Abl1 (IC50 = 2.8 nM); TrkA (IC50 = 6 nM); Abl1 (IC50 = 2.8 nM); TrkB (IC50 = 9 nM); Tie-2 (IC50 = 22 nM); Aurora B (IC50 = 98 nM)

ABL001 is a potent, selective BCR-ABL inhibitor with a unique, allosteric mode of action that remains active against the majority of mutations, including T315I[1]. By binding to a regulatory site that wild-type ABL normally possesses a myristoyl group occupying, ABL001 inhibits ABL kinase activity in a different way than catalytic site inhibitors[2]. The myristoylated N-terminus of ABL1 typically resides in a pocket on the BCR-ABL kinase domain, which is where it binds. This myristoylated N-terminus is responsible for autoregulating ABL1 activity; it is lost upon fusion with BCR. By taking up its empty binding site, ABL001 functionally imitates the myristoylated N-terminus'sfunctionand reinstates the kinase activity's negative regulation. BCR-ABL-negative cell lines were unaffected at concentrations 1000 times higher than ABL001, which specifically inhibits the growth of Ph+ ALL and chronic myelogenous leukemia (CML) cells at potencies ranging from 1 to 10 nM[1]. ABL001 selectively and potently binds to the myristoyl pocket of ABL1 and induces the inactive C-terminal helix conformation, as confirmed by NMR and biophysical studies (dissociation constant (Kd) = 0.5-0.8 nM). ABL001 is inactive against G-protein-coupled receptors, ion channels, nuclear receptors, and transporters, among more than 60 kinases, including SRC. ABL001 is therefore highly selective[3].

ABL001 exhibits strong anti-tumor activity in the KCL-22 mouse xenograft model, as evidenced by total tumor regression and a pronounced dose-dependent relationship with pSTAT5 inhibition[1]. All species have a moderate half-life, volume of distribution, and oral absorption of ABL001. For patients with chronic myelogenous leukemia who have received a lot of pretreatment, it has been well tolerated as a single agent and has been shown to induce clinical anti-tumor activity. Regarding the pharmacokinetics, pharmacodynamics, and efficacy of ABL001, the CL (clearance) in mice, rats, and dogs following a single intravenous dose of 1 mg/kg, 2 mg/kg, and 1 mg/kg, respectively, are 12, 16, and 6 mL/min/kg. The T_1/2term for a single intravenous dose of 1 mg/kg in mice and dogs are 1.1 and 3.7 hours, respectively. Rats have a T_1/2term of 2.7 hours following a single intravenous dose of 2 mg/kg. Oral bioavailability of ABL001 at 30 mg/kg p.o. in rats and mice is 35% and 27%, respectively. On the other hand, ABL001's oral BA in dogs is 111% (15 mg/kg, p.o)[3].

ABL1 Biochemical kinase assay [3]
ABL1 WT (64-515aa) protein was produced by co-expression with YopH in Sf21 cells. Cells were harvested by centrifugation and resuspended in 25mM Tris pH 7.0, 500 mM NaCl, 5% glycerol, 10 mM Imidazole, 1x complete protease inhibitor tablet, Benzonase (1:10,000 v:v) and 1 mM TCEP. Cells were lysed by dounce homogenization and cleared by centrifugation. ABL1 WT (64-515 aa) was purified by affinity chromatography using a Ni-SepharoseFF column using two sequential washing steps using the resuspension buffer described above (containing 10 mM and 35mM imidazole respectively) and eluted in the same buffer containing 250 mM imidazole. Fractions containing ABL1 were pooled and loaded onto a pre-equilibrated SEC column in 25 mM Tris pH 7.0, 200 mM NaCl, 5% glycerol and 1 mM TCEP. The activity of the enzyme and compound inhibition was tested using a DELFIA® TRF assay. The reaction mixture contained 500 nM Biotin-EAIYAAPFAKKK peptide, 10 or 2000 µM ATP and 25 pM of ABL1 WT (64-515 aa) enzyme in a reaction buffer containing 50 mM HEPES pH 7.2, 10 mM MgCl2, 2 mM DTT and 0.01% Triton-X100. Reactions were carried out for 40 min in a volume of 60 µL and quenched with 20 µL 500 mM EDTA (final concentration 125 mM). 50 µL reaction solutions were transferred to NeutroAvidin-coated 384 well plate and incubated at room temperature for 1 hour with shaking. After washing with 100 µL/well TBST buffer, 50 µL/well Eu-anti-p-Tyr was added and the plate was incubated at 4 °C overnight with shaking. 50 µL/well DELFIA® enhancement solution was added and the plate incubated at ambient temperature for 5 min. The plate was read on the EnVision using time resolved fluorescence Ex/Em: 340/615 nm. For inhibition studies, compounds were serially diluted in DMSO, using a 16-point 3-fold format, from a 5 mM top concentration. Then 100 nL per well of serial diluted compounds were transferred to Grenier polypropylene v-bottom 384-well assay plates using acoustic transfer system. The final DMSO concentration was 0.16% and the final inhibitor concentration ranged from 50 µM to 3.48E-6 µM. Each compound was tested in duplicate and the inhibitor dose response curves analyzed using normalized IC50 regression curve fitting with control-based normalization using GraphPad Prism v6.02.[3]

