Actions and Potential Therapeutic Applications of Growth Hormone – Releasing Hormone Agonists

In this article, we briefly review the identification of GHRH, provide an abridged overview of GHRH antagonists, and focus on studies with GHRH agonists. Potent GHRH agonists of JI and MR class were synthesized and evaluated biologically. Besides the induction of the release of pituitary GH, GHRH analogs promote cell proliferation and exert stimulatory effects on various tissues, which express GHRH receptors (GHRH-Rs). A large body of work shows that GHRH agonists, such as MR-409, improve pancreatic b -cell proliferation and metabolic functions and facilitate engraftment of islets after transplantation in rodents. Accordingly, GHRH agonists offer a new therapeutic approach to treating diabetes. Various studies demonstrate that GHRH agonists promote repair of cardiac tissue, producing improvement of ejection fraction and reduction of infarct size in rats, reduction of infarct scar in swine, and attenuation of cardiac hypertrophy in mice, suggesting clinical applications. The presence of GHRH-Rs in ocular tissues and neuroprotective effects of GHRH analogs in experimental diabetic retinopathy indicates their possible therapeutic applications for eye diseases. Other effects of GHRH agonists, include acceleration of wound healing, activation of immune cells, and action on the central nervous system. As GHRH might function as a growth factor, we examined effects of GHRH agonists on tumors. In vitro , GHRH agonists stimulate growth of human cancer cells and upregulate GHRH-Rs. However, in vivo , GHRH agonists inhibit growth of human cancers xenografted into nude mice and downregulate pituitary and tumoral GHRH-Rs. Therapeutic applications of GHRH analogs are discussed. The development of GHRH analogs should lead to their clinical use. Endocrinology 160: 1600 – 1612, 2019)

member of G protein-coupled receptors. Our group identified a splice variant of GHRH-Rs (SV1) in tumors and sequenced its cDNA (8,9). SV1 is found in most tumors and in normal tissues, possesses ligand-dependent and independent activity (10), and was shown to be functional as a receptor. Clinical studies established that GHRH can stimulate GH release in normal subjects (11) and in children with GH deficiency (12). Other investigations (13) proved the role of GHRH in the regulation of the GH secretion in animals, and clinical perspectives of GHRH were summarized by Gelato (14). The expression of GHRH gene and the presence of GHRH peptide were then also found not only in tumors, such as breast, ovarian, and endometrial cancers (15), but also in various extrahypothalamic tissues (16,17), hinting at other roles for GHRH, in addition to regulation of pituitary GH secretion.
Although GHRH was first identified in tumor tissues (2)(3)(4)(5), few investigators tried to explore the possible role of GHRH in carcinogenesis by the mid-1990s. At that time, our group, experienced in oncology from the work on analogs of LH-releasing hormone (LHRH), was already deeply engaged in cancer research, and we decided to initiate work on our project on the synthesis and evaluation of GHRH antagonists for possible use in cancer therapy (9). We also began a systematic synthesis of agonistic analogs of GHRH (18,19). As the biological activity of GHRH is confined to the N-terminal sequence of 29 amino acids, this sequence was used for the substitution and replacement of various amino acid residues in the solid-phase syntheses aimed at making both agonists and antagonists of GHRH (9). Our task to produce GHRH antagonists was facilitated by the previous demonstration by Robberecht et al. (20) that the replacement of Ala in position 2 of GHRH with D-arginine leads to antagonists. Our work between 1994 and 2017 resulted in several series of potent GHRH antagonists intended to inhibit tumor growth (9,(21)(22)(23). Antagonists of the Miami (MIA) series suppressed the growth after subcutaneous administration of diverse human cancer lines, xenografted into nude mice in microgram doses (23). Antagonists MIA-602 and MIA-690 were among the most potent antitumor analogs and also displayed anti-inflammatory activities (23).
