Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: a case study with melanocortin-4 receptor agonists.

1-[(2R)-2-([[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl]amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a lambda(max) at approximately 450 nm. The species responsible for this absorbance required the presence of beta-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the k(inact) and K(I) parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution alpha to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein.

Ghrelin, a stomach-derived orexigenic hormone, has stimulated great interest as a potential target for obesity control. Pharmacological evidence indicates that ghrelin's effects on food intake are mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the central nervous system. These include intracerebroventricular application of antibodies to neutralize NPY and AgRP, and the application of an NPY Y1 receptor antagonist, which blocks some of the orexigenic effects of ghrelin. Here we describe treatment of Agrp(-/-);Npy(-/-) and Mc3r(-/-);Mc4r(-/-) double knockout mice as well as Npy(-/-) and Agrp(-/-) single knockout mice with either ghrelin or an orally active nonpeptide ghrelin agonist. The data demonstrate that NPY and AgRP are required for the orexigenic effects of ghrelin, as well as the involvement of the melanocortin pathway in ghrelin signaling. Our results outline a functional interaction between the NPY and AgRP pathways. Although deletion of either NPY or AgRP caused only a modest or nondetectable effect, ablation of both ligands completely abolished the orexigenic action of ghrelin. Our results establish an in vivo orexigenic function for NPY and AgRP, mediating the effect of ghrelin.

Spiro(indoline-3,4'-piperidine) growth hormone secretagogues as ghrelin mimetics.

A series of small molecules derived from MK-0677, a potent synthetic GHS, mimicking the N-terminal Gly-Ser-O-(n-octanoyl)-L-Ser-Phe segment of ghrelin was synthesized and tested in a binding and in a functional assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Replacement of Phe in this tetrapeptide with a spiro(indoline-3,4'-piperidine) group, Gly-Ser with 2-aminoisobutyric acid, and O-(n-octanoyl)-L-Ser with O-benzyl-D-Ser provided synthetic GHS agonists with similar functional potency as ghrelin.

Orphan G-protein-coupled receptors and natural ligand discovery.

The superfamily of seven-transmembrane-domain G-protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Each of the approximately 1000 family members found in vertebrates responds to stimuli as diverse as hormones, neurotransmitters, odorants and light, which selectively activate intracellular signaling events mediated by heterotrimeric G proteins. Because GPCRs are centrally positioned in the plasma membrane to initiate a cascade of cellular responses by diverse extracellular mediators, it is not surprising that modulation of GPCR function has been successful in the development of many marketed therapeutic agents. It has become clear that GPCRs for which a natural activating ligand has not yet been identified (orphan GPCRs) might provide a path to discovering new cellular substances that are important in human physiology. The process of 'de-orphanizing' these novel proteins has accelerated significantly and opened up new avenues for research in human physiology and pharmacology.

Immune enhancing effect of a growth hormone secretagogue.

Growth hormone (GH) has been known to enhance immune responses, whether directly or through the insulin like growth factor-1, induced by GH. Recently a nonpeptidyl small m.w. compound, a GH secretagogue (GHS), was found to induce the production of GH by the pituitary gland. In this study, we examined the effect of GHS in immunological functions of 5- to 6-wk-old and 16- to 24-month-old mice. In young mice, we observed a significant increase in PBLs, but T and B cell-proliferative responses were not consistently enhanced. The old mice, treated with GHS for 3 wk, did not show increases in peripheral lymphocytes, but they exhibited a statistically significant increase in thymic cellularity and differentiation. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect, particularly in the old mice. We have also found that GHS promoted better thymic engraftment in bone marrow transplant of SCID mice. We found more cycling cells in the spleens of treated mice, suggesting that GHS may exert its immune enhancing effect by promoting cell division in lymphoid cells. These observations ascribe to GHS a novel therapy possible for aging, AIDS, and transplant individuals, whose immune functions are compromised.

Determination of brain and plasma drug concentrations by liquid chromatography/tandem mass spectrometry.

