URAT1; GLUT9

Compound KPH2f exhibited potent URAT1-inhibitory activity with IC50 of 0.24 mM, comparable to that of verinurad (IC50 ¼ 0.17 mM). KPH2f also inhibited GLUT9 with an IC50 value of 9.37 ± 7.10 mM, indicating the dual URAT1/GLUT9 targeting capability. In addition, KPH2f showed little effects on OAT1 and ABCG2, and thus was unlikely to cause OAT1/ABCG2-mediated drug-drug interactions and/or to neutralize the uricosuric effects of URAT1/GLUT9 inhibitors[1].

KPH2f (10 mg/kg) was effective in reducing serum uric acid levels and exhibited higher uricosuric effects in a mice hyperuricemia model, as compared to verinurad (10 mg/kg). Furthermore, KPH2f demonstrated favorable pharmacokinetic properties with an oral bioavailability of 30.13%, clearly better than that of verinurad (21.47%). Moreover, KPH2f presented benign safety profiles without causing hERG toxicity, cytotoxicity in vitro (lower than verinurad), and renal damage in vivo. Collectively, these results suggest that KPH2f represents a novel, safe and effective dual URAT1/GLUT9 inhibitor with improved druggabilities and is worthy of further investigation as an anti-hyperuricemic drug candidate[1].

14C-uric acid uptake inhibitory assay [1]
The HEK293 cells were seeded into poly-D-lysine (PDL) coated 96 well plate at a density of 1  105/well. The URAT1 plasmid (100ng/well) was transiently transfected into HEK293 cells by using lipofectamine 3000. After transfection for 24 h, the cells were incubated with uric acid uptake buffer with or without various concentration of tested compounds for 30 min. The uptake was initiated by adding 14C-uric acid at a final concentration of 25 mM for 15 min. Cells were then washed three times with ice-cold DPBS to terminate the reaction. Cell lysates were obtained by adding 100 ml of 0.1 M sodium hydroxide. Intracellular radioactivity was determined using liquid scintillation counter after adding 0.5 mL of scintillation fluid. Experiments were done in triplicates. Inhibition rates of tested compounds were calculated as follows: Specific inhibition ¼ [1-(CPMtest-CPM0)/(CPMcon-CPM0)]  100% Where CPMt is the radioactivity of the tested group, CPMcon is the intracelullar radioactivity of the control group. CPM0 is the radioactivity of the cells with empty vector without hURAT1.

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Inhibitory effects of compounds on ABCG2, OAT1 and GLUT9 [1]
The inhibitory effects of tested compounds on uric acid transport related transporters including GLUT9, ABCG2 and OAT1 were conducted to evaluate the selectively of tested compounds. GLUT9: 500 ng of pcDNA3.1 (þ)-GLUT9 plasmid was transient transfected into HEK293 cells in 24 well plates with lipofectamine 3000. After 24 h, the GLUT9 inhibitory effects of compounds were determined in HEK293-GLUT9 cells by the electrophysiological recordings currents using the whole-cell patch-clamp technique. A constant perfusion of solution with or without compounds was delivered by the perfusion device, allowing a rapid solution exchange. The currents were measured with a MultiClamp 700B patch-clamp amplifier/Digidata 1550B digitizer and calculated by pClamp 10 software, as previously reported by us.

Cell viability assay (MTT assay) [1]
The human kidney (HK2) cells were used to detect cytotoxicity and cell viability induced by compounds. HK2 Cells were plated into 96 well plate at a density of 5000/well. When the cell confluence reached 70%, cells were treated with series concentrations of compounds (0e200 mM) for 24 h incubation. 0.5 mg/mL MTT solution was added to each well and the plate was further incubated at 37 C for 2 h. Thereafter the mediumwas removed and 100 ml of DMSO was added to each well. The plate was shaken at 250 rpm for 30 min, after the formazan crystals was dissolved, the absorbance was determined at 570 nm[1].

