Michael Scherz

Promotion of sleep by targeting the orexin system in rats, dogs and humans

Orexins are hypothalamic peptides that play an important role in maintaining wakefulness in mammals. Permanent deficit in orexinergic function is a pathophysiological hallmark of rodent, canine and human narcolepsy. Here we report that in rats, dogs and humans, somnolence is induced by pharmacological blockade of both orexin OX1 and OX2 receptors. When administered orally during the active period of the circadian cycle, a dual antagonist increased, in rats, electrophysiological indices of both non-REM and, particularly, REM sleep, in contrast to GABAA receptor modulators; in dogs, it caused somnolence and increased surrogate markers of REM sleep; and in humans, it caused subjective and objective electrophysiological signs of sleep. No signs of cataplexy were observed, in contrast to the rodent, dog or human narcolepsy syndromes. These results open new perspectives for investigating the role of endogenous orexins in sleep-wake regulation.

2-Imino-thiazolidin-4-one Derivatives as Potent, Orally Active S1P1 Receptor Agonists

Sphingosine-1-phosphate (S1P) is a widespread lysophospholipid which displays a wealth of biological effects. Extracellular S1P conveys its activity through five specific G-protein coupled receptors numbered S1P(1) through S1P(5). Agonists of the S1P(1) receptor block the egress of T-lymphocytes from thymus and lymphoid organs and hold promise for the oral treatment of autoimmune disorders. Here, we report on the discovery and detailed structure-activity relationships of a novel class of S1P(1) receptor agonists based on the 2-imino-thiazolidin-4-one scaffold. Compound 8bo (ACT-128800) emerged from this series and is a potent, selective, and orally active S1P(1) receptor agonist selected for clinical development. In the rat, maximal reduction of circulating lymphocytes was reached at a dose of 3 mg/kg. The duration of lymphocyte sequestration was dose dependent. At a dose of 100 mg/kg, the effect on lymphocyte counts was fully reversible within less than 36 h. Pharmacokinetic investigation of 8bo in beagle dogs suggests that the compound is suitable for once daily dosing in humans.

UROTENSIN II MEDIATES ERK1=2 PHOSPHORYLATION AND PROLIFERATION IN GPR14-TRANSFECTED CELL LINES

ABSTRACT Urotensin-II (U-II), a vasoactive cyclic neuropeptide, was recently identified as the natural ligand for the G-protein coupled receptor GPR14. The expression pattern of U-II and GPR14 are consistent with a role as a neurohormonal regulatory system in cardiovascular homeostasis. Urotensin-II induces a rapid and short-lasting rise in intracellular calcium in recombinant GPR14 expressing cells. In the present study we show that U-II induces signal transduction pathways leading to the long-lasting activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in chinese hamster ovary cells expressing human GPR14 (CHO-GPR14). Furthermore, we observed a growth-stimulating and PD98059 sensitive activity of U-II in CHO-GPR14 cells, but not CHO-K1 cells. The investigation of the GPR14 induced signal transduction pathways leading to ERK1/2 phosphorylation revealed a previously unsuspected role for Gi/o-protein coupling and showed an involvement of phospatidylinositol-3-kinase, phospholipase C and calcium channel mediated mechanisms. Our results suggest that U-II and its receptor GPR14 may be involved in long-lasting physiological effects such as cardiovascular remodeling.

Novel S1P1 Receptor Agonists – Part 1: From Pyrazoles to Thiophenes

From a high-throughput screening campaign aiming at the identification of novel S1P1 receptor agonists, the pyrazole derivative 2 emerged as a hit structure. Medicinal chemistry efforts focused not only on improving the potency of the compound but in particular also on resolving its inherent instability issue. This led to the discovery of novel bicyclo[3.1.0]hexane fused thiophene derivatives. Compounds with high affinity and selectivity for S1P1 efficiently reducing the blood lymphocyte count in the rat were identified. For instance, compound 85 showed EC50 values of 7 and 2880 nM on S1P1 and S1P3, respectively, had favorable pharmacokinetic properties in rat and dog, distributed well into brain tissue, and efficiently and dose dependently reduced the blood lymphocyte count in the rat. After oral administration to spontaneously hypertensive rats, the S1P1 selective compound 85 showed no effect on mean arterial blood pressure and affected the heart rate during the wake phase of the animals only.

Novel S1P1 Receptor Agonists - Part 2: From Bicyclo[3.1.0]hexane-Fused Thiophenes to Isobutyl Substituted Thiophenes

Previously, we reported on the discovery of a novel series of bicyclo[3.1.0]hexane fused thiophene derivatives that serve as potent and selective S1P1 receptor agonists. Here, we discuss our efforts to simplify the bicyclohexane fused thiophene head. In a first step the bicyclohexane moiety could be replaced by a simpler, less rigid cyclohexane ring without compromising the S1P receptor affinity profile of these novel compounds. In a second step, the thiophene head was simplified even further by replacing the cyclohexane ring with an isobutyl group attached either to position 4 or position 5 of the thiophene. These structurally much simpler headgroups again furnished potent and selective S1P1 agonists (e.g., 87), which efficiently and dose dependently reduced the number of circulating lymphocytes upon oral administration to male Wistar rats. For several compounds discussed in this report lymphatic transport is an important route of absorption that may offer opportunities for a tissue targeted approach with minimal plasma exposure.