A method is described for the evaluation of drug concentrations in plasma and brain from treated rats. The analyte is recovered from plasma or brain homogenate by liquid-liquid extraction and subsequently analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A simple experimental protocol renders the procedure valuable for obtaining information rapidly on brain penetration and plasma exposure of specific classes of compounds. This methodology has been applied to evaluate brain penetration with 30 different compounds from the same discovery program. In an attempt to increase throughput in our screening efforts, mixture dosing was evaluated. Results from single compound administration were compared with results following administration of a mixture of four compounds. Preliminary results, with specific classes of compounds, show no major differences (ranking order) in brain or plasma concentrations between mixture dosing and single compound administration, suggesting that mixture dosing could be applicable to brain penetration studies in the drug discovery phase.

Modeling directed design and biological evaluation of quinazolinones as non-peptidic growth hormone secretagogues.

Quinazolinone derivatives were synthesized and evaluated as non-peptidic growth hormone secretagogues. Modeling guided design of quinazolinone compound 21 led to a potency enhancement of greater than 200-fold compared to human growth hormone secretagogue affinity of a screening lead 4.

Ligand activation domain of human orphan growth hormone (GH) secretagogue receptor (GHS-R) conserved from Pufferfish to humans.

Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.

Growth hormone releasing substances: types and their receptors.

A series of structurally diverse growth hormone (GH) releasing substances have been synthesized that are distinct from the naturally occurring GH releasing hormone (GHRH). These synthetic molecules range from the family of GH releasing peptides and mimetics such as MK-0677. The physiological importance of these molecules and their receptor is exemplified by studies in the elderly. For example, when MK-0677 was administered chronically to 70- to 90-year-old subjects, once daily, the age-related reduced amplitude of GH pulses was reversed to that of the physiological profile typical of young adults. In 1996, the synthesis of (35)S-MK-0677 was reported and used as a ligand to characterize a common receptor (GH secretagogue receptor [GHS-R]) for the GH releasing substances. The GHS-R is distinct from the GHRH receptor. Subsequently, the GHS-R gene was cloned and shown to encode a unique G-protein coupled receptor with a deduced protein sequence that was 96% identical in human and rat. Because of the physiological importance of the GHS-R, a search for family members (FMs) was initiated and its molecular evolution investigated. Three FMs GPR38, GPR39 and FM3 were isolated from human genomic libraries. To accelerate the identification of other FMs, a vertebrate organism with a compact genome distant in evolutionary terms from humans was exploited. The pufferfish (Spheroides nephelus) genome provides an ideal model for the discovery of human genes. Three distinct full-length clones encoding proteins of significant sequence identity to the human GHS-R were cloned from the pufferfish. Remarkably, the pufferfish gene with highest sequence homology to the human receptor was activated by the hexapeptide and non-peptide ligands. These intriguing results show that the structure and function of the ligand binding pocket of the human GHS-R has been highly conserved in evolution ( approximately 400 million years) and strongly suggests that an endogenous natural ligand has been conserved. This new information is consistent with a natural ligand for the GHS-R playing a fundamentally important and conserved role in physiology.

Receptor for motilin identified in the human gastrointestinal system.

Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was found to be 52 percent identical to the human receptor for growth hormone secretagogues. The macrolide antibiotic erythromycin also interacted with the cloned motilin receptor, providing a molecular basis for its effects on the human GI tract. The motilin receptor is expressed in enteric neurons of the human duodenum and colon. Development of motilin receptor agonists and antagonists may be useful in the treatment of multiple disorders of GI motility.

Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and nonpeptide secretagogues.

