GHRF/growth hormone-releasing factor

Examorelin (Hexarelin) is a synthetic growth hormone-releasing peptide that exerts cardioprotective effects. Regulation of autophagy is known to be cardioprotective so this study examined the role of autophagy and potential regulatory mechanisms in hexarelin-elicited anti-cardiac hypertrophic action in cardiomyocytes subjected to hypertrophy. H9C2 cardiomyocytes were subjected to hypertrophy by angiotensin-II (Ang-II). Autophagic light chain-3 (LC3) and cytoskeletal proteins were determined by immunofluorescence assay. Autophagy was also detected using monodansylcadaverine (MDC) for autophagic vacuole visualization and Cyto-ID staining for autophagic flux measurement. Molecular changes were analysed by Western blotting and qRT-PCR. Apoptosis was evaluated using flow cytometry and TUNEL assay. ATP content and CCK-8 assay were used in assessing enhanced cell survival whilst oxidative stress was analysed by measuring malondialdehyde(MDA) and superoxide dismutase(SOD) levels. Ang-II induced cardiomyocyte hypertrophy, oxidative stress, apoptosis and decreased cell survival, all of which were significantly suppressed by Examorelin (Hexarelin) treatment which also enhanced autophagy in hypertrophic H9C2 cells. Furthermore, inhibition of hexarelin induced autophagy by 3-methyladenine (3MA) abolished the anti-hypertrophic function of hexarelin and also abrogated the protection of hexarelin against cell survival inhibition and apoptosis. Conversely, the application of autophagy stimulator rapamycin in H9C2 hypertrophic cells inhibited apoptosis, cell survival and reduced cell size as well. Additionally, hexarelin regulated the upstream signalling of autophagy by inhibiting the phosphorylation of mammalian target of rapamycin(mTOR). We propose that hexarelin plays a novel role of attenuating cardiomyocyte hypertrophy and apoptosis via an autophagy-dependent mechanism associated with the suppression of the mTOR signalling pathway.

Acute myocardial ischemia and reperfusion injury (IRI) underly the detrimental effects of coronary heart disease on the myocardium. Despite the ongoing advances in reperfusion therapies, there remains a lack of effective therapeutic strategies for preventing IRI. Growth hormone secretagogues (GHS) have been demonstrated to improve cardiac function, attenuate inflammation and modulate the autonomic nervous system (ANS) in models of cardiovascular disease. Recently, we demonstrated a reduction in infarct size after administration of Examorelin (Hexarelin)/HEX, in a murine model of myocardial infarction. In the present study we employed a reperfused ischemic (IR) model, to determine whether HEX would continue to have a cardioprotective influence in a model of higher clinical relevance. Myocardial ischemia was induced by transient ligation of the left descending coronary artery (tLAD) in C57BL/6 J mice followed by HEX (0.3 mg/kg/day; n = 20) or vehicle (VEH) (n = 18) administration for 21 days, first administered immediately prior-to reperfusion. IR-injured and sham mice were subjected to high-field magnetic resonance imaging to assess left ventricular (LV) function, with HEX-treated mice demonstrating a significant improvement in LV function compared with VEH-treated mice. A significant decrease in interstitial collagen, TGF-β1 expression and myofibroblast differentiation was also seen in the HEX-treated mice after 21 days. HEX treatment shifted the ANS balance towards a parasympathetic predominance; combined with a significant decrease in cardiac troponin-I and TNF-α levels, these findings were suggestive of an anti-inflammatory action on the myocardium mediated via HEX. In this model of IR, HEX appeared to rebalance the deregulated ANS and activate vagal anti-inflammatory pathways to prevent adverse remodelling and LV dysfunction. There are limited interventions focusing on IRI that have been successful in improving clinical outcome in acute myocardial infarction (AMI) patients, this study provides compelling evidence towards the translational potential of HEX where all others have largely failed [2].

