Abstract
Aficamten (CK-3773274) is a cardiac myosin inhibitor in development for the treatment of hypertrophic cardiomyopathy (HCM), a commonly inherited heart condition often characterized as a disease of the sarcomere. Aficamten reduces pathologic cardiac hypercontractility by selectively binding to an allosteric site on cardiac myosin. To characterize the pharmacology and toxicology of aficamten, a series of nonclinical repeated dose studies were conducted. In a 10-day repeated dose pharmacology study in Sprague Dawley rats, aficamten produced dose-dependent reductions in left ventricular fractional shortening (FS) which were fully reversible within 24 h. Aficamten did not change the ratio of heart weight to tibia length (HW/TL) or left ventricular posterior wall (LVPW) thickness at any dose tested. At a supratherapeutic dose of 6 mg/kg/day, there was a significant increase in interventricular septum (IVS) thickness. Aficamten did not affect mRNA expression of the cardiac injury biomarkers BNP, β-MHC, or ANP. In repeated dose Good Laboratory Practice (GLP) regulatory toxicology studies in Sprague Dawley rats for up to 6 months and beagle dogs for up to 9 months, the primary adverse findings at supratherapeutic doses were consistent across all studies and observed in the heart consisting of atrial/ventricular dilatation that correlated with increased heart weights. These findings were largely reversible and consistent with excessive on-target pharmacology associated with cardiac myosin inhibition. The reversible nature of aficamten-associated adverse effects is supportive of its clinical safety as this property suggests that these findings, should they occur in humans, may also be reversible, limiting long-term human cardiac risk.
Keywords
Introduction
Hypertrophic cardiomyopathy (HCM) is a cardiac disorder distinguished by a marked diversity in clinical presentation, natural history, and prognosis. 1 HCM is one of the most commonly inherited heart conditions and generally manifests in an autosomal dominant pattern in patients. 2 While the estimated carrier prevalence affects up to about 1 in 500 individuals, the estimated prevalence of clinically manifest HCM is likely much less, since all carriers do not develop disease. One analysis of a large integrated claims database of 160 million covered lives estimated an incidence of about 1 in 3,195 for clinically identified HCM. 3 The true prevalence of clinically manifest disease is likely somewhere in between the cited prevalence rates since not all patients necessarily require medical attention. HCM is clinically characterized by either normal or increased left-ventricular ejection fraction (LVEF), diastolic dysfunction, reduced left ventricular (LV) chamber volume and cardiac LV hypertrophy in a LV that is not dilated. 4 Most patients develop LV outflow tract (LVOT) obstruction, termed obstructive hypertrophic cardiomyopathy (oHCM), which occurs in approximately 70% of patients with phenotypic HCM. 5 The structural changes and hemodynamics responsible for the LVOT obstruction are well defined and involve a complex interchange between the anterior mitral valve leaflet and asymmetric septal hypertrophy, both defining the confines of the LVOT. The LVOT is narrowed because of abnormal mitral valve systolic anterior motion (SAM) and septal contact due to flow drag of the anterior mitral valve leaflet, resulting in the development of an increased LVOT pressure gradient. LVOT obstruction leads to markedly elevated LV pressures and secondary mitral valve regurgitation.6–10 Additional clinical manifestations of HCM include an elevated risk for syncope, development of ventricular arrhythmias and sudden cardiac death, heart failure due to diastolic dysfunction, palpitations, and stroke due to atrial fibrillation. 11
HCM develops from pathogenic sequence variants that occur in multiple genes that encode proteins of the cardiac sarcomere, the force generating unit of cardiac muscle. Myosin is the molecular motor of muscle cells that converts chemical energy of ATP hydrolysis into the mechanical force required for muscle contraction. 12 The autosomal dominant mutations associated with approximately 50% of familial HCM primarily occur in the myosin heavy chain beta (MHC-β) isoform (encoded by the MYH7 gene) and cardiac myosin-binding protein C (cMYBP-C) (encoded by the MYBPC3 gene). 13 These pathogenetic variants alter myosin protein function and increase energy consumption, evoke hypercontractility and lead to impaired cardiac relaxation. 14 Cardiomyocyte disarray, extracellular matrix modification, microvascular dysfunction, and the subsequent development of myocardial fibrosis, are the main histopathological features of HCM. 15
The main approaches to the treatment of oHCM are a combination of medical management and septal reduction therapy. Contemporary guideline-directed pharmacologic treatment options include the use of beta blockers, calcium channel blockers, and the class Ia antiarrhythmic drug, disopyramide; however, the magnitude of benefit to patients is variable and more invasive treatments such as surgical myomectomy or septal ablation are often necessary to treat individuals with marked functional cardiac deficits and associated symptoms. 16 While the current clinical management strategies for oHCM have resulted in some patients achieving reduced morbidity, the mechanisms of pharmacologic agents currently in use are not sufficiently selective and do not always provide clinical benefit. Additionally, patient benefit from beta blockers, calcium channel blockers and disopyramide is often limited by poor tolerability resulting from the inherent pleiotropic properties associated with these drugs, including alterations in vascular tone, cardiac conduction, chronotropy, and inotropy. 17 More recently, small molecule cardiac myosin inhibitors that selectively ameliorate the maladaptive hypercontractility associated with HCM potentially addressing the underlying pathophysiology of the disease have been developed, and Mavacamten (Camzyos®) is currently approved and marketed for the treatment of oHCM.
Mavacamten was approved in 2022 as the first cardiac myosin inhibitor for use for the improvement of functional capacity and symptoms in patients with symptomatic oHCM. 18 In a variety of in vitro and in vivo nonclinical models, mavacamten was shown to produce concentration- and dose-dependent decreases in sarcomere force production and reduced cardiac function.19,20 Early clinical studies of oHCM patients showed that mavacamten reduced the LVOT pressure gradient (LVOT-G). 21 In the Phase 3 clinical trial EXPLORER-HCM (identified https://www.clinicaltrials.gov, NCT03470545), mavacamten was superior to placebo for improving exercise capacity and health status in oHCM patients, illustrating the benefit of reducing LVOT gradients that are the characteristic feature of oHCM. 22 In this phase 3 clinical trial, seven patients on mavacamten had a transient reduction in LVEF below 50%, resulting in three patients undergoing temporary discontinuation of treatment (versus two patients in placebo group). Additionally, four patients treated with mavacamten had an LVEF below 50% at the conclusion of treatment period. Mavacamten has a terminal elimination half-life (t1/2) of 8 h in rats, 161 h in dogs, and approximately 9 days in humans. 23 A dose titration is imposed for the dosing of mavacamten along with a Risk Evaluation Mitigation Strategies (REMS) program by the US Food and Drug Administration. Per its drug label, dosing of mavacamten is guided by echocardiography and any excessive cardiac myosin inhibition (i.e., LVEF below 50%) are handled by interrupting dosing and rechecking LVEF every 4 weeks until LVEF is ≥ 50% before resumption of dosing at a lower dose with monitoring of LVEF every three months for risk mitigation. 24 Altogether, direct inhibition of cardiac myosin to relieve hypercontractility is a proven targeted pharmacological approach to improve function and symptoms in patients with oHCM. 25 Further, reduction of LVOT obstruction decreases the intraventricular systolic pressure and may also prevent progression of hypertrophy and fibrosis, which could translate into a potential disease modifying benefit for oHCM patients.
