hGHS-R1a ( Ki = 7 nM )

Binding affinities were measured via competitive binding assays in HEK293 cells over-expressing the cloned human GHS-R1a (hGHS-R1a) using [125I]-ghrelin as a radioligand. Capromorelin was found to be 17-fold less potent than ghrelin, the endogenous ligand for the GHS-R1a (see Table 1). The binding activity of Capromorelin was shown to be stereospecific, with the 3aR-isomer of the pyrazolinone-piperidine heterocyclic ring preferred over the 3aS-isomer. Removal of the N-methyl group on the PP group led to a two-fold loss in activity. The N-ethyl PP derivative 5b was equipotent to ghrelin in the assay.[1]

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Despite exhibiting a 30-fold range of binding affinities, Capromorelin and analogues 5b and 5c stimulated GH release with similar potencies (see Table 1). The 3aS-diastereomer of Capromorelin (5a) showed significantly weaker activity, with an EC50 value greater than 1 μM. Interestingly, both the 3aR- and 3aS-diastereomers of compound 3 (structural precursors of PP dipeptides) potently stimulated GH secretion (EC50s<25 nM), though the 3aS-diastereomer was slightly preferred.9 With the exception of 5a, the binding affinities of the GHSs did not predict activity in the cellular assay, possibly because of the potentially confounding effects of protein binding in the whole cell assay.

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Capromorelin induces the release of growth hormone in rat pituitary cell cultures with EC50 of 3 nM[1].

The in vivo GH activities of the PP dipeptide analogues were measured in an anesthesized rat model following iv administration. Capromorelin and compounds 5b and 5c stimulated GH secretion after a single 1 mg/kg dose, though the mean GH peak heights for Capromorelin and 5c were significantly higher than the mean GH peak height for 5b (data not shown). Capromorelin and 5c showed similar dose–response relationships in the model, with ED50 values less than 0.05 mg/kg iv. The weaker in vivo activity of the N-ethyl PP derivative 5b was attributed to its increased lipophilicity, which could have reduced the amount of unbound drug in the plasma compartment capable of interacting with the GHS-R1a[1].
The oral activity of Capromorelin was examined in a dog model that was found to be predictive of GHS activity in humans. Following a single 1 mg/kg po dose, Capromorelin rapidly increased plasma GH levels, with a maximum peak height of 73 ng/mL. Significant GHS activity was observed at doses as low as 0.05 mg/kg. When administered at 1 mg/kg po for 5 days, Capromorelin stimulated GH secretion on the first and last days of the study; however, the post-dose GH response on day 5 was somewhat attenuated.

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The food consumption of dogs treated with capromorelin (30 mg/mL) is significantly higher than that of dogs treated with a placebo. More than the placebo group, all dogs in the capromorelin group gained weight by 0.52 kg[1]. In rodent models, capromolelin demonstrates improved intestinal absorption and superior pharmacoK_inetic characteristics, such as high bioavailabilities in two animal species [F(dog)=44%, F(rat)=65%]. Reference [2]. With an ED50 of 0.05 mg/kg iv, capromolelin stimulates the release of growth hormone in a model of anesthetized rats[2].

Binding assay[1]
Membranes were prepared from HEK293 cells (ATCC) stably transfected with the human GHS-R1a receptor cDNA in the plasmid pcDNA3.1neo. Competition radioligand binding assays were performed in 96-well format with GF/C filters pre-soaked in 0.3% polyethyleneimine. Assays were performed at room temperature for 1 h in duplicate using 50 pM [125I]-ghrelin and 1 μg membrane per well in 50 mM HEPES, pH 7.4, 10 mM MgCl2, 0.2% bovine serum albumin and the following protease inhibitors: 100 μg/mL bacitracin, 100 μg/mL benzamidine, 5 μg/mL aprotinin, 5 μg/mL leupeptin. The membranes were harvested and washed three times with ice-cold wash buffer containing 50 mM HEPES, pH 7.4 and 10 mM MgCl2. IC50 and Ki values were determined using Prism by Graphpad™. The Kd of [125I]-ghrelin at membranes expressing human GHS receptors was calculated to be 0.2 nM. No detectable binding was observed in un-transfected HEK293 cell membranes (data not shown).[1]

