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| Targets |
T-type calcium channels (TTCCs): Ca3.1, Ca3.2, Ca3.3 - blocker; IC50 (human): Ca3.1 = 6.4 nM (95% CI: 5.8-11), Ca3.2 = 18 nM (95% CI: 16-52), Ca3.3 = 7.5 nM (95% CI: 5.4-10); IC50 (rat): Ca3.1 = 16 nM (13-20), Ca3.2 = 13 nM (10-16), Ca3.3 = 1.8 nM (1.4-2.4); IC50 (mouse): Ca3.1 = 6.4 nM (5-10), Ca3.2 = 40 nM (31-56); IC50 (dog): Ca3.1 = 20 nM (17-25), Ca3.2 = 33 nM (26-48), Ca3.3 = 3.4 nM (2.9-4.2); IC50 (cynomolgus): Ca3.2 = 19 nM (14-31). [1]
Ca1.2 - weak blocker; IC50 = 2420 nM (human). [1] hERG (hK11.1) - blocker; IC50 = 5.5 μM. [1] |
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| ln Vitro |
Cav3.1, Cav3.2, Cav3.3, and Cav1.2 are all blocked by apinocaltamide (Compound 66b), with IC50 values of 6.4, 18, 7.5, and 2410 nM, respectively. The recombinant channel hCav3.3 is efficiently blocked by apinocaltamide with a notable voltage dependence (Kr≈1500 nM and Ki≈20 nM). With an IC50 of 5.5 μM, apinocaltamide inhibits current flow across hKv11.1-hERG channels [1]. Moreover, apinocaltamide inhibits P450 enzymes; its IC50 values for CYP2C8, CYP2D6, CYP2C9, CYP2C19, CYP3A4, and CYP2B6 are 14, 15, 22, 25, 51, and 52 μM, respectively [1].
Apinocaltamide blocked human TTCCs with IC50 values of 6.4 nM (Ca3.1), 18 nM (Ca3.2), and 7.5 nM (Ca3.3) in FLIPR assays; selectivity against Ca1.2 was moderate (IC50 = 2420 nM). [1] Apinocaltamide showed voltage-dependent block of hCa3.3 with Kd ≈ 1500 nM at negative holding voltages and Kd ≈ 20 nM at more depolarized voltages. Block of Ca1.2, Ca1.3, and Ca2.1 was <20% at 10 μM from negative holding voltages. [1] Apinocaltamide blocked hERG channels with IC50 = 5.5 μM. Inhibition of other channels except hKv1.5 and hKv4.3/hKChIP2 (~30% at 10 μM) was <20% at 10 μM. Block of sodium and potassium channels in rat cortical neurons was <5% and <15% respectively at 10 μM. [1] In off-target screening against 273 proteins at 10 μM, Apinocaltamide showed >50% inhibition for: calcium sensing receptor (84%), hERG (60%), tachykinin 2 receptor (60%), urotensin 2 receptor (60%), neuromedin U receptor 1 (56%), sigma 1 receptor (54%), and LPA3 receptor (55%). [1] Apinocaltamide was negative in the Ames test and showed low covalent binding to protein (72 pmol/mg·h) upon metabolic activation by human liver microsomes (compared to 207 pmol/mg·h for compound 66e). [1] |
| ln Vivo |
Apinocaltamide (66b, 100, and 300 mg/kg, orally administered, assessed 12 hours later) efficiently reduces the total amount of time mice experience absence-like seizures [1].