Aficamten (CK-3773274) is a next-in-class selective inhibitor of cardiac myosin that binds to a distinct allosteric binding site in the motor domain different than that of mavacamten; like mavacamten, aficamten reduces the number of force-producing actin-myosin cross-bridges.26,27 Aficamten was designed to reduce cardiac hypercontractility with a shallow pharmacokinetic (PK)/pharmacodynamic (PD) relationship, contributing to a wider pharmacodynamic window, and to provide a human t1/2 suitable for once a day (qd) dosing combined with rapid reversibility. 26 The human t1/2 of aficamten enables steady state drug concentrations to be reached within 2 weeks of dosing and substantial reversibility of its effect within a few days. The mechanism of action for aficamten has been shown to be the stabilization of the motor in the post-power stroke, non-force producing state, which confers the decreased hypercontractility in nonclinical models. 27 Aficamten is selective for the cardiac isoform of myosin by approximately five-fold compared to its most similar homolog, fast skeletal myosin, and has no appreciable activity on smooth muscle myosin.26,27 Aficamten reduces cardiac myocyte fractional shortening (FS) without changing the intracellular calcium transient. In healthy rats and dogs, and both murine and feline models of HCM, single doses of aficamten reduced cardiac contractility in a shallow dose- and concentration-dependent manner.26-29 The pharmacological effect of fractional shortening following administration of single doses of aficamten were similar between rats and dogs in response to this mechanism of action. 26
In this manuscript, we describe the reversibility of the effects of aficamten on cardiac functional parameters after short-term, repeated dosing in pharmacological studies of healthy rats and dogs and characterize its safety margin. Concurrently, subchronic and chronic Good Laboratory Practice (GLP) regulatory toxicology studies in rats and dogs were conducted not only to characterize target organ toxicity and establish no-observed-adverse effect levels (NOAELs) associated with aficamten but also to determine the reversibility of any potential toxicological effects that may be associated with prolonged, excessive cardiac myosin inhibition. The GLP toxicology studies detailed in this manuscript were conducted in accordance with ICH guidelines and were intended to support development and the proposed indication of aficamten.
Pharmacology and Toxicology Study Methods
The pharmacology studies were conducted in accordance with the best available scientific principles but were not intended to be in full compliance with GLP, while the toxicology studies described were performed in accordance with the U.S. Department of Health and Human Services, Food and Drug Administration, United States Code of Federal Regulations, Title 21, Part 58: Good Laboratory Practice for Nonclinical Laboratory Studies.
Justification for Test System and Number of Animals in Studies
The Sprague Dawley (SD) rat was chosen as the rodent animal species for the conduct of pharmacology and toxicology studies and the beagle dog was chosen as the non-rodent animal species for the repeated dose toxicology studies as they are accepted species for nonclinical efficacy and toxicity testing by regulatory agencies. The total number of animals used in each of these toxicology studies was selected to ensure that the study was sufficiently powered to properly characterize the effects of aficamten, while minimizing the number of animals utilized. 30 As cardiac myosin is highly conserved across mammalian species and adverse findings in the nonclinical studies detailed below were associated with excessive on-target pharmacology, the findings identified in the rat and dog studies are relevant for assessing human risk. 31
Animals
In the 10-day pharmacology studies, male Sprague Dawley Crl:CD(SD) rats were used, while in the 28-day, 13-week and 26-week repeated dose toxicology studies in rats, both male and female Sprague Dawley Crl:CD(SD) rats were used. Animals were obtained from Charles Rivers Laboratories (St. Constant, or Montreal, Quebec, Canada). All animals were 8 weeks old and weighed between 220–350 g for males and 160–260 g for females at the initiation of dosing. A minimum acclimation period of 3 days for pharmacology studies and 10 days for toxicology studies occurred between animal transfer and the start of treatment to acclimate the animals to the laboratory environment.
In the 28-day, 13-week and 39-week repeated dose toxicology studies in dogs, male and female naïve beagle dogs were received from Marshall BioResources (North Rose, New York). The animals were 6 to 8 months old and weighed between 6–10 kg for the males and 6–9 kg for the females at the initiation of dosing. A minimum acclimation period of 11 days was allowed between animal transfer and the start of treatment to accustom the animals to the laboratory environment.
Animal Husbandry
Housing
All SD rats used in pharmacology studies were individually housed on wood chip bedding in static filter-topped shoebox style cages. All SD rats used in toxicology studies were group housed together (up to three animals of the same sex and same dosing group together) in polycarbonate cages containing appropriate bedding equipped with an automatic watering valve. Similarly, beagle dogs were socially housed together (up to three animals of the same sex and same dosing group) in stainless steel cages equipped with an automatic watering valve as specified in Code of Federal Regulation of the USDA Animal Welfare Act and as described in the Guide for the Care and Use of Laboratory Animals.32–36 For both rat and dog studies, the housing conditions described were maintained throughout the study, and animals were socially housed except for times when they were separated for designated study procedures/activities.
Environmental Conditions
In all SD rat studies, room temperatures were maintained between 19° and 26°C, while for beagle dog studies room temperatures were maintained between 18° and 29°C. For both species, the relative room humidity was maintained between 30 and 70% and a 12-h light/12-h dark cycle was maintained, except when interrupted for designated procedures.
Food and Water
SD rats used in pharmacology studies were given Rodent Diet 5001 (24% protein) (LabDiet, Richmond, IN) while rats used in toxicology studies were given PMI Nutrition International Certified Rodent Chow No. 5CR4 (14% protein) (Shoreview, MN) ad libitum throughout each study. Similarly, for beagle dogs, PMI Nutrition International Certified Canine Chow No. 5007 (25% protein) was provided (300 g) as a single daily ration throughout the study. The food ration was offered up to 2 h after dosing and was left in the cage for a period of 2 to 4 h and then removed. For all studies, municipal tap water after treatment by reverse osmosis and dichlorination was freely available to each animal via an automatic watering system.
Animal Enrichment and Veterinary Care
SD rats in both pharmacology and toxicology studies were provided with items such as a hiding device and a chewing object and beagle dogs were provided with a floor toy, except during study procedures/activities. Food supplements were provided as necessary. Veterinary care was available throughout the course of these studies.
Experimental Design and Analysis
Rat GLP Toxicology Study Designs.
Conc = concentration; M: males; F: females; TK: toxicokinetic.
aMain Study animals were dosed (dose volume 10 mL/kg) for either 28 days, 13 weeks, or 26 weeks, and underwent terminal procedures on Day 29, Day 92, and Day 183, respectively.
bRecovery Study animals in the subchronic toxicology studies were dosed for 28 days or 13 weeks, retained for 28 days (not dosed), and underwent terminal procedures on Day 57 or Day 120, respectively. In the 26-week chronic study, Recovery Study animals were dosed for 26 weeks, retained for 2 months (not dosed), and underwent terminal procedures on Day 239.
cTK Study animals were used for toxicokinetic evaluation on Days 1 and 28.
dGroups were subdivided into replicate Cohorts, according to their assignment (Main Study, Recovery Study, and TK Study), with dosing initiation staggered over consecutive days.
eControl animals were given the Control Article/Vehicle [0.5% (w/v) hydroxypropylmethylcellulose and 0.1% (w/v) Tween 80 in Ultrapure Water] only.
fTK Study animals were used for toxicokinetic evaluation on Days 1, 45, and 91.
gTK Study animals were used for toxicokinetic evaluation on Days, 1, 91, and 182.
Dog GLP Toxicology Study Designs.
