March 2016

Modified GRF, or modGRF, is a shortened form of the growth-hormone releasing hormone (GHRH). GHRH is also known as the GH-releasing factor, or GRF. GRF is released from the hypothalamus to bind GHRH receptors (GHRH-R) in the pituitary gland, which results in the release of GH¹. modGRF is made up of 29 amino acids (1-29), whereas full GRF possesses 44. Some studies on the structure of modGRF indicate that amino acids 13-21 alone are essential for GHRH-R binding, as deleting amino acids outside this region decreased receptor affinity by a maximum of 5%, whereas deletion or substitution within the 13-21 chain decreased affinity to less than 1%². On the other hand, the alteration of either terminus also had a deleterious effect on GHRH-R affinity². modGRF may also be termed sermorelin. Treatment with a form of modGRF resulted in a four-fold increase in GH release in an in vitro study using rat pituitary cells in culture³. This peptide may have beneficial roles in animal growth, fat metabolism and hormonal regulation. It may also modulate IGF1 activity, due to its effects on GH¹. This implies another role for modGRF in immune system regulation¹. Based on its physiological and biochemical properties, modGRF is regarded as a viable experimental, and also possibly therapeutic, agent.  Experimental uses of modGRF GRF may also instigate pain relief in response to inflammation, due to its actions on pituitary receptors. An animal study demonstrated the hormone's effects on a rat model of this (i.e. endotoxin-induced hyperalgesia). Rats treated with intraperitoneal GRF 300 minutes before experimental endotoxin administration exhibited reduced, dose-dependent thermal and mechanical hyperalgesia⁴. Despite this, GRF may not have significant effects on the concentrations of pro-inflammatory biomarkers such as NFkappaB, interleukins or TNFalpha⁴. There is some evidence that GHRH activation results in the recruitment of immune-system cells. This receptor may also play a role in the promotion of granulocyte proliferation⁵. modGRF is also associated with the release of histamine in rodent mast cell culture studies⁶.  GHRH-R Activation and its Role in Disease The receptor for GRF and its analogs, GHRH-R, has been observed as being overexpressed on many cancer cell lines⁷. GRF may also promote the expression of epidermal growth factor-type receptors on murine prostate cancer cell lines⁸. Its receptors may also play a role in the progression and proliferation of the cells associated with atypical, treatment-resistant breast cancer types⁹. GHRH-R activation may also promote general cell proliferation and impair normal cellular apoptosis^1,10. modGRF also significantly increased the concentrations of vascular endothelial growth factor (VEGF), a marker of potential neovascularization (or new blood vessel growth) in an in vitro study¹¹. These properties may be exploited by tumors in order to enhance survival and avoid cell death. Therefore, modGRF may be used to validate the effects of emerging GHRH-R antagonists currently proposed as anticancer drugs. An example of these demonstrated the ability to significantly inhibit the growth of prostate cancer cells grafted onto nude mice¹⁰. On the other hand, the inhibition of GHRH-R was shown to have a negative effect in a mouse model of acetaminophen-induced liver damage¹².   References:

1. Qin YJ, Chan SO, Chong KK, et al. Antagonist of GH-releasing hormone receptors alleviates experimental ocular inflammation. Proceedings of the National Academy of Sciences of the United States of America. 2014;111(51):18303-18308.
2. Gaudreau P, Boulanger L, Abribat T. Affinity of human growth hormone-releasing factor (1-29)NH2 analogues for GRF binding sites in rat adenopituitary. Journal of medicinal chemistry. 1992;35(10):1864-1869.
3. Jette L, Leger R, Thibaudeau K, et al. Human growth hormone-releasing factor (hGRF)1-29-albumin bioconjugates activate the GRF receptor on the anterior pituitary in rats: identification of CJC-1295 as a long-lasting GRF analog. Endocrinology. 2005;146(7):3052-3058.
