ghrelin receptor ( Ki = 0.7 nM ); ghrelin receptor ( EC50 = 0.74 nM )
Ghrelin receptor (Growth hormone secretagogue type 1α receptor) (Agonist, EC₅₀ = 0.74 nM in FLIPR assay; Binding affinity K_i = 0.70 nM) [1]

Anamorelin (ANAM) exhibits strong agonist activity on the ghrelin receptor in the FLIPR assay, with an EC50 value of 0.74 nM. Anamorelin exhibits negligible antagonistic effects at 1,000 nM and higher concentrations. Anamorelin exhibits a binding affinity constant (Ki) of 0.70 nM for the ghrelin receptor in the binding experiments. Anamorelin (ANAM) is also shown to bind to the ghrelin receptor with a high affinity in the competition assay using radiolabeled ibutamoren (IC50=0.69 nM). Anamorelin has a dose-dependent stimulatory effect on GH release in rat pituitary cells, and its potency (EC50) is 1.5 nM. Anamorelin is tested for its ability to bind to more than 100 different receptors, ion channels, transporters, and enzymes. An NK₂ functional assay shows no functional activity, despite anamorelin's demonstrated binding to the tachykinin neurokinin 2 (NK₂) site (IC50=0.021 μM).

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In a fluorescent imaging plate reader (FLIPR) assay using HEK293 cells stably expressing human ghrelin receptor, Anamorelin showed significant agonist activity with an EC₅₀ of 0.74 nM (95% CI 0.50–1.12), comparable to natural ghrelin (EC₅₀ = 0.67 nM). No significant antagonist activity was observed up to 1000 nM. [1]
In a binding assay using membrane preparations from BHK cells expressing human ghrelin receptor and radiolabeled ghrelin, Anamorelin bound to the receptor with high affinity, exhibiting a K_i of 0.70 nM (95% CI 0.55–0.96), similar to ghrelin (K_i = 0.58 nM). [1]
In a competition binding assay using radiolabeled ibutamoren (^35^S-MK-677), Anamorelin demonstrated high affinity binding to the ghrelin receptor with an IC₅₀ of 0.69 nM. [1]
In primary rat pituitary cells, Anamorelin (0.01 nM – 10 µM) stimulated growth hormone (GH) release in a dose-dependent manner with an EC₅₀ of 1.5 nM. [1]
In a screening panel against over 100 receptors, ion channels, transporters, and enzymes, Anamorelin (10 µM) showed weak binding to L-type calcium channels (benzothiazepine and phenylalkylamine sites), the serotonin transporter, and sodium channels. It demonstrated binding to the tachykinin neurokinin 2 (NK₂) receptor (IC₅₀ = 0.021 µM) but showed no functional activity in a subsequent NK₂ functional assay. The compound exhibited high selectivity for the ghrelin receptor at nanomolar concentrations. [1]

Anamorelin (ANAM) increases food intake and body weight in rats from Day 2 to Day 7 of treatment significantly when given orally at a dose of 3, 10, or 30 mg/kg once daily, in comparison to the vehicle control. Each dose level causes a dose-dependent cumulative change in food intake and weight gain, which is significant (P<0.05) when compared to the control at all dose levels. A dose-dependent rise in plasma GH levels and GH A_UC0-6h is observed in rats administered a single oral dose of 3, 10, or 30 mg/kg of anamorelin[1].

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In rats, oral administration of Anamorelin once daily for 6 days at doses of 3, 10, and 30 mg/kg significantly and dose-dependently increased both food intake and body weight compared to the vehicle control group. The effects on food intake were apparent from Day 2 of treatment in the 10 and 30 mg/kg groups. [1]
In rats, a single oral dose of Anamorelin (3, 10, 30 mg/kg) induced a dose-dependent increase in plasma growth hormone (GH) levels and the area under the curve from 0 to 6 hours (AUC_0-6h). Peak GH concentrations (C_max) were reached between 0.5 and 2 hours post-dose. The maximum increase in GH concentration ranged from 2.3-fold (3 mg/kg) to 4.1-fold (30 mg/kg) above control levels. The GH AUC_0-6h was significantly higher than control at the 10 and 30 mg/kg doses. [1]
In pigs, a single oral dose of Anamorelin (3.5 mg/kg) caused a significant and acute rise in plasma GH levels. [1]
In pigs receiving repeated once-daily oral administration of Anamorelin (1 mg/kg/day) for 7 days, an acute GH release was observed after both the first and seventh doses, although the response was considerably reduced by the seventh dose. [1]
In a crossover study in pigs, treatment with Anamorelin (1 mg/kg/day) for 7 days significantly elevated mean plasma insulin-like growth factor-1 (IGF-1) concentrations by 21% compared to the control treatment period. [1]