Conc = concentration; M: males; F: females; - = not applicable.
aMain Study animals were dosed (dose volume 5 mL/kg) once daily for 28 days, 13 weeks, or 39-weeks and were sent to terminal procedures on Day 29, Day 92, and Day 274, respectively.
bRecovery Study animals in the subchronic toxicology studies were dosed for 28 days or 13 weeks, retained for 28 days (not dosed), and underwent terminal procedures on Day 57 or Day 120, respectively. In the 39-week chronic study, Recovery Study animals were dosed for 39 weeks, retained for 2 months (not dosed), and underwent terminal procedures on Day 330.
cGroups were divided into two cohorts, Cohort 1 (males) and Cohort 2 (females) and dosing initiation of Cohort 2 was staggered by 1 day.
dToxicokinetic samples were collected from Main Study and Recovery Study animals on Days 1, 7, and 28.
eControl animals were given the Control/Vehicle [0.5% (w/v) hydroxypropylmethylcellulose and 0.1% (v/v) Tween 80 in Ultrapure Water] only.
fGroups 1 to 4 were divided into four replicates: male Main Study and Recovery Study animals, and female Main Study and Recovery Study animals; with a dosing initiation staggered by 1 day.
gToxicokinetic samples were collected from Main Study and Recovery Study animals on Days 1, 14, 45, and 91.
hInterim Study animals were euthanized on Day 183.
iToxicokinetic samples were collected from Main Study and Recovery Study animals on Days 1, 91, 182, and 273.
Data and Statistical Analysis
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two-sided tests. Numerical data collected was analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV, or SEM) were reported. Values were also expressed as a percentage of pre-dose or control values, when appropriate.
Datasets with at least three groups were compared using an overall one-way ANOVA F-test, two-way ANOVA, or Kruskal-Wallis test (when parametric assumptions were not met) at the 5% significance level. If the overall F-test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. Datasets with two groups (the designated control group and one other group) were compared using a t-test if Levene’s test was not significant or the Wilcoxon Rank-Sum test if it was significant. Graphpad Prism, Charles River Laboratories SRS, and Charles River Laboratories Nevis software were used for statistical analyses.
Chemicals and Reagents
The dosing formulations of aficamten ((R)-N-(5-(5-ethyl-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl)-1-methyl-1H-pyrazole-4-carboxamide) (CAS 2364554-48-1) and vehicle (0.5% (w/v) hydroxypropylmethylcellulose (HPMC) and 0.1% (v/v) Tween®80 in ultrapure water) were prepared once weekly. The dosing formulations were stored in a refrigerator set to maintain 4°C, protected from light. The vehicle was used to prepare aficamten dosing formulations. An adequate amount of the vehicle was dispensed daily for administration to control animals.
Reagents and chemicals used in all studies were obtained from Dow Chemical Co. (Midland, MI, USA) and Sigma-Aldrich (St. Louis, MO, USA). Details for other materials used and suppliers are provided in specific sections.
Dose Formulation Analysis
In all pharmacology and toxicology studies, dose formulation samples were collected for concentration and homogeneity analysis using a validated high-performance liquid chromatography (HPLC) analytical procedure. Concentration results were considered acceptable when mean sample concentration results were within or equal to ± 15% of the nominal concentration. Each individual sample concentration result was considered acceptable when it was within or equal to ± 20% of the nominal concentration. Homogeneity results were considered acceptable when the relative standard deviation (RSD) of the mean value at each sampling location was ≤ 5%. Aficamten dosing preparations were stable at room temperature and in a refrigerator set to maintain 4°C, protected from light.
Administration of Test Materials
Both aficamten and vehicle were administered once daily by oral gavage in pharmacology studies to SD rats for 10 days and in toxicology studies to either SD rats or beagle dogs for 28 days, 13 weeks, and 26 or 39 weeks. The dose volume for each animal (10 mL/kg in rats and 5 mL/kg in dogs) was based on the most recent body weight measurement. The doses were given using a syringe with an attached gavage cannula and dosing formulations were stirred for at least 30 min prior to and continuously during dose administration.
In-life Procedures, Observations, and Measurements
All in-life procedures, observations, and measurements including laboratory evaluations were performed for all Main Study and Recovery Study animals. All toxicokinetic (TK) animals were weighed and any abnormal clinical observations recorded.
Mortality/Moribundity Checks
Throughout all studies, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Clinical Observations
Cageside observations were performed once daily, throughout the dosing and recovery periods. These observations include detailed assessment of respiration, motor activities, reflexes, muscle tone, and skin. During the dosing period, these observations were performed 1 h ± 30 min post-dose.
Body Weights and Food Consumption
Animals were weighed individually weekly, starting during Week 1 (pre-study). A fasted weight was recorded on the day of necropsy for all toxicology animals. Food consumption was quantitatively measured weekly starting on Day 1 (pre-dose) and continuing throughout the dosing and recovery periods.
Ophthalmic Examinations
In toxicology studies, ophthalmology (funduscopic and biomicroscopic) examinations were performed on all Main Study and Recovery Study animals once pre-study and at the completion of dosing. The mydriatic used was 1% tropicamide (Mydriacyl, Alcon Labs, Fort Worth, TX).
Electrocardiology
In beagle dog toxicology studies, electrocardiograms (ECG) were recorded once during pre-study period, on multiple days during the treatment period, and at the end of the recovery period. ECG recordings collected during the treatment period were made about 1 to 4 h after dosing. All animals had tracings recorded using limb leads I, II, III, aVR, aVL, and aVF. Individual tracings were recorded using a standard recording speed of 50 mm/sec and standard sensitivity. The recordings were evaluated (qualitatively and quantitatively) by a veterinary cardiologist. All waveforms were qualitatively evaluated to detect rhythm or conduction disturbances or other abnormalities of the P-QRS-T waves. A quantitative evaluation was conducted on the 28 day and 39-week dog toxicology studies and consisted of the manual measurement of the heart rate (HR) and the PR, QRS and QT intervals of the ECG. The QT interval was corrected for HR (QTc) using Van de Water’s correction formula (QTcV = QT−0.087[(60/Heart Rate)−1])). 37
Echocardiographic Assessments
In the pharmacology studies, an echocardiographic assessment was performed in animals prior to the first oral dose (pre-dose) of aficamten, then 2 h post-dose on days 4 and 10 and on Day 11 (24 h after the last dose of aficamten was administered). The treatment duration provided multi-day, dose-response pharmacological data that was intended to provide contextual information to subsequent long term toxicology studies. SD rats were placed under anesthesia (1–5% isoflurane, FORANE), and maintained within a surgical plane via nose cone delivery throughout the procedure. Core body temperature was maintained at 37°C with a heating pad. Once anesthetized, the chest area was shaved and cleaned with 70% ethanol and ultrasound gel was applied. Using a GE Vivid7 ultrasound machine (GE Healthcare, Chicago, IL), a 10 MHz probe was placed at the level of the papillary muscles and 2D M-mode images of the left ventricle were captured. Images and measurements were collected from the long axis view. In vivo percent FS was determined by analysis of the M-mode images using the GE Vivid7 ultrasound software. Heart rate, diastolic and systolic cardiac dimensions, left ventricular posterior wall thickness, and intraventricular septum wall thickness were also determined.
