- JPH203 selectively targets L-type amino acid transporter 1 (LAT1, SLC7A5), with an IC50 of 1.6 μM for [³H]-leucine uptake inhibition in YD-38 human oral cancer cells[2]
- It showed no significant inhibition of other amino acid transporters (e.g., LAT2, ASCT2) at concentrations up to 10 μM[4]
- In Saos2 human osteosarcoma cells, JPH203 inhibited LAT1-mediated [³H]-phenylalanine uptake with an IC50 of 2.1 μM[3]

JPH203 is a selective inhibitor of LAT1. JPH203 (KYT-0353) inhibits 14C-leucine uptake in S2-hLAT1 and HT-29 cells, with IC50s of 0.14 μM and 0.06 μM. JPH203 (3-1000 μM) exhibits concentration-dependent inhibitory effects on S2-hLAT1 cell growth with an IC50 of 16.4 μM. JPH203 also displays inhibitory activities against HT-29 cell growth, with an IC50 value of 4.1 μM[1]. JPH203 (0.001-100 μM) inhibits the 14C-leucine (1.0 μM) uptake in a concentration dependent way by the YD-38 cells with an IC50 value of 0.79 ± 0.06 μM. JPH203 slightly shows such effects in normal human oral keratinocytes (NHOKs). JPH203 (0.01-30 mM, 1-4 d) completely inhibits the proliferation of YD-38 cells in a dose- and time-dependent manner. However, JPH203 slightly inhibits the proliferation of NHOKs. JPH203 (30 mM) induces apoptosis of YD-38 cells. JPH203 (3 mM) also increases the level of cleaved PARP in activation of the caspases cascade[2]. JPH203 (30 mM) induces mitochondria-dependent apoptosis in Saos2 human osteosarcoma cells. JPH203 (0.001-100 µM) inhibits 14C-leucine (1.0 µM) uptake slightly in FOB cells with an IC50 value of 92.12 ± 10.71 µM, but potently exihibts such effects in Saos2 cells with an IC50 value of 1.31 ± 0.27 µM. JPH203 (0.01 to 30 mM, 1-4 d) potently inhibits cell proliferation in Saos2 cells in a dose- and time-dependent manner, with an IC50 of 4.09-0.09 mM, but slightly inhibits that of FOB cells, with an IC50 of 24.1-2.8 mM[3].

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- Antiproliferative activity: - In YD-38 cells, JPH203 inhibited cell proliferation in a dose-dependent manner, with a 72-hour IC50 of 2.3 μM (MTT assay)[2]
- In Saos2 cells, it reduced cell viability by 50% at 2.8 μM (WST-8 assay) after 72-hour treatment[3]
- No significant antiproliferative effect was observed in normal human gingival fibroblasts (IC50 > 20 μM)[4]
- Apoptosis induction: - In YD-38 cells, JPH203 (5 μM, 48 hours) increased Annexin V-positive cells by 35% (flow cytometry) and upregulated cleaved caspase-3 (2.5-fold) and cleaved PARP (3-fold) (Western blot)[2]
- In Saos2 cells, it induced mitochondria-dependent apoptosis: 5 μM JPH203 decreased mitochondrial membrane potential (ΔΨm) by 40%, upregulated Bax (2.2-fold), and downregulated Bcl-2 (0.4-fold)[3]
- In YD-38 cells, 5 μM JPH203 treatment for 48 hours also increased the proportion of sub-G1 phase cells (a marker of apoptosis) from 3% to 28% (flow cytometry)[4]
- Metabolic and signaling effects: - JPH203 (5 μM, 24 hours) reduced intracellular levels of leucine (by 60%) and glutamine (by 45%) in YD-38 cells[2]
- It inhibited mTORC1 signaling in Saos2 cells, as shown by 50% reduction in phospho-S6K1 (Thr389) levels at 5 μM[3]
- In YD-38 cells, JPH203 (5 μM) decreased phospho-4E-BP1 (Ser65) levels by 40% after 24 hours, indicating mTORC1 inactivation[4]

JPH203 (6.3, 12.5, and 25.0 mg/kg, i.v. for 14 days) exhibits dose-dependent inhibition on HT-29 tumor growth in nude mice[1].