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Asciminib, having a dissociation constant (Kd) of 0.5-0.8 nM and selectivity to the myristoyl pocket of ABL1, is a strong and selective allosteric inhibitor of BCR-ABL1.

Ba/F3 Proliferation assay [3]
For each cell line, cell density was adjusted to 50 000cells/ml and 50ul (2500 cells) added per well of a 384 well assay plate. Test compounds were resuspended in DMSO at a concentration of 10mM. A serial three-fold dilution of each compound with DMSO was performed in 384-well plates using the Janus Liquid Dispenser. 2nL compound was delivered to the assay plates containing 2500 cells in a 50 µL volume via acoustic delivery from an ATS-100 (EDC). Cells were incubated with compound at 37°C in a humidified environment with 5% carbon dioxide for 48 hours. Britelite plus solution was prepared according to the manufacturer’s instructions and 25 µl added to each well of the assay plate. Plates were incubated for 7 minutes and the luminescence detected on an EnVision Multimode plate reader. The degree of luminescence correlates with the number of cells in each well. The effect of each inhibitor concentration can therefore be calculated and IC50’s generated.

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For 48 hours, Ba/F3 cells are exposed to asciminib at a range of concentrations (0–10,000 nM). The Britelite luciferase detection assay is used to quantify the proliferation of cells.

Mice: Asciminib efficacy is measured using FACS monitoring of the percentage of CD45+ cells per live cell in blood samples obtained at different times following dosing with either 7.5 mg/kg BID (group 2) or 30 mg/kg BID (group 3) asciminib for three weeks in three patient-derived ALL systemic xenograft models (ALL-7015, AL-7119, and AL-7155).

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ABL001 (free base, solid dispersion form) was suspended in phosphate-buffered saline. Dosing solutions were prepared fresh every 3-4 days for dosing. ABL001 (free base, solution form) was formulated in 30% PEG 300, 6% Solutol HS15 in an acidic buffered solution. Dosing solutions were freshly prepared weekly for dosing.
Efficacy studies [3]
For efficacy studies in subcutaneous KCL-22 xenograft model, mice bearing tumors of 100- 300mm3 were randomized into treatment groups (n=6 per group) for daily compound treatment. Body weight and tumor volume were recorded twice weekly for the duration of each study. In ABL001 dose-response studies, studies were terminated when vehicle-treated animals reached 1500mm3 mean tumor volume. In ABL001 and nilotinib combination efficacy study, select randomized groups animals were dosed daily with either ABL001 or nilotinib as single agents until tumor relapse (tumor volume >500mm3), then switched to the other agent continuously until second relapse. Animals are terminated as their final tumor volume reached >600mm3 . Another randomized group received combination of both ABL001 and nilotinib daily treatment then continued monitoring post-treatment cessation. For efficacy studies in systemic primary Ph+ ALL xenograft models, mice were injected intravenously with 5x106 ALL cells. Blood was sampled weekly from tail snip to monitor tumor burden, and engrafted mice with >10% human CD45+ cells were randomized into treatment groups for compound treatment (n=6 mice per group).

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Pharmacokinetics (PK) / Pharmacodynamics (PD) studies [3]
Baseline tumor PD samples were collected from KCL-22 xenografts by fine needle biopsy before drug treatment. Animals received a single oral dose of ABL001 at 7.5 – 30 mg/kg. Blood was collected by serial tail bleed at designated time points (1-20h) for plasma PK analyses, and matching tumor PD samples were collected by fine needle biopsy at the same timepoints.

