xanthine oxidoreductase (XOR) (IC50 = 5.3 nM; Ki = 5.7 nM)

Topiroxostat（FYX-051，化合物 39）具有强大且持久的作用，已通过 XOR-Topiroxostat 复合晶体学研究得到验证。已观察到 Topiroxostat 和 XOR 之间的结合活性受到 Topiroxostat 氰基的显着影响。 XOR 的 Asn 768 和 Topiroxostat 的氰基形成氢键，这是造成这种情况的原因[1]。

在氧酸钾诱导的高尿酸血症大鼠模型中，托吡司他（FYX-051；0.03-10 mg/kg；口服；持续1小时；雄性Wistar/ST品系大鼠）治疗显示出强烈且持久的降尿酸作用[2 ]。 Topiroxostat（FYX-051，化合物 39）的 Cmax 为 4.62 μg/mL（3 mg/kg），生物利用度为 69.6%。此外，Topiroxostat 的 t1/2 值为 19.7 小时[1]。

抑制剂对XO的时间依赖性抑制和XO抑制剂复合物的稳定性。[2]
FYX-051在空气饱和条件下显示出对尿酸盐形成的时间和浓度依赖性抑制作用（图2A）。XOR本身对FYX-051进行初级羟基化产生的2-羟基-FYX-051也会引起时间和浓度依赖性抑制，尽管由于2-羟基-FY-051的亲和力较低，因此需要相对大量的2-羟基-FeX-051来实现有效的抑制（图2B），如稳态实验所示（见下文）。我们还估计。。

Animal/Disease Models: Male Wistar/ST strain rats (7 weeks old) injected with potassium oxonate[2]
Doses: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg
Route of Administration: Oral administration; for 1 hour
Experimental Results: Caused a dose-dependent decrease in serum urate levels with an extremely low ED50 of 0.15 mg/kg, evaluated at 1 h after oral administration.

Absorption, Distribution and Excretion
The time to reach peak plasma concentration of 229.9 ng/mL was 0.67 hour following a single oral dose of 20mg topiroxostat. The oral bioavailability in male rats was 69.6% after oral administration of a single dose of 1mg/kg.
Urinary excretion and fecal excretion of radiolabeled topiroxostat are 30.4% and 40.9% of total dose of 1mg/kg administered to rats, respectively. Within 24 h after a single oral administration of 120mg of topiroxostat, the main metabolites of topiroxostat, N-oxide, N1-gluculonide, and N2-gluculonide, are excreted into urine about 4.8, 43.3, and 16.1 % of the dose, respectively. Unchanged topiroxostat and the hydroxide metabolite was 0.1% or less.
The distribution of 14C-topiroxostat (20, 200, and 2000 ng/mL) in human blood cells was 6.7% to 12.8%.
The apparent total body clearance rate is 89.5 L/h and the renal clearance rate is 17.4 mL/h following a single oral dose of 20mg topiroxostat.

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Metabolism / Metabolites
Topiroxostat is mainly inactivated by hepatic metabolism. 2-hydroxy topiroxostat is formed from primary hydroxylation of the drug by xanthine oxidase and still retains an inhibitory activity on the enzyme. Topiroxostat N-oxide is another major metabolite that can be detected in plasma and urine. It is determined that the N-oxide and hydroxide metabolites are pyridine N-oxide and pyridine 2 (or 6)-hydroxide, respectively. Topiroxostat is mainly inactivated by hepatic metabolism where it undergoes glucuronidation. The metabolism of topiroxostat to N1-and N2-glucuronide conjugates is mainly mediated by UGT1A1, 1A7, and 1A9, with UGT1A9 being the most predominant.
FYX-051 has known human metabolites that include 4-[2-[(3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxyoxan-2-yl]-5-pyridin-4-yl-1,2,4-triazol-3-yl]pyridine-2-carbonitrilium and (2S,3S,4S,5R)-6-[3-(2-cyanopyridin-4-yl)-5-pyridin-4-yl-1,2,4-triazol-1-yl]-3,4,5-trihydroxyoxane-2-carboxylic acid.

