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 ratio and blood cell distribution[2]
Table 1 shows that the plasma protein binding percentage and blood cell distribution of dotinurad were similar in all the investigated species. There was no species difference in these parameters.

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Metabolism[2]
The results of the in vitro comparative metabolism study of 14C-FYU-981 in cryopreserved hepatocytes are presented in Table 3. The production ratios of glucuronide (7.3, 3.8, and 3.5%) and sulfate (3.8, 3.6 and 1.8%) metabolites of 14C-FYU-981 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, suggesting that the CL of 14C-FYU-981 was lower in human than it was in rat hepatocytes. The percentage of detected metabolites in samples collected after administration of 14C-FYU-981 to rats, monkeys, and humans are presented in Table 4.
In rat, monkey, and human plasma, the parent compound was detected as a major component, accounting for 81.9, 92.0, and 80.9% radioactivity, respectively. Levels of the methyl DCHB, sulfate, and 6-hydroxy metabolites were comparably high in the plasma samples of all tested species. In rat and human samples, levels of the methyl DCHB metabolite were the highest (2.8 and 5.1%, respectively), whereas the sulfate metabolite level was highest (1.5%) in monkeys. In rat urine, the percentage of 14C-FYU-981 was 2.2% and the sulfate of DCHB was detected as a major metabolite, accounting for 21.9%, followed by DCHB (12.2%) and a sulfate (11.7%). In monkey and human urine, 14C-FYU-981 was detected at 5.0 and 1.3%, respectively and the glucuronide was a major metabolite (37.0 and 51.8%, respectively), followed by the sulfate (26.7 and 23.4%, respectively). In feces, the levels of the 6-hydroxy, DCHB, and sulfonate metabolites and the parent compound were higher than those of other metabolites. The recovery of radioactivity from rat, monkey, and human fecal samples were 79.9, 78.8, and 67.5%, respectively. These values were slightly low. However, the radioactivity that could not be recovered was less than 4.6% as a percent of dose. The metabolite profile of the monkey was more similar to the human profile than it was to the rat profile.

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Excretion[2]
Fig. 3 shows the mean cumulative excretion of radioactivity into the urine, feces, and expired air and the total recovery. The urinary radioactivity levels were 68.7, 83.7, and 86.4% of the dose in rats, monkeys, and humans, respectively, whereas the corresponding levels in the feces were 22.7, 6.7, and 7.9% of the dose, respectively, for up to 168 h in both samples.
In the rat, monkey, and human samples, the excreted percentage radioactivity in expired air was 4.2, 7.8, and 5.0% of the dose for up to 96 h and the total recovered radioactivity was satisfactory at 95.5, 98.2, and 99.4%, respectively. The major route of excretion was the urine, and after accounting for the metabolite profile, the radioactivity was mainly excreted as the sulfate of DCHB in rats and as the glucuronide in monkeys and humans.

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CYP inhibition study[2]
Table 5 shows the result of evaluation of the inhibitory effect of dotinurad on major CYP isoforms (CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) in human liver microsomes. Dotinurad inhibited CYP2C9 with an inhibitory constant (Ki) value of 10.4 μmol/L. According to DDI guidelines [13], the R value calculated from the Ki and unbound Cmax after 7-day repeated administration of 4 mg dotinurad (7.0 nmol/L: unpublished data) was 1.00. Therefore, it was predicted that the risk of DDI via CYP inhibition was low.

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

Dotinurad is under investigation in clinical trial NCT03372200 (Febuxostat-controlled, Double-blind, Comparative Study of FYU-981 in Hyperuricemia With or Without Gout).

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The factors that maintain a high plasma drug concentration include high bioavailability (BA), low Vd, and low CL/F. The BA of dotinurad in rats and monkeys were 86.9 and 91.0%, respectively, and CL/F in human (0.013 L·h-1·kg-1) was lower than in rats (0.052 L·h-1·kg-1) and in 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, which was calculated using excreted percentage radioactivity of urine and expired air. Although the BA of dotinurad in humans has not been evaluated, it is expected to be high. Vd/F values of dotinurad in rats and monkeys were nearly equal to the extracellular fluid volume (0.297 and 0.208 L/kg, respectively). Furthermore, the Vd/F of dotinurad in humans was almost the same as that in rats and monkeys (Table 1). This result could be attributable to certain factors such as the similar Vd of rats and monkeys, which was almost constant following administration of doses of 0.3–3 mg/kg in rats (data not shown). In addition, the plasma protein binding was almost the same in rats, monkeys, and humans. The CL of dotinurad seems to be mainly mediated by metabolism, because the excretion pathways of dotinurad is mainly through the urine as metabolites (Fig. 3 and Table 4). In rats and monkeys the CL/F values were considerably lower than the hepatic blood flow values, which were 3.3 and 2.6 L·h-1·kg-1, respectively, indicating that the CL of dotinurad was low. These results indicated that high BA, low Vd/F, and CL/F leads to high plasma concentration of dotinurad at a low administration dose.[2]