The activity of the early series of agonists of GHRH with C-terminal agmatine (19) or JI class analogs with ornithine in positions 12 and 21 (18) was evaluated by the stimulation of release of GH in vitro and in vivo in rats. The agonistic GHRH activity was increased further in our analogs of MR series (24). We synthesized nearly 100 analogs of this series with modifications of the GHRH(1-29)NH 2 backbone, N-and C-terminals, and the substitutions of Apa 30 or Gab 30 C-terminal groups (24). Their GHRH activities were compared with the analog JI-38 of the previous series (18) and with GHRH(1-29)NH 2 . Agonists with N-terminal N-Me-Tyr and C-terminal methyl amide, including MR-403, MR-406, MR-409, and MR-410, were ;100 times more potent on subcutaneous administration than the parent hormone and had higher binding activity to the pituitary than GHRH(1-29)NH 2 (24). Analogs with C-terminal agmatine, such as MR-356, and analog MR-502 with C-terminal Gab 30 -NH 2 had much higher intravenous activity than JI-38 (24). In view of high endocrine activity, which would be difficult to increase further, these analogs of the MR series were deemed to be suitable for development in various medical fields and evaluation of their clinical applications. Initially, the JI class and subsequently, the MR series of GHRH agonists were used to evaluate potential clinical applications in diverse fields, including endocrinology and oncology (1), cardiology (25)(26)(27)(28)(29)(30), angiogenic therapy with stem cells (31), diabetes (32,33), wound healing (34), ophthalmology, and other applications. The actions and potential therapeutic applications of GHRH analogs are summarized in Table 1, and key investigations will be now reviewed.

Effects of GHRH Agonists on Tumor Growth
In tests in vitro, GHRH agonist MR-409 promoted the cell viability of NCI-H446 small cell lung cancer (SCLC) and HCC827 and NCI-H460 non-SCLC cells, as well as of other cancer lines, including PANC-1 pancreatic, HCT-116 and HCT-15 colorectal, and J-82 bladder cancer cells (1). MR-409 also stimulated G1-to S-phase cell-cycle transition and cyclin D1 and D2 expression and reduced cell apoptosis, whereas GHRH antagonist MIA-602 suppressed cell viability and stimulated apoptosis. The exposure of lung cancer cells to agonist MR-409 significantly stimulated the production of cellular cAMP, whereas antagonist MIA-602 decreased it (1). The levels of pGHRH-R and SV1 in lung cancers were significantly upregulated after in vitro treatment with MR-409 (1). The stimulatory activities of GHRH agonist MR-409 on tumor cells in vitro appeared to be in accord with its activating effects on various tissues (25)(26)(27)(28)(29)(30)(31)(32)(33)(34).
Surprisingly, in vivo, the effects of GHRH agonists, such as MR-409, on tumor growth turned out to be opposite (1). Daily subcutaneous administration of 5 mg agonist MR-409 to mice bearing xenografts of NCI-H446, HCC827, and NCI-H460 lung cancers significantly inhibited tumor growth by 48% to 65% after treatment of 4 to 8 weeks (1) (Fig. 1). The reduction in activated signal transducer and activator of transcription 3 (STAT3) and p21-activated kinase 1 in tumors was similar to that produced by GHRH antagonists. In animals with HCC827 and H446 tumors treated with MR-409 for 8 weeks, there was a substantial reduction in serum IGF-1 levels, but no changes were seen in H460 tumors after treatment with MR-409 for only 4 weeks. Likewise, MR-409 decreased serum IGF-1 in C57BL/6 wild-type mice (49). Likewise, treatment of nude mice bearing NCI-N87 human stomach cancers with 5 mg/day of another GHRH agonist, MR-356, lowered serum IGF-1 levels and markedly decreased tumor growth by day 63 of therapy (49). We also evaluated the effects of agonist MR-409 on in vivo tumor growth of other human cancers (1). The cancer lines tested comprised prostatic (PC-3), triple-negative breast cancers (MDA-MB231 and MX-1), colorectal (HCT-116 and HCT-15), as well as pancreatic (CFPAC-1 and PANC-1), gastric (NCI-N87), and bladder (RT-4 and J-82) cancers, which all expressed GHRH-Rs (1). The extent of inhibition of tumor growth was similar to that produced by GHRH antagonists at the same dose (1,23,36,37). Administration of MR-409 to nude mice xenografted with these tumors for 1 to 2½ months markedly decreased tumor growth (1).