Antibodies raised against an intracellular and extracellular domain of the GH secretagogue receptor (GHS-R) confirmed that its topological orientation in the lipid bilayer is as predicted for G protein-coupled receptors with seven transmembrane domains. A strategy for mapping the agonist-binding site of the human GHS-R was conceived based on our understanding of ligand binding in biogenic amine and peptide hormone G protein-coupled receptors. Using site-directed mutagenesis and molecular modeling, we classified GHS peptide and nonpeptide agonist binding in the context of its receptor environment. All peptide and nonpeptide ligand classes shared a common binding domain in transmembrane (TM) region 3 of the GHS-R. This finding was based on TM-3 mutation E124Q, which eliminated the counter-ion to the shared basic N+ group of all GHSs and resulted in a nonfunctional receptor. Restoration of function for the E124Q mutant was achieved by a complementary change in the MK-0677 ligand through modification of its amine side-chain to the corresponding alcohol. Contacts in other TM domains [TM-2 (D99N), TM-5 (M213K, S117A), TM-6 (H280F), and extracellular loop 1 (C116A)] of the receptor revealed specificity for the different peptide, benzolactam, and spiroindolane GHSs. GHS-R agonism, therefore, does not require identical disposition of all agonist classes at the ligand-binding site. Our results support the hypothesis that the ligand-binding pocket in the GHS-R is spatially disposed similarly to the well characterized catechol-binding site in the beta2-adrenergic receptor.

Tripeptide growth hormone secretagogues.

A series of C-terminus capped dipeptides and tripeptides was synthesized as growth hormone (GH) secretagogues. Among them, tripeptide Aib-D-Trp-D-homoPhe-OEt showed low nanomolar activity in the rat pituitary assay. Thus, we have demonstrated that the GH secretagogue activity of the hexa-hepta-GH releasing peptides can be mimicked at the tripeptide level.

Synthesis and biological activities of phenyl piperazine-based peptidomimetic growth hormone secretagogues.

A new class of potent, orally active phenyl piperazine-based GH secretagogues have been discovered from attempts to mimic the arrangement of the phenyl substituent in the spiroindanyl piperidine and spiroindoline sulfonamide privileged structures of 4 and 1, respectively. The best of these compounds, 18 (EC50 = 2.8 nM) is nearly as potent as MK-0677 for releasing GH from rat pituitary cells.

Growth hormone secretagogue increases muscle strength during remobilization after canine hindlimb immobilization.

Twenty-two beagles were divided into two equal groups, and the right hindlimb of each animal was immobilized at 105 degrees of knee flexion by external fixation. After 10 weeks of fixation, the device was removed, allowing free mobility for the following 5 weeks. Each day throughout the 15 weeks, one group received a growth hormone secretagogue (treatment) at a dose of 5 mg/kg, and the other received a lactose placebo (control). At weeks 0, 10, and 15, strength as indicated by maximum isometric extension torque was measured in the right hindlimb, biopsies of the vastus lateralis muscle were taken, and the dogs were weighed. Weekly blood samples were analyzed for insulin-like growth factor-1, blood urea nitrogen, and creatine phosphokinase. Between weeks 0 and 10, tetanic torque declined by about 60% (p < 0.001) in both groups, with no significant difference between the groups (p > 0.7). Between weeks 10 and 15, tetanic torque in the treated group increased by 0.81 Nm; this was significantly greater than the increase of 0.25 Nm in the placebo group (p < 0.05). The diameters of slow (type-1) and fast (type-2) fibers measured from the vastus lateralis muscle followed the same trend. At all time points, fiber diameter correlated strongly with torque; this argues against nonmuscular causes such as nerve injury for strength loss. The mean levels of insulin-like growth factor-1 increased 100% by week 4 in the treated group and remained elevated by about 60% throughout the experiment. Levels of insulin-like growth factor-1 in the placebo group decreased 30% within week 1 and remained depressed throughout the experiment. Our interpretation of these data suggests that the growth hormone secretagogue elevated levels of serum insulin-like growth factor-1, which in turn increased the size and strength of the quadriceps muscle during remobilization. These data may ultimately have therapeutic application to humans during rehabilitation after prolonged inactivity.

Repeat administration of the GH secretagogue MK-0677 increases and maintains elevated IGF-I levels in beagles.

