An oral antisense oligonucleotide for PCSK9 inhibition.

Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein (LDL) cholesterol and are used for treatment of dyslipidemia. Current PCSK9 inhibitors are administered via subcutaneous injection. We present a highly potent, chemically modified PCSK9 antisense oligonucleotide (ASO) with potential for oral delivery. Past attempts at oral delivery using earlier-generation ASO chemistries and transient permeation enhancers provided encouraging data, suggesting that improving potency of the ASO could make oral delivery a reality. The constrained ethyl chemistry and liver targeting enabled by N-acetylgalactosamine conjugation make this ASO highly potent. A single subcutaneous dose of 90 mg reduced PCSK9 by >90% in humans with elevated LDL cholesterol and a monthly subcutaneous dose of around 25 mg is predicted to reduce PCSK9 by 80% at steady state. To investigate the feasibility of oral administration, the ASO was coformulated in a tablet with sodium caprate as permeation enhancer. Repeated oral daily dosing in dogs resulted in a bioavailability of 7% in the liver (target organ), about fivefold greater than the plasma bioavailability. Target engagement after oral administration was confirmed by intrajejunal administration of a rat-specific surrogate ASO in solution with the enhancer to rats and by plasma PCSK9 and LDL cholesterol lowering in cynomolgus monkey after tablet administration. On the basis of an assumption of 5% liver bioavailability after oral administration in humans, a daily dose of 15 mg is predicted to reduce circulating PCSK9 by 80% at steady state, supporting the development of the compound for oral administration to treat dyslipidemia.

PCSK9 rs11591147 R46L loss-of-function variant protects against liver damage in individuals with NAFLD.

BACKGROUND AND AIMS: The proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis, and its inhibition represents an effective therapy to lower low-density lipoprotein cholesterol (LDL-C) levels. In this study, we examined the impact of the PCSK9 rs11591147 loss-of-function (LOF) variant on liver damage in a multicenter collection of patients at risk of nonalcoholic steatohepatitis (NASH), in clinical samples and experimental models. METHODS: We considered 1874 consecutive individuals at risk of NASH as determined by histology. The SNP rs11591147, encoding for the p.R46L variant of PCSK9, was genotyped by TaqMan assays. We also evaluated 1) PCSK9 mRNA hepatic expression in human liver, and 2) the impact of a NASH-inducing diet in mice with hepatic overexpression of human PCSK9. RESULTS: Carriers of PCSK9 rs11591147 had lower circulating LDL-C levels and were protected against nonalcoholic fatty liver disease (NAFLD) (OR: 0.42; 95% CI: 0.22-0.81; P = .01), NASH (OR: 0.48; 95% CI: 0.26-0.87; P = .01) and more severe fibrosis (OR: 0.55; 95% CI: 0.32-0.94; P = .03) independently of clinical, metabolic and genetic confounding factors. PCSK9 hepatic expression was directly correlated with liver steatosis (P = .03). Finally, liver-specific overexpression of human PCSK9 in male mice drives NAFLD and fibrosis upon a dietary challenge. CONCLUSIONS: In individuals at risk of NASH, PCSK9 was induced with hepatic fat accumulation and PCSK9 rs11591147 LOF variant was protective against liver steatosis, NASH and fibrosis, suggesting that PCSK9 inhibition may be a new therapeutic strategy to treat NASH.

Effects of a novel potent melanin-concentrating hormone receptor 1 antagonist, AZD1979, on body weight homeostasis in mice and dogs.

