JNK2; Akt
Ret receptor tyrosine kinase (including Ret/Ptc1 oncoprotein, MEN2A-associated RET mutants C634R and C634W) [1][2].

Following 72 hours of treatment, RPI-1's growth-inhibitory effect on TPC-1 cells is susceptible, with an IC50 of 5.1 μM. In TPC-1 cells, RPI-1 (7.5–60 μM) prevents Ret/Ptc1 autophosphorylation. In TPC-1 cell culture conditions, RPI-1 inhibitory effects result in the inhibition of pathways involving JNK2 and AKT[1].
In NIH3T3 cells expressing the Ret mutant, the RPI-1 IC50 value is 3.6 µM compared to 16 µM in non-transfected NIH3T3 cells, and in RET mutant-transfected and H-RAS-transfected NIH3T3 cells, it was 2.4 µM and 26 µM, respectively, for colony formation in soft agar. After 24 hours of RPI-1 treatment, Ret protein and tyrosine phosphorylation in NIH3T3 cells expressing the Ret mutant were undetectable. RPI-1 inhibits TT cell proliferation, Ret tyrosine phosphorylation, Ret protein expression, and PLCgamma, ERKs, and AKT activation[2].

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In human papillary thyroid carcinoma TPC-1 cells (spontaneously expressing RET/PTC1), RPI-1 inhibited cell proliferation with an IC50 of 5.1 ± 0.4 μM after 72 h treatment. A prolonged growth inhibitory effect persisted for days, suggesting a cytostatic effect [1].

- RPI-1 treatment (60 μM, 72 h) of TPC-1 cells induced accumulation of cells in the G2 phase of the cell cycle, increased expression of cyclins A and B1, partial hyperphosphorylation of pRb, and strong induction of p21WAF1. Mitosis-specific phosphopeptides were undetectable [1].

- In TPC-1 cells, RPI-1 abolished tyrosine phosphorylation of Ret/Ptc1 (both p59 and p64 isoforms) in a dose-dependent manner (complete dephosphorylation at >15 μM after 72 h; inhibition detectable after 10 min). It also disrupted the association of Ret/Ptc1 with Shc and PLCγ, and reduced tyrosine phosphorylation of PLCγ. The Shc docking site (pTyr451 on Ret/Ptc1, corresponding to pTyr1062) was dephosphorylated. p66Shc expression was down-regulated, p52Shc tyrosine dephosphorylated, while p46Shc was unaffected [1].

- In TPC-1 cells, RPI-1 inhibited activation of JNK2 (54 kDa) and AKT (Ser473 phosphorylation) in a dose-dependent manner, but did not affect JNK1, p42/p44 ERKs, or p38 MAPK. Activation of JNK and AKT pathways was also inhibited in NIH3T3PTC1 transfectants [1].

- RPI-1 (60 μM, 72 h) reduced telomerase activity by approximately 85% in TPC-1 cells. In Ret/Ptc1-negative NPA cells, telomerase activity was reduced by about 65%. No direct inhibition of telomerase was observed when cell extracts were exposed to the drug in vitro [1].

- In NIH3T3 fibroblasts transfected with the MEN2A-associated RET C634R mutant (NIH3T3MEN2A cells), RPI-1 inhibited cell proliferation with an IC50 of 3.6 μM (95% CI = 1.8 to 5.4 μM) after 72 h, compared to 16 μM (95% CI = 12.3 to 19.7 μM) in parental NIH3T3 cells and 14.1 μM (95% CI = 8.0 to 20.2 μM) in H-RAS-transfected NIH3T3 cells [2].

- In soft agar colony formation assays, RPI-1 gave an IC50 of 2.4 μM (95% CI = 0.8 to 4.0 μM) for NIH3T3MEN2A cells, and 26 μM (95% CI = 17 to 35 μM) for H-RAS-transfected NIH3T3 cells. Parental NIH3T3 cells do not form colonies in soft agar [2].

