Discovery, development, and clinical proof of mechanism of LY3463251, a long-acting GDF15 receptor agonist.

GDF15 and its receptor GFRAL/RET form a non-homeostatic system that regulates food intake and body weight in preclinical species. Here, we describe a GDF15 analog, LY3463251, a potent agonist at the GFRAL/RET receptor with prolonged pharmacokinetics. In rodents and obese non-human primates, LY3463251 decreased food intake and body weight with no signs of malaise or emesis. In a first-in-human study in healthy participants, single subcutaneous LY3463251 injections showed a safety and pharmacokinetic profile supporting further clinical development with dose-dependent nausea and emesis in a subset of individuals. A subsequent 12-week multiple ascending dose study in overweight and obese participants showed that LY3463251 induced significant decreases in food intake and appetite scores associated with modest body weight reduction independent of nausea and emesis (clinicaltrials.gov: NCT03764774). These observations demonstrate that agonism of the GFRAL/RET system can modulate energy balance in humans, though the decrease in body weight is surprisingly modest, suggesting challenges in leveraging the GDF15 system for clinical weight-loss applications.

The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL.

Growth/differentiation factor 15 (GDF15), also known as MIC-1, is a distant member of the transforming growth factor-ß (TGF-ß) superfamily and has been implicated in various biological functions, including cancer cachexia, renal and heart failure, atherosclerosis and metabolism. A connection between GDF15 and body-weight regulation was initially suggested on the basis of an observation that increasing GDF15 levels in serum correlated with weight loss in individuals with advanced prostate cancer. In animal models, overexpression of GDF15 leads to a lean phenotype, hypophagia and other improvements in metabolic parameters, suggesting that recombinant GDF15 protein could potentially be used in the treatment of obesity and type 2 diabetes. However, the signaling and mechanism of action of GDF15 are poorly understood owing to the absence of a clearly identified cognate receptor. Here we report that GDNF-family receptor α-like (GFRAL), an orphan member of the GFR-α family, is a high-affinity receptor for GDF15. GFRAL binds to GDF15 in vitro and is required for the metabolic actions of GDF15 with respect to body weight and food intake in vivo in mice. Gfral-/- mice were refractory to the effects of recombinant human GDF15 on body-weight, food-intake and glucose parameters. Blocking the interaction between GDF15 and GFRAL with a monoclonal antibody prevented the metabolic effects of GDF15 in rats. Gfral mRNA is highly expressed in the area postrema of mouse, rat and monkey, in accordance with previous reports implicating this region of the brain in the metabolic actions of GDF15 (refs. 4,5,6). Together, our data demonstrate that GFRAL is a receptor for GDF15 that mediates the metabolic effects of GDF15.

Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody.

Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site. In vitro exposure of PCSK9 to furin resulted in degradation of PCSK9 bound to LY, whereas cleavage was blocked by other mAbs. These other mAbs caused a significant accumulation of serum PCSK9 and displayed a shorter duration of LDL-cholesterol lowering than LY when administered to mice expressing the WT human PCSK9. In mice expressing a noncleavable variant of human PCSK9, LY behaved like a cleavage-blocking mAb, in that it caused significant PCSK9 accumulation, its duration of LDL lowering was reduced, and its clearance (CL) from serum was accelerated. Thus, LY neutralizes PCSK9 and allows its proteolytic degradation to proceed, which limits PCSK9 accumulation, reduces the CL rate of LY, and extends its duration of action. PCSK9 mAbs with this property are likely to achieve longer durability and require lower doses than mAbs that cause antigen to accumulate.

