in about 5 minutes and a second dose of 40-100 mg/m 2 of Compound I is administered in 12 hours, hi another embodiment, a first dose of 10 mg/m 2 of Compound I is administered in about 5 minutes and a second dose of 40-100 mg/m 2 of Compound I is administered in 18 hours.
  • a first dose of 10 mg/m 2 of Compound I is administered in about 5 minutes and a second dose of 40-100 mg/m 2 of Compound I is administered in 24 hours, hi another aspect of the present invention, methods of using Compound I comprise administering one or more doses of Compound I as an infusion or an injection, hi one embodiment, Compound I is administered as a 5 minute infusion, hi another embodiment, Compound I is administered as a 5 minute bolus injection, hi anotherembodiment, Compound I is administered as a 15 minute infusion, hi another embodiment, Compound I as a 1 -hour infusion, m other embodiments, Compound I is administered as a 3-hour infusion, hi other embodiments, Compound I is administered as a 6-hour infusion, hi other embodiments, Compound I is administered as an 8-hour infusion, hi other embodiments, Compound I is administered as a 10-hour infusion.
  • Compound I is administered as a 12-hour infusion. In other embodiments, Compound I is admininistered as an 18-hour infusion, hi still other embodiments, Compound I is administered as a 24-hour infusion.
  • methods of using Compound I comprise administering a daily dose of Compound I of 10-20 mg/m 2 as a 1-hour infusion for five days every three weeks. In another embodiment, the daily dose is 15-25 mg/m . In another embodiment, the daily dose is 20-30 mg/m 2 . In another embodiment, the daily dose is 25-35 mg/m 2 . In another embodiment, the daily dose is 30-40 mg/m 2 . In another embodiment, the daily dose is 35-45 mg/m 2 .
  • the daily dose is 40-50 mg/m 2 .
  • the daily dose is 50-60 mg/m .
  • the daily dose is 60-70 mg/m 2 .
  • the daily dose is 70-80 mg/m 2 .
  • cancer can be disease of skin tissues, organs, blood, and vessels, including, but not limited to, sarcomas; cancers of the bladder, bone or blood, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis, throat, uterus and hematologic malignancies, such as leukemias, lymphomas and myelomas.
  • the myeloma is multiple myeloma.
  • the lymphoma is Non-Hodgkin's lymphoma, B-cell lymphoma, mantle cell lymphoma, and Hodgkin's disease (also called Hodgkin's lymphoma), hi certain embodiments, the leukemia is chronic lymphocytic leukemia (CLL), chronic myeloid leukemia
  • the cancer comprises solid tumor, hi certain embodiments, the cancer can be relapsed, refractory or resistant to conventional therapy.
  • Compound I may exert anti-cancer effects by decreasing expression of cytokines and survival factors that promote and maintain malignancies (short half-life transcripts and proteins most affected by transcriptional blockade) and by inhibiting deregulated proliferation. For hematological malignancies in particular, decrease of cytokine expression may interfere with tumor-stromal interaction in the bone marrow that are necessary for maintaining these diseases.
  • multiple myeloma is a disease caused by the monoclonal proliferation of malignant plasma cells and their migration to and expansion within the bone marrow environment.
  • Compound I is a potent, selective inhibitor of CDKs 7 and 9 that inhibits both cell cycle progression and transcription.
  • Data provided herein demonstrate that Compound I abrogates dysregulated proliferation as demonstrated by inhibition of colony formation by RPMI 8226 cells.
  • Compound I also inhibits the phosphorylation of ser5 and ser2 of RNA Polymerase II CTD, consistent with mechanism-based inhibition of CDKs 7 & 9.
  • Compound I is formulated for parenteral (e.g., intravenous (PV)) administration for the treatment of cancer.
  • the API of a formulation for IV administration may be a 2: 1 salt of Compound I as a free base with L-tartaric acid ("Compound I API"), and is manufactured under Good Manufacturing Practice (GMP) controls.
  • GMP Good Manufacturing Practice
  • the molecular formula of Compound I API is C I 7 H 24 N 4 O 2 S 2 ⁇ (C 4 H 6 O O ) 1Z2 and its molecular weight is 455.5 Daltons.
  • Compound I API is a white non-hygroscopic powder that melts between 233 0 C and 240 0 C.
  • Compound I API is slightly soluble in water, propylene glycol and methanol, very slightly soluble in ethanol and polyethylene glycol 400 (PEG 400), and practically insoluble in acetone and acetonitrile.
  • the pH of a saturated solution of Compound I API in water at 25 0 C is 5.1.
  • the Compound I API pKa values are estimated to be 8.4 and 11. Dosage amounts used in the compositions and methods provided herein are based on Compound I free base rather than any particular salt form.
  • a formulation for administration of Compound I is exemplified to illustrate such formulations useful according to the invention.
  • Compound I is prepared as a clear, colorless to light-yellow aqueous solution (50 mg/vial (5 mg/mL) free base). It is supplied in 10 mL Type I glass vials. A 6% fill overage is included for vial-needle- syringe (VNS) withdrawal loss.
  • VNS vial-needle- syringe
  • Each vial contains 53.0 mg of Compound I active free base, 95.4 mg sodium chloride (solubilizer), 3.39 mg L-tartaric acid (buffer, pH 4.0), and water.
  • the vials of the Compound I solution should be stored under refrigeration (2 0 C to 8 0 C) and protected from light.
  • the diluted solution can be stored refrigerated or at room temperature (RT), but must be used within 26 hours of dilution.
  • This formulation of Compound I is diluted with 0.9% Sodium Chloride Injection (Normal Saline) to the desired concentrations prior to administration (Compound I API can be diluted to between 0.1 and 1.0 mg/mL). Note that this formulation is suitable for administration by oral or parenteral (e.g., intravenous) routes.
  • liquid formulations may be employed, e.g., formulations with different excipients and formulations having different concentrations of Compound I free base.
  • this Compound I formulation illustrates that a formulation may be prepared that is readily adapted for use in administering the first dose and the second dose of Compound I by simple dilution in the medium of choice for parenteral administration.
  • different formulations, at least differing by concentration of Compound I can be provided for each of the first dose and second dose of Compound I.
  • Such dilutions and alternative formulations may be prepared according to methods known to those of skill in the art.
  • Compound I may be used as a single agent according to methods of the invention, or alternatively other therapies, chemotherapeutic agents, or other anti-cancer agents may be used in combination with Compound I to treat proliferative diseases and cancer.
  • the invention is directed to a method of treating cancer comprising administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m 2 and a second active agent.
  • a dose of Compound I of about 1-150 mg/m 2 and a second active agent.
  • “in combination with” does not necessarily indicate that Compound I is physically combined with the second active agent prior to administration; in fact, whether it is possible to physically combine Compound I and the second active agent will depend on the identity of the second active agent.
  • the second active agent will be compatible with Compound I in the particular formulation to be used, it is preferred that Compound I not be physically combined with the second active agent prior to administration.
  • the intravenous administration may be in the same FV line, or in separate IV lines.
  • the 1-150 mg/m 2 dose of Compound I is administered once a week.
  • the second active agent may be administered at a different frequency than the 1-150 mg/m 2 dose of Compound I, or it may be administered at the same frequency as the 1- 150 mg/m 2 dose of Compound I.
  • the second active agent is administered at the same frequency as Compound I, but on a staggered schedule as compared with Compound I.
  • a staggered schedule refers to a waiting period between the end of administration of the second active agent or Compound I and the start of administration of Compound I or the second active agent, respectively.
  • the second active agent may be administered prior to the start of administration of Compound I, subsequently to the completion of the administration of Compound I, concurrently with Compound I, or simultaneously with Compound I.
  • the administration of the second active agent is completed 24 hours prior to administration of Compound I. hi another embodiment, the administration of the second active agent is completed 48 hours prior to administration of Compound I. hi another embodiment, the administration of the second active agent is completed 72 hours prior to administration of Compound I. In one embodiment, the administration of the second active agent is completed within 24 hours after administration of Compound I. In another embodiment, the administration of the second active agent is completed within 48 hours after administration of Compound I. In another embodiment, the administration of the second active agent is completed within 72 hours after administration of Compound I. In another embodiment, both Compound I and the second active agent are present at the same time in the subject.