Luminal drug concentrations in the renal proximal tubules could be regarded as the unbound plasma concentrations. Although lower plasma protein binding ratio would lead to higher concentrations in the renal lumen, this would also persistently affect the pharmacological by increasing the renal CL. The plasma protein binding of dotinurad was 99.4% in humans. This value might seem high as the pharmacological target of the drug is the renal tubules. However, this value suggests an adequate balance between the pharmacological effects and PK with a long duration. In fact, effects on serum urate levels and renal urate excretion of dotinurad were apparently saturable at a dose >5 mg, and PK/PD modeling and simulation of dotinurad (simple maximal effect model) indicated that the plasma concentration at the half-maximal effect of dotinurad was 3.8 nM (196 ng/mL corresponding to Cmax at a dose of 2 mg). Clinical dosage of benzbromarone is 50 mg (12.5-fold of dotinurad), even though IC50 value of benzbromarone (0.190 μM) in urate transport via URAT1 is approximately 5-fold that of dotinurad (0.0372 μM). The CL/F and Vd/F of benzbromarone in humans were approximately 0.053 L·h-1·kg-1, and 0.4 L/kg, respectively (calculated from dosage, AUC0-24 and T1/2). Although BA of dotinurad and benzbromarone remains unclear, if BA of dotinurad and benzbromarone were similar, the CL and Vd of dotinurad would be predicted to be lower than benzbromarone. Therefore, these results suggested that dotinurad has an ideal PK profile for a UA rather than benzbromarone for efficient drug delivery to its target.[2]

In addition, dotinurad potently inhibits URAT1 and exhibits high selectivity for other urate secretory transporters such as ABCG2, organic anion transporter 1 (OAT1, SLC22A6), and OAT3 (SLC22A8); consequently, it is designated a SURI. The pharmacological and PK characteristic of this agent both contribute to be its continuous serum uric acid-lowering activity at low doses (0.5–4 mg). In addition, at pharmacologically active concentrations, dotinurad did not inhibit drug transporters, such as multiple drug resistance 1 (MDR1), OATPs, and MATEs (recommended in DDI guidelines), and drug metabolizing enzymes such as CYPs (e.g., CYP2C9, which is inhibited by benzbromarone) and UGTs (data not shown). Furthermore, the contribution of MDR1 and ABCG2 for absorption of dotinurad from the intestine was low, as dotinurad has high absorption. In a rat in vivo study, dotinurad liver concentration was approximately 2-fold of plasma concentration (data not shown), and Vd/F in human was low. Therefore, this suggested that the contribution of OATP1B1 and OATP1B3 for uptake of dotinurad to the liver was low. If dotinurad was a substrate of these drug transporters, the affinity of dotinurad to these drug transporters would be low when considering IC50 of dotinurad on uptake of typical substrates by these drug transporters (MDR1 > 200 μM, ABCG2 74.7 μM, OATP1B1 11.5 μM, OATP1B3 > 200 μM). Therefore, the risk of DDIs with dotinurad is expected to be low as it exhibits pharmacological effects at low doses.[2]

Chronic kidney disease (CKD) has become a global public health issue and uric acid (UA) remains a major risk factor of CKD. As the main organ for the elimination of UA, kidney owned a group of urate transporters in tubular epithelium. Kidney disease hampered the UA excretion, and the accumulation of serum UA in return harmed the renal function. Commercially, there are three kinds of agents targeting at urate-lowering, xanthine oxidoreductase inhibitor which prevents the production of UA, uricosuric which increases the concentration of UA in urine thus decreasing serum UA level, and uricase which converts UA to allantoin resulting in the dramatic decrement of serum UA. Of note, in patients with CKD, administration of above-mentioned agents, alone or combined, needs special attention. New evidence is emerging for the efficacy of several urate-lowering drugs for the treatment of hyperuricemia in patients with CKD. Besides, loads of novel and promising drug candidates and phytochemicals are in the different phases of research and development. As of today, there is insufficient evidence to recommend the widespread use of UA-lowering therapy to prevent or slow down the progression of CKD. The review summarized the evidence and perspectives about the treatment of hyperuricemia with CKD for medicinal chemist and nephrologist.[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.)