BACKGROUND AND PURPOSE: Melanin-concentrating hormone (MCH) is an orexigen, and while rodents express one MCH receptor (MCH1 receptor), humans, non-human primates and dogs express two MCH receptors (MCH1 and MCH2 ). MCH1 receptor antagonists have been developed for the treatment of obesity and lower body weight in rodents. However, the mechanisms for the body weight loss and whether MCH1 receptor antagonism can lower body weight in species expressing both MCH receptors are not fully understood. EXPERIMENTAL APPROACH: A novel recently identified potent MCH1 receptor antagonist, AZD1979, was studied in wild type and Mchr1 knockout (KO) mice and by using pair-feeding and indirect calorimetry in diet-induced obese (DIO) mice. The effect of AZD1979 on body weight was also studied in beagle dogs. KEY RESULTS: AZD1979 bound to MCH1 receptors in the CNS and dose-dependently reduced body weight in DIO mice leading to improved homeostasis model assessment-index of insulin sensitivity. AZD1979 did not affect food intake or body weight in Mchr1 KO mice demonstrating specificity for the MCH1 receptor mechanism. In DIO mice, initial AZD1979-mediated body weight loss was driven by decreased food intake, but an additional component of preserved energy expenditure was apparent in pair-feeding and indirect calorimetry studies. AZD1979 also dose-dependently reduced body weight in dogs. CONCLUSION AND IMPLICATIONS: AZD1979 is a novel potent MCH1 receptor antagonist that affects both food intake and energy expenditure. That AZD1979 also lowers body weight in a species expressing both MCH receptors holds promise for the use of MCH1 receptor antagonists for the treatment of human obesity.

GPR103 antagonists demonstrating anorexigenic activity in vivo: design and development of pyrrolo[2,3-c]pyridines that mimic the C-terminal Arg-Phe motif of QRFP26.

GPR103, a G-protein coupled receptor, has been reported to have orexigenic properties through activation by the endogenous neuropeptide ligands QRFP26 and QRFP43. Recognizing that central administration of QRFP26 and QRFP43 increases high fat food intake in rats, we decided to investigate if antagonists of GPR103 could play a role in managing feeding behaviors. Here we present the development of a new series of pyrrolo[2,3-c]pyridines as GPR103 small molecule antagonists with GPR103 affinity, drug metabolism and pharmacokinetics and safety parameters suitable for drug development. In a preclinical obesity model measuring food intake, the anorexigenic effect of a pyrrolo[2,3-c]pyridine GPR103 antagonist was demonstrated. In addition, the dynamic 3D solution structure of the C-terminal heptapeptide of the endogenous agonist QRFP26(20-26) was determined using NMR. The synthetic pyrrolo[2,3-c]pyridine antagonists were compared to this experimental structure, which displayed a possible overlay of pharmacophore features supportive for further design of GPR103 antagonists.

A modeling approach for compounds affecting body composition.

Body composition and body mass are pivotal clinical endpoints in studies of welfare diseases. We present a combined effort of established and new mathematical models based on rigorous monitoring of energy intake (EI) and body mass in mice. Specifically, we parameterize a mechanistic turnover model based on the law of energy conservation coupled to a drug mechanism model. Key model variables are fat-free mass (FFM) and fat mass (FM), governed by EI and energy expenditure (EE). An empirical Forbes curve relating FFM to FM was derived experimentally for female C57BL/6 mice. The Forbes curve differs from a previously reported curve for male C57BL/6 mice, and we thoroughly analyse how the choice of Forbes curve impacts model predictions. The drug mechanism function acts on EI or EE, or both. Drug mechanism parameters (two to three parameters) and system parameters (up to six free parameters) could be estimated with good precision (coefficients of variation typically <20 % and not greater than 40 % in our analyses). Model simulations were done to predict the EE and FM change at different drug provocations in mice. In addition, we simulated body mass and FM changes at different drug provocations using a similar model for man. Surprisingly, model simulations indicate that an increase in EI (e.g. 10 %) was more efficient than an equal lowering of EI. Also, the relative change in body mass and FM is greater in man than in mouse at the same relative change in either EI or EE. We acknowledge that this assumes the same drug mechanism impact across the two species. A set of recommendations regarding the Forbes curve, vehicle control groups, dual action on EI and loss, and translational aspects are discussed. This quantitative approach significantly improves data interpretation, disease system understanding, safety assessment and translation across species.