Our studies also revealed that the treatment of mice bearing HCC827, H460, and H446 lung cancers with the agonist MR-409 induced a downregulation of GHRH-Rs in the pituitary gland and tumors, as measured by Western blot (1). The levels of both pGHRH-R and SV1 were reduced (Fig. 2). The reduction of GHRH-Rs in tumors was similar to that produced by GHRH antagonists MIA-602 (23,36). In contrast to findings observed in vitro, the levels of effector proteins of cell cycle (cell-cycle regulators), the expressions of cyclin D1, and D2, cyclin-dependent kinases, and activated STAT3 were reduced by the therapy with agonist MR-409, but conditional inhibitor for cyclindependent kinase 4/6 was upregulated (1).
It has been shown that GHRH, produced locally in tumors, might function as an autocrine/paracrine growth factor in lung and other cancers (2,9,35,(53)(54)(55). In vitro, GHRH agonists promote cell growth (1). Induction of tumor formation or proliferation by parenteral therapy with GHRH agonists would nullify their planned use in a wide range of medical fields. Our findings of inhibitory action of the GHRH agonist on tumors in vivo dispel these fears (1). The hindering effects of repeated administration of MR-409 and other GHRH agonists on the proliferation of various human tumors exerted in vivo relieve our grave concerns about stimulating growth of cancers with this class of compounds in the course of their clinical use in cardiology, diabetes, or other diseases and conditions. Moreover, in view of their suppressive action on tumor growth and the decline in IGF-1 level, GHRH agonists could find some applications in oncology, for instance, the potentiation of anticancer activity of traditional chemotherapeutic drugs, such as doxorubicin (56). The inhibition of tumor growth in vivo by the GHRH agonist MR-409 seems similar to that induced by GHRH antagonists MIA-602 or MIA-690 (36). This blocking of tumor growth by agonists of GHRH, which at first may seem paradoxical, appears to be caused mainly by the downregulation of the pGHRH-Rs and tumoral GHRH-Rs (1). These phenomena are thus analogous and correspond to the well-known downregulation of receptors for LHRH, induced by therapy with agonistic analogs of LHRH (57). It is thoroughly documented clinically that this event leads to suppression of the levels of LH, FSH (, and sex steroids and inhibition of growth of prostatic and breast cancers [reviewed in Schally et al. (57)]. Thus, short-term activation of GHRH-Rs by GHRH agonists in vitro leads to stimulatory effects, but chronic, long-term, persistent exposure to theses ligands produces downregulation of receptors and inhibitory effects, as pointed out by Kiaris and Chatzistamou (58). The detailed mechanism of action of downregulation of GHRH-Rs by GHRH analogs must continue to be investigated, as other phenomena, such as cell differentiation, could be also involved in inhibition of tumor growth (23,56).

Effects of GHRH Analogs on Diabetes
Diabetes currently affects more than 400 million people worldwide, with an incidence expected to rise dramatically (59). In both type 1 diabetes (T1D) and type 2 diabetes (T2D), the supply of insulin-producing tissue/cells is inadequate. Strategies aimed at the prevention of pancreatic b-cell loss and an increase in insulin production are among important approaches to treat diabetes.