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The human GHS-R1a receptor cDNA in the plasmid pcDNA3.1neo is transfected into HEK293 cells (ATCC) to produce membranes. Competition radioligand binding assays are run in 96-well format using GF/C filters that have been pre-soaked in 0.3% polyethyleneimine. Tests are run in duplicate at room temperature for one hour, employing 50 pM [¹²⁵I]-ghrelin and 1 μg membrane per well in 50 mM HEPES, pH 7.4, 10 mM MgCl₂, 0.2% bovine serum albumin, and the subsequent protease inhibitors: 100 μg/mL benzamidine, 100 μg/mL bacitracin, 5 μg/mL aprotinin, and 5 μg/mL leupeptin. After harvesting the membranes, they are rinsed three times in an ice-cold buffer that has pH 7.4, 50 mM HEPES, and 10 mM MgCl₂. Prism by Graphpad^TM is used to calculate the IC50 and K_i values. It is determined that the K_d of [¹²⁵I]-ghrelin at membranes expressing human GHS receptors is 0.2 nM.

Functional activity in vitro (GH release in rat pituitary cell cultures)[1]
Primary pituitary cell cultures were established by enzymatic dissociation of anterior pituitary glands from 6-week-old male Wistar rats. Cells were suspended in Dulbecco's modified Eagle's medium (DMEM, 4.5 g/L glucose supplemented with 1 mM sodium pyruvate, 1% MEM non-essential amino acids, 10% heat-inactivated horse serum, 2.5% fetal bovine serum plus antibiotics), plated at 1×105 cells per well in 24-well tissue culture plates and incubated in a humidified 5% CO2/95% air incubator at 37 °C. Hormone release was performed 3–4 days after plating. Cell cultures were rinsed twice and then equilibrated at 37 °C in release medium (DMEM with 25 mM HEPES buffer, pH 7.4 and 5 mg/mL bovine serum albumin) for 30 min. This medium was aspirated and replaced with pre-warmed release medium containing test agents. After a 15 min incubation period at 37 °C, the medium was removed and assayed for GH. Results are expressed as mean±SEM of quadruplicate wells. Groups were compared by unpaired Student's 2-tailed t-test unless otherwise indicated. The data are reported as the concentration necessary to produce a half-maximum response (EC50). MK-0677 was used as a reference standard in control plates.

Pharmacokinetics of Capromorelin in rats[1]
Eight adult female Sprague–Dawley rats were prepared for use by surgical implantation of a cannula in the femoral vein while under methoxyflurane anesthesia the day before study initiation. Study rats were fasted overnight prior to dosing, allowed free access to water and housed in standard polycarbonate rodent cages. The rodents were allowed access to food 4 h post-dose. The dose (intravenous and oral) was prepared as a 0.5 mg/mL base equivalent (0.5 mgA/kg) solution of Capromorelin in deionized water. Four Sprague–Dawley rats (weighing 0.280–0.305 kg) received an iv dose of 1 mgA/kg of Capromorelin. The dose was administered via the femoral vein catheter followed by a 1 mL infusion of normal saline to rinse the catheter. Blood samples (0.5 mL) were taken from the femoral vein catheter pre-dose, 0.083, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose and transferred into heparinized microtainers. The blood volume withdrawn was replaced with normal saline. Plasma was harvested from the centrifuged microtainers and stored frozen at −70 °C in 500 μL polypropylene microcentrifuge tubes. Four Sprague–Dawley rats (weighing 0.275–0.290 kg) received an oral dose of 1 mgA/kg Capromorelin. The dose was administered via an 18-gauge, 3 in., curved gavage needle. Blood samples (0.5 mL) were taken from the femoral vein catheter pre-dose, 0.083, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose and transferred into heparinized microtainers. The blood volume withdrawn was replaced with normal saline. Plasma was harvested from the centrifuged microtainers and stored frozen at −70 °C in 500 μL polypropylene microcentrifuge tubes.

