RESUMEN
The Industry Pharmacogenomics Working Group has an interest in attaining a better understanding of global requirements for sample collections intended for pharmacogenetics research. To have adequately powered pharmacogenetics studies representative of the clinical trial population, it is important to collect DNA samples from a majority of consenting study participants under many institutional review board/ethics committee (IRB/EC) jurisdictions. A survey was distributed to gather information from local and central IRBs/ECs. The survey included questions related to the approval of pharmacogenetics studies, collection and banking of samples, and return of data to subjects. A total of 204 responses were received from global IRBs/ECs with pharmacogenetic experience. The data show that requirements for approval of pharmacogenetic research differ between IRBs/ECs within and between countries but not between regions of the United States. A better understanding of differing requirements should facilitate global sample collection of DNA for pharmacogenetics research and may provide the basis for harmonized regulations for collection of genetic samples in the future.
Asunto(s)
ADN/análisis , Comités de Ética en Investigación/estadística & datos numéricos , Farmacogenética/métodos , Ensayos Clínicos como Asunto/métodos , Recolección de Datos , Diseño de Fármacos , Humanos , Manejo de Especímenes/métodosRESUMEN
Pharmacogenetic association studies have the potential to identify variations in DNA sequence which impact drug response. Identifying these DNA variants can help to explain interindividual variability in drug response; this is the first step in personalizing dosing and treatment regimes to a patient's needs. There are many intricacies in the design and analysis of pharmacogenetic association studies, including having adequate power, selecting proper endpoints, detecting and correcting the effects of population stratification, modeling genetic and nongenetic covariates accurately, and validating the results. At this point there are no formal guidelines on the design and analysis of pharmacogenetic studies. The Industry Pharmacogenomics Working Group has initiated discussions regarding potential guidelines for pharmacogenetic study design and analyses (http://i-pwg.org) and the results from these discussions are presented in this paper.
Asunto(s)
Industria Farmacéutica/tendencias , Farmacogenética/métodos , Proyectos de Investigación/tendencias , Industria Farmacéutica/normas , Determinación de Punto Final , Humanos , Guías de Práctica Clínica como Asunto , Control de Calidad , Proyectos de Investigación/normasRESUMEN
A new analogue of recombinant human growth hormone (hGH), hGH des(1-6,14) was expressed in Escherichia coli, refolded and purified to homogeneity. The mutation decreased the hormone's ability to bind lactogenic and somatogenic receptors through its site 1, and almost completely abolished its ability to bind these receptors through site 2, as evidenced by both binding and gel-filtration experiments. More specifically, the binding to prolactin receptors (PRLRs) from various species or their soluble recombinant extracellular domains (ECDs) was decreased 1.5-4-fold, whereas the binding to hGH receptor (hGHR) was decreased 10-85-fold. These changes caused an almost total loss of hormone agonistic activity in several in vitro bioassays and subsequently, the hGH des(1-6,14) analogue acquired antagonistic properties. This antagonistic activity was dependent upon modification of site 1. In those cases in which the binding was reduced only slightly, e.g. binding to rabbit PRLRs, hGH des(1-6,14) acted as a strong antagonist, whereas in others in which the binding of site 1 was reduced to a higher degree, such as other PRLRs and, in particular, hGHR, the antagonistic activity was correspondingly weaker. Circular dichroism spectra of the analogue suggested that these changes do not result from a decrease in overall alpha-helix content, but rather from minor local structural modifications at the N-terminus.
Asunto(s)
Hormona del Crecimiento/farmacología , Hormona de Crecimiento Humana/análogos & derivados , Hormona de Crecimiento Humana/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Animales , Unión Competitiva , Bovinos , División Celular/efectos de los fármacos , Línea Celular , Clonación Molecular , Escherichia coli/genética , Femenino , Hormona del Crecimiento/genética , Hormona del Crecimiento/aislamiento & purificación , Hormona de Crecimiento Humana/genética , Humanos , Técnicas In Vitro , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Pliegue de Proteína , Conejos , Receptores de Prolactina/genética , Receptores de Prolactina/metabolismo , Receptores de Somatotropina/genética , Receptores de Somatotropina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
Rat placental lactogen-I (rPL-I), expressed in rat uteri at midpregnancy, belongs to the GH/PRL/cytokine family of hormones (Wallis, 1992), all members of which exhibit a similar mechanism of receptor activation. Lactogenic activity of rPL-I as determined by Nb2 lymphoma cell bioassay was slightly higher than that of hGH. rPL-I was capable also of stimulating beta-casein production in mouse HC-11 cells. Competitive binding experiments using [125I]hGH or [125I]bPL and purified prolactin receptor extracellular domains (PRLR-ECDs) from rat (r), rabbit (rb), and bovine (b) showed rPL-I to be 12, 40, and 7-fold, respectively, less effective than hGH when competing with [125I]hGH. Displacement of [125I]bPL by rPL-I from rPRLR-ECD and rbPRLR-ECD was also 4-, and 30-fold less effective, respectively, than that by bPL. rPL-I did not compete at all with [125I]bPL for binding to bPRLR-ECD. In contrast, competitive binding experiments with [125I]hGH to a microsomal fraction of Nb2 cells showed rPL-I to be slightly more active than hGH. The stoichiometries of the complexes formed by rPL-I with rbPRLR-ECD, rPRLR-ECD, and bPRLR-ECD were studied by gel filtration. Whereas a 1:1 complex was formed between rPL-I and rPRLR-ECD and rbPRLR-ECD, no complex could be detected between rPL-I and bPRLR-ECD. The present results support the hypothesis that transient forms of a homodimer complex may be sufficient to initiate the biological signal, despite its short half-life.