Laboratory Evaluations
Clinical Pathology
In toxicology studies, whole blood was collected from the jugular vein during the dosing period and from the abdominal aorta after isoflurane anesthesia (study termination). Urine was collected from individual animals placed in metabolism cages overnight. Animals were fasted overnight before blood sampling (for clinical chemistry) and food was removed during the urine collection procedure.
Hematology and Coagulation
In toxicology studies, whole blood samples were analyzed for hematological parameters including red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, red blood cell distribution width, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, reticulocyte count, platelet count, and white blood cell count. Analysis of absolute neutrophil, lymphocyte, monocyte, eosinophil, basophil, and large unstained cell counts were conducted. Whole blood samples were also processed for plasma and analyzed for coagulation parameters including activated partial thromboplastin time (aPTT), fibrinogen, and prothrombin time (PT).
Clinical Chemistry and Urinalysis
In toxicology studies, whole blood samples were processed for serum and analyzed for alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), gamma-glutamyltransferase (GGT), creatine kinase (CK), total bilirubin, urea nitrogen, creatinine, calcium, phosphorus, lactate dehydrogenase (LD), total protein, albumin, globulin, albumin/globulin ratio (A/G ratio), glucose, cholesterol, triglycerides, sodium, potassium, and chloride. In toxicology studies, urine samples were processed and analyzed for color, appearance/clarity, specific gravity, volume, pH, protein, glucose, bilirubin, ketones, and blood.
Cardiac Biomarkers
Cardiac Troponin I (cTnI) Biomarker
In the 13-week rat and dog toxicology studies, whole blood samples (target volume of ∼0.5 mL) were collected in serum separator tubes, processed for serum and analyzed for Cardiac Troponin I (cTnI). Analyses were conducted using a validated electrochemiluminescence method (Meso Scale Diagnostics, LLC., Rockville, MD) that relied on a SULFO-TAG Anti-rat cTnl Antibody for rat samples and a validated dog cTnI ELISA method (Cat# CTNl-4-HS, Life Diagnostics, Inc., West Chester, PA) for dog samples.
Methods for Cardiac Hypertrophy Assessment by Heart Mass and Tibia Length
In pharmacology studies, following the final echocardiographic measurement, hearts were excised, cleaned, weighed, and snap frozen in liquid nitrogen. Tibia length was measured with calipers. Heart mass normalized to tibia length was utilized as a measure of cardiac hypertrophy.
Assessment of Cardiac Hypertrophy Markers by Real Time Quantitative PCR
In pharmacology studies, cardiac tissues were homogenized using FastPrep®-24 and FastRNA PRO™ Green kit (MP Biomedicals, Irvine, CA). RNA was extracted by phenol-chloroform following the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA). Relative gene expression levels were analyzed using the Taqman method on a QuantStudio™) 6 Flex System (Applied BioSystems, Waltham, MA), and normalized to a GAPDH internal control. The following Taqman probes were used: Nppa, Rn00664637_g1, Nppb, Rn00580641_m1, Myh7, Rn01488777_g1, GAPDH, Rn01775763_g1.
Bioanalysis and Toxicokinetic Evaluation
In pharmacology studies, whole blood samples (∼0.10 mL) were obtained via a free jugular vein venipuncture on Day 4 and Day 10 at 1-, 4-, and 8-h post-dose. In toxicology studies, whole blood (target volumes of up to ∼0.25 mL in rats and ∼0.5 mL in dogs) was collected by jugular venipuncture. Samples were collected pre-dose and at multiple timepoints up to 24 h post-dose on the first and last day of study and mid-study for the 3-month and chronic toxicology studies. All tubes contained K2EDTA as anticoagulant and were pre-chilled on crushed wet ice.
Bioanalytical Sample Processing and Analysis
Blood samples were gently inverted to ensure adequate mixing and placed on crushed wet ice until centrifugation, which was carried out within 60 min of collection. All samples were centrifuged for 10 min in a refrigerated centrifuge (set to maintain 4°C) at 1200g. The resultant plasma was separated, transferred to uniquely labeled clear polypropylene tubes and frozen immediately over dry ice. Samples were stored in a freezer set to maintain −70°C or colder. All plasma samples from toxicology studies were analyzed for the concentration of aficamten using a validated LC MS/MS method and Analyst software from AB Sciex (Sciex, Redwood, City, CA).
Toxicokinetic Evaluation
In toxicology studies, toxicokinetic parameters were estimated using Phoenix WinNonlin™ pharmacokinetic (PK) software (Certara, Princeton, NJ). A non-compartmental approach consistent with the oral route of administration was used for parameter estimation. All parameters were generated from mean aficamten plasma concentrations. PK and TK parameters included tmax (the time after dosing at which the maximum concentration was observed), Cmax (the maximum observed concentration measured after dosing), AUC(0–24) (the area under the concentration versus time curve from the start of dose administration to the time after dosing at which the last quantifiable concentration was observed, using the linear trapezoidal method), RAUC (the AUC(0–24) on the day plasma concentrations were evaluated divided by the AUC(0–24) on Day 1), and t1/2.
Terminal Study Procedures
In toxicology studies, Main Study and Recovery Study animals that survived until scheduled euthanasia had a terminal body weight recorded. All animals underwent abdominal aorta exsanguination after isoflurane anesthesia and blood sample collection. All animals were subjected to a complete necropsy examination, which included evaluation of the musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Organ Weights
In pharmacology studies, the hearts were identified and weighed at the end of the study dosing period. In toxicology studies, the organs identified and weighed at necropsy for all scheduled euthanasia animals included the brain, epididymis, adrenal gland, pituitary gland, prostate gland, thyroid gland, heart, kidneys, liver, lung, ovary, spleen, testis, thymus, and uterus. Paired organs (adrenal and thyroid glands, kidney, ovary, and testis) were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Tissue Collection, Histology and Histopathology
Standard Tissues Collected for Histopathology Evaluation in Aficamten GLP Toxicology Studies a .
aTissue samples were preserved in 10% neutral buffered formalin (NBF) unless otherwise noted.
bPreserved in Davidson’s fixative.
cPreserved in modified Davidson’s fixative.
Results
Pharmacological Effects and Reversibility of Aficamten in Rats
A 10-Day Echocardiography Assessment and Plasma Concentrations of Aficamten in Rat
In previous studies, healthy rats and dogs given single doses of aficamten exhibited decreased left ventricular contractility in a dose- and concentration-dependent manner.26,27 In rats, a single oral dose of aficamten at doses up to 4 mg/kg reduced left ventricular fractional shortening (FS) by up to 70% (as a percentage from pre-dose baseline) at 1-h post-dose (data not shown). In dogs, single oral doses of aficamten at doses up to 3 mg/kg reduced LVEF up to 50% from baseline at 2 h post-dose (data not shown). In relation to aficamten’s half-life in rats and dogs, FS returned to near pre-dose baseline values at 24 and 48 h in rats and dogs, respectively. 26
Heart Rate (Beats/Min) in the Rat Echocardiography Study.
Values shown are mean ± SEM (n = 3 males).
Effect of Aficamten on Fractional Shortening in the Rat Echocardiography Study.
Echocardiographic measurements were performed in male rats at pre-dose, 2 h post-dose on Day 4 and Day 10, and on Day 11 (24 h after last dose). Values shown as mean ± SEM (n = 3 males). *P < .05 vs. Vehicle.