In the competition assay, membranes are supplemented with ³⁵S-MK-677 and anamorelin (ANAM) concentrations ranging from 1 pM to 10 μM. To ascertain nonspecific binding, 10 μM nonlabeled MK-677 is added. The samples are applied to GF/B filters, which have been pretreated with 0.5% PEI, and the mixture is incubated at 30°C for 60 minutes. The filters are then counted using an OptiPhase counter after being cleaned in 0.9% NaCl[1].

For the rat pituitary cell GH release assay, pituitaries were dissected from male Sprague-Dawley rats. The neurointermediate lobes were removed, and the remaining tissue was minced and enzymatically dissociated using trypsin and DNase. The resulting cell suspension was filtered, washed, and seeded at a density of 4×10^4 cells per well. Cells were cultured for 3 days. After washing and pre-incubation, cells were stimulated with various concentrations of Anamorelin (0.01 nM to 10 µM) for 15 minutes. The culture medium was then collected, and GH content was quantified using a recombinant GH enzyme-linked immunosorbent assay (ELISA). [1]

Rats: Rats are separated into four groups for the purpose of assessing food intake and body weight: Anamorelin 3 mg/kg (n = 7), 10 mg/kg (n = 7), or 30 mg/kg (n = 7), or vehicle control (n = 8). 100 μL blood samples are taken prior to and 0.25, 0.5, 1, 2, 3, 4, 5, and 6 hours following a single dose. Rats are put to sleep using a 64.8 mg/kg dose of sodium pentobarbital. In order to collect blood, a catheter equipped with a three-way cock to allow extra blood to return, an extension tube, and a 1 mL sampling syringe is placed inside the left femoral artery and filled with heparinized saline solution. A Rat Growth Hormone EIA kit and microplate reader are used to immunochemically measure the levels of growth hormone in plasma. There are two measurement takes place. The time course of GH plasma concentrations is assessed, as well as the area under the GH concentration curve from 0 to 6 hours (A_UC0-6h) postdose.
Pig: Anamorelin is administered intraperitoneally (IDI) using a dosing catheter to six groups of pigs each. Blood is drawn for the GH stimulation profile at 15, 30, 45, 60, and 120 minutes after dosing, as well as at 30 and 15 minutes before. The animals were given either a single dose (3.5 mg/kg) or a once-daily administration (1 mg/kg) of anamorelin for seven days. Following the first and seventh doses, stimulation profiles were taken. In order to measure IGF-1 levels, pigs are given either a placebo or anamorelin (1 mg/kg/day) for seven days, after which they switch between the two treatments for an additional seven days. Every day, right before the dose, a single blood sample is obtained.

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For rat food intake and body weight studies, 7-week-old male Cri-CD (SD) rats were individually housed. Animals were stratified by body weight and baseline food intake and then randomly assigned to groups (n=7 per group). Anamorelin HCl was dissolved in distilled water to concentrations of 6, 2, and 0.6 mg/mL, corresponding to doses of 30, 10, and 3 mg/kg, respectively. The vehicle control was distilled water. Drugs or vehicle were administered orally once daily at a volume of 5 mL/kg for 6 consecutive days using a flexible tube attached to a syringe. Body weight and leftover food (for calculating daily food intake) were measured daily. [1]
For the rat GH study, animals (n=7-8 per group) were anesthetized with sodium pentobarbital (64.8 mg/kg) and a catheter was inserted into the femoral artery. Following a single oral dose of Anamorelin (3, 10, 30 mg/kg in distilled water) or vehicle, blood samples (100 µL) were collected at multiple time points up to 6 hours post-dose. Plasma GH levels were measured using a rat GH ELISA kit. [1]
For pig studies, female Danish Landrace pigs (40-45 kg) were surgically implanted with intragastric dosing catheters and vascular catheters for blood sampling under general anesthesia. After a recovery period, pigs received Anamorelin via the gastric catheter. In one study, a single oral dose of 3.5 mg/kg was administered, and blood was sampled for GH profiling. In another study, pigs received once-daily oral doses of 1 mg/kg/day for 7 days, and GH profiles were assessed after the first and seventh doses. For IGF-1 assessment, a crossover design was used where pigs received either Anamorelin (1 mg/kg/day) or placebo for 7 days, then treatments were switched for another 7 days. Blood for IGF-1 measurement was sampled daily before dosing. [1]