  • the second active agent is administered on the same day as (i.e., within 24 hours of) the 1-150 mg/m 2 dose of Compound I. In another embodiment, the second active agent is administered immediately prior to Compound I. In another embodiment, the second active agent is administered immediately after Compound I. In one example where Compound I is given as a 1-hour infusion, the infusion of Compound I would start immediately after administration of the second active agent is complete.
  • the second active agent administered in combination with Compound I is an anticancer cytotoxic agent
  • Compound I is administered on a staggered schedule with respect to the anticancer cytotoxic agent.
  • Compound I is administered 24 hours after the end of the administration of the anticancer cytotoxic agent.
  • the second active agent may be administered using a different length infusion than is used for Compound I.
  • the second active agent is administered in a 5-15 minute intravenous infusion, hi another embodiment, the second active agent is administered as a 1-hour infusion, hi another embodiment, the second active agent is administered as a 24-hour infusion.
  • the second active agent administered in combination with Compound I is a DNA-damaging agent, hi another embodiment, the second active agent administered in combination with Compound I is cisplatin. In another embodiment, the second active agent administered in combination with Compound I is carboplatin.
  • the invention is directed to a method of treating cancer comprising administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m 2 once a week, and a dose of a second active agent given at least once every four weeks.
  • the second active agent given at least once every four weeks is cisplatin.
  • the second active agent given at least once every four weeks is carboplatin.
  • the method for treating cancer comprises administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m once a week, and a dose of cisplatin of about 1-30 mg/m 2 administered once a week.
  • the method for treating cancer comprises administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m 2 once a week, and a dose of cisplatin of about 5-60 mg/m 2 every 2 weeks. In another embodiment comprises administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m 2 once a week, and a dose of cisplatin of about 10-100 mg/m 2 every 3 weeks. In one embodiment, the method comprises administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m 2 once a week, and a dose of cisplatin of about 15-120 mg/m 2 every 4 weeks.
  • the invention is directed to a method for treating cancer, comprising administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m 2 once a week, and a dose of carboplatin of about 60-225 mg/m 2 administered once a week.
  • the method for treating cancer comprises administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m 2 once a week, and a dose of carboplatin of about 125-250 mg/m 2 every 2 weeks.
  • the method comprises administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m 2 once a week, and a dose of carboplatin of about 250-500 mg/m 2 every 4 weeks.
  • the method for treating cancer comprises administering to a mammal having cancer a dose of Compound I of about 1-150 mg/m 2 once a week and an amount of carboplatin sufficient to achieve an AUC of about 4-7 mg/mL x min, wherein the amount of carboplatin is administered once a week.
  • AUC is well known as the Area Under the Curve in a graphical plot of drug concentration over time.
  • AUC is a pharmacokinetic parameter that represents the total amount of a drug present in plasma or blood, irrespective of the rate at which it is absorbed.
  • the methods of the invention are expected to be useful in treating hematologic cancers that are refractory or resistant to other cancer treatments regimens, including single agent regimens and combination regimens.
  • certain embodiments methods of the invention involve treating a subject having a B- cell malignancy that is refractory or resistant to treatment with alemtuzumab; bortezomib; cyclophosphamide; dexamethasone; doxorubicin; pegylated doxorubicin; liposomal doxorubicin; fludarabine; galiximab; lenalinomide; melphalan; prednisone; rituximab; thalidomide; vincristine.
  • the methods of the invention may be used to treat B cell malignancies that are refractory or resistant to combinations of anticancer agents such as fludarabine and alemtuzumab; fludarabine and cyclophosphamide; fludarabine, cyclophosphamide, and rituximab; fludarabine and rituximab; cyclophosphamide, vincristine, and prednisone; cyclophosphamide, vincristine, prednisone, and rituximab; melphalan and prednisone; melphalan, prednisone and thalidomide; or other combinations of such agents.
  • anticancer agents such as fludarabine and alemtuzumab; fludarabine and cyclophosphamide; fludarabine, cyclophosphamide, and rituximab; fludarabine and rituximab; cyclo
  • the invention is directed to a method of treating a hematologic cancer, comprising subjecting a human having a hematologic cancer to at least one 28-day treatment cycle, wherein each treatment cycle comprises administering a dose of about 15-150 mg/m 2 on each of days 1, 8, and 15 of the treatment cycle.
  • each dose of Compound I consists of a first dose of Compound I of about 5-50 mg/m 2 which is administered intravenously in about 5-30 minutes, and a second dose of Compound I of about 10-100 mg/m 2 administered intravenously in about 4-24 hours.
  • the treatment may be repeated, wherein the human is subjected to at least 2, 3, 4, 5, 6 or 28-day treatment cycles.
  • the cycles, if repeated, include a 13-day observation period following the day 15 dose of Compound I.
  • succeeding treatment cycles may be initiated on the day following the completion of the prior 28- day cycle, i.e., essentially contiguously (temporally).
  • the first dose and the second dose in each dose of Compound I are administered essentially contiguously.
  • the first dose of Compound I may be accomplished by injecting a solution of Compound I (e.g., a formulation such as that disclosed herein) into an intravenous piggy back bag of normal saline which is connected to the main intravenous tubing.
  • a solution of Compound I e.g., a formulation such as that disclosed herein
  • infusions are accomplished using an infusion pump.
  • bolus injections of a solution of Compound I may be pushed intravenously via an injection port.
  • a syringe pump may be used.
  • the second dose of Compound I may be administered by injecting a specified amount of a liquid formulation of Compound into the main intravenous bag of normal saline.
  • an infusion pump is employed for ensuring delivery of the second dose over the prescribed period.
  • Compound I and pharmaceutically acceptable compositions comprising Compound I can be employed in complementary combination therapies with other active agents or medical procedures.
  • Compound I and pharmaceutically acceptable compositions thereof can be administered concurrently with, prior to, or subsequent to, one or more other desired active agents or medical procedures.
  • the particular combination of therapies (agents or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved.
  • the therapies employed may achieve a desired effect for the same disorder (for example, Compound I may be administered concurrently with another active agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects).
  • Non- limiting examples of such agents and procedures include surgery, radiotherapy (e.g., gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioisotopes), endocrine therapy, biologic response modifiers (interferons, interleukins, and tumor necrosis factor (TNF) to name a few examples), hyperthermia and cryotherapy, agents to attenuate any adverse effects (e.g., antiemetic agents), and other approved chemotherapeutic anticancer agents.
