A Novel Ghrelin Receptor Agonist, for the Treatment of Cancer Anorexia-Cachexia Syndrome: Preclinical Profile
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J Cachexia Sarcopenia Muscle DOI 10.1007/s13539-014-0159-5 ORIGINAL ARTICLE Anamorelin HCl (ONO-7643), a novel ghrelin receptor agonist, for the treatment of cancer anorexia-cachexia syndrome: preclinical profile Claudio Pietra & Yasuhiro Takeda & Naoko Tazawa-Ogata & Masashi Minami & Xia Yuanfeng & Elizabeth Manning Duus & Robert Northrup Received: 5 June 2014 /Accepted: 8 August 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Increases in GH and IGF-1 levels were observed following Background Anamorelin HCl (ANAM) is a novel, orally ANAM administration in pigs. active, ghrelin receptor agonist in clinical development for Conclusion ANAM is a potent and highly specific ghrelin the treatment of cancer cachexia. We report in vitro and receptor agonist with significant appetite-enhancing activity, in vivo studies evaluating the preclinical pharmacologic pro- leading to increases in FI and BW, and a stimulatory effect on file of ANAM. GH secretion. These results support the continued investiga- Methods Fluorescent imaging plate reader and binding assays tion of ANAM as a potential treatment of cancer anorexia- in HEK293 and baby hamster kidney cells determined the cachexia syndrome. agonist and antagonist activity of ANAM, and its affinity for the ghrelin receptor. Rat pituitary cells were incubated with Keywords Anamorelin HCl . Ghrelin receptor agonist . ANAM to evaluate its effect on growth hormone (GH) re- Cancer anorexia-cachexia syndrome . Body weight . Growth lease. In vivo, rats were treated with ANAM 3, 10, or hormone 30 mg/kg, or control orally, once daily for 6 days to evaluate the effect on food intake (FI) and body weight (BW), and once to assess GH response. In pigs, single (3.5 mg/kg) or contin- uous (1 mg/kg/day) ANAM doses were administered to assess 1 Introduction GH and insulin-like growth factor (IGF-1) response. Results ANAM showed significant agonist and binding ac- Cancer anorexia-cachexia syndrome (CACS) was recently “ tivity on the ghrelin receptor, and stimulated GH release defined by an international expert consensus group as a in vitro. In rats, ANAM significantly and dose-dependently multifactorial syndrome characterized by an ongoing loss of increased FI and BW at all dose levels compared with control, skeletal mass (with or without loss of fat mass) that cannot be and significantly increased GH levels at 10 or 30 mg/kg doses. fully reversed by conventional nutritional support and leads to progressive functional impairment” [1]. CACS has severe consequences, such as reduction of treatment tolerance, re- duction of response to therapy, and shortened survival, and * C. Pietra ( ) can adversely affect a person’s quality of life [1–6]. The Helsinn Healthcare SA, Research and Preclinical Department, 6915 Lugano, Switzerland pathogenesis of CACS is complex and not completely under- e-mail: [email protected] stood, but altered metabolism, and reduced food intake con- : : tribute to loss of muscle mass and decrease in body weight Y. Ta ked a N. Tazawa-Ogata M. Minami [7–9]. ONO Pharmaceutical Co., Ltd, Discovery Research Laboratories, Osaka, Japan Ghrelin is an octanoylated 28-amino acid peptide gastric hormone with widespread peripheral expression and is the X. Yuanfeng endogenous ligand for the ghrelin receptor (formally known HD Biosciences, Shanghai, China as the growth hormone [GH] secretagogue type 1α receptor) E. M. Duus : R. Northrup [10]. The effects of ghrelin on signaling nutrient availability to Helsinn Therapeutics Inc., Bridgewater, NJ, USA the central nervous system, upregulating food intake, lowering J Cachexia Sarcopenia Muscle energy expenditure, and stimulating GH release are relevant in (DMSO) to make 30 mM stock. On the test day, serial dilu- the management of diseases such as CACS. In patients with tions had been prepared starting from the stock solution, and CACS, elevated levels of ghrelin are often observed [11, 12] test solutions were obtained by diluting the stock solutions 1/ perhaps as a physiological response to the decrease of body 200 in assay buffer (DMSO final concentration 0.5 %). weight and food intake. For the in vivo rat and pig studies, ANAM (Helsinn) was The administration of ghrelin in animal studies has resulted stored frozen at −20 °C under protection from light until use, in an improvement in food intake, body weight, and lean body when it was dissolved and serially diluted in distilled water to mass (LBM) retention [13, 14]. Several clinical trials in hu- concentrations of 6, 2, and 0.6 mg/mL for administration. man cancer patients have shown that ghrelin was well toler- These working solutions were stored protected from light at ated and increased food intake [15, 16] and appetite scores 5 °C and used within 6 days. [17]. However, due to its short