In WAG/Rij rat model of absence-like epilepsy, Apinocaltamide at 10 mg/kg po significantly decreased cumulative duration of absence-like seizures by 93% over 12 h (p<0.001, paired t test) and completely suppressed seizures over the first 6 h following administration. Brain free concentration 1 h post-administration at 10 mg/kg po in Wistar rats: Ctotal,brain = 1529 ± 116 nM, Cubrain = 49.8 ± 3.9 nM, Cfree,plasma = 72 ± 11 nM; total plasma Cmax = 462 nM at 10 mg/kg po; free brain concentrations were above IC50 values for TTCCs. [1] In audiogenic seizure-sensitive juvenile DBA/2J mouse model, Apinocaltamide at 100 mg/kg po significantly decreased seizure severity (p<0.001, Mann-Whitney test) at 1 h and 3 h post-administration. Brain free concentration at 1 h post-administration was 476 ± 121 nM. [1] In cardiovascular telemetry studies, Apinocaltamide induced minimal to slight decreases in heart rate in rats and monkeys, slight increase in dogs, no effect in guinea pigs; blood pressure decreased slightly in rats and monkeys, unchanged in dogs and guinea pigs; PR interval increased up to 13% in rats with low incidence (one to five per 15000 heart beats) of second-degree AV blocks (Wenckebach or Mobitz II); no changes in QTc or QRS intervals in any species. [1] |
| Enzyme Assay |
FLIPR (Fluorescence Imaging Plate Reader) calcium flux assay: The assay measured compound potency against T-type calcium channels (Ca3.1, Ca3.2, Ca3.3) and Ca1.2. IC50 values were determined using concentration-response curves with 95% confidence intervals. For the final compound Apinocaltamide, IC50 values were determined across human, rat, mouse, dog, and cynomolgus channels. Data were obtained with a FLIPR assay; see Supporting Information for details. [1]
Patch-clamp electrophysiology on recombinant channels: Apinocaltamide was assessed against a panel of human cardiac and neuronal ion channels (hCa3.3, hCa1.2, hCa1.3, hCa2.1; hKv1.5, hKv4.3/hKChIP2, hKv7.1/minK, hKv7.2/7.3, hKv11.1-hERG, hHCN4, hKir2.1, hKir3.1/3.4; hNav1.1, hNav1.2, hNav1.5, hNav1.6) using patch-clamp techniques. Voltage-dependency of block was assessed for hCa3.3 (Kd ≈ 1500 nM at negative holding voltages, Kd ≈ 20 nM at depolarized voltages). hERG block was measured with IC50 = 5.5 μM. [1] Off-target screening: 273 off-target proteins were screened at 10 μM Apinocaltamide using radioligand-binding assays and functional FLIPR assays, including GPCRs, enzymes, transporters, and nuclear receptors. Inhibition >50% was noted for seven targets. [1] CYP450 inhibition assay: Apinocaltamide was tested for inhibition of main P450 enzymes (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4) with IC50 values determined. No time-dependent P450 inhibition was observed. [1] |
| Cell Assay |
FLIPR calcium flux assay: Recombinant cell lines expressing TTCCs (Ca3.1, Ca3.2, Ca3.3) or Ca1.2 were used. Cells were loaded with calcium-sensitive fluorescent dye, and compound was added at various concentrations. Calcium influx was measured upon membrane depolarization. IC50 values were calculated from concentration-response curves. For Apinocaltamide, IC50 values were determined across multiple species (human, rat, mouse, dog, cynomolgus). [1]
Patch-clamp electrophysiology: Recombinant ion channels were expressed in mammalian cells. Whole-cell or inside-out patch-clamp recordings were performed to measure current blockade by Apinocaltamide. Voltage-dependency of block was assessed for hCa3.3. Selectivity against other calcium channels (Ca1.2, Ca1.3, Ca2.1) and potassium/sodium channels was evaluated at 10 μM. [1] CYP450 inhibition assay: Apinocaltamide was incubated with human liver microsomes and specific CYP isoform substrates. IC50 values were determined for eight CYP isoforms. Time-dependent inhibition was assessed with 30-minute preincubation. [1] Ames test: Apinocaltamide was tested in Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation (phenobarbital/beta-naphthoflavone induced rat liver S9 fraction). A compound was considered positive if a biologically relevant increase in revertants exceeded threshold by 2-fold compared to solvent controls. Apinocaltamide was negative. [1] Covalent binding assay: Apinocaltamide was incubated with human liver microsomes and NADPH; covalent binding to protein was measured (72 pmol/mg·h). [1] |
| Animal Protocol |
Animal/Disease Models: Male juvenile DBA/2J mice (22-24 days old) [1]
