URAT1/Urate transporter 1 (IC50 = 3.6 µM)

Dotinurad, a novel selective urate reabsorption inhibitor (SURI), has potent inhibitory effects at low doses on the uptake of urate by urate transporter 1 (URAT1, solute carrier family 22 member 12 [SLC22A12]), localized at the apical membrane of renal proximal tubular cells.[2]
Dotinurad inhibits organic ion transporters in the proximal tubule, especially URAT1, but maybe also interacting with OATs. [1]

This study sought to clarify the pharmacokinetic (PK) profile of dotinurad. In rats, monkeys, and humans, the apparent distribution volume (0.257, 0.205, and 0.182 L/kg, respectively) and oral clearance (0.054, 0.037, and 0.013 L·h-1·kg-1, respectively) of dotinurad were very low, whereas plasma and luminal concentrations were adequately maintained at high levels. In addition, species differences were scarcely observed with plasma protein binding of 99.4%. The main metabolite was dotinurad glucuronide (no specific metabolites in humans), and percentage excretion of unchanged dotinurad was low in all the investigated species. The risk of drug interaction with dotinurad was expected to be low, because it weakly inhibits metabolic enzymes such as cytochrome P450 (CYP). In conclusion, low-dose dotinurad exhibited excellent pharmacological effects as well as ideal PK properties as a SURI.[2]

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In the rat and monkey, the time to achieve Cmax (Tmax) of dotinurad and TRA after administration were comparatively fast (<1.83 h). The Tmax and Cmax of the TRA were faster and higher, respectively, than those of Dotinurad (FYU-981) in humans because 14C-FYU-981 was administered in solution, whereas dotinurad was administered as a tablet. The oral clearance (CL/F: 0.054, 0.037 and 0.013 L·h−1·kg−1) and apparent Vd (Vd/F: 0.257, 0.205, and 0.182 L/kg) of Dotinurad (FYU-981) were very low in rats, monkeys, and humans, respectively.[2]

Plasma protein binding and blood cell distribution ratios of 14C-Dotinurad (FYU-981)[2]
Rat, monkey, and human plasma and blood samples (n = 3) were collected on the day of study. A test sample was prepared by adding the 14C-Dotinurad (FYU-981) solution to each plasma and blood sample (final concentration: 1 μg/mL). To determine the radioactivity concentration in the plasma and the blood, an aliquot of each test sample was dissolved in tissue solubilizer SOLUENE-350 (only blood test sample was decolorized with benzene saturated with benzoyl peroxide) and mixed with scintillator HIONIC-FLUOR. The remaining test samples were incubated in a water bath at 37 °C for 5 min. Aliquots of the plasma test sample were injected into ultracentrifugation devices and centrifuged (1800×g, 37 °C, 10 min) to recover the filtrate. The filtrate was mixed using the scintillator. The radioactivity of processed plasma test sample and filtrate were measured using a liquid scintillation counter. Plasma protein binding ratio was calculated from the radioactivity concentration in the plasma and filtrate. On the contrary, the blood test sample was collected in a capillary tube to determine the hematocrit value (Ht). The remaining blood test sample was centrifuged (8000×g, 4 °C, 5 min) to separate plasma. An aliquot of the plasma was dissolved in tissue solubilizer and mixed using a scintillator. The radioactivity of processed blood test sample and separated plasma was measured using LSC. The blood cell distribution of radioactivity was calculated from the Ht and radioactivity concentration in the whole blood and plasma.

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CYP inhibition study[2]
The CYP inhibitory effects of dotinurad were evaluated according to the Human and Animal Bridging Research Organization (HAB) protocol. The concentrations of dotinurad evaluated in this inhibition study were 1, 2.5, 5, 10, 25, 50, and 100 μM. The analytical methods for metabolites of CYPs and calculation method of inhibitory effect from HAB protocol were modified. Chromatography was performed using CAPCELL PAK C18 UG 120 column (4.6 × 250 mm, 5 μm) for the studies of CYP1A2, CYP2B6, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and Mightysil RP-18GP column (4.6 × 250 mm, 5 μm) for the study of CYP2C9. Each IC50 was calculated by activity ratio (%), that is, activity in presence of dotinurad divided by activity in absence of dotinurad.