half-life (approximately 30 min) and intravenous administration, the clinical effective- 2.2 Animals ness of ghrelin remains limited. Anamorelin HCl (ONO-7643; ANAM) is a potent and To isolate the rat pituitary cells, Sprague-Dawley male albino selective novel ghrelin receptor agonist that mimics the N- rats (250 g±25 g) were purchased from Møllegaard, Lille terminal active core of ghrelin [18](Fig.1). ANAM has the Skensved, Denmark. Rats were housed in group cages at advantage of being orally active and having a longer half-life 19–24 °C with a 12-h light cycle. (approximately 7 h) than ghrelin [19, 20]. Similar to ghrelin, For the in vivo experiments, 7-week-old male Crl:CD (SD) ANAM stimulates neuroendocrine responses and can induce rats (Charles River Japan, Inc.) were housed individually rapid positive effects on appetite and metabolism, which can under controlled temperature (24±2 °C), humidity (55± lead to an increase in body weight and LBM [21, 22]. The 15 %), outside air ventilation (15±5 cycles/h), and light/dark compound is currently undergoing evaluation as a potential cycle (12 h). Animals were acclimatized for at least 1 week agent for the treatment of CACS in Phase III clinical trials in before use in experiments. Animals were provided solid feed non-small cell lung cancer (NSCLC) [23]. (CRF-1, Oriental Bioservices Co. Ltd.) and tap water ad The aim of the present manuscript is to describe the pre- libitum. clinical profile of ANAM, both in vitro and in vivo, by Female slaughter pigs (Danish Landrace) with an initial evaluating its agonist effects on the ghrelin receptor and its weight of 40–45 kg at first dosing were acclimatized 1 week activities on food intake, body weight gain, and GH and before blood sampling (vena jugularis and arteria carotis) and insulin-like growth factor 1 (IGF-1) secretion. dosing (intragastric) catheters were inserted under general anesthesia (propofol/isoflurane). The animals were then given at least 5 days to recover before the commencement of the 2Materialsandmethods study period. Experiments were performed in compliance with the “Guidance for Animal Experiments” established by 2.1 Cell lines and reagents Research Headquarters, ONO Pharmaceutical Co., Ltd. HEK293 and baby hamster kidney (BHK) cell lines were 2.3 In vitro fluorescent imaging plate reader and binding obtained from ATCC. HDB then engineered the cell lines to assays express human ghrelin receptor, and the cell line was main- tained under standard conditions and authenticated with func- HEK293 cells stably expressing recombinant human ghrelin tional calcium influx and immunoprecipitation-1 assays every receptor (HEK293/GRLN) were used in the fluorescent im- 2weeks. aging plate reader (FLIPR) assay. One day before the test, For the in vitro assays, ghrelin (Sigma) and ANAM cells were seeded at a density of 1.5×104/well in a Matrigel® (Helsinn) were diluted with 100 % dimethyl sulfoxide coated 384-well plate with 30 μLofcompleteDulbecco’s Modified Eagle’s Medium, and incubated at 37 °C in 5 % H C CH OOCH CO for 22–26 h. On the test day, 4× loading dye was added 3 3 H 3 2 N R R N into each well (10 μL per well for 384-well plates). Assay H N NNCH 3 3 plates were incubated at 37 °C in the dark for 30 min. The dye H CH O 3 content was then removed by centrifugation at 300 rpm for .HCI 30 s, and 30 μLHanks’ Balanced Salt Solution/Hepes with HN 1 mM probenecid was added with Platemate Matrix (low- speed setting; Thermo). The plate was then placed in FLIPR Tetra (Molecular Device) and 10 μL 4× working concentra- Fig. 1 Chemical structure of anamorelin tions of test compounds were added by FLIPR (agonist J Cachexia Sarcopenia Muscle mode). Fluorescence signal was detected with FLIPR at room buffer was then exchanged and the cells were incubated for temperature according to standard settings for 10 min before 15 min with concentrations of ANAM ranging from 0.01 nM cells were exposed to 10 μL 5× working concentrations of to 10 μM. The medium was then decanted and analyzed for agonists (compound added by FLIPR). The fluorescence sig- GH content in a recombinant (r)GH ELISA test system (Novo nal was detected in the subsequent 3 min (antagonist mode). Nordisk). BHK cells stably expressing human ghrelin receptor were used for binding assay membrane preparations. Cells were 2.5 Assessment of food intake and body weight cultured under continuous antibiotics selection. Cells were collected when confluence reached 90 %. Gentle cell dissoci- Four groups of seven rats were selected from a pool of 40 rats ate buffer (PBS/EDTA) was used to dissociate cells. Upon according to body weight, food intake, and general condition dissociation, the cells were homogenized (8,000 rpm for 30 s, during a 6-day period before grouping, and received ANAM three times) in assay buffer. The homogenates were centri- at 3, 10, or 30 mg/kg, or vehicle control. In order to keep fuged at 4,000g at 4 °C for 15 min.