Asunto(s)
Lactógeno Placentario/metabolismo , Receptores de Prolactina/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Caseínas/biosíntesis , Bovinos , División Celular/efectos de los fármacos , Línea Celular , Femenino , Técnicas In Vitro , Radioisótopos de Yodo , Ratones , Mitógenos/farmacología , Lactógeno Placentario/farmacología , Embarazo , Conejos , Ratas , Receptores de Prolactina/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal , Especificidad de la EspecieRESUMEN
The cDNA of the extracellular domain of bovine prolactin receptor (bPRLR-ECD) was cloned and expressed at high yield as an insoluble protein in Escherichia coli. This protein was solubilized, refolded and purified to > 98% homogeneity yielding 80 mg of monomeric fraction per 2 litres of induced culture. Its molecular mass was 25.7 kDa, as determined by SDS-PAGE in the absence of reducing agent and 24 kDa by gel filtration on a Superdex column. Binding experiments revealed that bPRLR-ECD binds to human (h) GH (hGH) with high affinity, whereas its affinity for ovine (o) or bovine (b) prolactins (PRLs) was lower and for bovine placental lactogen (bPL) very low. The affinity of bPRLR-ECD for the latter three hormones was, however, much higher than that of membrane-embedded or solubilized bPRLR. The stoichiometries of interaction of bPRLR-ECD with hGH, oPRL, bPRL and bPL were determined by gel-filtration chromatography. Even at a 3:1 ECD excess, only 1:1 complexes were detected at microM concentrations of ECD and ligand. At an up to 32-fold dilution, the complexes with oPRL, bPRL, and particularly bPL, underwent progressive dissociation, whereas the complex with hGH remained stable. Although all four hormones exhibited nearly identical activity in the Nb2 lymphoma cell bioassay, the ability of bPRLR-ECD to inhibit hormonal mitogenic activities differed, generally reflecting its affinity for the respective hormones. In view of these and previous results, we suggest that, unlike in the GH:GHR-ECD interaction, neither the stoichiometry of interaction of bovine or other PRLR-ECDs nor the affinity constants can predict the biological potency of the different lactogenic hormones.
Asunto(s)
Prolactina/metabolismo , Receptores de Prolactina/aislamiento & purificación , Animales , Secuencia de Bases , Bovinos , Cromatografía en Gel , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Femenino , Hormona del Crecimiento/metabolismo , Humanos , Datos de Secuencia Molecular , Lactógeno Placentario/metabolismo , Ratas , Receptores de Prolactina/genética , Receptores de Prolactina/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Ovinos , Especificidad de la EspecieRESUMEN
Two variants of rabbit prolactin receptor extracellular domain (rbPRLR-ECD) were prepared using insect/baculovirus (amino acids 1-198) and E. coli (amino acids 4-210) expression systems. Bovine PRLR-ECD (bPRPL-ECD amino acids 1-210) and human growth hormone receptor ECD (hGHR-ECD amino acids 1-246) were also prepared using E. coli expression system. All four proteins were purified as monomers with > 95% homogeneity. Their affinity for various lactogenic and somatogenic hormones was determined by binding assays. The stoichiometry of complex formation with these hormones was studied by gel filtration on a Superdex 75 column, and bioactivity was determined by in vitro bioassays. The results summarized in this paper indicate that, in contrast to hGHR-ECD, in which the ability to form a 2:1 complex with hGH is indicative of the biological activity of the hormone, the ability or inability of prolactin and placental lactogen to form 2:1 complexes with rb or bPRLR-ECD cannot predict their biological activity. This conclusion does not preclude however, hormone- or antibody-induced dimerization of the membrane-embedded receptor.
