Bcr-Abl1 tyrosine kinase (IC50 = 7 nM)

BCR-ABL1 tyrosine autophosphorylation was potently inhibited in Ba/F3 cells expressing BCR-ABL1, BCR-ABL1L248V, BCR-ABL1Y253H, or BCR-ABL1E255V upon vodobatinib (K0706; 0-2000 nM) treatment [1].

As of 15 Jul 2020, 31 CP-CML pts received vodobatinib at doses of 12 to 240 mg; 16 pts (9 males) in ponatinib treated (PT) cohort [7 (44%) ponatinib was the immediate prior TKI] and 15 pts (7 males) in the ponatinib naïve (PN) cohort. The baseline demographics and disease history are represented in Table 1.[2]
Efficacy: Median duration of treatment was 17.3 (0.6-36) and 14.8 (0.5- 42) months in the Ponatinib treated and naive groups, respectively; 11 pts in the PT group [2 in Deep molecular response (DMR), 3 in MMR; 5 in MCyR (2 in CCyR and 3 in PCyR); 1 in stable disease] and 10 pts in the PN group (2 in DMR, 4 in MMR and 3 in CCyR, 1 in stable disease) are continuing on treatment. Overall efficacy outcomes are included in Tables 2 and 3.[2]
Of 16 PT pts, 2 (13%) pts, both with double mutations, had disease progression. Of 15 PN pts, 4 (26%) pts (with baseline mutation of T315I at 48 mg, Y253H at 66 mg, F317L and E255V mutation at 174 mg) progressed.[2]

Sanger sequencing of BCR-ABL1 kinase domain[1]
DNA isolated from Ba/F3 BCR-ABL1 expressing lysates was used as a template for amplification of BCR-ABL1 kinase domain. Amplification (Phusion High-Fidelity DNA Polymerase) was performed using a two-step PCR to excluded endogenous ABL1. PCR products were electrophoresed on a 1% agarose gel to confirm amplification, purified, and Sanger sequenced.

Cell proliferation IC50 determination[1]
IL-3 dependent murine Ba/F3 cells cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% L-glutamine and IL-3 from WEHI-conditioned media were infected with retrovirus expressing p210 BCR-ABL1 (MSCV-IRES-GFP). Selection of infected cells was performed by IL-3 withdrawal. Clinically relevant BCR-ABL1 point mutations were introduced using site-directed mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit). Retrovirus generated using calcium phosphate transfection of 293FT cells and harvesting the supernatant. Ba/F3 BCR-ABL1 and BCR-ABL1-mutants were plated in 96-well plates (2x103 cells/well) and incubated with indicated inhibitor for 72 hours. Inhibitor concentrations: K0706, nilotinib, dasatinib, bosutinib and ponatinib (0, 4.9, 9.8, 19.5, 39.1, 78.1, 156.3, 315.5, 625, 1250, 2500, and 5000 nmol/L); Retested sensitive cell lines (IC50 ≤ 3) from original screen at (0, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 10, and 200 nmol/L). Proliferation was measured using methanethiosulfonate (MTS)-based viability assay (CellTiter 96 AQueous One Solution, Promega). IC50 values are reported as the mean of three independent experiments performed in quadruplicate.

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BCR-ABL1 tyrosine phosphorylation immunoblot analysis [1]
Ba/F3 cells expressing native and mutated BCR-ABL1 were plated in 24-well plates (2x106 cells/well) and cultured in 1 mL RPMI complete medium with titrated concentrations of TKI (0, 15.6, 32.3, 62.5, 125, 250, 500, 1000, and 2000 nmol/L) for 4 hours. Cells were washed with cold PBS and lysed in M-PER (Mammalian protein extraction reagent) containing phosphatase and protease inhibitor cocktails on ice for 10 min. Samples were diluted 1:1 in SDS-Page loading buffer (2x Laemmli sample buffer) supplemented with 2-mercaptoethanol and denatured by boiling for 10 min at 95°C. Lysates were separated on 4-20% Tris-Glycine (Mini-PROTEAN TGX) gradient gels, transferred and immunoblotted using anti-c-Abl (Ab-3) mouse mAb (24-21), anti-phospho-c-Abl (Tyr204) (C42B5) rabbit mAb, and anti-β-actin (D6A8) rabbit mAb.

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Cell-based resistance assay [1]
Native BCR-ABL1 expressing Ba/F3 cells were treated with N-ethyl-N-nitrosourea (ENU, 50 μg/mL) overnight, pelleted, resuspended in fresh RPMI complete medium, and plated in 96-well plates (1.25x105 cells/well) supplemented with graded concentrations of K0706 (400, 800, 1600, and 3200 nmol/L) in quintuplicates. ENU was inactivated overnight with a sodium thiosulfate (20% w/v) and sodium hydroxide (100 mmol/L) and safely disposed of according to protocol. Wells were monitored for outgrowth, visually, using a microscope every two days for 30 days. Fifty outgrowth colonies from each treatment group were randomly selected and expanded in 24-well plates containing an equivalent concentration of K0706 as the original 96-well plate. Expanded cells in 24-well plates were harvested by centrifugation and stored at -80°C.

