RESUMEN
A series of NN703 analogues with lysine mimetics combined with naphthyl- or biphenylalanine in the core has been prepared and tested in vitro in a rat pituitary cell based assay and subsequently in vivo in pigs in a single dose at 50 nmol/kg. Re-introduction of certain pharmacophores in the C-terminal of NN703, which were originally removed during optimisation for oral bioavailability, led to unexpectedly potent compounds in vitro as well as in vivo.
Asunto(s)
Dipéptidos/farmacología , Hormona del Crecimiento/efectos de los fármacos , Hormonas/farmacología , Indoles/farmacología , Oligopéptidos/farmacología , Receptores Acoplados a Proteínas G , Compuestos de Espiro/farmacología , Administración Oral , Animales , Disponibilidad Biológica , Células Cultivadas/citología , Células Cultivadas/metabolismo , Dipéptidos/química , Dipéptidos/metabolismo , Hormona del Crecimiento/metabolismo , Hormonas/síntesis química , Indoles/química , Indoles/metabolismo , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/metabolismo , Hipófisis/citología , Ratas , Receptores de Superficie Celular/metabolismo , Receptores de Ghrelina , Compuestos de Espiro/química , Compuestos de Espiro/metabolismo , Relación Estructura-Actividad , PorcinosRESUMEN
Repeated administration of growth hormone secretagogues (GHSs) has proven to be a delicate matter owing to development of tolerance. The aim of the present study was to define conditions during which the responsiveness to the orally active NN703 was maintained over several days. Growing pigs were fitted with stomach and vascular catheters, permitting unstressed intragastric administrations and blood sampling. NN703 or vehicle was administered once daily. When NN703 was given at a dose of 18 mg/kg, there was a massive acute increase in plasma growth hormone (GH) levels, but this was only seen on the first day of administration. A dose of 1.8 mg/kg did not cause a significant acute increase in plasma GH concentrations, whereas stimulation of pulsatile GH release was sustained over a 4-d period. During the first 7 h following injection of vehicle, the area under the curve of plasma GH was 1211+/-144 (microg/[L x 7 h]), but increased to 1770+/-269 and 1824+/-198 (microg/[L x 7 h]) on the first and fourth day of NN703 administration, respectively. Deconvolution analysis of the 7-h profiles revealed that the GH mass per burst as well as the GH burst amplitude were significantly (p < 0.001) increased during treatment with NN703, which led to an increase in pulsatile GH secretion rate (p < 0.001). Insulin-like growth factor-1 plasma concentrations increased steadily during NN703 administration (p < 0.01) and decreased after termination of treatment. The sustained increase in GH pulsatility observed with low-dose NN703 treatment suggests that development of tolerance to this GHS may be obviated by minimization of dose.
Asunto(s)
Dipéptidos/administración & dosificación , Hormona del Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/análisis , Periodicidad , Animales , Hormona del Crecimiento/sangre , Cinética , Estómago/efectos de los fármacos , PorcinosRESUMEN
Growth hormone secretagogues (GHSs) are synthetic compounds that induce GH release in several species, including man. The aim of the current study was to identify hypothalamic GHS receptor (GHS-R) agonists. This led to the discovery of adenosine as a GHS-R agonist. We demonstrate that adenosine as well as the A1 adenosine receptor agonist N6-R-phenylisopropyladenosine (R-PIA) induce calcium responses, with EC50 values of 50 nM and 0.5 nM, respectively, in cells which express recombinant human GHS-R. However, neither compound induces a calcium response in nontransfected cells. Binding experiments show that adenosine and the GHS compound MK-0677 bind to membranes from GHS-R expressing cells with nearly identical Bmax values (2.6 +/- 0.1 x 10(-10) mol/mg protein for adenosine and 2.0 +/- 0.3 x 10(-10) mol/mg protein for MK-0677). However, no binding to membranes from nontransfected cells could be detected. Furthermore, we show that the IC50 values for inhibition of the adenosine, R-PIA, and GHS induced calcium responses by the GHS-R antagonist [D-Arg1, D-Phe5, D-Trp7,9, D-Leu11]-substance P are similar. These findings strongly suggest that adenosine and R-PIA are agonists of the GHS-R. Interestingly, neither adenosine nor R-PIA were able to induce GH release from rat pituitary cells in vitro. The implications of the latter finding is discussed.
