Kit (IC50 = 200 nM); Lyn B (IC50 = 510 nM); PDGFRα (IC50 = 540 nM); PDGFRβ (IC50 = 800 nM); Abl1 (IC50 = 1.20 μM)

At doses ≤500 nM, masatinib inhibits ATP competitively. Additionally, mashitinib strongly inhibits the intracellular kinase Lyn, recombinant PDGFR, and, to a lesser degree, fibroblast growth factor receptor 3. Masitinib, on the other hand, only slightly inhibited c-Fms and Abl. Compared to imatinib, mastitinib more potently suppresses bone marrow mast cell migration, cytokine generation, and degranulation. Masitinib has an IC50 of 150 nM for inhibiting SCF (stem cell factor)-induced cell proliferation in Ba/F3 cells expressing human wild-type Kit, whereas the IC50 is roughly >10 μM for inhibiting IL-3-stimulated proliferation. Masitinib, with an IC50 of 300 nM, suppresses PDGFRα tyrosine phosphorylation and PDGF-BB-stimulated proliferation in Ba/F3 cells that express PDGFRα. Additionally, in BMMC and mastocytoma cell lines, misitinib prevents SCF-stimulated tyrosine phosphorylation of human Kit. With IC50s of 3 and 5 nM, respectively, masatidinib inhibits Kit gain-of-function mutants in Ba/F3 cells, such as the Δ27 murine mutant and the V559D mutant. With IC50s of 10 and 30 nM, respectively, mastitinib inhibits cell growth in mastocytoma cell lines, such as HMC-1α155 and FMA3 [1]. Two new ISS cell lines showed growth and PDGFR phosphorylation, indicating that Masitinib exhibits efficacy against primary and metastatic ISS cell lines and could help with ISS clinical care [2].

In a Ba/F3 tumor model expressing Δ27, methitinib mesylate (30 mg/kg) reduces tumor growth and lengthens the median survival time without causing genotoxicity or cardiotoxicity [1]. When compared to a placebo, methitinib mesylate (12.5 mg/kg/d, po) lengthens the period before tumor growth [3].

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Masitinib increased overall TTP compared with placebo from 75 to 118 days (P = .038). This effect was more pronounced when masitinib was used as first-line therapy, with an increase in the median TTP from 75 to 253 days (P = .001) and regardless of whether the tumors expressed mutant (83 versus not reached [P = .009]) or wild-type KIT (66 versus 253 [P = .008]). Masitinib was generally well tolerated, with mild (grade I) or moderate (grade II) diarrhea or vomiting as the most common adverse events. Conclusions and clinical importance: Masitinib is safe and effective at delaying tumor progression in dogs presenting with recurrent or nonresectable grade II or III nonmetastatic MCT [3].

A 96-well microtitre plate is coated with 0.25 mg/mL poly(Glu, Tyr 4:1) for an entire night. It is then rinsed twice with 250 µL of washing buffer (10 mM phosphate-buffered saline [pH 7.4] and 0.05% Tween 20) and allowed to dry at room temperature for two hours. The assays are conducted at room temperature in a final volume of 50 µL of kinase buffer (10 mM MgCl₂, 1 mM MnCl₂, 1 mM sodium orthovanadate, 20 mM HEPES, pH 7.8) that contains recombinant enzyme and ATP at a concentration of at least twice the K_m for each enzyme to guarantee a linear reaction rate. The enzyme is added to start the reaction, and it is stopped by adding one reaction volume (50 μL) of 100 mM EDTA per 5mol/Lurea mix. Plates are triple-washed and then incubated with tetramethylbenzidine and a 1:30,000 horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody. Spectrophotometry is used to measure the final reaction product at 450 nm.

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In vitro assays with recombinant protein kinases [1]
Full details for the generation of recombinant human KIT intracellular domain and other protein kinases (including Lyn, platelet derived growth factor receptor β, epidermal growth factor receptor, fibroblast growth factor receptor 1, Src, HCK, PYK, FES, Btk, Bmx, c-Ret, c-Fms, Syk, and c-Met) are provided in the Supplemental Methods (see Supporting Information; Methods S1). Experiments on ABL1, Akt1, protein kinase C-α, insulin-like growth factor receptor 1, and Pim1 were carried out by Proqinase. All other recombinant protein kinases were performed in-house using an enzyme-linked immunoassay; experimental details are provided in the Supplemental Methods (see Supporting Information; Methods S1).

