Growth hormone releasing peptides (GHRPs) are short molecules developed to mimic the growth hormone secretagogue (GHS, or ghrelin) as the name suggests. Interest in these small sequences began in the early 1980s. At this time, GHRP-6 was regarded as the most potent of these compounds¹. GHRP-6 is active at the GHS receptor (GHS-R) and may therefore activate or regulate a number of different pathways in animal systems. Ghrelin and/or GH may mediate effects on metabolism, appetite, cardiac function and/or protection and cell survival. Therefore, GHRP-6 may be a useful reagent in animal models of various conditions, ranging from ischemia to obesity. GHRP-6 may be administered by injection, topically or orally. However, there are other ghrelin mimetics that may be more effective when administered by the latter route¹. Intranasal delivery systems for this peptide have also been proposed². GHRP-6 has limited stability and availability as a topical treatment, unless modified or conjugated to another small molecule³. One group recently reported improvements in these properties through the conjugation of GHRP-6 to biotin, and that this formulation elicited significantly increased collagen synthesis in cultured skin cells³. The same group then applied this compound to cultured myoblasts, and reported that this treatment resulted in significant increases in their differentiation to myocytes when compared to control cells³. This was thought to be associated with increased collagen-I synthesis³. Treatment with GHRP-6-biotin was also associated with significant increases in intracellular ATP and lactate compared to controls, indicating that treated cells used more energy than non-treated cells³. These findings may align with others that indicate GH may regulate the expression of various genes involved in myoblast differentiation through IGF signaling⁴. Many researchers have concluded that GHS and its mimetics also act to prevent apoptosis in a number of cell types⁵. Apoptosis in pituitary cells, as well as hypothalamic and cerebellar astrocytes, has been observed as a side-effect of diabetes⁵. This condition may also result in a decrease in GH expression⁵. Therefore, GHRP-6 may be used in relevant studies to assess the effects of GH or ghrelin on both diabetes and diabetes-induced cell death. A 2011 study compared a daily regimen of either GHRP-6, (150μg/kg) this dose of the same combined with approximately 8IU insulin, insulin alone or a placebo on apoptosis in rats with experimentally-induced diabetes⁵. Rats with this condition exhibited significantly more apoptosis in the cell types listed above compared to control animals. As individual treatments, GHRP-6 and insulin did not significantly affect cell death after eight weeks. However, the ‘combined’ treatment was associated with significant decreases in apoptosis and in the concentrations of glial fibrillary acidic protein, a marker of nerve cell damage⁵. GHRP-6 may also enhance the survival of heart muscle cells following experimentally-induced cardiac disease⁶. A single treatment of 400μg/kg in an animal model of acute myocardial infarction was associated with significant reductions in cardiac injuries and oxidative stress compared to controls⁶. GHRP-6 may also be used to assess the putative roles of GH in other processes, most notably aging and hormonal dysregulation⁷. References:

1. DeVita RJ. Small molecule mimetics of GHRP-6. Expert opinion on investigational drugs. 1997;6(12):1839-1843.
2. Pontiroli AE. Peptide hormones: Review of current and emerging uses by nasal delivery. Advanced drug delivery reviews. 1998;29(1-2):81-87.
3. Lim CJ, Jeon JE, Jeong SK, et al. Growth hormone-releasing peptide-biotin conjugate stimulates myocytes differentiation through insulin-like growth factor-1 and collagen type I. BMB Reports. 2015;48(9):501-506.
4. Florini JR, Ewton DZ, Coolican SA. Growth hormone and the insulin-like growth factor system in myogenesis. Endocrine reviews. 1996;17(5):481-517.
5. Granado M, Garcia-Caceres C, Tuda M, Frago LM, Chowen JA, Argente J. Insulin and growth hormone-releasing peptide-6 (GHRP-6) have differential beneficial effects on cell turnover in the pituitary, hypothalamus and cerebellum of streptozotocin (STZ)-induced diabetic rats. Molecular and cellular endocrinology. 2011;337(1-2):101-113.
6. Berlanga J, Cibrian D, Guevara L, et al. Growth-hormone-releasing peptide 6 (GHRP6) prevents oxidant cytotoxicity and reduces myocardial necrosis in a model of acute myocardial infarction. Clinical science (London, England : 1979). 2007;112(4):241-250.
7. Zouboulis CC, Makrantonaki E. Hormonal therapy of intrinsic aging. Rejuvenation research. 2012;15(3):302-312.

What is CJC-1295 No DAC? CJC-1295 is an isoform of the growth hormone releasing hormone (GHRH), also popularly known as GH-releasing factor or GRF. This protein is produced in the hypothalamus of many animals, and targets receptors on somatotrophic cells in the pituitary when released¹. It may be applied as a treatment for growth disorders, or with the intention of tissue mass enhancement; in which case it is bound to a drug-affinity complex (or DAC) which is intended to control its release over time when administered. However, the peptide as referred to in the title above is CJC-1295 NO-DAC, or modified GRF without DACⁱ. Therefore, it may also be referred to as modGRF, or the first 29 amino acids that comprise the original GRF moleculeⁱ. Native GRF, on the other hand, may have as many as 44 amino acids², although residues 1-29 have been shown to exert all the biological properties associated with this protein³. modGRF weighs just under 3.4kDa, is synthetically produced and is suitable for research use onlyⁱ. The main function of modGRF is to enhance the release of GH, as mentioned above. Therefore, it may be useful in studies of growth and development. Some studies indicate that modGRF has pharmacokinetic and pharmacokinetic properties that are superior to endogenous GHRH⁴. It may have increased activity in vivo and a greater half-life compared to un-modified GRF⁴. Human GRF (or GRF1-44) may be regarded as a ‘reference’ GRF, and was used as a template for many GRF analogs. However, its modification in respect to the isoforms of other species may confer benefits. For example, substituting the first two amino acids for those of the murine sequence reportedly inhibits metabolism by a peptidase, thus improving stability in cell culture studies². Additionally, replacing the eighth residue in the human sequence (asparagine) with serine, (as in the rat sequence) threonine (mouse) or glutamine (synthetic) results in further stability and enhanced conformity to one peptide conformation². Substituting alanine for the original glycine at the fifteenth residue is thought to increase receptor binding². Another measure of GRF optimization is truncation, as outlined above. This may make improve many factors of potency for this molecule². GRF has other targets beside GH. The administration of CJC-1295 has resulted in significant increases in the release of IGF-1³. Endogenous GRF, GH and IGF-1 form a regulatory ‘axis’ that may also control pro-inflammatory mechanisms when activated⁵. The peptide has also demonstrated the ability to reduce growth deficits in a mouse model of GHRH knockout. Knockout mice treated with 2µg CJC-1295 daily for five weeks achieved significantly increased total body weight and length compared to placebo-treated knockout animals⁶. The body weight and length of knockout mice treated daily with CJC-1295 was also comparable to ‘normal’ untreated mice⁶. Therefore, modGRF may also have profound effects on growth in experimental animals. It may also be used to assess the abnormal response to GRF that has been observed in rodent models of obesity⁷. Rats with genetically-induced obesity have exhibited significant decreases in GH release following treatment with GRF in a time-dependent manner (i.e. at eight weeks following birth compared to six weeks)⁷. In summary, modGRF is a useful lab-grade peptide that may be applied to studies of growth, development and metabolic disorders. References:

1. Sackmann-Sala L, Ding J, Frohman LA, Kopchick JJ. Activation of the GH/IGF-1 axis by CJC-1295, a long acting GHRH analog, results in serum protein profile changes in normal adult subjects. Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society. 2009;19(6):471-477.
2. Campbell RM, Stricker P, Miller R, et al. Enhanced stability and potency of novel growth hormone-releasing factor (GRF) analogues derived from rodent and human GRF sequences. Peptides. 1994;15(3):489-495.
3. Ionescu M, Frohman LA. Pulsatile Secretion of Growth Hormone (GH) Persists during Continuous Stimulation by CJC-1295, a Long-Acting GH-Releasing Hormone Analog. The Journal of Clinical Endocrinology & Metabolism. 2006;91(12):4792-4797.
4. 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.
5. Qin YJ, Chan SO, Chong KKL, 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.
6. Alba M, Fintini D, Sagazio A, et al. Once-daily administration of CJC-1295, a long-acting growth hormone-releasing hormone (GHRH) analog, normalizes growth in the GHRH knockout mouse. American journal of physiology. Endocrinology and metabolism. 2006;291(6):E1290-1294.
7. Renier G, Gaudreau P, Deslauriers N, Petitclerc D, Brazeau P. Dynamic of the GRF-induced GH response in genetically obese Zucker rats: influence of central and peripheral factors. Regulatory peptides. 1990;28(1):95-106.

1. CJC-1295 NO-DAC Product Page. Bluesky Peptides. 2016

Follistatin 344 (or FS-344) acts as a precursor to the protein follistatin when administered to animals as a laboratory-grade peptide. Mature follistatin regulates the release of follicle-stimulating hormone (FSH) through the inhibition of activin¹. Naturally, activin (also known as activin-A) is classically viewed as an FSH-level promoter, although it may have other roles in accordance to the part of the body in which it is present. Therefore, FS-344 may be present in any tissue that also contains activin (according to some studies on rats). These include skeletal muscle, several vital organs, the thymus and part of the cerebral cortex, although the majority of follistatin molecules may be found in the gonads and anterior pituitary². Follistatin also has other targets in vivo, the best-studied of which are myostatin and growth differentiation factor-11 (GDF-11)¹. This may be due to the close homogeneity in structure between activin and these additional proteins³. Myostatin and GDF-11 were also perceived as similar in function at one time, although more up-to-date research indicates that they have wildly different roles in mammalian systems³. Myostatin is associated with muscle development after birth, whereas GDF-11 has more diverse functions in development³. This suggests a diverse range of future applications for follistatin and for derivatives such as FS-344. There are a number of applications for FS-344 in the laboratory and in animal studies. These may mostly concern experiments on the roles of activin in various tissues, due to the influence of follistatin on this protein. FS-344 is composed of all the amino acids that can comprise the protein, whereas species-specific isoforms are spliced or further processed into slightly smaller variants (e.g. FS-315)⁴. The presence of FS-344 mRNA was originally confirmed in porcine studies⁵. It has also been found in rats, and stood out at the time as the most abundant messenger RNA associated with follistatin precursors (of which there are indeed others, e.g. FS-317) in this animal². FS-344 also distinguishes itself through a novel 27-amino acid tail at the C-terminus, which other smaller follistatin precursors lack⁵. Follistatin derivatives have also recently attracted attention as research agents in murine models of muscle growth and development^1,6. This is due to the inhibitory effects of the protein on myostatin, which has a role in the negative regulation of cardiac and skeletal muscle mass³. Some groups have also reported results suggesting roles for follistatin and/or its derivatives in inflammatory processes, scar tissue formation, hair growth and tissue re-modeling^4,7,8. The anti-inflammatory effects of follistatin are most likely to be due to its binding of activin, which has been shown to be released in response to animal models of immune-system challenge⁸. Follistatin may also reduce mortality in these animals, due to the neutralization of activin and, thus, increased inflammation⁸. However, activin may also promote tissue re-modeling. Therefore, follistatin may be used to selectively modulate tissue regeneration, leading to the improved control of wound healing and fibrosis in animals affected by disorders of the relevant processes⁸. Few formal studies have been conducted to elucidate the pharmacokinetics or pharmacodynamics of FS-344. However, a study of the same of the splice-variant FS-315 suggests that follistatin exhibits rapid clearance and limited distribution⁴. Therefore, local (e.g. intramuscular for muscle mass and/or regeneration studies) as opposed to systemic administration may be advisable when incorporating FS-344 into a study. References:

1. Yaden BC, Croy JE, Wang Y, et al. Follistatin: a novel therapeutic for the improvement of muscle regeneration. J Pharmacol Exp Ther. 2014;349(2):355-371.
2. Michel U, Albiston A, Findlay JK. Rat follistatin: gonadal and extragonadal expression and evidence for alternative splicing. Biochem Biophys Res Commun. 1990;173(1):401-407.
3. Walker RG, Poggioli T, Katsimpardi L, et al. Biochemistry and Biology of GDF11 and Myostatin: Similarities, Differences, and Questions for Future Investigation. Circulation research. 2016;118(7):1125-1142.
4. Datta-Mannan A, Yaden B, Krishnan V, Jones BE, Croy JE. An engineered human follistatin variant: insights into the pharmacokinetic and pharmocodynamic relationships of a novel molecule with broad therapeutic potential. J Pharmacol Exp Ther. 2013;344(3):616-623.
5. Shimasaki S, Koga M, Esch F, et al. Porcine follistatin gene structure supports two forms of mature follistatin produced by alternative splicing. Biochem Biophys Res Commun. 1988;152(2):717-723.
6. Tsuchida K. Myostatin inhibition by a follistatin-derived peptide ameliorates the pathophysiology of muscular dystrophy model mice. Acta Myol. 2008;27:14-18.
7. Fumagalli M, Musso T, Vermi W, et al. Imbalance between activin A and follistatin drives postburn hypertrophic scar formation in human skin. Experimental dermatology. 2007;16(7):600-610.
8. de Kretser DM, O'Hehir RE, Hardy CL, Hedger MP. The roles of activin A and its binding protein, follistatin, in inflammation and tissue repair. Molecular and cellular endocrinology. 2012;359(1-2):101-106.