Doses: 100, 300 mg/kg, 1 hour or 3 hrs (hrs (hours)) before exposure to stimulation. Route of Administration: Orally for 12 hrs (hrs (hours)) Experimental Results: Cumulative duration of absence seizures was diminished by 93% over the next 12 hrs (hrs (hours)). WAG/Rij rat model of absence-like epilepsy: Male WAG/Rij rats were previously implanted with telemetry transmitters allowing continuous EEG recording of spontaneous seizures in freely moving undisturbed animals. Apinocaltamide or vehicle (10% PEG400 + 90% MC 0.5%) was administered by oral gavage at the beginning of the night active period at 10 mg/kg. Cumulative duration of absence seizures was measured over 6 h and 12 h periods. Data expressed as mean ± SEM (n=6-8 per group); statistical analysis by paired t test. [1] Audiogenic seizure-sensitive juvenile DBA/2J mouse model: Juvenile DBA/2J mice were exposed to an auditory stimulus of maximum 60 s or until the mouse showed tonic extension of the hind limbs. Test was performed 1 or 3 h following oral administration of Apinocaltamide at 100 mg/kg or vehicle. Seizure severity was assessed via behavioral scale: stage 0=normal, stage 1=wild running, stage 2=clonic seizure, stage 3=tonic extension of hind limbs. Statistical analysis by Mann-Whitney test (n=8-10 per group). Blood and brain samples collected at end of test for concentration measurement. [1] Cardiovascular telemetry studies: ECG/BP telemetry was performed in conscious freely moving rats (normotensive Wistar and spontaneously hypertensive SHR), Dunkin-Hartley guinea pigs, Beagle dogs, and Cynomolgus monkeys following single oral administration of Apinocaltamide at doses of 10-30 mg/kg (depending on species). Mean arterial pressure (MAP), heart rate (HR), PR, QRS, and QTc intervals were monitored over 24 h. [1] |
| ADME/Pharmacokinetics |
Apinocaltamide in Wistar rats (iv 1 mg/kg): CL = 2.2 mL/min/kg, t1/2 = 4 h; (po 10 mg/kg): Cmax = 462 nM, F = 75%. [1]
Apinocaltamide in mice: fu = 0.01 (unbound fraction); CLint (liver microsomes) = 181 μL/min/mg protein; predicted CL = 1.1 mL/min/kg; observed CL = 13 mL/min/kg. [1] Apinocaltamide in rats: fu = 0.03; CLint (liver microsomes) = 159 μL/min/mg protein; predicted CL = 2.2 mL/min/kg; observed CL = 29 mL/min/kg. [1] Apinocaltamide in dogs: fu = 0.04; CLint (liver microsomes) = 1.8 μL/min/mg protein; predicted CL = 0.08 mL/min/kg; observed CL = 0.9 mL/min/kg. [1] Apinocaltamide in monkeys: fu = 0.047; CLint (liver microsomes) = 9.0 μL/min/mg protein; predicted CL = 0.5 mL/min/kg; observed CL = 5.5 mL/min/kg. [1] Human PK prediction: fu = 0.026; CLint (liver microsomes) = 3.7 μL/min/mg protein; predicted CL = 0.21 mL/min/kg by allometric scaling from observed CL in mouse, rat, dog, and monkey with correction for species differences in plasma protein binding. [1] Solubility: Apinocaltamide aqueous solubility at pH 7 = 10 mg/L; FaSSIF = 103 mg/L; FeSSIF = 104 mg/L. [1] MDR1-MDCK permeability: PappA→B = 18×10⁻⁶ cm/s, PappB→A = 54×10⁻⁶ cm/s, ratio B→A/A→B = 3.0 (at 1 μM). [1] |
| Toxicity/Toxicokinetics |
No time-dependent CYP450 inhibition was observed. IC50 values for CYP inhibition: 1A2 >100 μM, 2A6 >100 μM, 2B6 = 52 μM, 2C8 = 14 μM, 2C9 = 22 μM, 2C19 = 5 μM, 2D6 = 5 μM, 3A4 = 1 μM. [1]
Cardiovascular effects: In rats, Apinocaltamide (30 mg/kg po) caused -7% MAP, -10% HR, +13% PR; in SHR (30 mg/kg): -8% MAP, -13% HR, +13% PR; in guinea pigs (30 mg/kg): no significant effects on MAP, HR, PR, QRS, QTc; in dogs (10 mg/kg): +15% HR, no change in MAP, PR, QRS, QTc; in cynomolgus monkeys (100 mg/kg): -13% MAP, +17% HR. PR interval increased up to 13% in rats with low incidence of second-degree AV blocks (one to five per 15000 heart beats); no changes in QTc or QRS intervals in any species. [1] Low covalent binding: 72 pmol/mg·h upon metabolic activation by human liver microsomes. [1] |
| References | |
| Additional Infomation |
Apinocaltamide is a secondary amide formed by the condensation of the carboxyl group of {4-[1-(trifluoromethyl)cyclopropyl]phenyl}acetic acid with the amino group of 6-[(3-amino-1H-pyrazol-1-yl)methyl]pyridin-3-onitrile. It is a selective, orally effective T-type calcium channel blocker, and its potential as a novel treatment for epilepsy is currently being investigated. It has dual effects of anticonvulsant and T-type calcium channel blocking. Apinocaltamide belongs to the pyrazole, secondary amide, cyclopropane, organofluorine, nitrile, pyridine, and benzene classes. Apinocaltamide is currently being investigated in the clinical trial NCT03239691 (a study evaluating the efficacy of ACT-709478 in patients with photosensitive epilepsy).
