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

Topiroxostat (FYX-051, compound 39) has strong and longer-lasting effects that have been verified by XOR-Topiroxostat complex crystallographic investigation. The binding activity between Topiroxostat and XOR has been observed to be significantly influenced by the cyano group of Topiroxostat. Asn 768 of XOR and the cyano group of Topiroxostat have formed a hydrogen bond, which is responsible for this[1].

In a rat model of potassium oxonate-induced hyperuricemia, topiroxostat (FYX-051; 0.03-10 mg/kg; oral administration; for 1 hour; male Wistar/ST strain rats) treatment demonstrates a strong and long-lasting hypouricemic effect[2]. Topiroxostat (FYX-051, compound 39) has a Cmax of 4.62 μg/mL (3 mg/kg) and a bioavailability of 69.6%, respectively. Additionally, Topiroxostat's t1/2 value is 19.7 hours[1].

Time-Dependent Inhibition of XO with Inhibitors and the Stability of XO-Inhibitor Complexes. [2]
FYX-051 displayed time- and concentration-dependent inhibition of urate formation under air-saturated conditions (Fig. 2A). 2-Hydroxy-FYX-051, generated by primary hydroxylation of FYX-051 by XOR itself, also caused time- and concentration-dependent inhibition, although a relatively large amount of 2-hydroxy-FYX-051 was necessary to achieve potent inhibition (Fig. 2B) because of the lower affinity of 2-hydroxy-FYX-051, as determined from steady-state experiments (see below). We also estimated..

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
Following a single oral dose of 20 mg topirocustat, the time to reach peak plasma concentration (229.9 ng/mL) was 0.67 hours. In male rats, the oral bioavailability of topirocustat after a single oral dose of 1 mg/kg was 69.6%. The amounts of radiolabeled topirocustat excreted in urine and feces were 30.4% and 40.9% of the total dose of 1 mg/kg in rats, respectively. Within 24 hours following a single oral dose of 120 mg topirocustat, the major metabolites N-oxide, N1-glucuronide, and N2-glucuronide were excreted in urine, accounting for approximately 4.8%, 43.3%, and 16.1% of the total dose, respectively. The content of unconverted topirocustat and its hydroxide metabolites was 0.1% or less.
The distribution of 14C-topiroxistat in human blood cells (at concentrations of 20, 200, and 2000 ng/mL) ranged from 6.7% to 12.8%.
After a single oral dose of 20 mg topiroxistat, the apparent systemic clearance was 89.5 L/h, and the renal clearance was 17.4 mL/h.
Metabolism/Metabolites

Topiroxistat is primarily inactivated by hepatic metabolism. 2-Hydroxytopiroxistat is generated by primary hydroxylation of the drug by xanthine oxidase, retaining its inhibitory activity against this enzyme. Topiroxistat N-oxide is another major metabolite, detectable in plasma and urine. The N-oxide and hydroxide metabolites were determined to be pyridine N-oxide and pyridine 2 (or 6)-hydroxide, respectively. Topiroxistat is primarily inactivated by hepatic metabolism, mainly through glucuronidation. Topiroximate is metabolized into N1- and N2-glucuronide conjugates primarily via UGT1A1, 1A7, and 1A9, with UGT1A9 being the most prevalent. Known human metabolites of FYX-051 include 4-[2-[(3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxyoxacyclohexane-2-yl]-5-pyridin-4-yl-1,2,4-triazol-3-yl]pyridin-2-nitriles and (2S,3S,4S,5R)-6-[3-(2-cyanopyridin-4-yl)-5-pyridin-4-yl-1,2,4-triazol-1-yl]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid.

---

Biological Half-Life
Mean Half-Life: The half-life of topirocustat after a single oral dose of 20 mg is 5 hours on an empty stomach. The half-life of the molybdenum(IV)-topirocustat complex is approximately 20.4 hours. Compound 39/topirocustat exhibits weak CYP3A4 inhibitory activity (18.6%); its Cmax and bioavailability are as high as 4.62 μg/mL (3 mg/kg) and 69.6%, respectively. Furthermore, the half-life of compound 39 (19.7 h) is greater than that of compound 2 (0.97 h). Since compound 39 is mainly excreted in the urine in monkeys and humans as triazole N1- and N2-glucuronide, it is expected to be safe for patients with renal insufficiency. [1]