GHRP-2 vs GHRP-6 Growth hormone releasing peptides (GHRPs) are relatively small molecules associated with the ability to affect the growth hormone secretagogue (GHS) system in some animals. Most GHRPs are made of six amino acids, and were developed with the goal of GHS enhancement and/or study. Their sequences are chosen for the ability to interact with the growth hormone secretagogue receptor (GHS-R) and to elicit some of the downstream effects of growth hormone release (e.g. increased IGF-1 levels). Therefore, either may be useful when applied to studies investigating the various potential roles of the GHS (also known as ghrelin) in mammalian systems. These are thought to include cardioprotection, the prevention of dysfunctional angiogenesis, the control of leptin signalling and cholesterol levels in circulation ^1-3. GHRP-2 and GHRP-6 are two particularly well-studied synthetic GHS analogs. They are comparable in size (818 and 873Da respectively^i,ii). They are both available as high-quality research-grade compunds, available as lyophilized white solids^i,ii. Both peptides are associated with the classic effects of GHS-R activation (i.e. significant increases in GH and IGF-1 concentrations) and may also be associated with significant increases in GHS-R mRNA levels in cardiac tissues⁴. They have similar sequences, but have structural differences⁵. Both peptides have been found to act in tandem with GH-releasing factor (GRF or GHRH) to release GH from the rat pituitary⁵. Both also have a lysine residue at the NH-terminal, although in the case of GHRP-6, this may be substituted for D-Lys, to generate an isoform that antagonizes the GHS-R⁶. Therefore, GHRP-6 may have a greater range of application compared to GHRP-2. Both peptides are thought to be associated with beneficial effects in response to events such as cardiac ischemia, as mentioned above. A study assessed this property using four GHRP variants, including GHRP-2 and (non-DLys) GHRP-6. These were administered subcutaneously to rats that underwent experimentally-induced chronic heart failure, and to identical control animals, at 100µg/kg twice daily for three weeks (or 100µg/kg saline as a placebo). Cardiomyocyte apoptosis (a marker of chronic heart failure) was significantly increased in placebo-treated animals compared to those treated with both GHRP-2 and GHRP-6⁴. Treatment with both ghrelin mimetics also resulted in significant decreases in the stress-related hormones norepinephrine and renin compared to placebo-treated and control animals.  GHRP-2 and -6 also had similar and significant effects on the levels of atrial natriuretic peptide, aldosterone and angiotensin-II compared to placebo-treated animals⁴. The two peptides also exhibited significant decreases in the abnormal left ventricular dimensions associated with chronic heart failure when compared to that in placebo-treated animals⁴. Treatment with both peptides also resulted in significantly improved ejection fractions compared to placebo⁴. Both peptides were also associated with non-significant improvements in diastolic wall (albeit posterior only) thickness post-surgery compared to placebo-treated animals. Both peptides elicited similar effects on GH, IGF-1 and the GSH-R concentrations in experimental animals, as might be expected⁴. These results indicate that GHRP-2 and -6 demonstrated similar abilities to improve cardiac function by significantly reducing stress and cardiomyocyte death in a model of chronic heart failure. However, neither peptide was associated with significant heart wall remodeling, as may have been expected⁴. In conclusion, GHRP-2 and GHRP-6 may have similar effects in the main locations of the GHS-R, i.e. the pituitary and in cardiac tissues. However, GHRP-6 has greater flexibility in terms of structure and receptor-binding activity when compared to GHRP-2. References:

1. Berlanga J, Cibrian D, Guevara L, et al. Growth-hormone-releasing peptide 6 (GHRP6) prevents oxidant cytotoxicity and reduces myocardial necrosis in a model of acute myocardial infarction. Clinical science (London, England : 1979). 2007;112(4):241-250.
2. Madhavadas S, Kutty BM, Subramanian S. Amyloid beta lowering and cognition enhancing effects of ghrelin receptor analog [D-Lys (3)] GHRP-6 in rat model of obesity. Indian journal of biochemistry & biophysics. 2014;51(4):257-262.
3. Zaniolo K, Sapieha P, Shao Z, et al. Ghrelin modulates physiologic and pathologic retinal angiogenesis through GHSR-1a. Investigative ophthalmology & visual science. 2011;52(8):5376-5386.
4. Xu XB, Pang JJ, Cao JM, et al. GH-releasing peptides improve cardiac dysfunction and cachexia and suppress stress-related hormones and cardiomyocyte apoptosis in rats with heart failure. American journal of physiology. Heart and circulatory physiology. 2005;289(4):H1643-1651.