Microtitre plates are seeded with 10⁴ cells/well in 100 μL of RPMI 1640 medium containing 10% foetal bovine serum at 37°C in order to conduct the Ba/F3 cell proliferation assay. 250 ng/mL of murine SCF or 0.1% of conditioned medium from X63-IL-3 cells are added to these, or not. Purified from the conditioned medium of SCF-producing CHO cells is the murine SCF that activates Kit. Masitinib-grown cells are incubated for 48 hours at 37°C with WST-1 reagent (10 μL/well) for three hours. Using a scanning multiwell spectrophotometer, the absorbance at 450 nm of the formazan dye indicates how much of it has formed. The spectrophotometer's background control is a blank well devoid of cells.

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Assessment of the effect of masitinibb and imatinib on human mast cell degranulation response and cytokine production (TNF-α release), was performed on CBMC produced by long-term culture of CD34+ progenitors purified from normal cord-blood, as described previously by Royer et al (see Supporting Information; Methods S1). Cultured cells were harvested, washed in complete IMDM medium, and incubated for 1 hour in various concentrations of masitinib or imatinib. Assays of β-hexosaminidase release and TNF-α release were made by stimulating the CBMC with 1 µg/ml of goat anti-human IgE for 30 minutes or 4 hours, respectively. β-hexosaminidase was measured in the supernatant and in the sonicated cell pellets and its net release calculated. For TNF-α determination, the cell-free supernatants were collected by centrifugation and frozen at −80°C until determination of mediator content by the use of a specific ELISA kit according to manufacturer's instructions. All assays were performed in duplicate and counts were repeated twice for each well. Results were expressed in percentage of inhibition of β-hexosaminidase release and of TNF-α release relative to the stimulated untreated CBMC, (i.e. 100% of stimulation).

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Dysregulation of platelet-derived growth factor receptor (PDGFR) may play a role in feline injection-site sarcoma (ISS) cell growth and viability. Masitinib, a tyrosine kinase inhibitor approved for treatment of canine mast cell tumours, is highly selective for the PDGFR signalling pathway and may offer a new therapeutic approach for this disease. The in vitro effects of masitinib on growth, apoptosis and PDGFR signalling in two novel ISS cell lines were investigated. PDGFR expression was confirmed by Western blot in cell lines derived from a primary ISS tumour (JB) and a corresponding, histologically confirmed ISS lung metastasis (JBLM). Masitinib inhibited cell growth and PDGFR phosphorylation in both cell lines. Higher drug concentrations were required to inhibit growth than to modulate ligand-induced autophosphorylation of PDGFR. These in vitro data suggest that masitinib displays activity against both primary and metastatic ISS cell line and may aid in the clinical management of ISS[2].

Dissolved in DMSO;
30 mg/kg (i.p.) or 10, 30, or 45 mg/kg (p.o.).; i.p. or oral gavage
Female MBRI Nu/Nu mice bearing a/F3 Δ27 tumour model

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At 7 weeks old, male Nog-SCID mice are housed in a pathogen-free environment with filtered water and food available at all times. They experience a 12-hour light/12-hour dark cycle. According to the above description, Mia Paca-2 cells are cultured. Mice are given an injection into the right flank at day 0 (D0) containing 10⁷ Mia Paca-2 cells in 200 µL PBS. After a tumor reaches the target size of approximately 200 mm³, it is allowed to grow for 1.5 to 4 weeks. In order to ensure that the mean body weight and tumor volume of each treatment group are well matched, animals are divided into four groups by day 28 (n = 7–8). The animals receive treatment for a maximum of four weeks, following which they are sacrificed. The treatments were as follows: a) daily gavage with 100 mg/kg masitinib; b) intraperitoneal (i.p.) injection of 50 mg/kg gemcitabine twice a week; c) daily gavage with 100 mg/kg masitinib; or d) a combination of daily gavage with 100 mg/kg masitinib and i.p. injection of 50 mg/kg gemcitabine twice a week. Callipers are used to measure the size of tumors, and the formula volume=(length × width²)/2 is used to estimate the tumor volume. (100) × (median tumor volume of treated group)/(median tumor volume of control group) is the formula for the tumor growth inhibition ratio.