The change in fractional shortening produced by aficamten after 10 days of daily oral dosing is completely reversed within 24 h after administration of the last dose (Day 11). Aficamten significantly reduced fractional shortening in a dose-dependent manner to a similar extent after 4 and 10 days of dosing. Approximately 24 h after the last dose, fractional shortening returned to baseline (pre-dose) levels. Data are expressed as mean ± SEM (N = 3 males/dose group). *P < .05 vs vehicle. Groups: Vehicle (black); aficamten (1 mg/kg/day, neon green); aficamten (3 mg/kg/day, green); aficamten (6 mg/kg/day, dark green).
There was no statistically significant difference in the heart weight to tibia length (HW/TL) ratio (Figure 2), or interventricular septum (IVS) thickness between aficamten and vehicle-treated animals after 10 days of dosing (Figure 3 and Table 6). At the end of the study, markers of cardiac hypertrophy, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and myosin heavy-chain beta (β-MHC) were measured by qPCR.38,39 Relative to vehicle treatment, there no significant increase in ANP mRNA at all aficamten dose levels (Figure 4). BNP and β-MHC mRNA levels were unchanged with aficamten treatment (Figure 4). Collectively, while there was no overall change in heart weight, an early sign of adverse cardiac remodeling despite 10 days of high dose (6 mg/kg/day) aficamten that reduced FS by ∼75%. The effect of 10 days of daily reductions in fractional shortening by aficamten does not affect cardiac size. Cardiac size was assessed as heart wet mass weight normalized to tibia length (HW/TL) after 10 days of daily oral dosing. Data are expressed as mean ± SEM (N = 6 males/dose group). Groups: Vehicle (black); aficamten (1 mg/kg/day, neon green); aficamten (3 mg/kg/day, green); aficamten (6 mg/kg/day, dark green). Relative to pre-dose values, interventricular septal wall thickness is only significantly higher after 10 days of daily oral aficamten dosing in the high dose (6 mg/kg/day) group. Data are expressed as mean ± SEM (N = 3 males/dose group). *P < .05 vs vehicle by two-way ANOVA. Groups: Vehicle (black); aficamten (1 mg/kg/day, neon green); aficamten (3 mg/kg/day, green); aficamten (6 mg/kg/day, dark green). Effects of Aficamten on Interventricular Septal Wall Thickness at End Diastole (IVSd) in the Rat Echocardiography Study. IVSd: Interventricular septum wall thickness at end diastole. Echocardiographic measurements in male rats were performed at pre-dose, 2 h post-dose on Day 4 and Day 10, and on Day 11 (24 h after last dose). Values shown as mean ± SEM (n = 3 males). Note that *P < .05 vs. 6 mg/kg/day pre-dose value by two-way ANOVA. Aficamten daily oral dosing for 10 days does not significantly alter mRNA markers of cardiac hypertrophy. The mRNA biomarkers of cardiac hypertrophy, assessed 24 h after the last aficamten dose, included (A) = atrial natriuretic peptide (ANP), (B) = brain natriuretic peptide (BNP) and (C) = β-myosin heavy chain (β-MHC). Data are expressed as mean ± SEM (N = 6 males/dose group). Groups: Vehicle (black); aficamten (1 mg/kg/day, neon green); aficamten (3 mg/kg/day, green); aficamten (6 mg/kg/day, dark green).
Total Aficamten Plasma Concentrations (ng/mL) in the Rat Echocardiography Study.
Values shown as mean ± SEM (n = 2 males per timepoint).
Toxicological Effects of Aficamten in Rats and Dogs
All dose formulations were within or equal to the acceptance criteria of the theoretical concentration and the homogeneity of the concentrations was confirmed for all formulations.
A 28-Day Oral Gavage Toxicity and Toxicokinetic Study in Rats with a 28-Day Recovery Period (GLP)
In a 28-day GLP study, rats received either vehicle or aficamten (0.5, 1.5, 3, or 9 mg/kg/day) once daily for 28 consecutive days. On Day 10, one high-dose (9 mg/kg/day) male was found dead with no clinical observations noted prior to death. Marked acute liver necrosis was noted and microscopically characterized by large bands of coagulative necrosis. Liver necrosis was considered the cause of death of this animal. This adverse effect is likely the result of passive congestion resulting from the prolonged, excessive reduction in cardiac contractility and low cardiac output that develops with high (suprapharmacological) doses of aficamten. All other animals survived dosing.
Terminal Heart Weights Percent Difference of Control Group in Rat GLP Aficamten General Toxicology Studies.
Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group—P ≤ .05.
aAll values are expressed as percent (%) difference of control group means.
Total Plasma Exposures of Aficamten in the Rat and Dog GLP General Toxicology Studies at the NOAEL.
A 13-Week Oral Gavage Toxicity and Toxicokinetic Study in Rats with a 28-Day Recovery Period (GLP)
In a 13-week study, rats received either vehicle or aficamten (1.5, 3, or 6 mg/kg/day) once daily for 13 weeks. Two toxicology male rats at 6.0 mg/kg/day and one toxicokinetic (TK) Study female at 1.5 mg/kg/day were found dead on Days 21, 86, and 91, respectively. In one of the two males dosed at 6.0 mg/kg/day, a marked diffuse acute hemorrhage of the lungs was observed for which the origin remained unclear. The cause of death of the other male dosed at 6.0 mg/kg/day remained undetermined; however, following necropsy, this animal had cardiac changes like those seen in terminal animals at the same dose. The cause of death of the TK Study animal remained undetermined.
Mean Cardiac Troponin I Concentrations in Rat GLP 13-Week General Toxicology Study.
Lower Limit of Quantitation (LLOQ) = 40.6 pg/mL, <LLOQ will be assigned as 40.6/2 (20.3 pg/mL) for statistical analysis purposes.
aCollected from Recovery Study Animals at the end of the recovery period.
Summary of Microscopic Findings: Scheduled Euthanasia (Day 92).
aNumbers in parentheses represent the number of animals with the finding.
Terminal Lung Weights Percent Difference of Control Group in Rat Aficamten General Toxicology Studies.
Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group—P ≤ .05.
aAll values expressed as percent difference of control group means.
bAll Groups excluded from statistical analysis (N < 3 in Control).
Systemic exposure to aficamten was generally similar between males and females (female-to-male ratios ranged from 1.02 to 1.57 for Cmax and from 0.796 to 1.19 for AUC(0–24)), therefore data were combined. At 1.5 mg/kg/day Cmax of aficamten generally occurred at 1-h post-dose on all occasions. At 3 mg/kg/day, Cmax of aficamten occurred at 1-h post-dose on all occasions, except for males on Day 1 where Cmax was observed at 6 h. At 6 mg/kg/day, Cmax was observed at 0.5-h post-dose on all occasions, except for males on Day 1 and Day 45 where Cmax was observed at 4 h. The t1/2 of aficamten was estimated between 3.6 and 4.7 h on Day 1, between 5.6 and 7.4 h on Day 45, and between 6.2 and 8.7 h on Day 91. Systemic exposure to aficamten, as assessed by Cmax and AUC(0–24), increased approximately in proportion with increasing dose across the dose range tested (from 0.5 to 6 mg/kg/day). Following repeat administration of aficamten, a slight increase in exposure was observed, as assessed by AUC(0–24), relative to Day 1 at all dose levels with RAUC values ranging from 1.40 to 1.54 on Day 45 and from 1.58 to 1.79 on Day 91. The NOAEL was 3 mg/kg/day for males and 6 mg/kg/day for females. At the NOAEL, on Day 91, the mean Cmax was 4700 ng/mL and 10800 ng/mL and the mean AUC(0–24) was 73000 ng∙h/mL and 99200 ng∙h/mL in males at 3 mg/kg/day and females at 6 mg/kg/day, respectively (Table 9).