Asunto(s)
Adenosina/farmacología , Hormona del Crecimiento/metabolismo , Receptores de Droga/agonistas , Animales , Calcio/metabolismo , Cromatografía Líquida de Alta Presión , Cricetinae , Femenino , Colorantes Fluorescentes , Fura-2 , Humanos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Indoles/farmacología , Hipófisis/citología , Hipófisis/metabolismo , Ratas , Ratas Wistar , Receptores de Droga/genética , Espectrofotometría Ultravioleta , Compuestos de Espiro/farmacología , TransfecciónRESUMEN
Based on NN703, low molecular weight growth hormone secretagouges (GHSs) with a reduced number of hydrogen binding sites were designed by removal of the C-terminal amide group. The compounds were highly potent in combination with high efficacy in a rat pituitary cell assay, being characterized with EC(50) values down to 0.8 nM. Selected compounds were tested in in vivo animal models. The oral bioavailability in dogs was 16-44%. Also, the ED(50) values of the compounds were determined both in dog and swine.
Asunto(s)
Dipéptidos/química , Dipéptidos/farmacología , Hormona del Crecimiento/metabolismo , Tiofenos/química , Tiofenos/farmacología , Administración Oral , Animales , Sitios de Unión , Disponibilidad Biológica , Perros , Evaluación Preclínica de Medicamentos/métodos , Femenino , Hormona del Crecimiento/efectos de los fármacos , Hidrógeno , Masculino , Imitación Molecular , Peso Molecular , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , PorcinosRESUMEN
A series of GH secretagogues based on modifications in the C-terminal of NN703 is reported. The C-terminal N-methyl amide of NN703 has been replaced with alkylated hydrazides in order to decrease the volume of distribution and identify GH secretagogues with shorter duration of action. Most of the prepared compounds show high potency in a rat pituitary assay. Subsequent to an initial in vivo screening in dogs, four compounds were selected for further pharmacological and pharmacokinetic evaluation. The four compounds showed oral bioavailability around 35% and equipotency in vitro compared to NN703. The relationship between lipophilicity and volume of distribution is discussed and it is speculated whether the lower volume of distribution is attributed to the observed higher in vivo potency and shorter plasma elimination half-life.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Hormonas/síntesis química , Hidrazinas/síntesis química , Oligopéptidos/síntesis química , Animales , Dipéptidos/química , Dipéptidos/farmacología , Perros , Hormona del Crecimiento/metabolismo , Hidrazinas/farmacología , Estructura Molecular , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , RatasRESUMEN
The discovery of a new class of compounds that stimulate the release of growth hormone (GH) in a manner distinctly different from growth hormone-releasing hormone (GHRH) is advancing the understanding of the mechanisms that control GH secretion. These compounds, the GH secretagogues, act at both pituitary and hypothalamic levels, and might even elicit effects in the CNS and peripheral systems. A receptor with high affinity for the GH secretagogues has been identified and several observations suggest the presence of additional receptors. The existence of these specific endogenous receptors could indicate that the mechanism of GH release is not yet fully understood. Several potential indications have been explored clinically and, as some of these compounds are orally active, they could offer attractive alternatives to recombinant human growth hormone (hGH) in treating GH disorders such as growth hormone deficiency (GHD), age-related conditions, obesity and catabolic conditions.
RESUMEN
NN703 is a novel orally active GH secretagogue (GHS) derived from ipamorelin. NN703 stimulates GH release from rat pituitary cells in a dose-dependent manner with a potency and efficacy similar to that of GHRP-6. The effect is inhibited by known GHS antagonists, but not by a GH-releasing hormone antagonist. Binding of (35)S-MK677 to the human type 1A GHS receptor (GHS-R 1A) stably expressed on BHK cells was inhibited by GHRP-6 and MK677 as expected. NN703 was also able to inhibit the binding of (35)S-MK677. However, the observed K(i) value was lower than expected, as based on the observed potencies regarding GH release from rat pituitary cells. Similarly, the effect of NN703 on the GHS-R 1A-induced inositol phosphate turnover in these cells showed a lower potency, when compared with GHRP-6 and MK677, than that observed in rat pituitary cells. The effect of i.v. administration of NN703 on GH and cortisol release was studied in swine. The potency and efficacy of NN703 on GH release were determined to be 155+/-23 nmol/kg and 91+/-7 ng GH/ml plasma respectively. A 50% increase of cortisol, compared with basal levels, was observed for all the tested doses of NN703, but no dose-dependency was shown. The effect of NN703 on GH release after i. v. and oral dosing in beagle dogs was studied. NN703 dose-dependently increased the GH release after oral administration. At the highest dose (20 micromol/kg), a 35-fold increase in peak GH concentration was observed (49.5+/-17.8 ng/ml, mean+/-s.e.m.). After a single i.v. dose of 1 micromol/kg the peak GH plasma concentration was elevated to 38.5+/-19.6 ng/ml (mean+/-s.e.m.) approximately 30 min after dosing and returned to basal level after 360 min. The oral bioavailability was 30%. The plasma half-life of NN703 was 4.1+/-0.4 h. A long-term biological effect of NN703 was demonstrated in a rat study, where the body weight gain was measured during a 14-day once daily oral challenge with 100 micromol/kg. The body weight gain was significantly increased after 14 days as compared with a vehicle-treated group. In summary, we here describe an orally active and GH specific secretagogue, NN703. This compound acts through a similar mechanism as GHRP-6, but has a different receptor pharmacology. NN703 induced GH release in both swine and dogs after i.v. and/or p.o. administration, had a high degree of GH specificity in swine and significantly increased the body weight gain in rats.