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Pharmacokinetics of Capromorelin in dogs[1]
Four adult beagle dogs (2 males and 2 females weighing 8.9–12.9 kg) were fasted overnight prior to dosing but were allowed free access to water. The dogs were allowed access to food 4 h post-dose. The iv dose was prepared as a 10 mgA/mL solution of Capromorelin in deionized water and filter sterilized. The oral dose was prepared as a 2 mgA/mL solution of Capromorelin in deionized water. Four beagle dogs (weighing 8.9–12.9 kg) received an oral dose of 1 mgA/kg of Capromorelin. The dose was administered via an oral gavage tube. Blood samples (2 mL) were taken from the jugular vein pre-dose, 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose. Blood samples were collected directly into heparinized vacutainers. Plasma was harvested from the centrifuged vacutainers and stored frozen at −70 °C in 500 μL polypropylene microcentrifuge tubes. Following a 2 day wash-out period, the same four beagle dogs received an iv dose of 1 mgA/kg of Capromorelin. The dose was administered via the cephalic vein. Blood samples (2 mL) were taken from the jugular vein pre-dose, 0.083, 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose. Blood samples were collected directly into heparinized vacutainers. Plasma was harvested from the centrifuged vacutainers and stored frozen at −70 °C in 500 μL polypropylene microcentrifuge tubes.

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The study evaluated a flavored oral solution containing 30 mg/mL of capromorelin in comparison to a matched placebo-flavored oral solution treatment that was given for four days and contained all the formulation's ingredients but no capromorelin. Two groups of dogs are randomly assigned; Group 1 receives a placebo (0.1 mL/kg), while Group 2 receives 3.0 mg/kg. Each day, at around nine in the morning, both groups receive the same treatment. Day 0 is the first day of medication. A syringe inserted into the mouth's corner is used to administer both the test medication and the placebo. When calculating doses, the Day 0 weight is utilized.

Pharmacokinetic properties of two PP dipeptide GHSs, Capromorelin and 5c, were measured in female Sprague–Dawley rats. For Capromorelin, plasma clearance (CL), volume of distribution (Vd) and plasma elimination half-life (t1/2) following a 1 mgA/kg iv dose were 34±5 mL/min/kg, 1.7 L/kg and 0.79±0.21 h respectively. Capromorelin was rapidly absorbed after a 1 mgA/kg oral (po) dose, reaching maximum systemic concentrations (Cmax) of 329±158 ng/mL in 0.25 h. The unbound plasma free fraction was high (Fu=27%), possibly explaining the robust GHS activity in the in vivo anesthesized rat model. Oral bioavailability was 65%, far greater than the reported bioavailabilities of other peptidyl or peptidomimetic GHSs in the literature. Compound 5c was detected as a circulating metabolite, though exposure generally represented less than 10% of parent drug. Pharmacokinetic values for 5c were similar to those for Capromorelin. Following a 1 mgA/kg iv dose, systemic clearance was high and volume of distribution was moderate, resulting in a short half-life (CL=56.7 mL/min/kg; Vd=2.6 L/kg; t1/2=0.83 h). Oral bioavailability was low, most likely due to the combination of incomplete intestinal absorption and high CL (F=12%).[1]
As the only PP GHS with superior bioavailability and good absorption properties in a rat model, Capromorelin was selected for pharmacokinetic evaluation in the dog. After a 1 mgA/kg iv dose, the plasma clearance of the compound was 19±5 mL/min/kg, the volume of distribution was 2.0±0.4 L/kg and the elimination half-life was 1.3 h. Mean values for Cmax and Tmax following a 1 mgA/kg po dose were 180±66 ng/mL and 1 h. The plasma free fraction of drug was 51% and the bioavailability was 44%. Compound 5c was also identified as a circulating plasma metabolite.[1]
Pharmacokinetic characterization of Capromorelin in rats and dogs revealed short plasma elimination half-lives. In dogs, the short half-life was attributed to a moderate volume of distribution and a moderate-to-high clearance due in part to de-methylation of the PP ring and oxidation of both the benzyl group in the (d)-O-Bn-Ser moiety and a methyl group in the Aib moiety (data not shown). The compound did not undergo proteolytic degradation in plasma. The des-methyl metabolite 5c exhibited a similarly short half-life, but because of its lower rat bioavailability, it did not offer any advantages over Capromorelin. A short half-life was considered pharmacologically desirable for stimulating GH secretion (rather than increasing IGF-1 levels) and preventing attenuation of the post-dose GH response during repeat administration.[1]

