Bcr-Abl (IC50 = 30 nM); AMPK
Nilotinib primarily targets the Bcr-Abl fusion protein, a constitutively active tyrosine kinase that drives the proliferation of Philadelphia chromosome-positive (Ph+) leukemic cells. It binds to and stabilizes the inactive conformation of the kinase domain of the Abl portion of the Bcr-Abl fusion protein, thereby inhibiting the constitutive kinase activity of Bcr-Abl and blocking the downstream signaling pathways that promote leukemic cell proliferation and survival. In addition to its primary target, nilotinib also inhibits several other receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR), mast/stem cell growth factor receptor Kit (c-Kit), and to a lesser extent, colony-stimulating factor 1 receptor (CSF-1R) and discoidin domain-containing receptor 1 (DDR1). This multi-kinase inhibition profile contributes to its therapeutic effects and may also be responsible for some of its observed off-target activities and side effects. The compound demonstrates significant potency against wild-type BCR-ABL, with an IC₅₀ of less than 30 nM, and maintains activity against most BCR-ABL point mutants that confer imatinib resistance. Nilotinib shows high selectivity for Bcr-Abl over other kinases such as Src and LYN, with IC₅₀ values greater than 5,000 nM for both, indicating a favorable selectivity profile that contributes to its clinical utility.

Nilotinib inhibits proliferation, migration, and actin filament formation, as well as the expression of α-SMA and collagen in activated HSCs. Nilotinib induces apoptosis of HSCs, which is correlated with reduced bcl-2 expression, increases p53 expression, cleavage of PARP, as well as increases expression of PPARγ and TRAIL-R. Nilotinib also induces cell cycle arrest, accompanied by increased expression of p27 and downregulation of cyclin D1. Interestingly, Nilotinib not only inhibits activation of PDGFR, but also TGFRII through Src. Nilotinib significantly inhibits PDGF and TGFβ-simulated phosphorylation of ERK and Akt. Furthermore, PDGF- and TGFβ-activated phosphorylated form(s) of Abl in human HSCs are inhibited by Nilotinib. Nilotinib inhibits most imatinib-resistant Bcr-Abl mutations, except for T315I. Nilotinib inhibits PDGF-DD-mediated ERK1/2 activation, basal and PDGF-DD-mediated activation of PDGFRβ and Akt, and schwannoma proliferation. Nilotinib is more potent than imatinib, exerting its maximal inhibitory effect at concentrations lower than steady-state trough plasma levels. Nilotinib also significantly reduces the expression levels of the genes for TGF-β1 and platelet-derived growth factor (PDGF). Nilotinib treatment also significantly inhibits the PDGF-induced proliferation of lung fibroblasts. Nilotinib inhibits the proliferation of Ba/F3 cells expressing p210- and p190-Bcr-Abl, or K562 and Ku-812F cells with IC50 values ≤12 nM.
Kinase Assay: The novel, selective Abl inhibitor, Nilotinib (AMN107), is designed to interact with the ATP-binding site of BCR-ABL with a higher affinity than Imatinib. In addition to being significantly more potent compared with Imatinib (IC50<30 nM), Nilotinib also maintains activity against most of the BCR-ABL point mutants that confer Imatinib resistance
Cell Assay: Human primary Schwann and schwannoma cells are seeded on precoated 96-well plates. Nilotinib is added 40 minutes before stimulation with 100 ng/mL PDGF-DD, and cells are cultured for 72 hours (3 days). Because the half-life of Nilotinib is 18 hours, one-half of the originally added concentrations are added freshly every day. In addition to DAPI staining and determination of the total cell number, the more sensitive and accurate BrdU incorporation method is used to detect proliferating cells. Total cell amount (DAPI) and number of dividing cells (BrdU-positive) are blindly counted using an inverted fluorescent microscope and 200 × magnification. All cells in every well are counted. The total cell number per well differed between various cell batches and is 100–300 cells/well.
In vitro, nilotinib demonstrates exceptional potency and efficacy against Bcr-Abl-driven cellular models. It inhibits Bcr-Abl autophosphorylation and cell proliferation in Ba/F3 cells expressing wild-type or mutant Bcr-Abl, with IC₅₀ values ranging from 7 to 155 nM for autophosphorylation and 13 to 51 nM for cell proliferation. Nilotinib is significantly more potent than imatinib, with an IC₅₀ of less than 30 nM against wild-type BCR-ABL, and it maintains activity against most BCR-ABL point mutants that confer imatinib resistance. The compound demonstrates significant antitumor efficacy against gastrointestinal stromal tumor (GIST) xenograft lines and imatinib-resistant GIST cell lines. In imatinib-sensitive parent cell lines GK1C and GK3C, imatinib shows IC₅₀ values of 4.59±0.97 µM and 11.15±1.48 µM, respectively; in contrast, imatinib-resistant cell lines GK1C-IR and GK3C-IR exhibit imatinib resistance with IC₅₀ values of 11.74±0.17 µM and 41.37±1.07 µM, respectively. Nilotinib effectively overcomes this resistance, demonstrating activity in these resistant cell lines. Beyond its anticancer effects, nilotinib has shown promise in non-oncological contexts, including neuroprotective effects in models of Alzheimer's disease, where it reduces midbrain Bcr-Abl autophosphorylation, amyloid-β levels, and neuronal loss, while improving autophagosome clearance and reversing cognitive deficits. It also exhibits renoprotective effects, reducing serum creatine levels, renal profibrotic gene expression, and tubulointerstitial damage in a rat model of chronic kidney disease.

