Development of Growth Hormone Secretagogues

http://www.100md.com 内分泌进展 2005年第3期

     The Huffington Center on Aging, Department of Molecular and Cellular Biology and Department of Medicine, Baylor College of Medicine, Houston, Texas 77030

    Abstract

    The GH secretagogues (GHS) were developed by reverse pharmacology. The objective was to develop small molecules with pharmacokinetics suitable for once-daily oral administration that would rejuvenate the GH/IGF-I axis. Neither the receptor nor the ligand that controlled pulse amplitude of hormone release was known; therefore, identification of lead structures was based on function. I reasoned that GH pulse amplitude could be increased by four possible mechanisms: 1) increasing GHRH release; 2) amplifying GHRH signaling in somatotrophs of the anterior pituitary gland; 3) reducing somatostatin release; and 4) antagonizing somatostatin receptor signaling. Remarkably, the GHS act through all four mechanisms to reproduce a young adult physiological GH profile in elderly subjects that was accompanied by increased bone mineral density and lean mass, modest improvements in strength, and improved recovery from hip fracture. Furthermore, restoration of thymic function was induced in old mice. The GHS receptor (GHS-R) was subsequently identified by expression cloning and found to be a previously unknown G protein-coupled receptor expressed predominantly in brain, pituitary gland, and pancreas. Reverse pharmacology was completed when the cloned GHS-R was exploited to identify an endogenous agonist (ghrelin) and a partial agonist (adenosine); ghsr-knockout mice studies confirmed that GHS are ghrelin mimetics.

    I. Introduction

    II. Drug Discovery by Reverse Pharmacology

    A. Selection of a model

    B. Identifying leads by selected functional screening assays

    III. Identification of Substituted Benzolactams as the First Nonpeptide GHS

    A. Chemistry

    B. In vivo studies in animal models

    C. Clinical evaluation of L-692,429

    D. Benzolactams with improved potency and oral bioavailability

    IV. Search for New Structural Leads: Privileged Structure Design Leading to Development of MK-0677

    A. Chemistry

    B. Potency and selectivity of L-163,191 in vivo

    C. L-163,255 accelerates recovery from limb immobilization in dogs

    D. L-163,255 stimulates thymic function in old mice

    V. Clinical Efficacy of MK-0677

    A. Potency and selectivity

    B. Efficacy in catabolic states

    C. Efficacy in obese subjects

    D. Treatment of osteoporosis

    E. Treatment of elderly hip fracture patients

    VI. Central Mechanism of GHS Action

    VII. Characterization and Cloning of the GHS-R

    A. Binding studies with [35S]MK-0677

    B. Identification of the GHS-R by expression cloning

    C. Structural studies and ligand binding to the GHS-R

    D. GHS-R is expressed in brain centers besides those regulating GH release

    VIII. Additional GHS

    IX. Identification of GHS-R Endogenous Ligands

    A. Ghrelin and adenosine

    B. Ghrelin and the GHS-R are expressed in hypothalamic centers that regulate energy balance

    X. GHS-R Is Essential for the Orexigenic and GH-Releasing Properties of Ghrelin

    XI. Summary

    I. Introduction

    HORMONES ARE GENERALLY released episodically as peaks of differing amplitude with frequencies dependent upon biological rhythms (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). The traditional pharmaceutical approach to hormone replacement involves pharmacological treatment with the hormone. However, this results in a hormone profile in the blood that is not episodic and therefore fails to provide replacement that mimics normal physiology. Upon joining Merck Research Laboratories in 1987, I initiated a project designed to replace hormones in a physiological way by normalizing the function of the underlying regulatory feedback pathways. The GH axis was selected as the initial target because the frequency of episodic GH release is highly conserved across species, and a decline in pulse amplitude during aging is well documented (21). Hence, learning how to manipulate GH pulsatility in animal models gave promise of results that should translate to humans.

    When I initiated the project, the mechanism by which the amplitude of GH pulsatility is fine-tuned was unknown. However, it was well accepted that GHRH was the hypothalamic hormone that stimulated GH release from the pituitary gland and that overstimulation of GH release was prevented by negative feedback regulation mediated by somatostatin. Hence, it seemed likely that GH pulse amplitude would be increased by stimulating GHRH release from hypothalamic neurons, by amplifying GHRH signaling in somatotrophs of the anterior pituitary gland, and/or by attenuating somatostatin-mediated negative feedback. Because the concentration of somatotrophs in the anterior pituitary gland is higher than the concentration of GHRH neurons in the hypothalamus, and in our hands the former were easier to culture, we focused on identifying small molecules that would amplify GH release from the pituitary gland. Having decided that the function we sought for the new drug was to increase the pulse amplitude of episodic GH release, the next step was to establish methods that would identify a lead molecule.

    II. Drug Discovery by Reverse Pharmacology

    A. Selection of a model

    During an extensive literature search to seek clues to the identity of compounds that might have potential in amplifying pulsatile GH release, we discovered the work of Bowers and Momany (22) describing the synthesis of a class of small synthetic peptides based on C-amidated met- and leu-enkephalins. Their studies culminated in the development of a synthetic hexapeptide, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6; Fig. 1, structure 1) that stimulated GH release in vitro and in vivo by an unknown mechanism (23). The small size of this peptide seemed ideal for the design of a peptidomimetic; therefore, my laboratory investigated the mechanism of action of GHRP-6 to determine whether it might be a suitable prototype of a drug capable of increasing the amplitude of episodic GH release.

    Upon investigating its mechanism of action, we determined that GHRP-6 appeared to act through a novel receptor. Its activity was not blocked by the opiate receptor antagonist naloxone; furthermore, it was not a GHRH receptor agonist or a somatostatin receptor (sst) antagonist (24, 25, 26). Subsequently, my laboratory showed that GHRP-6 had two very important properties that made it an ideal prototype for the design of small molecules that would increase the amplitude of endogenous GH pulsatility; remarkably, in pituitary cells GHRP-6 amplified the GHRH signal transduction pathway and behaved as a functional antagonist of somatostatin (24, 27, 28).

    We sought a drug candidate with high oral bioavailability and pharmacokinetics suitable for once daily administration. Although GHRP-6 itself had properties consistent with an amplifier of GH release, GHRP-6 had poor oral bioavailability (0.3%) and short in vivo half-life (20 min) in humans (29). Furthermore, as a peptide it did not readily lend itself to optimization of pharmacokinetic properties by medicinal chemistry. Hence, GHRP-6 was selected only as a model structure; our objective was to design a nonpeptide mimetic. The probability of identifying a nonpeptide mimetic of GHRH was considered low because native GHRH is a 44-amino acid peptide, and the smallest known homolog to exhibit biological activity was a 29-mer (30, 31).

    The GHRP-6 template also seemed ideal because it had been demonstrated that nonpeptide antagonists of small peptides could be designed based on a benzodiazepine template (32). However, an issue to be overcome at this time (1989) was the perceived difficulty of designing nonpeptide agonist mimetics. Fortunately, despite considerable resistance from some quarters, with the help of a few enthusiastic medicinal chemists, our efforts met with early success (33). Indeed, the design of GHRP-6 mimetics was considered a milestone achievement, because once we showed that such nonpeptide mimetics could be made, many more examples were forthcoming. For example, nonpeptide agonists of the peptides cholecystokinin, angiotensin, somatostatin, and melanocortin were developed (34, 35, 36, 37, 38).

    B. Identifying leads by selected functional screening assays

    We sought lead structures that would amplify the GH-releasing capacity of GHRH on somatotrophs and/or would functionally antagonize somatostatin. However, the conventional approach for identifying lead structures through high-volume screening of chemical libraries was not feasible. Although our model was GHRP-6, the receptor activated by GHRP-6 was unknown. Furthermore, the binding of radiolabeled GHRP-6 did not yield the high-affinity, limited capacity binding to pituitary membranes that was characteristic of a specific GHRP-6 receptor. We were also frustrated in our attempts to identify established cell lines that transduced a signal in response to GHRP-6. It was therefore necessary to screen for function using primary cultures of rat pituitary cells, using GH secretion as the endpoint.

    To determine the specificity of GH-releasing lead structures by discriminating between pituitary cell membrane depolarizing agents, GHRH mimetics, and GHRP-6 mimetics, it was necessary to develop secondary and tertiary counterscreens. The counterscreens were based on the use of GHRH and GHRP-6 antagonists, GHRH/GHRP-6 cross- desensitization assays, and characterization of signal transduction pathways. Hence, the selectivity of prototype GHRP-6 nonpeptide mimetic lead structures was determined according to the following: 1) GH-releasing activity on primary rat pituitary cells should be blocked by antagonists of GHRP-6 but not by GHRH antagonists (33); 2) like GHRP-6, but in contrast to GHRH, signal transduction should be mediated by phospholipase C, resulting in production of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (25); 3) lead structures should produce should produce a synergistic effect on both GHRH-stimulated cAMP production and release of GH from pituitary cells (24); 4) activity of lead structure on pituitary cells should be blocked by preincubation with GHRP-6 but not GHRH (26); and 5) a lead structure should behave as a functional antagonist of somatostatin by causing depolarization of somatotrophs (27, 39).

    III. Identification of Substituted Benzolactams as the First Nonpeptide GHS

    A. Chemistry

    To model the GHRP-6 structure, potential nonpeptide lead structures were selected by focusing on a benzodiazepine-like template containing aromatic substitutions. Based on structure activity relationships derived from the GHRPs, it was clear that a basic amine at position 1 was critical for GHRP stimulation of GH release. Aromatic amino acids were preferred at positions 2, 4, and 5, and the location of D-Trp at position 2 transformed the original opioid peptide to a GHS (22). These parameters were used to select nonpeptide compounds from the Merck & Co. chemical sample collection for evaluation in the rat pituitary cell assay. From directed screening of approximately 100 compounds, a substituted racemic benzolactam (Fig. 2, structure 2) was identified that over a series of concentrations increased GH secretion from rat pituitary cells to yield an EC50 of 3 μM. An increase in potency was afforded by substituting tetrazole for the carboxylic acid side chain. Synthesis of the R-enantiomer furnished the first potent nonpeptide GHS (L-692,429; Fig. 2, structure 3), which displayed an EC50 of 60 nM. The S-enantiomer was inactive, suggesting that the biological activity of L-692,429 was receptor mediated. The discovery of L-692,429 was considered a seminal breakthrough because it showed that a small molecule peptidomimetic agonist for GHRP-6 could be designed (33).