A 26-Week Oral Gavage Toxicity and Toxicokinetic Study in Rats with an 8-Week Recovery Period (GLP)
In a 26-week study, rats received either vehicle or aficamten (0.25, 0.5, or 1.5 mg/kg/day) once daily for 26 weeks. There were two preterminal deaths in the Main Study and three preterminal deaths in the TK Study. The cause of death of the Main Study animals was related to the gavage procedure based upon the gross observations (perforation of the stomach or lungs and the hemorrhages observed in the thymus, lungs, and base of the heart). Similarly, none of the TK Study animal deaths were considered related to aficamten based on the low number of deaths in the study, the absence of gross findings in these animals and the lack of aficamten-related pathology findings including increased heart weights or dilated cardiomyopathy in the study. There were no aficamten-related effects on any parameters or endpoints evaluated in this study, including heart weight (Table 8) either at the end of the dosing or recovery interval.
Systemic exposure to aficamten was generally similar between males and females (female-to-male ratios ranged from 0.93 to 1.3 for Cmax and from 0.55 to 1.4 for AUC(0-24)). The Cmax occurred at 1-h post-dose on all occasions and at all dose levels for the sex combined profiles. The t1/2 of aficamten in the sex combined group was estimated between 4.4 and 5.6 h on Day 1, between 6.5 and 9.2 h on Day 91, and between 7.9 and 9.1 h on Day 182. Systemic exposure to aficamten, as assessed by Cmax and AUC(0–24), increased proportionally with dose levels. Following repeat administration of aficamten, a slight increase in exposure was observed, as assessed by AUC(0–24), on Days 91 and 182 relative to Day 1 at all dose levels with RAUC values ranging from 1.36 to 1.52 on Day 91 and from 1.43 to 1.74 on Day 182.
Under the conditions of the study, aficamten was well tolerated in male and female rats at levels up to 1.5 mg/kg/day. There were no target organ effects at any dose level tested. Based on these results, the no-observed-effect level (NOEL) was 1.5 mg/kg/day. At the NOEL, on Day 182, the mean Cmax was 2780 ng/mL and 3340 ng/mL and the mean AUC(0–24) was 45000 ng∙h/mL and 24900 ng∙h/mL in males and females at 1.5 mg/kg/day, respectively (Table 9).
A 28-Day Oral Gavage Toxicity and Toxicokinetic Study in the Beagle Dog with a 28-Day Recovery Period (GLP)
Terminal Heart Weights Percent Difference of Control Group in Dog GLP Aficamten General Toxicology Studies.
Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group—P ≤ .05.
aAll values are expressed as percent (%) difference of control group means.
bAll groups excluded from statistical analysis (N < 3 in Control).
28-Day Dog Toxicology Study: Summary of Electrocardiology Evaluations.
Values shown as mean ± SD (n = 6).
Bolded values indicate significant difference from control. Significantly different from control group 1 value: a = P ≤ .05, b = P ≤ .01, c = P ≤ .001 (Dunn), d = P ≤ .05, e = P ≤ .01, and f = P ≤ .001 (Dunnett).
AAll Groups excluded from statistical analysis (N < 3 in Control).
Systemic exposure to aficamten (Cmax and AUC(0–24)) increased in an approximately dose-proportional manner with increasing dose in both sexes over the dose range evaluated; there were no apparent sex-related differences (<2-fold) in exposure with female-to-male ratios ranging between 0.63 and 1.1 for Cmax and between 0.65 and 1.1 for AUC(0–24). The Cmax of aficamten was observed between 0.25- and 1-h post-dose on Days 1 and 28 with a median tmax of 0.25 or 0.5 h). The short tmax values indicated that absorption was rapid, and was not significantly different between dose groups, sampling days, or genders. Systemic exposure to aficamten increased approximately in proportion with increasing dose in the dose range tested (0.25 to 2 mg/kg/day). Following repeat administration, exposure to aficamten, as assessed by AUC(0–24), did not change with repeated dosing on Day 28 relative to Day 1 at 0.25, and 0.5 mg/kg/day; however, the AUC(0–24) was approximately two-fold higher following repeated dosing at 1 and 2 mg/kg/day on Day 28 when compared to Day 1, and the mean RAUC values ranged between 1.83 and 2.47. Under the conditions of this study, the NOAEL for aficamten was 2 mg/kg/day. Systemic exposure (Cmax and AUC(0–24)) to aficamten at the NOAEL on Day 28 was 820 ng/mL and 6980 ng∙h/mL, respectively, in males and 746 ng/mL and 5300 ng∙h/mL, respectively, in females (Table 9).
A 13-Week Oral Gavage Toxicity and Toxicokinetic Study in Beagle Dogs with a 4-Week Recovery Period (GLP)
Mean Cardiac Troponin I Concentrations in Dog GLP 13-Week General Toxicology Study.
Lower Limit of Quantitation (LLOQ) = 200.00 pg/mL, <LLOQ was assigned as 200.00/2 (100.00 pg/mL).
aCollected from Recovery Study Animals at the end of the recovery period.
When compared to controls, moderate, reversible lower mean body weight gains were noted in both male and female aficamten-treated groups (Figure 5). These changes were not dose-dependent but were significant during the Main Study (Day 1 to Day 91) only in animals at the highest dose group (2 mg/kg/day). As the food consumption was similar amongst groups, the changes in body weight gains were not attributed to a lower food intake. The lower body weight gains were not associated with any other clinical observations, and they were considered non-adverse. The aficamten-related changes in hematology parameters (decreases in reticulocyte and red blood cell mass parameters [red blood cell count, hemoglobin and hematocrit]) and the aficamten-related changes in a clinical chemistry parameter (decreases in alkaline phosphatase) were limited to 2 mg/kg/day, were minimal to moderate in severity, were not associated with any histological correlates, and were not observed following the 4-week recovery period. Given that the changes in clinical parameters were transient, of low magnitude, and fully reversible, they were considered non-adverse. In the aficamten 13-week GLP Toxicology study in dogs, moderate and reversible lower mean body weight gains were noted in aficamten-treated groups relative to controls. Body weight changes were significant during the Main Study only in the high dose group (2 mg/kg/day). As food consumption was similar across groups, body weight changes were not attributed to a lower food intake.
Qualitative ECG evaluation revealed normal ECGs but elevated heart rates (sinus tachycardia) in four animals dosed at 2 mg/kg/day at Week 6 and 13. During ECG recordings, which were collected at the approximate tmax (0.5 to 2 h post-dose), a high dose heart rate effect was observed; however, this effect was reversible in one animal assigned to recovery following the 4-week recovery period.
Dog GLP 13-Week General Toxicology Study: Summary of Gross Pathology Findings-Main Study (Day 92).
Dog GLP 13-Week General Toxicology Study: Summary of Gross Pathology Findings-Recovery Study (Day 120).