In vitro comparative metabolism study of 14C-Dotinurad (FYU-981) in cryopreserved hepatocytes[2]
Pooled male rat and monkey cryopreserved hepatocytes and pooled mixed gender human cryopreserved hepatocytes were from Sekisui XenoTech. Hepatocyte suspensions were prepared using Hepatocyte Isolation Kit (K2000) manufactured by Sekisui XenoTech. The hepatocyte suspension (final concentration: 1 × 106 cells/mL) from each species or Krebs-Henseleit Buffer (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 6.3 mM HEPES, and 11 mM d-glucose, pH 7.4; control group) was added to a 24-well plate and pre-incubated in a CO2 incubator (37 °C, 5% CO2, 95% air) for 10 min. The reaction commenced by the addition of 14C-Dotinurad (FYU-981) solution (final concentration: 10 μM), and the mixture was incubated in the CO2 incubator for 0 and 1 h. After the incubation, acetonitrile (volume equal to the mixture) was added to the incubation mixture to terminate the reaction. After termination, the mixture was stirred and centrifuged (1800×g, 4 °C, 5 min) to separate the supernatant. The collected supernatant was evaporated to dryness using an evaporator. After drying, the residue was dissolved in N-methyl-2-pyrrolidione/initial mobile phase (1:1, v/v) by sonication. The solution was centrifuged (15000×g, 4 °C, 5 min), and the obtained supernatant was injected into HPLC-radioactivity detection (RAD) with a setup consisting of a Shimadzu 10A HPLC system, and 625 TR flow scintillation analyzer. The HPLC analytical condition was referred as “Sample Analysis”.

The animal studies were conducted at SMD (all radioisotope and monkey studies) or FY (rat study). Male Sprague-Dawley (SD, Crj:CD[SD]IGS) rats, aged 6 weeks were purchased from Charles River Laboratories. After a 1-week acclimatization period, the animals were used for the study at 7 weeks old. Male cynomolgus monkeys, aged 3 years, were purchased from Hamri Co., Ltd. Dotinurad (FYU-981) (including 14C-FYU-981) was suspended in 0.5% methylcellulose and administered orally (p.o.) at a dose of 1 mg/kg to starved rats or monkeys.[2]

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Dotinurad (FYU-981) concentration in rat, monkey, and human plasma were analyzed using a validated method at FY. The rat and monkey plasma samples were deproteinized, using methanol containing an internal standard (F12994), and analyzed using liquid chromatography-tandem mass spectrometry and ultraperformance-LC, respectively. The standard curve of dotinurad ranged between 1 and 300 ng/mL for rat plasma and 30–3000 ng/mL for monkey plasma. The human plasma samples were processed using solid-phase extraction in a 96-well plate format and analyzed using LC-MS/MS. The standard curve of dotinurad ranged between 1 and 1000 ng/mL for human plasma. Dotinurad and matrix constituents in human plasma were separated using an Inertsil ODS-3 (2.1 × 150 mm 3 μm) at 50 °C with a mobile phase of 5 mmol/L ammonium acetate (pH 4) in water and methanol (50:50, v/v). The total flow rate was set at 0.18 mL/min. Ionization was conducted in the turbo ion spray and negative ion modes. Dotinurad was analyzed as [M-H]- ions in the multiple reaction monitoring mode (transitions: dotinurad 356.0/159.9 and internal standard F12994 341.1/145.1).[2]

Plasma protein binding and hematocytic distribution [2] Table 1 shows that the plasma protein binding and hematocytic distribution of dotinurard were similar in all studied species, and these parameters were not different among different species. Metabolism [2] Table 3 lists the results of an in vitro comparative metabolic study of 14C-FYU-981 in cryopreserved hepatocytes. The production rates of 14C-FYU-981 glucuronide (7.3%, 3.8%, and 3.5%) and sulfate (3.8%, 3.6%, and 1.8%) metabolites were high in rat, monkey, and human hepatocytes. The level of 14C-FYU-981 in human hepatocytes was slightly higher than that in rat hepatocytes, indicating that the clearance rate of 14C-FYU-981 in human hepatocytes was lower than that in rat hepatocytes. Table 4 lists the percentage of metabolites detected in samples collected after injection of 14C-FYU-981 into rats, monkeys, and humans. In rat, monkey, and human plasma, the parent compound was detected as the major component, accounting for 81.9%, 92.0%, and 80.9% of the radioactivity, respectively. Levels of methylDCHB, sulfate, and 6-hydroxy metabolites were relatively high in plasma samples from all tested species. The highest levels of methylDCHB metabolites were observed in rat and human samples (2.8% and 5.1%, respectively), while the highest level of sulfate metabolites was observed in monkeys (1.5%). In rat urine, ¹⁴C-FYU-981 accounted for 2.2%, with DCHB sulfate being detected as the major metabolite at 21.9%, followed by DCHB (12.2%) and sulfate (11.7%). In monkey and human urine, the detection rates of ¹⁴C-FYU-981 were 5.0% and 1.3%, respectively, with glucuronide being the main metabolite (37.0% and 51.8%, respectively), followed by sulfate (26.7% and 23.4%, respectively). In feces, the levels of 6-hydroxy, dichlorobenzothiophene (DCHB), and sulfonate metabolites, as well as the parent compound, were higher than other metabolites. The recovery rates of radioactivity from rat, monkey, and human fecal samples were 79.9%, 78.8%, and 67.5%, respectively, which were slightly low. However, the percentage of unrecovered radioactivity was less than 4.6% of the dose. The metabolite profile of monkeys was more similar to that of humans than that of rats.

