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]
Apinocaltamide targets T-type calcium channels, specifically the Cav3.1, Cav3.2, and Cav3.3 channel subtypes, with IC50 values of 6.4 nM, 18 nM, and 7.5 nM, respectively. These channels are voltage-gated calcium channels that play critical roles in regulating neuronal excitability and rhythmic burst firing. By blocking T-type calcium channels, Apinocaltamide reduces abnormal neuronal firing patterns associated with absence seizures and other forms of generalized epilepsy. The compound penetrates the blood-brain barrier, enabling direct action on central nervous system targets.

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].

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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]
In vitro, Apinocaltamide demonstrates potent inhibition of T-type calcium channels with IC50 values of 6.4 nM for Cav3.1, 18 nM for Cav3.2, and 7.5 nM for Cav3.3 in electrophysiological assays. The compound shows selectivity for T-type calcium channels over other voltage-gated calcium channel subtypes. In patch-clamp electrophysiology studies using HEK-293 cells expressing recombinant human T-type channels, Apinocaltamide produces concentration-dependent inhibition of calcium currents. The compound's inhibitory effects are reversible upon washout, indicating a non-covalent binding mechanism.

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]
In vivo, Apinocaltamide has demonstrated anticonvulsant efficacy in animal models of epilepsy. In rodent models of absence epilepsy, oral administration of the compound significantly reduces the frequency and duration of spike-wave discharges. The compound also shows efficacy in maximal electroshock seizure and pentylenetetrazole-induced seizure models. Pharmacodynamic studies demonstrate a correlation between plasma and brain concentrations of Apinocaltamide and its anticonvulsant effects. The compound's brain penetration enables therapeutic efficacy at doses that are well-tolerated systemically.

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]
In vitro enzyme/receptor binding assays for Apinocaltamide typically involve radioligand binding studies using cell membranes expressing recombinant T-type calcium channels. [³H]-labeled T-type channel ligands are used to measure competitive displacement by Apinocaltamide. Alternatively, patch-clamp electrophysiology is employed to directly measure the compound's effects on calcium currents through recombinant Cav3.1, Cav3.2, and Cav3.3 channels expressed in HEK-293 or CHO cells. Selectivity profiling includes testing against other voltage-gated calcium channels (L-type, N-type, P/Q-type) to confirm target specificity.

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]
In vitro cellular assays for Apinocaltamide utilize HEK-293 or CHO cells stably or transiently expressing recombinant human Cav3.1, Cav3.2, or Cav3.3 T-type calcium channels. Whole-cell patch-clamp electrophysiology is the primary method for assessing compound activity. Cells are voltage-clamped at a holding potential of -100 mV, and calcium currents are evoked by depolarizing voltage steps to -30 mV. Apinocaltamide is applied at concentrations ranging from 0.1 nM to 10 µM, and the percentage of current inhibition is calculated. IC50 values are determined from concentration-response curves.

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)).

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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]
In vivo animal experiments for Apinocaltamide typically involve rodent models of epilepsy. In the genetic absence epilepsy rat from Strasbourg (GAERS) model, the compound is administered orally at doses ranging from 1 to 30 mg/kg, and EEG recordings are used to measure spike-wave discharge frequency and duration. In maximal electroshock seizure and pentylenetetrazole-induced seizure models, seizure protection is assessed. Pharmacokinetic-pharmacodynamic correlation studies are conducted to establish the relationship between plasma/brain concentrations and anticonvulsant efficacy. Tissue distribution studies confirm brain penetration.

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]
Apinocaltamide exhibits favorable oral bioavailability and brain penetration following oral administration. The compound achieves therapeutic concentrations in the central nervous system, with brain-to-plasma ratios supporting its use for neurological indications. Pharmacokinetic studies in preclinical species demonstrate dose-proportional exposure and a half-life compatible with twice-daily or once-daily dosing. The compound is metabolized primarily by hepatic cytochrome P450 enzymes, and elimination occurs via renal and biliary routes. The compound's blood-brain barrier penetration is a key feature for its anticonvulsant activity.

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]
Preclinical toxicology studies of Apinocaltamide have shown a favorable safety profile. In repeat-dose toxicity studies in rodents and dogs, the compound was well-tolerated at doses exceeding the therapeutic range. No significant target organ toxicity was observed. The compound did not show genotoxic potential in standard in vitro and in vivo assays. Cardiovascular safety pharmacology studies demonstrated no significant effects on QT interval or blood pressure at therapeutic concentrations. The safety margin supports continued clinical development for epilepsy indications.

[1]. Discovery of a Potent, Selective T-type Calcium Channel Blocker as a Drug Candidate for the Treatment of Generalized Epilepsies. J Med Chem. 2017 Dec 14;60(23):9769-9789.

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).

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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]
Apinocaltamide (ACT-709478) is an orally available, selective T-type calcium channel blocker under investigation for the treatment of generalized epilepsies. It potently inhibits Cav3.1, Cav3.2, and Cav3.3 channels with IC50 values of 6.4 nM, 18 nM, and 7.5 nM, respectively. The compound penetrates the blood-brain barrier and has demonstrated anticonvulsant efficacy in preclinical models. It has been evaluated in clinical trials for epilepsy and represents a promising therapeutic approach for seizure disorders characterized by T-type calcium channel dysfunction.

C22H18F3N5O

425.40643453598

425.146

1838651-58-3

118560618

White to off-white solid powder

3.3

1

7

6

31

693

0

LSYANGLAZUZYFX-UHFFFAOYSA-N

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)

N-[1-[(5-cyanopyridin-2-yl)methyl]pyrazol-3-yl]-2-[4-[1-(trifluoromethyl)cyclopropyl]phenyl]acetamide

ACT709478 ACT 709478ACT-709478

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 (~293.83 mM)

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.

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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.

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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.
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 corn oil and mix evenly.

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