    B. In vivo studies in animal models

    In vivo potency was generally evaluated in dogs because of the ease of sequential blood sampling and the ability to conduct crossover dose-response studies in single animals. Intravenous administration of L-692,429 (0.1–1 mg/kg) showed dose-dependent stimulation of GH release to a maximum of 90-fold that of basal levels within 30 min after administration (40). After 120 min, GH levels returned to baseline. Besides GH, relatively moderate increases in cortisol and ACTH were observed, but no changes in prolactin, LH, insulin, T3, or T4 were noted.

    C. Clinical evaluation of L-692,429

    L-692,429 underwent preclinical safety and toxicology studies to test the concept that nonpeptide mimetics of GHRP-6 would be effective in humans. After demonstration of its safety, L-692,429 was administered iv to the targeted clinical population: young adults, elderly adults, obese subjects, and patients treated with glucocorticoids (41). A dose-dependent increase in GH release was observed in all subjects after single iv administration of L-692,429. Elderly subjects, obese individuals, and those dosed with prednisone were less responsive than healthy young adults, but all groups produced a robust response. To evaluate the potential of a long-acting GHS, L-692,429 was infused for 24 h, and serum GH levels were measured every 15 min to assess effects on the amplitude of episodic GH release (42). L-692,429 increased GH-pulse amplitude during the infusion. Based on these encouraging results, the medicinal chemistry effort was increased and focused on developing a compound with improved oral bioavailability and extended half-life.

    D. Benzolactams with improved potency and oral bioavailability

    In parallel with clinical evaluation of L-692,429, investigations of structure activity relationships around structure 2 (Fig. 2) continued because, although the oral bioavailability of L-692,429 and serum half-life were superior to GHRP-6, the overall pharmacokinetic properties were not suitable for once daily oral administration. Enhanced potency was demonstrated by N-terminal derivatization with the (R)-2- hydroxypropyl moiety (Fig. 3, structure 4, L-692,585; EC50 = 3 nM). The in vivo potency of L-692,585 was much greater than L-692,429, with a minimum effective iv dose of 0.01 mg/kg. Transient increases in IGF-I were observed 6 h after a single dose, returning to baseline within 24 h (43). L-692,585 was also infused into guinea pigs for 12 h, and GH levels were measured at 10-min intervals (44). Increased pulse amplitude of GH release was observed; interestingly, L-692,585 administration initiated GH pulsing, showing that GHS are capable of resetting the GH self-entraining 3-h pulse frequency.

    Although with new designs the in vitro and in vivo potency of the benzolactams continued to improve, optimization of oral bioavailability in dogs proved challenging. On the hypothesis that limited oral bioavailability might be due to zwitterion formation, the tetrazole group was replaced by a carboxamide moiety (Fig. 3, structure 5; EC50 = 3 nM), which significantly improved oral bioavailability and furnished a highly potent drug candidate (45).

    IV. Search for New Structural Leads: Privileged Structure Design Leading to Development of MK-0677

    A. Chemistry

    In parallel with developing structure activity relationships for the benzolactams, alternative structural leads were sought. Evans et al. (32) had suggested that a useful approach to designing receptor agonists and antagonists was to derivatize frequently occurring units. These recurring structural units were termed "privileged structures" and had been recognized earlier by Ariens et al. (46) as hydrophobic double ring systems that contributed to receptor binding of many antagonists of biogenic amines.

    It was hypothesized by Patchett et al. (47) that derivatizing privileged structures with amino acids and small peptides might furnish agonists or antagonists for peptide receptors. The spiroindanylpiperidine was speculated to be a privileged structure, and one of the first new lead structures that screened positive in the rat pituitary cell GH release assay was structure 6 (Fig. 4). Although this molecule was a mixture of four diastereomers, it had relatively good potency (EC50 = 50 nM) (47). Structure 6 lacked high oral bioavailability, but this was improved in a D-tryptophan analog where the ureidoquinuclidine was replaced by the aminoisobutyric acid moiety (Fig. 4, structure 7; EC50 = 14 nM).

    Potency and oral bioavailability were optimized by replacing D-Trp by O-benzyl-D-serine and incorporating a methanesulfonylamido group to furnish L-163,191 (Fig. 5, structure 8; EC50 = 1.3 nM) (47). As a spiropiperidine, L-163,191 represented a new structural class of GHRP-6 mimetics. Although L-163,191 evolved from a privileged structure, it was highly selective even at concentrations above 10 μM (28, 47).

    Piperazine analogs of L-163,191 (48) and substituted spiroindanes (49) were also developed and had excellent activity in the rat pituitary cell assay; two examples of these are shown in Fig. 6 (structure 9, EC50 = 6.3 nM; and structure 10, EC50 = 1.9 nM). Compound 10 had comparable oral bioavailability to L-163,191, but shorter serum half-life in dogs (1.7 h vs. 4.7 h). Subsequent design of high-potency GHS involved replacement of the privileged structure component with 3,3-substituted piperidine (50).

    B. Potency and selectivity of L-163,191 in vivo

    In dogs, L-163,191 proved to have oral bioavailability of more than 60% and released GH reproducibly at an oral dose of 0.125 mg/kg (47). Selectivity was excellent and was similar to that previously observed for GHRPs and the benzolactam GHS. Chronic studies in dogs (once daily for 14 d) showed a significant attenuation of the GH response on d 2. Although attenuated, the increase in GH was maintained from d 2–14 and was characterized by increased amplitude of the pulsatile profile. IGF-I increased by up to 126% and was sustained during treatment with L-163,191; the daily predose IGF-I levels were always higher than in placebo-treated dogs (51). Interestingly, the modest increase in cortisol that was observed after acute dosing was not evident during chronic dosing. By contrast, when dogs were dosed with L-163,191 on alternate days, neither the GH response nor the cortisol response was attenuated, which correlated with a lack of sustained increase in IGF-I (51). Subsequent studies indicated that IGF-I feeds back negatively on GHRH neurons to attenuate GHRH release (51). Having established excellent potency, selectivity, and oral bioavailability, as well as appropriate pharmacokinetics suitable for once daily oral dosing, safety assessment studies were initiated. L-163,191 was found to have an excellent safety profile and entered clinical development as MK-0677.

    C. L-163,255 accelerates recovery from limb immobilization in dogs

    An analog of MK-0677, L-163,255 (Fig. 7, structure 11; EC50 = 1.5 nM), was used extensively in animal studies to evaluate the potential clinical utility of MK-0677. A study was designed to evaluate whether L-163,255 treatment would provide benefit during and after limb immobilization (52). The right hind-limbs of beagles were pinned for 10 wk, after which the pins were removed to allow the dogs to move freely for 5 wk. Throughout this experiment, the dogs were treated orally with placebo or L-163,255 once daily. IGF-I levels decreased in the placebo group, and the decrease was accompanied by weight loss (1.13 ± 0.19 kg). In the L-163,255 group, IGF-I increased by 60%, and weight loss was marginal (0.21 ± 0.20). Muscle strength in the immobilized limb was compared in each group by isometric torque. After 10 wk of immobilization, an equal decline in strength was observed in both groups. At wk 15, muscle strength had increased by 16% in the placebo group and by 43% in the group treated with L-163,255 (52). The improved strength correlated with the diameter of the vastus lacteralis muscle, indicating that the GHS benefited rehabilitation.

    D. L-163,255 stimulates thymic function in old mice

    The effect of the MK-0677 analog on thymic function in old mice was investigated because GH is known to enhance immune responses, either directly or through IGF-I (53, 54, 55, 56, 57). Koo et al. (58) reported beneficial effects of restoring GH levels on the immune system of old mice by administering L-163,255. They showed that chronic treatment was accompanied by partial reversal of the age-related shrinkage of the thymus and of reduced T cell production. The advantage of rejuvenating the GH/IGF-I axis was illustrated by implanting aggressively growing tumors into the mice. Treatment with the GHS reduced the rate of growth and metastases of the tumors and increased longevity of the mice (58). L-163,255 did not inhibit growth of the tumor cells in vitro; therefore, we concluded that its stimulatory effect on the mouse immune system caused inhibition of tumor growth.

    V. Clinical Efficacy of MK-0677

    A. Potency and selectivity

    Human studies with acutely administered MK-0677 revealed that GH secretion was stimulated dose dependently with a threshold oral dose of 5 mg. Transient increases in cortisol were also observed, similar to that seen with L-692,429. However, during chronic administration, as had been observed in dogs (59), IGF-I levels increased, resulting in attenuation of the GH response together with an absence of an increase in cortisol (60).

    To determine efficacy and specificity in older subjects, elderly men and women were treated orally with placebo or MK-0677 (10- or 25-mg doses) once daily for 14 d (60). Before dosing and again on d 14, GH concentrations were measured in serum at 20-min intervals for 24 h to determine pulse amplitude and frequency of release. On d 14, increased peak amplitude and 24-h GH AUC was observed without changes in pulse frequency; IGF-I was increased 40 and 60% by 10- and 25-mg doses, respectively. Serum samples collected at 20-min intervals before dosing and on d 14 were also assayed for cortisol and prolactin. Cortisol pulse amplitude, frequency, and 24-h AUC were unchanged by either 10- or 25-mg MK-0677 treatment. At the 25-mg dose, prolactin increased by 24%; small dose-dependent increases in mean fasting glucose levels were also noted; however, these changes were within the normal range. It was therefore concluded that MK-0677 had acceptable selectivity.

    B. Efficacy in catabolic states

    MK-0677 was considered to have potential for treating catabolic states. When a catabolic state is induced by dietary caloric restriction, nitrogen loss is accompanied by a decrease in IGF-I and an increase in GH (20, 61). The increase in GH is due to reduction in IGF-I-mediated negative feedback on GH release, and the decrease in circulating IGF-I is probably caused by reduced sensitivity of IGF-I producing cells in the liver to GH stimulation. However, GH resistance is not complete because treatment with exogenous GH increases IGF-I and promotes nitrogen retention (62, 63).

