Typically, for injectable use, sterile aqueous solutions or dispersion of, and sterile powders of, a compound used in the disclosed methods for the extemporaneous preparation of sterile injectable solutions or dispersions are appropriate.

For nasal administration, the compounds used in the disclosed methods can be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.

For buccal or sublingual administration, the compounds used in the disclosed methods can be formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine, as tablets, lozenges or pastilles.

For rectal administration, the compounds used in the disclosed methods can be formulated in the form of suppositories containing a conventional suppository base such as cocoa butter.

EXAMPLES

The following examples are intended to be illustrative and are not meant in any way to limit the scope of the disclosure.

Example 1. In Vitro Cytotoxicity (IC₅₀) Assay

Cells were seeded in triplicate at 10,000 per 80 uL, into 96-well plates 24 hours before compound overlay and cultured at 37° C. and 5% CO₂. Compound (I) was prepared as a 10 mM stock in 100% DMSO and diluted with RPMI 1640 cell growth medium containing 10% FBS such that the final concentrations ranged from 50 pM to 15 uM. Aliquots (20 uL) from each concentration were overlaid to 80 uL of pre-seeded cells to achieve final concentrations of 10 pM to 3 uM. After 3 days, cell viability in each well was assessed by alamarBlue assay according to the manufacturer's instruction (ThermoFisher Scientific). Absorbance was read at 570 nm using a SpectraPlus microplate reader (Molecular Devices Corporation). alamarBlue absorbance values were adjusted by subtracting the average of the baseline readings from untreated cells assessed one day after cell seeding. The percentage (%) of relative inhibition of cell viability was calculated by comparing to DMSO-treated cells. IC₅₀s were calculated using GraphPad PRISM software (GraphPad Software Inc.). The results are listed in Table 1 below and FIG. 1.

TABLE 1

Response to
Compound (I)

Cell Line
Cell Type
Compound (I)
IC₅₀(nM)
TP53 Status
PTEN Status

RAJI
NHL, B-Cell
death
0.1
R213Q,
WT

Burkitt

Y234H

DAUDI
NHL, B-Cell
death
0.2
R213*
V175G

Burkitt

G266E

JURKAT
T-ALL
death
0.6
multiple
multiple

mutations
mutations

KOPN-8
B-ALL
death
0.7
R248Q (het)
WT

THP-1
AML M5
death
1.4
p.R174fs*3
WT

(het)

TF-1
AML M6
death
2.2
p.I251fs (het)
WT

splice site

MOLT-4
T-ALL
death
4.1
R306*
p.K267fs*9

REH
B-ALL
death
5.1
R181C
R173C

MV4-11
AML M5
death
5.7
L344
WT

mutation

OCI-LY10
NHL, ABC-
death
6.8
K319*
splice site

DLBCL

HL-60
AML M2
death
8.4
null
promoter

hypermethylation

SU-DHL-8
NHL,
death
12
Y234N,
WT

DLBCL

R249G

K-562
CML
mitotic defects +

p.Q136fs*13
WT

arrest

OCI-LY18
NHL, GCB-
mitotic defects +

WT
WT

DLBCL
arrest

OCI-AML4
AML M4
mitotic defects +

WT
WT

arrest

OCI-LY3
NHL, ABC-
mitotic defects +

WT
WT

DLCBL
arrest

NB4
AML M3
mitotic defects +

K319, L344
WT

arrest

mutations

NALM-16
B-ALL
mitotic defects +

WT
WT

arrest

OCI-AML2
AML M4
mitotic defects +

WT
WT

arrest

RS4;11
B-ALL
mitotic defects +

I254T (het)
WT

arrest

OCI-LY8
NHL, GCB-
mitotic defects +

WT
WT

DLBCL
arrest

NALM-6
B-ALL
mitotic defects +

WT
WT

arrest

OCI-AML3
AML M4
mitotic defects +

WT
WT

arrest

Example 2. Compound (I) Fumarate Inhibits Tumor Growth in MOLT-4 ALL Model

Drug and test system

Drug Name/s
Compound (I)

Batch reference/s
Fumarate batch 16

Dosage Groups (mg/kg)
Compound (I) 7.5 mg/kg qd

Compound (I) 13.5 mg/kg 2-on/5-off

Vincristine 0.5 mg/kg IP, qw

Dosing Volume
10 mL/kg

Formulation
Compound (I) will be dissolved/

suspended in water before dosing.