Multiple escalating doses of vodobatinib (once daily) in 28-day cycles were evaluated in a 3+3 study design. The primary objective was determination of the maximal tolerated dose (MTD) or recommended phase 2 dose (RP2D) along with safety and a secondary objective was to evaluate anti-leukemic activity. Dose escalation involved dose doubling until 2 pts in a cohort experienced Grade 2 toxicity, or 1 pt experienced Grade 3 or 4 toxicity, after which dose escalation was reduced to 40% increments. Treatment continued until unacceptable toxicity, disease progression (PD), consent withdrawal, or death.[2]

Among patients receiving panatinib, the most common treatment-interventional adverse events (TEAEs) (all grades) included nausea (4 cases, 25%) and diarrhea (3 cases, 25%). Other common TEAEs included thrombocytopenia (3 cases, 19%), rash (3 cases, 19%), non-cardiac chest pain (3 cases, 19%), elevated amylase (3 cases, 19%), and falls (3 cases, 19%). Ten patients (63%) reported ≥ grade 3 TEAEs, including one case each of anemia, lymphopenia, falls, skull fracture, spinal fracture, elevated lipase, fluid overload, syncope, dyspnea, and hypertension. Voldobatinib-related adverse events included elevated amylase, elevated lipase, dyspnea, fluid overload, thrombocytopenia, and neutropenia. Adverse events of grade ≥3 during treatment reported in more than one patient included neutropenia (2 cases, 13%), elevated amylase (2 cases, 13%), and thrombocytopenia (2 cases, 13%). [2] In patients with PN, the most frequently reported adverse events during treatment (all grades) included myalgia (5 cases, 33%) and back pain (4 cases, 27%). Other common adverse events during treatment included thrombocytopenia (4 cases, 27%) and nasopharyngitis (3 cases, 20%). Seven (47%) patients reported adverse events (TEAEs) of grade ≥3 during treatment (1 case of anemia, 1 case of pneumonia, 1 case of neutropenia, 1 case of gout, 1 case of hypokalemia, 1 case of thrombocytopenia, 1 case of elevated liver and pancreatic enzymes, 1 case of dementia, and 1 case of amnesia). AEs associated with vordobatinib included elevated alanine aminotransferase, elevated bilirubin, amnesia, neutropenia, and thrombocytopenia. No grade ≥ 3 events were reported in more than one patient. [2] Overall, three cardiovascular TEAEs were reported in two patients (one in the PT group and one in the PN group), all of which were considered unrelated to vordobatinib. Three patients died during the study: one in the PT group died from disease progression; one died from pneumonia (suspected COVID-19); and one died from… Intracranial hemorrhage occurred in the PN group. This intracranial hemorrhage event (grade 5 adverse event) was considered to be possibly associated with disease progression to the extramedullary blast crisis and was influenced by disease progression to extramedullary lesions. Two dose-limiting toxicities were reported at the highest dose of 240 mg. The next lower dose level of 204 mg was determined to be the maximum tolerated dose (MTD) and was well-tolerated in patients with chronic phase chronic myeloid leukemia (CP-CML) who had previously received extensive treatment. [2]

[1]. BCR-ABL1 tyrosine kinase inhibitor K0706 exhibits preclinical activity in Philadelphia chromosome-positive leukemia. Exp Hematol. 2019 Sep;77:36-40.e2.

[2]. Phase 1 Trial of Vodobatinib, a Novel Oral BCR-ABL1 Tyrosine Kinase Inhibitor (TKI): Activity in CML Chronic Phase Patients Failing TKI Therapies Including Ponatinib. Session: 632: Chronic Myeloid Leukemia: Therapy: CML: New and Beyond.

Vodobatinib is an orally bioavailable Bcr-Abl tyrosine kinase inhibitor (TKI) with potential antitumor activity. After administration, vodobatinib selectively targets and binds to the Bcr-Abl fusion oncoprotein, including various Bcr-Abl mutants, such as those carrying the "gatekeeper" resistance mutation T315I. This inhibits the proliferation of tumor cells expressing Bcr-Abl. The Bcr-Abl fusion protein is an aberrantly activated tyrosine kinase produced by certain leukemia cells. T315I is an amino acid substitution at position 315 of the ABL1 portion of the Bcr-Abl fusion protein tyrosine kinase, where threonine (T) is mutated to isoleucine (I). It plays a crucial role in resistance to certain chemotherapeutic agents, and its expression is associated with poor prognosis.

C27H20CLN3O2

453.919605255127

453.124

C, 71.44; H, 4.44; Cl, 7.81; N, 9.26; O, 7.05

1388803-90-4

89884852

White to off-white solid powder

1.4±0.1 g/cm3

748.6±60.0 °C at 760 mmHg

406.5±32.9 °C

0.0±2.5 mmHg at 25°C

1.701

5.73

2

3

4

33

774

0

ClC1=CC=CC(C)=C1C(NNC(C1C=CC(C)=C(C#CC2=CN=C3C=CC=CC3=C2)C=1)=O)=O

ZQOBVMHBVWNVBG-UHFFFAOYSA-N

InChI=1S/C27H20ClN3O2/c1-17-10-12-22(26(32)30-31-27(33)25-18(2)6-5-8-23(25)28)15-20(17)13-11-19-14-21-7-3-4-9-24(21)29-16-19/h3-10,12,14-16H,1-2H3,(H,30,32)(H,31,33)

2-chloro-6-methyl-N'-[4-methyl-3-(2-quinolin-3-ylethynyl)benzoyl]benzohydrazide

Vodobatinib; K-0706; K0706; SCO-088; K0706; Vodobatinib [USAN]; K-0706; N8Q12KU2SW; 2-Chloro-6-methyl-N'-(4-methyl-3-(quinolin-3-ylethynyl)benzoyl)benzohydrazide; K0706;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~275.38 mM)

Solubility in Formulation 1: 2.08 mg/mL (4.58 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)