The effects of GHRH on the stimulation of the secretion of the endocrine pancreas were demonstrated in the late 1980s (60,61). Direct stimulatory action of human GHRH(1-44)NH 2 on the release of insulin and glucagon from perfused dog pancreas and isolated rat islets of Langerhans, as well as the effects on glucose homeostasis in diabetic mice and normal rats, was reported by Hermansen et al. (60) and Bailey et al. (61). The mechanism of action of the effect of GHRH on insulin release was also investigated (62). Following the demonstration that the GHRH-R and its SV1 were expressed in INS-1 cells, a rat b-cell line derived from rat insulinoma, and rat and human pancreatic islets, Ludwig et al. (32) reported that GHRH agonist JI-36, one of our early analogs, could function as a potential effector for survival and proliferation of pancreatic islets. This agonist significantly increased cell proliferation, reduced cell apoptosis, and promoted glucose-responsive insulin secretion in vitro. In vivo, JI-36 improved engraftment and metabolic function of rat islets following transplantation under the kidney capsule of streptozotocin (STZ)-induced diabetic nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice (32). Further studies showed that JI-36 facilitated the function of rat islets encapsulated in a bioartificial pancreas after implantation into rats with STZ-induced diabetes (44). Following the development of the MR series of GHRH analogs (24), the activity of several MR series compounds on b-cell proliferation was tested compared with that of JI-36 (33,46). Analog MR-403 significantly increased cell viability and proliferation, reduced apoptosis on INS-1 cells, and promoted viability of rat islets when in coculture with rat adrenal cells in which the expression of the GHRH-R was demonstrated. Intra-adrenal transplantation of rat islets pretreated with MR-403 resulted in a rapid normoglycemia in STZ-induced NOD/SCID mice (46). Furthermore, analogs MR-356, MR-409, and MR-502 displayed higher potency on cell viability and stimulated more efficiently the expression of cellular insulin, IGF-1, GHRH-Rs, and glucoseresponsive insulin secretion in INS-1 cells (33). Agonist MR-409 also induced activation of phosphorylation of ERK and protein kinase B (AKT) and significantly increased the levels of cellular cAMP and the phosphorylation of cAMP response element-binding protein in INS-1 cells (33). Treatment of rat islets with agonist MR-409 significantly increased islet size and the expression of insulin. An in vivo study revealed that the maximal therapeutic benefits with respect to the efficiency of engraftment, ability to reach normoglycemia, gain in body weight, response to high glucose challenge, and induction of higher levels of serum insulin and IGF-1 were observed when STZ-induced NOD/SCID mice were transplanted with rat islets preconditioned with the GHRH agonist, MR-409, and received additional treatment with MR-409 post-transplantation (33). The study provides evidence for the beneficial action of synthetic GHRH agonists on pancreatic b-cells in vitro and in vivo (33). The results of a preliminary clinical trial with the GHRH agonist tesamorelin in patients with T2D support the safety of clinical use of GHRH agonists (52). Although the treatment with tesamorelin for 12 weeks did not alter insulin response or glycemic control, the levels of total cholesterol and non-high-density lipoprotein cholesterol decreased significantly (52). The use of GHRH agonists offers a physiologically and therapeutically simple and convenient approach to increase the viability of islet cells. Long-acting GHRH agonists may fulfill the requirements of effective drugs for improvement of engraftment of islets following transplantation. The synthetic GHRH agonists appear to allow the reduction of the islet mass needed to treat a diabetic status by transplantation (44). This effect would be a useful addition to the armamentarium for the b-cell replacement or other types of cellbased therapy for management of diabetes mellitus (63, 64) (Table 1).
Dyslipidemia, an elevation of triglyceride-rich lipoproteins in plasma, frequently accompanies diabetes mellitus and represents an important component of the disease, imposing cardiovascular risk and correlating with renal dysfunction. Romero et al. (43) reported that GHRH might play an important role in dyslipidemia associated with T1D. In STZ-induced rat T1D, GHRH-R expression was found to be upregulated in the distal small intestine, a tissue involved in chylomicron synthesis. Treatment of T1D rats with the GHRH antagonist, MIA-602, at a dose that did not affect plasma GH levels significantly reduced triglyceride-rich lipoprotein, as well as markers of renal injury, and improved endothelial-dependent vasorelaxation. MIA-602 restored glucagon-like peptide 1 actions on hyperlipidemia and hyperglucagonemia in T1D rats. These findings revealed a previously unidentified pathway in T1D mediated by GHRH, associated with impaired glucagon-like peptide 1 signaling and hyperlipidemia. Inhibition of GHRH signaling by the use of GHRH antagonists might be a promising approach to improve the dyslipidemia, kidney damage and cardiovascular diseases associated with T1D and probably T2D (43). Whereas the loss of islets is predominant in T1D, T2D is a long-term metabolic disorder characterized by dysfunction of b-cells and/or insulin resistance occurring primarily within the muscles, liver, and fat tissue. The proportion between insulin resistance and dysfunction of b-cells varies among individuals with T2D. Dyslipidemia may play a pathological role in the early stages of T2D. Further studies to reveal the effects of GHRH analogs in the development of T2D and insulin resistance in appropriate animal models of T2D would therefore be particularly important and informative (Table 1).