    MK-0677 increases GH and IGF-I, but IGF-I-mediated negative feedback prevents MK-0677 from producing major increases in GH and IGF-I that are possible by administering exogenous GH. However, by increasing the pulse amplitude of endogenous GH release to mimic normal physiology, it was believed that benefits would be achieved by administering MK-0677. To test for efficacy, a once daily oral dose (25 mg) of MK-0677 was administered to healthy young men subjected to short-term diet-induced nitrogen wasting (64). Treatment with MK-0677 for 7 d produced a sustained increase in serum concentrations of GH and IGF-I as well as reversal of nitrogen wasting. Hence, GHS might prove useful for treating wasting associated with HIV and cancer.

    C. Efficacy in obese subjects

    Obesity is associated with refractoriness to stimulation of GH release by classical stimuli; however, we had shown that acute iv administration of L-692,429 was capable of eliciting a robust GH response in obese subjects (65). Therefore, it was important to determine whether chronic oral treatment with MK-0677 would have sustained effects on the GH/IGF-I axis. Healthy obese males were treated once daily with 25 mg MK-0677 for 8 wk. Increased basal metabolic rate was observed after 2 wk, but not after 4 wk of treatment. Sustained increases in serum levels of GH, IGF-I, and IGF binding protein-3 occurred, as well as a 3-kg gain in weight. Dual energy x-ray absorptiometry scanning revealed that weight gain was explained by an increase in fat-free mass. Neither total nor visceral fat was affected. Although food intake was not measured, the sustained increase in fat-free mass is likely explained by increased appetite and deposition of calories into lean tissue. The finding that MK-0677 increased weight as lean mass, coupled with reversal of nitrogen loss observed in the diet-induced catabolism study described in Section V.B. (64), suggest that long-acting GHS, like MK-0677, would provide benefit to cancer cachexia patients.

    D. Treatment of osteoporosis

    Because GH is known to stimulate bone turnover and activate osteoblast activity, it was postulated that administration of MK-0677 together with an inhibitor of bone resorption would increase the rate of bone formation. Hence, the combination should provide enhanced benefit in women with postmenopausal osteoporosis. The effects of MK-0677, alone and in combination with the bisphosphonate alendronate, were evaluated during 12 and 18 months of treatment in a multicenter, randomized, double-blind, placebo-controlled study. A total of 292 women (ages, 64–85 yr) with low femoral neck bone mineral density (BMD) participated in the study (66). To evaluate efficacy, serum IGF-I, biochemical markers of bone formation (osteocalcin and bone-specific alkaline phosphatase), and a marker of bone resorption [urinary N-telopeptide cross-links (NTx)], as well as BMD were measured. Women were randomly assigned to one of four daily regimens for 12 months: MK-0677 (25 mg) plus alendronate (10 mg); alendronate (10 mg); MK-677 (25 mg); or double placebo (66). Those patients receiving MK-0677 alone or placebo through month 12 received MK-0677 (25 mg) plus alendronate (10 mg) for months 12–18. Other patients remained on their assigned therapy.

    MK-0677, with or without alendronate, increased IGF-I levels by 39 and 45%, respectively. Similarly, osteocalcin and urinary NTx increased by 22 and 41% relative to placebo (P < 0.05). The combination of MK-0677 and alendronate increased bone formation compared with alendronate alone, reduced the effect of alendronate on resorption (NTx), and increased BMD at the femoral neck by 4.2% compared with 2.5% with alendronate alone (P < 0.05). However, the combination did not enhance BMD of the lumbar spine, total hip, or total body beyond that achieved with alendronate alone. Before interpreting these results, it is important to remember that the study was discontinued after 18 months, which is too short a time to see optimal effects of stimulating the GH/IGF-I axis. The beneficial effect of GH treatment on BMD occurs slowly because GH stimulates bone remodeling. Indeed, GH administration to GH-deficient individuals leads first to a reduction followed by an increase in BMD, and the increase is not usually seen until after 2 yr of therapy (67). The observed increase in BMD at the femoral neck after 18 months of treatment with MK-0677 should be considered encouraging because this may translate to prevention of fractures at a sight of frequent occurrence in the elderly.

    E. Treatment of elderly hip fracture patients

    Based on results derived from studies showing that dogs treated with a close structural analog of MK-0677 recovered more quickly than placebo-treated dogs after immobilization of their hind limb, the potential benefit of MK-0677 treatment on recovery in elderly hip fracture patients was evaluated (68). A placebo-controlled, randomized, double-blind trial was implemented at 13 acute care hospitals and rehabilitation centers in England, Sweden, Denmark, Belgium, Switzerland, Canada, and the United States. A total of 161 hip fracture patients were recruited at 3 to 14 d after surgery or not more than 18 d after fracture. The entry criteria included consenting patients aged 65 and older who were ambulatory before their fracture, medically stable, and mentally competent. Patients with multiple fractures, severe trauma, diabetes mellitus, cancer, uncontrolled hypertension, congestive heart failure, or total hip replacement in the involved extremity were excluded.

    Patients were randomly assigned to daily treatment with MK-0677 or placebo for 6 months and then followed for an additional 6 months after completing therapy (69). IGF-I levels were measured, and each patient was evaluated from 6 to 26 wk using a panel of functional performance measures. These evaluations included outcome measures such as changes in the Sickness Impact Profile for Nursing Homes (SIP-NH) and ability to live independently. MK-0677 treatment caused an 84% increase in serum IGF-I. There were no significant changes between MK-0677 and placebo in improving functional performance measures or in the overall SIP-NH score, but MK-0677 treated patients showed greater improvement compared with placebo in three of four lower extremity functional performance measures, in the physical domain of the SIP-NH, and in the ability to live independently. Although it is uncertain whether clinically significant outcomes on physical function were achieved, making clear conclusions from functional studies in clinical trials with hip fracture patients is complicated by the lack of validated outcome measures and absence of a baseline assessment. Indeed, present functional performance measures may not be sufficiently responsive to be used as an endpoint in small intervention studies.

    VI. Central Mechanism of GHS Action

    Besides direct stimulation of the anterior pituitary gland resulting in increased GH release, the GHS also act on the central nervous system (41, 69). It was shown that in common with GHRP-6, both the benzolactam and spiropiperidine GHS increased the activity of GHRH and neuropeptide-Y (NPY) neurons in the arcuate nucleus as well as nonadrenergic neurons in the area postrema (70, 71, 72, 73, 74, 75, 76, 77). A series of studies established that depending on the species, GHS stimulate GHRH release or inhibit somatostatin release from arcuate neurons (28, 78, 79, 80). Hence, GHS-induced amplification of GH pulsatility is explained by induction of GHRH release from the hypothalamus, by synergistic stimulation of GH release from somatotrophs by the joint actions of GHRH and GHS, and by attenuation of hypothalamic somatostatin release and antagonism of somatostatin action on somatotrophs (28).

    To investigate the potential of GH-mediated negative feedback on MK-0677 activation of arcuate neurons and the mechanisms involved, we generated mice with the somatostatin receptor subtype 2 (sst2) gene deleted (81). Treatment of wild-type mice with MK-0677 caused activation of c-Fos in the arcuate nucleus. However, pretreatment with GH activated c-Fos in the periventricular nucleus (PeN) but prevented MK-0677-induced activation of c-Fos in arcuate neurons. In sst2 –/– mice, GH pretreatment again increased c-Fos expression in the PeN but failed to inhibit activation of c-Fos by MK-0677. These results are consistent with GH-mediated negative feedback of GHS action being regulated by GH stimulation of somatostatin neurons in the PeN that inhibit activity of arcuate neurons through sst2 (Fig. 8). Intriguingly, hyperstimulation of the GH/IGF-I axis by high doses of GHS is prevented by IGF-I-mediated negative feedback (41). These collective observations, coupled with the demonstration that both the amplitude of GH pulsatility and IGF-I levels in elderly subjects are restored to those of young adults, but not higher, indicate that a preexisting uncharacterized physiological pathway is activated by GHS (41).

    VII. Characterization and Cloning of the GHS-R

    A. Binding studies with [35S]MK-0677

    The striking results obtained in the clinic with MK-0677 emphasized the need to understand the regulatory mechanisms involved by identifying a receptor for the GHS. Characterization of the MK-0677 receptor was accomplished by substituting 32S with 35S in MK-0677 to produce a radiolabeled molecule with specific activity of 1100 Ci/mmol (82). Incubation of porcine pituitary gland membranes with [35S]MK-0677 showed high-affinity (Kd = 140 pM), limited-capacity binding (Bmax = 8 fmol/mg protein) (83). This concentration of binding sites is extraordinarily low; the concentration is even lower in rat pituitary membranes (2 fmol/mg protein). Binding was also characterized in hypothalamic membranes where the concentration of binding sites was approximately 3-fold higher. Neither GHRH nor somatostatin decreased [35S]MK-0677 binding. The dependence of binding upon Mg2+ and allosteric inhibition of binding by GTP-S suggested that the unknown GHS-R was a G protein-coupled receptor (GPCR) (83).

    To determine whether MK-0677 binding to pituitary membranes was specific to somatotrophs, a biotinylated homolog of MK-0677 (Fig. 9, structure 12), was synthesized to determine localization by fluorescence/immunofluorescence. To investigate whether this bulky analog of MK-0677 was biologically active, it was evaluated in binding and signal transduction assays. Despite its increased bulk, the biotinylated derivative was a potent inhibitor of [35S]MK-0677 binding (IC50 = 0.2 nM) and an efficient stimulator of GH release from rat pituitary cells (EC50 = 2.5 nM) (41). To investigate the cell specificity of binding, cultured rat pituitary cells were incubated (3 min at 37 C) with compound 12 and then treated with avidin-Texas red. GH-containing cells were observed with fluorescein-tagged GH antibodies. Dual fluorophor and confocal microscopy showed that only cells containing GH were labeled with Texas red, indicating that MK-0677 selectively binds to somatotrophs.