  • radiotherapy e.g., gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioisotopes
  • endocrine therapy e.g., gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioisotopes
  • biologic response modifiers interferons, interleukins,
  • chemotherapeutic anticancer agents that may be used as second active agents in combination with Compound I include, but are not limited to, alkylating agents (e.g., mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide), antimetabolites (e.g., methotrexate), aurora kinase inhibitors, purine antagonists and pyrimidine antagonists (e.g., 6-mercaptopurine, 5-fluorouracil, cytarabine, gemcitabine), spindle poisons (e.g., vinblastine, vincristine, vinorelbine, paclitaxel), podophyllotoxins (e.g., etoposide, irinotecan, topotecan), antibiotics (e.g., doxorubicin, daunorubicin, bleomycin, mitomycin), nitrosoureas (e.g., carmustine,
  • Some specific anticancer agents that can be used in combination with Compound I include, but are not limited to: azacitidine (e.g., Vidaza ® ); bortezomib (e.g., Velcade ® ); capecitabine (e.g., Xeloda ® ); carboplatin (e.g., Paraplatin ® ); cisplatin (e.g., Platinol ® ); cyclophosphamide (e.g., Cytoxan , Neosar ® ); cytarabine (e.g., Cytosar ® ), cytarabine liposomal (e.g., DepoCyt ® ), cytarabine ocfosfate or other formulations of the active moiety; doxorubicin, doxorubicin hydrochloride (e.g., Adriamycin ® ), liposomal doxorubic
  • anticancer agents that can be used in combination with Compound I include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adalimumab (e.g., Humira ® ); adozelesin; alitretinoin (e.g., Panretin ® ); altretamine (hexamethylmelamine; e.g., Hexalen ® ); ambomycin; ametantrone acetate; aminoglutethimide (e.g., Cytadren ® ); amonafide malate (e.g., Xanafide ® ); amsacrine; anastrozole (e.g., Arimidex ® ); anthramycin; asparaginase (e.g., Kidrolase ® , Elspar ® ); asperlin; azetepa; azo
  • Omnitarg ® pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride (e.g., Matulane ® ); puromycin; puromycin hydrochloride; pyrazofurin; R-roscovitine (seliciclib); riboprine;; safingol; safingol hydrochloride; semustine; pumprazene; sorafenib (e.g., Nexavar ® ); sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin (e.g., Zanosar ® ); sulofenur; sunitinib malate (e.g., Sutent ® ); talisomycin;
  • anticancer agents that can be used in combination with Compound I include, but are not limited to: 20-epi-l,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone acetate; acylfulvene, (hydroxymethyl)acylfulvene; adecypenol; ALL-TK antagonists; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; anagrelide (e.g., Agrylin ® ); andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP- DL-PT
  • Gleevec ® imiquimod (e.g., Aldara ® ), and other cytokine inducers; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons such as interferon alpha (e.g., Intron ® A); pegylated interferon alfa-2b
  • interleukins such as IL-2 (aldesleukin, e.g., Proleukin ® ); iobenguane; iododoxorubicin; 4-ipomeanol; iroplact; irsogladine; isobengazole; isohomohalicondrin B; jasplakinolide; kahalalide F; lamellarin-N triacetate; leinamycin; lenograstim; lentinan sulfate; leptolstatin; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lonidamine; loxoribine
  • IL-2 aldesleukin, e.
  • O6-benzylguanine O6-benzylguanine; octreotide (e.g., Sandostatin ® ); octreotide acetate (e.g.,
  • Sandostatin LAR ® okicenone; oligonucleotides; onapristone; oracin; osaterone; oxaliplatin (e.g., Eloxatin ® ); oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; panaxytriol; panomifene; parabactin; pazelliptine; peldesine; pentosan polysulfate sodium; pentostatin (e.g., Nipent ® ); pentrozole; perflubron; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; piloca ⁇ ine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum-triamine complex; propyl bis- acri
  • Nolvadex ⁇ tamoxifen methiodide; tauromustine; tazarotene; tellurapyrylium; telomerase inhibitors; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; titanocene bichloride; topsentin; translation inhibitors; tretinoin (all-trans retinoic acid, e.g., Vesanoid ® ); triacetyluridine; turosteride; tyrosine kinase inhibitors; ty ⁇ hostins; UBC inhibitors; ubenimex;
  • the second active agent is a supportive care agent, such as an antiemetic agent or erythropoiesis stimulating agents.
  • antiemetic agents include, but are not limited to, phenothiazines, butyrophenones, benzodiazapines, corticosteroids, serotonin antagonists, cannabinoids, and NKl receptor antagonists.
  • phenothiazine antiemetic agents include, but are not limited to, prochlorperazine and trimethobenzamide.
  • butyrophenone antiemetic agents include, but are not limited to, haloperidol.
  • benzodiazapine antiemetic agents include, but are not limited to, lorazepam.
  • corticosteroid antiemetic agents include, but are not limited to, dexamethasone.
  • serotonin receptor (5-HT3 receptor) antagonist antiemetic agents include, but are not limited to, dolasetron mesylate (e.g., Anzemet ® ), granisetron (e.g., Kytrir), itasetron, ondansetron (e.g., Zofran ® ), palonosetron (e.g., Aloxi ® ) ramosetron, tropisetron (e.g., Navoban ® ), batanopride, dazopride, renzapride.
  • cannabinoid antiemetic agents include, but are not limited to, dronabinol.
  • NKl receptor antagonists include, but are not limited to, aprepitant (e.g., Emend ® ).
  • Other supportive care agents include agents that stimulate erythropoiesis or other hematopoietic processes, such as epoetin alfa (e.g., Epogen ® , Procrit ® ); G-CSF and recombinant forms such as filgrastim (e.g., Neupogen ® ), pegfilgrastim (e.g., Neulasta ® ), and lenofilgrastim; darbepoetin alfa (e.g., Aranesp ® ); and GM-CSF and recombinant forms such as sargramostim (e.g., Leukine ® ) or molgramostim.
  • epoetin alfa e.g., Epogen ® , Procrit ®
  • G-CSF and recombinant forms such as filgrastim (e.g., Neupogen ® ), pegfilgrastim (e.g., Neulasta
  • chemoprotectant agents such as amifostine (e.g., Ethyol ® ), dexrazoxane (e.g., Zinecard ® ), leucovorin (folinic acid), and mesna (e.g., Mesnex ® ); thrombopoeitic growth factors such as interleukin-11 (IL-11, oprelvekin, e.g., Neumega ® ); bisphosphonates such as pamidronate disodium (e.g., Aredia ® ), etidronate disodium (e.g., Didronel ® ) and zoledronic acid (e.g., Zometa ® ); and TNF antagonists, such as infliximab (e.g., Remicade ;.
  • amifostine e.g., Ethyol ®
  • dexrazoxane e.g., Zinecard ®
  • Tumor lysis syndrome may be expected in the treatment of hematologic cancers, and supportive care treatment(s) to mitigate or prevent TLS or its component symptoms may be administered to patients treated with Compound I according to the invention.
  • Treatments suitable for preventing or mitigating TLS include, for example, allopurinol (e.g., Zyloprim ® ), rasburicase (e.g., Elitek ® ), and sodium polystyrene sulfonate (e.g., Kayexalate ® ).
  • Doses and dosing regimens of Compound I together with other active moieties and combinations thereof should depend on the specific indication being treated, age and condition of a patient, and severity of adverse effects, and may be adjusted accordingly by those of skill in the art. Examples of doses and dosing regimens for other active moieties can be found, for example, in Physician 's Desk Reference, and will require adaptation for use in the methods of the invention.
  • active moieties mentioned herein as second active agents may be identified as free active moieties or as salt forms (including salts with hydrogen or coordination bonds) or other as non-covalent derivatives (e.g., chelates, complexes, and clathrates) of such active moieties, it is to be understood that the given representative commercial drug products are not limiting, and free active moieties, or salts or other derivative forms of the active moieties may alternatively be employed. Accordingly, reference to an active moiety should be understood to encompass not just the free active moiety but any pharmacologically acceptable salt or other derivative form that is consistent with the specified parameters of use.
  • the invention is directed to a method of determining whether a patient is responding to treatment with Compound I comprising measuring a difference in the PBMC level before and after treatment with the compound of at least one RNA transcript selected from the group consisting of: Cyclin Dl, Cyclin D2, XIAP, BCI-X L , BAG-I, MCI-I , c-myc, and VEGF, in each case as normalized to a transcript that is not modulated by inhibition of RNA polymerase II.
  • RNA transcript selected from the group consisting of: Cyclin Dl, Cyclin D2, XIAP, BCI-X L , BAG-I, MCI-I , c-myc, and VEGF
  • the invention is directed to A method of determining whether a patient is responding to treatment with Compound I comprising measuring a difference in the PBMC level before and after treatment with the compound of at least one protein selected from the group consisting of: Cyclin Dl, Cyclin D2, XIAP, BcI- X L , BAG-I, MCI-I, and VEGF, in each case as normalized to a protein not modulated by inhibition of RNA polymerase II.
  • Example 1 In Vivo Activity Studies As described in the examples herein, Compound I is a selective inhibitor of CDK 2, 7, and 9 that shows potent activity against cellular and xenograft models of hematological malignancies. Compound I inhibits in vivo the phosphorylation of Ser2 and Ser5 of RNA Pol II CTD, consistent with mechanism-based inhibition of CDKs 7 and 9. A sustained apoptotic response (indicated by PARP cleavage) is observed in vivo after administration of Compound I. Pro-apoptotic effects of Compound I may be explained in some instances by the down-regulation of short half-life survival proteins like McI-I.
  • Compound I shows potent anti-tumor activity as evidenced by multiple tumor regressions after intermittent well tolerated treatment schedules.
  • the activity of Compound I as a single agent was studied in the human cell line RPMI 8226 (ATCC: CCL- 155) established as subcutaneous xenografts in nu/nu female mice.
  • animals were randomized by tumor volume and distributed into groups often animals each. Treatments were initiated when tumors averaged about 200 mm 3 in volume. End points for each group were determined based on body weight nadir, adverse clinical observations, or tumor volumes exceeding maximum threshold of 2000 mm 3 .
  • Responses were assessed by tumor growth inhibition (TGI) and tumor growth delay (TGD). TGI and TGD in the treatment group were evaluated against the vehicle control group.
  • TGI tumor growth inhibition
  • TGD tumor growth delay
  • Compound I was administered intraperitoneally (IP) once every day for 5 days (qd > ⁇ 5) at a dose of 30 mg/kg or 15 mg/kg.
  • IP intraperitoneally
  • the change in tumor volume over time is shown in Figure 3 A, and body weight as a percent of initial over time is shown in Figure 3B.
  • Compound I dosed as a single agent qd ⁇ 5 at 30 mg/kg had significant single agent activity and significantly delayed the tumor growth compared to the vehicle.
  • Example 2 Compound I Activity in Leukemia and Multiple Myeloma Xenograft
  • Compound I qdx5 at the MTD (30 mg/kg) resulted in 121% and 130% TGI in HL-60 and RPMI 8226 xenografts, respectively.
  • Compound I showed potent anti-tumor activity against HL-60 xenografts using an intermittent q4d schedule at MTD and X A MTD, with 119% and 86% TGI, respectively.
  • 30 mg/kg Compound I was administered qdx5 in the HL-60 xenograft model complete responses were observed in 7 out of 10 animals on day 78; q4d administration of Compound I for 3 doses resulted in complete responses in 6 out of 10 animals on day 78.
  • Example 3 Compound I induces a potent in vivo inhibition of RNA pol II and a sustained pro-apoptotic response in human xenografts
  • Figure 1 shows the results of tumor-bearing mice that were dosed IP with a single injection of Compound I. Tumors were collected at 2, 6, and 24 hours post-dose. Forty milligrams of total proteins was separated on 4-12% Tris-glycine NuPAGE gels (Invitrogen). After transfer, nitrocellulose membranes were probed for RNA pol II pSer2 CTD (Abeam 5095), RNA pol II pSer5 CTD (Abeam 5131), MCL-I (BD Pharmingen 554103), cleaved PARP (Cell Signaling 9541), and b-actin (Sigma A- 2228).
  • RNA pol II is significantly modulated after a single dose of 15 or 30 mg/kg Compound I.
  • pSer2 CTD is more sensitive than pSer5 to inhibition after Compound I treatment.
  • Drug-induced decrease in the levels of pSer2 CTD and pSer5 CTD is already evident 6 hr after Compound I administration and sustained at least for 24 hr after 30 mg/kg as a single dose.
  • MCL-I protein levels are decreased to differing extents in the 3 xenografts with MV 4-11 > HL-60 > RPMI 8226. Decrease in the level of MCL-I protein is sustained for 24 hr after 30 mg/kg Compound I in MV 4-11 and HL-60.
  • PARP cleavage indicative of apoptosis, is evident after a single 15 and 30 mg/kg Compound I dose. Increased levels of PARP cleavage are evident as early as 6 hours post-dose and sustained at least for 24 hours. Overall, PARP cleavage does not correlate with decrease in MCL-I protein levels in these 3 xenografts.
  • Example 4 Compound I Induces Regression of Leukemia and Multiple Myeloma Xenografts
  • Compound I is well tolerated and highly efficacious in HL-60 and RPMI 8226 tumor bearing mice after intermittent or daily treatment schedules. Multiple long term regressions of xenografts were observed in all the models investigated to date.
  • Five of nine RPMI 8226 tumor-bearing mice were still tumor free 78 days after administration of 5 daily doses of 30 mg/kg Compound I.
  • Example 5 - hi Vitro Colony Growth Assay
  • the RPMI 8226 cell line was cultured in RPMI 1640 medium (Cellgro) with 10% FBS. Following treatment with a dilution series of Compound I, cells were washed twice with fresh medium, counted and plated (500 cells/mL) into a 96-well plate coated with poly-L-lysine. Following 7 day incubation, colonies were stained with Hoechst 33342 and counted using an ArrayScan ® High Content Screening (HCS) device. As shown in Figure 3 C, the cells were incubated with Compound I for 4 hours, 8 hours, 16 hours, and 24 hours.
  • HCS High Content Screening
  • Compound I to induce cell death (through apoptosis) in RPMI 8226 cells in a dose- and time-dependent manner was measured by FACS (fluorescence activated cell sorting). This assay also gives the limited ability to distinguish apoptotic cell death from necrotic cell death.
  • a TUNEL assay was performed using the APO-BrdU TUNEL Assay Kit (#A-23210,
  • DPBS Dulbecco's phosphate buffered saline
  • Figure 4A depicts results for RPMI 8226 cells treated for 8 hr with 300 nM Compound I or with DMSO vehicle, (top panel and bottom panel, respectively). It can be seen from Figure 4A that after the 8 hr exposure to Compound I, 51% of the cells are normal (cell population Ml), and 49% of the cells are apoptotic (cell population M2). Corresponding values for the DMSO control were 83% and 17%, respectively. Thus Compound I induces apoptosis in multiple myeloma cells after only 8 hours exposure.
  • Figure 4B depicts results for RPMI 8226 cells treated for 6 hours with Compound I (top panels) or DMSO (bottom panels).
  • results were obtained without (left panels) and with (right panels) a 2-hour washout following the treatment with the Compound I or vehicle. It can be seen that a 6-hour exposure to Compound I is sufficient to cause apoptosis, as compared with DMSO treatment. For cells treated with Compound I, after a 2-hour washout the observed proportion of apoptotic cells is greater than before the washout, likely indicating that Compound I has effected an apoptotic program in the cells. For cells treated with DMSO vehicle, the proportion of apoptotic cells observed is the same after the 2-hour washout as compared with before the washout.
  • Cells were collected into 15 mL conical tubes and pelleted by centrifugation at 1200 rpm at 4 °C for 5 min. Medium was aspirated and cells were resuspended in ice-cold IX DPBS (Cellgro #21-031-CV) and 1 mL of resuspended cells was transferred to a 1.5 mL tube. Cells were pelleted by centrifugation at 1200 rpm at 4 °C for 5 min. The DPBS was aspirated and the cells were resuspended in 1 mL of ice-cold annexin binding buffer (10 mM HEPES, 140 niM NaCl, 2.5 niM CaCl 2 , pH 7.4).
  • ice-cold annexin binding buffer 10 mM HEPES, 140 niM NaCl, 2.5 niM CaCl 2 , pH 7.4
  • Annexin binding buffer was aspirated and cell pellet was resuspended in 100 ⁇ L of annexin labeling solution [prepared from 1 mL annexin binding buffer, 51 ⁇ L annexin V-FITC (BD Pharmingen #556419), 1.9 ⁇ L propidium iodide (Molecular Probes #V-13244)]. Contents were transferred to a 1 mL FACS tubes and incubated for at least 20-min. at RT, protected from light, with occasional mixing to keep cells suspended. Just prior to analysis, add an additional 100 ⁇ L of annexin binding buffer to samples.
  • Figure 4C shows results of exposure of the RPMI 8226 cells to Compound I for 6 hours followed by a 36-hour washout in the propidium iodide/ Annexin V staining assay. As can be seen from the figure, as little as a 6-hour exposure to 300 nM Compound I is sufficient to cause the vast majority of multiple myeloma cells to undergo apoptosis by 36 hours after the exposure.
  • the time-dependent effect of exposure of RPMI 8226 cells with 300 nM Compound I is illustrated in Figure 4D. The first row of the figure shows cells treated with
  • the second row of the figure shows results for RPMI 8226 cells treated with DMSO for 2 hours, 4 hours, and 6 hours, respectively. It can be seen that increasing the length of exposure to Compound I but not to DMSO increases the proportion of cells that are apoptotic as compared with normal cells.
  • the third row of the figure shows results for cells treated with Compound I for 6 hours followed by a washout for 2 hours, 18 hours, and 42 hours, respectively.
  • the fourth row of the figure shows results for cells treated with DMSO for 6 hours followed by a washout for 2 hours, 18 hours, and 42 hours, respectively.
  • Example 9 Inhibition of Cellular Cdk9 and Cdk7 Activity Cells were treated for 16 hr with a serial dilution of Compound I, fixed and permeabilized with 100% methanol. Primary antibodies used were anti-phospho RNA polymerase II serine2 (Abeam #ab5095) or anti-phospho RNA polymerase II serine5 (Abeam #ab5131).
  • the secondary antibody was AlexaFluor 488 anti-rabbit IgG (Invitrogen #A11008). Nuclei were stained with Hoechst 33342 (Invitrogen #3570). Fluorescence was assessed by HCS using a Cellomics ArrayScan ® device. Results from the assay are shown in Figure 5. As can be seen from the figure, Compound I effects a dose-dependent inhibition of cdk9 (Figure 5A) and cdk7 ( Figure 5B) with an EC 5O of approximately 200 nM for each.
  • Example 10 Effect of Serum on Compound I Activity (MTT Cytotoxicity Assay) The effect of human serum binding and bovine serum binding on Compound I and flavopiridol activity was measured using EC 50 values in an MTT proliferation assay.
  • RPMI 8226 cells were seeded into 96-well plates at a density of 1000 cells/well in a volume of 50 ⁇ L of RPMI 1640 medium containing either 10% (volume/volume) human serum or fetal bovine serum. Cells were incubated overnight at 37 °C/5% CO 2 .
  • Stock solutions of Compound I or flavopiridol were diluted in DMSO to a 10 niM concentration.
  • This 10 mM solution was then use to make a 3 -fold dilution series with seven dilutions ranging in concentration from 10 mM - 0.014 mM.
  • This IOOOX (final concentration) series was diluted 500X using RPMI 1640/10% serum (either human or fetal bovine) to yield a 2X (final concentration) series.
  • Fraction of live cells value of sample well - average (background) (reported as % activity) average (value of DMSO only control) - average (background).
  • the background wells contained no cells, and the DMSO only control contained no compound. Results from the experiment are shown in the following table, and in Figure 6A.
  • Example 11 Ser2-CTD RNA Polymerase II Phosphorylation hi this experiment, RPMI 8226 cells were grown in medium containing either 10% FBS or 10% human serum. The cells were then treated with a titration of either Compound I or flavopiridol to determine if human serum has an effect on the activity of either compound compared to bovine serum. The cells were then analyzed by HCS for RNA Pol II CTD pSer2 levels to determine CDK9 activity.
  • RPMI 1640 medium + 10% FBS RPMI 1640 medium + 10% FBS
  • RPMI medium + 10% human serum The cells were then seeded into poly-L-lysine 96-well plates at 10,000 cells/well under the same growth conditions as above, and grown overnight. Cells were treated with eleven 3-fold dilutions of Compound I or flavopiridol, starting at 10 ⁇ M (100 ⁇ L final volume in medium with either FBS or human serum), and incubated for either 6 hrs or 16 hrs. Cells were fixed by adding 10 ⁇ L 37% formaldehyde directly to the medium in each well and incubating 15 min.
  • Cells were incubated 1 hr in 50 ⁇ L secondary antibody + 1 ⁇ g/mL Hoechst in 1% BSA/PBS. Cells were washed twice in 50 ⁇ L PBS. Cells were analyzed using the ArrayScan. Cells were identified by Hoechst stain on channel 1. Phospho-Ser2 levels were measured on channel 2 (488 nm). Mean average fluorescence intensity was calculated for each well based on 1000 cells.
  • Compound I is a 3 -fold more potent inhibitor of cdk9 than flavopiridol in human serum.
  • Figure 6 B-C show plots of cdk9 activity (shown as a percent of DMSO control activity) plotted against the micromolar concentration of compound used.
  • the inhibition of cdk9 in human or bovine serum by Compound I is shown in Figure 6B, and the inhibition of cdk9 in human or bovine serum by flavopiridol is shown in Figure 6C.
  • Example 12 Compound I Effects on Target Activity and PARP Cleavage in RPMI 8226 cells
  • the pSer2 levels and PARP cleavage in RPMI 8226 cells was measured by western blotting. Lysates were made from cells previously treated with Compound I in a dose-dependent manner for 8 and 16 hours as indicated in Figure 7 A. Cells were lysed in a MPER (Pierce # 78501) lysis buffer containing Protease inhibitors (Pierce # 78415), and Phosphatase inhibitors (Pierce # 78420). Ten micrograms protein sample in 15 ⁇ L was loaded and run on 4-12% Bis-Tris gel, in MES running buffer. Primary and secondary antibodies were used as provided in the tables below. A PVDF membrane was used for the blot. Primary antibodies
  • Example 13 Compound I Effects on Survival Proteins in RPMI 8226 cells
  • Lysates were made from RPMI 8226 cells previously treated with 1 ⁇ M Compound I for 1.5, 3, 5, 8, 16, and 24 hours.
  • the lysis buffer was MPER (Pierce # 78501) containing Protease inhibitors (Pierce # 78415) and Phosphatase inhibitors (Pierce # 78420).
  • Anti-rabbit (Zymed 626120) 1:5,000 5% milk/TBST 1 hr ( ⁇ ) RT
  • RNA pol II pSer2 CTD abam #5095; total RNA pol II, Covance #MMS126R; McI-I, Santa Cruz Biotech #sc-12756; XIAP, CST#2042; BCL-X, BD #610747; Bcl-2, CST #2876; PARP, CST #9542; b-actin, Sigma #A2228.
  • Figure 7C and 7D shows results from RPMI 8226 cells treated with 300 nM Compound I or DMSO, and analyzed at various time intervals. The same cells were used for both blots. As can be seen in Figure 7C, exposure to 300 nM Compound I for 2 hours, 4 hours, or 6 hours results in a decrease in the level of RNA pol II pSer2. Further, there is a time-dependent increase in the level of cleaved PARP as compared with full-length (FL) PARP. Shown in Figure 7D are the corresponding blots for XIAP, BCI-X L , MCI-I and Bcl-2.
  • RNA Transcript Levels in RPMI 8226 cells RPMI 8226 cells were exposed to 300 nM Compound I under a variety of dosing conditions, including chronic exposure as well as conditions where compound was removed and the cells allowed to continue growth in the absence of compound. The cells were harvested at different treatment time points and their RNA isolated using ArrayGrade Total RNA Isolation kit (Super Array).
  • RNA was then subjected to microarray analysis using Oligo GEArray system (SuperArray).
  • the microarray data are presented in Figure 8 A as a ratio of gene transcript levels in the compound-treated cells compared to the corresponding transcript levels in untreated cells.
  • a Compound I-driven increase in a specific gene transcript will appear as a bar above the line; a decrease in a specific gene transcript will appear as a bar below the line.
  • transcript levels generally decrease with constant exposure to Compound I. Following removal of the compound, transcript levels appear to either increase or return to a basal level.
  • Transcript levels of certain genes were also measured by RT-PCR. RNA was isolated from treated cells using RNAqueous 4PCR kit (Ambion #AM1914) and cDNA synthesized using cDNA Reverse Transcription Kit (Applied Biosystems #4374966). cDNA samples were subjected to RT-PCR analysis following standard methods using a TaqMan device and gene expression kits (Applied Biosystems). Results are shown in Figure 8B-C for RPMI 8226 cells treated with 300 nM
  • Relative Quantification is calculated as a ratio of transcript levels in Compound I-treated cells compared to DMSO control. All samples were normalized to 18S rRNA, an RNA polymerase I transcript that is not modulated by inhibition of RNA polymerase II. It can be seen in Figure 8B that levels of cyclin Dl, eye Im D2, McI-I, XIAP, and VEGF transcripts are decreased after a 6 hour exposure to Compound I. A time-course experiment is shown in Figure 8C. It can be seen that, after exposure to Compound I for 2 hours, 4 hours, and 6 hours, there is a time-dependent decrease in the levels of Cyclin D2, XIAP, and VEGF transcripts.
  • Example 15 Target Modulation in Peripheral Blood Mononuclear Cells of Human Patients hi an open-label, multi-center, dose-escalation clinical trial of Compound I infusion in patients having advanced solid malignancies, the correlative science component assessed target modulation (TM) consistent with CDK inhibition in peripheral blood mononuclear cells (PBMC); explored pharmacodynamic (PD) relationships with dose, pharmacokinetics (PK) and outcome; anddetermined a maximum-tolerated dose (MTD).
  • TM target modulation
  • PBMC peripheral blood mononuclear cells
  • PD pharmacodynamic
  • PK pharmacokinetics
  • MTD maximum-tolerated dose
  • Compound I inhibits transcriptional initiation and elongation by blocking the phosphorylation of Ser5 and Ser2 of the CTD of RNA pol II by CDK7 and CDK9, respectively.
  • Short half-life (Ti /2 ) transcripts and proteins are maximally affected by transient exposure to Compound I.
  • / 2 survival signaling proteins by Compound I was also recently demonstrated in a multiple myeloma cell line (RPMI 8226) (Conroy et al. AACR 2007).
  • Clinical evidence of Compound I target modulation was demonstrated ex vivo in peripheral blood mononuclear cells from treated cancer patients (Hawtin et al., Haematologica 2007; 92, suppl 1). Patients having solid tumors were treated with intravenous infusions of Compound I over 1 hour, and blood samples were collected.
  • RNA Pol II CTD pSer2 and pSer5 decreased, indicating inhibition of CDKs 9 and 7, with uniform TM at 3 hr post-dose; the rate and extent of TM increased with dose, though on Day 4 there was some evidence of target rebound. A dose-dependent decrease in actin was observed on Day 1. MCL-I survival-protein reduction was first noted at 48 mg/m 2 . TM was sustained with Compound I levels below IC io.
  • PBMC Peripheral blood mononuclear cells collected from patients were isolated and frozen on site. Duplicates were analyzed by Western blot. Lysates were generated from the PBMC pellets using standard protocols. Protein quantification was performed using the Protein Assay Kit (BioRad).
  • Anti-pS2 RNA pol II CTD (Abeam ab5095) at 1/15,000 dilution in milk
  • Anti-pS5 RNA pol II CTD (Abeam ab5131) at 1/500 dilution in milk
  • Anti-MCL-1 antibody (CST #4572) at 1/500 dilution in milk
  • SIGMA milk Anti-actin
  • Blots were washed and probed with secondary antibodies from Zymed (Anti-rabbit at 1/5,000 in milk; Anti-mouse at 1/3000 in milk) for 1 hour at RT. ECL-plus (Pierce) was used as reagent, following manufacturer's instructions. Blots were exposed to film and scanned for documentation. For stripping blots, the Restore Western blot stripping buffer (Pierce) was used. Membranes were then rinsed in sterile water and blocked for 1 hour in 5% milk at RT, prior to incubation with primary antibody. Representative western blots showing target modulation by Compound I are shown in Figure 9.
  • Cdk9 modulation data for an ocular melanoma patient who received a 48 mg/m 2 dose of Compound I is shown in Figure 9A.
  • cdk9 is inhibited in a human patient in vivo, as evidenced by decreased phosphorylation of the C-terminal domain (CTD) of RNA Polymerase II, and by decreased survival factor McI-I levels.
  • CCD C-terminal domain
  • McI-I levels survival factor
  • Figure 9B shows a decrease in the level of phosphorylated Ser2 of RNA the CTD of RNA Polymerase II for a patient having parotid malignancy with lung metastases that was treated with 24 mg/m 2 of Compound I.
  • the corresponding plasma levels of Compound I in the ocular melanoma patient are shown in the table below. PK levels were assessed by LC/MS/MS.
  • cdk9 modulation was observed in humans at lower dosage levels than were seen in in vitro experiments performed in RPMI 8226 cells, where the IC 50 was 0.25-0.3 ⁇ M for levels of phosphorylated Ser2 of the CTD of RNA polymerase II.
  • the whole blood cell counts for the ocular melanoma patient are shown in the following table.
  • Figure 9 C-E shows results from a patient having colorectal cancer with liver metastases dosed at 24 mg/m 2 . On days 1 and 2, a reduction in the amount of phosphorylated Ser2 of RNA pol II is observed; however, there may have been a rebound of this protein on Day 4.
  • Figure 9D shows results from a patient having melanoma. Dosing was at 65 mg/m 2 on day 1 ; however, because the patient exhibited Grade 3 neutropenia on day 2, the patient was not dosed that day.
  • Compound I is a potent, selective inhibitor of cyclin dependent kinase (CDK)s 2, 7, and 9 that inhibits both cell cycle progression and transcription and is currently in Phase I clinical trials for the treatment of hematological malignancies. It was desired to maintain the plasma level of Compound I above 0.3 ⁇ M for 6 hr, which is the concentration required for 90% cell killing in vitro. Results obtained in the clinical trials CAl 74001, CAl 74002, and CAl 74006 show that the PK of Compound I is well characterized. Using the experimentally determined PK parameters, a model was developed to predict Compound I plasma-concentration-time profiles. Figure 1OA shows a depiction of the model.
  • CDK cyclin dependent kinase
  • the administration schedule for the Compound I formulation described herein above was optimized to maintain plasma concentrations above the IC 90 (> 0.3 ⁇ M) for 6 hours.
  • Compound I pharmacokinetics were fit to a 3 -compartment infusion model with a 1 st order elimination rate.
  • the model parameters were as follows: Vl : 4785 mL/m 2 , k 2] : 1.048 L/hr, k 31 : 0.106 L/hr, a: 13.596 L/hr, ⁇ : 0.622 L/hr, ⁇ : 0.063 L/hr.
  • Compound I formulation has been administered to patients having multiple myeloma or CLL on a once weekly for three weeks, every 28 days schedule.
  • Compound I formulation has been safely administered to three patients, including two patients with CLL and one patient with multiple myeloma, at a dose of 22 mg/m 2 , with 10 mg/m 2 given as a 15-minute loading infusion and 12 mg/m 2 given as a 6-hour maintenance infusion.
  • Plasma concentration data for each of the two CLL patients, as compared with the simulated plasma concentration for this schedule, is provided in Figures 10B- C. Patient samples were taken before, during and after the first maintenance infusion. It can be seen in the figures that the predicted plasma concentration of Compound I over time is similar to the observed plasma concentration of Compound I over time in the patients on this infusion schedule.
  • Figure 1OD shows a comparision of this schedule with the 15-minute loading infusion/6-hour maintenance infusion schedule. Both simulations were fit to a 3-compartment infusion model with a 1 st order elimination rate, and model parameters used for the simulations shown in Figure 1OD were the same as the model parameters used for the simulations shown in Figure 1OA, as described above.
  • the inset in Figure 1OD shows a comparison of Compound I concentration in the first hour. Examples of doses according to a 5 minute bolus loading /6-hour maintenance dose schedule finding use in solid tumor and hematologic malignancies are shown in Table IB.
  • these doses may be administered weekly.
  • the 5 minute bolus dose/6-hour maintenance dose is administered once a week for three weeks.