5. Cheng J, Wu TJ, Butler B, Cheng K. Growth hormone releasing peptides: a comparison of the growth hormone releasing activities of GHRP-2 and GHRP-6 in rat primary pituitary cells. Life sciences. 1997;60(16):1385-1392.
6. Luque EM, Torres PJ, de Loredo N, et al. Role of ghrelin in fertilization, early embryo development, and implantation periods. Reproduction (Cambridge, England). 2014;148(2):159-167.
7. GHRP-2 Product Page. Bluesky Peptides. 2016
8. GHRP-6 Product Page. Bluesky Peptides. 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.

  Introduction Exemestane is a steroidal aromatase inhibitor (AI)¹. Unlike many other AIs, its bond to the aromatase protein is irreversible². Aromatase is associated with the final step in estrogen synthesis². Therefore, exemestane may be used to impair the production of estrogen in the study and treatment of cancers associated with the abnormal function of this hormone and/or its receptors². Exemestane may also demonstrate the efficiency of combined therapies (as opposed to monotherapies) proposed to address these diseases³. It is highly hydrophilic, and may react with the free thiol groups on the cysteine residues of many proteins².  Exemestane and Estrogen-Specific Cancers A combination of cisplatin and exemestane elicited the greatest response to treatment in a rat model of ovarian cancer³. The administration of this combination (in female Wistar rats with experimentally-induced ER-positive tumors) also resulted in significant reductions in a marker of angiogenesis, although a combination of the GnRH agonist triporelin and cisplatin was superior in this respect³. Exemestane and Other Cancers This peptide may also be effective in the treatment of other cancers with which aromatase expression is thought to be associated. This includes malignant pleural mesothelioma (MPM). A study on the effect of exemestane on cultured MPM cells found that the peptide elicited dose-dependent reductions in the proliferation and metabolic activity of these cells, which were significant at higher (35μM or more) doses⁴. This was found to be associated with a modulation of cAMP, and thus CD44, that resulted from treatment with exemestane⁴.  Exemestane and Pain Biology Many researchers have observed that treatment with exemestane and other similar AIs results in the side effect of pain. This is thought to be associated with certain TRPA channels in animals. The TRPA family of proteins is also associated with the neurobiological response to irritants such as wasabi (or its active ingredient, AITC)⁵. A study investigated the response of the TRPA1 channel, expressed on cell lines, to exemestane and other peptides in its category. Treatment with exemestane elicited the highest calcium response from the channels compared to that with anastrozole and letrozole, and resulted in an EC₅₀ of 58μM, compared to 134 for anastrozole and 69 for letrozole¹. All responses were significantly different compared to an identical placebo treatment¹. These effects were abolished by a selective antagonist for TRPA1¹. These effects were confirmed using cultured mouse and rat dorsal root ganglion neurons on which TRPA1s are present (i.e. 'pain' neurons). Exemestane and other AIs elicited similar calcium responses, with an EC₅₀ of 82μM for exemestane¹. The responses in normal mouse neurons were then compared to those from TRPA1-deficient mice. This resulted in significantly greater responses in the normal cells to exemestane, compared to that in the 'deficient' cells¹.  Exemestane and Antioxidant Pathways Exemestane has been found to have remarkable homology with inducer proteins associated with the genes regulated by the Keap1/Nrf2/ARE signaling pathway². In other words, the peptide may have potential as an antioxidant. Some researchers have demonstrated the ability of exemestane to activate the enzymes NQO1 and HO1, which may be associated with reduced damage in cultured cells following exposure to UV and hypoxia². It can also protect cells against oxidative damage caused by 4-hydroxynonenal - which is also an activator of the TRPA1 channel^2,6. Therefore, exemestane may protect against cancer development and oxidative stress, albeit with the drawback of hyperalgesia and/or various types of pain in vivo.  References:

1. Fusi C, Materazzi S, Benemei S, et al. Steroidal and non-steroidal third-generation aromatase inhibitors induce pain-like symptoms via TRPA1. Nature communications. 2014;5:5736.
2. Liu H, Talalay P. Relevance of anti-inflammatory and antioxidant activities of exemestane and synergism with sulforaphane for disease prevention. Proc Natl Acad Sci U S A. 2013;110(47):19065-19070.
3. Tkalia IG, Vorobyova LI, Grabovoy AN, Svintsitsky VS, Tarasova TO. The antitumor efficacy of cisplatin in combination with triptorelin and exemestane therapy for an ovarian cancer ascites model in Wistar rats. Experimental oncology. 2015;37(1):30-35.