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Excretion[2]
Figure 3 shows the average cumulative excretion of radioactive material in urine, feces, and exhaled gases, as well as the total recovery rate. In rats, monkeys, and humans, the urinary radioactivity levels were 68.7%, 83.7%, and 86.4% of the dose, respectively, while the corresponding fecal radioactivity levels were 22.7%, 6.7%, and 7.9% of the dose (both samples over 168 hours). In rat, monkey, and human samples, the percentage of radioactive material excreted in exhaled breath was 4.2%, 7.8%, and 5.0% of the dose (over 96 hours), respectively, with total radioactivity recoveries of 95.5%, 98.2%, and 99.4%, all satisfactory. The primary route of excretion was urine. Metabolite analysis showed that the radioactive material was primarily excreted as DCHB sulfate in rats, and as glucuronide in monkeys and humans.

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CYP Inhibition Study [2]
Table 5 shows the evaluation results of dotinurara's inhibitory effect on major CYP isoenzymes (CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) in human liver microsomes. The inhibitory constant (Ki) of dotinurara against CYP2C9 was 10.4 μmol/L. According to the Drug Interaction (DDI) guidelines [13], the R value calculated based on Ki and free Cmax was 1.00 after 7 consecutive days of administration of 4 mg dotinurara (7.0 nmol/L: unpublished data). Therefore, the risk of drug interaction through CYP inhibition is predicted to be low.

[1]. Eur J Med Chem. 2019 Mar 15;166:186-196.;
[2]. Drug Metab Pharmacokinet. 2020 Jun;35(3):313-320.