    B. Identification of the GHS-R by expression cloning

    The extraordinarily low abundance of MK-0677 binding sites in the pituitary gland frustrated efforts to clone the GHS-R. Therefore, an expression-cloning strategy using Xenopus oocytes was adopted based on evidence that MK-0677 binds to a GPCR that couples through phospholipase C (83). A cDNA expression library was prepared from mRNA isolated from porcine pituitary gland. Pools of cRNA prepared from the pituitary library were coinjected into Xenopus oocytes with cRNA encoding G11 and cRNA encoding aequorin (84). Most importantly, reproducible detection of positive pools by activation of aequorin bioluminescence after addition of MK-0677 was absolutely dependent upon coinjection of G11 cRNA; other G-subunits such as Go, Gq, Ga13, G16, Gi1, and Gi3 given singly or in combination, were unable to reproducibly restore MK-0677 responsiveness. By reducing the complexity of each positive pool, a single clone activated by MK-0677 was identified. Sequencing of the clone showed that the receptor was a new orphan GPCR, which we named the GHS-R. The closest homology of the open reading frame with other GPCRs was to neurotensin (35%) and TRH (29%) receptors (84). Southern blotting indicated that a single highly conserved gene was present in the human, chimpanzee, bovine, rat, mouse, and pufferfish genomes (84, 85). Fluorescence in situ hybridization showed that the human GHS-R mapped to band 3Q26.2 (41).

    Cloning of the porcine GHS-R was followed by cloning the human and rat homologs from the respective cDNA libraries. In each case, two mRNA species were identified; one encoded a full-length GPCR with seven transmembrane (TM) domains, and the other lacked TM6 and TM7 (84, 86). The former was designated GHS-R1a and the latter GHS-R1b. The swine cDNA pituitary library also contained a GHS-R1b (84). The nucleotide sequences of the human and swine GHS-R1a and -1b are identical from the methionine translation initiation codon to Leu265 where the GHS-R1b sequence diverges from 1a and is fused to a short conserved reading frame of 24 amino acids followed by a stop codon. Inspection of the genomic sequences showed that GHS-R1a is encoded by two exons. TM1–5 are encoded by exon-1 and TM6 and 7 by exon-2; the intron contains a stop codon explaining the production of GHS-R1b mRNA by alternative processing of pre-mRNA. The human and swine GHS-R1a are 93% identical at the amino acid level, and the rat GHS-R1a is 96% identical to human (86). [35S]MK-0677 binding studies on membranes from cells expressing GHS-R1a and 1b showed that only GHS-R1a bound MK-0677 with high affinity. Similarly, studies designed to measure MK-0677 or GHRP-6 activation of GHS-R1b through aequorin bioluminescence in transfected cells or injected oocytes indicated that only GHS-R1a was an active receptor. The function of GHS-R1b remains to be defined.

    C. Structural studies and ligand binding to the GHS-R

    Antibodies selectively raised to the extracellular and intracellular domains established the topological orientation of the GHS-R expressed in HEK293 cells (Fig. 10). By molecular modeling and site-directed mutagenesis of the GHS-R, combined with binding and activation data obtained for each mutant with GHS of different structures, the ligand binding pocket was mapped. All of the synthetic GHS share a common binding domain in TM3, which is based on mutation E124Q that eliminates the counter-ion to a shared basic amine present in all the GHS. Confirmation of this essential interaction was demonstrated by rescue of function of the E124Q mutant by modifying MK-0677 through replacement of its side chain -NH2 with -OH (87). Analysis of data generated with other GHS-R mutants revealed contact points in TM2 (D99N), TM5 (M213K), TM6 (H280F), and extracellular loop 1 that were specific for different peptide, benzolactam, and spiroindane GHS. Hence, activation of the GHS-R does not require that the agonist binds to an identical pocket.

    D. GHS-R is expressed in brain centers besides those regulating GH release

    Tissue specificity of GHS-R expression could not be determined by Northern blot analysis; 10 μg of polyA+ RNA isolated from pituitary glands failed to produce a positive signal. The abundance of the GHS-R cDNA in a nonamplified swine pituitary gland library was estimated by PCR to be approximately 1 in 300,000; because of the low concentration of GHS-R transcripts, localization of expression was determined by ribonuclease protection assays and in situ hybridization (84, 88). GHS-R expression was predominant in the anterior pituitary gland and specific regions of the brain. In agreement with electrophysiology studies and c-Fos activation induced by GHS, GHS-R expression is observed in the arcuate nucleus. More interesting is that the GHS-R is also expressed in regions besides those generally considered to be involved in GH release. For example, expression is seen in the anterior hypothalamus, suprachiasmatic nucleus, supraoptic nucleus, ventromedial hypothalamus, dentate gyrus, CA2 and CA3 regions of the hippocampal structures, tuberomamillary nucleus, pars compacta of substantia nigra, ventral tegmental area, dorsal raphe nuclei, and median raphe nuclei (88). Recently, GHS-R expression has also been measured in T cells and in the thymus (89).

    The sites of expression of the GHS-R in the brain and thymus could have profound significance to aging. Clinical studies in humans have shown that GHS activation of the GHS-R rejuvenates the GH/IGF-I axis. It therefore seems reasonable to speculate that specific central nervous system functions might also be restored in elderly subjects. The hippocampus is enriched with neurotransmitter systems that affect memory and learning, and the substantia nigra and ventral tegmental areas are centers for the dopaminergic systems in the midbrain that affect motor control and reinforcement behavior. Furthermore, the dorsal and median raphe nuclei are enriched with serotonergic neurons that project to areas implicated in nociception and affective behaviors. GHS-R expression in the thymus and on T cells suggests that in elderly subjects immune function might be restored by treatment with GHS. Indeed, we have already seen that L-163,255 benefited thymic function in old mice (58).

    VIII. Additional GHS

    After disclosure of the peptidomimetics L-692,429 and MK-0677, as well as cloning of the MK-0677 receptor at Merck, extensive medicinal chemistry studies were initiated around GHRP-6. These were the subject of a comprehensive review (90). In more recent developments, additional structures were described (91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101). Hansen et al. (93) described NN703 (Fig. 11, structure 13), which has oral bioavailability of 30% in dogs and a plasma half-life of 4.1 h. NN703 dose-dependently increases GH release in both swine and dogs, has a high degree of GH specificity, and significantly increases weight gain. Subsequently, NN703 was shown to have oral activity in humans, but with markedly reduced potency compared with MK-0677 (98). CP-424,391 (Fig. 11, structure 14) is a novel, orally active pyrazolidinone-piperidine GHS that shows excellent in vitro and in vivo potency (101). Chronic oral administration to dogs induced dose-dependent increases in GH, IGF-I, and weight gain. CP-424,391 was subsequently evaluated in humans for treating clinical conditions that could benefit from augmentation of GH and IGF-I levels.

    Nagamine et al. (96) described oxindole derivatives as a distinct new structural class of orally active GHS (Fig. 11, structure 15). In the rat pituitary cell GH release assay the oxoindole SM-130686 stimulates GH release with an EC50 of 6.3 nM. Despite its structural difference, SM-130686 is a potent inhibitor of [35S]MK-0677 binding to the GHS-R (IC50 = 1.2 nM). Twice daily oral administration to rats for 9 d produces significant increases in GH, IGF-I, body weight, and fat-free mass. The increase in fat-free mass in rats is remarkable, and evidence was presented suggesting that SM13086 behaves as a partial agonist.

    IX. Identification of GHS-R Endogenous Ligands

    A. Ghrelin and adenosine

    Cloning of the GHS-R allowed the engineering of cell lines stably expressing the GHS-R, which were essential for identification of endogenous GHS-R ligands. Two endogenous ligands were identified in fractionated tissue extracts; ghrelin was found in stomach extracts (102) and adenosine in hypothalamic extracts (103, 104). In contrast to ghrelin and the synthetic GHS-R agonists, adenosine failed to stimulate GH release from pituitary cells.

    The GHS-R signal transduction pathways activated by ghrelin and adenosine are distinct (105). Ghrelin is a full agonist of the GHS-R and triggers intracellular second messengers coupled to a heterotrimeric G protein complex involving G11, which results in activation of phospholipase C signaling. Adenosine is a partial agonist of the GHSR-1a, acting through a binding pocket distinct from that of ghrelin (104). Analysis of the pathways involved in the regulation of GHS-R signaling showed that adenosine, in a dose-dependent manner, induces calcium mobilization from IP3-sensitive intracellular stores, but does not affect the formation of inositol phosphates. The calcium-mobilizing activity is blocked when the GHS-R-expressing cells are preincubated with cholera toxin, with MDL-12,330A, an inhibitor of adenylate cyclase, and with the protein kinase A blocker H-89. Adenosine also stimulated cAMP production in GHS-R- expressing cells. Based on these data, it was proposed that adenosine activates a GHS-R signaling pathway involving adenylate cyclase and protein kinase A resulting in phosphorylation of the IP3 receptor. Hence, depending on the agonist, the GHS-R is capable of activating different intracellular second-messenger systems. However, the synthetic ghrelin mimetics like MK-0677 and GHRP-6 share the same signal transduction pathway as ghrelin. Ghrelin, as a closer biochemical and biological mimic of the synthetic GHS-R ligands, became the focus of subsequent research.

    The production of ghrelin in the stomach focused attention on ghrelin’s potential role in obesity. Plasma ghrelin levels are influenced by nutritional status and are believed to regulate GH, appetite, and fat deposition (106, 107, 108, 109, 110). Intriguingly, low circulating ghrelin levels correlate with sustained weight loss and reduced appetite in obese humans after gastric bypass surgery (111). However, causality remains to be determined; neither ghrelin- nor ghsr- knockout mice have a lean phenotype, and neither is resistant to diet-induced obesity (112, 113). The association with obesity in humans was surprising because administration of the synthetic GHS-R ligand, MK-0677, to obese subjects increased lean mass rather than fat deposition (114).

    B. Ghrelin and the GHS-R are expressed in hypothalamic centers that regulate energy balance

    We generated ghrelin-knockout mice and in collaboration with Cowley and Horvath addressed the question of whether ghrelin was expressed in hypothalamic areas involved in regulating energy balance (112, 115). Intriguingly, immunohistochemistry showed that ghrelin production was localized to a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei (Fig. 12) (115). Notably, this unique distribution does not overlap with known hypothalamic cell populations.

    The results of these studies are consistent with ghrelin-expressing neurons sending efferents onto key hypothalamic circuits that include those producing NPY, agouti-related protein (AGRP), proopiomelanocortin (POMC), and CRH (115). Binding studies with biotinylated ghrelin showed localization of receptors in the arcuate nucleus, lateral anterior hypothalamus, and paraventricular nucleus that are mainly associated with presynaptic boutons. Axon terminals that bound ghrelin were frequently found to contain NPY. Electrophysiological recordings from neurons showed that ghrelin stimulated the activity of arcuate NPY neurons and indeed mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus. We proposed that release of ghrelin stimulates the release of orexigenic peptides and neurotransmitters, establishing a novel regulatory circuit controlling energy homeostasis (Fig. 12). The binding data showing localization of expression of ghrelin in axons adjacent to presynaptic nerve terminals, and results of electrophysiology studies support the notion that ghrelin modulates neurotransmission. These experiments describe an anatomical basis for pre- and postsynaptic interactions between ghrelin and NPY/AGRP, POMC, and CRH circuits (Fig. 12). In collaboration with Chen et al. (116), we confirmed the involvement of NPY/AGRP neurons by showing that like Ghsr-knockout mice, agrp/npy-double knockout mice are insensitive to the orexigenic effects of ghrelin.

    The observation of ghrelin-secreting neurons in the hypothalamus was controversial because many laboratories had failed to demonstrate ghrelin mRNA expression in brain sections by in situ hybridization. We addressed this issue by performing RT-PCR on RNA isolated from the brains of wild-type and ghrelin –/– mice using two sets of primers: one directed to amplify ghrelin transcripts; the other designed to amplify lacZ transcripts expressed in the disrupted ghrelin locus of the knockout mice. In wild-type mice, a weak signal corresponding to ghrelin transcripts was produced by the ghrelin primers, but not by lacZ primers. In contrast, RT-PCR on RNA from the brains of ghrelin –/– mice failed to produce a signal with ghrelin primers but produced a weak signal with lacZ primers. For comparison, the expression of ghrelin mRNA in the brain is at least 100-fold lower than in the stomach. We concluded that ghrelin is expressed at very low levels in the brain, but that mRNA abundance is too low to be detectable by in situ hybridization.

    X. GHS-R Is Essential for the Orexigenic and GH-Releasing Properties of Ghrelin

    Having discovered ghrelin using cells engineered to express the GHS-R, it was assumed that the GHS-R was the physiologically relevant receptor for ghrelin. However, this assumption was based on indirect evidence, and suggestions had been made for existence of unknown ghrelin receptor subtypes (117). Like the synthetic ligands, ghrelin stimulates GH release and appetite. However, as a 28-amino acid peptide containing a unique octanoyl modification (102), ghrelin is structurally different from MK-0677 and the synthetic peptide agonists used to characterize and clone the GHS-R. Furthermore, molecular modeling studies comparing structural features assigned from proton nuclear magnetic resonance spectroscopy of GHS-R ligands illustrate similarities, but these are not predictive of the known binding characteristics of the GHS (118). Accordingly, direct evidence was needed before the GHS-R could be named the ghrelin receptor.

    To determine directly whether the ghsr encoded the ghrelin receptor that mediated ghrelin’s in vivo orexigenic and GH-releasing properties (113, 115), we generated ghsr-knockout mice to investigate their phenotype and to determine their response to a ghrelin challenge. The ghsr –/– and wild-type mice are visibly indistinguishable in physical appearance and activity; therefore, it seems unlikely that ghrelin plays a dominant role in determining growth and body composition. This conclusion is subject to the caveat that an alternative pathway(s) might be compensating for the lack of ghrelin signaling. However, IGF-I levels are slightly lower and body weight is modestly reduced in ghsr –/– mice, which supports our hypothesis that the GHS-R is a subtle enhancer of function (41). In contrast to wild-type mice, when ghsr –/– mice were treated with ghrelin, ghrelin stimulated neither appetite nor GH release. These results showed unambiguously that the GHS-R is the ghrelin receptor that: 1) regulates the activity of GHRH neurons and GH release (113); and 2) maintains normal IGF levels during aging of young adults (113).

    The characteristics of ghsr-null mice added credence to our initial hypothesis that ghrelin mimetics were modest amplifiers of biological function (41). We had speculated, based on evidence from electrophysiology studies (77), that an endogenous GHS-R agonist regulates function by controlling the "gain" or "setpoint" of hypothalamic neurons. During aging, the gain appears to be reduced, which correlates with a decline in ghrelin levels (119, 120). Evidently, the gain can be restored by administering GHS-R agonists exogenously (121). Indeed, chronic treatment with GHS does not produce major increases in GH and IGF-I levels in young animals; nevertheless, in old rodents and elderly humans, chronic treatment is sufficient to restore GH pulse amplitude and IGF-I to levels seen in young adults (58, 60). Restoration of the GH/IGF-I axis in old mice increases the cellularity of the thymus, inhibits tumor growth and metastases, and increases longevity (58). Therefore, modest effects on neuronal activity translate to significant functional benefits on overall physiology.

    XI. Summary

    The reverse pharmacology approach for drug discovery is a unique example of the process of "bench to bedside" that was based on first establishing what function the drug should have and then understanding mechanistically how that function could be implemented by a small molecule. In this case I reasoned that amplification of endogenous GH pulsatility could be established by amplifying GHRH action and/or attenuating the negative feedback properties of somatostatin. Remarkably, the resulting research led to discovery of a small molecule that had both of these properties, as well as having two additional desirable properties: first, the property of stimulating GHRH release by modifying the membrane potential of GHRH neurons; and second, that of antagonizing somatostatin release from the hypothalamus.

    To complete the circle of reverse pharmacology, after a potent amplifier of pulsatile GH release was identified and tested in the clinic, we used this molecule to characterize and clone the receptor involved (GHS-R). The GHS-R was shown to be highly specific for GHS and is a new orphan GPCR that had little homology to any known GPCRs. To close the loop, endogenous GHS were sought, which was made possible by the availability of GHS-R cDNA clones. Using cells engineered to stably express the GHS-R, tissue extracts were screened and fractionated, which led to the isolation of the new hormone, ghrelin, in stomach extracts by Kojima and co-workers (102), and identification by my laboratory of adenosine as a partial agonist in hypothalamic extracts.

    Footnotes

    First Published Online April 6, 2005

    Abbreviations: AGRP, Agouti-related protein; BMD, bone mineral density; GHRP-6, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2; GHS, GH secretagogue(s); GHS-R, GHS receptor; GPCR, G protein-coupled receptor; IP3, inositol 1,4,5-triphosphate; NPY, neuropeptide-Y; NTx, N-telopeptide cross-links; PeN, periventricular nucleus; POMC, proopiomelanocortin; SIP-NH, Sickness Impact Profile for Nursing Homes; sst, somatostatin receptor; TM, transmembrane.

    References

    Boyar RM, Nogeire C, Fukushima D, Hellman L, Fishman J 1977 Studies of the diurnal pattern of plasma corticosteroids and gonadotropins in two cases of feminizing adrenal carcinoma: measurements of estrogen and corticosteroid production. J Clin Endocrinol Metab 44:39–45

    Spratt DI, O’Dea LS, Schoenfeld D, Butler J, Rao PN, Crowley Jr WF 1988 Neuroendocrine-gonadal axis in men: frequent sampling of LH, FSH, and testosterone. Am J Physiol 254:E658—E666

    Veldhuis JD, King JC, Urban RJ, Rogol AD, Evans WS, Kolp LA, Johnson ML 1987 Operating characteristics of the male hypothalamo-pituitary-gonadal axis: pulsatile release of testosterone and follicle-stimulating hormone and their temporal coupling with luteinizing hormone. J Clin Endocrinol Metab 65:929–941

    Lejeune-Lenain C, Van Cauter E, Desir D, Beyloos M, Franckson JR 1987 Control of circadian and episodic variations of adrenal androgens secretion in man. J Endocrinol Invest 10:267–276

    Winters SJ, Troen P 1982 Episodic luteinizing hormone (LH) secretion and the response of LH and follicle-stimulating hormone to LH-releasing hormone in aged men: evidence for coexistent primary testicular insufficiency and an impairment in gonadotropin secretion. J Clin Endocrinol Metab 55:560–565

    Winters SJ, Troen P 1986 Testosterone and estradiol are co-secreted episodically by the human testis. J Clin Invest 78:870–873

    Penny R, Goebelsmann U 1984 Effect of estradiol on plasma melatonin levels. J Endocrinol Invest 7:55–57

    West CD, Mahajan DK, Chavre VJ, Nabors CJ, Tyler FH 1973 Simultaneous measurement of multiple plasma steroids by radioimmunoassay demonstrating episodic secretion. J Clin Endocrinol Metab 36:1230–1236

    Rosenfield RL, Helke JC 1974 Small diurnal and episodic fluctuations of the plasma free testosterone level in normal women. Am J Obstet Gynecol 120:461–465

    Ehara Y, Siler T, VandenBerg G, Sinha YN, Yen SS 1973 Circulating prolactin levels during the menstrual cycle: episodic release and diurnal variation. Am J Obstet Gynecol 117:962–970

    Refetoff S, Van Cauter E, Fang VS, Laderman C, Graybeal ML, Landau RL 1985 The effect of dexamethasone on the 24-hour profiles of adrenocorticotropin and cortisol in Cushing’s syndrome. J Clin Endocrinol Metab 60:527–535

    Westerlund J, Gylfe E, Bergsten P 1997 Pulsatile insulin release from pancreatic islets with nonoscillatory elevation of cytoplasmic Ca2+. J Clin Invest 100:2547–2551

    Vermeulen A, Deslypere JP, Kaufman JM 1989 Influence of antiopioids on luteinizing hormone pulsatility in aging men. J Clin Endocrinol Metab 68:68–72