Systemic exposure to aficamten (Cmax and AUC(0–24)) increased in an approximately dose-proportional manner with increasing dose in both sexes over the dose range evaluated; there were no apparent sex-related differences (<2-fold) in exposure with female-to-male ratios ranging between 0.69 and 1.2 for Cmax and between 0.61 and 1.3 for AUC(0–24). The Cmax of aficamten was observed at 0.5-h post-dose on all occasions at 0.5 and 1 mg/kg/day in both males and females. The Cmax was observed generally at 0.5- or 1-h post-dose at 2 mg/kg/day on all occasions in males and females. The median tmax was observed at 0.5-h post-dose on all dose levels and on all occasions in males and females, indicating a rapid absorption of aficamten. Systemic exposure to aficamten, as assessed by Cmax and AUC(0–24), increased approximately in proportion with increasing dose in the dose range tested (from 0.5 to 2 mg/kg/day). Following repeat administration of aficamten, an increase in exposure, as assessed by AUC(0–24), was observed on Days 14, 45, and 91 relative to Day 1 at all dose levels, with mean accumulation ratios ranging from 1.5 to 2.1 on Day 14, from 1.6 to 2.2 on Day 45, and from 1.8 to 2.6 on Day 91. Under the conditions of the study, the NOAEL was 1 mg/kg/day for both male and females. Systemic exposure (Cmax and AUC(0–24)) to aficamten at the NOAEL on Day 91 was 387 ng/mL and 3330 ng∙h/mL, respectively, in males and 430 ng/mL and 3670 ng∙h/mL, respectively, in females (Table 9).
A 39-Week Oral Gavage Toxicity and Toxicokinetic Study in Beagle Dogs with an 8-Week Recovery Period (GLP)
39-Week Dog Toxicology Study: Summary of Electrocardiology Evaluations.
Values shown as mean ± SD (n = 6).
Bolded values indicate significant difference from control. Significantly different from control group 1 value: a = P ≤ .05, b = P ≤ .01, c = P ≤ .001 (Dunn), d = P ≤ .05, e = P ≤ .01, and f = P ≤ .001 (Dunnett).
AAll Groups excluded from statistical analysis (N < 3 in Control).
Systemic exposure to aficamten, as assessed by AUC(0–24), increased with increasing dose levels in an approximately dose proportional manner, except for females on Day 273 between 0.25 and 0.5 mg/kg/day, where the exposure was greater than dose proportional. Systemic exposure to aficamten was generally similar between males and females, except on Days 91, 182, and 273 at 0.25 mg/kg/day and on Day 182 at 1 mg/kg/day, where the exposure to aficamten was slightly higher in males. Female-to-male ratios ranged from 0.70 to 1.1 for Cmax and from 0.62 to 1.1 for AUC(0–24). Following repeated administration of aficamten, an increase in exposure, as assessed by AUC(0–24), was observed at all dose levels on Days 91, 182, and 273 relative to Day 1 with mean RAUC values ranging from 1.83 to 3.13. The Cmax was generally observed at 0.5-h post-dose on all occasions and at all dose levels except for two animals on Day 182 at 0.5 mg/kg/day, for which Cmax was observed at 1 h post-dose. Based on the absence of any adverse effect, the NOAEL was 1 mg/kg/day for both males and females. Systemic exposure (Cmax and AUC(0–24)) to aficamten at the NOAEL on Day 273 was 537 ng/mL and 5040 ng∙h/mL, respectively, in males and 412 ng/mL and 4040 ng∙h/mL, respectively, in females (Table 9).
Discussion and Conclusions
Hypertrophic cardiomyopathy is a complex clinical syndrome resulting from changes in ventricular and valvular structure, which can lead to the impairment of the ability of the ventricle to fill with or eject blood. The clinical impact can be mild in some patients but often progresses to significant symptoms and functional burden. In some, disease progression may even result in cardiac transplant. Cardiac myosin inhibition has been validated as a targeted pharmacological approach to reduce hypercontractility and LVOT obstruction in patients with oHCM, which can lead to meaningful improvement in patient symptoms and function. 25
Aficamten is an allosteric inhibitor of cardiac myosin and was designed to reduce cardiac hypercontractility, which underlies diseases such as HCM. In vitro studies show aficamten is more selective for inhibition of cardiac myosin compared to fast skeletal myosin and has no activity on smooth muscle myosin.26,27 Studies in bovine cardiac myofibrils showed that aficamten directly reduced adenosine triphosphatase (ATPase) activity (IC50 = 1.26 µM) and lacked activity on smooth muscle myosin ATPase (IC50 > 39 μM), an indicator of biochemical selectivity and activity. The functional effect of aficamten is the inhibition of cardiac myofibrillar ATPase activity manifesting as a reduction in cellular contractility with the pharmacodynamic effect translating to a reduction in FS in isolated rat ventricular myocytes without any changes to the intracellular Ca2+ transients.26,27 In echocardiography studies, single doses of aficamten decreased left ventricular contractility in a dose- and concentration-dependent manner; these effects were fully reversible as plasma concentrations of aficamten decreased. 26 Secondary pharmacology studies found a low potential for off-target binding of aficamten. 26 In PK studies, aficamten exhibited moderate-to-high oral bioavailability (55.1% to 78.8% in fasted male rats and 45.2% in fasted male dogs), a low plasma clearance, and moderate-to-high volume of distribution. 26 Aficamten has high protein binding in rat plasma (98.4%), and moderate protein binding across most other nonclinical species, including dog (75.1%).26,41 Overall, aficamten possesses the desired profile of myosin selectivity defined by biochemical and in vitro assays, an optimal PK profile and nonclinical primary pharmacodynamic (PD) activity and lacks secondary “off-target” pharmacological activity. These properties were supportive of advancing aficamten into clinical development.
An important goal of the nonclinical safety evaluation includes a characterization of adverse toxicological effects with respect to target organs, dose dependence, relationship to exposure, and the potential reversibility of findings. 42 An objective of these current studies was to evaluate the reversibility of the effects of aficamten on cardiac functional parameters after repeated dosing in pharmacology studies and end-organ adverse effects in repeated dose toxicology studies in healthy rats and dogs. The GLP toxicology studies detailed in this manuscript were conducted in accordance with ICH guidelines and were intended to support development and the proposed indication for aficamten.
Assessing reversibility is an important goal of any toxicology study. All aficamten toxicology studies included cohorts of animals that were dosed in parallel to the Main Study animals, termed the Recovery group. These animals were no longer dosed after completion of the designated study dosing durations (i.e., 28-days, 13 weeks, or 6 and 9 months), and were given a washout phase of a specific duration, termed the recovery phase or reversibility phase. 43 For the aficamten studies, these recovery phase durations ranged between 4 and 8 weeks, during which time animals were observed without exposure to aficamten. The inclusion of this recovery phase allows for the assessment of the reversibility of drug-induced effects, that is, whether any adverse effects observed at the end of the dosing phase either partially or fully recover, persist, or worsen. The demonstration of partial or full reversibility of aficamten-related toxicity suggests that a particular finding may similarly be reversible if observed in humans. Conversely, the persistence or progression of adverse findings may represent an additional potential safety risk in humans, depending on the nature of the finding. Inclusion of a recovery phase also allows for the identification of any potential delayed toxicity that may develop following cessation of dosing. Delayed toxicity can include immune reactions, or delayed organ damage. The recovery period may also provide insight into mechanisms associated with developed toxicity. 44 Thus, an understanding of the reversibility of findings, such as is seen with aficamten in the nonclinical safety studies conducted, has important implications within the overall nonclinical development safety assessment. 45
The effects of aficamten were found to be reversible in repeated dose pharmacology studies. In healthy rats, 10 days of daily oral dosing produced consistent dose-related reductions in systolic function, with the highest dose evaluated (6 mg/kg) reducing fractional shortening by ∼75% after 4 and 10 days of dosing. The effect of 10 days of dosing on fractional shortening was completely reversible, returning to pre-dose baseline values 24 h after the last aficamten dose (Figure 1). Protracted, disproportionate reductions in cardiac contractility may lead to compensatory cardiac hypertrophy and ventricular enlargement as a response to preserve sufficient stroke volume and cardiac output. 46 mRNA expression of brain natriuretic peptide (BNP) and myosin heavy chain, markers of cardiac hypertrophy, changed significantly after 10 days of dosing (Figure 4). Overall, 10 days of daily, excessive pharmacological dosing with aficamten in rats produced persistent decreases in fractional shortening that did not lead to the development of cardiac hypertrophy or ventricular enlargement, and upon cessation of dosing, cardiovascular function returned to pre-dose levels within 24 h.
