In vitro, acoziborole exhibits efficacy against strains of Trypanosoma brucei, such as T. b. Rhodesia and T. b. Acoziborole demonstrated strong efficacy against representative T in whole-cell tests. b. Brussels, T. b. Rhodesia and T. b. strains of Gambiae. Following a 72-hour incubation period, the IC50 value of Acoziborole for the parasite strains was around 0.07 µg/mL to 0.37 µg/mL. As in Tb. Acoziborole's minimum inhibitory concentration (MIC) for the Brucei S427 strain is 0.6 µg/mL, which is roughly double the strain's IC50. At drug concentrations up to 50 µg/mL, no discernible decrease of cell growth was seen in in vitro mammalian cell (L929 mouse cell line) experiments, in contrast to the strong action of acoziborole against trypanosomes. Using the P450-Glo test, the ability of Acoziborole to inhibit CYP enzymes was assessed for the human isoforms of CYP3A4, CYP1A2, CYP2C19, CYP2C9, and CYP2D6. In these tests, acetazolamide's IC50 values were greater than 10 µM [1].

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Acoziborole exhibited potent activity against T. b. brucei 427 with an IC₅₀ of 0.292 ± 0.019 μg/mL (n=9) and an MIC of 0.6 μg/mL. Against T. b. rhodesiense STIB 900, IC₅₀ = 0.294 μg/mL (n=1). Against T. b. gambiense 108R, IC₅₀ = 0.165 μg/mL; against T. b. gambiense 40R, IC₅₀ = 0.363 μg/mL; against T. b. gambiense ITMAP 141267, IC₅₀ = 0.092 μg/mL; against T. b. gambiense Drani, IC₅₀ = 0.129 μg/mL; against T. b. gambiense DAL 1402, IC₅₀ = 0.065 μg/mL. No significant inhibition of mammalian cell proliferation was observed in L929 mouse fibroblast cells at concentrations up to 50 μg/mL (IC₅₀ > 50 μg/mL). The primary oxidative metabolite SCYX-3109 was inactive at 10 μM. [1]

Time-kill assay: Acoziborole displayed concentration-dependent trypanocidal activity with rapid onset. At 1.25 μg/mL (about 2× MIC), >50% reduction in viability occurred within 8 h, and >99% of parasites were killed within 24 h of continuous exposure. [1]

Irreversibility assay: A short exposure (10–12 h) to Acoziborole at approximately 5× IC₅₀ produced irreversible effects on trypanosome survival. [1]

Acoxiborole at an intravenous dose of 4.3 mg/kg had a substantial elimination half-life (t1/2) of 26.6 hours, systemic clearance (CL) of 0.089 L/h/kg, volume of distribution (Vdss) of 1.69 L/kg, and an area under the concentration-time curve (AUC0-24 h) of 48 h·μg/mL in mice that were not infected. At 13.4 mg/kg, which is the least effective dose in the mouse phase 2 HAT model, acoziboral was absorbed quickly, as evidenced by a plasma Cmax of 6.96 µg/mL six hours after administration. The oral clearance (Cl/F) value was 0.163 L/h/kg, the AUC0–24 h was 82 h·μg/mL, and the absolute oral bioavailability was 55%. Cmax climbed to 9.8 μg/mL and AUC0-24 h was 113 h·μg/mL following an oral dose of 26 mg/kg, which corresponds to the dose of 100% cure rate in the mouse phase 2 HAT model. When oral acoziboral was administered at a nominal dose of 25 mg/kg (the dose at which the mouse cure rate was 100%), the Cmax of uninfected rats increased approximately 2-fold compared to mice, and the oral clearance rate increased approximately 4-fold (AUC0-24 hours 291h·μg/mL and CL/F=0.092L/kg/h). Similar to mice, it takes the same amount of time (tmax=8 hours) to achieve maximum concentration. Acoziborole 2 mg/kg (IV) on study day 1 and 10 mg/kg (NG) on study day 8 were administered to uninfected male and female cynomolgus monkeys. Upon intravenous administration, the Vdss of AUC0-24 h and AUC0-inf are 0.656 L/kg, and the area under the concentration-time curve is 78.8, respectively. Acoziborole demonstrates excellent plasma pharmacokinetics, with a CL of 0.022 L/h/kg. 94.4 and h•μg/mL.[1]

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In a mouse model of acute (stage 1) HAT (T. b. brucei EATRO 110), oral administration of Acoziborole once daily for 4 days at doses as low as 5 mg/kg/day (5 mg/kg QD or 2.5 mg/kg BID) produced 100% cure rates (animals remained parasite-free for >30 days). A single intraperitoneal dose of 25 mg/kg also produced 100% cure. Oral doses of 2.5 mg/kg QD or 1.25 mg/kg BID were not curative. [1]