Nilotinib reduces collagen deposition and α-SMA expression in CCl4 and BDL-induced fibrosis. Nilotinib could induce HSC undergoing apoptosis, which is correlated with downregulation of bcl-2. Nilotinib attenuates the extent of lung injury and fibrosis. Nilotinib therapy significantly reduces the levels of hydroxyproline on days 14 and 21, which is accompanied by decreased expression levels of transforming growth factor (TGF)-β1 and PDGFRβ. AMN107 prolongs survival of mice injected with Bcr-Abl-transformed hematopoietic cell lines or primary marrow cells, and prolongs survival in imatinib-resistant CML mouse models.

The novel, selective Abl inhibitor, Nilotinib (AMN107), is designed to interact with the ATP-binding site of BCR-ABL with a higher affinity than Imatinib. In addition to being significantly more potent compared with Imatinib (IC50<30 nM), Nilotinib also maintains activity against most of the BCR-ABL point mutants that confer Imatinib resistance.
In vitro enzyme/receptor binding assays for nilotinib typically involve kinase inhibition studies using purified recombinant kinases and radiolabeled ATP. The compound's binding affinity and inhibitory activity against Bcr-Abl and other kinases are assessed through competitive binding assays that measure the displacement of radiolabeled ATP or the inhibition of kinase-mediated phosphorylation of peptide substrates. For Bcr-Abl, the assay measures the inhibition of autophosphorylation or the phosphorylation of exogenous substrates using recombinant Bcr-Abl protein. The IC₅₀ values for wild-type and mutant Bcr-Abl are determined by incubating the enzyme with varying concentrations of nilotinib in the presence of ATP and a suitable substrate, followed by detection of phosphorylated products using scintillation counting or ELISA-based methods. Selectivity profiling is conducted using panels of kinases to determine the compound's specificity, with IC₅₀ values for off-target kinases such as Src and LYN typically exceeding 5,000 nM. Binding affinity is further characterized using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure the thermodynamic parameters of the nilotinib-kinase interaction.

