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

When it comes to HER2-negative cell lines (BT474, SK-OV-3), parotinib is quite effective, while its inhibitory effects are less pronounced when it comes to MDA-MB-231. It blocks the actions of BT474 and SK-OV. -3Pyrotinib cells have respective IC50 values of 5.1 and 43 nM[1].

Pyrotinib's effective bioavailability was found in 20.6%, 43.5%, and 13.5% of nude mice, sediments, and dogs, respectively. At dosages of 5 mg/kg, 10 mg/kg, and 20 mg/kg, respectively, the TGI% (growth inhibition) of pyrotinib on day 21 was 109%, 157%, and 159%. Pyrotinib demonstrated TGI% on day 21 in the ovarian xenograft model SK-OV-3 of 2%, 12%, and 83% at doses of 2.5 mg/kg, 5 mg/kg, and 10 mg/kg, respectively). This additional evidence supports Pyrotinib's strong in vivo anti-tumor effectiveness at 10 mg/kg [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)

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Preclinical toxicity and toxicokinetic (TK) studies[1]
Prior to chronic toxicity studies on rat and dog, dose escalations were investigated in both rats and dogs to determine approximate maximum tolerated dose (MTD), respectively. Then, a rat chronic toxicity study (5, 20, 100 mg/kg) up to 182 days was conducted, followed by a dog chronic toxicity study (3, 10, 30/45 mg/kg) up to 272 days. The corresponding TK and accumulation factor data as well as major findings are discussed in preclinical toxicity section. It should be noted that the highest dose was changed from 45 mg/kg to 30 mg/kg after 15 days due to observed adverse effect during the dog chronic toxicity studies.

[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 is under investigation in clinical trial NCT03756064 (Neoadjuvant Study of Pyrotinib in Patients With HER2 Positive Breast Cancer).
Pyrotinib is an orally bioavailable, dual kinase inhibitor of the epidermal growth factor receptor (EGFR 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.

C32H31CLN6O3

583.079945802689

582.214

C, 65.92; H, 5.36; Cl, 6.08; N, 14.41; O, 8.23

1269662-73-8

(Rac)-Pyrotinib;1246089-97-3;Pyrotinib dimaleate;1397922-61-0

51039030

Typically exists as light yellow to yellow solids at room temperature

5.4

2

8

10

42

960

1

CCOC1=C(C=C2C(=C1)N=CC(=C2NC3=CC(=C(C=C3)OCC4=CC=CC=N4)Cl)C#N)NC(=O)/C=C/[C@H]5CCCN5C

SADXACCFNXBCFY-IYNHSRRRSA-N

InChI=1S/C32H31ClN6O3/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)42-20-23-7-4-5-13-35-23/h4-5,7,9-13,15-17,19,24H,3,6,8,14,20H2,1-2H3,(H,36,37)(H,38,40)/b12-10+/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

SHR-1258; SHR1258; SHR-1258; CJN36EQM0H; (R,E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-3-(1-methylpyrrolidin-2-yl)acrylamide; SHR1258; 1269662-73-8 (free base); 2-Propenamide, N-(4-((3-chloro-4-(2-pyridinylmethoxy)phenyl)amino)-3-cyano-7-ethoxy-6-quinolinyl)-3-((2R)-1-methyl-2-pyrrolidinyl)-, (2E)-; SHR 1258

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)

DMSO : ~10 mg/mL (~17.15 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.)