Asunto(s)
Espacio Extracelular/metabolismo , Lactógeno Placentario/metabolismo , Prolactina/metabolismo , Receptores de Prolactina/metabolismo , Animales , Bovinos , Escherichia coli/genética , Humanos , Conejos , Receptores de Prolactina/genética , Receptores de Somatotropina/genética , Receptores de Somatotropina/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Human growth hormone (GH) is an anabolic hormone required for normal growth. In addition, human GH affects the metabolism of proteins, carbohydrates and fats and possesses lactogenic effects. Although the GH receptor has recently been cloned, it is not clear whether the diverse biological activities of human GH are transduced through a single or through several types of related receptors. To address this question, 15 recombinant analogues of human GH were prepared and tested in bioassays in vitro and in vivo, in which the hormone action is mediated through lactogen or somatogen receptors. The results clearly suggest that recombinant analogues of human GH that recognize either somatogen or lactogen receptors, or both, but have selectively modified post-receptor effects, are helpful in elucidating the diverse biological activities of GH. These differences are most probably due to minor structural variability in GH and lactogen receptors in different organs and/or species. Genetic engineering of human GH may lead to production of modified analogues with changed and narrower specificities. One of the possible applications would be for a human GH analogue devoid of diabetogenic activity.
Asunto(s)
Hormona del Crecimiento/química , Hormona del Crecimiento/farmacología , Mutagénesis Sitio-Dirigida , Animales , Bioensayo , Caseínas/biosíntesis , Bovinos , Femenino , Hormona del Crecimiento/genética , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Ratones , Conejos , Ratas , Ratas Wistar , Receptores de Péptidos/metabolismo , Receptores de Somatotropina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Relación Estructura-ActividadRESUMEN
Four analogues of human growth hormone (hGH) mutated by site-directed mutagenesis at the 54-74 loop-Met-hGH(P59A), Met-hGH(P61A), Met-hGH(P59A,P61A) and Met-hGH(Des 62-67) were analyzed for: (1) their biological activity mediated through lactogenic receptors using rat lymphoma Nb2-11C cell proliferation and mouse mammary gland HC-11 cell beta-casein synthesis bioassays and (2) their ability to interact with recombinant hGH binding protein (hGHBP). The analogues Met-hGH(P59A), Met-hGH(P61A) and Met-hGH(P59A,P61A) partially lost their activity relative to native hGH in the HC-11, but not in the Nb2-11C cell bioassay. These analogues were nevertheless capable of forming a 1:2 complex with a recombinant hGH binding protein (hGHBP), despite the fact that the affinity of Met-hGH(P61A) and Met-hGH(P59A,P61A) analogues had decreased 8- and 14-fold, respectively. Met-hGH(Des 62-67) failed to form 1:1 or 1:2 complexes with hGHBP and did not compete with [125I]hGH for binding to hGHBP. It lost all biological activity in HC-11 cells, but retained 0.4% of its activity, in the Nb2-11C cell proliferation bioassay. These results confirm the involvement of Pro-61 in the hGH binding and activity mediated through somatogenic receptors, while the activity mediated through two different types of lactogenic receptors was selectively modified. These findings emphasize the fact that lactogen receptors in different species or organs are not identical.