Human primary Schwann and schwannoma cells are seeded on precoated 96-well plates. Nilotinib is added 40 minutes before stimulation with 100 ng/mL PDGF-DD, and cells are cultured for 72 hours (3 days). Because the half-life of Nilotinib is 18 hours, one-half of the originally added concentrations are added freshly every day. In addition to DAPI staining and determination of the total cell number, the more sensitive and accurate BrdU incorporation method is used to detect proliferating cells. Total cell amount (DAPI) and number of dividing cells (BrdU-positive) are blindly counted using an inverted fluorescent microscope and 200 × magnification. All cells in every well are counted. The total cell number per well differed between various cell batches and is 100–300 cells/well.
In vitro cell-based assays for nilotinib primarily utilize cell lines expressing wild-type or mutant Bcr-Abl to evaluate its antiproliferative and signaling inhibitory effects. The most commonly used model is the Ba/F3 cell system, where cells are engineered to express wild-type or various mutant forms of Bcr-Abl. Cells are cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and appropriate selection antibiotics, then seeded in 96-well plates and treated with serial dilutions of nilotinib for 48-72 hours. Cell proliferation is assessed using MTT, CCK-8, or [³H]-thymidine incorporation assays, and IC₅₀ values for growth inhibition are calculated. Bcr-Abl autophosphorylation is evaluated by Western blot analysis using phospho-specific antibodies against Bcr-Abl (Tyr245) and its downstream signaling molecules such as STAT5, CRKL, and ERK. Flow cytometry is employed to assess cell cycle distribution and apoptosis induction using propidium iodide staining and Annexin V-FITC staining, respectively. For imatinib-resistant cell lines (GK1C-IR and GK3C-IR), the assays demonstrate that nilotinib effectively overcomes resistance, with IC₅₀ values significantly lower than those of imatinib. In GIST cell lines, nilotinib's activity is evaluated through similar proliferation and signaling assays, confirming its efficacy against both imatinib-sensitive and imatinib-resistant lines.

BALB/cSLc-nu/nu mice with GIST xenograft (GK1X, GK2X and GK3X)[2]
40 mg/kg
Oral gavage; daily; 4 weeks
In vivo animal studies for nilotinib are conducted in various mouse and rat models to evaluate its antitumor efficacy, pharmacokinetics, and pharmacodynamic effects. For efficacy studies, immunocompromised mice (such as BALB/cSLc-nu/nu) bearing subcutaneous GIST xenografts are treated with nilotinib via oral gavage at doses of 40 mg/kg daily for 4 weeks. Tumor volume is measured twice weekly using calipers, and tumor growth inhibition (TGI) is calculated relative to vehicle-treated controls. Pharmacodynamic endpoints include assessment of Bcr-Abl phosphorylation in tumor tissues by immunohistochemistry and Western blot, as well as evaluation of downstream signaling markers. In the indomethacin-induced enterocolitis rat model, nilotinib is administered orally to assess its mucosal healing effects, with macroscopic and microscopic pathologic scores evaluated, and PDGFRα and PDGFRβ levels measured in colonic tissues. For Alzheimer's disease studies, the Tg2576 transgenic mouse model is used, with nilotinib administered at 1 mg/kg to evaluate its effects on amyloid-β levels, neuronal loss, autophagosome clearance, and cognitive function through behavioral tests such as the Morris water maze. In the 5/6 nephrectomy-induced chronic kidney disease rat model, nilotinib's renoprotective effects are assessed by measuring serum creatine levels, renal profibrotic gene expression, and tubulointerstitial damage, with survival rates monitored over the study period. Dose-finding and pharmacokinetic studies are conducted in healthy animals to determine appropriate dosing regimens for efficacy studies and to characterize the compound's absorption, distribution, metabolism, and excretion (ADME) profile.

