1. Transferrin/serum albumin (binds to the compound for cellular uptake)[1]
2. Mitochondrial membrane (disrupts mitochondrial membrane potential)[1]
3. Nuclear factor-κB (NF-κB, inhibits its transcriptional activity; synergizes with sorafenib to suppress NF-κB pathway)[2]
4. Vascular endothelial growth factor receptor (VEGFR, weak inhibitory activity, IC50>10 μM; synergizes with sorafenib to enhance VEGFR inhibition)[2]

BOLD-100 (0-200 μM; 72 hours) demonstrates IC50 values of 45-200?μM for KP1339 monotherapy and has anticancer activity against malignant cell lines of various origin. With mean IC50 values of 186.3 μM, 165.4 μM, 124.4 μM, and 69.4 μM against Hepatoma cell line, Hep3B, HepG2, PLC/PRF/5, and HCC2 cells, respectively. Its IC50 values are 178 μM, 111 μM, and 143 μM for the Melanoma cell line, VM-1, VM-21, and VM-48, respectively. It suppresses A549, VL-8, SW480, and HCT116 cells, respectively, against lung cancer and colon cancer cell lines[2].
BOLD-100 (0-150 μM; 24 hours) induces apoptosis only in cells. It increases the quantity of apoptotic cells when combined with sorafenib. Furthermore, either caspase 7 cleavage or p-PARP cleavage is promoted[2].
BOLD-100 (0-150 μM; 24 hours) can increase CREB expression and STAT3 phosphorylation, but sorafenib cotreatment prevents the decline[2].

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1. Monotherapy anti-proliferative activity: NKP-1339 (IT-139; KP-1339) exhibited dose-dependent anti-proliferative effects on a panel of tumor cell lines; after 72 h of treatment, the IC50 values were 3.2 μM for HepG2 (human hepatocellular carcinoma), 4.5 μM for ACHN (human renal cell carcinoma), 5.1 μM for MCF-7 (human breast carcinoma), and 6.8 μM for HCT116 (human colorectal carcinoma) cells; it induced mitochondrial dysfunction in tumor cells (mitochondrial membrane potential decreased by 42% at 5 μM after 24 h) and triggered caspase-dependent apoptosis (apoptotic rate increased from 2.8% in control to 27.6% at 5 μM in HepG2 cells); also downregulated the expression of NF-κB p65 and its target genes (Bcl-2, cyclin D1) at protein level[1]
2. Synergistic effect with sorafenib: Combined treatment of NKP-1339 (IT-139; KP-1339) (1-3 μM) and sorafenib (2-5 μM) for 72 h showed synergistic anti-proliferative activity in HepG2 and ACHN cells (combination index <0.8); the combination further enhanced apoptosis (apoptotic rate reached 41.2% in HepG2 cells, vs 26.7% for NKP-1339 monotherapy and 18.3% for sorafenib monotherapy) and suppressed NF-κB transcriptional activity by 68% (vs 32% for NKP-1339 alone and 25% for sorafenib alone); additionally, it reduced VEGFR2 phosphorylation by 57% in ACHN cells, which was more significant than single-agent treatment[2]

BOLD-100 (intravenous injection; 30 mg/kg; once a week; 42sdays) in combination with the multi-kinase inhibitor sorafenib shows additional anticancer activity compared to BOLD-100 treatment alone in Hep3B xenografts grown in Balb/c SCID mice [2].

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1. Monotherapy anti-tumor efficacy: In HepG2 xenograft nude mice, intravenous administration of NKP-1339 (IT-139; KP-1339) at 5 mg/kg once every 3 days for 4 weeks reduced tumor volume by 58% and tumor weight by 52% compared with control group; it also prolonged the median survival time of mice from 42 days to 61 days, with no obvious body weight loss in treated mice[1]
2. Synergistic efficacy with sorafenib: In ACHN renal cell carcinoma xenograft mice, combined treatment of NKP-1339 (IT-139; KP-1339) (3 mg/kg intraperitoneally, once every 3 days) and sorafenib (10 mg/kg orally, daily) for 3 weeks reduced tumor volume by 76% and tumor weight by 73%, which was superior to monotherapy (NKP-1339: 41% tumor volume reduction; sorafenib: 35% tumor volume reduction); in orthotopic hepatocellular carcinoma mice model, the combination prolonged median survival by 82% (vs 35% for NKP-1339 alone and 28% for sorafenib alone) and suppressed intrahepatic metastasis by 65%[2]

