Safety, pharmacokinetics and target engagement of novel RIPK1 inhibitor SAR443060 (DNL747) for neurodegenerative disorders:Randomized, placebo-controlled, double-blind phase I/Ib studies in healthy subjects and patients

RIPK1 is a master regulator of inflammatory signaling and cell death and increased RIPK1 activity is observed in human diseases, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). RIPK1 inhibition has been shown to protect against cell death in a range of preclinical cellular and animal models of diseases. SAR443060 (previously DNL747) is a selective, orally bioavailable, central nervous system (CNS)-penetrant, small-molecule, reversible inhibitor of RIPK1. In three early-stage clinical trials in healthy subjects and patients with AD or ALS (NCT03757325 and NCT03757351), SAR443060 distributed into the cerebrospinal fluid (CSF) after oral administration and demonstrated robust peripheral target engagement as measured by a reduction in phosphorylation of RIPK1 at serine 166 (pRIPK1) in human peripheral blood mononuclear cells compared to baseline. RIPK1 inhibition was generally safe and well-tolerated in healthy volunteers and patients with AD or ALS. Taken together, the distribution into the CSF after oral administration, the peripheral proof-of-mechanism, and the safety profile of RIPK1 inhibition to date, suggest that therapeutic modulation of RIPK1 in the CNS is possible, conferring potential therapeutic promise for AD and ALS, as well as other neurodegenerative conditions. However, SAR443060 development was discontinued due to long-term nonclinical toxicology findings, although these nonclinical toxicology signals were not observed in the short duration dosing in any of the three early-stage clinical trials. The dose-limiting toxicities observed for SAR443060 preclinically have not been reported for other RIPK1-inhibitors, suggesting that these toxicities are compound-specific (related to SAR443060) rather than RIPK1 pathway-specific

Dealing with doping. A plea for better science, governance and education

First paragraph: A participant from the ancient Greek Olympics stepping into Tokyo's Olympic Stadium at the opening of the Games on 23 July 2021 would find the pageantry and competition familiar. Since doping-like behavior was prevalent at the time, the athlete would be confused by the efforts to detect the use of substances in the body fluids of his fellow athletes. The anti-doping lab at the 2016 Summer Games in Rio de Janeiro operated on a 24-hour basis, requiring 5'500m2 of space for its 30 mass spectrometry instruments and 700 staff. Tokyo's organizers have announced an even more elaborate operation. Current anti-doping efforts are extensive, expensive, and complex. They not only concern the Olympic Games but competitive sport in general, also targeting non-Olympic athletes and, increasingly, amateurs

Erythropoietin doping in cycling: Lack of evidence for efficacy and a negative risk-benefit 1 Erythropoietin doping in cycling: Lack of evidence for efficacy and a negative risk-benefit