Vehicle control group
Water (10 mL/kg)

Positive control Group
Vincristine diluted in PBS before dosing

Route of administration
PO (oral), daily for Compound (I), IP

(intraperitoneal injection), weekly for vincristine

Tumor cell type and line
MOLT-4 acute lymphoblastic leukemia

Number of cells and
1 × 10⁷cells in a volume of 0.1 ml

volume injected

Tumor Cell Culture:

The human acute lymphoblastic leukemia cell line MOLT-4 was acquired from American Type Culture Collection (Manassas, VA, USA) and grown in RPMI medium containing 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate. The media was supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 0.11% sodium pyruvate. The tumor cells were maintained in a humidified environment of 5% CO₂and 95% air at 37° C. Cells were verified to be free of mouse pathogens by IMPACT IV PCR analysis (IDEXX RADIL, Colombia, MO, USA) before injection.

Animals and Tumor Cells:

Female SCID mice were purchased from the Ontario Cancer Institute and received and acclimated at the MaRS-TMDT Animal Resources Centre for 2 weeks prior to the start of the experiment. The mice were fed ad libitum autoclaved water and Rodent Lab Diet (Harlan Teklad LM-485) consisting of 19% crude protein, 5% crude fat, and 5% crude fiber. Mice were housed in microisolator cages and maintained in an environment with a 12-hour light cycle at 20-22° C. and 40-60% humidity. On the day of implantation, MOLT-4 cells were harvested and resuspended with serum free DMEM to a concentration of 1×10⁸/mL and each mouse was injected subcutaneously with a volume of 0.1 mL containing 1×10⁷MOLT-4 cells in the right rear flank.

Controls and Dosing:

Animals were dosed Compound (I) via oral gavage in a volume of 10 mL/kg. The animals were dosed with a 2.25 mm×50 mm curved gavage needle affixed to a 1 mL syringe. Vincristine was dosed by IP injection once per week.

in-Life Observations:

Toxicity was evaluated by body weight measurements and clinical observations. The mice were observed daily for overt signs of any adverse, treatment related side effects, and clinical signs of toxicity. Acceptable toxicity was set at a group mean body weight loss of less than 20% during the study and not more than one treatment related death in a group. Animals were monitored for body weights and tumor growth where tumor volumes were calculated using the formula: tumor volume=width²×length/2.

Results

The study evaluated Compound (I) fumarate administered by oral gavage for 21 days, either daily at 7.5 mg/kg or 2 days on/5 days off at 13.5 mg/kg, in comparison to the standard-of-care agent vincristine, dosed in 4 weekly intraperitoneal injections of 0.5 mg/kg, in the MOLT-4 acute myeloid leukemia xenograft model in SCID (severe combined immunodeficiency) mice. Xenografts were established by subcutaneously injecting 1×10⁷tumor cells into the right flank of female SCID mice. Treatment was initiated 9 days following implantation when tumor volumes reached a mean volume of approximately 250 mm³.

By day 13, the average volume of the control tumors was 1754 mm³, averaging 683% tumor growth. On day 13 the average tumor growth inhibition in the vincristine treated arm was 18% (p=0.21). Compound (I) oral dosing daily at 7.5 mg/kg resulted in an average of 77% tumor regression (p=2.5*10⁻⁶) with regression observed in 7 of 8 tumors, and dosing 2-on/5-off at 13.5 mg/kg resulted in an average of 28% regression (p=6.5*10⁻⁶), with regression observed in 7 of 8 tumors. After 21 days of dosing, tumors continued to regress, with maximum antitumor efficacy observed on Day 25, with 8 of 8 complete regressions in the daily dosing arm and 6 of 8 complete regressions in the 2-on/5-off arm (average 96% regression). Tumor regrowth eventually occurred in all animals, with the last animal taken off study due to excessive tumor burden on Day 68.

The result is shown in FIG. 2 and FIG. 3. Specifically, FIG. 2 shows tumor volume (MOLT-4) in SCID mice vs treatment day. FIG. 3 shows the body weight (SCID mice) vs treatment day. In all, Compound (I) is very efficacious and potentially curative in the MOLT-4 acute lymphoblastic leukemia xenograft model.

Protocol Design

Batch/
Salt
Bio-equiv.