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Both Examorelin (Hexarelin) and its natural analog ghrelin exert comparable cardioprotective activities. A single dose of ghrelin administered at the very acute phase after experimental myocardial infarction positively affects cardiac function in chronic heart failure. Therefore, this study aimed to determine whether a single dose of oral Examorelin (Hexarelin) has the same effect in the chronic disease phase. Myocardial infarction or sham operation was generated by left coronary artery ligation in male C57BL/6J mice, which subsequently received one dose of hexarelin or vehicle treatment by oral gavage 30 min after operation. Although the mortality within 14 days after myocardial infarction did not differ between the groups, hexarelin treatment protected cardiac function in the chronic phase as evidenced by higher ejection fraction and fractional shortening, as well as lower lung weight/body weight and lung weight/tibial length ratios, compared with vehicle treatment. Hexarelin treatment concurrently lowered plasma epinephrine and dopamine levels, and shifted the balance of autonomic nervous activity toward parasympathetic nervous activity as evidenced by a smaller low/high-frequency power ratio and larger normalized high-frequency power on heart rate variability analysis. The results first demonstrate that one dose of oral hexarelin treatment potentially protects chronic cardiac function after acute myocardial infarction, and implicate that activating growth hormone secretagogue receptor 1a might be beneficial for cardioprotection, although other mechanism may also be involved[3].

Cytokine and Cardiac troponin (CnT)-I determination [2]
Blood samples were collected 24 h and 21 days post tLAD ligation or sham procedure. The blood was allowed to clot and samples were centrifuged. The serum was immediately removed and stored at −80 °C until assayed. The serum concentrations of CnT-I, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were measured at 24 h and 21 days post-operatively using a MILLIPLEX® map Assay according to the manufacturer’s instructions.

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HRV analysis [2]
HRV analysis was performed 21 days post tLAD ligation or sham procedure. ECG signals were recorded using a physiological analyzing system. Mice were anaesthetized with isoflurane and ECG signals were recorded for a minimum of 20 min once the heart rate (HR) had stabilized. ANS function was examined by power spectral analysis of HRV where HR was used to generate a power spectral density curve using a fast Fourier transformation.

Treatment administration [2]
Examorelin (Hexarelin)(0.3 mg/kg/day) or VEH was administered SC to each mouse immediately prior to reperfusion. Similarly, mice undergoing the sham procedure also received either VEH or Examorelin (Hexarelin) treatment. This dose was chosen based on previous studies demonstrating a cardioprotective effect. All mice then received their respective treatments once daily throughout the 21-day study period.

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Examorelin (Hexarelin) and vehicle administration [3]
Examorelin (Hexarelin) (600 μg per mouse) or vehicle was administered to 24 mice each by oral gavage 30 min after the MI procedure (hexarelin- or vehicle-treated group), and 10 mice after sham operation were also received oral vehicle administration (sham-operated group). The dose of oral hexarelin was chosen to be as effective as 40 μg/kg subcutaneous administration, which is approximately equimolar with that of ghrelin in the previous study.

Regarding the pharmacokinetic properties of Examorelin (Hexarelin), existing research data are primarily derived from animal studies and limited clinical observations. In canine studies following intravenous injection (1 µg/kg), the drug exhibited a half-life of approximately 120 minutes, a plasma clearance of approximately 4.28 mL/min/kg, and a steady-state volume of distribution of approximately 387.7 mL/kg, indicating relatively wide distribution and moderate clearance in the body. Furthermore, in clinical studies of patients with growth hormone deficiency, whether administered intravenously or subcutaneously, the drug dose-dependently stimulated growth hormone secretion, typically reaching peak plasma concentrations approximately 30 minutes after administration, demonstrating good bioactivity and absorption characteristics.

With respect to toxicity, based on available preclinical and clinical study data, Examorelin is generally well-tolerated at therapeutic doses. Published literature has not clearly reported significant genotoxicity, carcinogenicity, or reproductive toxicity associated with the drug, nor have specific organ toxicities (such as hepatorenal toxicity) been warned against. In previous clinical trials, subjects did not report serious adverse events or obvious toxic side effects, indicating a relatively high safety profile for short-term use. However, as a growth hormone secretagogue, theoretical long-term use still warrants attention to potential risks related to metabolism, the cardiovascular system, and tumorigenesis. Nevertheless, the specific safe dosage range and long-term toxicity data require further systematic investigation to be fully elucidated.

[1]. Hexarelin protects cardiac H9C2 cells from angiotensin II-induced hypertrophy via the regulation of autophagy. Pharmazie. 2019 Aug 1;74(8):485-491.
[2]. Hexarelin targets neuroinflammatory pathways to preserve cardiac morphology and function in a mouse model of myocardial ischemia-reperfusion. Biomed Pharmacother. 2020 Jul:127:110165.
[3]. One dose of oral hexarelin protects chronic cardiac function after myocardial infarction. Peptides. 2014 Jun;56:156-62.