Cardioprotective Effects of GHRH
In 1988, Hasegawa et al. (65) showed that GHRH-at that time, defined as GH-releasing factor-displayed a direct positive inotropic effect in guinea pig papillary muscles. A decade later, we started to investigate in detail the cardiovascular effects of GHRH, also considering evidence on the cardioprotective activities of other neuropeptides, such as corticotropin-releasing factor and urocortins, or GH secretagogues, such as ghrelin and its analogs (66)(67)(68). We first examined the potential cardioprotective effects of GHRH and demonstrated the ability of GHRH(1-44)NH 2 to prevent apoptosis induced by serum deprivation and isoproterenol in adult rat ventricular myocytes (ARVMs) and H9c2 rat cardiomyocytes (CMs), expressing pGHRH-R. These effects were abolished by the GHRH antagonist JV-1-36, indicating receptor-mediated mechanisms. Accordingly, underlying signaling included an increase in adenylyl cyclase/cAMP/protein kinase A (PKA) and activation of MAPK ERK1/2 and phosphatidylinositol 3-kinase/ Akt pathways. GHRH also counteracted isoproterenolinduced elevation of the proapoptotic protein, inducible cAMP early repressor. In agreement with the in vitro findings, in isolated rat hearts subjected to ischemia/ reperfusion, preischemic administration of GHRH improved heart function and strongly reduced infarct size after reperfusion through GHRH-R and phosphatidylinositol 3-kinase/Akt-mediated mechanisms (69). We later demonstrated that postischemic treatment with GHRH also reduced infarct size and contractile dysfunction in the same model. The mechanisms included activation of the reperfusion injury salvage kinases and the survivoractivating factor-enhancement pathway (70). GHRH also promoted the phosphorylation of endothelial nitric oxide synthase and AMP-activated protein kinase and preserved postischemic nicotinamide adenine dinucleotide levels. Overall, these findings suggested potential therapeutic implication for GHRH and its analogs in myocardial infarction (MI) and heart failure (HF). Consistent with these findings, Ma et al. (31) recently showed that pretreatment of mesenchymal stem cells (MSCs) with GHRH agonist JI-34 enhanced viability and mobility of MSCs in vitro, along with increased STAT3 activity. In vivo, JI-34-pretreated MSCs showed improved engraftment into ischemic limbs and superior proangiogenic effects (31). Furthermore, Kiscsatári et al. (47) demonstrated that GHRH agonists JI-34 and MR-356 can alleviate radiation-induced damage in rat CMs. In addition, Shen et al. (48) reported that MR-409 alleviates in vivo smooth muscle cell-mediated aortic calcification in osteoprotegerin knockout mice through an increase in cAMP/PKA signaling and inhibition of inflammatory pathways.
In 2010, Kanashiro-Takeuchi et al. (26) performed a series of studies testing the hypothesis the GHRH agonists ameliorate myocardial injury as a result of acute infarction in the rat. Administration of agonist JI-38 to rats with MI, immediately following ligation of the left anterior descending coronary artery led to a reduction in infarct size and offset the decline in ejection fraction relative to placebo (Fig. 3). Importantly, administration of JI-38 for 4 weeks did not elevate either GH or IGF-1 (26). Subsequently, Kanashiro-Takeuchi et al. (25) tested whether JI-38 produced reversed remodeling post-MI in the rat model. In similar fashion to the previous study, animals were subjected to left anterior descending ligation, but administration of JI-38 was only begun 4 weeks following cardiac injury, at a time that ventricular remodeling had already matured. Importantly, even in the setting of cardiac enlargement, administration of JI-38 stimulated reverse remodeling. This was accompanied by a reduction in tissue fibrosis. Coadministration of a GHRH-R antagonist offset the impact of the agonist on the improvement in ventricular structure and function, demonstrating that this is a GHRH-R-mediated phenomenon. These two studies performed in the rat formed the basis for testing the impact of GHRH analogs in a large animal infarction model (29). In this study, female Yorkshire pigs were randomized to receive either placebo or the GHRH agonist MR-409. The dominant finding was that stimulation of the GHRH-R signaling pathway led to a 22% reduction in myocardial scar mass compared with an increase in the placebo group (Fig. 4). Accordingly, the results collectively revealed an important and potentially clinically relevant improvement in parameters of left-ventricular (LV) function and remodeling in two mammalian species. These findings support the consideration of clinical testing of GHRH agonists for the treatment of myocardial injury in humans.
Pathological cardiac hypertrophy, in response to hypertension or MI, is one of the main risks for arrhythmias, dilated cardiomyopathy, and HF (71). The role of GHRH and its agonists was studied in different in vitro and in vivo models of cardiac hypertrophy. Gesmundo et al. (30) recently demonstrated that GHRH(1-44)NH 2 counteracts phenylephrine (PE)-induced hypertrophy in H9c2 cells, ARVMs, and CMs, derived from human induced pluripotent stem cell-CMs by inhibition of the expression of hypertrophic genes and regulation of hypertrophic pathways. In H9c2 cells, GHRH blocked the a1-adrenergic receptor/Gq/phospholipase Cb hypertrophic pathway, which was elevated by PE. GHRH also attenuated the phosphorylation of prohypertrophic phospholamban at Thr17, dependent on Ca 21 /calmodulin-dependent protein kinase II. These effects were blocked by the GHRH antagonist JV-1-36, suggesting GHRH-R-mediated mechanisms; accordingly, GHRH elevated Ga s /cAMP/ PKA signaling, along with phosphorylation of anti-hypertrophic phospholamban at Ser16. Importantly, in H9c2 cells, ARVMs, and induced pluripotent stem cell-CMs, the anti-hypertrophic effects of GHRH included blockade of PE-induced expression of the exchange protein directly activated by cAMP1, a key player in hypertrophy and HF (72).
In vivo, the role of the GHRH agonistic analog MR-409 was studied in mice subjected to transverse aortic constriction (TAC), a model of pressure overload hypertrophy and HF. TAC mice treated for 2 weeks with MR-409 showed reduced hypertrophy and improved cardiac function compared with untreated mice. Furthermore, in agreement with the results in vitro, MR-409 reduced the size of CMs in TAC hearts; inhibited the expression of hypertrophic genes, including the exchange protein directly activated by cAMP1; and upregulated sarco/endoplasmic reticulum Ca 21 ATPase 2a protein that was reduced by TAC. Moreover, CMs isolated from TAC mice showed normalization of contractile responses after chronic treatment with MR-409. Overall, the results of this study suggest therapeutic use of GHRH analogs in pathological hypertrophy and HF (30) ( Table 1).

Effects of GHRH in Eye Diseases
GHRH and GHRH-Rs have been identified in several ocular tissues. In particular, Dubovy et al. (73) have analyzed postmortem human eyes and demonstrated the presence of GHRH and GHRH-Rs in different human ocular tissues, such as the following: cornea epithelium and endothelium, ciliary body, trabecular meshwork, optic nerve, and neuroretina. These studies validated the potential relevance of modulation of the GHRH-GHRH-Rs signaling in human ocular pathologies. Analogs of GHRH and GHRH-Rs have been studied in a number of eye diseases. Chu et al. (38) have shown that antagonists of GHRH promote apoptosis in retinoblastoma cells, and Qin et al. (39) determined that GHRH antagonists could mitigate ocular surface inflammation in experimental uveitis and in pterygium (40). Similar results were obtained However, of major relevance for this review are the findings of Thounaojam et al. (50), revealing that the GHRH agonistic analog MR-409 exerted neuro-and vascularprotective effects in early experimental diabetic retinopathy through a mechanism involving antioxidative and anti-inflammatory activities. In these studies, the authors showed that the expression of GHRH-R was significantly downregulated in both human postmortem retinas of diabetic donors, as well as in a rat model of T1D. Accordingly, maintenance of retinal GHRH signaling by systemic administration of MR-409 in diabetic rats halted hyperglycemia-induced retinal vascular permeability and death of retinal neurons, the retinal ganglion cells (50). Interestingly, anti-permeability effects of GHRH agonists have been previously documented in response to bacterial toxin-induced pulmonary edema (74). The observed neuroprotective effects of GHRH on retinal ganglion cells are supported by historical studies demonstrating the presence and importance of the GHRH/GH axis for the maintenance of retinal ganglion cell survival (75,76). Moreover, clinical studies have reported retinal neurovascular abnormalities in congenital GH deficiency (77). Taken together, these studies underscore the importance of the GHRH-GHRH-R signaling in ocular diseases and warrant further studies to characterize/validate better the effects of GHRH agonistic analogs for retinal neuroprotection (Table 1).