Protein Binding
The average protein binding rate of radiolabeled (14C)-topirostat to human plasma was >97.5% at 20 ng/mL, 98.8% at 200 ng/mL, and 98.4% at 2000 ng/mL. The binding to serum albumin was most significant, ranging from 92.3% to 93.2%, while the average protein binding rates to α1-acidic protein and γ-globulin were 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 used to treat and control hyperuricemia and gout. Xanthine oxidase, or xanthine oxidoreductase (XOR), regulates purine metabolism, and inhibiting this enzyme effectively lowers serum uric acid levels. Xanthine oxidase inhibitors are divided into two classes: purine analogs, such as [DB00437] and [DB05262]; and non-purine drugs, including Topiroxostat. Although [DB00437] is considered a first-line drug for treating hyperuricemia, it is often accompanied by side effects and is not very effective at lowering uric acid levels at the recommended dose. Renal complications are the main comorbidity limiting [DB00437] treatment, so dose reduction is recommended. Studies have shown that Topiroxostat and its metabolites are not affected by renal complications and may therefore be effective in patients with chronic kidney disease. Topiroxostat has been approved for treatment in Japan since 2013 under the brand names Topiloric and Uriadec, taken orally twice daily. Drug Indications In Japan, Topiroxostat is used to treat gout and hyperuricemia. Mechanism of Action Uric acid synthesis depends on the activity of xanthine oxidase, which catalyzes the conversion of hypoxanthine to xanthine, which is then converted to uric acid. The active site of xanthine oxidase uses molybdenum ions as cofactors, which exhibit different redox states upon binding to substrates. When substrates such as hypoxanthine or xanthine bind to xanthine, xanthine oxidase hydroxylates them, reducing the molybdenum ion from hexavalent molybdenum (Mo(VI)) to tetravalent molybdenum (Mo(IV)). When the hydroxylated substrate (xanthine or uric acid) dissociates from the active site, the molybdenum ion is re-oxidized to the hexavalent state. Topiroxostat interacts with multiple amino acid residues in the solvent channel and forms a reaction intermediate through covalent bonding of the oxygen atom to the molybdenum (IV) ion. It can also form hydrogen bonds with the molybdenum (VI) ion, indicating that it has multiple inhibitory modes against xanthine oxidase. Enhanced binding to xanthine oxidase delays the dissociation of topirocustat from the enzyme. The metabolite 2-hydroxytopirocustat, formed by primary hydroxylation of topirocustat by xanthine oxidase, also inhibits the enzyme's activity in a time- and concentration-dependent manner. In vitro experiments showed that topirocustat inhibits ATP-binding cassette transporter G2 (ABCG2), a membrane protein responsible for renal reabsorption of uric acid and intestinal secretion of uric acid. Our previous research found that 2-[2-(2-methoxyethoxy)ethoxy]-5-[5-(2-methyl-4-pyridyl)-1H-[1,2,4]triazol-3-yl]benzonitrile (2) is 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 focused on their in vivo activity in reducing serum uric acid levels in rats. Therefore, we found that 3-(2-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole (FYX-051, compound 39) [corrected] is one of the most potent XOR inhibitors; it exhibits strong in vivo activity, weak CYP3A4 inhibitory activity, and better pharmacokinetic properties than compound 2. Compound 39 is currently undergoing a phase II clinical trial. [1]

---

4-[5-(pyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridin-2-nitrile (FYX-051) is a potent inhibitor of bovine lactoxine oxidoreductase (XOR). Steady-state kinetic studies showed that FYX-051 initially acted as a competitive inhibitor with a Ki value of 5.7 × 10^-9 M. Minutes later, it forms a tight complex with XOR via a Mo-oxygen-carbon covalent bond, consistent with previous reports (Proc Natl Acad Sci USA 101:7931-7936, 2004). Therefore, FYX-051 is a hybrid inhibitor exhibiting both structural and mechanistic inhibitory properties. The FYX-051-XOR complex has a half-life of 20.4 hours, but enzyme activity is not fully recovered. The study found that this was due to the XOR-mediated conversion of FYX-051 to 4-[5-(2-hydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridin-2-onitrile (2-hydroxy-FYX-051), and the formation of 6-hydroxy-4-[5-(2-hydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridin-2-onitrile (dihydroxy-FYX-051) and 4-[5-(2,6-dihydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]-6-hydroxypyridin-2-onitrile (trihydroxy-FYX-051) during the 72-hour incubation period. A distinct charge-transfer absorption band was observed concurrently with the formation of the trihydroxy-FYX-051-XOR complex. Crystallographic analysis of the charge-transfer complex showed that a Mo-nitrogen-carbon bond was formed between the molybdenum atom of XOR and the nitrile group of trihydroxy-FYX-051. FYX-051 showed a potent and sustained uric acid-lowering effect in a rat model of potassium oxocyanate-induced hyperuricemia, and therefore is expected to become a candidate drug 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

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)

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

 (Please use freshly prepared in vivo formulations for optimal results.)