GHS/growth hormone secretagogue (Ki =7 nM for hGHS-R1a; EC50 = 3 nM in rat pituicyte )

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.

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.

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).

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.

This study determined the pharmacokinetic properties of two PP dipeptide growth hormone-releasing hormone (GHS) agents—capromorelin and 5c—in female Sprague-Dawley rats. Following intravenous injection of 1 mgA/kg capromorelin, the plasma clearance (CL), volume of distribution (Vd), and elimination half-life (t1/2) were 34 ± 5 mL/min/kg, 1.7 L/kg, and 0.79 ± 0.21 h, respectively. After oral administration of 1 mgA/kg, capromorelin was rapidly absorbed, reaching its maximum systemic concentration (Cmax) of 329 ± 158 ng/mL within 0.25 h. The high free plasma concentration (Fu = 27%) may explain its significant GHS activity in an anesthetized rat model. The oral bioavailability was 65%, significantly higher than the bioavailability reported for other peptide or peptide-like growth hormone-releasing hormones (GHS) in the literature. Compound 5c was detected as a circulating metabolite, but its exposure was generally less than 10% of the parent drug. The pharmacokinetic parameters of 5c were similar to those of capromorelin. After intravenous injection of 1 mgA/kg, the systemic clearance was high and the 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). The oral bioavailability was low, likely due to the combined effect of incomplete intestinal absorption and high clearance (F=12%). [1] Capromorelin was the only peptide growth hormone-releasing hormone (PP GHS) with excellent bioavailability and good absorption in a mouse model, and was therefore selected for pharmacokinetic evaluation in dogs. After intravenous injection of 1 mgA/kg, 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. After oral administration of 1 mgA/kg, the mean Cmax and Tmax were 180 ± 66 ng/mL and 1 h, respectively. The plasma free fraction of the drug was 51%, and the bioavailability was 44%. Compound 5c was also identified as a circulating plasma metabolite. [1] Pharmacokinetic characterization of capprorelin in rats and dogs showed that it had a short plasma elimination half-life. In dogs, the short half-life of the compound was due to its moderate volume of distribution and moderate to high clearance, partly due to the demethylation of the PP ring and the oxidation of the benzyl group in the (d)-O-Bn-Ser moiety and the methyl group in the Aib moiety (data not shown). The compound did not undergo proteolytic degradation in plasma. The half-life of the demethylated metabolite 5c was also short, but it did not have an advantage over capprorelin due to its lower bioavailability in rats. Pharmacologically, a shorter half-life is believed to stimulate growth hormone (GH) secretion (rather than increase insulin-like growth factor-1 (IGF-1) levels) and prevent a weakening of the GH response after repeated administration. [1]

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

Capromorelin is being investigated in the clinical trial NCT00527046 (Effects of oral growth hormone secretagogues in elderly adults with functional impairment).
See also: Capmorelin tartrate (salt form).

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A novel pyrazolinone-piperidine dipeptide derivative was synthesized and its activity as a growth hormone secretagogue (GHS) was evaluated. Both analogues, capmorelin (5, CP-424391-18, hGHS-R1a K(i)=7 nM, rat pituitary EC(50)=3 nM) and its demethylated analogue 5c (hGHS-R1a K(i)=17 nM, rat pituitary EC(50)=3 nM), increased plasma growth hormone levels in anesthetized rat models with ED(50) values less than 0.05 mg/kg intravenously. Capmorelin exhibits enhanced intestinal absorption in rodent models and possesses excellent pharmacokinetic properties, including high bioavailability in both animals [65% in rats and 44% in dogs]. This short-acting growth hormone analog is orally active in canine models and has been selected as a candidate drug for the treatment of musculoskeletal weakness in the elderly. [1]

C28H35N5O4

505.6086

505.268

193273-66-4

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

216208

Typically exists as solid at room temperature

1.3±0.1 g/cm3

1.621

4.07

2

6

9

37

878

2

O=C1[C@@]2(C([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])C(C([H])([H])C([H])([H])N(C([C@@]([H])(C([H])([H])OC([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])N([H])C(C(C([H])([H])[H])(C([H])([H])[H])N([H])[H])=O)=O)C2([H])[H])=NN1C([H])([H])[H]

KVLLHLWBPNCVNR-SKCUWOTOSA-N

InChI=1S/C28H35N5O4/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/h4-13,22H,14-19,29H2,1-3H3,(H,30,35)/t22-,28-/m1/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; 193273-66-4; Capimorelin; Capromorelin [INN]; CP-424391; CP-424,391; UNII-0MQ44VUN84; 0MQ44VUN84;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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