Evaluation of urate lowering effects of compounds in vivo [1]
Mice were feed one week to adapt to the environment before experiments. Mice then were randomly divided into 6 groups. Control group (n ¼ 8), model group (n ¼ 12), 3 compound groups (SG1C, KP, KPH2F) with 10 mg/kg treatment (n ¼ 8) and positive group (verinurad) with 10 mg/kg treatment (n ¼ 8). The method of hyperuricemia induction was conducted as we previous reported. Potassium oxonate (PO), a uricase inhibitor, was subcutaneous injected into mice at 400 mg/kg in 0.5% CMC-Na and 600 mg/kg of hypoxanthine was oral gavage in model and compound groups, 0.5 h after PO injection, the mice received 10 mg/kg compounds by oral gavage, while the control group was treatmented with 0.5% CMC. 3 h after the drug administration, blood samples were obtained from the orbital vein. The samples were then centrifuged (3000g, 10 min) to obtain serum for further analysis. Urine samples were collected after drug administration for 24 h in metabolic cages. The serum and urine uric acid level were determined by uric acid assay kit.

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In vivo pharmacokinetic study [1]
The male Sprague-Dawley rats (300 ± 20 g) were provided by Laboratory Animal Center of Southern Medical University. 24 male rats were divided into 4 groups for intravenous (5 mg/kg) and oral administration (5 mg/kg) of KPH2F and verinurad. KPH2F and verinurad were dissolved with normal saline for intragastric and intravenous administration. Blood samples (0.5 mL) were collected into heparinized tubes from the orbital vein at 2min, 5 min, 15 min, 30min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h. All the samples were immediately centrifuged at 8000 rpm for 5 min, and then the plasma were stored at 80 C until analysis. The samples were quantified with the LC-MS/MS system. 100 mL of plasma samples were spiked with 10 mL of the internal standard solution (testosterone, 1 mg/mL). The mixture was extracted with 600 mL of ethyl acetate. The supernatant was combined twice in succession and evaporated to dryness at 40e50 C. The residue was reconstituted in 400 mL of mobile phase (50% methanol) and then centrifuged at 14000 rpm for 15 min. A 300 mL aliquot of the resulting solution was injected into the LC-MS/MS system for analysis. Related pharmacokinetic parameters were calculated using DAS 2.0 software. The pharmacokinetic parameters determined included the elimination half-life (t1/2), time of peak plasma concentration (Tmax), maximum plasma concentration (Cmax), area under the concentrationetime curve (AUC), mean residence time (MRT) and bioavailability (F).

The pharmacokinetic properties of KPH2f and verinurad were assessed in SD rats. The results are summarized in Table 5 and Fig. 5. After a single 5 mg/kg i.v. dose of KPH2f, the half-life (t1/2), time-tomaximum- concentration (Tmax), maximum concentration (Cmax) and mean residence time (MRT) values were 4.19 h, 0.17 h, 11093.32 ng/mL and 4.80 h respectively. At the dose of 5 mg/kg (oral), KPH2f was rapidly absorbed with a Tmax of 0.50 h, a t1/2 of 5.14 h, an MRTof 3.17 h, a Cmax of 7649.04 ng/mL, and an area under curve (AUC) of 5656.03 ng/mLh. Notably, KPH2f exhibited a reasonable oral bioavailability of 30.13%, which is clearly better than that of verinurad (21.47%) and is sufficient for an oral drug candidate. Overall, KPH2f demonstrated more favorable drug-like properties when compare to verinurad. [1]

In vitro cytotoxicity assay [1]
KPH2f was selected for evaluating the in vitro cytotoxicity to normal cells by a well-established MTT assay. Human kidney HK2 cells were incubated with various concentrations of KPH2f for 24 h, and cell viabilities were measured. As shown in Fig. 6, KPH2f exhibited little or essentially no cytotoxicity to the HK2 cells with IC50 values of 207.56 mM and 167.24 mM for 24 h and 48 h incubation, respectively. While the cytotoxicity of verinurad was stronger (IC50 ¼ 197.45 mM and 108.78 mM for 24 h and 48 h incubation, respectively) than KPH2f. These results suggest that KPH2f has a benign cytotoxicity profile and is unlikely to cause toxicity at therapeutic concentrations (e.g. <50 mM).