Nilotinib is well absorbed after oral administration with good bioavailability. Unchanged nilotinib is the main circulating form of the drug in serum, with no metabolites contributing to its pharmacological activity. The drug has a half-life of approximately 17 hours, and greater than 90% of the administered dose is eliminated in the feces. The mean clearance in patients with newly diagnosed Ph+ CML in chronic phase is 20.2 L/h. Nilotinib is extensively metabolized in the liver, primarily by oxidation and hydroxylation, mainly via the cytochrome P450 enzyme CYP3A4. CYP2C8 plays a minor role in its metabolism. Nilotinib is a competitive inhibitor of several CYP enzymes, including CYP3A4, CYP2C8, CYP2C9, CYP2D6, and UGT1A1, which has significant implications for drug-drug interactions. Co-administration with rifampicin (a potent CYP3A4 inducer) increases nilotinib clearance approximately 5-fold, while co-administration with ketoconazole (a CYP3A4 inhibitor) increases nilotinib exposure and the occurrence of side effects. Nilotinib is transported by both ABCB1 (P-glycoprotein) and ABCG2 (BCRP) efflux transporters; overexpression of ABCG2 protects cells from nilotinib toxicity, whereas overexpression of ABCB1 only slightly decreases nilotinib toxicity. However, other studies suggest ABCB1 is the main transporter, with ABCG2 having a lesser role. These transporter interactions may influence tissue distribution and drug resistance mechanisms.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
While the levels of nilotinib in breast milk appear to be low, and one breastfed infant experienced no adverse reactions while the mother was taking nilotinib, long-term data are currently unavailable. Because nilotinib binds to plasma proteins at a rate as high as 98%, the levels in breast milk are likely low. However, there are few reports of experience with nilotinib use during lactation, so alternative medications may be preferred, especially when breastfeeding newborns or premature infants. The National Comprehensive Cancer Network (NCCN) guidelines recommend avoiding breastfeeding during nilotinib treatment, and the manufacturer recommends discontinuing breastfeeding two weeks after the last dose.
◉ Effects on Breastfed Infants
A woman with chronic myeloid leukemia continued taking nilotinib (dosage not specified) for the first 20 months of pregnancy, throughout pregnancy, and during lactation (duration of lactation not specified). No adverse reactions were reported in her breastfed infant.
◉ Impact on breastfeeding and breast milk
As of the revision date, no relevant published information was found.
The toxicity profile of nilotinib is well-characterized from preclinical and clinical studies. Common adverse effects associated with nilotinib therapy include hematological toxicities such as thrombocytopenia, neutropenia, and anemia, which are manageable with dose adjustments and supportive care. Non-hematological toxicities include rash, headache, pruritus, nausea, fatigue, and musculoskeletal pain. Of particular clinical concern is the potential for QT interval prolongation, a cardiotoxic effect that requires regular electrocardiographic monitoring during treatment. Nilotinib has also been associated with hepatotoxicity, manifesting as elevations in liver enzymes (ALT, AST, and bilirubin), and pancreatic enzyme elevations (lipase and amylase). In preclinical studies, nilotinib has shown neuroprotective and renoprotective effects at certain doses, suggesting that its toxicity profile may be context-dependent and dose-related. The drug's interaction with CYP3A4 and efflux transporters can lead to significant drug-drug interactions that may increase the risk of toxicity when co-administered with other medications that affect these pathways. In chronic kidney disease models, nilotinib administration improves renal function and survival, indicating that its toxicity profile may be favorable in certain disease contexts.

[1]. Beneficial effects of combining nilotinib and imatinib in preclinical models of BCR-ABL+ leukemias. Blood. 2007 Mar 1;109(5):2112-20.

[2]. Antitumor effect of the tyrosine kinase inhibitor Nilotinib on gastrointestinal stromal tumor (GIST) and Imatinib-resistant GIST cells. PLoS One. 2014 Sep 15;9(9):e107613.

[3]. Mucosal healing effect of nilotinib in indomethacin-induced enterocolitis: A rat model. World J Gastroenterol. 2015 Nov 28;21(44):12576-85.

Nilotinib hydrochloride monohydrate is the monohydrate-hydrochloride form of nilotinib. Nilotinib is an orally bioavailable aminopyrimidine derivative belonging to the Bcr-Abl tyrosine kinase inhibitor class and possessing antitumor activity. Nilotinib is designed to overcome imatinib resistance caused by Bcr-Abl kinase mutations. After administration, nilotinib binds to the kinase domain of the Abl portion of the Bcr-Abl fusion protein and stabilizes its inactive conformation, thereby inhibiting the constitutive kinase activity of the Bcr-Abl protein. This inhibits Bcr-Abl-mediated proliferation of Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) cells. Nilotinib can also inhibit other receptor tyrosine kinases such as platelet-derived growth factor receptor (PDGF-R; PDGFR), mast cell/stem cell growth factor receptor Kit (c-Kit), colony-stimulating factor 1 receptor (CSF-1R; CSF1R), and discoid domain receptor 1 (DDR1), but with weaker inhibitory effects.

