EGFR (IC50 = 13 nM); HER2 (IC50 = 38 nM)
Epidermal growth factor receptor (EGFR) (IC₅₀ = 0.013 μM in enzymatic assay). [1]
Human epidermal growth factor receptor 2 (HER2) (IC₅₀ = 0.045 μM in enzymatic assay). [1]

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].

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Pyrotinib inhibited EGFR and HER2 kinase activities in enzymatic assays with IC₅₀ values of 0.013 μM and 0.045 μM, respectively. [1]
In cell proliferation assays, pyrotinib inhibited the growth of HER2-dependent cancer cell lines. IC₅₀ values: BT474 breast cancer cells (HER2+++) = 5.1 nM; SK-OV-3 ovarian cancer cells (HER2+++) = 43.0 nM; MDA-MB-231 breast cancer cells (HER2-) = 3500 nM. For comparison, the reference compound HKI-272 showed IC₅₀ values of 4.0 nM (BT474), 19.6 nM (SK-OV-3), and 4850 nM (MDA-MB-231). [1]
Three major metabolites of pyrotinib were characterized: M1 (O-depicolyl), M2 (O-depicolyl and pyrrolidine lactam), and M5 (pyrrolidine lactam). M1 and M2 exhibited strong activity at EGFR but not at HER2; M5 showed very weak potency at EGFR and no activity at HER2. [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].

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In BT-474 human breast cancer xenograft model in nude mice, pyrotinib (2.5, 5, and 10 mg/kg, oral, once daily) significantly inhibited tumor growth in a dose-dependent manner. Tumor growth inhibition (TGI) was observed at all doses tested without mortality or significant body weight loss (<5% relative to vehicle controls). At 10 mg/kg, pyrotinib showed robust antitumor efficacy. [1]
In SK-OV-3 ovarian cancer xenograft model, pyrotinib (2.5, 5, and 10 mg/kg, oral, once daily) showed TGI of 2%, 12%, and 83% on day 21, respectively. [1]
In phase I clinical studies in patients with HER2-positive metastatic breast cancer, pyrotinib (80-480 mg once daily) demonstrated antitumor activity. The objective response rate (ORR) was 55.6% for the 320 mg dose cohort and 87.5% for the 400 mg dose cohort. Median progression-free survival (PFS) was 31.9 weeks and 59.7 weeks, respectively. [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].

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EGFR kinase assay: A human-derived recombinant protein (cell signaling technology, #7908) was reacted with peptide substrate at different concentrations of test compounds in a buffer solution containing 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 minutes. Activity was determined by a time-resolved fluorescence method. [1]
HER2 kinase assay: The HER2 kinase assay kit (Invitrogen, #PV3366) was used with protein substrate (Tyr 87) at different concentrations of test compounds in buffer containing 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 minutes. Activity was 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].

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Cell proliferation inhibition assay: Cancer cells (A431, SK-BR-3, NCI-N87) were seeded at a suitable concentration (e.g., 5000 cells/mL medium) and incubated until 85% confluency. Test compounds were added at 6-7 concentrations, and cells were cultured for 72 hours. Cell proliferation inhibition was determined by the sulforhodamine B (SRB) method. IC₅₀ values were calculated from inhibition rates. [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)

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Animals: Nude mice (BALB/Ca-nude, 6-7 weeks, female) were used for efficacy studies; Sprague Dawley rats and beagle dogs were used for PK studies. All animals were treated in accordance with institutional guidelines. [1]
In vivo efficacy study: Nude mice were hypodermically inoculated with BT-474 human breast cancer cells or SK-OV-3 ovarian cancer cells. After tumors reached 150-250 mm³, mice were randomly divided into groups and dosed once daily with pyrotinib (2.5, 5, 10 mg/kg) or vehicle. Tumor volume (V = 1/2 × a × b², where a = length, b = width) and body weight were measured 2-3 times per week. Tumor growth inhibition (TGI) was calculated. [1]
In vivo PK study: Test compounds were administered via intravenous (i.v.) and intragastric (i.g.) routes to mice, rats, and dogs to obtain bioavailability. Plasma samples were collected at pre-dose and various time points post-dose (0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h for i.v.; 1.0, 2.0, 3.0, 3.5, 4, 4.5, 5, 6, 8, 12, 24 h for rats i.g.; 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24 h for dogs i.g.). [1]
Human PK study: Healthy subjects received a single oral administration of 240 mg pyrotinib maleate tablet. Plasma, urine, and feces samples were collected at specified time intervals up to 96 hours post-dose. A dose-escalation study (80, 160, 240, 320, 400 mg) was conducted for phase I. [1]
Tissue distribution study: Rats were administered pyrotinib at 3 mg/kg; tissues were collected at various time points for concentration analysis. [1]