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Biological Half-Life
The mean half life of topiroxostat after a single oral dose of 20mg topiroxostat is 5 hours under fasting condition. The complex of molybdenum (IV)- topiroxostat has an approximate half life of 20.4 hours.

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Compound 39/topiroxostat exhibited a weak CYP3A4-inhibitory activity (18.6%); its Cmax and bioavailability were as high as 4.62 μg/mL (3 mg/kg) and 69.6%, respectively. Moreover, the t1/2 value of 39 was greater (19.7 h) than that of compound 2 (0.97 h). Since 39 is mainly excreted in the urine as triazole N1- and N2-glucuronides in monkeys and humans, it is expected to be a safe drug in patients with renal impairment.[1]

Protein Binding
The mean protein binding of radiolabeled (14C)-topiroxostat in human plasma is >97.5% at 20ng/mL, 98.8% at 200ng/mL, and 98.4% at 2000ng/mL. Binding to serum albumin is most predominant with 92.3-93.2%, and mean protein binding to α1-acid protein and γ-globulin is 12.3% to 16.8% and 34.7% to 40.4%, respectively.

[1]. Sato T, et al. Discovery of 3-(2-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole, FYX-051 - a xanthine oxidoreductase inhibitor for the treatment of hyperuricemia [corrected]. Bioorg Med Chem Lett. 2009 Nov 1;19(21):6225-9.
[2]. Matsumoto K, et al. FYX-051: a novel and potent hybrid-type inhibitor of xanthine oxidoreductase. J Pharmacol Exp Ther. 2011 Jan;336(1):95-103.

Topiroxostat is a selective xanthine oxidase inhibitor developed for treatment and management of hyperuricemia and gout. Xanthine oxidase, or xanthine oxidoreductase (XOR), regulates purine metabolism, and inhibition of the enzyme results in efficacious reduction of serum urate levels. Xanthine oxidase inhibitors are classified into two groups; purine analogs such as [DB00437] and [DB05262], and non-purine agents which includes topiroxostat. While [DB00437] is considered a first-line therapy in treating hyperuricemic conditions, it is often associated with side effects and ineffective in reducing uric acid levels under recommended dosing regimens. Renal complications are major comorbidities that limit the [DB00437] therapy as dose reductions are recommended. Topiroxostat and its metabolites are shown to be unaffected by renal complications, thus may be effective in patients with chronic kidney diseases. Approved for therapeutic use in Japan since 2013, topiroxostat is marketed under the name Topiloric and Uriadec and is orally administered twice daily.

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Drug Indication
Indicated for the treatment of gout and hyperurcemia in Japan.

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Mechanism of Action
Uric acid synthesis depends on the action of xanthine oxidase activity in the conversion of hypoxanthine to xanthine, followed by the conversion of xanthine to uric acid. Xanthine oxidase consists of a molybdenum ion as cofactor in the active center that has different redox states upon substrate binding. When a substrate such as hypoxanthine or xanthine binds, xanthine oxidase hydroxylates it and molybdenum ion is reduced from hexavalent, Mo(VI), to tetravalent form, Mo(IV). Molybdenum ion is reoxidized into hexavalent state once the hydroxylated substrate, xanthine or uric acid, dissociates from the active site. Topiroxostat is shown to interact with multiple amino acid residues of the solvent channel and additionally forms a reaction intermediate by covalent binding with molybdenum (IV) ion via an oxygen atom. It also forms hydrogen bonds with molybdenum (VI) ion, suggesting that it has multiple inhibition modes to xanthine oxidase. Enhanced binding interactions to xanthine oxidase achieves delayed dissociation of topiroxostat from the enzyme. 2-hydroxy-topiroxostat, the metabolite formed by primary hydroxylation of topiroxostat by xanthine oxidase, also causes time and concentration-dependent inhibition of the enzyme. Topiroxostat is shown to inhibit ATP-binding cassette transporter G2 (ABCG2) in vitro, which is a membrane protein responsible for recovering uric acid in the kidneys and secreting uric acid from the intestines.