Oral baclofen reduces visceral pain-related pseudo-affective responses to colorectal distension in rats: relation between plasma exposure and efficacy.

OBJECTIVE: We previously showed that activation of GABA(B) receptors by intravenous baclofen reduces pseudo-affective responses to colorectal distension in rats. Here we evaluate the potential clinical significance of these observations. MATERIAL AND METHODS: Clinically relevant colorectal distension protocols were used to assess the effects of oral baclofen on visceromotor and autonomic cardiovascular responses in conscious rats. Plasma levels of baclofen were monitored to provide clinical relevance to the doses used. Conscious female Sprague-Dawley rats were subjected to repeated noxious colorectal distension (12 × 80 mmHg), ascending-phasic colorectal distension (10-80 mmHg, 10 mmHg increments) or ramp colorectal distension (10 min ramp at 8 mmHg/min). Visceromotor and cardiovascular responses (mean arterial blood pressure and heart rate) were monitored. Pain-related response thresholds were assessed using ascending-phasic and ramp colorectal distension. RESULTS: Baclofen (1-10 µmol/kg, p.o.) reduced the visceromotor response to colorectal distension, reaching a 40% maximal inhibition (p < 0.05). The highest dose (10 µmol/kg, p.o.) also inhibited pain-related cardiovascular responses in telemetrized rats (50-55% reduction in colorectal distension-evoked hypertensive and tachycardic responses; p < 0.05). Similar thresholds for pain-related visceromotor responses were determined during ramp or ascending-phasic colorectal distension (34.1 ± 1.9 and 31.7 ± 3.2 mmHg, respectively). Baclofen (10 µmol/kg, p.o.) increased thresholds to 71.1 ± 3.7 and 77.5 ± 1.8 mmHg during ramp and ascending-phasic colorectal distension, respectively (p < 0.001). Plasma levels of baclofen were 3.3 ± 0.2 µmol/l at 90 min post-dosing, corresponding to the end of the colorectal distension procedure. CONCLUSIONS: Oral baclofen, at plasma levels similar to those reported safe and within a therapeutic range in humans, produced significant visceral anti-nociceptive effects in rats.

Role of tachykinin NK(1) and NK(2) receptors in colonic sensitivity and stress-induced defecation in gerbils.

The pharmacology of tachykinin NK receptors varies greatly among species. The aim of the present study was to assess the role of NK(1) and NK(2) receptors in mediating colorectal distension-evoked nociception and psychological stress-induced defecation in gerbils, a species with human-like NK receptor pharmacology. The effects of the selective NK(1) and NK(2) receptor antagonists, aprepitant and saredutant, on acute (1 h) restraint stress-evoked defecation and plasma adenocorticotropin (ACTH) levels in gerbils were assessed. The effects of antagonists alone or in combination on colorectal distension-evoked visceral pain in conscious gerbils were evaluated using the visceromotor response as a surrogate marker of pain. Restraint stress increased fecal pellet output 2-3-fold and plasma ACTH levels 9-fold. Aprepitant inhibited the defecatory and endocrine responses to stress by 50%, while saredutant completely normalized the same parameters. Visceral pain responses during colorectal distension were attenuated by both compounds, but aprepitant (19+/-6% inhibition, P<0.01) was slightly more effective than saredutant (10+/-9% inhibition, P<0.05). A combination of both compounds resulted in an additive effect (30+/-10% inhibition, P<0.01). The results demonstrate that NK(1) and NK(2) receptors are involved in stress-related colonic motor alterations and visceral pain responses in gerbils and that combined antagonism provides enhanced inhibition of visceral pain responses. This suggests that for therapeutic use in for instance functional gastrointestinal disorders, dual NK(1)/NK(2) receptor antagonists may provide better clinical outcome than selective compounds.