Vepafestinib is a pharmacologically advanced RET-selective inhibitor with high CNS penetration and inhibitory activity against RET solvent front mutations.

RET receptor tyrosine kinase is activated in various cancers (lung, thyroid, colon and pancreatic, among others) through oncogenic fusions or gain-of-function single-nucleotide variants. Small-molecule RET kinase inhibitors became standard-of-care therapy for advanced malignancies driven by RET. The therapeutic benefit of RET inhibitors is limited, however, by acquired mutations in the drug target as well as brain metastasis, presumably due to inadequate brain penetration. Here, we perform preclinical characterization of vepafestinib (TAS0953/HM06), a next-generation RET inhibitor with a unique binding mode. We demonstrate that vepafestinib has best-in-class selectivity against RET, while exerting activity against commonly reported on-target resistance mutations (variants in RETL730, RETV804 and RETG810), and shows superior pharmacokinetic properties in the brain when compared to currently approved RET drugs. We further show that these properties translate into improved tumor control in an intracranial model of RET-driven cancer. Our results underscore the clinical potential of vepafestinib in treating RET-driven cancers.

IGF-1 prevents simvastatin-induced myotoxicity in C2C12 myotubes.

Statins are generally well tolerated, but treatment with these drugs may be associated with myopathy. The mechanisms of statin-associated myopathy are not completely understood. Statins inhibit AKT phosphorylation by an unclear mechanism, whereas insulin-like growth factor (IGF-1) activates the IGF-1/AKT signaling pathway and promotes muscle growth. The aims of the study were to investigate mechanisms of impaired AKT phosphorylation by simvastatin and to assess effects of IGF-1 on simvastatin-induced myotoxicity in C2C12 myotubes. C2C12 mouse myotubes were exposed to 10 µM simvastatin and/or 10 ng/mL IGF-1 for 18 h. Simvastatin inhibited the IGF-1/AKT signaling pathway, resulting in increased breakdown of myofibrillar proteins, impaired protein synthesis and increased apoptosis. Simvastatin inhibited AKT S473 phosphorylation, indicating reduced activity of mTORC2. In addition, simvastatin impaired stimulation of AKT T308 phosphorylation by IGF-1, indicating reduced activation of the IGF-1R/PI3K pathway by IGF-1. Nevertheless, simvastatin-induced myotoxicity could be at least partially prevented by IGF-1. The protective effects of IGF-1 were mediated by activation of the IGF-1R/AKT signaling cascade. Treatment with IGF-1 also suppressed muscle atrophy markers, restored protein synthesis and inhibited apoptosis. These results were confirmed by normalization of myotube morphology and protein content of C2C12 cells exposed to simvastatin and treated with IGF-1. In conclusion, impaired activity of AKT can be explained by reduced function of mTORC2 and of the IGF-1R/PI3K pathway. IGF-1 can prevent simvastatin-associated cytotoxicity and metabolic effects on C2C12 cells. The study gives insight into mechanisms of simvastatin-associated myotoxicity and provides potential targets for therapeutic intervention.

Statins Trigger Mitochondrial Reactive Oxygen Species-Induced Apoptosis in Glycolytic Skeletal Muscle.

AIMS: Although statins are the most widely used cholesterol-lowering agents, they are associated with a variety of muscle complaints. The goal of this study was to characterize the effects of statins on the mitochondrial apoptosis pathway induced by mitochondrial oxidative stress in skeletal muscle using human muscle biopsies as well as in vivo and in vitro models. RESULTS: Statins increased mitochondrial H2O2 production, the Bax/Bcl-2 ratio, and TUNEL staining in deltoid biopsies of patients with statin-associated myopathy. Furthermore, atorvastatin treatment for 2 weeks at 10 mg/kg/day in rats increased H2O2 accumulation and mRNA levels and immunostaining of the Bax/Bcl-2 ratio, as well as TUNEL staining and caspase 3 cleavage in glycolytic (plantaris) skeletal muscle, but not in oxidative (soleus) skeletal muscle, which has a high antioxidative capacity. Atorvastatin also decreased the GSH/GSSG ratio, but only in glycolytic skeletal muscle. Cotreatment with the antioxidant, quercetin, at 25 mg/kg/day abolished these effects in plantaris. An in vitro study with L6 myoblasts directly demonstrated the link between mitochondrial oxidative stress following atorvastatin exposure and activation of the mitochondrial apoptosis signaling pathway. INNOVATION: Treatment with atorvastatin is associated with mitochondrial oxidative stress, which activates apoptosis and contributes to myopathy. Glycolytic muscles are more sensitive to atorvastatin than oxidative muscles, which may be due to the higher antioxidative capacity in oxidative muscles. CONCLUSION: There is a link between statin-induced mitochondrial oxidative stress and activation of the mitochondrial apoptosis signaling pathway in glycolytic skeletal muscle, which may be associated with statin-associated myopathy.

Simvastatin induces mitochondrial dysfunction and increased atrogin-1 expression in H9c2 cardiomyocytes and mice in vivo.

Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10 µmol/L simvastatin for 24 h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transport chain (ETC). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5 mg/kg/day simvastatin for 21 days was associated with a 5 % drop in heart weight as well as impaired activity of several enzyme complexes of the ETC and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy.

Contractile function and energy metabolism of skeletal muscle in rats with secondary carnitine deficiency.

The consequences of carnitine depletion upon metabolic and contractile characteristics of skeletal muscle remain largely unexplored. Therefore, we investigated the effect of N-trimethyl-hydrazine-3-propionate (THP) administration, a carnitine analog inhibiting carnitine biosynthesis and renal reabsorption of carnitine, on skeletal muscle function and energy metabolism. Male Sprague-Dawley rats were fed a standard rat chow in the absence (CON; n = 8) or presence of THP (n = 8) for 3 wk. Following treatment, rats were fasted for 24 h prior to excision of their soleus and EDL muscles for biochemical characterization at rest and following 5 min of contraction in vitro. THP treatment reduced the carnitine pool by ∼80% in both soleus and EDL muscles compared with CON. Carnitine depletion was associated with a 30% decrease soleus muscle weight, whereas contractile function (expressed per gram of muscle), free coenzyme A, and water content remained unaltered from CON. Muscle fiber distribution and fiber area remained unaffected, whereas markers of apoptosis were increased in soleus muscle of THP-treated rats. In EDL muscle, carnitine depletion was associated with reduced free coenzyme A availability (-25%, P < 0.05), impaired peak tension development (-44%, P < 0.05), and increased glycogen hydrolysis (52%, P < 0.05) during muscle contraction, whereas PDC activation, muscle weight, and water content remained unaltered from CON. In conclusion, myopathy associated with carnitine deficiency can have different causes. Although muscle atrophy, most likely due to increased apoptosis, is predominant in muscle composed predominantly of type I fibers (soleus), disturbance of energy metabolism appears to be the major cause in muscle composed of type II fibers (EDL).

The AKT/mTOR signaling pathway plays a key role in statin-induced myotoxicity.

Statins are drugs that lower blood cholesterol levels and reduce cardiovascular morbidity and mortality. They are generally well-tolerated, but myopathy is a potentially severe adverse reaction of these compounds. The mechanisms by which statins induce myotoxicity are not completely understood, but may be related to inhibition of the AKT signaling pathway. The current studies were performed to explore the down-stream effects of the statin-associated inhibition of AKT within the AKT signaling pathway and on myocyte biology and morphology in C2C12 myotubes and in mice in vivo. We exposed C2C12 myotubes to 10 µM or 50 µM simvastatin, atorvastatin or rosuvastatin for 24 h. Simvastatin and atorvastatin inhibited AKT phosphorylation and were cytotoxic starting at 10 µM, whereas similar effects were observed for rosuvastatin at 50 µM. Inhibition of AKT phosphorylation was associated with impaired phosphorylation of S6 kinase, ribosomal protein S6, 4E-binding protein 1 and FoxO3a, resulting in reduced protein synthesis, accelerated myofibrillar degradation and atrophy of C2C12 myotubes. Furthermore, impaired AKT phosphorylation was associated with activation of caspases and PARP, reflecting induction of apoptosis. Similar findings were detected in skeletal muscle of mice treated orally with 5 mg/kg/day simvastatin for 3 weeks. In conclusion, this study highlights the importance of the AKT/mTOR signaling pathway in statin-induced myotoxicity and reveals potential drug targets for treatment of patients with statin-associated myopathies.

Reductive stress impairs myoblasts mitochondrial function and triggers mitochondrial hormesis.

Even though oxidative stress damage from excessive production of ROS is a well known phenomenon, the impact of reductive stress remains poorly understood. This study tested the hypothesis that cellular reductive stress could lead to mitochondrial malfunction, triggering a mitochondrial hormesis (mitohormesis) phenomenon able to protect mitochondria from the deleterious effects of statins. We performed several in vitro experiments on L6 myoblasts and studied the effects of N-acetylcysteine (NAC) at different exposure times. Direct NAC exposure (1mM) led to reductive stress, impairing mitochondrial function by decreasing maximal mitochondrial respiration and increasing H2O2production. After 24h of incubation, the reactive oxygen species (ROS) production was increased. The resulting mitochondrial oxidation activated mitochondrial biogenesis pathways at the mRNA level. After one week of exposure, mitochondria were well-adapted as shown by the decrease of cellular ROS, the increase of mitochondrial content, as well as of the antioxidant capacities. Atorvastatin (ATO) exposure (100µM) for 24h increased ROS levels, reduced the percentage of live cells, and increased the total percentage of apoptotic cells. NAC exposure during 3days failed to protect cells from the deleterious effects of statins. On the other hand, NAC pretreatment during one week triggered mitochondrial hormesis and reduced the deleterious effect of statins. These results contribute to a better understanding of the redox-dependant pathways linked to mitochondria, showing that reductive stress could trigger mitochondrial hormesis phenomenon.