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| ln Vitro |
Ko143 (10 nM) dramatically lowers the IC50 of MTX on mouse G2 and HEK G2 cells by a factor of 2.5. ABC transporter function is not inhibited by Ko143 (1-100 μM) metabolites [1]. Ko143, an FTC analog, overcomes drug resistance in human IGROV1/T8 cells and mouse MEF3.8/T6400 cells chosen with SKF 104864A. At zero, one, or eight times the EC90 of 25 nM, Ko143 can be utilized [2]. In Madin-Darby canine kidney (MDCK) 2-BCRP421CC (wild-type) and MDCK2-BCRP421AA (mutant) cells, Ko143 inhibits BCRP-mediated ZD 4522 trafficking [3].
Ko143 potently inhibited ABCG2-mediated efflux of fluorescent substrates (MTX, PPA, Hoechst 33342, JC-1, P-18) in human and mouse cells at nanomolar concentrations, with significant species differences for MTX, Hoechst, and P-18 (Table 3). In cytotoxicity assays, Ko143 at 10 nM significantly sensitized human ABCG2 cells to MTX (2.5-fold reduction in IC50, P<0.01) and mouse ABCG2 cells (P<0.001). At ≥1 μM, Ko143 sensitized human ABCB1 cells to paclitaxel (P<0.0001) and human ABCC1 cells to doxorubicin (P<0.001), and mouse ABCB1 cells to paclitaxel (P<0.01). Flow cytometry showed that Ko143 at 20–100 μM significantly increased accumulation of rh123 in ABCB1 cells and CAM in ABCC1 cells, confirming inhibition of these transporters at high concentrations. ATPase assays revealed that Ko143 inhibited ABCG2 ATPase (IC50 = 9.7 nM) but stimulated ABCB1 ATPase activity in a biphasic manner (peak stimulation at 10 μM, IC50 for stimulation ~2.7 μM). Radiolabeled [³H]Ko143 showed 2-fold higher accumulation in ABCG2-expressing cells than in parental cells, which was displaced by FTC or cold Ko143, indicating specific binding to ABCG2; no such effect was seen for ABCB1 or ABCC1 cells. The hydrolytic metabolite (Ko143 acid) had negligible inhibitory activity on all three transporters except weak inhibition of ABCG2 at 100 μM. Concentrations of Ko143 above 5 μM were cytotoxic to HEK cells after 72 h. [1] |
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| ln Vivo |
In mice, Ko143 (10 mg/kg, po) raises SKF 104864A's oral availability [2]. Ko143 has a major impact on ZD 4522's pharmacokinetics in rats [3].
In Vivo: In rats, intravenous administration of Ko143 (dose not specified in the text, but given as a pretreatment for 7 consecutive days, once daily? Actually the protocol says "Ko143 (intravenously)" given for 7 consecutive days, two times per day? The text: "group four and five were firstly given 80mg/kg of ursolic acid (orally) and Ko143 (intravenously, inhibitor of BCRP) respectively for 7 consecutive days, two times per day" - but dose of Ko143 is not stated. However, the pharmacokinetic results showed that pretreatment with Ko143 significantly increased Cmax, AUC0-t, and AUC0-∞ of rosuvastatin, and decreased CLz/F compared to rosuvastatin alone (all P<0.05). No significant effect on Tmax or T1/2. The data are presented in Table 2: Cmax 4.18±0.18 vs 2.23±0.22 mg/L; AUC0-t 62.72±6.34 vs 31.99±1.93 mg·L⁻¹·min⁻¹; AUC0-∞ 80.17±9.56 vs 40.92±3.10; CLz/F 0.25±0.03 vs 0.49±0.04 L·h⁻¹·kg⁻¹. [3] |
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| Enzyme Assay |
ATPase assays were performed using crude membranes isolated from Hi-five insect cells expressing human ABCG2 or ABCB1. Membrane vesicles were incubated in ATPase assay buffer (50 mM MES-Tris pH 6.8, 50 mM KCl, 5 mM sodium azide, 1 mM EGTA, 1 mM ouabain, 10 mM MgCl₂, 2 mM dithiothreitol) with varying concentrations of Ko143 with or without beryllium fluoride (0.2 mM beryllium sulfate plus 2.5 mM NaF). ATP hydrolysis was measured by estimating inorganic phosphate release after incubation with 5 mM ATP, following a previously described method. Basal activity (without Ko143) was normalized to 100%, and percent change in activity was plotted against Ko143 concentration to determine IC50. For ABCG2, Ko143 decreased ATPase activity with an IC50 of 9.7 nM; for ABCB1, Ko143 stimulated ATPase activity with a biphasic response, reaching maximum stimulation at 10 μM (2-fold over basal) and an IC50 for stimulation of 2.7 μM. [1]
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| Cell Assay |
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| Animal Protocol |