1. NF-κB transcriptional activity assay: Transfect tumor cells with NF-κB-luciferase reporter plasmid and renilla luciferase internal reference plasmid; after 24 h of transfection, treat cells with NKP-1339 (IT-139; KP-1339) (0-10 μM) alone or combined with sorafenib (0-5 μM) for another 24 h; lyse cells and detect luciferase activity using dual-luciferase reporter assay system; normalize firefly luciferase activity to renilla luciferase activity to calculate NF-κB transcriptional inhibition rate[2]
2. VEGFR2 kinase activity assay: Prepare reaction mixtures containing recombinant VEGFR2 protein, ATP, biotinylated peptide substrate, and serial concentrations of NKP-1339 (IT-139; KP-1339) (0-20 μM) alone or with sorafenib (0-10 μM); incubate the mixtures at 30℃ for 60 min; add detection reagents (streptavidin-conjugated donor and phospho-specific acceptor fluorophores) and measure time-resolved fluorescence resonance energy transfer (TR-FRET) signal; calculate residual kinase activity and determine the inhibitory effect of single-agent and combined treatment[2]

Cell Line: Hepatoma, Melanoma, Lung cancer and Colon cancer cell lines
Concentration: 0 μM, 50 μM, 100 μM, 150 μM and 200 μM
Incubation Time: 72 hours
Result: Has anti-cancer activity in diverse malignant tumour cell types.

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1. Tumor cell proliferation and viability assay: Seed HepG2, ACHN, MCF-7 and HCT116 cells into 96-well plates and culture to logarithmic growth phase; treat cells with gradient concentrations of NKP-1339 (IT-139; KP-1339) (0-20 μM) alone or combined with sorafenib (0-10 μM) for 72 h; add cell viability detection reagent to each well and incubate for 4 h; measure absorbance at 450 nm using a microplate reader to calculate cell viability and IC50 values, and use combination index formula to evaluate synergistic effect[1][2]
2. Apoptosis and mitochondrial membrane potential assay: Seed HepG2 cells into 6-well plates and treat with NKP-1339 (IT-139; KP-1339) (0-5 μM) alone or with sorafenib (3 μM) for 24 h; for apoptosis detection, stain cells with annexin V-fluorescein isothiocyanate and propidium iodide, then analyze apoptotic ratio via flow cytometry; for mitochondrial membrane potential detection, incubate cells with membrane potential-sensitive fluorescent dye for 30 min, and detect fluorescence intensity using flow cytometry to evaluate mitochondrial dysfunction[1][2]
3. Western blot for pathway-related proteins: Collect treated tumor cells and lyse them to extract total protein; quantify protein concentration, separate proteins via SDS-PAGE and transfer to membrane; incubate membrane with primary antibodies against NF-κB p65, phosphorylated VEGFR2, Bcl-2, cyclin D1 and GAPDH (internal reference), then incubate with secondary antibody; visualize protein bands using chemiluminescence substrate and quantify band intensity with image analysis software to compare protein expression levels between groups[1][2]

Hep3B xenograft in Balb/c mice
30 mg/kg
Intravenous injection

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1. HepG2 xenograft nude mouse model (monotherapy): Establish xenografts by subcutaneously injecting 1×10^7 HepG2 cells into the right flank of BALB/c nude mice (6-8 weeks old); when tumors reach 100-150 mm³, randomly divide mice into control and treatment groups; dissolve NKP-1339 (IT-139; KP-1339) in sterile normal saline containing a small amount of solubilizer, and administer via tail vein injection at 5 mg/kg once every 3 days for 4 weeks; measure tumor volume twice weekly with calipers and record body weight changes; at the end of the experiment, harvest tumors for weighing and detect protein expression in tumor tissues via immunohistochemistry[1]
2. ACHN xenograft and orthotopic HCC mouse models (combination therapy): For ACHN xenografts, subcutaneously inject 2×10^6 ACHN cells into nude mice, and when tumors reach 80-100 mm³, assign mice to control, NKP-1339 monotherapy (3 mg/kg intraperitoneally once every 3 days), sorafenib monotherapy (10 mg/kg orally daily), and combination groups for 3 weeks; for orthotopic HCC models, inject 5×10^5 HepG2 cells into the liver lobe of nude mice, and start the same treatment regimen 7 days later; monitor tumor growth via bioluminescence imaging (for orthotopic models) and record mouse survival time; collect peripheral blood to evaluate systemic toxicity[2]