Dotinurad is currently undergoing clinical trial NCT03372200 (a double-blind, comparative study with febuxostat as a control, comparing the efficacy of FYU-981 in hyperuricemia with and without gout). Factors maintaining high plasma drug concentrations include high bioavailability (BA), low volume of distribution (Vd), and low clearance/fraction of free (CL/F). Dotinurad's bioavailability in rats and monkeys was 86.9% and 91.0%, respectively, while the CL/F in humans (0.013 L·h⁻¹·kg⁻¹) was lower than that in rats (0.052 L·h⁻¹·kg⁻¹) and monkeys (0.037 L·h⁻¹·kg⁻¹). Furthermore, a human mass balance study showed that at least 91.4% of Dotinurad was absorbed after oral administration, calculated as a percentage of radioactive excretion in urine and exhaled breath. Although the bioavailability (BA) of dotinurara in humans has not been assessed, it is expected to be high. The volume of distribution/fraction of distribution (Vd/F) values of dotinurara in rats and monkeys were nearly equal to the extracellular fluid volume (0.297 and 0.208 L/kg, respectively). Furthermore, the Vd/F values of dotinurara in humans were nearly identical to those in rats and monkeys (Table 1). This result may be attributed to factors such as the similarity of the volume of distribution (Vd) in rats and monkeys, and the fact that the Vd values remained almost unchanged after administration of doses of 0.3–3 mg/kg in rats (data not shown). In addition, plasma protein binding was nearly identical in rats, monkeys, and humans. The clearance (CL) of dotinurara appears to be primarily mediated by metabolism, as dotinurara is mainly excreted in the urine as metabolites (Figure 3 and Table 4). In rats and monkeys, the CL/F values were significantly lower than those of hepatic blood flow (3.3 and 2.6 L·h⁻¹·kg⁻¹, respectively), indicating low clearance of Dotinurad. These results suggest that high bioavailability, low volume of distribution/blood flow ratio (Vd/F), and CL/F lead to higher plasma concentrations of Dotinurad at low doses. [2]
Drug concentrations in the proximal tubules of the kidney can be considered as free plasma concentrations. While lower plasma protein binding results in higher drug concentrations in the tubules, this also has a sustained effect on pharmacological action by increasing renal clearance. Dotinurad has a plasma protein binding rate of 99.4% in humans. This value may seem high given that the drug's pharmacological target is the renal tubules. However, this value suggests a proper balance between pharmacological action and pharmacokinetics, and a longer duration of action. In fact, the effect of Dotinurad on serum uric acid levels and renal uric acid excretion appears to be saturated at doses above 5 mg. Pharmacokinetic/pharmacodynamic modeling and simulation (simple maximum effect model) of Dotinurad indicated a half-maximal plasma concentration of 3.8 nM (equivalent to a Cmax of 196 ng/mL at a 2 mg dose). Although the IC50 value of benzbromarone for uric acid transport via URAT1 (0.190 μM) is approximately 5 times that of Dotinurad (0.0372 μM), the clinical dose of benzbromarone remains 50 mg (12.5 times that of Dotinurad). The CL/F and Vd/F of benzbromarone in humans are approximately 0.053 L·h⁻¹·kg⁻¹ and 0.4 L/kg, respectively (calculated based on dose, AUC0-24, and T1/2). Although the bioavailability of Dotinurad and benzbromarone is not well understood, if their bioavailability is similar, the CL and Vd of Dotinurad are expected to be lower than those of benzbromarone. Therefore, these results indicate that Dotinurad has a more desirable PK profile for uric acid drugs compared to benzbromarone, enabling more efficient drug delivery to the target. [2]
In addition, Dotinurad effectively inhibits URAT1 and exhibits high selectivity for other uric acid secretion transporters such as ABCG2, organic anion transporter 1 (OAT1, SLC22A6) and OAT3 (SLC22A8); therefore, it has been designated as SURI (severe uric acid deficiency). The pharmacological and pharmacokinetic characteristics of the drug contribute to its sustained reduction of serum uric acid levels at low doses (0.5–4 mg). Furthermore, at pharmacologically active concentrations, Dotinurad does not inhibit drug transporters such as multidrug resistance protein 1 (MDR1), organophosphorus transporter (OATP) and MATE (recommended by the drug interaction guideline), nor does it inhibit drug-metabolizing enzymes such as CYP (e.g., CYP2C9, which can be inhibited by benzbromarone) and UGT (data not shown). Furthermore, due to the high absorption rate of Dotinurad, the contributions of MDR1 and ABCG2 to its intestinal absorption are also very low. In rat studies, the liver concentration of Dotinurad was approximately twice the plasma concentration (data not shown), and its volume of distribution/distribution factor (Vd/F) in humans was low. Therefore, this suggests that OATP1B1 and OATP1B3 contribute little to the hepatic uptake of Dotinurad. If Dotinurad is a substrate of these drug transporters, given the IC50 values of Dotinurad for typical substrates of these drug transporters (MDR1 > 200 μM, ABCG2 74.7 μM, OATP1B1 11.5 μM, OATP1B3 > 200 μM), Dotinurad's affinity for these drug transporters should be low. Therefore, given that Dotinurad exerts its pharmacological effects at low doses, the risk of drug interactions is expected to be low. [2]
Chronic kidney disease (CKD) has become a global public health problem, and uric acid (UA) remains a major risk factor for CKD. As the main organ for clearing uric acid, the kidneys contain a group of uric acid transport proteins in their renal tubular epithelial cells. Kidney disease can hinder the excretion of uric acid (UA), and the accumulation of serum uric acid can in turn impair kidney function. Currently, there are three main types of commercially available uric acid-lowering drugs: xanthine oxidoreductase inhibitors, whose mechanism of action is to inhibit the production of uric acid; uricosuric agents, whose mechanism of action is to increase the concentration of uric acid in urine, thereby reducing serum uric acid levels; and uricase, whose mechanism of action is to convert uric acid into allantoin, thereby significantly reducing serum uric acid levels. It is worth noting that for patients with chronic kidney disease (CKD), the use of the above drugs alone or in combination should be approached with extreme caution. Increasing evidence suggests that a variety of uric acid-lowering drugs are effective in treating hyperuricemia in patients with CKD. In addition, a large number of novel and promising candidate drugs and phytochemicals are in different stages of development. To date, there is insufficient evidence to support the widespread use of uric acid-lowering therapy to prevent or delay the progression of CKD. This review summarizes the evidence and opinions on the treatment of CKD with hyperuricemia for the reference of medicinal chemists and nephrologists. [1]

C14H9CL2NO4S

358.1966

356.962

C, 46.95; H, 2.53; Cl, 19.79; N, 3.91; O, 17.87; S, 8.95

1285572-51-1

1285572-51-1;

51349053

Typically exists as solid at room temperature

2.9

1

4

1

22

538

0

VOFLAIHEELWYGO-UHFFFAOYSA-N InChi Code

InChI=1S/C14H9Cl2NO4S/c15-9-5-8(6-10(16)13(9)18)14(19)17-7-22(20,21)12-4-2-1-3-11(12)17/h1-6,18H,7H2

(3,5-dichloro-4-hydroxyphenyl)(1,1-dioxidobenzo[d]thiazol-3(2H)-yl)methanone

FYU-981; FYU 981; Dotinurad; 1285572-51-1; (3,5-dichloro-4-hydroxyphenyl)(1,1-dioxidobenzo[d]thiazol-3(2H)-yl)methanone; (3,5-dichloro-4-hydroxyphenyl)-(1,1-dioxo-2H-1,3-benzothiazol-3-yl)methanone; 305EB53128; Urece; FYU981; Urece

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.)