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Animals: Nude mice (BALB/Ca-nude, 6-7 weeks, female) were used for efficacy studies; Sprague Dawley rats and beagle dogs were used for PK studies. All animals were treated in accordance with institutional guidelines. [1]
In vivo efficacy study: Nude mice were hypodermically inoculated with BT-474 human breast cancer cells or SK-OV-3 ovarian cancer cells. After tumors reached 150-250 mm³, mice were randomly divided into groups and dosed once daily with pyrotinib (2.5, 5, 10 mg/kg) or vehicle. Tumor volume (V = 1/2 × a × b², where a = length, b = width) and body weight were measured 2-3 times per week. Tumor growth inhibition (TGI) was calculated. [1]
In vivo PK study: Test compounds were administered via intravenous (i.v.) and intragastric (i.g.) routes to mice, rats, and dogs to obtain bioavailability. Plasma samples were collected at pre-dose and various time points post-dose (0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h for i.v.; 1.0, 2.0, 3.0, 3.5, 4, 4.5, 5, 6, 8, 12, 24 h for rats i.g.; 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24 h for dogs i.g.). [1]
Human PK study: Healthy subjects received a single oral administration of 240 mg pyrotinib maleate tablet. Plasma, urine, and feces samples were collected at specified time intervals up to 96 hours post-dose. A dose-escalation study (80, 160, 240, 320, 400 mg) was conducted for phase I. [1]
Tissue distribution study: Rats were administered pyrotinib at 3 mg/kg; tissues were collected at various time points for concentration analysis. [1]

Pyrotinib showed favorable drug-like physicochemical properties with LogD₇.₄ = 2.68 and solubility of 13.2 μM in FaSSIF (pH 6.5). Bioavailability was 20.6% in nude mice, 43.5% in rats, and 13.5% in dogs. [1]
Half-life (t₁/₂): Mouse = 4.2 h; Rat = 3.0 h; Dog = 3.2 h; Human = 15.0-20.9 h. [1]
Plasma clearance (CL): Mouse = 37.2 mL/min/kg; Rat = 31.9 mL/min/kg; Dog = 28.0 mL/min/kg; Human predicted = 21.5 mL/min/kg. [1]
Human PK: Single oral administration of 80-400 mg showed tₘₐₓ = 4.0-5.5 h, t₁/₂ = 15.0-20.9 h, Cₘₐₓ = 32.3-179 ng/mL, AUC₀₋ₜ = 510-3260 ng·h/mL. Good linear PK characteristics were observed. Food enhanced bioavailability with 45.5% increase in AUC₀₋ₜ and 80.6% increase in Cₘₐₓ at 320 mg under fed vs. fasted conditions. [1]
Metabolism: Pyrotinib was primarily metabolized by CYP3A4 (>75% consumption), and to a lesser extent by CYP1B1, CYP2C8, CYP2C19, CYP2D6, and CYP3A5. Major metabolites identified: M1 (O-depicolyl), M2 (O-depicolyl and pyrrolidine lactam), and M5 (pyrrolidine lactam). Fecal excretion was the major elimination route in rats. [1]

Pyrotinib demonstrated favorable safety profiles in preclinical studies. In rats, chronic toxicity study (182 days, once daily) showed NOAEL = 5 mg/kg, MTD > 100 mg/kg. Major findings at MTD included slower body weight gain, decreased food consumption, partial hair loss, slight/mild atrophy of small intestine villi, and decreased triglyceride, total protein, and albumin. [1]
In dogs, chronic toxicity study (272 days, once daily) showed NOAEL = 3 mg/kg, MTD > 30 mg/kg. Major findings at MTD included reduced motor activity, decreased body weight and food consumption, emesis, fluid feces, mild degeneration of seminiferous tubules, and epididymal oligospermia. No deaths occurred in any dose groups. [1]
CYP inhibition safety margins: For CYP3A4, margins >81-fold based on total human Cₘₐₓ at 400 mg; for CYP2C19, margins >60-fold. hERG safety margins: >16-fold based on total human Cₘₐₓ. Using free Cₘₐₓ (fraction unbound = 0.75% in human), safety margins are considerably larger. [1]
Clinical safety: In healthy subjects, pyrotinib was well tolerated with only grade I adverse events; no dose-limiting toxicity was observed up to 400 mg. In patients, the most common adverse events were grade I/II gastrointestinal symptoms (diarrhea). MTD was determined as 400 mg per day; the 480 mg cohort experienced dose-limiting toxicity (grade III diarrhea). [1]

[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 of pyrotinib, a highly bioavailable oral dual kinase inhibitor that inhibits epidermal growth factor receptor (EGFR, ErbB1, or HER-1) and human epidermal growth factor receptor 2 (ErbB2 or HER-2), exhibiting potential antitumor activity. After oral administration, pyrotinib binds to and inhibits the activity of EGFR and HER2, thereby suppressing tumor growth and angiogenesis, and inducing regression in EGFR/HER2-expressing tumor cells. EGFR and HER2 are receptor tyrosine kinases upregulated in various tumor cell types and play important roles in tumor cell proliferation and tumor angiogenesis.

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Pyrotinib (SHR1258) is a novel, orally available, irreversible EGFR/HER2 dual tyrosine kinase inhibitor developed for the treatment of HER2-positive breast cancer. It contains an α,β-unsaturated carbonyl moiety as a Michael acceptor for covalent binding with the target enzymes (Cys-805 of HER2). The lead optimization process identified pyrotinib with overall advantages in potency, PK profiles, antitumor efficacy, and safety. Preclinical studies demonstrated robust antitumor effects in HER2-overexpressing xenograft models and favorable safety windows. Phase I clinical studies showed acceptable safety profile, favorable PK properties, and encouraging antitumor responses (ORR up to 87.5% at 400 mg) in patients with HER2-positive advanced breast cancer. Pyrotinib is currently under further clinical development, including combination studies with capecitabine. [1]

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.)