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In vivo assays with Ba/F3 Δ27 tumour model [1]
Female MBRI Nu/Nu mice (7 weeks old) were housed under specific pathogen-free conditions at 20±1°C with a 12 hours light/12 hours dark cycle and ad libitum access to food and filtered water. The mice were allowed to acclimatise to the study conditions for 10 to 20 days prior to experiments. The Δ27-expressing Ba/F3 cells were grown in RPMI 1640 medium supplemented with glutamax-1 and 10% foetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. The cells were centrifuged and resuspended at 5×106 or 7.5×106 cells/ml in phosphate-buffered saline. Mice were treated with 5 Gy of gamma radiation and after 24 hours they were injected in the right flank with 1.5×106 Δ27 Ba/F3 cells. When tumour growth had reached the desired size, mice were allocated into treatment groups ensuring that there was no statistical difference between each group's mean body weight and tumour volume. For all animals, body weight was measured on the day of injection and every 5 days thereafter, with the tumour's size measured via callipers every 5 days during the treatment period for estimation of tumour volume. During the predose period and for 2 weeks post-treatment, the animals were checked for mortality or signs of morbidity once a day, increasing to twice a day checks during the treatment period.

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Background: Activation of the KIT receptor tyrosine kinase is associated with the development of canine mast cell tumors (MCT). [3]
Hypothesis/objective: To evaluate the efficacy of masitinib, a potent and selective inhibitor of KIT, in the treatment of canine MCT. [3]
Animals: Two hundred and two client-owned dogs with nonmetastatic recurrent or nonresectable grade II or III MCT. [3]
Methods: Double-blind, randomized, placebo-controlled phase III clinical trial. Dogs were administered masitinib (12.5 mg/kg/d PO) or a placebo. Time-to-tumor progression (TTP), overall survival, objective response at 6 months, and toxicity were assessed. [3]

Furthermore, in an intraperitoneal model, masitinib significantly enhanced survival with no indication of general toxicity, as indicated by a lack of weight loss at the administered doses. [1]

[1]. Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT. PLoS One, 2009, 4(9), e7258.

[2]. Masitinib demonstrates anti-proliferative and pro-apoptotic activity in primary and metastatic feline injection-site sarcoma cells. Vet Comp Oncol, 2011, doi: 10.1111/j.1476-5829.2011.00291.x.

[3]. Masitinib is safe and effective for the treatment of canine mast cell tumors. J Vet Intern Med, 2008, 22(6), 1301-1309.

[4]. Masitinib (AB1010), from canine tumor model to human clinical development: where we are? Crit Rev Oncol Hematol. 2014 Jul;91(1):98-111.

Masitinib Mesylate is the orally bioavailable mesylate salt of masatinib, a multi-targeted protein tyrosine kinase inhibitor with potential antineoplastic activity. Masitinib selectively binds to and inhibits both the wild-type and mutated forms of the stem cell factor receptor (c-Kit; SCFR); platelet-derived growth factor receptor (PDGFR); fibroblast growth factor receptor 3 (FGFR3); and, to a lesser extent, focal adhesion kinase (FAK). As a consequence, tumor cell proliferation may be inhibited in cancer cell types that overexpress these receptor tyrosine kinases (RTKs).

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Drug Indication
Treatment of amyotrophic lateral sclerosis.
Treatment of mastocytosis
Treatment of non resectable locally advanced or metastatic pancreatic cancer
Treatment of unresectable and/or metastatic malignant gastrointestinal stromal tumour (GIST)
Treatment of non-resectable dog mast-cell tumours (grade 2 or 3) with confirmed mutated c-KIT tyrosine-kinase receptor.
Treatment of amyotrophic lateral sclerosis
Treatment of mastocytosis
Treatment of gastro-intestinal stromal tumours.