    Kitamura N, Shigeno C, Shiomi K, Lee K, Ohta S, Sone T, Katsushima S, Tadamura E, Kousaka T, Yamamoto I 1990 Episodic fluctuation in serum intact parathyroid hormone concentration in men. J Clin Endocrinol Metab 70:252–263

    Licinio J, Negrao AB, Mantzoros C, Kaklamani V, Wong ML, Bongiorno PB, Mulla A, Cearnal L, Veldhuis JD, Flier JS, McCann SM, Gold PW 1998 Synchronicity of frequently sampled, 24-h concentrations of circulating leptin, luteinizing hormone, and estradiol in healthy women. Proc Natl Acad Sci USA 95:2541–2546

    Mantzoros CS, Ozata M, Negrao AB, Suchard MA, Ziotopoulou M, Caglayan S, Elashoff RM, Cogswell RJ, Negro P, Liberty V, Wong ML, Veldhuis J, Ozdemir IC, Gold PW, Flier JS, Licinio J 2001 Synchronicity of frequently sampled thyrotropin (TSH) and leptin concentrations in healthy adults and leptin-deficient subjects: evidence for possible partial TSH regulation by leptin in humans. J Clin Endocrinol Metab 86:3284–3291

    Boesgaard S, Hagen C, Hangaard J, Andersen AN, Eldrup E 1990 Effect of dopamine and a dopamine D-1 receptor agonist on pulsatile thyrotrophin secretion in normal women. Clin Endocrinol (Oxf) 32:423–431

    Genter P, Berman N, Jacob M, Ipp E 1998 Counterregulatory hormones oscillate during steady-state hypoglycemia. Am J Physiol 275:E821—E829

    Juhl CB, Porksen N, Sturis J, Hansen AP, Veldhuis JD, Pincus S, Fineman M, Schmitz O 2000 High-frequency oscillations in circulating amylin concentrations in healthy humans. Am J Physiol Endocrinol Metab 278:E484—E490

    Ho KY, Veldhuis JD, Johnson ML, Furlanetto R, Evans WS, Alberti KG, Thorner MO 1988 Fasting enhances growth hormone secretion and amplifies the complex rhythms of growth hormone secretion in man. J Clin Invest 81:968–975

    Ho KY, Evans WS, Blizzard RM, Veldhuis JD, Merriam GR, Samojlik E, Furlanetto R, Rogol AD, Kaiser DL, Thorner MO 1987 Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab 64:51–58

    Bowers CY, Momany F, Reynolds GA, Chang D, Hong A, Chang K 1981 Structure-activity relationships of a synthetic pentapeptide that specifically releases growth hormone in vitro. Endocrinology 106:663–667

    Bowers CY, Momany FA, Reynolds GA, Hong A 1984 On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology 114:1537–1545

    Cheng K, Chan WW-S, Butler BS, Barreto A, Smith RG 1989 The synergistic effects of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on GRF stimulated growth hormone release and intracellular cAMP accumulation in rat primary pituitary cell cultures. Endocrinology 124:2791–2797

    Cheng K, Chan WW-S, Butler BS, Barreto A, Smith RG 1991 Evidence for a role of protein kinase-C in His-D-Trp-Ala-Trp-D-Phe-Lys-NH2-induced growth hormone release from rat primary pituitary cells. Endocrinology 129:3337–3342

    Blake AD, Smith RG 1991 Desensitization studies using perifused rat pituitary cells show that growth hormone releasing hormone and His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 stimulates growth hormone release through distinct receptor sites. J Endocrinol 129:11–19

    Leonard RJ, Chaung L-YP, Pong S-S 1991 Ionic conductances of identified rat somatotroph cells studies by perforated patch recording are modulated by growth hormone secretagogues. Biophys J 59:254 (Abstract)

    Smith RG, Pong S-S, Hickey GJ, Jacks TM, Cheng K, Leonard RJ, Cohen CJ, Arena JP, Chang CH, Drisko JE, Wyvratt Jr MJ, Fisher MH, Nargund RP, Patchett AA 1996 Modulation of pulsatile GH release through a novel receptor in hypothalamus and pituitary gland. Recent Prog Horm Res 51:261–286

    Bowers CY, Alster DK, Frentz JM 1992 The growth hormone-releasing activity of a synthetic hexapeptide in normal men and short statured children after oral administration. J Clin Endocrinol Metab 74:292–298

    Ling N, Baird A, Wehrenberg WB, Ueno N, Munegumi T, Brazeau P 1984 Synthesis and in vitro bioactivity of C-terminal deleted analogs of human growth hormone-releasing factor. Biochem Biophys Res Commun 123:854–861

    Campbell RM, Lee Y, Rivier J, Heimer EP, Felix AM, Mowles TF 1991 GRF analogs and fragments: correlation between receptor binding, activity and structure. Peptides 12:569–574

    Evans BE, Rittle KE, Bock MG, DiPardo RM, Freidinger RM, Whitter WL, Lundell GF, Veber DF, Anderson PS, Chang RSL, Lotti VJ, Cerino DJ, Chen TB, Kling PJ, Kunkel KA, Springer JP, Hirshfield J 1988 Methods for drug discovery: development of potent, selective orally effective cholecystokinin antagonists. J Med Chem 31:2235–2246

    Smith RG, Cheng K, Schoen WR, Pong S-S, Hickey GJ, Jacks TM, Butler BS, Chan WW-S, Chaung L-YP, Judith F, Taylor AM, Wyvratt Jr MJ, Fisher MH 1993 A nonpeptidyl growth hormone secretagogue. Science 260:1640–1643

    Henke BR, Aquino CJ, Birkemo LS, Croom DK, Dougherty Jr RW, Ervin GN, Grizzle MK, Hirst GC, James MK, Johnson MF, Queen KL, Sherrill RG, Sugg EE, Suh EM, Szewczyk JW, Unwalla RJ, Yingling J, Willson TM 1997 Optimization of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepines as potent, orally active CCK-A agonists. J Med Chem 40:2706–2725

    Perlman S, Schambye HT, Rivero RA, Greenlee WJ, Hjorth SA, Schwartz TW 1995 Non-peptide angiotensin agonist. Functional and molecular interaction with the AT1 receptor. J Biol Chem 270:1493–1496

    Yang L, Berk SC, Rohrer SP, Mosley RT, Gui L, Arison BH, Birzin ET, Hayes EC, Mitra SW, Parmar RM, Cheng K, Wu T-J, Butler BS, Foor R, Pasternak A, Pat Y, Silva M, Freidinger RM, Smith RG, Chapman K, Shaeffer JM, Patchett AA 1998 The design and biological activities of potent peptidomimetics selective for somatostatin receptor subtype-2. Proc Natl Acad Sci USA 95:10836–10841

    Rohrer SP, Birzin ET, Mosley RT, Berk SC, Hutchins SM, Shen DM, Xiong Y, Hayes EC, Parmar RM, Foor F, Mitra SW, Degrado SJ, Shu M, Klopp JM, Cai SJ, Blake A, Chan WW, Pasternak A, Yang L, Patchett AA, Smith RG, Chapman KT, Schaeffer JM 1998 Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry. Science 282:737–740

    Sebhat IK, Martin WJ, Ye Z, Barakat K, Mosley RT, Johnston DB, Bakshi R, Palucki B, Weinberg DH, MacNeil T, Kalyani RN, Tang R, Stearns RA, Miller RR, Tamvakopoulos C, Strack AM, McGowan E, Cashen DE, Drisko JE, Hom GJ, Howard AD, MacIntyre DE, van der Ploeg LH, Patchett AA, Nargund RP 2002 Design and pharmacology of N-[(3R)-1,2,3,4-tetrahydroisoquinolinium-3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine (1), a potent, selective, melanocortin subtype-4 receptor agonist. J Med Chem 45:4589–4593

    Pong S-S, Chaung L-YP, Smith RG, GHRP-6 (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) stimulates growth hormone secretion by depolarization in rat pituitary cell cultures. Program of the 73rd Annual Meeting of The Endocrine Society, Washington, DC, 1991, p 88 (Abstract)

    Hickey GJ, Jacks TM, Judith F, Taylor J, Clark JN, Smith RG 1994 In vivo efficacy and specificity of L-692,429, a novel nonpeptidyl growth hormone secretagogue in beagles. Endocrinology 134:695–701

    Smith RG, Van der Ploeg LH, Howard AD, Feighner SD, Cheng K, Hickey GJ, Wyvratt Jr MJ, Fisher MH, Nargund RP, Patchett AA 1997 Peptidomimetic regulation of growth hormone secretion. Endocr Rev 18:621–645

    Chapman IM, Hartman ML, Pezzoli SS, Thorner MO 1996 Enhancement of pulsatile growth hormone secretion by continuous infusion of a growth hormone-releasing peptide mimetic, L-692,429, in older adults. A clinical research center study. J Clin Endocrinol Metab 81:2874–2880

    Jacks TM, Hickey GJ, Judith F, Taylor J, Chen HY, Krupa DA, Feeney WP, Schoen WR, Ok D, Fisher MH, Wyvratt Jr M, Smith RG 1994 Effects of acute and repeated intravenous administration of L-692,585, a novel nonpeptidyl growth hormone secretagogue, on plasma growth hormone, IGF-I, ACTH, cortisol, prolactin, insulin and thyroxine (T4) levels in beagles. J Endocrinol 143:399–406

    Fairhall KM, Mynett A, Smith RG, Robinson ICAF 1995 Consistent GH responses to repeated injections of GH-releasing hexapeptide (GHRP-6) and the nonpeptide GH secretagogue, L-692,585. J Endocrinol 145:417–426

    DeVita RJ, Bochis R, Frontier AJ, Kotliar A, Fisher MH, Schoen WR, Wyvratt MJ, Cheng K, Chan WW-S, Butler B, Jacks TM, Hickey GJ, Schleim KD, Leung L, Chen Z, Chiu LS-H, Feeney WP, Cunningham PK, Smith RG 1998 A potent, orally bioavailable benzazepinone growth hormone secretagogue. J Med Chem 41:1716–1728

