EGFR (IC50 = 13 nM); HER2 (IC50 = 38 nM)

In HER2-dependent cell lines (BT474, SK-OV-3), parotinib dimaleate exhibits strong inhibitory effects; in HER2-negative cell lines (MDA-MB-231), it is less effective. BT474 and SK-OV-3 cells are inhibited by pyrotinib dimaleate, with IC50 values of 5.1 and 43 nM, respectively. Pyrotinib maleate exhibits the same high selectivity as HKI-272 when tested in a panel of several kinases, including KDR, c-Kit, PDGFRβ, c-Src, and C-Met (IC50 for c-Src is 790 nM, others >3000 nM) [1].

Pyrotinib maleate's tolerable bioavailability in rats, dogs, and naked mice was found to be 20.6%, 43.5%, and 13.5%, respectively. The physicochemical properties of pyrotinib maleate are similar to those of drugs. It also shows a relatively high oral exposure in human subjects (oral; t1/2=15 hours), and its half-life is shorter than that of preclinical animal species, such as rats (iv; t1/2= 1.56 h; ig; t1/2= 2.52 h) and mice (iv; t1/2=4.42 h; ig; t1/2=3.38 h) [1].

The EGFR/HER2 kinase inhibition assays were utilized to determine the in vitro activity of the compounds. The half maximal inhibitory concentration IC50 (the concentration of the tested compound showing 50% inhibition of the enzyme activity) of each compound was measured by incubating a series of concentrations of the tested compounds with a specific enzyme and substrate. The EGFR kinase assay used a human-derived recombinant protein, which reacted with the peptide substrate at different concentrations of test compounds in a buffer solution containing a mixture of 60 mM HEPES (pH 7.5), 5 mM MgCl2, 5 mM MnCl2, 3 μM Na3VO4, 1.25 M DTT and 20 μM ATP at 25 °C for 45 min. The HER2 Kinase Assay Kit was reacted with the protein substrate (Tyr 87) at different concentrations of tested compounds in a buffer solution containing a mixture of 60 mM HEPES (pH 7.5), 5 mM MgCl2, 5 mM MnCl2, 3 μM Na3VO4,1.25 M DTT and 20 μM ATP at 25 °C for 60 min. Both EGFR and HER2 kinase activities were determined by a time-resolved fluorescence method[1].

The general procedures of the in vitro cell proliferation inhibition assays were performed on cancer cells (A431, SK-BR-3 and NCI-N87) at a suitable concentration (e.g., 5000 cells/mL medium). Then the cells were incubated in a carbon dioxide (5% CO2) incubator until they reached 85% confluency, subsequently, cell culture medium was replaced by fresh one with test compounds added in a series of concentrations (generally 6 to 7 concentrations). The cells were then put back to the incubator and cultured continuously. After 72 h, the activity of the test compounds for inhibiting the cell proliferation was determined by a sulforhodamine B (SRB) method. The IC50 values were calculated by the data of inhibition rates of serial concentrations of test compounds[1].

In vivo efficacy studies were performed on BALB/Ca-nude mice (6 to 7 weeks, female) from SLAC. Nude mice were hypodermic inoculated BT-474 human breast cancer cell or SK-OV-3 ovarian cancer cell. After tumor grew to 150–250 mm3, mice were randomly divided into groups and dosed once daily. The volume of tumors and the weight of the mice were measured and recorded for 2–3 times per week. The volume of tumor (V) was calculated as V = 1/2 xaxb2 (a: length of tumors, b: width of tumors). Tumor growth inhibition (TGI) was calculated as: TGI (%) = 100 − (VT − VT0) / (VC − VC0) ∗ 100%; where VT0 and VT are the tumor volumes of the beginning and finish days of dosed groups, respectively; and VC0 and VC are the tumor volumes of the beginning and finish days for the control group, respectively. In the case of tumor regression, TGI was calculated as: TGI (%) = 100 − (VT − VT0) / VT0 ∗ 100.[1]