Group
N
Test Article
supplier
form
ratio
Vehicle
Route, schedule, and dose

1
6
Vehicle
N/A
N/A
N/A
water
PO QD + IP QW

2
6
Compound
16
fumarate
0.82
water
PO, 7.5 mg/kg QD × 21

(I)

3
6
Compound
16
fumarate
0.82
water
PO, 13.5 mg/kg 2-on/

(I)

5-off × 3 (21 days)

4
6
Vincristine
Sigma
Sulfate
0.89
PBS
IP, 0.5 mg/kg QW × 4

Example 3. Compound (I) Fumarate Inhibits Tumor Growth in MV4-11 AML Model

Drug and test system

Drug Name/s
Compound (I)

Batch reference/s
Fumarate batch 16

Dosage Groups (mg/kg)
Compound (I)-16 7.5 mg/kg qd

Compound (I)-16 13.5 mg/kg 2-on/5-off

Vincristine 0.5 mg/kg IP, qw

Dosing Volume
10 mL/kg

Formulation
Compound (I) will be dissolved/

suspended in water before dosing.

Vehicle control group
Water (10 mL/kg)

Positive control Group
Vincristine diluted in PBS before dosing

Route of administration
PO (oral), daily for Compound (I), IP

(intraperitoneal injection), weekly for vincristine

Tumor cell type and line
MV4-11 acute myeloid leukemia

Number of cells and
1 × 10⁷cells in a volume of 0.1 ml

volume injected

Tumor Cell Culture:

The human acute myeloid leukemia cell line MV4-11 was acquired from American Type Culture Collection (Manassas, VA, USA) and grown in RPMI medium containing 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate. The media was supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 0.11% sodium pyruvate. The tumor cells were maintained in a humidified environment of 5% CO₂and 95% air at 37° C. Cells were verified to be free of mouse pathogens by IMPACT IV PCR analysis (IDEXX RADIL, Colombia, MO, USA) before injection.

Animals and Tumor Cells:

Female SCID mice were purchased from the Ontario Cancer Institute and received and acclimated at the MaRS-TMDT Animal Resources Centre for 2 weeks prior to the start of the experiment. The mice were fed ad libitum autoclaved water and Rodent Lab Diet (Harlan Teklad LM-485) consisting of 19% crude protein, 5% crude fat, and 5% crude fiber. Mice were housed in microisolator cages and maintained in an environment with a 12-hour light cycle at 20-22° C. and 40-60% humidity. On the day of implantation, MV4-11 cells were harvested and resuspended with serum free DMEM to a concentration of 1×10⁸/mL and each mouse was injected subcutaneously with a volume of 0.1 mL containing 1×10⁷MV4-11 cells in the right rear flank.

Controls and Dosing:

Animals were dosed Compound (I) via oral gavage in a volume of 10 ml/kg. The animals were dosed with a 2.25 mm×50 mm curved gavage needle affixed to a 1 ml syringe. Vincristine was dosed by IP injection once per week.

in-Life Observations:

Toxicity was evaluated by body weight measurements and clinical observations. The mice were observed daily for overt signs of any adverse, treatment related side effects, and clinical signs of toxicity. Acceptable toxicity was set at a group mean body weight loss of less than 20% during the study and not more than one treatment related death in a group. Animals were monitored for body weights and tumor growth where tumor volumes were calculated using the formula: tumor volume=width²×length/2.

Results

The study evaluated Compound (I) fumarate administered by oral gavage, either daily at 7.5 mg/kg or 2 days on/5 days off at 13.5 mg/kg, in comparison to the standard-of-care agent vincristine, dosed in 4 weekly intraperitoneal injections of 0.5 mg/kg, in the MV4-11 acute myeloid leukemia xenograft model in SCID (severe combined immunodeficiency) mice. Xenografts were established by subcutaneously injecting 1×10⁷tumor cells into the right flank of female SCID mice. Treatment was initiated 14 days following implantation when tumor volumes reached a mean volume of approximately 90 mm³. Daily oral dosing of Compound (I) at 7.5 mg/kg was halted after 14 days due to excessive body weight loss. Animals were dosed in the 2-on/5-off schedule for 21 days. Dosing of Compound (I) by either schedule completely regressed tumors by day 10; by this point the average volume of the control tumors was 589 mm³. By day 13, the average volume of the control tumors was 878 mm³, averaging 1666% tumor growth over the 13 days. On day 13 the average tumor growth inhibition in the vincristine treated arm was 85% (p=0.14).

Animals remained tumor-free until Day 24 in the 2 days-on/5 days-off arm, and Day 74 in the daily dosing arm. Four of 6 animals in the daily dosing arm remained tumor free until the last measurement time point (Day 128).

The result is shown in FIG. 4 and FIG. 5. Specifically, FIG. 4 shows tumor volume (MV4-11) in SCID mice vs treatment day. FIG. 5 shows the body weight (SCID mice) vs treatment day. In all, Compound (I) is very efficacious and potentially curative in the MV4-11 acute myeloid leukemia xenograft model.

Protocol Design

Batch/
Salt
Bio-equiv.