We have reported that MK-0677 is a novel, orally active GH secretagogue that stimulates an immediate and long-lasting increase in serum GH levels in dogs. Significant elevations in IGF-I levels were associated with the increased GH secretion. Cortisol secretion was also increased following MK-0677 administration. In the current study, we determined the effect of repeat oral administration of MK-0677 on GH, IGF-I and cortisol levels; we also investigated if the GH and cortisol responses to MK-0677 are influenced by circulating IGF-I concentrations. Following the initial oral administration of MK-0677, GH secretion (area under the time-response curve (AUC) ng/ml per h) was increased 7.9- to 9.8-fold (1.0 mg/kg), 5.6-fold (0.5 mg/kg) or 3.9-fold (0.25 mg/kg). With repeat MK-0677 administration, the GH response was decreased by 41-77%; GH concentrations remained significantly above control in the 0.5 mg/kg and 1.0 mg/kg groups. Individual beagle GH profiles indicated that the increased GH concentration was associated with an amplified GH pulsatile profile. Serum IGF-I levels were significantly increased over control levels at all dosage levels by 480 min on the first day of MK-0677 administration. With repeated administration, IGF-I levels were increased up to 126% and remained elevated through 14 days, the longest treatment period evaluated. While daily MK-0677 administration appeared to increase IGF-I levels over 24 h, as evidenced by significant increases in the pretreatment IGF-I levels on days 4-14, no such increase was noted with alternate day MK-0677 administration; thus the dosage regimen modulated circulating IGF-I levels. MK-0677 stimulated increases in cortisol secretion (AUC microgram/dl per h) on the first day of treatment. A decreased cortisol response was observed following repeated daily treatment with MK-0677; in contrast, with alternate day treatment, no decrease in cortisol response to MK-0677 occurred. A marked increase in circulating IGF-I concentrations following administration of exogenous GH resulted in a significant decrease in both the GH and cortisol response to MK-0677 compared with control animals. Our findings suggested, therefore, that circulating IGF-I concentrations regulate GH and cortisol response to MK-0677. In summary, chronic oral administration of MK-0677 was associated with significant increases in GH and IGF-I levels that were maintained for the duration of the treatment. The GH profile following MK-0677 administration consisted of episodic increases above control. Compared with day 1, repeated daily treatment with MK-0677 resulted in an attenuated GH response that was associated with an increase in circulating IGF-I levels. The cortisol response was similarly reduced during chronic MK-0677 treatment, suggesting that IGF-I mediated negative feedback on both the GH and cortisol axes. The fact that similar attenuation of the GH and cortisol responses to MK-0677 on day 1 was observed if IGF-I levels were increased by treating animals with exogenous GH suggested that the attenuated response to MK-0677 that occurred during chronic treatment was mediated by increases in IGF-I rather than desensitization to MK-0677. Thus, a regulatory feedback loop apparently prevents hyperstimulation of the GH axis by MK-0677. We conclude that MK-0677 offers the potential of an orally active GH secretagogue that can maintain elevated IGF-I levels when administered chronically.

MK-0677, a potent, novel, orally active growth hormone (GH) secretagogue: GH, insulin-like growth factor I, and other hormonal responses in beagles.