Animal Protocol: Sprague-Dawley rats (260-280 g) were randomly divided into groups (n=6). In the Ko143 treatment group, rats received Ko143 intravenously for 7 consecutive days, twice per day (exact dose not specified). On the 7th day, after Ko143 injection, rosuvastatin (50 mg/kg) was orally administered. Blood samples were collected from the femoral artery at 0.5, 1, 1.5, 3, 5, 8, 10, 12, and 24 hours post-dose. Plasma was separated and stored at -20°C until analysis. Rosuvastatin concentrations were determined by LC-MS with pitavastatin as internal standard. Pharmacokinetic parameters were calculated using DAS 2.0 software. [3]
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| ADME/Pharmacokinetics |
In rat plasma, Ko143 was rapidly hydrolyzed with a half-life of approximately 12 minutes at room temperature, and no parent compound remained after 60 minutes. The addition of the esterase inhibitor sodium fluoride (NaF) completely prevented hydrolysis, indicating enzymatic degradation. Ko143 acid (the hydrolytic metabolite) showed negligible inhibitory activity on ABCG2, ABCB1, or ABCC1 except weak ABCG2 inhibition at 100 μM. Plasma protein binding of Ko143 is reported to be 92–95% (from literature, not measured in this study). [1]
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| Toxicity/Toxicokinetics |
Ko143 exhibited cytotoxicity at concentrations greater than 5 μM in HEK-293 cells after 72-hour incubation. No other toxicity data (e.g., in vivo) were reported. [1]
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| References | ||
| Additional Infomation |
LSM-6260 is a β-carboline compound, belonging to the tert-butyl ester class.
Ko143 is a tert-butyl ester and is rapidly hydrolyzed by plasma esterases to its carboxylic acid metabolite, which is inactive. Despite being a potent ABCG2 inhibitor at nanomolar concentrations, it lacks specificity for ABCG2 at higher micromolar concentrations, also inhibiting ABCB1 and ABCC1. This non-selectivity should be considered when using Ko143 in both in vitro and in vivo experiments, especially for PET imaging studies where high local concentrations may occur. The study emphasizes the need for a specific ABCG2 substrate or inhibitor for reliable imaging of transporter function. [1] |
| Molecular Formula |
C26H35N3O5
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|---|---|
| Molecular Weight |
469.582
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| Exact Mass |
469.257
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| CAS # |
461054-93-3
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| PubChem CID |
10322450
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
689.8±55.0 °C at 760 mmHg
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| Melting Point |
147ºC
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| Flash Point |
371.0±31.5 °C
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| Vapour Pressure |
0.0±2.2 mmHg at 25°C
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| Index of Refraction |
1.597
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| LogP |
2.42
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
34
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| Complexity |
794
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| Defined Atom Stereocenter Count |
3
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| SMILES |
CC(C)C[C@H]1C2=C(C[C@@H]3N1C(=O)[C@@H](NC3=O)CCC(=O)OC(C)(C)C)C4=C(N2)C=C(C=C4)OC
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| InChi Key |
NXNRAECHCJZNRF-JBACZVJFSA-N
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| InChi Code |
InChI=1S/C26H35N3O5/c1-14(2)11-20-23-17(16-8-7-15(33-6)12-19(16)27-23)13-21-24(31)28-18(25(32)29(20)21)9-10-22(30)34-26(3,4)5/h7-8,12,14,18,20-21,27H,9-11,13H2,1-6H3,(H,28,31)/t18-,20-,21-/m0/s1
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| Chemical Name |
tert-butyl 3-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2'
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| Synonyms |
Ko-143 Ko-143 Ko-143
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~212.96 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.5 mg/mL (5.32 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 ultrasonication (<50°C). For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 2.5 mg/mL (5.32 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1296 mL | 10.6478 mL | 21.2956 mL | |
| 5 mM | 0.4259 mL | 2.1296 mL | 4.2591 mL | |
| 10 mM | 0.2130 mL | 1.0648 mL | 2.1296 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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