Asunto(s)
Dipéptidos/farmacología , Hormona del Crecimiento/efectos de los fármacos , Hipófisis/efectos de los fármacos , Administración Oral , Animales , Disponibilidad Biológica , Dipéptidos/administración & dosificación , Dipéptidos/química , Dipéptidos/farmacocinética , Perros , Hormona del Crecimiento/metabolismo , Humanos , Masculino , Hipófisis/metabolismo , Ratas , Ratas Sprague-Dawley , Porcinos , Aumento de Peso/efectos de los fármacosRESUMEN
Somatostatin [somatotropin release-inhibiting factor (SRIF)] is a cyclic tetradecapeptide that is a potent inhibitor of growth hormone (GH) secretion from the anterior pituitary. In addition to the inhibitory effects on GH-release, SRIF-14 and SRIF-28, a 28-amino acid form of SRIF extended from the N-terminal end, inhibit the release of a variety of other peptides including glucagon, insulin, and gastrin, and both peptides act as neurotransmitters and neuromodulators in the central nervous system and the periphery. SRIF exerts its potent inhibitory effects following binding to high affinity SRIF receptors (ssts) that have been identified on target tissues. The recent cloning of five ssts has confirmed that the effects of SRIF are mediated by a family of G protein-coupled receptors (sst1-5). Based on structural and pharmacological properties sst2, sst3, and sst5 belong to the SRIF1 receptor subclass, and the sst1 and sst4 subtypes comprise the SRIF2 subclass. The major difference between these two subclasses is that SRIF1 receptors bind octapeptide and hexapeptide SRIF-14 analogs with high affinity, while SRIF2 receptors bind these analogs with drastically reduced affinity. A screening program was initiated to identify a lead nonpeptide with affinity for sst1-5 receptors. The search focused on a scaffold with the following attachments: (1) a heteroaromatic nucleus to mimic the Trp8 residue, (2) a nonheteroaromatic nucleus to mimic Phe7, and (3) a primary amine or other basic group to mimic the Lys9 residue of SRIF-14. Using these criteria, a novel thiourea (NNC 26-9100, 17) was discovered as a structural lead. The key fragments in this compound are a heteroaromatic moiety (pyridine), an aromatic group, and a basic imidazole group connected through a thiourea scaffold. Compound 17 exhibited a Ki = 6 nM at sst4 receptors with a 100-fold sst4/sst2 selectivity and was shown to be a full agonist at this receptor subtype. This article will review the literature on the design and development of nonpeptide somatostatin receptor ligands and the therapeutic potential of these agents. Furthermore, our work on the development of 2-pyridylthioureas as sst4 receptor agonists will be described.
Asunto(s)
Receptores de Somatostatina/efectos de los fármacos , Somatostatina/agonistas , Tiourea/farmacología , Animales , Humanos , Proteínas de la Membrana , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/antagonistas & inhibidores , Somatostatina/metabolismo , Somatostatina/fisiología , Tiourea/análogos & derivadosRESUMEN
The development and pharmacology of a new potent growth hormone (GH) secretagogue, ipamorelin, is described. Ipamorelin is a pentapeptide (Aib-His-D-2-Nal-D-Phe-Lys-NH2), which displays high GH releasing potency and efficacy in vitro and in vivo. As an outcome of a major chemistry programme, ipamorelin was identified within a series of compounds lacking the central dipeptide Ala-Trp of growth hormone-releasing peptide (GHRP)-1. In vitro, ipamorelin released GH from primary rat pituitary cells with a potency and efficacy similar to GHRP-6 (ECs) = 1.3+/-0.4nmol/l and Emax = 85+/-5% vs 2.2+/-0.3nmol/l and 100%). A pharmacological profiling using GHRP and growth hormone-releasing hormone (GHRH) antagonists clearly demonstrated that ipamorelin, like GHRP-6, stimulates GH release via a GHRP-like receptor. In pentobarbital anaesthetised rats, ipamorelin released GH with a potency and efficacy comparable to GHRP-6 (ED50 = 80+/-42nmol/kg and Emax = 1545+/-250ng GH/ml vs 115+/-36nmol/kg and 1167+/-120ng GH/ml). In conscious swine, ipamorelin released GH with an ED50 = 2.3+/-0.03 nmol/kg and an Emax = 65+/-0.2 ng GH/ml plasma. Again, this was very similar to GHRP-6 (ED50 = 3.9+/-1.4 nmol/kg and Emax = 74+/-7ng GH/ml plasma). GHRP-2 displayed higher potency but lower efficacy (ED50 = 0.6 nmol/kg and Emax = 56+/-6 ng GH/ml plasma). The specificity for GH release was studied in swine. None of the GH secretagogues tested affected FSH, LH, PRL or TSH plasma levels. Administration of both GHRP-6 and GHRP-2 resulted in increased plasma levels of ACTH and cortisol. Very surprisingly, ipamorelin did not release ACTH or cortisol in levels significantly different from those observed following GHRH stimulation. This lack of effect on ACTH and cortisol plasma levels was evident even at doses more than 200-fold higher than the ED50 for GH release. In conclusion, ipamorelin is the first GHRP-receptor agonist with a selectivity for GH release similar to that displayed by GHRH. The specificity of ipamorelin makes this compound a very interesting candidate for future clinical development.
Asunto(s)
Hormona del Crecimiento/metabolismo , Hormonas/farmacología , Oligopéptidos/farmacología , Hormona Adrenocorticotrópica/sangre , Anestesia , Animales , Área Bajo la Curva , Gonadotropinas/sangre , Hormona del Crecimiento/sangre , Hormona Liberadora de Hormona del Crecimiento/sangre , Hormonas/química , Hidrocortisona/sangre , Masculino , Conformación Molecular , Oligopéptidos/química , Hipófisis/citología , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Somatostatina/efectos de los fármacos , Estimulación Química , Relación Estructura-Actividad , PorcinosRESUMEN
Utilizing NNC 26-9100 (11) as a structural lead, a variety of nonpeptide derivatives of somatostatin were synthesized and evaluated for sst2 and sst4 receptor binding affinity. A novel thiourea scaffold was utilized to attach (1) a heteroaromatic nucleus to mimic the Trp8 residue, (2) a nonheteroaromatic nucleus to mimic Phe7, and (3) a primary amine or other basic group to mimic the Lys9 residue of somatostatin. Displacement studies were carried out using membranes from cell lines expressing ssts [BHK cells (sst4) and HEK 293 cells (sst2)] utilizing [125I]Tyr11-SRIF as the radioligand. Several thioureas (11, 38, 39, 41, and 42) and the urea 66 exhibited Ki values of less than 100 nM. The thioureas 11 (Ki = 6 nM) and 41 (Ki = 16 nM) and the urea 66 (Ki = 14 nM) are believed to be the most potent nonpeptide sst4 agonists known. Since the thiourea 11 and the urea 66 exhibit high sst4 selectivity, these novel nonpeptide derivatives may be useful tools for studying the sst4 receptor. Studies are currently in progress to evaluate the therapeutic potential of NNC 26-9100 (11) in the treatment of glaucoma.