Conclusion: Modulating autonomic nervous system imbalances by enhancing parasympathetic tone has emerged as a promising therapeutic approach for ischemic heart disease and angina. The vagus nerve (VN) is considered to play a central role in the action of the growth hormone-releasing hormone agonist (GHS) and to act as an endogenous mechanism regulating immune responses and inflammation. In this study, we demonstrated that Examorelin (Hexarelin) treatment modulates the autonomic nervous system (ANS) and influences the ischemia-reperfusion (IR)-induced inflammatory response. Our results suggest that Examorelin (Hexarelin) may have significant implications for balancing the sympathetic and parasympathetic nervous systems and modulating adverse inflammatory pathways following acute myocardial infarction (AMI). Pharmacological stimulation of the VN with Examorelin (Hexarelin) may provide a novel cardioprotective strategy against ischemia-reperfusion injury (IRI) by inhibiting myofibroblast activation and left ventricular (LV) remodeling in AMI. [2]
Clinical Significance: Research in the field of cardioprotection has been plagued by numerous failures stemming from the failure to translate effective treatment strategies for preventing myocardial ischemia-reperfusion injury discovered in basic science laboratories into clinical applications. A major reason for this failure is the inappropriate use of experimental animal models. In our previous studies, we have clearly demonstrated the cardioprotective effect of Examorelin (Hexarelin) in a mouse model of permanent myocardial infarction (MI); however, the translational application prospects of this model are limited. In this study, using a clinically relevant model, Examorelin (Hexarelin) was administered before myocardial reperfusion (removal of coronary artery ligation sutures), and the results showed that Examorelin (Hexarelin) has good application prospects as an emerging drug for the prevention of myocardial ischemia-reperfusion injury (IRI). This protocol can be implemented in reperfusion centers. Therefore, this study makes an important contribution to the field of cardioprotection. In summary, although no improvement in overall mortality was observed, a single oral dose of Examorelin (Hexarelin) during the acute phase of myocardial infarction (MI) can still protect cardiac function during the chronic phase. In addition, it can reduce plasma adrenaline and dopamine levels and shift the balance of autonomic nervous activity towards parasympathetic activity. This study provides the first clear evidence to support oral Examorelin (Hexarelin) as a potential treatment for acute MI to protect cardiac function in the chronic phase. [3]

C47H58N12O6

887.040220000001

886.46

C, 63.64; H, 6.59; N, 18.95; O, 10.82

140703-51-1

6918297

H-His-D-Trp(2-Me)-Ala-Trp-D-Phe-Lys-NH2; L-histidyl-2-methyl-D-tryptophyl-L-alanyl-L-tryptophyl-D-phenylalanyl-L-lysinamide

HXAWFK

Crystalline solid at room temperature

1.322 g/cm3

1403.6ºC at 760 mmHg

802.7ºC

0mmHg at 25°C

1.66

5.392

11

9

23

65

1600

6

CC1=C(C2=CC=CC=C2N1)C[C@@H](NC([C@@H](N)CC3=CN=CN3)=O)C(N[C@H](C(N[C@H](C(N[C@@H](C(N[C@H](C(N)=O)CCCCN)=O)CC4=CC=CC=C4)=O)CC5=CNC6=CC=CC=C56)=O)C)=O

RVWNMGKSNGWLOL-GIIHNPQRSA-N

InChI=1S/C47H58N12O6/c1-27-34(33-15-7-9-17-37(33)54-27)23-41(58-44(62)35(49)22-31-25-51-26-53-31)45(63)55-28(2)43(61)57-40(21-30-24-52-36-16-8-6-14-32(30)36)47(65)59-39(20-29-12-4-3-5-13-29)46(64)56-38(42(50)60)18-10-11-19-48/h3-9,12-17,24-26,28,35,38-41,52,54H,10-11,18-23,48-49H2,1-2H3,(H2,50,60)(H,51,53)(H,55,63)(H,56,64)(H,57,61)(H,58,62)(H,59,65)/t28-,35-,38-,39+,40-,41+/m0/s1

L-Lysinamide, L-histidyl-2-methyl-D-tryptophyl-L-alanyl-L-tryptophyl-D-phenylalanyl-

EP-23905; MF-6003; EP23905; Hexarelin; Examorelin; 140703-51-1; Examorelin [INN]; examorelina; examoreline; L-Lysinamide, L-histidyl-2-methyl-D-tryptophyl-L-alanyl-L-tryptophyl-D-phenylalanyl-; EP-23,905; MF6003; EP 23905; MF 6003

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)

Water or PBS (pH 7.2): 10 mg/ml
DMSO: 30 mg/ml
DMF: 30 mg/ml
Ethanol: 5 mg/ml

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.)