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Our previous study identified 2-[2-(2-methoxyethoxy)ethoxy]-5-[5-(2-methyl-4-pyridyl)-1H-[1,2,4] triazol-3-yl]benzonitrile (2)[corrected]as a safe and potent xanthine oxidoreductase (XOR) inhibitor for the treatment of hyperuricemia. Here, we synthesized a series of 3,5-dipyridyl-1,2,4-triazole derivatives and, in particular, examined their in vivo activity in lowering the serum uric acid levels in rats. As a result, we identified 3-(2-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole (FYX-051, compound 39) [corrected] to be one of the most potent XOR inhibitors; it exhibited an extremely potent in vivo activity, weak CYP3A4-inhibitory activity and a better pharmacokinetic profile than compound 2. Compound 39 is currently being evaluated in a phase 2 clinical trial.[1]

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4-[5-(Pyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine-2-carbonitrile (FYX-051) is a potent inhibitor of bovine milk xanthine oxidoreductase (XOR). Steady-state kinetics study showed that it initially behaved as a competitive-type inhibitor with a K(i) value of 5.7 × 10(-9) M, then after a few minutes it formed a tight complex with XOR via a Mo-oxygen-carbon atom covalent linkage, as reported previously (Proc Natl Acad Sci USA 101:7931-7936, 2004). Thus, FYX-051 is a hybrid-type inhibitor exhibiting both structure- and mechanism-based inhibition. The FYX-051-XOR complex decomposed with a half-life of 20.4 h, but the enzyme activity did not fully recover. This was found to be caused by XOR-mediated conversion of FYX-051 to 4-[5-(2-hydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine-2-carbonitrile (2-hydroxy-FYX-051), as well as formation of 6-hydroxy-4-[5-(2-hydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine-2-carbonitrile (dihydroxy-FYX-051) and 4-[5-(2,6-dihydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]-6-hydroxypyridine-2-carbonitrile (trihydroxy-FYX-051) during prolonged incubation for up to 72 h. A distinct charge-transfer band was observed concomitantly with the formation of the trihydroxy-FYX-051-XOR complex. Crystallographic analysis of the charge-transfer complex indicated that a Mo-nitrogen-carbon bond was formed between molybdenum of XOR and the nitrile group of trihydroxy-FYX-051. FYX-051 showed a potent and long-lasting hypouricemic effect in a rat model of potassium oxonate-induced hyperuricemia, and it seems to be a promising candidate for the clinical treatment of hyperuricemia.[2]

C13H8N6

248.24

248.081

C, 62.90; H, 3.25; N, 33.85

577778-58-6

Topiroxostat-d4;2732868-49-2

5288320

White to off-white solid powder

1.5±0.1 g/cm3

594.7±60.0 °C at 760 mmHg

175.3±18.1 °C

0.0±1.7 mmHg at 25°C

1.697

1.35

91.14

1

5

2

19

344

0

N1([H])C(C2C([H])=C([H])N=C([H])C=2[H])=NC(C2C([H])=C([H])N=C(C#N)C=2[H])=N1

UBVZQGOVTLIHLH-UHFFFAOYSA-N

InChI=1S/C13H8N6/c14-8-11-7-10(3-6-16-11)13-17-12(18-19-13)9-1-4-15-5-2-9/h1-7H,(H,17,18,19)

4-(5-pyridin-4-yl-1H-1,2,4-triazol-3-yl)pyridine-2-carbonitrile

FYX051; FYX-051; 577778-58-6; topiloric; 4-(5-PYRIDIN-4-YL-1H-1,2,4-TRIAZOL-3-YL)PYRIDINE-2-CARBONITRILE; Topiroxostat [INN]; TOPIROXOSTAT [MI]; FYX 051; Trade names: Topiloric; Uriadec; Topiroxostat.

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)

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
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示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
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注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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