Asunto(s)
Aminopiridinas/síntesis química , Receptores de Somatostatina/agonistas , Somatostatina/agonistas , Tiourea/análogos & derivados , Tiourea/síntesis química , Aminopiridinas/química , Aminopiridinas/metabolismo , Aminopiridinas/farmacología , Animales , Línea Celular , Cricetinae , Glaucoma/tratamiento farmacológico , Humanos , Proteínas de la Membrana , Ensayo de Unión Radioligante , Receptores de Somatostatina/biosíntesis , Relación Estructura-Actividad , Tiourea/química , Tiourea/metabolismo , Tiourea/farmacologíaRESUMEN
A new series of GH secretagogues derived from ipamorelin is described. In an attempt to obtain oral bioavailability, by reducing the size and the number of potential hydrogen-bonding sites of the compounds, a strategy using the peptidomimetic fragment 3-(aminomethyl)benzoic acid and sequential backbone N-methylations was applied. Several compounds from this series release GH with high in vitro potency and efficacy in a rat pituitary cell assay and high in vivo potency and efficacy in anesthetized rats. The tetrapeptide NNC 26-0235 (3-(aminomethyl)benzoyl-D-2Nal-N-Me-D-Phe-Lys-NH2) shows, following iv administration, comparable in vivo potency to ipamorelin, GHRP-2, and GHRP-6 with an ED50 in swine at 2 nmol/kg. NNC 26-0235 demonstrated a 10% oral bioavailability in dogs, and NNC 26-0235 and ipamorelin were able to increase basal GH level by more than 10-fold after oral administration of a dose of 1.8 and 2.7 mg/kg, respectively. The tripeptide NNC 26-0323 (3-(aminomethyl)benzoic acid-N-Me-D-2Nal-N-Me-D-Phe-ol) which showed moderate in vitro potency but lacked in vivo potency demonstrated a 20% oral bioavailability in rats.
Asunto(s)
Hormona del Crecimiento/metabolismo , Hormonas/síntesis química , Oligopéptidos/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Perros , Femenino , Hormonas/química , Hormonas/farmacocinética , Hormonas/farmacología , Técnicas In Vitro , Inyecciones Intravenosas , Espectroscopía de Resonancia Magnética , Masculino , Imitación Molecular , Oligopéptidos/química , Oligopéptidos/farmacocinética , Oligopéptidos/farmacología , Hipófisis/citología , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , PorcinosRESUMEN
A novel class of growth hormone-releasing compounds with a molecular weight in the range from 500 to 650 has been discovered. The aim of this study was to obtain growth hormone secretagogues with oral bioavailability. By a rational approach we were able to reduce the size of the lead compound ipamorelin (4) and simultaneously to reduce hydrogen-bonding potential by incorporation of backbone isosters while retaining in vivo potency in swine. A rat pituitary assay was used for screening of all compounds and to evaluate which compounds should be tested further for in vivo potency in swine and oral bioavailability, fpo, in dogs. Most of the tested compounds had fpo in the range of 10-55%. In vivo potency in swine after iv dosing is reported, and ED50 was found to be 30 nmol/kg of body weight for the most potent compound.
Asunto(s)
Hormona del Crecimiento/metabolismo , Hormonas/síntesis química , Oligopéptidos/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Perros , Evaluación Preclínica de Medicamentos , Femenino , Hormonas/química , Hormonas/farmacocinética , Hormonas/farmacología , Inyecciones Intravenosas , Espectroscopía de Resonancia Magnética , Masculino , Modelos Moleculares , Imitación Molecular , Oligopéptidos/química , Oligopéptidos/farmacocinética , Oligopéptidos/farmacología , Hipófisis/citología , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , PorcinosRESUMEN
Aspirin is widely used for its analgesic, antiinflammatory and antipyretic properties. Among its disadvantages are the relatively narrow therapeutic margin, its irritancy towards the gastric mucosa, and occasionally patient hypersensitivity towards aspirin. As part of our effort to develop prodrugs without these liabilities eleven new title compounds have been isolated and characterized. These 'superaspirin' candidates were subjected to non-enzymatic hydrolysis for a first rapid screening in vitro. Only 2-(2,6-dimethoxybenzyloxy)-2-methyl-4H-1,3-benzodioxin-4-one (4c) was observed to act as an exclusive aspirin prodrug, while 2-(2-methoxybenzyloxy)-2-methyl-4H-1,3-benzodioxin-4-one (4b) and 2-(2-ethoxybenzyloxy)-2-methyl-4H-1,3- benzodioxin-4-one (4d) were shown to release both aspirin 6 and salicylic acid 7. Subsequently, these three candidates were further characterized by investigation of the pH profile of their hydrolysis rates.
Asunto(s)
Aspirina/análogos & derivados , Alcoholes Bencílicos/síntesis química , Dioxinas/síntesis química , Profármacos/síntesis química , Alcoholes Bencílicos/química , Cromatografía Líquida de Alta Presión , Dioxinas/química , Hidrólisis , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Profármacos/químicaRESUMEN
The fifty-four known title compounds are fully documented and their chemical and biological properties discussed in detail. Special attention is devoted to their potential as aspirin and salicylic acid prodrugs, respectively.