Asunto(s)
Proteínas Portadoras/metabolismo , Hormona del Crecimiento/genética , Mutagénesis Sitio-Dirigida , Animales , Sitios de Unión , Caseínas/biosíntesis , Línea Celular , Hormona del Crecimiento/análogos & derivados , Hormona del Crecimiento/química , Linfoma , Glándulas Mamarias Animales/efectos de los fármacos , Ratones , Proteínas de Neoplasias/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Human growth hormone (hGH) inhibits alpha 1(I) collagen gene expression in cultured avian skin fibroblasts resulting in a decrease in the amount of collagenase-digestible proteins (CDP) in the medium. In addition, a synergism exists between GH and insulin-like growth factor-I (IGF-I) in their effect on CDP. Four N-terminal modified hGH analogs were tested for their ability to affect collagen metabolism in these cells. The truncated analog Des-7 hGH(R8M, D11A) was found to be a strong antagonist of the hGH-induced inhibition of the collagen synthesis but by itself did not inhibit collagen alpha 1(I) gene expression or modify the CDP appearance in the medium. Some synergism between Des-7 hGH and IGF-I was observed. The analog Met-hGH(R19H, L20P), in which Arg19 was replaced by histidine, and Leu20 by proline was only partially potent compared with the native hormone in causing inhibition of collagen gene expression, in attenuating CDP appearance in the medium, and in antagonizing hGH. However, this analog was as potent as hGH in its ability to synergize with IGF-I. The importance of His18 was assessed by testing the response to Met-hGH(H18D), in which His18 was replaced by Asp, and to Met-hGH(H18Q), in which His18 was replaced by glutamine (as in chicken GH sequence). Substitution of His18 by a negatively charged amino acid abolished all the hormone activities tested whereas substitution with glutamine restored only part of the activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Colágeno/biosíntesis , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/análogos & derivados , Piel/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Pollos , Sinergismo Farmacológico , Fibroblastos/metabolismo , Hormona del Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Datos de Secuencia Molecular , Piel/citología , Piel/metabolismo , Relación Estructura-ActividadRESUMEN
Recent work with various point and deletion mutants of human GH (hGH) has suggested that the proximal N-terminal end of the hormone molecule is important for its growth promoting action. This study was conducted to examine the growth promoting, diabetogenic, and insulin-like activities of two N-terminal mutants of hGH, the deletion mutant Des-7 hGH (met8, ala11), and a chimeric mutant of bovine GH (bGH) and hGH containing the N-terminal 13 amino acids of bGH (met, ala 1-13/14-191, asp11). The CD spectra of these mutants are similar to that of wild-type hGH and they retain lactogenic activity on Nb2 lymphoma cells, whereas their ability to bind to somatogenic receptors on IM-9 lymphocytes and bovine liver membranes is markedly reduced. In this study, growth promoting activity of the mutants was assessed using the 9-day weight gain test in hypophysectomized rats. Des-7 hGH had a potency of 0.03 IU/mg protein in this assay, whereas the potency of the bGH/hGH chimera was 0.71 IU/mg. Diabetogenic activity was tested in the ob/ob mouse, using the elevation of fasting blood glucose and the worsening of glucose tolerance after a 3-day course of treatment as end-points. Both Des-7 hGH and the bGH/hGH chimera had reduced diabetogenic activity compared to that of biosynthetic wild-type hGH, consistent with their reduced growth activity. Insulin-like activity was assessed by testing the in vitro ability of the mutants to stimulate [14C] glucose oxidation by epididymal adipose tissue of hypophysectomized rats. Des-7 hGH had about 1% the activity of wild-type hGH, whereas the chimera was about 20% as active. When Des-7 hGH was added to the incubation medium along with wild-type hGH in ratios of 5, 12.5, or 25:1 (Des-7 hGH:hGH), the insulin-like action of hGH was significantly inhibited, indicating that the mutant is a modest antagonist of the insulin-like action of hGH. When the ability of Des-7 hGH to compete with [125I] hGH for binding to isolated rat adipocytes was tested, the mutant was about 10% as effective as wild-type hGH. Thus, Des-7 hGH appears to be more effective in binding to adipocyte GH receptors than in triggering an insulin-like response, perhaps accounting for its modest antagonistic activity. The results of this study suggest that the proximal N-terminal end of the hGH molecule is involved in the expression of the growth promoting, diabetogenic and insulin-like activities of GH.
Asunto(s)
Glucemia/metabolismo , Hormona del Crecimiento/química , Insulina/fisiología , Aumento de Peso/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/ultraestructura , Animales , Femenino , Hormona del Crecimiento/genética , Hormona del Crecimiento/fisiología , Masculino , Ratones , Ratones Mutantes , Ratones Obesos , Mutación/genética , Conformación Proteica , Ratas , Receptores de Somatotropina/efectos de los fármacos , Receptores de Somatotropina/fisiologíaRESUMEN
Recombinant human growth hormone was administered orally to carp and serum levels of absorbed bioactive hormone were investigated using a highly sensitive Nb2 rat lymphoma cell bioassay and radioimmunoassay. Serum levels of bioactive hGH reached maximum values 30 min after oral intubation and then gradually decreased. Co-administration of the hormone with deoxycholate to fasted carp resulted in up to a 1000-fold increase in absorption compared to aqueous solutions of the hormone, but had no effect on the kinetics of the absorption process. Absorption of the hormone in starved fish was significantly greater than in fed fish. A linear dose-response relationship was observed for hGH in starved fish and the level of absorption in fed fish was influenced by the time interval from the last meal. The ratio of bioactive to immunoactive hGH in fasted fish indicated little loss of bioactivity and also that deoxycholate may be protective against hGH degradation. The present study demonstrates for the first time that biologically active hGH is absorbed in the common carp after oral intubation. Furthermore, the use of a biological detergent dramatically increased the extent of hGH absorption. Additional studies are required to establish the appropriate conditions (diet composition, feeding level, and frequency, etc.) in which polypeptide hormones could be introduced orally to fish.