4. Nuvoli B, Germoni S, Morosetti C, et al. Exemestane blocks mesothelioma growth through downregulation of cAMP, pCREB and CD44 implicating new treatment option in patients affected by this disease. Molecular cancer. 2014;13:69.
5. Kurganov E, Zhou Y, Saito S, Tominaga M. Heat and AITC activate green anole TRPA1 in a membrane-delimited manner. Pflugers Archiv : European journal of physiology. 2014;466(10):1873-1884.
6. Trevisani M, Siemens J, Materazzi S, et al. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc Natl Acad Sci U S A. 2007;104(33):13519-13524.

Introduction  CJC-1295 is an analog of the human growth-hormone releasing factor (also known as GHRH)^1,2. This synthetic peptide has selectivity for albumin (through a covalent bond with its 34th amino acid residue), thus increasing its half-life once administered². Such a property confers advantages for CJC-1295 as a research compound and possible future therapeutic agent over human GHRH. CJC-1295 has demonstrated the ability to increase the AUC for human growth hormone (hGH) that was four-fold compared to that elicited by hGHRH. Its activity can be detected in laboratory animals for a number of days after administration². The administration of CJC-1295 may also result in the increased release of insulin-like growth factor 1 (IGF-1)¹. It may also have a positive effect on the proliferation of somatotroph cells¹. CJC-1295 possesses the distinguishing feature of a modified lysine residue at the C-terminus².  CJC-1295 and Growth Promotion  Treatment with CJC-1295 may restore growth rates to nearly normal in animal models of growth hormone deficiency. A study compared the effects of 2μg subcutaneous CJC1295 given once every 24, 48 or 72 hours for five weeks in week-old GH-knockout mice¹. Similar groups of knockout and normal mice injected once daily with saline were used as controls. Knockout mice who received 2μg CJC-1295 every 24 hours exhibited significant increases in bodyweight and length compared to those injected every 48 or 72 hours, or to knockout controls¹. The weights and lengths of the '24 hour' group were not significantly different from those of the normal control mice¹. These growth-related values observed in the '48-' and '72-' hour groups were also significantly different to those in the control knockout group¹. The '24 hour' group exhibited a 13-fold increase in pituitary development (as measured by total RNA detection) compared to knockout control mice¹. This group also had an eleven-fold increase in pituitary GH expression (as indicated by mRNA detection) compared to knockout controls¹. In addition, the '24-hour' group had significantly greater concentrations of IGF-1 compared to normal controls¹. This indicates the potential of CJC-1295 to affect growth and somatotroph proliferation, provided the dose used is administered in line with the half-life of albumin (approximately 24 hours in mice)².  CJC-1295 and the Neuronal Regulation of the Pituitary  This compound may also have a role in the determination of the nerve-cell activity (e.g. which receptors are involved, etc.) underpinning the normal function of the pituitary gland, which is not as yet fully understood. For example, it is not clear how the cells of the anterior pituitary are regulated, although muscarinic acetylcholine receptors appear to be involved in this³. Animals that do not express the M₃ subtype of this receptor in brain cells exhibit a dwarfed phenotype with significantly reduced GH release and thus impaired anterior pituitary development and function⁴. 'Knockout' mice were treated with 2μg subcutaneous CJC-1295 daily for eight weeks, with similarly-treated normal mice as controls⁴. Body length and anterior pituitary size were not significantly different between these two groups⁴. GH and IGF-1 concentrations in the CJC-1295-treated knockout mice were increased at least two-fold compared to identical untreated knockout controls⁴. This supports the theory that neurons associated with the release of GHRH in the brain express M₃ muscarinic receptors. References: 1. Alba M, Fintini D, Sagazio A, et al. Once-daily administration of CJC-1295, a long-acting growth hormone-releasing hormone (GHRH) analog, normalizes growth in the GHRH knockout mouse. American Journal of Physiology - Endocrinology and Metabolism. 2006;291(6):E1290-E1294. 2. 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. 3. Giustina A, Veldhuis JD. Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human. Endocrine reviews. 1998;19(6):717-797. 4. Gautam D, Jeon J, Starost MF, et al. Neuronal M(3) muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(15):6398-6403.