  • the 5-minute bolus /6-hour maintance dose is administerered on Days 1, 8, and 15, on a 28-day schedule.
  • a dose of 10 mg/m 2 administered as a 15-minute infusion is predicted to result in a C max of 0.6 ⁇ g/mL.
  • the same dose given as a 5-minute IV bolus is predicted to result in a C max of 1.3 ⁇ g/mL, double the C ma ⁇ predicted for the 15-minute infusion.
  • This C max will only be sustained for a short duration of time, since
  • Compound I inhibited 80% of RNA synthesis. Again, Compound I was about 30-fold more potent than flavopiridol at inhibiting transcription.
  • Example 18 Isolation of Primary CLL cells and peripheral blood mononuclear cells Peripheral blood from human CLL patients or normal donors was collected in heparin vacutainer tubes and centrifuged at 1500 rpm for 15 min to separate the patient plasma. The plasma (upper layer) was removed and saved for cell culture.
  • the lower layer was diluted with phosphate-buffered saline (PBS), and the mononuclear cells were isolated by Ficoll ® density-gradient centrifugation (Atlanta #140150).
  • the isolated cells were cultured at 1 x 10 7 cells/mL in RPMI 1640 medium (Cellgro #10- 040-CV) containing 10% autologous plasma or human blood type AB serum (Sigma- Aldrich #H4522) when the patient plasma was not adequate.
  • Example 19 - Toxicity of Compound I in primary CLL cells in vitro Toxicity was assessed by annexin V/propidium iodide (PI) staining.
  • CLL cells (5 x 10 5 ) suspended in 500 ⁇ L annexin binding buffer (BD Biosciences #556454) in FACS tubes (BD Labware #352058) were pelleted by centrifugation at 1500 rpm for 5 min. Media was aspirated and the cell pellet was resuspended in 200 ⁇ L annexin V binding buffer with 5 ⁇ L Annexin V-FITC (BD Pharmingen #556419), and incubated in dark for 15 min at RT. At the end of incubation, 300 ⁇ L annexin V binding buffer and 5 ⁇ L PI (50 ⁇ g/mL) (Sigma Aldrich #P4864) were added to samples. Cells were then analyzed on a BD FACSCalibur.
  • FIG. 1 IA shows a comparison of the effects of concentrations of flavopiridol and Compound I on cell death in CLL cells.
  • Figure 1 IB compares the levels of cell death induced by different concentrations of Compound I in CLL cells and in three different samples of human PBMC. The toxicity of Compound I against CLL cells is selective, having only modest effects on PBMC at higher concentrations.
  • Example 20 Effect of Compound I on RNA synthesis
  • RNA synthesis was measured by quantitating incorporation of [ 3 H]uridine into the perchloric acid-insoluble materials. Briefly, after incubation with Compound I at designed time and concentrations, CLL cells were labeled for 60 min at 37 0 C with [ 3 H]uridine (Moravek Biochemical #MT799) at (20 ⁇ Ci/mL) in a 15 mL centrifuge tube. At the end of incubation, 10 mL of ice-cold PBS was added to the tubes. The cells were centrifuged at 1500 rpm at 4 0 C for 5 min and washed again with 10 mL ice-cold PBS.
  • Example 21 Inhibition of RNA Polymerase II Phosphorylation by Compound I Inhibition of phosphorylation of RNA polymerase II (pol II) was evaluated by immunoblotting using antibodies specific for the phosphorylated Ser2 and Ser5 sites of the CTD of RNA polymerase II, which are substrates of Cdk9 and Cdk7 respectively.
  • the primary CLL cells (—2.75 ⁇ 10 7 ) were collected and lysed by sonication in 200 ⁇ L buffer containing 25 niM HEPES (pH 7.5), 300 mM NaCl, 1.5 mM MgCl 2 , 0.5% sodium deoxycholate, 20 mM /3-glycerophosphate, 1% Triton X-100, 0.1% SDS, 0.2 mM EDTA (pH 8), 0.5 mM DTT, 1 mM sodium orthovanadate (pH 10), 1 mM phenylmethylsulfonyl fluoride, 20 ⁇ g/mL aprotinin, and 20 ⁇ g/mL leupeptin.
  • Protein content in the lysate was determined using the Bio-Rad DC Protein Assay kit according to the manufacturer's instructions (Bio-Rad #5000112).
  • Cell lysate proteins (20 ⁇ g) were loaded to 4-12% Bis-Tris gel (Bio-Rad #3450124) and separated by SDS-polyacrylamide gel electrophoresis in MOPS running buffer (Bio- Rad #1610788), and then electro-transferred to a nitrocellulose membrane (GE Osmonics Labstore # EP2HY00010).
  • the membranes were blocked for 1 hr in PBS containing 5% nonfat dried milk and then incubated with primary antibodies for 3 hr, followed by incubation 1 hr with secondary antibodies labeled with fluorescent probes.
  • the membranes were scanned with the LI-COR Odyssey infrared imaging system to obtain image and quantitation.
  • the levels of the phosphorylation were normalized to total pol II, and the relative phosphorylation of time-matched controls was calculated.
  • the antibodies used are listed in the tables below. Primary antibodies
  • Example 22 Effect of Compound I on the mRNA level of anti-apoptotic proteins
  • the mRNA levels of anti-apoptotic proteins such as McI-I, XIAP and Bcl-2 were measured by Real-time quantitative PCR.
  • Total cellular RNA was isolated from the primary CLL cells using the RNeasy mini kit (Qiagen #74104) with DNase digestion (Qiagen #79254) to completely remove the genomic DNA.
  • Total RNA (20-50 ng) was used for the one-step real-time PCR reaction in the TaqMan ® One-Step RT-PCR Master Mix (Applied Biosystems #4313803).
  • PCR reaction was carried out in a 25 ⁇ L volume on 96-well optical reaction plate (Applied Biosystems #N8010560) for 30 min at 48 0 C for the reverse transcription reaction, followed by 10 min at 95 0 C for initial denaturing, then followed by 40 cycles of 95 0 C for 15 seconds and 60 0 C for 2 min in the 7900HT Sequence Detection System (Applied Biosystems).
  • the relative gene expression was analyzed by the Comparative threshold (Ct) method using 18S ribosomal RNA as endogenous control, after confirming that the efficiencies of the target and the endogenous control amplifications were approximately equal.
  • the primers and probes used are listed below: mRNA Vendor Catalog #
  • Compound I reduced the mRNA levels of the short-lived anti-apoptotic proteins McI- 1 and XIAP, in primary CLL cells exposed to the compound for 6 hours or 24 hours, as shown in Figure 14 .
  • the results are presented relative to the gene expression of the time-matched controls.
  • Example 23 Compound I Effects on Anti-Apoptotic Proteins in Primary CLL Cells After incubation of primary CLL cells with Compound I for 6 or 24 hr at different concentrations, the cells were collected to measure the expression of anti-apoptotic proteins such as McI-I, XIAP and Bcl-2, and another short-lived oncogene, Myc, by immunoblotting, as described above for pol II studies in detail. The quantitative levels of the proteins were normalized to actin, and expressed as a percentage of time- matched controls. Primary antibodies
  • Example 25 - PARP cleavage correlates with Annexin V assay After incubation with Compound I or Flavopiridol (F) for 6 or 24 hours at different concentrations, toxicity was assessed by annexin V/PI staining (as described above at Example 19);_cleavage of PARP, an indicator of apoptosis, was assessed by immunoblotting.
  • the antibodies used are listed below: Primary antibodies
  • Percent cell death measured by annexin/PI staining generally approximated the percent cell death assessed by cleaved PARP and was Compound I-concentration dependent.
  • Example 26 Compound I Effects on Apoptosis. Survival Proteins, and Growth of Mantle Cell Lymphoma Cell Lines
  • MCL Mantle cell lymphoma
  • the biology of MCL is characterized by the t(l 1 : 14)(ql3;q32) translocation, which leads to the over-expression of Cyclin Dl which sustains the malignancy.