The literature indicates that the half-life of Anamorelin is about 7 hours, which is longer than the half-life of natural ghrelin, which is about 30 minutes. [1]

Selective screening showed that the weak binding to non-targets (L-type calcium channels, serotonin transporters, sodium channels) observed at a concentration of 10 µM was unlikely to be clinically significant unless a large overdose occurred. [1] Referring to another preclinical study (cited in the literature discussion section), which administered anamarin daily for 28 consecutive days in a mouse xenograft model of lung cancer, the study found that despite elevated growth hormone (GH) levels, tumor growth was not promoted. The authors also noted that formal carcinogenicity studies in tumor-free rats and mice also supported the conclusion that anamarin does not stimulate tumor growth. [1] (This literature does not include data on acute toxicity, median lethal dose (LD50), organ toxicity, drug interactions, or plasma protein binding.) [1]

[1]. Anamorelin HCl (ONO-7643), a novel ghrelin receptor agonist, for the treatment of cancer anorexia-cachexiasyndrome: preclinical profile. J Cachexia Sarcopenia Muscle. 2014 Dec;5(4):329-37.

Anamoline is a synthetic, orally bioavailable, small-molecule auxin-releasing peptide analog with appetite-stimulating and anabolic effects. Anamoline binds to and stimulates the central growth hormone-secreting peptide receptor (GHSR), thereby mimicking the appetite-stimulating and growth hormone-releasing effects of auxin-releasing peptide. Stimulation of the GHSR may also reduce the production of pro-inflammatory cytokines TNF-α and interleukin-6, which may play a direct role in cancer-related anorexia. Drug Indications Studied for the treatment of cachexia and anorexia. For the treatment of anorexia, cachexia, or unintended weight loss in adult patients with non-small cell lung cancer (NSCLC). Anamoline hydrochloride (ONO-7643) is a novel, orally active synthetic auxin-releasing peptide receptor agonist that mimics the N-terminal active core of the endogenous hormone auxin-releasing peptide. [1]
Its mechanism of action includes stimulating appetite (food intake), promoting weight gain, and stimulating the release of growth hormone (GH) and insulin-like growth factor-1 (IGF-1). [1]
This drug is currently in clinical development for the treatment of cancer anorexia-cachexia syndrome (CACS). As of the time of this publication, the drug is undergoing a phase III clinical trial in patients with advanced non-small cell lung cancer (NSCLC) complicated with CACS. [1]

C31H42N6O3

546.70359

546.331

C, 63.85; H, 7.43; Cl, 6.08; N, 14.41; O, 8.23

249921-19-5

Anamorelin hydrochloride; 861998-00-7; Anamorelin Fumarate; 339539-92-3; 249921-19-5

9828911

white to off-white solid powder

1.2±0.1 g/cm3

1.619

2.98

3

5

9

40

904

2

[H][C@](CC1=CNC2=CC=CC=C21)(NC(C(C)(C)N)=O)C(N3CCC[C@@](C(N(C)N(C)C)=O)(CC4=CC=CC=C4)C3)=O

VQPFSIRUEPQQPP-MXBOTTGLSA-N

InChI=1S/C31H42N6O3/c1-30(2,32)28(39)34-26(18-23-20-33-25-15-10-9-14-24(23)25)27(38)37-17-11-16-31(21-37,29(40)36(5)35(3)4)19-22-12-7-6-8-13-22/h6-10,12-15,20,26,33H,11,16-19,21,32H2,1-5H3,(H,34,39)/t26-,31-/m1/s1

2-amino-N-[(2R)-1-[(3R)-3-benzyl-3-[dimethylamino(methyl)carbamoyl]piperidin-1-yl]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]-2-methylpropanamide

ONO7643; ONO-7643; ONO 7643; RC 1291; RC1291; RC-1291

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~182.9 mM)
Ethanol: ~50 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.57 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.57 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.57 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)