Introduction Albuterol is historically linked to bronchodilation, as it activates the beta-2 subtype of the adrenergic receptor (β2-AR). It is available as two discrete isomers; S-albuterol and R-albuterol (also known as albuterol). A treatment consisting of a mixture of these isomers may have a complementary effect on therapeutic corticosteroids and a beneficial effect on inflammation, but purified S-albuterol lacks this property¹. Albuterol elicits generally beneficial effects in lung tissue through the modulation of different ions, occurring as a result of β2-AR activation. However, these same effects may precipitate into adverse events when albuterol is introduced into different tissues that also possess adrenergic receptors.  Albuterol and Cardiovascular Effects  High-dose albuterol is associated with increased risks of tachycardia and tachypnea (accelerated heart rate and breathing) and with ventricular arrythmia. This may be associated with sharp decreases in cellular potassium as a result of β2-AR activation, which may lead to increased weakness in various muscle tissue types². These effects may result in acute myocardial injury in severe cases². On the other hand, albuterol may also increase intracellular calcium in these cells, which may be associated with increased contractility in cardiac muscle³. Albuterol and Respiratory Conditions Treatment with albuterol is a well-established method in the alleviation of conditions such as asthma. Albuterol at concentrations of 10⁷M to 10⁶M significantly reduced the contraction in isolated guinea pig trachea mediated by 10⁷M to 10³M insulin⁴. The administration of this compound may also address conditions such as acute respiratory distress syndrome (ARDS) and other acute lung conditions through the regulation of sodium/potassium-ATPase (Na/K-ATPase)⁵. This enzyme is involved in the reduction of Na⁺ concentrations in alveolar spaces, thus contributing to the control of fluid accumulation and swelling in these areas of lung tissue⁵. Recent research has improved the understanding of how albuterol regulates this ATPase. A study using rat alveolar cells demonstrated the ability of the molecule to induce the influx of intracellular calcium through calcium release-activated calcium (CRAC) channels. This in turn enhances the aggregation of Na/K-ATPase at the plasma membrane of alveolar cells, which is also mediated by β2-AR activation⁵. Albuterol may also be useful in trials that determine the genes that may be involved in respiratory function and health (e.g. airway responsiveness or the regulation of inflammation). The administration of albuterol failed to change lung resistance in mice with an extra copy of the Plp gene, although it had a negative effect on this measure in normal and carrier mice⁶. This indicates a role for the gene in responsiveness. Similarly, albuterol may also be used in the study of conditions that may be associated with increased risks of respiratory dysfunction. For example, congenital cryptorchidism (or retention of the testicles in the abdominal cavity) may be comorbid with asthma symptoms in some species⁷. Treatment with albuterol significantly reduced methacholine-resistance in both rats with this condition and corresponding control animals⁷. This treatment also resulted in the increased down-regulation of interleukin-4 and -6 in the lungs of rats affected by cryptorchidism⁷. This indicates a role for albuterol in the control of respiratory inflammation for animals with co-morbid conditions. Albuterol may also be used in trials assessing novel treatments for airway constriction and other respiratory symptoms⁸.  References: 1. Ameredes BT, Calhoun WJ. Levalbuterol versus albuterol. Current allergy and asthma reports. 2009;9(5):401-409. 2. Matos J, Jenni S, Fischer N, Bienz H, Glaus T. [Myocardial damage and paroxysmal ventricular tachycardia in a dog after Albuterol intoxication]. Schweizer Archiv fur Tierheilkunde. 2012;154(7):302-305. 3. Ogrodnik J, Niggli E. Increased Ca(2+) leak and spatiotemporal coherence of Ca(2+) release in cardiomyocytes during beta-adrenergic stimulation. The Journal of physiology. 2010;588(Pt 1):225-242. 4. Sharif M, Khan BT, Ajmal K, Anwar MA. Acute effect of insulin on guinea pig airways and its amelioration by pre-treatment with salbutamol. JPMA. The Journal of the Pakistan Medical Association. 2014;64(8):932-935. 5. Keller MJ, Lecuona E, Prakriya M, et al. Calcium release-activated calcium (CRAC) channels mediate the beta(2)-adrenergic regulation of Na,K-ATPase. FEBS letters. 2014;588(24):4686-4693. 6. Rodriguez E, Sakowski L, Hobson GM, et al. Plp1 gene duplication inhibits airway responsiveness and induces lung inflammation. Pulmonary pharmacology & therapeutics. 2015;30:22-31. 7. Rodriguez E, Barthold JS, Kreiger PA, et al. The orl rat is more responsive to methacholine challenge than wild type. Pulmonary pharmacology & therapeutics. 2014;29(2):199-208. 8. Donovan C, Simoons M, Esposito J, Ni Cheong J, Fitzpatrick M, Bourke JE. Rosiglitazone is a superior bronchodilator compared to chloroquine and beta-adrenoceptor agonists in mouse lung slices. Respiratory research. 2014;15:29.