  • the transcript and protein of Cyclin Dl are both short-lived, and thus may be targeted by Compound I, providing further biological context to test this strategy, hi addition, Cdk2, which is highly expressed in MCL cells, is also blocked by Compound I.
  • Cdk2 which is highly expressed in MCL cells
  • MCL cell lines (Granta 519, Jeko-1, Mino and SP-53) were used in this study.
  • the cells were maintained in DMEM (Cellgro # 10-017-CV) with 20% FBS (Atlanta #S11150) (Granta-519); RPMI 1640 (Cellgro #10-040-CM) with 10% FBS (Jeko-1); RPMI 1640 (ATCC #30-2001) with 20% FBS (Mino); or RPMI 1640 (Cellgro) + 20% FBS (SP-53).
  • RNA synthesis was measured by quantitating incorporation of [ 3 H] uridine into the perchloric acid-insoluble materials. Briefly, after incubation with Compound I at designated times and concentrations, MCL cells were labeled for 30 min at 37 0 C with [ 3 H]uridine (Moravek Biochemical #MT799) at 1 ⁇ Ci/mL in a 15 mL centrifuge tube. At the end of incubation, 10 mL of ice-cold PBS were added to the tubes. The cells were centrifuged at 1500 rpm at 4 0 C for 5 min and washed again with 10 mL ice-cold PBS.
  • Example 29 Compound I-induced changes of RNA polymerase II phosphorylation Inhibition of phosphorylation of RNA polymerase II (pol II) was evaluated by immunoblotting using antibodies specific for the phosphorylated Ser2 and Ser5 sites of the C-terminal domain of RNA polymerase II, which are substrates of Cdk9 and Cdk7 respectively.
  • the MCL cells ( ⁇ 5 * 10 6 ) were collected and lysed by sonication in 500 ⁇ L buffer containing 25 mM HEPES (pH 7.5), 300 mM NaCl, 1.5 mM MgCl 2 , 0.5% sodium deoxycholate, 20 mM ⁇ -glycerophosphate, 1% Triton X-100, 0.1% SDS, 0.2 mM EDTA (pH 8), 0.5 mM DTT, 1 mM sodium orthovanadate (pH 10), 1 mM phenylmethylsulfonyl fluoride, 20 ⁇ g/mL aprotinin, and 20 ⁇ g/mL leupeptin.
  • Protein content in the lysate was determined using the Bio-Rad DC Protein Assay kit according to the manufacturer's instructions (Bio-Rad #5000112).
  • Cell lysate proteins (20 ⁇ g) were loaded to 4-12% Bis-Tris gel (Bio-Rad #3450124) and separated by SDS-polyacrylamide gel electrophoresis in MOPS running buffer (Bio- Rad #1610788), and then electro-transferred to a nitrocellulose membrane (GE Osmonics Labstore # EP2HY00010).
  • the membranes were blocked for 1 hr in PBS containing 5% nonfat dried milk and then incubated with primary antibodies for 3 hr, followed by incubation 1 hr with secondary antibodies labeled with fluorescent probes.
  • the membranes were scanned with the LI-COR Odyssey infrared imaging system to obtain images and quantitation. Phosphorylation levels were normalized to total pol ⁇ , and the relative phosphorylation of time-matched controls was calculated. As Cdk7 also activates Cdk2, phosphorylation of Cdk2 at Thrl ⁇ O was also investigated by immunoblotting. Primary and secondary antibodies used were: Primary antibodies
  • Example 30 Effect of Compound I on the mRNA level of anti-apoptotic proteins
  • the mRNA levels of anti-apoptotic proteins such as McI-I and Cyclin Dl were measured by Real-time quantitative PCR.
  • Total cellular RNA was isolated from the primary MCL cells using the RNeasy mini kit (Qiagen #74104) with DNase digestion (Qiagen #79254) to completely remove the genomic DNA.
  • Total RNA (20-50 ng) was used for the one-step real-time PCR reaction in the TaqMan ® One-Step RT-PCR Master Mix (Applied Biosystems #4313803).
  • PCR reaction was carried out in a 25 ⁇ L volume on 96-well optical reaction plate (Applied Biosystems #N8010560) for 30 min at 48°C for the reverse transcription reaction, followed by 10 min at 95 °C for initial denaturing, then followed by 40 cycles of 95 °C for 15 seconds and 60 0 C for 2 min in the 7900HT Sequence Detection System (Applied Biosystems).
  • the relative gene expression was analyzed by the Comparative threshold (Ct) method using 18S ribosomal RNA as endogenous control, after confirming that the efficiencies of the target and the endogenous control amplifications were approximately equal. The results were presented as the percentage of gene expression of the time-matched controls.
  • the primers and probes used here were listed below:
  • FIGS 18B and 19-21 show that Compound I decreases expression levels of these genes.
  • Example 31 Compound I effects on protein expression and induction of PARP cleavage
  • McI-I and Bcl-2 and other short-lived oncoproteins such as Cyclin Dl and Myc, generally as described in Example 23.
  • the quantitative levels of the proteins were normalized to actin, and expressed relative to time-matched controls. Cleavage of
  • PARP an indicator of apoptosis
  • Apoptosis was induced within 24 hr in all cell lines except for Granta, as shown by PARP cleavage, as well as the emergence of SubGl populations (Example 32, below). Apoptosis was strongly induced by 0.3 ⁇ M Compound I in Jeko and Mino cells after a 24 hr exposure, despite their deficiency in p53 function. Although apoptosis was not detected in Granta cells, clonogenic assays showed a strong, time- dependent inhibition of colony formation, with an IC 50 of about 50 nM after 24 hr incubation, indicating that Compound I effectively impaired MCL repopulating ability, as shown above in Figure 18 A.
  • Exponentially growing MCL cells were incubated with Compound I for 4 and 24 hr. About 5 x 10 5 cells were collected, washed with ice-cold PBS (pH 7.4) and fixed in 2.5 mL 70% ethanol. Fixed cells were washed twice with cold PBS before incubation in 300 ⁇ L PBS with 15 ⁇ g/mL propidium iodide (Sigma- Aldrich #P4864) and 2.5 ⁇ g/mL DNase-free RNase A (Roche, #11119915001). Fluorescence was measured on a Becton Dickinson FACSCalibur flow cytometer. Figure 22 shows that, except in Granta cells, a SubGl population emerged in all of the MCL lines upon 24 hr exposure to higher concentrations of Compound I.

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Abstract

L'invention concerne des procédés de traitement du cancer faisant appel à l'utilisation de N-[5-[[[5-(1,1-diméthyléthyl)-2-oxazolyl]méthyl]thio]-2-thiazolyl]-4-pipéridinecarboxamide. L'invention concerne également des procédés de traitement du cancer utilisant le composé susmentionné en combinaison avec d'autres thérapies.
PCT/US2007/025095 2006-12-08 2007-12-07 Procédés de traitement du cancer Ceased WO2008073304A2 (fr)

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US60/923,432 2007-04-13
US97151307P 2007-09-11 2007-09-11
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WO2012078492A1 (fr) * 2010-12-06 2012-06-14 Celgene Corporation Traitement d'association avec du lénalidomide et un inhibiteur de cdk pour traiter le myélome multiple

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US6515004B1 (en) * 1999-12-15 2003-02-04 Bristol-Myers Squibb Company N-[5-[[[5-alkyl-2-oxazolyl]methyl]thio]-2-thiazolyl]-carboxamide inhibitors of cyclin dependent kinases
WO2006101846A1 (fr) * 2005-03-16 2006-09-28 Aventis Pharmaceuticals Inc. Regime posologique de flavopiridol destine au traitement de cancer, notamment cll

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012078492A1 (fr) * 2010-12-06 2012-06-14 Celgene Corporation Traitement d'association avec du lénalidomide et un inhibiteur de cdk pour traiter le myélome multiple
US20140031325A1 (en) * 2010-12-06 2014-01-30 Celgene Corporation Combination therapy with lenalidomide and a cdk inhibitor for treating multiple myeloma

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