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In vivo PK and human PK studies[1] Animals utilized for preclinical studies include nude mice, rats and dogs. All animals were treated in accordance with Institutional Guide for the Care and Use of Laboratory Animals. Nude mice (around 20 g, 9 males and 9 females) were purchased from Sino-British Sippr/BK Lab Animal Co. Ltd. (Shanghai) (SCXK 2013-0016), Sprague Dawley (SD) rats (200–250 g, 3 males and 3 females) from Shanghai SLAC Laboratory Animal Co., LTD (SYXK 2003-0029), and beagle dogs (9–13 kg, 2 males and 2 females) from Beijing Marshall Biotechnology Co., Ltd. (SCXK 2009-0002), respectively. Briefly, test compounds were administrated in both intravenous (i.v.) and intragastric (i.g.) for mice, rats and dogs in order to obtain their bioavailability. Plasma samples of nude mice, SD rats and dogs were collected at pre-dose and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h after the IV administration, plasma samples of SD rats were collected at pre-dose and 1.0, 2.0, 3.0, 3.5, 4, 4.5, 5, 6, 8, 12, 24 h and plasma samples of beagle dogs were collected at pre-dose and 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24 h after the i.g. dose. Human sample collection was conducted in Teda International Cardiovascular Hospital with study protocol approved by the ethics committee of the hospital. Written informed consent was obtained from the subjects enrolled in this study. Ten healthy subjects participated in the study for human PK and metabolite identification as well as elimination study. After an overnight fast, each subject received a single oral administration of 240 mg pyrotinib maleate tablet. Plasma samples were collected at pre-dose and 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 12, 24, 36, 48, 72, and 96 h post-dose. Urine samples were collected at pre-dose and 0–4, 4–8, 8–12, 12–24, 24–36, 36–48, 48–72, and 72–96 h post-dose. Feces samples were collected at pre-dose and 0–24, 24–48, 48–72, and 72–96 h post-dose. All the samples were preserved at − 80 °C until analysis. A range of five doses (80, 160, 240, 320 and 400 mg) was conducted for Phase I dose escalation study. All PK and TK parameters were calculated throughout a non-compartmental model using Phoenix WinNonlin software (5.2)

[1]. Discovery and development of Pyrotinib: A novel irreversible EGFR/HER2 dual tyrosine kinase inhibitor with favorable safety profiles for the treatment of breast cancer. Eur J Pharm Sci. 2017 Jan 21. pii: S0928-0987(17)30043-X.

Pyrotinib Dimaleate is the dimaleate ester of pyrotinib, an orally bioavailable, dual kinase inhibitor of the epidermal growth factor receptor (EGFR, ErbB1 or HER-1) and the human epidermal growth factor receptor 2 (ErbB2 or HER-2), with potential antineoplastic activity. Upon oral administration, pyrotinib binds to and inhibits both EGFR and HER2, which may result in the inhibition of tumor growth and angiogenesis, and tumor regression in EGFR/HER2-expressing tumor cells. EGFR and HER2 are receptor tyrosine kinases that are upregulated in various tumor cell types and play major roles in tumor cell proliferation and tumor vascularization.

C36H35CLN6O7

815.22

814.236

C, 61.84; H, 5.05; Cl, 5.07; N, 12.02; O, 16.02

1397922-61-0

Pyrotinib;1269662-73-8

72710866

Typically exists as light yellow to yellow solids at room temperature

6

16

14

58

1080

1

CCOC1=C(C=C2C(=C(C=NC2=C1)C#N)NC3=CC(=C(C=C3)OCC4=CC=CC=N4)Cl)NC(=O)/C=C/[C@@H]5N(CCC5)C.C(=C\C(=O)O)\C(=O)O.C(=C\C(=O)O)\C(=O)O

ZUZJPRMSUMMELD-ZLWATVBPSA-N

InChI=1S/C32H31ClN6O3.C4H4O4/c1-3-41-30-17-27-25(16-28(30)38-31(40)12-10-24-8-6-14-39(24)2)32(21(18-34)19-36-27)37-22-9-11-29(26(33)15-22)42-20-23-7-4-5-13-35-235-3(6)1-2-4(7)8/h4-5,7,9-13,15-17,19,24H,3,6,8,14,20H2,1-2H3,(H,36,37)(H,38,40)1-2H,(H,5,6)(H,7,8)/b12-10+2-1-/t24-/m1./s1

(R,E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-3-(1-methylpyrrolidin-2-yl)acrylamide maleate

SHR-1258; SHR1258; SHR-1258 dimaleate; Pyrotinib Maleate; 1397922-61-0; 85KUE857XM; 2-Propenamide, N-(4-((3-chloro-4-(2-pyridinylmethoxy)phenyl)amino)-3-cyano-7-ethoxy-6-quinolinyl)-3-((2R)-1-methyl-2-pyrrolidinyl)-, (2E)-, (2Z)-2-butenedioate (1:2); (Z)-but-2-enedioic acid;(E)-N-[4-[3-chloro-4-(pyridin-2-ylmethoxy)anilino]-3-cyano-7-ethoxyquinolin-6-yl]-3-[(2R)-1-methylpyrrolidin-2-yl]prop-2-enamide; UNII-85KUE857XM; SHR 1258.

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)

H2O : ≥ 106 mg/mL (~130.03 mM)
DMSO : ~100 mg/mL (~122.67 mM)

Solubility in Formulation 1: 2.5 mg/mL (3.07 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 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 (3.07 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)