Introduction Epithalon (also referred to as epitalon or AEDG) is a small peptide made of four amino acids (i.e. a sequence of Ala-Glu-Asp-Gly). Some researchers, particularly those involved in the isolation of this tetrapeptide, claim that epithalon confers beneficial effects in the areas of cancer prevention, the treatment of age-related physiological changes and nerve cell activity¹. Epithalon may have some advantages as a research peptide, mostly due to its relatively small mass. However, its role in the management of states such as tumorigenesis and aging is uncertain at best.  Epithalon and its purported effects in Cancer Development Some scientists have claimed that epithalon may prevent tumor development in animal models of carcinogenesis. A study used an inbred mouse strain to assess this property². 61 female mice were treated with 0.5μg/ml subcutaneous epithalon five days a week for 26 weeks. The number of spontaneously-forming tumors in these mice was compared to those in 56 identical mice receiving saline as a control. The mice receiving epithalon developed fewer tumors compared to controls, and exhibited no metastases compared to three in the control mice². However, no significant differences were reported here². Treatment with epithalon was shown to have no effect in the rate of malignant lymphoma in another study using a mouse line susceptible to this condition³. Other researchers have claimed that 0.1μg epithalon given five times a week prevented spontaneous tumor formation in rats living in ambient environmental light, but not those exposed to standard laboratory or constant illumination⁴. Epithalon and Aging Aging in animals is characterized by an increased susceptibility to DNA damage, (e.g. mutations) shorter telomeres (which essentially measure the ability of the cell to replicate its DNA in the course of proliferation) and other deficiencies in normal processes that protect cells from damage and death. There are some reports claiming evidence that epithalon may conserve cell survival through the restoration of telomere and telomerase structure and function, which degrade as a cell ages⁵. The authors of another study claimed a significant (20%) decrease in the chromosomal aberration (another marker of aging) of mice carrying mutations that promote aging (SAMP-1 mice) compared to identical untreated controls⁶. Another study compared female SAMR-1 and SAMP-1 mice treated with either melatonin (a neurotransmitter that is also linked to increased protection against aging and age-related physiological irregularities) or epithalon five times weekly for four weeks to untreated control mice (although the group size was unreported)³. These authors observed no difference between the treated or control animals of either strain in metabolic parameters. However, they claimed that both epithalon and melatonin alleviated irregularities in the estrous cycle of the treated animals compared to controls³. The last 10% of the SAMP-1 mice to die exhibited an increased rate of aging and a decreased mean life span compared to SAMR-1 mice, although treatment with epithalon or melatonin resulted in increased mean and maximum lifespan in these SAMP-1 mice compared to their untreated controls³. A study of aging under different lighting conditions claimed that, while treatment with 0.1μg epithalon five times a week did not affect the survival of rats living under standard laboratory illumination regimens, it did increase the maximum survival times of rats living under either constant or ambient (i.e. natural to the surrounding environment) illumination by 24 and 95 days respectively⁴. References: 1. Vanhee C, Moens G, Van Hoeck E, Deconinck E, De Beer JO. Identification of the small research tetra peptide Epitalon, assumed to be a potential treatment for cancer, old age and Retinitis Pigmentosa in two illegal pharmaceutical preparations. Drug testing and analysis. 2015;7(3):259-264. 2. Kossoy G, Anisimov VN, Ben-Hur H, Kossoy N, Zusman I. Effect of the synthetic pineal peptide epitalon on spontaneous carcinogenesis in female C3H/He mice. In vivo (Athens, Greece). 2006;20(2):253-257. 3. Anisimov VN, Popovich IG, Zabezhinskii MA, et al. [Effect of epitalon and melatonin on life span and spontaneous carcinogenesis in senescence accelerated mice (SAM)]. Voprosy onkologii. 2005;51(1):93-98. 4. Vinogradova IA, Bukalev AV, Zabezhinski MA, Semenchenko AV, Khavinson V, Anisimov VN. Effect of Ala-Glu-Asp-Gly peptide on life span and development of spontaneous tumors in female rats exposed to different illumination regimes. Bulletin of experimental biology and medicine. 2007;144(6):825-830. 5. Khavinson V, Bondarev IE, Butyugov AA. Epithalon peptide induces telomerase activity and telomere elongation in human somatic cells. Bulletin of experimental biology and medicine. 2003;135(6):590-592. 6. Rosenfeld SV, Togo EF, Mikheev VS, Popovich IG, Khavinson V, Anisimov VN. Effect of epithalon on the incidence of chromosome aberrations in senescence-accelerated mice. Bulletin of experimental biology and medicine. 2002;133(3):274-276.