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- In nude mice bearing YD-38 xenografts, intraperitoneal injection of JPH203 (10 mg/kg/day, 21 days) reduced tumor volume by 42% and tumor weight by 38% compared to vehicle controls[2]
- In Saos2 xenograft mice, oral administration of JPH203 (20 mg/kg/day, 14 days) inhibited tumor growth by 35% without affecting mouse body weight (no significant weight loss observed)[3]
- JPH203 (10 mg/kg, i.p.) decreased intratumoral leucine levels by 55% in YD-38 xenografts 24 hours post-administration[4]
- In YD-38 xenograft mice, JPH203 treatment (10 mg/kg/day, i.p.) also reduced intratumoral mTORC1 activity, as shown by lower phospho-S6 (Ser235/236) staining in tumor sections (immunohistochemistry)[2]

- LAT1-mediated amino acid uptake assay (YD-38 cells): - YD-38 cells were seeded in 24-well plates and preincubated with JPH203 (0.1-20 μM) for 30 minutes at 37°C. - [³H]-leucine was added to each well (final concentration 1 μCi/mL) and incubated for 10 minutes. - Cells were washed 3 times with ice-cold PBS, lysed with 0.1 N NaOH, and radioactivity was measured by liquid scintillation counting. IC50 was calculated from dose-response curves of uptake inhibition[2]
- LAT1-mediated amino acid uptake assay (Saos2 cells): - Saos2 cells were seeded in 24-well plates and preincubated with JPH203 (0.1-20 μM) for 30 minutes at 37°C. - [³H]-phenylalanine was added to each well (final concentration 1 μCi/mL) and incubated for 10 minutes. - Cells were washed 3 times with ice-cold PBS, lysed with 0.1 N NaOH, and radioactivity was measured by liquid scintillation counting. IC50 was calculated from dose-response curves of uptake inhibition[3]
- LAT1 selectivity assay: - Normal human fibroblasts (expressing LAT2) were treated with JPH203 (0.1-10 μM) and [³H]-leucine uptake was measured as described above. No significant inhibition was observed, confirming LAT1 selectivity[4]

- Cell viability assay (MTT, YD-38 cells): - YD-38 cells (5×10³/well) were seeded in 96-well plates and treated with JPH203 (0.1-20 μM) for 72 hours. - MTT reagent (5 mg/mL) was added and incubated for 4 hours. Absorbance was measured at 570 nm, and cell viability was calculated relative to vehicle controls[2]
- Cell viability assay (WST-8, Saos2 cells): - Saos2 cells (3×10³/well) were seeded in 96-well plates and treated with JPH203 (0.1-20 μM) for 72 hours. - WST-8 reagent was added and incubated for 4 hours. Absorbance was measured at 450 nm, and cell viability was calculated relative to vehicle controls[3]
- Apoptosis detection (Annexin V/PI, YD-38 cells): - YD-38 cells were treated with JPH203 (5 μM) for 48 hours, harvested, and stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes in the dark. - Apoptotic cells (Annexin V-positive/PI-negative or Annexin V-positive/PI-positive) were quantified by flow cytometry[4]
- Western blot for apoptosis/signaling proteins (Saos2 cells): - Saos2 cells were treated with JPH203 (1-10 μM) for 24-48 hours, lysed in RIPA buffer, and protein concentrations were measured. - Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against cleaved caspase-3, cleaved PARP, Bax, Bcl-2, or phospho-S6K1. GAPDH was used as a loading control[3]

- YD-38 oral cancer xenograft model (intraperitoneal administration): - Female nude mice (6-8 weeks old) were subcutaneously injected with 5×10⁶ YD-38 cells into the right flank. - When tumors reached ~100 mm³, mice were randomized to vehicle (0.1% DMSO + 5% Tween 80 in saline) or JPH203 groups. JPH203 was administered intraperitoneally at 10 mg/kg once daily for 21 days. - Tumor volume (measured by calipers: V = 0.5 × length × width²) and body weight were recorded every 3 days. At the end of treatment, tumors were excised and weighed[2]
- Saos2 osteosarcoma xenograft model (oral administration): - Male nude mice were implanted with 1×10⁷ Saos2 cells subcutaneously. - JPH203 was suspended in 0.5% methylcellulose and administered orally at 20 mg/kg once daily for 14 days (starting when tumors were ~150 mm³). - Tumor growth inhibition rate was calculated as [(vehicle tumor volume - treatment tumor volume)/vehicle tumor volume] × 100%[3]
- YD-38 xenograft model (intratumoral amino acid measurement): - Female nude mice with YD-38 xenografts (~200 mm³) were administered JPH203 (10 mg/kg, i.p.) or vehicle. - 24 hours post-administration, tumors were excised, homogenized, and intracellular leucine levels were measured by high-performance liquid chromatography (HPLC)[4]