Serum PCSK9 is associated with multiple metabolic factors in a large Han Chinese population.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that regulates cholesterol metabolism through low-density lipoprotein receptor (LDLR) degradation. Gain-of-function and loss-of-function mutations within PCSK9 gene lead to hypercholesterolemia or hypocholesterolemia respectively. Studies in the U.S. and Canada reported a correlation between multiple metabolic factors and circulating PCSK9 concentrations. However, there is no data available on circulating PCSK9 levels in Chinese. A sandwich ELISA assay was applied to measure serum PCSK9 levels in a Chinese population of 2719 adults from Nanjing district, China, which represents a large and uniform ethnic population of Han Chinese. Serum PCSK9 levels ranged from 12.85 to 222.50 ng/ml with a mean concentration of 69.35 ng/ml in this population. Serum PCSK9 levels were slightly higher in women than in men. Compared to premenopausal women, postmenopausal women had significantly higher PCSK9 levels. Serum PCSK9 levels were correlated with multiple metabolic variables including age, BMI, total cholesterol, LDL cholesterol, triglycerides, fasting blood glucose, systolic blood pressure (SP) and diastolic blood pressure (DP) in this population. After stepwise regression analysis, there was a significant positive association between serum PCSK9 levels and total cholesterol, triglycerides and SP in men. In women, there was a positive correlation between PCSK9 levels and total cholesterol, age and DP. Our study indicates that the serum PCSK9 level may be a biomarker of metabolic status and cardiovascular disease.

Central H3R activation by thioperamide does not affect energy balance.

The central histamine 3 receptor (H3R) is a presynaptic autoreceptor that regulates neuronal release and synthesis of histamine, and is thought to play a key role in controlling numerous central nervous system (CNS)-mediated parameters, including energy homeostasis. Thioperamide, the prototypical selective H3R antagonist, was used to examine the role that H3R plays in regulating energy balance in vivo. Thioperamide was administered either intraperitoneally or orally to rats and the pharmacokinetic parameters were examined along with central H3R binding and histaminergic system activation. Food intake and metabolic parameters of either route of thioperamide administration were likewise examined. In a dose-dependent manner, both the intraperitoneal and oral route of administration resulted in similar ex vivo binding curves and tele-methylhistamine dose-response curves despite the route of administration. However, only intraperitoneal administration of 30 mg/kg thioperamide resulted in a significant decrease in 24-h food intake (60% lower than control) and respiratory quotient (RQ), while the oral route of delivery did not. Moreover, the decrease in RQ with the 30 mg/kg ip administration also decreased energy expenditure (EE) thus resulting in an unchanged energy balance. The decrease in food intake and EE was coupled with a conditioned taste aversion with the 30-mg/kg ip administration. These data indicate that the activation of the central H3R system by thioperamide does not play a direct role in decreasing food intake or altering energy homeostasis.

Estrogen receptor-mediated repression of human hepatic lipase gene transcription.

Estrogen replacement therapy in women decreases hepatic lipase (HL) activity, which may account for the associated increase in HDL cholesterol. To investigate whether estrogen decreases HL transcription, transient cotransfection assays with HL promoter and estrogen receptor-alpha (ERalpha) expression constructs were performed in HepG2 cells. 17beta-estradiol (E(2)) decreased transcription driven by the -1557/+41 human HL promoter by up to 50% at 10(-7) M. Mutation of ERalpha by deletion of its transactivation domains or ligand-binding domain eliminated E(2)-induced repression of the promoter, whereas deletion of the DNA-binding domain of ERalpha resulted in a 7-fold activation by E(2). The E(2)-induced repression was maintained after mutation of a potential estrogen-response element in the promoter. The region of estrogen responsiveness was localized to -1557/-1175 of the HL promoter by deletion analysis. Mutation of an AP-1 site at -1493 resulted in a partial loss of E(2)-induced repression, similar to that caused by deletion of nucleotides -1557 to -1366. Gel shift assays with nuclear extracts from E(2)-treated HepG2 cells stably expressing ERalpha demonstrated an increase in binding to an AP-1 consensus oligonucleotide. The AP-1 activator, phorbol 12-myristate 13-acetate, inhibited the HL promoter by greater than 50%. Collectively, the data suggest that estrogen represses the transcription of the HL gene, possibly through an AP-1 pathway.