- 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 selectively targets the L-type amino acid transporter 1 (LAT1 or SLC7A5). It is a non-transportable, competitive inhibitor with a Ki of 38.7 nM. The compound exhibits greater than 167-fold selectivity for LAT1 over LAT2, another member of the L-type amino acid transporter family. LAT1 is highly expressed in many tumor cells, making it an attractive target for cancer therapy. By inhibiting this transporter, JPH203 blocks the uptake of leucine and other essential amino acids that are critical for cancer cell growth and proliferation.

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]
In vitro, JPH203 potently inhibits 14C-leucine uptake in cells expressing LAT1. It shows IC50 values of 0.14 μM in S2-hLAT1 cells and 0.06 μM in HT-29 human colon adenocarcinoma cells. The compound also inhibits cell growth with IC50 values of 16.4 μM in S2 cells and 4.1 μM in HT-29 cells. In human oral cancer cells (YD-38) and leukemic cells, JPH203 similarly shows dramatic inhibition of leucine uptake (IC50 = 0.79 μM) and cell growth. These effects are concentration-dependent, demonstrating its potent antiproliferative activity across multiple cancer cell lines.

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]
In vivo, JPH203 has demonstrated antitumor activity in mouse xenograft models. It is active in HT-29 mouse xenograft models, where it inhibits tumor growth. The compound is typically administered at doses ranging from 6.3 to 25 mg/kg in these models. The in vivo efficacy is attributed to its ability to inhibit LAT1-mediated amino acid uptake in tumors, thereby depriving cancer cells of essential nutrients. These preclinical findings have supported the advancement of JPH203 into clinical trials as a potential anticancer agent.

- 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]
The in vitro enzyme/receptor binding (non-cellular) assay for JPH203 typically involves assessing its ability to inhibit the transport of radiolabeled substrates in membrane preparations or artificial systems. While it is a transporter inhibitor rather than a classical enzyme inhibitor, its binding affinity and inhibitory potency are often measured using cell-based uptake assays. The compound's Ki of 38.7 nM for LAT1 is determined through competitive binding studies. These cell-free or membrane-based assays provide a direct measure of the compound's interaction with the transporter protein.

- 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]
In vitro cellular assays for JPH203 measure its ability to inhibit 14C-L-leucine uptake in cells expressing LAT1. Typically, cells such as S2-hLAT1 (which overexpress human LAT1) or HT-29 human colon adenocarcinoma cells are incubated with the radiolabeled substrate in the presence of various concentrations of the compound. After a defined incubation period (e.g., 1 hour), the amount of radiolabeled leucine taken up by the cells is measured by liquid scintillation counting. The IC50 value is then calculated from the concentration-response curve. Cell growth inhibition is typically assessed after 96 hours of treatment using cell counting methods.

- 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 vivo animal studies for JPH203 have been conducted using mouse xenograft models, particularly with HT-29 human colon cancer cells. In these studies, immunodeficient mice are implanted with tumor cells, and after tumor establishment, JPH203 is administered at various doses (e.g., 6.3-25 mg/kg). Tumor growth is monitored over time, and endpoints such as tumor volume, tumor weight, and animal survival are measured. The compound's efficacy is evaluated by comparing tumor growth in treated versus control groups, demonstrating its antitumor activity in vivo.

JPH203 diHCl is orally bioavailable, as it has advanced to clinical trials as an oral therapeutic candidate. Its formulation as a dihydrochloride salt enhances its aqueous solubility. However, detailed pharmacokinetic parameters such as half-life, Cmax, and AUC are not extensively detailed in the available literature. As a substrate for LAT1, its distribution may be influenced by the expression of this transporter in various tissues. The compound's pharmacokinetic profile supports its development as an orally administered anticancer agent.

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]
Specific toxicity data for JPH203 diHCl are not extensively detailed in the available literature. As an LAT1 inhibitor that targets amino acid transport, its toxicity profile is likely related to its mechanism of action, potentially affecting normal cells that also express LAT1. However, the compound exhibits selectivity for LAT1 over LAT2, which may contribute to a favorable safety profile. Preclinical toxicology studies would typically be conducted to assess the compound's safety margin, but these specific data are not provided in the search results. Standard toxicological evaluations would be required for clinical development.

[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).
JPH203 (Nanvuranlat) has advanced to clinical development as a potential anticancer therapeutic. It has been evaluated in clinical trials, including Phase III studies, for the treatment of various cancers. The compound's mechanism of action involves inhibiting LAT1-mediated amino acid uptake, thereby starving cancer cells of essential nutrients. Its high selectivity for LAT1 over LAT2 and non-transportable, competitive inhibition profile make it a promising candidate for cancer therapy. The compound is not yet approved for clinical use and remains an investigational agent.

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.)