Definitive repeated dose toxicology studies conducted with aficamten in rats include studies up to 26-weeks in duration and in dogs up to 39-weeks in duration. There were limited aficamten-related clinical signs, ophthalmic findings, effects on body weight, food consumption, coagulation, clinical chemistry, or urinalysis throughout the dosing and recovery periods across all studies conducted. Most findings at the well tolerated doses, when observed, were considered incidental and unrelated to aficamten based upon the low frequency, sporadic incidence, and/or similarity to observations in controls and/or during the prestudy period.
The consistent target organ effect across all the studies in both rats and dogs given aficamten was primarily observed in the heart. This was observed at supratherapeutic concentrations and exceeded the NOAEL defined within each toxicology study. Although other organ effects occurred, they are likely secondary to effects observed in the heart. Cardiac effects included aficamten-related increases in heart weight, cardiac hypertrophy, bilateral ventricular dilatation with or without associated atrial dilatation and cardiomyocyte degeneration/necrosis with accompanying inflammatory cell infiltrates and/or fibroplasia. In rats, ventricular and atrial dilatation, when observed, occurred at increased frequency and severity in males as compared to females. The earlier sub-chronic toxicology studies conducted showed partial to complete reversibility of cardiac effects. In the chronic rat and dog studies, the reversibility periods were extended up to 8 weeks in duration, and observed aficamten-related Main Study findings were also partially or fully reversible.
In contemporary toxicology studies, cardiac muscle damage is primarily measured in terms of changes in cardiac troponin (i.e., cTnI) levels and cTnI is an established standard biochemical marker for the diagnosis of clinical myocardial ischemia.47,48 Similar to a lack of effect of aficamten on the injury biomarkers BNP and β-MHC observed in the repeated dose pharmacology studies, there were no aficamten-related changes in cTnI concentrations observed at any dose level administered during the dosing and recovery periods in the rat and dog 13-week toxicology studies suggested that there is likely no direct structural or permanent damage to cardiac muscle resulting from aficamten administration.
In addition to the reversible anatomic findings in the hearts of aficamten-treated dogs, reversible aficamten-related effects on heart rate were noted in dog toxicology studies. While heart rates were increased, the elevations were either within the normal range for the canine or were not evident on examination of the recovery ECGs. 40 ECG effects also included a non-adverse lengthening of the QTc interval in male dogs, but this effect was not evident in the recovery ECGs. While heart rate monitoring was not included in toxicology studies conducted with aficamten in rats, the anatomic and/or functional effects of aficamten in rats and dogs are likely to be related to the primary pharmacodynamic properties resulting from cardiac myosin inhibition. The effect on heart rate is likely compensatory, with the reduction in cardiac contractility reducing stroke volume, which may lead to increased heart rate to maintain cardiac output.
The excessive on-target pharmacology of cardiac myosin inhibitors at suprapharmacologic doses for a sustained period may cause excessive and prolonged reductions in cardiac systolic function that can lead to potential heart failure. 49 Single oral doses of the cardiac myosin inhibitor, aficamten, in normal Sprague Dawley rats have shown a dose-dependent reduction in left ventricular fractional shortening.26,27 While the effect from single doses in rats were fully reversible within 24 h, changes in FS in the 10-day repeated dose pharmacology study discussed were also reversible within 24 h. Similarly, across all toxicology studies conducted, the primary adverse finding revealed in all organ systems evaluated was observed in the heart. This adverse finding is the physiological response to the effects associated with the exaggerated pharmacological actions of allosteric inhibition of cardiac myosin by aficamten in normal hearts. Aficamten-associated increases in heart organ weight changes appear related to dose, duration of dosing and sex when the repeated dose toxicology studies are reviewed, and changes were observed to be reversible in both rats and dogs.
Terminal Lung Weights Percent Difference of Control Group in Dog GLP Aficamten General Toxicology Studies.
aAll values expressed as percent difference of control group means.
bAll Groups excluded from statistical analysis (N < 3 in Control).
The totality of data from these pharmacology and toxicology studies conducted demonstrated sufficient efficacy and safety to support the investigation of aficamten in clinical studies in adults. 54 The safety and efficacy of aficamten in humans was validated by a recent Phase 3 clinical trial, which assessed the safety and efficacy of aficamten at 5, 10, 15, or 20 mg in individuals with symptomatic oHCM (SEQUOIA-HCM, https://www.clinicaltrials.gov identified, NCT05186818). SEQUOIA-HCM met the primary endpoint of significantly improved exercise capacity relative to placebo as well as all 10 secondary endpoints and found the incidence of adverse events were similar between aficamten and placebo groups. 55 In this phase 3 clinical trial, seven patients on aficamten had a transient decrease in LVEF below 50% as assessed by the site, resulting in a per-protocol dose reduction. However, no patients on aficamten underwent a treatment interruption. 55
Summary
Aficamten is a novel, oral small molecule cardiac myosin inhibitor under development for the treatment of HCM. By directly inhibiting cardiac myosin to relieve hypercontractility and left ventricular outflow tract (LVOT) obstruction, aficamten provides a targeted pharmacological approach intended to improve functional capacity and symptoms. In a repeated dose pharmacology study in rats, aficamten produced dose-dependent reductions in left ventricular fractional shortening. The effects were fully reversible within 24 h. Similarly, repeated dose toxicology studies were conducted in rats and dogs for dosing intervals up to 9 months in duration. The findings suggest that excessive on-target pharmacology of cardiac myosin inhibitors associated with suprapharmacologic doses in normal hearts for sustained periods of time lead to excessive and prolonged reductions in cardiac systolic function. The primary adverse findings observed in the GLP regulatory toxicology studies were in the heart, which consisted of cardiac hypertrophy and ventricular enlargement, adaptive changes known to results from chronic depression of cardiac function. However, the findings observed were reversible following discontinuation of drug administration, even after 9 months of dosing. These data demonstrate that the nonclinical efficacy and safety of aficamten are supportive of its clinical development in humans.
Footnotes
Acknowledgments
We would like to thank Yangsong Wu for technical assistance.
Author Contributions
M.K.P., E.R.C., J.W., and D.T.H. conceived and designed the experiments. M.K.P., J.W., D.T.H., and B.R.W. conducted and analyzed the experiments. M.K.P. and D.T.H. reviewed the statistical analyses. M.K.P., D.T.H., and B.R.W. wrote the initial draft of the manuscript. B.P.M. and F.I.M. revised the manuscript. All authors have critically reviewed the manuscript and approved its final version.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: All authors are current or former employees of Cytokinetics and received financial compensation for their work.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding support was provided by Cytokinetics.
