Identification and evaluation of growth hormone antagonists

Abstract

States in which Growth hormone (GH) levels are elevated have been implicated in several physiological disorders such as acromegaly, diabetic complications and progression of malignancies. Classical treatments for acromegaly are surgery and radiotherapy but also pharmacological therapies exist. There is, however, a need for improved remedies and in the development of new drugs reliable preclinical in vitro and in vivo models are crucial.

GH, secreted from the pituitary, acts both directly and indirectly through induction of insulinlike growth factor-I (IGF-I) to promote tissue growth and regulate metabolism. GH induces IGF-I expression in the liver and peripheral target tissues but hepatic-derived IGF-I accounts for most circulating IGF-I. Therefore, the liver constitutes a valuable target organ for studies on GH-induced IGF-I production.

In the first part of this study, we used rodent primary hepatocytes for studies on pharmacological intervention of GH-induced IGF-I mRNA expression. We developed a 96well non-radioactive IGF-I mRNA quantification assay based on hybridization of sense and antisense RNA probes to replica membranes with crude hepatocyte lysates. The sense hybridization was used as an internal standard. The antagonistic properties of a set of GHreceptor binding compounds were evaluated and two compounds were found to reduce GHinduced IGF-I mRNA expression. Effects due to metabolic inhibition or toxicity were excluded using a cell proliferation assay. To investigate possible unspecific transcriptional effects, the mRNA levels of the housekeeping genes beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined. The antagonistic property of one compound, BVT-A, was confirmed by showing mitigated GH-induction of two additional GH regulated genes using RNase protection assays. Accordingly, this direct filter hybridization assay of hepatocyte lysates using non-radioactive sense and antisense probes can be used for quantitative mRNA measurements and could constitute a valuable tool in screening for pharmacologically active compounds. Using other probe sets, the assay can have a wider application in cellular screening for drugs affecting gene transcription.

In the second part of the study, we used GH-treated, hypophysectomized (HX) rats to circumvent the feedback mechanisms of the hypothalamo-pitutary axis on the GH/IGF-I system, to study in vivo effects of BVT-A. In addition to hepatic IGF-I expression, GH also regulates production of major binding proteins for IGF-I in serum, IGF binding protein-3 (IGFBP-3) and the acid labile subunit (ALS). As expected, one week of GH treatment of HX rats induced serum IGF-I, body weight and hepatic mRNA levels of IGF-I, IGFBP-3, ALS, and of IGF-I receptor and GH receptor. Co-treatment with the GH antagonist BVT-A suppressed the GH-induced rise in serum IGF-I and body weight and also reduced hepatic mRNA levels of IGF-I, IGFBP-3, ALS, IGF-I receptor and GH receptor. Thus, the GH substituted HX rat is a useful model for studies of GH receptor antagonists. Together, the results show that BVT-A is a GH antagonist acting both in vitro and in vivo.