Apinocaltamide (ACT-709478) was selected as a clinical candidate for the treatment of generalized epilepsies and entered phase I clinical trials. [1] T-type calcium channels are products of three genes (CACNA1G, CACNA1H, CACNA1I) yielding Ca3.1, Ca3.2, and Ca3.3 channels. Ca3.1 and Ca3.2 are expressed in heart and brain; Ca3.3 is exclusively expressed in brain. [1] TTCCs are expressed in neuronal networks involved in generation of spike-and-wave discharges (hallmark of absence seizures). Mutations in Ca3.1 or Ca3.2 genes have been described in human and animal models of absence epilepsy or other idiopathic generalized epilepsies; most Ca3.2 mutations are gain-of-function. [1] Mibefradil (1) was the prototypic TTCC blocker but is not brain-penetrant and blocks multiple channels (IC50: Ca3.1=64 nM, Ca3.2=130 nM, Ca3.3=130 nM, Ca1.2=250 nM, hERG=580 nM). MK-8998 (2a) and Z-944 (2b) are selective, brain-penetrant TTCC blockers that entered clinical trials. [1] The aminopyrazole metabolite of Apinocaltamide (57k) was identified as Ames-negative; several other aminopyrazole metabolites (57a, 14b, 57b, 57d) were Ames-positive. [1] Compound 66b (ACT-709478) showed lower covalent binding (72 pmol/mg·h) than compound 66e (207 pmol/mg·h) upon metabolic activation by human liver microsomes, triggering its selection for further characterization. [1] |
| Molecular Formula |
C22H18F3N5O
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|---|---|
| Molecular Weight |
425.40643453598
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| Exact Mass |
425.146
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| CAS # |
1838651-58-3
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| PubChem CID |
118560618
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| Appearance |
White to off-white solid powder
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
31
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| Complexity |
693
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
LSYANGLAZUZYFX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H18F3N5O/c23-22(24,25)21(8-9-21)17-4-1-15(2-5-17)11-20(31)28-19-7-10-30(29-19)14-18-6-3-16(12-26)13-27-18/h1-7,10,13H,8-9,11,14H2,(H,28,29,31)
|
| Chemical Name |
N-[1-[(5-cyanopyridin-2-yl)methyl]pyrazol-3-yl]-2-[4-[1-(trifluoromethyl)cyclopropyl]phenyl]acetamide
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| Synonyms |
ACT709478 ACT 709478ACT-709478
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
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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| Solubility (In Vitro) |
DMSO : ≥ 125 mg/mL (~293.83 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.89 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.89 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3507 mL | 11.7534 mL | 23.5067 mL | |
| 5 mM | 0.4701 mL | 2.3507 mL | 4.7013 mL | |
| 10 mM | 0.2351 mL | 1.1753 mL | 2.3507 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT04123288 | COMPLETED | Drug: ACT-709478 Drug: ACT-709478 |
Healthy Subjects | Idorsia Pharmaceuticals Ltd. | 2019-12-16 | Phase 1 |
| NCT03165097 | COMPLETED | Drug: ACT-709478 Drug: Placebo Drug: Midazolam Drug: ACT-709478 combined with midazolam |
Healthy Subjects | Idorsia Pharmaceuticals Ltd. | 2017-07-07 | Phase 1 |
| NCT03239691 | COMPLETED | Drug: ACT-709478 for oral use Drug: Placebo |
Photosensitive Epilepsy | Idorsia Pharmaceuticals Ltd. | 2017-10-06 | Phase 2 |
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