In a mouse model of stage 2 (CNS) HAT (T. b. brucei TREU 667), oral administration of Acoziborole once daily for 7 days starting on day 21 post-infection produced an 80% cure rate at 12.5 mg/kg and a 100% cure rate at 25 mg/kg. Cure was defined as lack of parasitemia for 180 days after the last dose and negative subinoculation of blood/brain homogenates into fresh animals. [1]

For trypanosome sensitivity: compounds were serially diluted in DMSO and added to 96-well plates (final concentrations 5 to 0.01 μg/mL). T. b. brucei (1×10⁴ parasites/well) or T. b. rhodesiense/gambiense (1×10³ cells/well) were added. After 72 h incubation, resazurin (20 μL of 25 mg/100 mL stock in PBS) was added and incubated for 4–6 h. Fluorescence was measured at excitation 530 nm and emission 590 nm. IC₅₀ values were determined using curve fitting. MIC was determined by visual inspection. [1]

For mammalian cytotoxicity: L929 mouse fibroblast cells were seeded at 2×10³ per well and handled similarly to the trypanosome assay. [1]

Time-kill assay: T. b. brucei were exposed to serial dilutions of compound (5 to 0.01 μg/mL) in HMI-9 media. At specified intervals, CellTiter Glo reagent was added to lyse parasites, incubated for 10 min in the dark, and luminescence quantified. [1]

Irreversibility assay: T. b. brucei (1×10⁵ parasites/well) were incubated with test compound (10 to 0.02 μg/mL) in 96-well V-bottom plates. At designated times, plates were centrifuged at 2,600×g for 5 min, supernatant aspirated, and parasites washed twice with drug-free media. Parasites were resuspended, and after 72 h incubation, resazurin was added to determine viability. [1]

For acute HAT model: Female Swiss Webster mice were injected i.p. with 2.5×10⁵ T. b. brucei EATRO 110 parasites. Treatment started 24 h post-infection. Acoziborole was given daily by oral gavage or i.p. injection. Compound was formulated in 2% ethanol:5% dextrose in sterile water, dose volume 200 μL (for a 25 g animal). Animals were monitored for 30 days; parasitemia checked weekly. [1]

For stage 2 CNS HAT model: Mice were infected i.p. with 1×10⁴ T. b. brucei TREU 667 parasites. Treatment started on day 21 post-infection. Acoziborole was given orally once daily for 7 days at doses of 12.5, 25, 50, or 100 mg/kg. Positive control: berenil (10 mg/kg i.p. single dose on day 4); negative control: berenil on day 21. Cure defined as no parasitemia for 180 days plus negative subinoculation into fresh mice. [1]

For pharmacokinetic studies: Compounds were formulated in 50% PEG400:20% ethanol:30% carboxymethylcellulose (0.5%) or 2% ethanol:5% dextrose in water. Doses were administered at volumes of 4 mL/kg (mice), 2 mL/kg (rats), or 1 mL/kg (cynomolgus monkeys). Blood samples from mice were collected via cardiac puncture under terminal anesthesia; from rats via vascular access port. Brain tissue collected after decapitation. CSF collected from rats via cisterna magna puncture. [1]

Acoziborole has a logD of 3.51, aqueous solubility of 25 μM in pH 7.4 PBS. [1]

Metabolic stability: intrinsic clearance <5 μL/min/mg protein and half-life >350 min in liver microsomes and S9 fractions from rodents, dog, monkey, and human. Primary metabolite SCYX-3109 (oxidative deboronation) accounted for <5% over 12 h. [1]

CYP inhibition: IC₅₀ >10 μM for CYP3A4, 1A2, 2C19, 2C9, 2D6 (specific values: CYP1A2 >100 μM, CYP2C19 23.1 μM, CYP2C9 23.5 μM, CYP2D6 21.1 μM, CYP3A4 47.4 μM). [1]

Plasma protein binding in mouse: unbound fraction (fu) = 0.3% at 0.6 μg/mL (MIC), rising to 3.2% at 15 μg/mL. In human plasma: fu = 1.3% at 1 μg/mL, 5.5% at 50 μg/mL. Brain tissue binding in mouse: fubrain ~5%. [1]

MDCK-MDR1 permeability: Papp A→B = 776 nm/sec; in presence of GF120918, Papp = 853 nm/sec; absorption quotient (AQ) = 0.09, indicating no P-gp substrate. [1]

Mouse PK (uninfected): i.v. 4.3 mg/kg – t½ = 26.6 h, CL = 0.089 L/h/kg, Vdss = 1.69 L/kg, AUC₀₋₂₄ = 48 h·μg/mL. Oral 13.4 mg/kg – Cmax = 6.96 μg/mL at 6 h, AUC₀₋₂₄ = 82 h·μg/mL, oral bioavailability = 55%. Oral 26 mg/kg – Cmax = 9.8 μg/mL, AUC₀₋₂₄ = 113 h·μg/mL. Brain AUC₀₋₂₄ after 13.4 mg/kg oral = 31.4 h·μg/mL; after 26 mg/kg = 68 h·μg/mL. Brain-to-plasma AUC ratio = 38% and 60%, respectively. [1]

Rat PK (uninfected): oral 25 mg/kg – Cmax = 18.2 μg/mL at 8 h, AUC₀₋₂₄ = 291 h·μg/mL, CL/F = 0.092 L/kg/h. Brain Cmax = 8.5 μg/mL, CSF Cmax = 1.08 μg/mL. Brain AUC₀₋₂₄ = 129 h·μg/mL, CSF AUC₀₋₂₄ = 14.6 h·μg/mL. Brain-to-plasma AUC ratio = 44%. [1]

Monkey PK: i.v. 2 mg/kg – CL = 0.022 L/h/kg, Vdss = 0.656 L/kg, AUC₀₋₂₄ = 78.8 h·μg/mL. Oral (nasogastric) 10 mg/kg – Cmax = 11 μg/mL at 9.5 h, CL/F = 0.025 L/h/kg, AUC₀₋ᵢₙf = 460 h·μg/mL, bioavailability = 89%. CSF concentration ~5% of plasma. [1]

Steady-state in infected mice (7 daily oral doses): at 12.5 mg/kg, brain Cmax = 6.54 μg/mL, brain AUC₀₋ᵢₙf = 34.2 h·μg/mL; at 25 mg/kg, brain Cmax = 11.98 μg/mL, brain AUC₀₋ᵢₙf = 81.5 h·μg/mL. Brain concentrations remained above MIC (0.6 μg/mL) for close to 24 h at 25 mg/kg. [1]

Acoziborole was well tolerated in mice at doses up to 100 mg/kg BID (200 mg/kg/day, 8× effective dose). [1]

hERG channel: no significant binding in radioligand assay (−6% at 10 μM); whole-cell patch clamp showed mean fractional block of 0.108±0.037 at 30 μM and 0.198±0.018 at 100 μM, indicating IC₅₀ >100 μM. [1]

Ames test (bacterial reverse mutation): negative up to 5000 μg/plate in S. typhimurium and E. coli strains with and without rat liver S9 fraction. [1]

Broad receptor/enzyme panel (over 100 targets): at 10 μM, no significant binding or inhibition was observed. [1]

Plasma protein binding (see ADME) – low affinity. [1]

Primary metabolite SCYX-3109: IC₅₀ for CYP3A4 inhibition = 7.5 μM; for other isoforms >100 μM. Estimated boric acid concentrations in plasma were below background levels (0.03–0.10 μg/g) and below NOAEL in rats. [1]


Additional Info:
Acoziborole is a C(3)-dimethyl benzoxaborole 6-carboxamide, derived from optimization of the lead SCYX-6759. The mechanism of trypanocidal action is unknown. It does not inhibit P-glycoprotein efflux and readily crosses the blood-brain barrier. The compound was selected for IND-enabling preclinical studies with expected phase 1 trials in 2011. Based on pharmacodynamic relationships in the mouse stage 2 HAT model, once-daily oral dosing is sufficient. Clinical dose estimation based on scaling from preclinical species is approximately 2.5 mg/kg. [1]

[1]. SCYX-7158, an orally-active benzoxaborole for the treatment of stage 2 human African trypanosomiasis. PLoS Negl Trop Dis. 2011 Jun;5(6):e1151.

Acoziborole belongs to the (trifluoromethyl)benzene family of compounds. SCYX-7158 has been used in research trials for the treatment of trypanosomiasis, parasitic diseases, protozoan infections, and African trypanosomiasis.

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C17H14BF4NO3

367.1066

367.1

1266084-51-8

44178354

White to off-white solid powder

3.433

2

7

2

26

545

0

PTYGDEXEGLDNAZ-UHFFFAOYSA-N

InChI=1S/C17H14BF4NO3/c1-16(2)12-6-4-10(8-14(12)18(25)26-16)23-15(24)11-5-3-9(19)7-13(11)17(20,21)22/h3-8,25H,1-2H3,(H,23,24)

4-fluoro-N-(1-hydroxy-3,3-dimethyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-2-(trifluoromethyl)benzamide

SCYX-7158 SCYX 7158SCYX7158 AcoziboroleOxaborole

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 : ≥ 125 mg/mL (~340.51 mM)

Solubility in Formulation 1: ≥ 8 mg/mL (21.79 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 80.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 8 mg/mL (21.79 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 80.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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