Myosin (IC50 = 1.4 μM)

Hypercontractility of the cardiac sarcomere may be essential for the underlying pathological hypertrophy and fibrosis in genetic hypertrophic cardiomyopathies. Aficamten (CK-274) is a novel cardiac myosin inhibitor that was discovered from the optimization of indoline compound 1. The important advancement of the optimization was discovery of an Indane analogue (12) with a less restrictive structure-activity relationship that allowed for the rapid improvement of drug-like properties[1].

---

Aficamten is an oral, selective, and allosteric cardiac myosin inhibitor designed to directly target the underlying hypercontractility associated with hypertrophic cardiomyopathy (HCM). It binds reversibly to a distinct allosteric pocket on the beta-cardiac myosin motor domain, stabilizing the myosin in a specific conformation known as the "super-relaxed" or weak actin-binding state. By locking myosin heads in this state, aficamten slows the rate of phosphate release during the ATP hydrolysis cycle, which prevents the conformational changes required for myosin to transition into the force-generating, strongly actin-bound state. This action effectively decreases the number of active actin-myosin cross-bridges participating in the contractile cycle, thereby reducing the force generated by myosin at the cardiac sarcomere and attenuating left ventricular outflow tract (LVOT) obstruction.

Aficamten is a next-generation indigenous myosin formulation that delivers a near-term human half-life suitable for once-daily dosing, achieves steady state within weeks, and displays a large domestic processing window and a clear PK/PD connection [1]. After oral treatment (mice 1 mg/kg, mice 2 mg/kg, rats 3 mg/kg, rats 8 mg/kg, dogs 1 mg/kg, and monkeys 1 mg/kg), Aficasten showed central Barrier bioavailability (model 98%, rat 55%, rat 58%, rat 79%, dog 45%, monkey 41%) [1]. Aficasten revealed a final elimination half-life (4.5 hours in monkey, 3.0 hours in monkey, 33.8 hours in dog, and 8.1 hours[1].

---

Aficamten exhibits a dose-dependent reduction in left ventricular contractility, resulting in significant improvements in LVOT obstruction and heart failure symptoms. Pharmacokinetically, aficamten has a half-life of approximately 3.4 days in humans, reaching steady-state concentrations within two weeks. In clinical trials, reductions in the LVOT gradient (LVOT-G) were observed within two weeks of initiating treatment and sustained through Week 24. At Week 24, the mean change from baseline in resting and Valsalva LVOT-G was -35 mmHg and -48 mmHg, respectively, for the aficamten group, compared to +4 mmHg and +2 mmHg, respectively, for the placebo group. Beyond hemodynamic changes, aficamten induces favorable cardiac remodeling. A cardiac magnetic resonance (CMR) substudy demonstrated significant reductions in left ventricular mass index, maximal wall thickness, and left atrial volume index after 24 weeks of treatment, alongside substantial declines in N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin I. Consistent with its mechanism, aficamten decreases left ventricular ejection fraction (LVEF); at Week 24, the mean change from baseline in LVEF was -7% in the aficamten group versus -2% in the placebo group. The drug's effects are reversible: four weeks after discontinuation of treatment, LVEF and Valsalva LVOT-G returned to baseline.

Cardiac Myofibrils Assay. [1]
To evaluate the effect of compounds on the ATPase activity of fulllength cardiac myosin in the context of the native sarcomere, skinned myofibril assays can be performed. Bovine cardiac myofibrils can be obtained by homogenizing bovine cardiac left ventricular tissue in the presence of a detergent such as triton X-100. Such treatment removes membranes and a majority of the soluble cytoplasmic proteins but leaves intact the cardiac sarcomeric acto-myosin apparatus. Myofibril preparations retain the ability to hydrolyze ATP in a Ca2+ regulated manner. ATPase activities of such myofibril preparations in the presence and absence of compounds can be assayed at Ca2+ concentrations activating to a defined fraction of the maximal rate (i.e. 25%, 75%). Small molecule agents were assessed for their ability to inhibit the steady-state ATPase activity bovine cardiac myofibrils using a pyruvate kinase and lactate dehydrogenase-coupled enzyme system that regenerates myosin-produced adenosine diphosphate (ADP) into adenosine triphosphate (ATP) by oxidizing the reduced form of nicotinamide adenine dinucleotide (NADH) to the oxidized form of nicotinamide adenine dinucleotide (NAD), producing an absorbance change at 340 nm. Myofibril ATPase assays were performed in PM12 buffer (12 mM Pipes, 2 mM MgCl2, 1 mM DTT, pH 6.8) supplemented with 60 mM KCl and ATP at approximately 3-10x the KM for the particular myofibril system (0.5 mM ATP for fast skeletal, 0.05 mM ATP for slow skeletal and cardiac). Calcium concentrations were controlled using 0.6 mM EGTA and sufficient CaCl2 to obtain the desired free calcium concentration (calculated using web resource http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm). Sodium azide (0.25 mM) was included in cardiac myofibril assays (where indicated) to suppress non-myosin ATPase activity. Non-myosin ATPase activity was subtracted from cardiac and slow skeletal myofibril assays (where indicated) by subtracting the ATPase activity in the presence of a saturating concentration of the non-selective myosin II inhibitor blebbistatin. Myofibrils were present at approximately 0.25 mg/mL (fast skeletal) or 1 mg/mL (slow skeletal, cardiac). Calcium concentrations were controlled using 0.6 mM EGTA and sufficient CaCl2 to obtain the desired free calcium concentration (calculated using web resource http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm). Absorbance measurements (340 nm) were carried out at approximately 25 °C using either an Envision or SpectraMax plate reader.