Summary Aim & Methods Imagine a medicine that is expected to have very limited effects based upon knowledge of pharmacology and (patho)physiology, is studied in the wrong population, with low quality studies that use a surrogate endpoint that relates to the clinical endpoint in a partial manner at most. Such a medicine would surely not be recommended. Recombinant human erythropoietin (rHuEPO) use to enhance performance in cycling is very common. A qualitative systematic review of the available literature was performed to look at the evidence for these ergogenic properties of this drug normally used to treat anaemia in chronic renal failure patients. Results The results of this literature search show there is no scientific basis to conclude rHuEPO has performance enhancing properties in elite cyclists. The reported studies have many shortcomings regarding translation of the results to professional cycling endurance performance. Additionally, the possibly harmful side-effects have not been adequately researched for this population but appear to be worrying at least. Conclusions Sport is big business The summer of 2012 was an intensive summer of sports. From all these events, it is clear that sports play a very important role in our society as it brings people together, gives pleasure, keeps people healthy and can bring professional athletes fame and honour. Sport has grown to be so important that large amounts of money are now involved and the will and pressure to win have steadily increased. Cheating has therefore become a threat to all sports, with some sports being more susceptible to it than others. Cheating by use of medicines has understandably taken place outside the realm of clinical pharmacology and evidence based medicine. We question if this is desirable, as uncontrolled use of a substance induces risks for the users, irrespective of such a substance being used legally or illegally. In this review we will focus on the use of recombinant human erythropoietin (rHuEPO) in cycling, a sport that has had many reports of cheating, culminating in the last decennia, with many suspicions and suspensions. We will address the question if the current available evidence even justifies the widespread use of this substance. Many of the big champions in cycling have been associated with, or suspended for use of (blood) doping. In the Tour de France of 1998 the entire Festina team, as well as the TVM team, were taken out of the race on suspicion of rHuEPO use. This Tour was later given the name 'Tour du Dopage' and many confessions of systematic doping (i.e. rHuEPO) use throughout the peloton were given. In spite of this, later champions in the Tour de France, Giro d'Italia and Vuelta a España have also been suspended because of proof of blood doping, but the Code of Silence also called 'omerta', was never broken. Seven years after the last of seven consecutive Tour de France wins, one of the most successful road cyclists ever, Lance Armstrong, has been suspended for life by the United States Anti-Doping Agency (USADA) on charges of doping (e.g. rHuEPO) use and trafficking in the biggest doping case ever, backed by confessions of many of his teammates. Physiology of erythropoietin Erythropoietin (EPO) is a (glyco)protein that is mainly involved in erythropoiesis, the (re-)generation of erythrocytes, or red blood cells. Red blood cells are cells without a nucleus and transport oxygen through the blood. Due to a lack of ability to repair themselves without a nucleus and other cellular machinery, erythrocytes have a life span of approximately 120 days in the circulation and after that need to be replaced. [2] The spleen removes the old erythrocytes (2-3 million every second) and to keep oxygen carrying capacity of the blood at a steady level, constant erythropoiesis is necessary. [3] These cells go through different stages, one of which is the burst-forming unit-erythroid (BFU-E). This cell type matures into a colony-forming unit-erythroid (CFU-E), which in turn forms the proerythroblast, which divides four times into 16 reticulocytes, later developing into mature red blood cells. Production and metabolism The kidneys are the main EPO producing organs in humans [13;14], where peritubular interstitial cells govern its production [15;16], which is highly regulated. Baseline EPO levels can increase up to 1,000-fold in low blood oxygen content, for example in severe anaemia. [3] EPO production is highly dependent on blood oxygen tension, with hypoxia increasing EPO production, irrespective of the cause of reduced tissue oxygen supply. [3] However, only moderately elevated serum concentrations of EPO seem to be sufficient to maintain an increased erythropoiesis rate. [17] The proposed oxygen-sensing mechanism regulating EPO production involves the hypoxia-inducible factor (HIF), a transcription factor. [29] The major elimination route for EPO seems to be EpoR mediated uptake and degradation Recombinant erythropoietin in disease As EPO plays an important role in regulating erythropoiesis, a major step in medicine was taken when recombinant EPO was first produced by Lin et al …and in sport. But does it work? The treatment immediately also got the attention of athletes. As rHuEPO increases red blood cell mass and exercise capacity in anaemic patients, it might have the same effect in the athlete's body, thereby enhancing performance. With this rationale athletes started using rHuEPO, and the use of rHuEpo was put on the International Olympic Committee's (IOC) list of prohibited substances already in 1990. Now the list has been expanded to all "Erythropoiesis-Stimulating Agents (ESA's) [e.g. erythropoietin (EPO), darbepoetin (dEPO), hypoxia-inducible factor (HIF) stabilizers, methoxy polyethylene glycol-epoetin beta (CERA), peginesatide (Hematide)]" What is endurance performance? Main determining factors The main determinants of aerobic endurance performance are maximal oxygen uptake (VO2max), the lactate threshold (LT) and work economy (C). [44] These three factors are now generally accepted as key factors in endurance performance VO2max is a prerequisite but not a sole determining factor VO 2 max, the maximal oxygen uptake, has traditionally been regarded as the most important measure in endurance performance. According to Fick's Law it is dependent on cardiac output and the arteriovenous oxygen difference. These in turn, are mainly dependent on total blood volume (BV), the main limiting factor of stroke volume, and total body haemoglobin (tHb). However, lung diffusing capacity, heart rate, distribution of the blood volume to working skeletal muscles and arterial O 2 extraction contribute to VO 2 max as well, as reviewed by Joyner et al It is more than the VO2max It is not VO 2 max, but power output at submaximal intensities such as the first (VT1) and second (VT2) ventilation threshold, or respiratory compensation point (RCP) that significantly differ between elite amateur and professional cyclists.[64;74] Thus factors other than VO 2 max play an important role in determining performance in professional and world-class cyclists. For example, when a published model Lactate Threshold (LT) We therefore now address the importance of LT in determining the performance of endurance athletes. LT, similar to VT1 or VT, is the intensity of work or VO 2 at which the blood lactate concentration gradually starts to increase [81] Because LT reflects an onset of anaerobic metabolism and the coinciding metabolic alterations [45;53], LT in turn determines the fraction of maximal aerobic power that can be sustained for an extended period. Several studies show that the VO 2 at this LT is highly related to performance, more so than VO 2 max. [45;47;63-65;67;68;82] Elite cyclists are reported to be able to reach LTs between 300 and 400W[63;77;83], or 70-85% VO 2 max (3.5-4.7 l/min) [65;67]. LT reflects a balance between the rate of lactate production in the muscles (and hence the rate of lactate influx to the blood) and clearance from the blood. In this balance another independent factor appears to play a role in endurance performance; difference in performance (time to fatigue) in cyclists with similar VO 2 max can be explained by %VO 2 max at LT, but an additional increase in performance in some athletes seems to be related to a high muscle capillary density. [45;65] A similar correlation between endurance performance and capillary density was found in another study by Coyle et al Lactate Turn Point (LTP) LTP is a distinct factor related also to lactate Economy (C) The third main factor contributing to endurance performance is assumed to be completely independent of VO 2 max and lactate-related factors and is called work economy or efficiency (C). It is the ratio between work output (speed, power) and oxygen cost. Running economy is commonly defined as the steady-rate VO 2 in millilitres per kilogram per minute at a standard velocity, cycling economy as the caloric expenditure at a given work rate. Several physiological and biomechanical factors influence C in trained or elite athletes. These include metabolic adaptations within the muscle such as increased mitochondria and oxidative enzymes, the ability of the muscles to store and release elastic energy by altering the mechanical properties of the muscles, and more efficient mechanics leading to less energy wasted on braking forces and excessive vertical oscillation [44]. C is a discriminator of endurance performance independently of VO 2 max in runners [48;68;91-93] Other factors Besides these main determinants several other factors were also reported to influence endurance performance. Heart rate for example, shows a rightward shift in its relationship with running speed [100] Also the quantity of muscle mass recruited for sustained power production can influence performance, as elite cyclists can use 20-25% more muscle mass in endurance tests, therefore reducing the stress and power production per fibre. [65;101] Additionally, peak power output has been shown to be a predictor of performance in a time tria