    Ariens EJ, Beld AJ, Rodrigues de Miranda JF, Simonis AM 1979 The pharmacon-receptor-effector concept, a basis for understanding the transmission of information in biological systems. In: O’Brien RD, ed. The receptors. New York and London: Plenum Press; 33–91

    Patchett AA, Nargund RP, Tata JR, Chen M-H, Barakat KJ, Johnston DBR, Cheng K, Chan WW-S, Butler BS, Hickey GJ, Jacks TM, Scleim K, Pong S-S, Chaung L-YP, Chen HY, Frazier E, Leung KH, Chui S-HL, Smith RG 1995 The design and biological activities of L-163,191 (MK-0677): a potent orally active growth hormone secretagogue. Proc Natl Acad Sci USA 92:7001–7005

    Barakat RJ, Cheng K, Chang WW-S, Butler BS, Jacks TM, Schleim KD, Hickey GJ, Smith RG, Patchett AA, Nargund RP 1998 Synthesis and biological activities of phenyl piperazine-based peptidomimetic growth hormone secretagogues. Bioorg Med Chem Lett 8:1431–1436

    Tata JR, Lu Z, Jacks TJ, Schleim KD, Cheng K, Wei L, Chan WW-S, Butler B, Tsu N, Leung K, Chiu S-HL, Hickey G, Smith RG, Patchett AA 1997 The design and synthesis of orally active short duration spiroindane growth hormone secretagogues. Biorg Med Chem Lett 7:2319–2324

    Yang L, Morriello G, Prendergast K, Cheng K, Jacks T, Chan WW, Schleim KD, Smith RG, Patchett AA 1998 Potent 3-spiropiperidine growth hormone secretagogues. Bioorg Med Chem Lett 8:107–112

    Hickey GJ, Jacks TM, Schleim K, Frazier E, Chen HY, Krupa DA, Feeney WP, Nargund RP, Patchett AA, Smith RG 1997 Repeat administration of the growth hormone secretagogue MK-0677 increases and maintains elevated IGF-I levels in beagles. J Endocrinol 152:183–192

    Lieber RL, Jacks TM, Mohler RL, Schleim K, Haven M, Cuizon D, Gershuni DH, Lopez MA, Hora Jr D, Nargund R, Feeney W, Hickey GJ 1997 Growth hormone secretagogue increases muscle strength during remobilization after canine hindlimb immobilization. J Orthop Res 15:519–527

    Napolitano LA, Lo JC, Gotway MB, Mulligan K, Barbour JD, Schmidt D, Grant RM, Halvorsen RA, Schambelan M, McCune JM 2002 Increased thymic mass and circulating naive CD4 T cells in HIV-1-infected adults treated with growth hormone. AIDS 16:1103–1111

    Nguyen BY, Clerici M, Venzon DJ, Bauza S, Murphy WJ, Longo DL, Baseler M, Gesundheit N, Broder S, Shearer G, Yarchoan R 1998 Pilot study of the immunologic effects of recombinant human growth hormone and recombinant insulin-like growth factor in HIV-infected patients. AIDS 12:895–904

    Murphy WJ, Durum SK, Longo DL 1992 Human growth hormone promotes engraftment of murine or human T cells in severe combined immunodeficient mice. Proc Natl Acad Sci USA 89:4481–4485

    Taub DD, Tsarfaty G, Lloyd AR, Durum SK, Longo DL, Murphy WJ 1994 Growth hormone promotes human T cell adhesion and migration to both human and murine matrix proteins in vitro and directly promotes xenogeneic engraftment. J Clin Invest 94:293–300

    Clark R 1997 The somatogenic hormones and insulin-like growth factor-1: stimulators of lymphopoiesis and immune function. Endocr Rev 18:157–179

    Koo GC, Huang C, Camacho R, Trainor C, Blake JT, Sirotina-Meisher A, Schleim KD, Wu TJ, Cheng K, Nargund R, McKissick G 2001 Immune enhancing effect of a growth hormone secretagogue. J Immunol 166:4195–4201

    Jacks T, Smith R, Judith F, Schleim K, Frazier E, Chen H, Krupa D, Hora D, Jr., Nargund R, Patchett A, Hickey G 1996 MK-0677, a potent, novel, orally active growth hormone (GH) secretagogue: GH, insulin-like growth factor I, and other hormonal responses in beagles. Endocrinology 137:5284–5289

    Chapman IM, Bach MA, Van Cauter E, Farmer M, Krupa DA, Taylor AM, Schilling LM, Cole KY, Skiles EH, Pezzoli SS, Hartman ML, Veldhuis JD, Gormley GJ, Thorner MO 1996 Stimulation of the growth hormone (GH)-insulin-like growth factor-I axis by daily oral administration of a GH secretagogue (MK-0677) in healthy elderly subjects. J Clin Endocrinol Metab 81:4249–4257

    Isley WL, Underwood LE, Clemmons DR 1983 Dietary components that regulate serum somatomedin-C concentrations in humans. J Clin Invest 71:175–182

    Snyder DK, Clemmons DR, Underwood LE 1988 Treatment of obese, diet-restricted subjects with growth hormone for 11 weeks: effects on anabolism, lipolysis, and body composition. J Clin Endocrinol Metab 67:54–61

    Manson JM, Wilmore DW 1986 Positive nitrogen balance with human growth hormone and hypocaloric intravenous feeding. Surgery 100:188–197

    Murphy MG, Plunkett LM, Gertz BJ, He W, Wittreich J, Polvino WM, Clemmons DR 1998 MK-0677, an orally active growth hormone secretagogue reverses diet-induced catabolism. J Clin Endocrinol Metab 83:320–325

    Kirk SE, Gertz BJ, Schneider SH, Hartman ML, Pezzoli SS, Wittreich JM, Krupa DA, Seibold JR, Thorner MO 1997 Effect of obesity and feeding on the growth hormone response to the GH secretagogue L-692,429 in young men. J Clin Endocrinol Metab 82:1154–1159

    Murphy MG, Weiss S, McClung M, Schnitzer T, Cerchio K, Connor J, Krupa D, Gertz BJ 2001 Effect of alendronate and MK-677 (a growth hormone secretagogue), individually and in combination, on markers of bone turnover and bone mineral density in postmenopausal osteoporotic women. J Clin Endocrinol Metab 86:1116–1125

    Gillberg P, Mallmin H, Petren-Mallmin M, Ljunghall S, Nilsson AG 2002 Two years of treatment with recombinant human growth hormone increases bone mineral density in men with idiopathic osteoporosis. J Clin Endocrinol Metab 87:4900–4906

    Bach MA, Rockwood K, Zetterberg C, Thamsborg G, Hebert R, Devogelaer JP, Christiansen JS, Rizzoli R, Ochsner JL, Beisaw N, Gluck O, Yu L, Schwab T, Farrington J, Taylor AM, Ng J, Fuh V 2004 The effects of MK-0677, an oral growth hormone secretagogue, in patients with hip fracture. J Am Geriatr Soc 52:516–523

    Smith RG, Cheng K, Pong S-S, Leonard RJ, Cohen CJ, Arena JP, Hickey GJ, Chang CH, Jacks TM, Drisko JE, Robinson ICAF, Dickson SL, Leng G 1996 Mechanism of action of GHRP-6 and nonpeptidyl growth hormone secretagogues. In: Bercu BB, Walker RF, eds. Growth hormone secretagogues. Serono Symposia. New York: Springer-Verlag; 147–163

    Dickson SL, Leng G, Robinson ICAF 1993 Systemic administration of growth hormone-releasing peptide (GHRP-6) activates hypothalamic arcuate neurones. Neuroscience 53:303–306

    Dickson SL, Leng G, Dyball REJ, Smith RG 1995 Central actions of peptide and nonpeptide growth hormone secretagogues in the rat. Neuroendocrinology 61:36–43

    Willesen MG, Kristensen P, Romer J 1999 Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat. Neuroendocrinology 70:306–316

    Dickson SL, Doutrelant-Viltart O, Dyball REJ, Leng G 1996 Retrogradely labelled neurosecretory neurones of the rat hypothalamic arcuate nucleus express Fos protein following systemic injection of growth hormone (GH)-releasing peptide. J Endocrinol 151:323–331

    Dickson SL, Luckman SD 1997 Induction of c-fos messenger ribonucleic acid in neuropeptide-Y and growth hormone (GH)-releasing factor neurones in the rat arcuate nucleus following systemic injection of the GH secretagogue, GH-releasing peptid-6. Endocrinology 138:771–777

    Leng G, Bailey ART, Dickson SL, Smith RG 1997 Electrophysiological studies of the arcuate nucleus: central actions of growth-hormone secretagogues. In: Bercu BB, Walker RF, eds. Growth hormone secretagogues in clinical practice. New York: Marcel Dekker, Inc.; 173–186

    Tannenbaum GS, Lapointe M, Beaudet A, Howard AD 1998 Expression of GH secretagogue-receptors by GH-releasing hormone neurons in the mediobasal hypothalamus. Endocrinology 139:4420–4423

    Bailey ART, Von Englehardt N, Smith RG, Leng G, Dickson SL 2000 Growth hormone secretagogue activation of the arcuate nucleus and brainstem occurs via a non-noradrenergic pathway. J Neuroendocrinol 12:191–198

    Guillaume V, Magnan E, Cataldi M, Dutour A, Sauze N, Renard M, Razafindraibe H, Conte-Devolx B, Deghenghi R, Lenaerts V, Oliver C 1994 Growth hormone (GH)-releasing hormone secretion is stimulated by a new GH-releasing hexapeptide in sheep. Endocrinology 135:1073–1076

    Fletcher TP, Thomas GB, Clarke I 1996 Growth hormone-releasing hormone and somatostatin concentrations in the hypophysial portal blood of conscious sheep during the infusion of growth hormone-releasing peptide-6. Domest Anim Endocrinol 13:251–258

    Drisko JE, Faidley TD, Zhang D, McDonald TJ, Nicolich S, Hora DF, Cunningham P, Li C, Rickes E, McNamara L, Chang C, Smith RG, Hickey GJ 1999 Administration of a nonpeptidyl growth hormone secretagogue, L-163,255, changes somatostatin pattern, but has no effect on patterns of growth hormone-releasing factor in the hypophyseal-portal circulation of the conscious pig. Proc Soc Exp Biol Med 222:70–77