Cryopreserved plated human hepatocyte assay.[1]
The plated human hepatocyte assay was performed at Eurofins Pharma Discovery Services. The plated hepatocyte assay used to determine the potential induction of CYP isoforms by CK-3773274 by measuring the mRNA levels of CYP1A2, 2B6 and 3A4 in human hepatocyte cultures was performed at Eurofins Pharma Discovery Services. Hepatocytes were thawed and plated into collagen-coated 96-well plates in S54 the plating medium at a density of 0.7 X 106 viable cells/mL. The hepatocytes were cultured at 37 °C and 5% CO2. At 4 h post plating, the hepatocytes are washed, followed by overnight recovery. After 24 h of plating, the hepatocytes were incubated with the test compound in the incubation medium, which is changed daily for total incubation of 72 h. On day 5 after plating, the hepatocytes were washed and lysed by RT-qPCR Sample Reparation Reagent. The compounds were tested in triplicates at each concentration with a final DMSO concentration of 0.1%. The cell lysate is used as the mRNA template and is reverse transcribed into cDNA by RTPCR using a mixture of oligo(dT) and random primers. The relative expression (fold difference over vehicle control) of each isoform is determined by singleplex two-step RT-qPCR using CYP1A2, CYP2B6 and CYP3A4 sequence specified primers, respectively, and GAPDH as the reference gene. The induction potential of each test compound was analyzed by comparing its fold induction to a pre-determined cutoff value for each lot of hepatocytes.

Pharmacokinetics. [1]
Pharmacokinetic parameters were determined in male and female SpragueDawley rats, n = 3, male beagle dogs, n = 3 (PO) and n =2 (IV), and male cynomolgus monkeys, n = 3, following single bolus intravenous and/or oral administration of compound. Blood samples were taken periodically beginning immediately before compound administration to 24 h after administration. The concentrations of compound in blood or plasma samples were analyzed by HPLC relative to a standard. Compounds were dosed orally either in solution or as suspensions to animals that were fasted for 12 h. Concentration of test article in skeletal muscle tissue was S56 determined by excision, weight determination and homogenization of tissue samples followed by compound extraction and HPLC quantitation relative to a standard in the same manner employed for blood and plasma determinations. Summary pharmacokinetic data were analyzed using WinNonlin®.

Absorption
After a single dose of 1 mg to 75 mg aficatin, and after multiple daily doses of 5 mg to 20 mg aficatin, the exposure of aficatin increases proportionally with the dose. The geometric mean cumulative ratio of aficatin is similar at different dose levels, ranging from 4.6 to 4.8. Steady state is expected to be reached after approximately 17 days of once-daily aficatin in patients with hypertrophic cardiomyopathy (oHCM). The time to peak concentration (Tmax) is 1.5 to 2.0 hours. Absorption of aficatin is rapid after oral administration. The bioavailability of aficatin after oral administration is unknown.
Elimination route: After a single dose of 20 mg of radiolabeled aficatin, 32% (0.06% unchanged aficatin) is excreted in the urine and 58% (5.1% unchanged aficatin) is excreted in the feces.
Volume of distribution
The volume of distribution is 313 liters. The plasma concentration-to-dose ratio of aficatin is 0.94.
Clearance
Total clearance is 2.6 L/h, with renal clearance less than 0.1% of total clearance.
Protein Binding
Aficatin binds to plasma proteins at approximately 90%.
Metabolism/Metabolites
Aficatin is extensively metabolized in the human body, primarily via CYP2C9, with CYP3A, CYP2D6, and CYP2C19 also contributing. Aficatin is mainly metabolized into two less pharmacologically active metabolites, CK-3834282 and CK-3834283, which account for approximately 56% and 103% of the parent drug concentration in plasma, respectively. Inactive metabolites undergo further biotransformation through various reactions, such as glucuronidation, sulfonation, microbial-mediated reduction, and amide hydrolysis.
Biological Half-Life
In patients with hypertrophic cardiomyopathy (oHCM), the median terminal half-life (t½) of aficartan is approximately 80 hours.

[1]. Discovery of Aficamten (CK-274), a Next-Generation Cardiac Myosin Inhibitor for the Treatment of Hypertrophic Cardiomyopathy. J Med Chem. 2021 Oct 14;64(19):14142-14152.

Drug Indication
Treatment of hypertrophic cardiomyopathy

C18H19N5O2

337.375763177872

337.153

C, 64.08; H, 5.68; N, 20.76; O, 9.48

2364554-48-1

139331495

White to light brown solid powder

2.1

1

5

4

25

490

1

CCC1=NC(=NO1)C2=CC3=C(C=C2)[C@@H](CC3)NC(=O)C4=CN(N=C4)C

IOVAZWDIRCRMTM-OAHLLOKOSA-N

InChI=1S/C18H19N5O2/c1-3-16-21-17(22-25-16)12-4-6-14-11(8-12)5-7-15(14)20-18(24)13-9-19-23(2)10-13/h4,6,8-10,15H,3,5,7H2,1-2H3,(H,20,24)/t15-/m1/s1

N-[(1R)-5-(5-ethyl-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl]-1-methyl-1H-pyrazole-4-carboxamide

CK-3773274; CK274; Aficamten [INN]; [USAN]; CK-3773,274; B1I77MH6K1; CK3773,274; CK 274; CK 3773274; CK-274; Aficamten

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 (~370.50 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.17 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 (6.17 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 (6.17 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.

---

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