15-lipoxygenase (15-LO) (no corresponding IC50/Ki value provided in the literature)
Ferroptosis (the compound inhibits ferroptosis with EC50 ≤98 nM) [1]
15-Lipoxygenase (15-LO). Utreloxastat functions as a potent inhibitor of the enzyme 15-LO, which is involved in the production of pro-inflammatory mediators and lipid peroxidation. By inhibiting 15-LO, the compound is designed to reduce the formation of reactive oxygen species (ROS), decrease the consumption of the antioxidant glutathione, and ultimately prevent ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation.

1. Cytochrome P450 (CYP) enzyme inhibition test: Utreloxastat weakly inhibited CYP1A2 and CYP2B6, with half maximal inhibitory concentration >5.3 μM; its inhibitory effect on other CYP450 enzymes including CYP2C8, CYP2C19, CYP3A4 and CYP4F2 was slight, with half maximal inhibitory concentration >46 μM. [1]
2. CYP enzyme induction test: When the concentration of Utreloxastat reached 20 μM in human hepatocytes, it induced the mRNA expression of CYP2B6 and CYP3A4, but did not induce CYP1A2. [1]
3. Anti-ferroptosis activity test: Utreloxastat inhibited ferroptosis with EC50 ≤98 nM and had no obvious impact on cell viability. It was about 70 times more potent than edaravone and more than 50 times more potent than riluzole in inhibiting ferroptotic death of human spinal astrocytes. [1]

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In vitro, Utreloxastat effectively reduces oxidative stress markers and prevents the depletion of reduced glutathione (GSH) in cellular models. By inhibiting 15-LO, the compound blocks the propagation of lipid peroxidation chains in biological membranes, thereby inhibiting ferroptosis. It has been shown that Utreloxastat at 20 microM can induce the mRNA expression of CYP2B6 and 3A4 in human hepatocytes.

1. Non-clinical pharmacokinetic performance in experimental animals: After oral administration to mice, Utreloxastat could be well distributed into brain tissue, and the brain-to-plasma concentration ratio was approximately 10-fold after reaching the peak plasma concentration. The compound showed good oral bioavailability in mice, rats and monkeys. [1]
2. Drug metabolism in rats: After single oral administration of ¹⁴C radiolabeled Utreloxastat, the proportion of prototype drug in total plasma radioactivity was less than 5%, and no prototype drug was detected in urine and bile of rats, indicating extensive metabolism of the drug in rats. [1]

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In vivo, Utreloxastat was designed to cross the blood-brain barrier and reduce oxidative stress in the CNS. However, in the phase 2 CARDINALS trial, the compound failed to meet its primary and secondary efficacy endpoints, which were to slow disease progression as measured by the ALSFRS-R scale. Consequently, the drug showed no significant effect on ALS progression.

1. CYP450 enzyme inhibition assay: Pooled human liver microsomes and recombinant human CYP enzymes were adopted to assess the inhibitory effect of Utreloxastat on multiple CYP isoforms including CYP1A2, 2B6, 2C8, 2C19, 3A4 and 4F2. The half maximal inhibitory concentration of each CYP subtype was determined to evaluate the strength of drug inhibition. [1]
2. CYP450 enzyme induction assay: Human hepatocytes were used as the test system. Utreloxastat at 20 μM was incubated with hepatocytes, then the mRNA expression levels of CYP1A2, CYP2B6 and CYP3A4 were detected to judge whether the drug could induce the expression of related enzymes. [1]

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A cell‑free 15‑LO activity assay is used to assess Utreloxastat's inhibitory potency. Recombinant human 15-lipoxygenase (1-5 nM) is incubated in a 100 mM Tris‑HCl buffer (pH 7.4) with varying concentrations of Utreloxastat (1 nM to 10 uM) for 10 minutes. The reaction is initiated by adding the substrate, linoleic acid (25 uM), and incubated for 10-30 minutes at 25degC. The resulting 13(S)-hydroxyoctadecadienoic acid (13-HODE) is quantified by an ELISA kit or by HPLC. The IC50 value is determined from the resulting inhibition curve.

1. Human spinal astrocyte ferroptosis inhibition assay: Human spinal astrocytes were used to establish a ferroptosis cell model. Different concentrations of Utreloxastat, edaravone and riluzole were added respectively. The cell death status was detected to calculate EC50 of Utreloxastat for inhibiting ferroptosis, and compare the anti-ferroptosis potency among three drugs. [1]

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To measure ferroptosis inhibition, HT-22 neuronal cells are seeded in 96-well plates (10,000 cells/well). After 24 hours, the media is replaced, and cells are pretreated with Utreloxastat at varying concentrations for 2 hours. Ferroptosis is induced by adding erastin (1 uM) or RSL3 (1 uM). After 12-24 hours, cell viability is measured using an MTT or CellTiter-Glo assay. Lipid peroxidation can be quantified by the C11-BODIPY581/591 fluorescent probe using flow cytometry or a fluorescence plate reader. A concentration-dependent reversal of erastin/RSL3-induced cell death indicates on‑target activity.