    Zheng H, Bailey ART, Jiang M-H, Honda K, Chen HY, Trumbauer ME, Van der Ploeg LHT, Schaeffer JM, Leng G, Smith RG 1997 Somatostatin receptor subtype-2 knockout mice are refractory to growth hormone negative feedback on arcuate neurons. Mol Endocrinol 11:1709–1717

    Dean DC, Nargund RP, Pong S-S, Chaung L-YP, Griffin PR, Melillo DG, Ellsworth RL, Van der Ploeg LHT, Patchett AA, Smith RG 1996 Development of a high specific activity sulfur-35-labeled sulfonamide radioligand that allowed the identification of a new growth hormone secretagogue receptor. J Med Chem 39:1767–1770

    Pong S-S, Chaung L-Y, Dean DC, Nargund RP, Patchett AA, Smith RG 1996 Identification of a new G-protein-linked receptor for growth hormone secretagogues. Mol Endocrinol 10:57–61

    Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong S-S, Chaung L-YP, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJS, Dean DC, Melillo DG, Patchett AA, Nargund RP, Griffin PR, DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LHT 1996 A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273:974–977

    Palyha OC, Feighner SD, Tan CP, McKee KK, Hreniuk DL, Gao Y-D, Schleim KD, Yang L, Morriello GJ, Nargund R, Patchett AA, Howard AD, Smith RG 2000 Ligand activation domain of human orphan growth hormone secretagogue receptor (GHS-R) conserved from pufferfish to humans. Mol Endocrinol 14:160–169

    McKee KK, Palyha OC, Feighner SD, Hreniuk DL, Tan CP, Phillips MS, Smith RG, Van der Ploeg LH, Howard AD 1997 Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors. Mol Endocrinol 11:415–423

    Feighner SD, Howard AD, Prendergast K, Palyha OC, Hreniuk DL, Nargund RP, Underwood D, Tata JR, Dean DC, Tan C, McKee KK, Woods JW, Patchett AA, Smith RG 1998 Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and non-peptide secretagogues. Mol Endocrinol 12:137–145

    Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ, Smith RG, Van der Ploeg LH, Howard AD 1997 Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 48:23–29

    Dixit VD, Schaffer EM, Pyle RS, Collins GD, Sakthivel SK, Palaniappan R, Lillard Jr JW, Taub DD 2004 Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J Clin Invest 114:57–66

    Nargund RP, Patchett AA, Bach MA, Murphy MG, Smith RG 1998 Peptidomimetic growth hormone secretagogues. Design considerations and therapeutic potential. J Med Chem 41:3103–3127

    Peschke B, Ankersen M, Hansen TK, Hansen BS, Lau J, Nielsen KK, Raun K 2000 New highly potent dipeptidic growth hormone secretagogues with low molecular weight. Eur J Med Chem 35:599–618

    Peschke B, Madsen K, Hansen BS, Johansen NL 1997 Aminomethylthiophene-2-carboxylic acids as dipeptide mimetic in new growth hormone secretagogues. Bioorg Med Chem Lett 7:1969–1972

    Hansen BS, Raun K, Nielsen KK, Johansen PB, Hansen TK, Peschke B, Lau J, Andersen PH, Ankersen M 1999 Pharmacological characterisation of a new oral GH secretagogue, NN703. Eur J Endocrinol 141:180–189

    Ankersen M, Johansen NL, Madsen K, Hansen BS, Raun K, Nielsen KK, Thogersen H, Hansen TK, Peschke B, Lau J, Lundt BF, Andersen PH 1998 A new series of highly potent growth hormone-releasing peptides derived from ipamorelin. J Med Chem 41:3699–3704

    Ankersen M, Peschke B, Hansen BS, Hansen TK 1997 Investigation of bioisosters of the growth hormone secretagogue L-692,429. Bioorg Med Chem Lett 7:1293–1298

    Nagamine J, Nagata R, Seki H, Nomura-Akimaru N, Ueki Y, Kumagai K, Taiji M, Noguchi H 2001 Pharmacological profile of a new orally active growth hormone secretagogue, SM-130686. J Endocrinol 171:481–489

    Peschke B, Hansen BS 1999 New growth hormone secretagogues: C-terminal modified sulfonamide-analogues of NN703. Bioorg Med Chem Lett 9:1295–1298

    Zdravkovic M, Christiansen T, Eliot L, Agersoe H, Thomsen MS, Falch JF, Sogaard B, Ynddal L, Ilondo MM 2001 The pharmacokinetics, pharmacodynamics, safety and tolerability following 7 days daily oral treatment with NN703 in healthy male subjects. Growth Horm IGF Res 11:41–48

    Peschke B, Ankersen M, Bauer M, Hansen TK, Hansen BS, Nielsen KK, Raun K, Richter L, Westergaard L 2002 The influence of conformational restriction in the C-terminus of growth hormone secretagogues on their potency. Eur J Med Chem 37:487–501

    Hansen TK, Ankersen M, Raun K, Hansen BS 2001 Highly potent growth hormone secretagogues: hybrids of NN703 and ipamorelin. Bioorg Med Chem Lett 11:1915–1918

    Pan LC, Carpino PA, Lefker BA, Ragan JA, Toler SM, Pettersen JC, Nettleton DO, Ng O, Pirie CM, Chidsey-Frink K, Lu B, Nickerson DF, Tess DA, Mullins MA, MacLean DB, DaSilva-Jardine PA, Thompson DD 2001 Preclinical pharmacology of CP-424,391, an orally active pyrazolinone-piperidine [correction of pyrazolidinone-piperidine] growth hormone secretagogue. Endocrine [Erratum (2001) 14:437] 14:121–132

    Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660

    Tullin S, Hansen BS, Ankersen M, Moller J, Von Cappelen KA, Thim L 2000 Adenosine is an agonist of the growth hormone secretagogue receptor. Endocrinology 141:3397–3402

    Smith RG, Griffin PR, Xu Y, Smith AG, Liu K, Calacay J, Feighner SD, Pong C, Leong D, Pomes A, Cheng K, Van der Ploeg LH, Howard AD, Schaeffer J, Leonard RJ 2000 Adenosine: a partial agonist of the growth hormone secretagogue receptor. Biochem Biophys Res Commun 276:1306–1313

    Carreira MC, Camina JP, Smith RG, Casanueva FF 2004 Agonist-specific coupling of growth hormone secretagogue receptor type 1a to different intracellular signaling systems. Role of adenosine. Neuroendocrinology 79:13–25

    Peino R, Baldelli R, Rodriguez-Garcia J, Rodriguez-Segade S, Kojima M, Kangawa K, Arvat E, Ghigo E, Dieguez C, Casanueva FF 2000 Ghrelin-induced growth hormone secretion in humans. Eur J Endocrinol 143:R11—R14

    Hataya Y, Akamizu T, Takaya K, Kanamoto N, Ariyasu H, Saijo M, Moriyama K, Shimatsu A, Kojima M, Kangawa K, Nakao K 2001 A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans. J Clin Endocrinol Metab 86:4552

    Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S 2001 A role for ghrelin in the central regulation of feeding. Nature 409:194–198

    Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR 2001 Ghrelin causes hyperphagia and obesity in rats. Diabetes 50:2540–2547

    Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR 2001 Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 86:5992

    Cummings DE, Weigle DS, Frayo RS, Breen PA, Ma MK, Dellinger EP, Purnell JQ 2002 Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 346:1623–1630

    Sun Y, Ahmed S, Smith RG 2003 Deletion of ghrelin impairs neither growth nor appetite. Mol Cell Biol 23:7973–7981

    Sun Y, Wang P, Zheng H, Smith RG 2004 Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl Acad Sci USA 101:4679–4684

    Svensson J, Lonn L, Jansson J-O, Murphy G, Wyss D, Krupa D, Cerchio K, Polvino W, Gertz B, Boseaus I, Sjostrom L, Bengtsson B-A 1998 Two-month treatment of obese subjects with the oral growth hormone (GH) secretagogue MK-677 increases GH secretion, fat-free mass, and energy expenditure. J Clin Endocrinol Metab 83:362–369

    Cowley MA, Smith RG, Diano S, Tschop M, Pronchuk N, Grove KL, Strasburger CJ, Bidlingmaier M, Esterman M, Heiman ML, Garcia-Segura LM, Nillni EA, Mendez P, Low MJ, Sotonyi P, Friedman JM, Liu H, Pinto S, Colmers WF, Cone RD, Horvath TL 2003 The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 37:649–661

    Chen HY, Trumbauer ME, Chen AS, Weingarth DT, Adams JR, Frazier EG, Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, Van Der Ploeg LH, Howard AD, MacNeil DJ, Qian S 2004 Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein. Endocrinology 145:2607–2612

    Papotti M, Ghe C, Cassoni P, Catapano F, Deghenghi R, Ghigo E, Muccioli G 2000 Growth hormone secretagogue binding sites in peripheral human tissues. J Clin Endocrinol Metab 85:3803–3807

    Silva Elipe MV, Bednarek MA, Gao YD 2001 1H NMR structural analysis of human ghrelin and its six truncated analogs. Biopolymers 59:489–501

    Liu YL, Yakar S, Otero-Corchon V, Low MJ, Liu JL 2002 Ghrelin gene expression is age-dependent and influenced by gender and the level of circulating IGF-I. Mol Cell Endocrinol 189:97–103

    Rigamonti AE, Pincelli AI, Corra B, Viarengo R, Bonomo SM, Galimberti D, Scacchi M, Scarpini E, Cavagnini F, Muller EE 2002 Plasma ghrelin concentrations in elderly subjects: comparison with anorexic and obese patients. J Endocrinol 175:R1–R5

    Smith RG, Feighner S, Prendergast K, Guan X, Howard A 1999 A new orphan receptor involved in pulsatile growth hormone release. Trends Endocrinol Metab 10:128–135(Roy G. Smith)

    http://www.100md.com/html/DirDu/2006/09/10/16/84/59.htm