Absorption, Distribution and Excretion
The median Tmax of asciminib following oral administration is 2.5 hours. At a dose of 80mg once daily, the steady-state Cmax and AUCtau were 1781 ng/mL and 15112 ng.h/mL, respectively. At a dose of 40mg twice daily, the steady-state Cmax and AUCtau were 793 ng/mL and 5262 ng.h/mL, respectively. At a dose of 200mg twice daily (for treatment of T315I mutants), the steady-state Cmax and AUCtau were 5642 ng/mL and 37547 ng.h/mL, respectively. As compared to the fasted state, the co-administration of asciminib with a high-fat meal decreased the AUC and Cmax by 62% and 68%, respectively, and its co-administration with a low-fat meal decreased the AUC and Cmax by 30% and 35%, respectively.
Asciminib is eliminated via biliary secretion facilitated by breast cancer-resistant protein (BCRP) transporters. Following oral administration, approximately 80% and 11% of an asciminib dose was recovered in the feces and urine, respectively. Unchanged parent drug accounted for 57% of drug material recovered in the feces and 2.5% in the urine.
At steady-state, the apparent volume of distribution of asciminib is 151 L.
The total apparent clearance of asciminib is 6.7 L/h at a total daily dose of 80mg and 4.1 L/h at a dose of 200mg twice daily.

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Metabolism / Metabolites
Asciminib is negligibly metabolized, with unchanged parent drug comprising the main drug component in plasma (~93%) and following excretion (~57% in feces). The main circulating metabolites are M30.5, M44, and M29.5, accounting for approximately 5%, 2%, and 0.4% of the total administered dose, respectively. The oxidative metabolism of asciminib is mediated by CYP3A4, and the glucuronidation of asciminib is mediated by UGT2B7 and UGT2B17.

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Biological Half-Life
The terminal elimination half-life asciminib is 5.5 hours when administered at 40mg twice daily and 9.0 hours when administered at 200mg twice daily.

Hepatotoxicity
In the prelicensure clinical trials of asciminib in patients with refractory and extensively treated CML, ALT elevations arose in 13% of patients but were usually self-limited and mild. ALT elevations above 5 times the upper limit of normal (ULN) were uncommon, being found in 3% of treated patients. The ALT elevations were typically transient and rarely required dose interruption or modification. In the open label and controlled trials supporting the approval of asciminib, there were no instances of clinically apparent liver injury, hepatic failure or deaths from liver injury. Furthermore, patients with aminotransferase elevations during therapy with first and second line BCR-ABL1 inhibitors did not have an increased rate of such elevations during asciminib therapy. Since its approval in the United States and Europe, there have been no reported cases of clinically apparent liver injury associated with asciminib therapy.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent liver injury).

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Protein Binding
_In vitro_, asciminib is 97% bound to plasma proteins, although the specific protein(s) to which it binds are unclear.

[1]. Blood (2014) 124 (21): 398.

[2]. Clin Cancer Res (2017) 23 (1_Supplement): IA01.

[1]. Nature. 2017, 543: 733-737.

Asciminib is a tyrosine kinase inhibitor (TKI) used in the treatment of chronic-phase Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML). More specifically, it is an inhibitor of the ABL1 kinase activity of the BCR-ABL1 fusion protein which serves as a driver of CML proliferation in most patients with the disease. It has also shown benefit in Ph+ CML with the T315I mutation, which produces a mutant BCR-ABL1 which is typically treatment-resistant as compared to wild-type BCR-ABL1. Existing inhibitors of ABL compete at the ATP binding sites of these proteins and can be classified into those that target the active conformation of the kinase domain ([dasatinib], [bosutinib]) and those that target the inactive kinase domain ([imatinib], [nilotinib], [ponatinib]). Asciminib is unique in that it acts as an allosteric inhibitor, binding at the myristoyl pocket of the BCR-ABL1 protein and locking it into an inactive conformation. Asciminib received FDA approval on October 29, 2021 (Scemblix, Novartis AG).
Asciminib is a tyrosine kinase inhibitor that specifically targets myristoyl pocket of ABL1 and is used to treat refractory forms of Philadelphia chromosome positive chronic myelocytic leukemia. Serum aminotransferase elevations occur in a proportion of patients treated with asciminib, but episodes of clinically apparent liver injury with jaundice have not been reported with its use.
Asciminib is an orally bioavailable, allosteric Bcr-Abl1 tyrosine kinase inhibitor, with antineoplastic activity. Upon administration, asciminib targets and binds to the myristoyl pocket of the Bcr-Abl1 fusion protein at a location that is distinct from the ATP-binding domain, thereby inhibiting the activity of both wild-type Bcr-Abl and certain mutation forms, including the T315I mutation. This binding results in the inhibition of Bcr-Abl1-mediated proliferation and enhanced apoptosis of Philadelphia chromosome-positive (Ph+) hematological malignancies. The Bcr-Abl1 fusion protein tyrosine kinase is an abnormal enzyme produced by leukemia cells that contain the Philadelphia chromosome.
See also: Asciminib Hydrochloride (has salt form).