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Determination of hERG-inhibitory activity [1]
Compounds that bind to hERG potassium channels with high affinities may induce QT prolongation, thus causing severe cardiotoxicity. Therefore, we tested the in vitro hERG inhibitory activity of KPH2f using a manual patch-clamp method. As shown in Fig. 7, KPH2f showed no inhibitory effects on hERG potassium channel at 50 mM, while the positive control cisapride inhibited the potassium channel completely at a concentration of 1 mM (100% reduction of the current), which is consistent with previous reports.

[1]. Discovery of novel verinurad analogs as dual inhibitors of URAT1 and GLUT9 with improved Druggability for the treatment of hyperuricemia. Eur J Med Chem. 2022;229:114092.

Among them, compound KPH2f exhibited the highest URAT1-inhibitory activity with IC50 value of 0.24 mM, comparable to that of verinurad (IC50 ¼ 0.17 mM). Mechanism study demonstrated that verinurad at 10 mM had no effects on GLUT9, while KPH2f inhibited GLUT9 effectively with an IC50 value of 9.37 ± 7.10 mM, suggesting a dual URAT1/GLUT9 targeting mechanism. In addition, KPH2f showed little inhibitory effects against OAT1 and ABCG2, two important transporters involved in urate secretion and the excretion of various drug molecules. Thus, it is unlikely for KPH2f to cause OAT1/ABCG2-mediated drug-drug interactions and/or to neutralize the uricosuric effects of URAT1/GLUT9 inhibitors. Importantly, KPH2f (10 mg/kg) was equally effective in reducing serum uric acid levels and exhibited higher uricosuric effects in a mice hyperuricemia model, as compared to verinurad (10 mg/kg). The higher uricosuric effects of KPH2f may be attributed to the dual URAT1/GLUT9 targeting capabilities, as compared to verinurad which is a single URAT1-targeting agent. Furthermore, KPH2f demonstrated favorable pharmacokinetic properties with an oral bioavailability of 30.13%, clearly better than that of verinurad (21.47%). Moreover, KPH2f presented benign safety profiles without causing hERG toxicity, cytotoxicity in vitro (lower than verinurad), and renal damage in vivo. Finally, molecular docking analysis using a homology model indicated that KPH2f and verinurad bound similarly to URAT1, and the flexible NH-linker contributed to the binding affinity of KPH2f to URAT1. Site directed mutagenesis study further confirmed the binding interactions between KPH2f and URAT1, and the residues F358 and R487 are unique for the binding of KPH2f to URAT1. Collectively, these results suggest that KPH2f represents a novel, safe and effective dual URAT1/GLUT9 inhibitor with improved druggabilities and is worthy of further investigation as an anti-hyperuricemic drug candidate.[1]

C24H17N3NAO2S

434.47

433.086

2760615-09-4

NA for KPH2f

163196377

Light yellow to light brown solid powder

1

6

6

31

642

0

N(C1=CN=CC=C1SCC1C=CC=CC=1C(=O)O)C1=CC=C(C2C=CC=CC1=2)C#N.[Na]

VFQCYGPPRZHSKX-UHFFFAOYSA-M

InChI=1S/C24H17N3O2S.Na/c25-13-16-9-10-21(20-8-4-3-6-18(16)20)27-22-14-26-12-11-23(22)30-15-17-5-1-2-7-19(17)24(28)29;/h1-12,14,27H,15H2,(H,28,29);/q;+1/p-1

sodium;2-[[3-[(4-cyanonaphthalen-1-yl)amino]pyridin-4-yl]sulfanylmethyl]benzoate

KPH2f; 2760615-09-4; CHEMBL5202967; URAT1/GLUT9 inhibitor; Sodium 2-(((3-((4-cyanonaphthalen-1-yl)amino)pyridin-4-yl)thio)methyl)benzoate

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.)