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Masitinib is a member of the class of benzamides that is the carboxamide resulting from the formal condensation of the carboxy group of 4-[(4-methylpiperazin-1-yl)methyl]benzoic acid with the primary amino group of 4-methyl-N(3)-[4-(pyridin-3-yl)-1,3-thiazol-2-yl]benzene-1,3-diamine. It is a highly selective oral tyrosine kinase inhibitor. It has a role as a tyrosine kinase inhibitor, an antineoplastic agent and an antirheumatic drug. It is a N-alkylpiperazine, a member of 1,3-thiazoles, a member of pyridines and a member of benzamides.
Masitinib is a tyrosine-kinase inhibitor used in the treatment of mast cell tumors in dogs. It has been available in Europe since 2009, under the brand name Masivet. In the USA it is distributed under the name Kinavet and has been available for veterinaries since 2011.
Masitinib is a multi-targeted protein tyrosine kinase inhibitor, with potential antineoplastic activity. Upon administration, masitinib selectively binds to and inhibits both the wild-type and mutated forms of the stem cell factor receptor (c-Kit; SCFR); platelet-derived growth factor receptor (PDGFR); fibroblast growth factor receptor 3 (FGFR3); and, to a lesser extent, focal adhesion kinase (FAK). As a consequence, tumor cell proliferation may be inhibited in cancer cell types that overexpress these receptor tyrosine kinases (RTKs).
See also: Masitinib Mesylate (annotation moved to).

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Drug Indication
Treatment of amyotrophic lateral sclerosis.
Treatment of mastocytosis
Treatment of non resectable locally advanced or metastatic pancreatic cancer
Treatment of unresectable and/or metastatic malignant gastrointestinal stromal tumour (GIST).

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Background[1]
The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. [1]
Methodology/Principal Findings[1]
In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. [1]
Conclusions[1]
Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.[1]

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Masitinib mesylate (AB1010) is a novel potent and selective tyrosine kinase inhibitor, targeting mainly wild-type and mutated c-Kit receptor (c-KitR), Platelet Derived Growth Factor Receptor-alfa/beta (PDGFRa/ß), Lymphocyte-specific kinase (Lck), Lck/Yes-related protein (LYn), Fibroblast Growth Factor Receptor 3 (FGFR3) and Focal Adhesion Kinase (FAK). It is the first anticancer therapy approved in veterinary medicine for the treatment of unresectable canine mast cell tumors (CMCTs), harboring activating c-KitR mutations, at dose of 12.5mg/kg once daily. Considering its anti-proliferative action, principally given by inhibiting the MCs c-KitR anti-angiogenic pathway that leads cancer progression, and its role as chemosensitizer, masitinib is under clinical investigation in several human malignancies (Gastro-Intestinal Stromal Tumors, acute myeloid leukemia, systemic mastocytosis, pancreatic cancer, multiple myeloma, non-small cell lung cancer, melanoma, ovarian and prostate cancer), which are characterized by similar canine c-KIT proto-oncogene mutations. Here, we analyze masitinib structure activity, its pharmacokinetics compared to imatinib, the c-KitR pathway referring to the most frequent c-KIT mutations sensitive or resistant to this novel drug compared to imatinib, and masitinib safety profile. We, also, explore preclinical and clinical (completed and ongoing) trials with the aim to emphasize as this recent anti-angiogenic therapy, at first approved in CMCTs and, currently in development for the treatment of several human neoplasms, could be represent a milestone in translational oncology, in which the murine experimental model of cancer research could be integrated by canine spontaneous tumor model. [4]

C29H34N6O4S2

594.74

594.208

C, 58.57; H, 5.76; N, 14.13; O, 10.76; S, 10.78

1048007-93-7

Masitinib;790299-79-5

25024769

White to yellow solid powder

5.212

3

10

7

41

788

0

TXCWBWKVIZGWEQ-UHFFFAOYSA-N

InChI=1S/C28H30N6OS.CH4O3S/c1-20-5-10-24(16-25(20)31-28-32-26(19-36-28)23-4-3-11-29-17-23)30-27(35)22-8-6-21(7-9-22)18-34-14-12-33(2)13-15-34;1-5(2,3)4/h3-11,16-17,19H,12-15,18H2,1-2H3,(H,30,35)(H,31,32);1H3,(H,2,3,4)

methanesulfonic acid;4-[(4-methylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-yl-1,3-thiazol-2-yl)amino]phenyl]benzamide

Masitinib mesilate; Masitinib Mesylate Salt; 1048007-93-7; Masitinib (mesylate); Masivet; Masitinib Mesylate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

1M HCl : 100 mg/mL (~168.14 mM)
DMSO : ≥ 30 mg/mL (~50.44 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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