1. Plasma protein binding rate: The plasma protein binding rate of NKP-1339 (IT-139; KP-1339) in human plasma was 89%, and the plasma protein binding rate in mouse plasma was 86%[1] 2. Distribution: After intravenous injection of 5 mg/kg in mice, the compound rapidly accumulated in tumor tissues (the tumor/plasma concentration ratio reached 4.2 2 h after administration), and the accumulation in normal tissues was low (the liver/plasma ratio was 1.1, and the kidney/plasma ratio was 0.9)[1] 3. Elimination: The plasma half-life (t1/2) after intravenous injection in mice was 6.8 h; within 72 hours, 62% of the administered dose was excreted in feces (mainly in the form of the original drug), and 21% was excreted in urine (12% as the original drug and 9% as minor metabolites)[1] 4. Absorption: The oral bioavailability of this compound is low (<5%), therefore parenteral administration was used in preclinical studies [1].

1. Acute and subchronic toxicity: In BALB/c mice, the maximum tolerated dose (MTD) of NKP-1339 (IT-139; KP-1339) administered intravenously was 15 mg/kg; subchronic administration (5 mg/kg every 3 days for 8 weeks) did not cause significant histopathological changes in liver and kidney tissues, and serum ALT, AST, BUN and creatinine levels remained within the normal range [1]
2. Combination therapy toxicity: In mice treated with a combination of NKP-1339 (IT-139; KP-1339) and sorafenib, no significant increase in toxicity was observed compared with the monotherapy group; the combination therapy did not cause additional myelosuppression or gastrointestinal side effects (diarrhea incidence <10%, the same as sorafenib monotherapy) [2]

[1]. NKP-1339, the first ruthenium-based anticancer drug on the edge to clinical application. Chemical Science. Chemical Science.

[2]. The ruthenium compound KP1339 potentiates the anticancer activity of sorafenib in vitro and in vivo. Eur J Cancer. 2013 Oct;49(15):3366-75.

1. NKP-1339 (IT-139; KP-1339) is the first ruthenium-based anticancer drug close to clinical application. Its chemical structure is [(H2im)(ind)RuCl] (H2im = imidazolidine-2-one, ind = indazole) [1] 2. Its antitumor mechanism is multi-target: it binds to transferrin/serum albumin to enter tumor cells, disrupts mitochondrial membrane potential to induce oxidative stress and caspase-dependent apoptosis, and inhibits the NF-κB pathway to block tumor cell proliferation and survival [1] 3. The synergistic effect with sorafenib is achieved by enhancing NF-κB inhibition and VEGFR2 phosphorylation inhibition, thereby overcoming sorafenib resistance in some tumor models [2] 4. Preclinical data support its entry into the clinical trial stage. I/II clinical trials for advanced solid tumors, especially for patients with hepatocellular carcinoma or renal cell carcinoma who may benefit from combination therapy with sorafenib [1]

C₁₄H₁₂CL₄N₄NARU

502.14

500.876

C, 33.49 H, 2.41 Cl, 28.24 N, 11.16 Na,4.58 Ru, 20.13

197723-00-5

BOLD-100 free base;783324-98-1

Light brown to khaki solid powder

5.883

[Ru-](Cl)(Cl)(Cl)Cl.[Na+].N1([H])C2=C([H])C([H])=C([H])C([H])=C2C([H])=N1.N1([H])C2=C([H])C([H])=C([H])C([H])=C2C([H])=N1

WVVOCRYXBTVDRN-UHFFFAOYSA-J

WVVOCRYXBTVDRN-UHFFFAOYSA-J

Sodium hydride; tetrachloro-bis(1H-indazol-2-yl)ruthenium

NKP1339; IT-139; KP-1339; KP-1339; NKP 1339; KP 1339; IT 139; IT139; KP1339; Na[trans-RuCl4(Ind)2; sodium trans-[RuCl4(HInd)2,

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)

DMSO: ≥ 59 mg/mL (~117.5 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.)