Three different up‐titration regimens of ponesimod, an S1P1 receptor modulator, in healthy subjects

Ponesimod is a selective S1P1 receptor modulator, and induces dose‐dependent reduction of circulating lymphocytes upon oral dosing. Previous studies showed that single doses up to 75 mg or multiple doses up to 40 mg once daily are well tolerated, and heart rate (HR) reduction and atrio‐ventricular conduction delays upon treatment initiation are reduced by gradual up‐titration to the maintenance dose. This single‐center, open‐label, randomized, multiple‐dose, 3‐treatment, 3‐way crossover study compared the tolerability, safety, pharmacokinetics, cardiodynamics, and effects on lymphocytes of 3 different up‐titration regimens of ponesimod in healthy male and female subjects. Up‐titration regimens comprised escalating periods of b.i.d. dosing (2.5 or 5 mg) and q.d. dosing (10 or 20 mg or both). After the third up‐titration period a variable‐duration washout period of 1–3 days was followed by re‐challenge with a single 20‐mg dose of ponesimod. Adverse events were transient and mild to moderate in intensity, not different between regimens. HR decrease after the first dose was greater than after all subsequent doses, including up‐titration doses. Little or no HR change was observed with morning doses of b.i.d. regimens, suggesting that 2.5 and 5 mg b.i.d. are sufficient to sustain cardiac desensitization for the 12‐hours dosing interval.

Reply to 'Promotion of sleep by targeting the orexin system in rats, dogs and humans'

Brisbare-Roch et al. reply: We agree with Tafti that animal and human orexin deficiency syndromes suggest a theoretical risk of cataplexy following administration of an orexin receptor antagonist. Our study involved healthy rodent, canine and human subjects, in whom a dual OX1/OX2 receptor antagonist that crosses the blood-brain barrier elicited somnolence and sleep, but not cataplexy, when administered as a single oral dose during the active period of the circadian cycle1. The absence of cataplexy in all three species was in contrast to the narcolepsy-cataplexy syndromes commonly observed in rodents, canines and humans. Thus it appears that the impact of acute and transient pharmacological antagonism of OX1 and OX2 receptors differs from that of a chronic and life-long deactivation induced by genetic or acquired orexin peptide or orexin receptor deficits. Our observations confirm those from other studies, and suggest that somnolence and cataplexy might be separable phenomena and that orexin deficit per se is not sufficient to cause cataplexy. Some narcoleptic patients do not develop cataplexy despite low CSF orexins2,3, and loss of hypothalamic factors besides orexins, such as neuronal activity–regulated pentraxin (Narp), dynorphin and glutamate, possibly contributes to the narcolepsy syndrome4–6. Many additional variables may have contributed to the occurrence of somnolence without cataplexy that we observed: the dosage, the degree of receptor antagonism, the relative inhibition at OX1 and OX2 receptors, and the duration of orexin receptor blockade. Concerning our experimental approaches, we are of the opinion that the methods described in our article, in particular for assessing the presence of cataplexy, fully allow the conclusions drawn. We also do not consider it critically important to demonstrate that our drug blocks the effects of exogenously administered orexins. Such experiments serve only to recapitulate agonist-antagonist interactions already demonstrated in vitro. Our goal, rather, was to assess the impact of our compound on endogenous orexins and the maintenance of wakefulness. Tafti states that ACT-078573 treatment of rats primarily decreased their locomotor activity during wakefulness, rather than inducing sleep. Our EEG data showed that waking time decreased in proportion to increased time in NREM sleep and shortened latency to NREM sleep; together with increased REM sleep time, this resulted in a net decrease in home cage activity. Our assessment of a physiological sleep pattern is based on the increase in both NREM and REM sleep observed with ACT078573 (Table 1). The behavioral assessment of sleep posture in dogs is a prima facie assessment of sleep and the distal muscle movements a surrogate of REM activity. Since the publication of our manuscript, we have reported early proof of concept for enhanced sleep efficiency following ACT-078573 administration at bedtime in patients with primary insomnia (www. actelion.com/uninet/www/www_main_p.nsf/ Content/me+04+Feb+2007). Tafti wonders why only two 25-min EEG recording periods were performed in the entry-into-man study. The duration and timing of EEG recording were chosen to accommodate the purposes of this phase I study, which were to assess the drug’s safety, tolerability and pharmacokinetics in healthy subjects. This is also a variant of a multiple sleep latency test (MSLT) that is used as a diagnostic tool to assess sleep-onset REM periods (SOREMP) in narcoleptic patients3. Full EEG evaluations are being and will be conducted in dedicated clinical trials, and will be published in due course. Finally, Tafti worries about short latency to sleep stage 2. First, short latency is not uncommon in healthy subjects7, as also evidenced in the placebo controls; second, the latency was shortened dose dependently, and we have no reason to believe that a shortened latency to stage 2 is undesirable. As this discussion indicates, there are still many open questions related to the effects of an orexin receptor antagonist, and in that sense our study can be considered as initial but essential groundwork. Our selective, dual OX1/OX2 receptor antagonist is a useful agent for deline ating the physiological and pathophysiological roles of endogenous orexins. We view the results of our proof-of-concept study with ACT-078573 as very promising and reinforcing the results described in our paper. The increase in both NREM and REM sleep with ACT-078573 may mimic a physiological sleep pattern better than the increase in only NREM sleep induced by previous hypnotic drugs. However, many more studies are needed before we can conclude whether dual orexin receptor antagonists will represent a safer and better class of sleep-promoting agents than currently available hypnotic drugs.