1. Repeat-dose toxicity study in rats and monkeys: Rats and monkeys were given repeated administration of Utreloxastat for 28 consecutive days to evaluate the in vivo safety of the drug. The no observed adverse effect level of rats was 100 mg/kg, and the converted human equivalent dose was 16 mg/kg. [1]
2. Non-clinical pharmacokinetic study in mice, rats and monkeys: The drug was orally administered to mice, rats and monkeys. After drug administration, blood samples and brain tissue samples were collected at preset time points to detect drug concentration, so as to evaluate oral bioavailability and tissue distribution characteristics, especially the distribution in brain tissue. [1]
3. Human single ascending dose (SAD) clinical trial: A total of 40 healthy subjects were divided into 4 cohorts, receiving single oral doses of 100 mg, 250 mg, 500 mg, 1000 mg Utreloxastat or placebo respectively. All drugs were taken after overnight fasting and medium-fat breakfast. Sampling was conducted on day 2, day 3 and day 4 after administration. Dose escalation was implemented after reviewing safety data of the previous cohort. [1]
4. Human multiple ascending dose (MAD) clinical trial: 30 healthy subjects were divided into 3 cohorts, continuously taking corresponding doses of Utreloxastat or placebo for 14 days: 250 mg twice daily, 500 mg once daily, 150 mg twice daily (days 1–6) then 150 mg once daily (days 7–14). All doses were given with medium-fat diet, and the twice-daily group took the evening dose 12 hours after the morning dose. [1]
5. Human food-effect (FE) clinical trial: 12 healthy subjects participated in a 3×3 crossover trial. Each subject received a single 500 mg dose of Utreloxastat under fasting, low-fat diet and high-fat diet conditions respectively. A 7-day washout period was set between different treatment periods. Sampling was performed on day 2, day 3 and day 4 after each administration. [1]

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C57BL/6 mice (8-10 weeks old, 20-25 g) are used to evaluate Utreloxastat's efficacy in a model of neuroinflammation. For 7 days, Utreloxastat (50, 100 mg/kg) or vehicle (0.5% methylcellulose) is administered by oral gavage. On day 7, 1 hour after the last dose, lipopolysaccharide (LPS, 5 mg/kg) is injected intraperitoneally to induce neuroinflammation. After 6 hours, the mice are euthanized. Brain tissue (cortex and hippocampus) is collected for Western blot analysis of inflammatory markers (e.g., Cox-2, iNOS) and for measuring oxidative stress markers (e.g., malondialdehyde, MDA).

1. Absorption (single dose): After single oral doses of 100–1000 mg Utreloxastat, the median time to peak plasma concentration (tmax) was about 4 hours (range: 1–6 hours). Maximum plasma concentration (Cmax) and area under the concentration-time curve (AUC) increased slightly in a greater-than-proportional manner with dose; the slope β values of Cmax, AUC0-t and AUC0-inf were 1.225, 1.257 and 1.223 respectively. [1]
2. Elimination half-life (single dose): The terminal half-life (t1/2) of single dose ranged from 20 hours to 25.3 hours. The drug presented a biphasic elimination characteristic, with the distribution half-life of about 1.8 hours. A secondary concentration peak appeared at about 10 hours in some subjects, and the occurrence rate rose with the increase of dose. [1]
3. Clearance (single dose): The apparent total body clearance (CL/F) ranged from 49.4 L/h to 81.9 L/h in the single-dose groups. [1]
4. Multiple-dose pharmacokinetics: After 14 days of continuous administration, the apparent clearance of Utreloxastat decreased, and the terminal half-life extended to ≥33 hours; partial subjects had a t1/2 of about 70 hours. The accumulation ratio (Racc) was about 1.5 for 150 mg twice/once daily group and 500 mg once daily group, and 2.32 for 250 mg twice daily group; the effective half-life was approximately 15 hours. The drug basically reached steady state after 7 days of administration. There was no significant difference in drug exposure between morning dose and evening dose. [1]
5. Food effect on absorption: Compared with fasting state, low-fat, medium-fat and high-fat diets all elevated Cmax and AUC of Utreloxastat, without changing tmax. Low-fat diet: Cmax GMR 180.25%, AUC0-t GMR 148.66%, AUC0-inf GMR 145.90%; Medium-fat diet: Cmax GMR 169.71%, AUC0-t GMR 161.01%, AUC0-inf GMR 162.25%; High-fat diet: Cmax GMR 241.30%, AUC0-t GMR 182.45%, AUC0-inf GMR 179.07%. [1]
6. Gender difference: No obvious difference in drug exposure was observed between male and female subjects under various dietary conditions. [1]
7. Excretion: The excretion of unchanged Utreloxastat via urine was extremely low, with the fraction excreted in urine (Fe) less than 0.23%, indicating the drug was mainly eliminated through metabolism or biliary excretion. [1]
8. Distribution (animal): After oral administration in mice, Utreloxastat had a brain-to-plasma ratio of about 10-fold, showing favorable brain distribution. [1]

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As a small molecule (MW 276.41), Utreloxastat is highly lipophilic (logP calculated >5), facilitating oral absorption and brain penetration. In a first-in-human study, following single oral doses, the time to maximum concentration (Tmax) was observed approximately 4 hours after dosing, with a terminal half-life ranging from 20 to 25.3 hours. Following multiple dosing, the terminal half-life was ≥33 hours. Exposure increases slightly over dose proportionally. Food increases Cmax and AUC without altering Tmax.

1. Single-dose safety in humans: Single oral dose up to 1000 mg Utreloxastat did not cause obvious safety risks in healthy volunteers. All adverse events were mild grade 1 and resolved completely before the end of the study. No serious adverse events or adverse events leading to drug withdrawal occurred. [1]
2. Multiple-dose safety in humans: Continuous administration of 500 mg once daily or 250 mg twice daily for 14 days had no obvious safety hazards. No clinically significant abnormal ECG results were found, and no QT interval prolongation exceeding 60 milliseconds was detected. [1]
3. Laboratory abnormality: Multiple doses of Utreloxastat caused decreased high-density lipoprotein (HDL) and total cholesterol, and the reduction degree of HDL increased with higher dose. This change was reversible after drug discontinuation and had no associated adverse events. [1]
4. Drug-drug interaction potential: Utreloxastat was a weak inhibitor of CYP1A2 and CYP2B6, and had minimal inhibitory effect on other CYP450 enzymes; it could induce the expression of CYP2B6 and CYP3A4 at 20 μM, which indicated potential drug-drug interaction risk related to CYP enzymes. [1]
5. Non-clinical repeated-dose toxicity: 28-day repeated-dose administration in rats and monkeys showed favorable safety results, supporting subsequent human trials. The starting dose for human trial was set at 100 mg, equivalent to one-tenth of the human equivalent dose converted from rat no observed adverse effect level. [1]

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In a first-in-human trial of 82 healthy volunteers, Utreloxastat was found to be safe and well tolerated across a wide dose range. No marked safety signal was observed following single doses up to 1000 mg and multiple doses over 14 days (500 mg once daily or 250 mg twice daily). In the phase 2 CARDINALS trial, the drug was again demonstrated to be safe and well tolerated, with no serious safety concerns reported. The development was halted due to lack of efficacy, not toxicity.

[1]. First-in-Human Studies of Pharmacokinetics and Safety of Utreloxastat (PTC857), a Novel 15-Lipooxygenase Inhibitor for the Treatment of Amyotrophic Lateral Sclerosis. Clin Pharmacol Drug Dev. 2023 Feb;12(2):141-151.

[2]. 2,3,5-TRIMETHYL-6-NONYLCYCLOHEXA-2,5-DIENE-1,4-DIONE FOR SUPPRESSING AND TREATING α-SYNUCLEINOPATHIES, TAUOPATHIES, AND OTHER DISORDERS. WO2020081879A2.

1. Disease background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease damaging motor neurons, leading to progressive paralysis and respiratory failure-induced death within 3–5 years after symptom onset. Only two drugs (riluzole, edaravone) are approved for ALS treatment currently with limited efficacy. Oxidative stress and 15-LO mediated ferroptosis are key pathological mechanisms of ALS. [1]
2. Mechanism of action: Utreloxastat is an alkyl-substituted cyclohexadienedione compound. It inhibits 15-LO, reduces oxidative stress, blocks glutathione depletion, and inhibits ferroptosis by converting the active Fe³⁺ of 15-LO into inactive Fe²⁺, so as to slow down neurodegeneration in ALS. [1]
3. Formulation: Utreloxastat was prepared with sesame oil and encapsulated into 50 mg hard gelatin capsules; placebo adopted the same capsule filled with pure sesame oil. [1]
4. Recommended clinical dose for follow-up study: Based on pharmacokinetic and safety data, 250 mg twice daily administered with food was selected as the dose for phase 2 clinical study in ALS patients. [1]
5. Trial ethics: All human clinical trials complied with the Declaration of Helsinki and local medical research regulations, and obtained approval from independent ethics committees. All subjects signed written informed consent before enrollment. [1]

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Utreloxastat is a CNS-penetrant, orally active 15-LO inhibitor designed to reduce oxidative stress and ferroptosis. While it showed a favorable safety profile and strong preclinical rationale, a phase 2 trial for ALS failed to show efficacy, leading to discontinued development (patent WO2020081879A2).

C18H28O2

276.41

276.208

C, 78.21; H, 10.21; O, 11.58

1213269-96-5

46174641

Light yellow to yellow solid powder

5.8

0

2

8

20

438

0

C1(=O)C(CCCCCCCCC)=C(C)C(=O)C(C)=C1C

IJWAQTHZBDBIID-UHFFFAOYSA-N

InChI=1S/C18H28O2/c1-5-6-7-8-9-10-11-12-16-15(4)17(19)13(2)14(3)18(16)20/h5-12H2,1-4H3

2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione

Utreloxastat; PTC-857; 1213269-96-5; PTC 857; Utreloxastat [INN]; WW8KZK2PZT; PTC857;

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)

DMSO: 50 mg/mL (180.89 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 1.25 mg/mL (4.52 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 66.67 mg/mL (241.20 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; Need ultrasonic and warming and heat to 49.6°C.

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