- In NIH3T3MEN2A cells, RPI-1 (10 μM) inhibited Ret tyrosine phosphorylation within 2 hours, and after prolonged treatment (24-72 hours) lowered expression of both mature (170 kDa) and precursor (150 kDa) Ret species. The compound also reversed the transformed morphology to a more flattened and ordered phenotype [2].

- In human medullary thyroid carcinoma TT cells (endogenously expressing RET C634W mutant), RPI-1 inhibited cell proliferation with an IC50 of 7.2 μM (95% CI = 6.6 to 7.8 μM) after 7 days of treatment. Growth arrest was observed at concentrations >10 μM [2].

- In TT cells, RPI-1 (24 h treatment) dose-dependently inhibited Ret tyrosine phosphorylation and Ret expression (170 and 150 kDa). It also reduced the binding of Ret to PLCγ, inhibited PLCγ tyrosine phosphorylation, and dephosphorylated the pY1062 docking site on Ret. Concomitantly, phosphorylation (activation) of ERKs and AKT was inhibited [2].

RPI-1 (50, 100 mg/kg; p.o.; twice a day for 10 days) inhibits TT xenograft tumor growth by 81%[2].

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In nude mice bearing subcutaneous human medullary thyroid carcinoma TT xenografts, oral administration of RPI-1 at 2 × 100 mg/kg/day (twice daily) for 10 days resulted in 75% tumor weight inhibition (P<.001 vs control) and 2/8 mice were tumor-free at the end of experiment (day 53). Prolonged treatment (40 days) with 2 × 100 mg/kg/day gave 81% tumor weight inhibition (P<.001) at day 66; 2/8 mice remained tumor-free at day 140. A lower dose of 2 × 50 mg/kg/day for 10 days gave 60% tumor weight inhibition (P = .003 vs control) [2].

- RPI-1 treatment (2 × 100 mg/kg/day for 40 days) significantly reduced plasma calcitonin levels in TT tumor-bearing mice compared to controls (P = .01). Calcitonin levels showed a strong linear correlation with tumor weight (r = 0.95, P<.001) [2].

Cell proliferation assay (TPC-1, NPA, K1, Nthy-ori 3-1): Cells were seeded in 6-well or 96-well plates, treated with RPI-1 or vehicle (DMSO, final 0.5%) for 72 h or longer. Cell density was measured by trypsinization and Coulter counter, or by sulforhodamine B colorimetric assay. IC50 values were calculated from dose-response curves [1].

- Anchorage-independent growth assay (soft agar): A cell suspension (15,000 cells/mL) in medium containing 0.33% agarose with RPI-1 or vehicle was layered onto a base layer of 0.5% agarose. After 8-10 days, colonies were stained and counted [2].

- Cell cycle analysis: TPC-1 cells treated with RPI-1 (60 μM, 72 h) were fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. For cyclin B1 biparametric analysis, cells were stained with anti-cyclin B1 antibody followed by FITC-secondary antibody, and counterstained with propidium iodide [1].

- Immunoprecipitation and Western blotting: Cells were lysed in SDS sample buffer or Triton X-100 lysis buffer. Protein concentration was determined by BCA method. For immunoprecipitation, extracts (0.5-5 mg protein) were incubated with anti-Ret or anti-PLCγ antibodies and protein A-agarose. Immunoprecipitates or whole-cell lysates (30-60 μg) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against phosphotyrosine, Ret, Shc, PLCγ, pY1062-Ret, pAKT, pERK, etc. Blots were stripped and reprobed for total proteins [1][2].

- Telomerase activity assay (TRAP): Cell extracts were prepared and telomerase activity measured by telomeric repeat amplification protocol using [32P]-labeled TS primer. Products were resolved on polyacrylamide gels, quantified by densitometry using an internal standard (ITAS) and TSR8 quantitation standard. Results expressed as percentage inhibition relative to untreated controls [1].

- RT-PCR for telomerase components: Total RNA was extracted, reverse transcribed, and amplified with specific primers for hTR, TEP1, hTERT, and β-actin. PCR products were run on agarose gels and visualized with ethidium bromide [1].

8- to 11-week-old female athymic nude CD-1 mice (bearing TT cells)[2]
50, 100 mg/kg
P.o.; twice a day for 10 days

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Xenograft model: Human TT medullary thyroid carcinoma cells (1.6 × 10⁷) were inoculated subcutaneously into the right flank of female athymic nude CD-1 mice (8-11 weeks old). Treatment started when tumors were measurable (mean ~50 mg, approximately 25 days after inoculation) [2].

- Drug formulation and administration: RPI-1 was dissolved in absolute ethanol and Cremophor EL (each 5% of final volume), stirred at 4°C until clear, then diluted with cold 0.9% NaCl solution (90% of final volume). The final concentration was 3.3 mg/mL. The drug was delivered orally by gavage, twice daily (every 12 h), at doses of 2 × 50 or 2 × 100 mg/kg/day. Control mice received vehicle (ethanol:Cremophor EL:0.9% NaCl = 5:5:90) [2].

- Tumor measurement: Tumor diameters were measured twice weekly with a caliper. Tumor weight (mg) was calculated as (d² × D)/2, where d and D are the shortest and longest diameters, assuming density = 1. Tumor weight inhibition (%) = 100 – (mean tumor weight of treated / mean tumor weight of control × 100) [2].

- Calcitonin assay: Blood was collected by orbital puncture under anesthesia. Plasma calcitonin levels were measured by immunoradiometric assay using two different anti-human calcitonin antibodies [2].

[1]. Inactivation of Ret/Ptc1 oncoprotein and inhibition of papillary thyroid carcinoma cell proliferation by indolinone RPI-1. Cell Mol Life Sci. 2003;60(7):1449-1459.

[2]. Cellular effects and antitumor activity of RET inhibitor RPI-1 on MEN2A-associated medullary thyroid carcinoma. J Natl Cancer Inst. 2004;96(13):1006-1014.

RPI-1 (1,3-dihydro-5,6-dimethoxy-3-[(4-hydroxyphenyl)methylene]-H-indol-2-one, formerly Cpd1) is a 2-indolinone derivative identified as a Ret tyrosine kinase inhibitor. It was previously shown to inhibit the transforming activity of Ret/Ptc1 in NIH3T3 fibroblasts [1][2].

- The compound inhibits Ret autophosphorylation and reduces Ret expression, possibly via proteasome-mediated degradation (as suggested for related inhibitors) [2].

- RPI-1 demonstrates selectivity for RET-transformed cells over H-RAS-transformed or normal cells. It is effective against both papillary thyroid carcinoma (RET/PTC1) and medullary thyroid carcinoma (MEN2A-associated RET mutants) models [1][2].

- The antitumor efficacy, oral bioavailability (though low), and tolerability support the therapeutic potential of RPI-1 for RET-driven thyroid cancers, especially medullary thyroid carcinoma which responds poorly to conventional chemotherapy [2].

C17H15NO4

297.31

297.1

C, 68.68; H, 5.09; N, 4.71; O, 21.53

269730-03-2

269730-03-2

1749978

Yellow to brown solid powder

1.3±0.1 g/cm3

533.6±50.0 °C at 760 mmHg

276.5±30.1 °C

0.0±1.5 mmHg at 25°C

1.660

3.08

2

4

3

22

442

0

OC1C=CC(/C=C2/C3C(=CC(OC)=C(OC)C=3)NC/2=O)=CC=1

JGSMCYNBVCGIHC-QPEQYQDCSA-N

InChI=1S/C17H15NO4/c1-21-15-8-12-13(7-10-3-5-11(19)6-4-10)17(20)18-14(12)9-16(15)22-2/h3-9,19H,1-2H3,(H,18,20)/b13-7-

(3Z)-3-[(4-hydroxyphenyl)methylidene]-5,6-dimethoxy-1H-indol-2-one

RPI1; RPI 1; RPI-1

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: 59~125 mg/mL (198.5~420.4 mM)
Ethanol: ~2 mg/mL (~6.7 mM)

Solubility in Formulation 1: 2.08 mg/mL (7.00 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.)