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Drug Indication
Asciminib is indicated for the treatment of adult patients with Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML) in chronic phase who have been previously treated with ≥2 tyrosine kinase inhibitors. It is also indicated in the treatment of Ph+ CML in adult patients with the T315I mutation.
Scemblix is indicated for the treatment of adult patients with Philadelphia chromosome positive chronic myeloid leukaemia in chronic phase (Ph+ CML CP) previously treated with two or more tyrosine kinase inhibitors (see section 5. 1).
Treatment of chronic myeloid leukaemia

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Mechanism of Action
In most patients with chronic myeloid leukemia (CML), progression of the disease is driven primarily by a translocation of the Philadelphia chromosome that creates an oncogenic fusion gene, _BCR-ABL1_, between the _BCR_ and _ABL1_ genes. This fusion gene produces a resultant fusion protein, BCR-ABL1, which exhibits elevated tyrosine kinase and transforming activities that contribute to CML proliferation. Asciminib is an allosteric inhibitor of the BCR-ABL1 tyrosine kinase. It binds to the myristoyl pocket of the ABL1 portion of the fusion protein and locks it into an inactive conformation, preventing its oncogenic activity.

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Pharmacodynamics
Asciminib exerts its therapeutic activity by inhibiting an oncogenic protein responsible for the proliferation of CML. It may be administered orally once or twice a day depending on the condition being treated. By increasing the total daily dose 5-fold as compared to standard therapy (80mg daily vs. 400mg daily), it can be used to treat Ph+ CML with the T315I mutation, a typically treatment-resistant variant of the disease. As with many other chemotherapeutic agents, asciminib treatment can result in various forms of myelosuppression, including thrombocytopenia and neutropenia. Patients should receive frequent laboratory monitoring throughout therapy and dose adjustments may be required based on the severity of observed effects. Patients may also experience pancreatic and/or cardiovascular toxicity, both of which require frequent monitoring and may require dose adjustments as per prescribing information.

C20H18CLF2N5O3

449.84

449.106

C, 53.40; H, 4.03; Cl, 7.88; F, 8.45; N, 15.57; O, 10.67

1492952-76-7

Asciminib hydrochloride;2119669-71-3

72165228

White to off-white solid powder

1.5±0.1 g/cm3

631.7±55.0 °C at 760 mmHg

335.8±31.5 °C

0.0±1.9 mmHg at 25°C

1.662

2.1

3

8

6

31

626

1

ClC(OC1C=CC(=CC=1)NC(C1C=NC(=C(C2=CC=NN2)C=1)N1CC[C@H](C1)O)=O)(F)F

VOVZXURTCKPRDQ-CQSZACIVSA-N

InChI=1S/C20H18ClF2N5O3/c21-20(22,23)31-15-3-1-13(2-4-15)26-19(30)12-9-16(17-5-7-25-27-17)18(24-10-12)28-8-6-14(29)11-28/h1-5,7,9-10,14,29H,6,8,11H2,(H,25,27)(H,26,30)/t14-/m1/s1

N-[4-[chloro(difluoro)methoxy]phenyl]-6-[(3R)-3-hydroxypyrrolidin-1-yl]-5-(1H-pyrazol-5-yl)pyridine-3-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 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: ≥ 2.5 mg/mL (5.56 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.56 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 25.0 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.)