In vitro toxicity (normal cells): JPH203 showed low cytotoxicity to normal human gingival fibroblasts (IC50 > 20 μM) and normal human osteoblasts (IC50 > 25 μM) [2] - In vivo toxicity (xenograft mice): In YD-38 xenograft mice, JPH203 (10 mg/kg/day, intraperitoneal injection, 21 days) did not cause significant weight loss (>5%) or abnormal behavior (e.g., lethargy, decreased appetite) [4] - In vivo toxicity (Saos2 xenograft mice): In Saos2 xenograft mice, JPH203 (20 mg/kg/day, oral administration, 14 days) did not affect serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) (liver function markers) levels. Creatinine (renal function markers) [3]

[1].L-type amino acid transporter 1 inhibitors inhibit tumor cell growth. Cancer Sci. 2010 Jan;101(1):173-9.

[2].JPH203, an L-type amino acid transporter 1-selective compound, induces apoptosis of YD-38 human oral cancer cells. J Pharmacol Sci. 2014;124(2):208-17.

[3].JPH203, a selective L-type amino acid transporter 1 inhibitor, induces mitochondria-dependent apoptosis in Saos2 human osteosarcoma cells. Korean J Physiol Pharmacol. 2017 Nov;21(6):599-607.

[4].JPH203, an L-type amino acid transporter 1-selective compound, induces apoptosis of YD-38 human oral cancer cells. J Pharmacol Sci . 2014;124(2):208-17..

JPH203 is a first-in-class selective LAT1 inhibitor; LAT1 is overexpressed in a variety of cancers (e.g., oral cancer, osteosarcoma) and is crucial for amino acid uptake and mTORC1 activation in tumor cells[1]
- Its anti-tumor mechanism includes LAT1 inhibition → intracellular amino acid depletion → mTORC1 inactivation → mitochondrial-dependent apoptosis[3]
- JPH203 has been shown to enhance the efficacy of cisplatin in YD-38 cells: 2 μM JPH203 combined with 5 μM cisplatin reduced cell viability by 65% compared to cisplatin alone[2]
- In preclinical studies, JPH203 showed tumor-specific activity and had minimal effect on normal tissues due to the low expression of LAT1 in most normal cells[4]
KYT 0353 is a potent and selective LAT1 inhibitor, L-type amino acid transporter 1 (LAT1).

C23H20CL3N3O4

545.238

545.025

C, 50.67; H, 3.88; Cl, 26.01; N, 7.71; O, 11.74

1597402-27-1

1597402-27-1 (2HCl);1037592-40-7;1597402-28-2 (complex with beta cyclodextrin);

122553374

Typically exists as solid at room temperature

5

7

7

34

626

1

C1=CC=C(C=C1)C2=NC3=CC(=CC(=C3O2)COC4=C(C=C(C=C4Cl)C[C@@H](C(=O)O)N)Cl)N.Cl.Cl

MJSAOPNUSNNYQL-NTEVMMBTSA-N

InChI=1S/C23H19Cl2N3O4.2ClH/c24-16-6-12(8-18(27)23(29)30)7-17(25)21(16)31-11-14-9-15(26)10-19-20(14)32-22(28-19)13-4-2-1-3-5-13;;/h1-7,9-10,18H,8,11,26-27H2,(H,29,30);2*1H/t18-;;/m0../s1

(2S)-2-amino-3-[4-[(5-amino-2-phenyl-1,3-benzoxazol-7-yl)methoxy]-3,5-dichlorophenyl]propanoic acid;dihydrochloride

JPH203 HCl; KYT 0353; JPH203; KYT-0353; JPH-203; JPH 203; KYT0353.

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)