4. Talhouk RS, Saade NE, Mouneimne G, Masaad CA, Safieh-Garabedian B. Growth hormone releasing hormone reverses endotoxin-induced localized inflammatory hyperalgesia without reducing the upregulated cytokines, nerve growth factor and gelatinase activity. Progress in neuro-psychopharmacology & biological psychiatry. 2004;28(4):625-631.
5. Khorram O, Yeung M, Vu L, Yen SS. Effects of [norleucine27]growth hormone-releasing hormone (GHRH) (1-29)-NH2 administration on the immune system of aging men and women. The Journal of clinical endocrinology and metabolism. 1997;82(11):3590-3596.
6. Estevez MD, Alfonso A, Vieytes MR, Louzao MC, Botana LM. Study of the activation mechanism of human GRF(1-29)NH2 on rat mast cell histamine release. Inflammation research : official journal of the European Histamine Research Society ... [et al.]. 1995;44(2):87-91.
7. Ziegler CG, Ullrich M, Schally AV, et al. Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells. Molecular and cellular endocrinology. 2013;371(0):189-194.
8. Munoz-Moreno L, Arenas MI, Carmena MJ, Schally AV, Prieto JC, Bajo AM. Growth hormone-releasing hormone antagonists abolish the transactivation of human epidermal growth factor receptors in advanced prostate cancer models. Investigational new drugs. 2014;32(5):871-882.
9. Perez R, Schally AV, Vidaurre I, Rincon R, Block NL, Rick FG. Antagonists of growth hormone-releasing hormone suppress in vivo tumor growth and gene expression in triple negative breast cancers. Oncotarget. 2012;3(9):988-997.
10. Stangelberger A, Schally AV, Rick FG, et al. Inhibitory effects of antagonists of growth hormone releasing hormone on experimental prostate cancers are associated with upregulation of wild-type p53 and decrease in p21 and mutant p53 proteins. The Prostate. 2012;72(5):555-565.
11. Stepien T, Sacewicz M, Lawnicka H, et al. Stimulatory effect of growth hormone-releasing hormone (GHRH(1-29)NH2) on the proliferation, VEGF and chromogranin A secretion by human neuroendocrine tumor cell line NCI-H727 in vitro. Neuropeptides. 2009;43(5):397-400.
12. Wang T, Hai J, Chen X, et al. Inhibition of GHRH aggravated acetaminophen-induced acute mice liver injury through GH/IGF-I axis. Endocrine journal. 2012;59(7):579-587.

Introduction  Hexarelin is a six-amino-acid peptide synthesized with the goal of studying modifications of the growth hormone (GH) pathway. It mimics the action of ghrelin by binding to the growth hormone secretagog receptor 1a (GHS-R1a). Ghrelin, by contrast, has 28 amino acid residues and may require acylation to bind to the GHS-R1a in some tissues¹. Synthetic GHSs such as hexarelin are thought to influence GH release via phospholipid-dependent protein kinase (PKC) signaling². This may affect development in infant animals, normal growth rates and other functions. It may be used in the lab to study these topics, as well as other functions of ghrelin, based on these properties. Ghrelin, hexarelin and other similar synthetic peptides that act as GHSs are known for their effects on appetite and muscle mass³. Hexarelin is also employed in the determination of GHS-R1a presence, locations and functions in new and less-studied species⁴. Hexarelin may elicit a stronger response to GHS-R1a activation (in terms of calcium release) compared to ghrelin in some species, although the ED₅₀ values of the two compounds may be similar⁴. This suggests that, while the gene for GHS-R1a may be conserved across species, the presence of ghrelin may not be. Hexarelin and Anabolic Effects on Muscle Hexarelin may instigate an increase in calcium influx through activation of the GHS-R1a, as outlined above. However, it may not have these effects in skeletal muscle. A study using isolated rat muscle fibers found that non-peptide GHSs, but not hexarelin or ghrelin, elicited an increase in intercellular calcium via GHS-R1a-independent pathways. On the other hand, hexarelin (50 or 100μM) has been found to enhance the contractility of isolated rat skeletal muscle in a significant, dose-dependent and calcium-independent manner⁵. This was associated with the activation of the GHS-R1a, as demonstrated by the use of D-Lys3-GHRP-6, which is an antagonist of the GHS-R1a⁵. Interestingly, this beneficial effect (i.e. modulating the mechanical threshold of muscle cell contraction) was only seen in tissue samples from younger rats. The administration of hexarelin to samples from older rats was unable to alleviate deficits in contractility associated with a deficiency in GH⁵. This may be due to the ability of hexarelin to elicit significant decreases in resting conductances conveyed by both chlorine and potassium, whereas muscular contractility essentially derives the most benefit from decreases in potassium-related conductance and increases in chlorine-related conductance⁵. Hexarelin, Ghrelin and their Differential Roles in Cardiovascular Tissues Some studies indicate the presence of the GHS-R1a in cardiac tissue, and, therefore, a role for ghrelin in heart tissue function¹. Other researchers have also claimed a similar role for hexarelin in vascular and cardiac cells, and that it is associated with beneficial effects on these tissues, particularly in cases of injury or disease^6-8. There is some evidence that hexarelin may also alleviate lesion development in mouse models of atherosclerosis¹. However, these effects are not associated with GHSR-1a, but with CD36, a cardiovascular receptor at which hexarelin may also be active¹. Hexarelin in Adipose Tissue There is some evidence that hexarelin is an agonist of CD36, a protein involved in the control of lipolysis and fatty acid breakdown in fat tissue⁹. Cultured murine adipose tissue was treated with 10μM hexarelin every 12 hours⁹. This resulted in the activation of CD36, which elicited the decreased expression of phosphoenolpyruvate carboxykinase (PEPCK). As PEPCK is associated with fatty acid breakdown, this indicates that CD36 regulates this process, and is activated by hexarelin⁹. References:

1. Benso A, Broglio F, Marafetti L, et al. Ghrelin and synthetic growth hormone secretagogues are cardioactive molecules with identities and differences. Seminars in vascular medicine. 2004;4(2):107-114.
2. Smith RG, Van der Ploeg LH, Howard AD, et al. Peptidomimetic regulation of growth hormone secretion. Endocrine reviews. 1997;18(5):621-645.
3. Liantonio A, Gramegna G, Carbonara G, et al. Growth hormone secretagogues exert differential effects on skeletal muscle calcium homeostasis in male rats depending on the peptidyl/nonpeptidyl structure. Endocrinology. 2013;154(10):3764-3775.
4. Kaiya H, Konno N, Kangawa K, Uchiyama M, Miyazato M. Identification, tissue distribution and functional characterization of the ghrelin receptor in West African lungfish, Protopterus annectens. General and comparative endocrinology. 2014;209:106-117.
5. Pierno S, De Luca A, Desaphy JF, et al. Growth hormone secretagogues modulate the electrical and contractile properties of rat skeletal muscle through a ghrelin-specific receptor. British journal of pharmacology. 2003;139(3):575-584.
6. Mao Y, Tokudome T, Kishimoto I, Otani K, Miyazato M, Kangawa K. One dose of oral hexarelin protects chronic cardiac function after myocardial infarction. Peptides. 2014;56:156-162.
7. Ma Y, Zhang L, Edwards JN, Launikonis BS, Chen C. Growth hormone secretagogues protect mouse cardiomyocytes from in vitro ischemia/reperfusion injury through regulation of intracellular calcium. PLoS One. 2012;7(4):e35265.
8. Xu X, Ding F, Pang J, et al. Chronic administration of hexarelin attenuates cardiac fibrosis in the spontaneously hypertensive rat. Am J Physiol Heart Circ Physiol. 2012;303(6):H703-711.
9. Wan Z, Matravadia S, Holloway GP, Wright DC. FAT/CD36 regulates PEPCK expression in adipose tissue. American journal of physiology. Cell physiology. 2013;304(5):C478-484.