Other Effects of GHRH and Its Analogs
Based on the demonstration that agonistic analogs of GHRH increase proliferation of human fibroblasts (45) (34). Skin regeneration was likely related to an increase in migration of fibroblasts and activation of a-smooth muscle actin. These findings suggest that GHRH analogs may be considered for promotion of the healing of skin wounds. Gallo et al. (79) showed that GHRH(1-44)NH 2 prevented apoptosis and atrophy in skeletal myotubes through inhibition of proteolytic pathways, indicating a role for GHRH agonists in muscle atrophy-associated diseases. With regard to the immune system, Khorram et al. (51,80) reported that the administration of a GHRH agonist [Norleucine27]GHRH (1-29)-NH 2 to elderly subjects resulted in activation of both immune cells (51) and somatotropic axis (80). Thus, GHRH agonists could be of therapeutic benefit for stimulation of compromised immune systems in aging persons (51). Moreover, it has been shown that GHRH(1-44)NH 2 can inhibit IL-6 and increase levels of IL-17 in peripheral blood mononuclear cells (81,82).
Interestingly, a recent study reported that adipocytes from morbidly obese subjects showed a higher expression of GHRH and a lower expression of GHRH-R compared with nonobese subjects. In vitro exposure of GHRH inhibited the differentiation of human MSCs to adipocytes and promoted lipolysis in human differentiated adipocytes. These effects were blocked when the expression of mRNA for the GH receptor was silenced, suggesting effects mediated by the GH or GH receptor (83). Furthermore, it has been shown that the GHRH agonist tesamorelin reduces visceral adipose tissue in HIV-infected patients and improves liver enzymes (84,85). These findings suggest therapeutic implications for GHRH agonists in obesity and metabolic dysfunctions.
Both the agonists and antagonists of GHRH show important effects on the central nervous system, and GHRH-Rs are present in the brain cortex and other brain areas (42). Thus, Baker et al. (86) showed that the GHRH agonist tesamorelin improves cognitive function in adults with mild cognitive impairment and in healthy older adults (Table 1). Furthermore, GHRH antagonists MIA-690 and MIA-602 exhibited the capability to inhibit amyloid aggregation and proteotoxicity and decreased the deterioration in cognitive performance in a transgenic mouse model of Alzheimer's disease (42).

Perspectives and Conclusions
The findings on GHRH analogs reviewed in this article appear to have profound implications. The antagonists of GHRH are being developed for therapy of cancers but should also be evaluated in further studies on dyslipidemia, ocular pathologies, and Alzheimer's disease. The agonists of GHRH warrant continued investigations in cardiology on their possible applications in myocardial injuries, HF, and pathological hypertrophy. GHRH agonists may also find possible uses in the field of diabetes for b-cell replacement and other types of cell-based therapy. Experimental research is also revealing possible beneficial effects of agonists of GHRH in eye diseases. These various potential beneficial effects of GHRH analogs are possible because of the presence of GHRH-Rs in many cells and tissues. We can no longer ignore the many possible clinical applications of GHRH analogs, the list of which is continuously extending.