[1]. Pyrazolinone-piperidine dipeptide growth hormone secretagogues (GHSs). Discovery of capromorelin. Bioorg Med Chem. 2003 Feb 20;11(4):581-90.

[2]. Capromorelin oral solution (ENTYCE?) increases food consumption and body weight when administered for 4 consecutive days to healthy adult Beagle dogs in a randomized, masked, placebo controlled study. BMC Vet Res. 2017 Jan 5;13(1):10.

Capromorelin is under investigation in clinical trial NCT00527046 (Effects Of An Oral Growth Hormone Secretagogue In Older Functionally Limited Adults).
See also: Capromorelin Tartrate (has salt form).

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Novel pyrazolinone-piperidine dipeptide derivatives were synthesized and evaluated as growth hormone secretagogues (GHSs). Two analogues, capromorelin (5, CP-424391-18, hGHS-R1a K(i)=7 nM, rat pituicyte EC(50)=3 nM) and the des-methyl analogue 5c (hGHS-R1a K(i)=17 nM, rat pituicyte EC(50)=3 nM), increased plasma GH levels in an anesthesized rat model, with ED(50) values less than 0.05 mg/kg iv. Capromorelin showed enhanced intestinal absorption in rodent models and exhibited superior pharmacokinetic properties, including high bioavailabilities in two animal species [F(rat)=65%, F(dog)=44%]. This short-duration GHS was orally active in canine models and was selected as a development candidate for the treatment of musculoskeletal frailty in elderly adults.[1]

C32H41N5O10

655.705

655.29

C, 58.62; H, 6.30; N, 10.68; O, 24.40

193273-69-7

193273-66-4; 193273-69-7 (tartrate)

9852610

White to off-white solid powder

1.388

6

12

12

47

1010

4

O=C1[C@@]2(CC3C=CC=CC=3)C(CCN(C([C@@H](COCC3C=CC=CC=3)NC(C(C)(C)N)=O)=O)C2)=NN1C.O[C@@H](C(=O)O)[C@H](C(=O)O)O

MJGRJCMGMFLOET-MYPSAZMDSA-N

InChI=1S/C28H35N5O4.C4H6O6/c1-27(2,29)25(35)30-22(18-37-17-21-12-8-5-9-13-21)24(34)33-15-14-23-28(19-33,26(36)32(3)31-23)16-20-10-6-4-7-11-20;5-1(3(7)8)2(6)4(9)10/h4-13,22H,14-19,29H2,1-3H3,(H,30,35);1-2,5-6H,(H,7,8)(H,9,10)/t22-,28-;1-,2-/m11/s1

N-[(2R)-1-[(3aR)-3a-benzyl-2-methyl-3-oxo-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-5-yl]-1-oxo-3-phenylmethoxypropan-2-yl]-2-amino-2-methylpropanamide;(2R,3R)-2,3-dihydroxybutanedioic acid

Capromorelin tartrate; CP424,391; CP-424391; CP-424,391; CP 424,391;CP 424391; CP424391; CP-424,391-18

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (~152.5 mM)
H2O: ~100 mg/mL (~152.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.81 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 (3.81 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 (3.81 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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Solubility in Formulation 4: 50 mg/mL (76.25 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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