MK-0677, a spiroindoline sulfonamide, is a novel, orally active GH secretagogue. The effects of MK-0677 on serum GH and other hormones after oral and iv single dose administrations in beagles were evaluated. After oral administration in a balanced eight-dog crossover study, treatment with MK-0677 significantly increased peak GH concentrations, with a 5.3-fold increase (mean +/- SEM, 10.5 +/- 1.9 ng/ml) at the 0.25 mg/kg dose, a 9.0-fold increase (18.0 +/- 3.3 ng/ml) at the 0.50 mg/kg dose, and a 15.8-fold increase (31.6 +/- 5.8 ng/ml) at the 1.0 mg/kg dose. Total GH release, expressed as the area under the curve, showed similar significant increases over the effect of the water placebo. A single oral 1 mg/kg dose in three dogs induced a mean GH peak of 27.6 +/- 1.5 ng/ml at 120 min, and GH levels remained elevated up to 360 min after treatment. Insulin-like growth factor I (IGF-I) levels were significantly increased by 30% at 480 min after treatment. Cortisol levels were increased 2.4-fold over pretreatment levels. After i.v. administration, compared to the saline control group which had a mean (+/- SEM) serum GH peak of 3.8 +/- 0.7 ng/ml, MK-0677 at 0.25 mg/kg significantly increased (P < 0.05) peak GH concentrations 20.4-fold (77.4 +/- 13.7 ng/ml). Total GH release, expressed as the area under the curve, showed a similar increase. The mean peak GH level was recorded 10 min after treatment, with GH levels elevated up to 180 min after treatment. IGF-I levels were significantly elevated by 25% 360 min after the administration of MK-0677. Cortisol levels were increased 2.3-fold over pretreatment levels. Insulin and glucose levels were higher, LH and PRL levels were unaltered, and T4 levels were marginally lower; the levels of each of these hormones remained within the normal ranges for dogs throughout the experiment. In summary, MK-0677 is a potent GH secretagogue that induces an immediate, large, long lasting increase in GH levels when administered orally or i.v. In contrast to GH-releasing peptide-6 and benzolactam secretagogues, GH levels were elevated up to 360 min after treatment, and this was associated with a significant increase in IGF-I levels. Cortisol levels were increased; however, the increases were modest compared to those in GH.

Growth hormone (GH) and insulin-like growth factor I responses after treatments with an orally active GH secretagogue L-163,255 in swine.

L-163,255 is a potent orally active spiropiperidine GH secretagogue. When administered iv or orally, L-163,255 caused GH to be increased in a dose-related manner, with a return to baseline by 90 min. After iv administrations of saline and L-163,255 at 1, 3, and 10 micrograms/kg, GH areas under the curves (GH AUCs) over 120 min were 377 +/- 136, 1151 +/- 413 (P < 0.05), 795 +/- 413 (P = NS), and 1770 +/- 416 ng.min/ml (P < 0.01), and peak GH concentrations were 8 +/- 3, 16 +/- 7 (P = NS), 17 +/- 5 (P = NS), and 43 +/- 12 ng/ml (P < 0.01), respectively. No changes in plasma cortisol concentrations were noted. After oral administrations at 3, 10, and 30 micrograms/kg, GH AUCs over 180 min were 1133 +/- 154, 1246 +/- 129 (P = NS), and 1551 +/- 210 ng.min/ml (P = NS), and peak GH concentrations were 7 +/- 2, 11 +/- 3 (P = NS), and 23 +/- 6 ng/ml (P < 0.01), respectively. After administration in feed, L-163,255 caused a dose-related increase in GH, with an initial peak observed at 60 min for both 30 and 300 micrograms/kg dose groups, and remained elevated above baseline through 180 min for the high dose group only. GH AUCS for 180 min posttreatment were 929 +/- 134 and 1897 +/- 244 ng.min/ml, and peak GH concentrations were 9 +/- 2 and 22 +/- 4 ng/ml for the 30 and 300 micrograms/kg doses prepared in 150 g feed, respectively. When provided in feed ad libitum over the 72-h period, mean plasma insulin-like growth factor I levels increased 15%, 62% (P < 0.01), and 109% (P < 0.01) in the untreated, treated with L-163,255 at 360 ppm, or treated with porcine somatotropin groups, respectively. Repeated iv administration of L-163,255 at 1 mg/kg once daily over 14 days resulted in an initial marked GH response, followed by a much reduced, but significantly elevated, GH response over the saline control values on subsequent treatment days. Repeated iv treatments with L-163,255 also resulted in an elevated insulin-like growth factor I level (approximately 60%) over that in saline controls. Compared to those in saline controls, plasma cortisol concentrations tended to be increased after the initial dose of L-163,255, but no significant increases were noted on days 7 and 14 in the L-163,255 group. The results of these studies indicate that L-163,255 is an orally active GH secretagogue suitable for long term efficacy studies in swine.

A receptor in pituitary and hypothalamus that functions in growth hormone release.

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.