Group
N
Test Article
supplier
form
ratio
Vehicle
Route, schedule, and dose

1
6
Vehicle
N/A
N/A
N/A
water
PO QD + IP QW

2
6
Compound
16
fumarate
0.82
water
PO, 7.5 mg/kg QD × 13

(I)

3
6
Compound
16
fumarate
0.82
water
PO, 13.5 mg/kg 2-on/

(I)

5-off × 3 (21 days)

4
6
Vincristine
Sigma
Sulfate
0.89
PBS
IP, 0.5 mg/kg QW × 4

Example 4. Phase 1 Study of Compound (I) Fumarate in Patients with Relapsed or Refractory Acute Myeloid Leukemia or Myelodysplastic Syndrome

This study (NCT number: NCT03187288) is a multi-center, phase 1, dose-escalation trial designed to assess the safety, tolerability, pharmacokinetics, and clinical benefit of treatment with Compound (I) fumarate administered orally over a range of doses in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) that is relapsed or refractory to current treatment or for which no curative therapy exists.

Results: 9 patients with relapsed or refractory AML were treated with escalating doses of Compound (I) (64 mg to 128 mg) administered orally once daily. Among them, 3 patients were dosed at 64 mg, 4 patients were dosed at 96 mg, and 2 patients were dosed at 128 mg. More detailed information with respect to the patients is provided in the table below. Of six patients evaluable for response, two (33%) achieved complete remission (CR), and 3 patients (50%) had stable disease (with one patient having a 78% reduction in marrow blast count). The dose limiting toxicity (DLT) was determined to be colitis at the 128 mg dose level. The 96 mg dose level was suggested for further exploration. However, only 4 patients were enrolled at the 96 mg dose level before the study was paused for enrollment and was amended.

Characteristics
N = 9

Age (years), median (range)
65 (61-78)

Gender

Male
6

Female
3

Diagnosis

AML
8 (89%)

MDS/MPN
1 (11%)

Number of prior therapies,
0 (0-4)

median (range)

Karyotype

Complex
6 (67%)

Del 5q
1 (11%)

Chromosome 7 abnormally
2 (22%)

Somatic mutations (n = 6)

TP53
2 (33%)

IDH1/2
1 (17%)

FLT3
0

NPM1
0

Conclusion: single agent Compound (I) has activity in patients with poor risk AML.

Example 5. Phase 1b/2 Study of Compound (I) Fumarate as a Single Agent or in Combination with Azacitidine or Decitabine in Patients with Acute Myeloid Leukemia, Myelodysplastic Syndrome or Chronic Myelomonocytic Leukemia

Part 1A Single Agent Dose Optimization Lead-In

The primary objectives of this study are (i) to assess the safety and tolerability, and identify the maximum tolerated dose (MTD) of Compound (I) administered orally in patients with acute myeloid leukemia (AML), Myelodysplastic Syndromes (MDS) or Chronic Myelomonocytic Leukemia (CMML); and (ii) to determine the Recommended Phase 2 Dose of Compound (I) in patients with AML, Myelodysplastic Syndromes (MDS) or Chronic Myelomonocytic Leukemia (CMML).

The secondary objectives of this study are (i) to determine the PK of Compound (I) administered orally in patients with AML, Myelodysplastic Syndromes (MDS) or Chronic Myelomonocytic Leukemia (CMML); (ii) to determine the Composite Complete Remission Rate, CRc (complete remission+complete remission with incomplete blood count recovery+complete remission with incomplete platelet count recovery [CR+CRi+CRp]) of Compound (I) in patients with AML; and (iii) to determine the Overall Response Rate (ORR, defined as Complete remission+Marrow CR+Partial remission+Hematologic Improvement (CR+mCR+PR+HI) in MDS or CMML.

Study Design and Methodology

In Part 1A, patients will receive oral Compound (I) daily, in the morning and in the absence of food, i.e. not eating for 2 h before or 1 h after taking their dose, for 21 days followed by 7 days off (28-day cycles), except on Day 1 and Day 21 of Cycle 1 when Compound (I) will be administered after an overnight fast. Water is permitted ad lib following 1 hour after the dose administration after the overnight fast; no food is allowed for at least 4 hours after the dose. The starting dose for Compound (I) is 32 mg and is based on preliminary clinical data. Patients who complete the initial cycle of therapy without evidence of significant toxicity or clinical evidence of progressive disease) may receive additional 28-day cycles of treatment at the same dose level for up to 6 cycles and if no PR or better is achieved, the Investigator should remove the patient from study following discussion with the Medical Monitor.