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Drug Indications
Tasigna is indicated for the treatment of: newly diagnosed adults and children with chronic-phase Philadelphia chromosome-positive chronic myeloid leukemia (CML), and children with chronic-phase Philadelphia chromosome-positive CML who are resistant to or intolerant of prior treatments, including imatinib. Tasigna is indicated for the treatment of: newly diagnosed adults and children with chronic-phase Philadelphia chromosome-positive chronic myeloid leukemia (CML); adults with chronic-phase and accelerated-phase Philadelphia chromosome-positive CML who are resistant to or intolerant of prior treatments (including imatinib). There are no efficacy data for patients with blast crisis CML; and children with chronic-phase Philadelphia chromosome-positive CML who are resistant to or intolerant of prior treatments (including imatinib).
Nilotinib (marketed as Tasigna®) is FDA-approved for the treatment of Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML) in chronic phase and accelerated phase in adult patients. It is indicated for patients with newly diagnosed Ph+ CML in chronic phase, as well as for those who are resistant or intolerant to prior therapy including imatinib. The recommended dose is 300 mg or 400 mg twice daily, administered orally on an empty stomach. Nilotinib represents a significant advancement in CML therapy, offering enhanced potency and activity against many imatinib-resistant Bcr-Abl mutants. The drug has also been investigated in preclinical models for potential applications beyond oncology, including neurodegenerative diseases such as Alzheimer's disease, where it has shown promise in reducing amyloid-β levels and improving cognitive function in transgenic mouse models. Additionally, nilotinib has demonstrated renoprotective effects in chronic kidney disease models. The compound's multi-kinase inhibitory profile, including activity against PDGFR, c-Kit, CSF-1R, and DDR1, suggests potential for further therapeutic exploration in other diseases where these kinases play pathogenic roles. Ongoing research continues to explore nilotinib's full therapeutic potential and its role in combination therapies for various malignancies and non-malignant conditions.

C28H25CLF3N7O2

584.0

583.171

C, 57.59; H, 4.32; Cl, 6.07; F, 9.76; N, 16.79; O, 5.48

923288-90-8

Nilotinib;641571-10-0;Nilotinib hydrochloride;923288-95-3; Nilotinib monohydrochloride monohydrate;923288-90-8;Nilotinib-d6;1268356-17-7;Nilotinib-d3;1215678-43-5;Nilotinib hydrochloride;923288-95-3; 641571-10-0; 923289-71-8 (hydrochloride dihydrate); 1277165-20-4 (dihydrochloride dihydrate)

16757572

White to off-white solid powder

6.812

4

10

6

41

817

0

Cl.O=C(C1C=C(NC2N=C(C3C=CC=NC=3)C=CN=2)C(C)=CC=1)NC1C=C(C(F)(F)F)C=C(N2C=C(C)N=C2)C=1.O

YCBPQSYLYYBPDW-UHFFFAOYSA-N

InChI=1S/C28H22F3N7O.ClH.H2O/c1-17-5-6-19(10-25(17)37-27-33-9-7-24(36-27)20-4-3-8-32-14-20)26(39)35-22-11-21(28(29,30)31)12-23(13-22)38-15-18(2)34-16-38;;/h3-16H,1-2H3,(H,35,39)(H,33,36,37);1H;1H2

4-methyl-N-[3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]benzamide;hydrate;hydrochloride

Nilotinib; AMN 107; AMN107; AMN-107; US brand name: Tasigna. Nilotinib HCl hydrate

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)

DMSO : ≥ 33 mg/mL (~56.51 mM)
H2O : < 0.1 mg/mL

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.)