Muscle velocity recovery cycles as pharmacodynamic biomarker: Effects of mexiletine in a randomized double‐blind placebo‐controlled cross‐over study

Abstract Measuring muscle velocity recovery cycles (MVRCs) is a method to obtain information on muscle cell excitability, independent of neuromuscular transmission. The goal was to validate MVRC as a pharmacodynamic (PD) biomarker for drugs targeting muscle excitability. As proof‐of‐concept, sensitivity of MVRC to detect effects of mexiletine, a voltage‐gated sodium channel (Nav) blocker, was assessed. In a randomized, double‐blind, two‐way crossover study, effects of a single pharmacologically active oral dose of 333 mg mexiletine was compared to placebo in 15 healthy male subjects. MVRC was performed predose, and 3‐ and 5‐h postdose using QTrac. Effects of mexiletine versus placebo were calculated using a mixed effects model with baseline as covariate. Mexiletine had significant effects on MVRC when compared to placebo. Early supernormality after five conditioning stimuli was decreased by mexiletine (estimated difference −2.78% [95% confidence interval: −4.16, −1.40]; p value = 0.0003). Moreover, mexiletine decreased the difference in late supernormality after five versus one conditioning stimuli (5XLSN; ED −1.46% [−2.26, −0.65]; p = 0.001). These results indicate that mexiletine decreases the percentage increase in velocity of the muscle fiber action potential after five conditioning stimuli, at long and short interstimulus intervals, which corresponds to a decrease in muscle membrane excitability. This is in line with the pharmacological activity of mexiletine, which leads to use‐dependent NaV1.4 blockade affecting muscle membrane potentials. This study shows that effects of mexiletine can be detected using MVRC in healthy subjects, thereby indicating that MVRC can be used as a tool to demonstrate PD effects of drugs targeting muscle excitability in early phase drug development

A leucine‐rich repeat kinase 2 (LRRK2) pathway biomarker characterization study in patients with Parkinson's disease with and without LRRK2 mutations and healthy controls

Abstract Increased leucine‐rich repeat kinase 2 (LRRK2) kinase activity is an established risk factor for Parkinson's disease (PD), and several LRRK2 kinase inhibitors are in clinical development as potential novel disease‐modifying therapeutics. This biomarker characterization study explored within‐ and between‐subject variability of multiple LRRK2 pathway biomarkers (total LRRK2 [tLRRK2], phosphorylation of the serine 935 (Ser935) residue on LRRK2 [pS935], phosphorylation of Rab10 [pRab10], and total Rab10 [tRab10]) in different biological sources (whole blood, peripheral blood mononuclear cells [PBMCs], neutrophils) as candidate human target engagement and pharmacodynamic biomarkers for implementation in phase I/II pharmacological studies of LRRK2 inhibitors. PD patients with a LRRK2 mutation (n = 6), idiopathic PD patients (n = 6), and healthy matched control subjects (n = 10) were recruited for repeated blood and cerebrospinal fluid (CSF) sampling split over 2 days. Within‐subject variability (geometric coefficient of variation [CV], %) of these biomarkers was lowest in whole blood and neutrophils (range: 12.64%–51.32%) and considerably higher in PBMCs (range: 34.81%–273.88%). Between‐subject variability displayed a similar pattern, with relatively lower variability in neutrophils (range: 61.30%–66.26%) and whole blood (range: 44.94%–123.11%), and considerably higher variability in PBMCs (range: 189.60%–415.19%). Group‐level differences were observed with elevated mean pRab10 levels in neutrophils and a reduced mean pS935/tLRRK2 ratio in PBMCs in PD LRRK2‐mutation carriers compared to healthy controls. These findings suggest that the evaluated biomarkers and assays could be used to verify pharmacological mechanisms of action and help explore the dose–response of LRRK2 inhibitors in early‐phase clinical studies. In addition, comparable α‐synuclein aggregation in CSF was observed in LRRK2‐mutation carriers compared to idiopathic PD patients

EpoR stimulates rapid cycling and larger red cells during mouse and human erythropoiesis

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor(−/−) mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress