Preclinical and clinical in vitro in vivo correlation of an hGH dextran microsphere formulation.

PURPOSE: To investigate the in vitro in vivo correlation of a sustained release formulation for human growth hormone (hGH) based on hydroxyethyl methacrylated dextran (dex-HEMA) microspheres in Pit-1 deficient Snell dwarf mice and in healthy human volunteers. MATERIALS AND METHODS: A hGH-loaded microsphere formulation was developed and tested in Snell dwarf mice (pharmacodynamic study) and in healthy human volunteers (pharmacokinetic study). RESULTS: Single subcutaneous administration of the microspheres in mice resulted in a good correlation between hGH released in vitro and in vivo effects for the hGH-loaded microsphere formulation similar to daily injected hGH indicating a retained bioactivity. Testing the microspheres in healthy volunteers showed an increase (over 7-8 days) in hGH serum concentrations (peak concentrations: 1-2.5 ng/ml). A good in vitro in vivo correlation was obtained between the measured and calculated (from in vitro release data) hGH serum concentrations. Moreover, an increased serum concentration of biomarkers (insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3) was found again indicating that bioactive hGH was released from the microspheres. CONCLUSIONS: Good in vitro in vivo correlations were obtained for hGH-loaded dex-HEMA microspheres, which is an important advantage in predicting the effect of the controlled drug delivery product in a clinical situations.

Non-hyperinsulinemic hypoglycemia in a patient with a gastrointestinal stromal tumor.

One of the authors (JvD) recently reported a case of non-islet cell tumor hypoglycemia caused by the production of "big" IGF-II by a gastrointestinal stromal tumor (GIST). Here, we report a patient with a GIST in whom non-hyperinsulinemic hypoglycemia was not attributable to aberrant tumoral IGF-II processing, but instead was caused by a combination of cachexia, renal and hepatic dysfunction, and tumoral glucose consumption.

Differential patterns of insulin-like growth factor-I and -II mRNA expression in medulloblastoma.

Insulin-like growth factors (IGFs) play an important role in tumour growth and development. We hypothesized that this is also the case for medulloblastomas, which are highly malignant cerebellar brain tumours usually occurring in children. In these tumours the expression patterns of IGF-I and -II mRNA were studied. Tumour specimens obtained from 12 children and two adults at diagnosis were hybridized in situ with digoxigenin-labelled cRNA probes for hIGF-I and hIGF-II mRNAs. In all cases, tumour cells showed abundant expression of IGF-I mRNA. Nine of the 14 tumours showed variable but significant IGF-II expression. In these tumours, the hybridization signal almost exclusively colocalized with a subpopulation of Ki-M1P positive cells that were identified as ramified microglia (RM) cells. In the five tumours without IGF-II expression, microglia/brain macrophages with a more rounded amoeboid-like morphology predominated. RM cells in normal cerebellar tissues, residing abundantly in areas of the white and, to a less extent, in the grey matter, were IGF-II mRNA-negative. These RM cells showed a thinner and more extensively branched appearance and were more evenly distributed than those encountered in medulloblastoma. Probably, during the transformation from the resting ramified towards the amoeboid morphology (or vice versa) IGF-II mRNA expression is only temporarily induced. The physiological meaning of the induction of IGF-II mRNA expression by these cells in medulloblastoma remains unclear but any IGF-II peptide synthesized could exert unfavourable mitogenic and antiapoptotic effects on adjacent tumour cells. However, in this relatively small number of cases we could not find any indications for a relationship between clinical characteristics of the various cases and the extent of IGF-II mRNA expression.

Short-term glucocorticoid treatment of prepubertal mice decreases growth and IGF-I expression in the growth plate.

The insulin-like growth factor (IGF) system is an important mediator of postnatal longitudinal growth, and the growth inhibiting effects of glucocorticoid (GC) treatment are suggested to be due to impaired action of the IGF system. However, the precise changes of the IGFs and the IGF-binding proteins (IGFBPs) in the growth plate, occurring upon short-term GC treatment have not been characterized. Prepubertal mice treated daily with dexamethasone (DXM) for 7 days, showed significant growth inhibition of total body length and weight and weight of the liver, thymus and spleen, whereas the weight of the kidneys was not affected. Analysis of the tibial growth plate showed that the total growth plate width significantly decreased to 84.5% of control values, caused by a significant decrease in the proliferative zone. The number of proliferating cell nuclear antigen (PCNA)-positive chondrocytes in the proliferative zone decreased significantly (to 40%) and TUNEL staining showed a significant 1.6-fold increase in apoptotic hypertrophic chondrocytes. In the growth plates, both IGF-I and IGF-II, as well as IGFBP-2 mRNAs were detected, mainly in the proliferative and prehypertrophic zones. DXM treatment significantly decreased the number of chondrocytes expressing IGF-I, whereas the number of chondrocytes expressing IGF-II and IGFBP-2 were not affected. The decrease in IGF-I expression in the growth plate indicates that GC treatment affects IGF-I at the local level of the growth plate, which could contribute to the GC-induced growth retardation.

IGF and IGF-binding protein expression in the growth plate of normal, dexamethasone-treated and human IGF-II transgenic mice.

Glucocorticoid (GC) treatment in childhood can lead to suppression of longitudinal growth as a side effect. The actions of GCs are thought to be mediated in part by impaired action of the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins (IGFBP-1 to -6). We have studied the effects of GCs on IGF and IGFBP expression at the local level of the growth plate, using non-radioactive in situ hybridization. We treated 3-week-old normal mice for 4 weeks with dexamethasone (DXM). We also treated human IGF-II (hIGF-II) transgenic mice in order to investigate whether IGF-II could protect against the growth retarding effect of this GC. DXM treatment resulted in general growth retardation in both mice strains, however, only in normal mice was tibial length decreased. In both normal and hIGF-II trangenic mice, the total width of the growth plate was not affected, whereas the width of the proliferative zone decreased as a result of the DXM treatment. Additionally, only in normal mice, the width of the hypertrophic zone thickened. Only expression of IGF-I, IGF-II and IGFBP-2 could be detected in the growth plates of 7-week-old normal mice. IGFBP-1, -3, -4, -5 and -6 mRNAs were not detected. DXM treatment of normal mice induced a significant 2.4-fold increase in the number of cells expressing IGF-I mRNA, whereas IGF-II and IGFBP-2 mRNA levels were not affected. In hIGF-II transgenic mice, IGF-I mRNA levels were significantly increased, while endogenous IGF-II and IGFBP-2 mRNAs were unaffected, compared to normal animals. DXM treatment of the hIGF-II transgenic mice induced a further increase of IGF-I mRNA expression, to a similar extent as in DXM-treated normal mice. The increase of IGF-I due to DXM treatment in normal mice might be a reaction in order to minimize the GC-induced growth retardation. Another possibility could be that the increase of IGF-I would contribute to the GC-induced growth retardation by accelerating the differentiation of chondrocytes, resulting in accelerated ossification. In the growth plates of hIGF-II transgenic mice, the higher basal level of IGF-I, might be responsible for the observed partial protection against the adverse effects of GCs on bone.

Human insulin-like growth factor (IGF) binding protein-1 inhibits IGF-I-stimulated body growth but stimulates growth of the kidney in snell dwarf mice.

The actions of insulin-like growth factor-I (IGF-I) are modulated by IGF binding proteins (IGFBPs). The effects of IGFBP-1 in vivo are insufficiently known, with respect to inhibitory or stimulatory actions on IGF-induced growth of specific organs. Therefore, we studied the effects of IGFBP-1 on IGF-I-induced somatic and organ growth in pituitary-deficient Snell dwarf mice. Human GH, IGF-I, IGFBP-1, and a preequilibrated combination of equimolar amounts of IGF-I and IGFBP-1 were administered sc during 4 weeks. Treatment with IGF-I alone induced a significant increase in body length (108% of control) and weight (112%) as well as an increase in weight of the submandibular salivary glands (135%), kidneys (124%), femoral muscles (111%), testes (129%), and spleen (126%) compared with saline-treated controls. IGFBP-1 alone induced a significant increase in weight of the kidneys (152% of control). Coadministration of IGF-I with IGFBP-1 neutralized the stimulating effects of IGF-I on body length and weight as well as on the femoral muscles and testes. In contrast, the weights of the submandibular salivary glands (143%) were not significantly different from those of IGF-I-treated animals, whereas the weights of the kidneys (171%) and spleen (156%) were significantly increased compared with IGF-I-treated mice. The effect of IGFBP-1 plus IGF-I on kidney weight was not significantly greater than the effect of IGFBP-1 alone. Western ligand blotting showed induction of the IGFBP-3 doublet as well as IGFBPs with molecular masses of 24 kDa, most probably IGFBP-4, by human GH, IGF-I alone, and IGF-I in combination with IGFBP-1. Our data show that coadministration of IGFBP-1 inhibits IGF-I-induced body growth of GH-deficient mice but significantly stimulates the growth promoting effects of IGF-I on the kidneys and the spleen. These data warrant further investigation because differences in concentrations of IGFBP-1 occurring in vivo may influence IGF-I-induced anabolic processes.

Expression of insulin-like growth factor II (IGF-II) and histological changes in the thymus and spleen of transgenic mice overexpressing IGF-II.

Previously, transgenic mice were constructed overexpressing human insulin-like growth factor II (IGF-II) under control of the H2kb promoter. The IGF-II transgene was highly expressed in thymus and spleen, and these organs showed an increase in weight. In the current study we have analyzed the sites of IGF-II mRNA expression, the distribution of IGF-II, IGF-I, and both IGF receptors, and histomorphometrical changes in thymus and spleen. With in situ mRNA hybridization, expression of the IGF-II transgene is found with high intensity in the thymic medulla and in the white pulp/marginal zone of the spleen, whereas there were scattered positive cells in the thymic cortex and in the splenic red pulp. Hybridization was restricted to non-lymphocytic cells. Immunohistochemistry revealed intense IGF-II peptide staining with the same distribution as IGF-II mRNA. There was additional intense IGF-II staining of all elements in the splenic red pulp (including trabeculae) and diffuse, low level staining in the thymic cortex. These findings were not observed in control mice. In the thymic medulla, most IGF-II producing cells co-labelled with keratin, whereas a minor population also stained for the monocyte/ macrophage marker MOMA-2. In the spleen, co-labelling of IGF-II producing cells was found with MOMA-1 (marginal zone), or with the dendritic cell marker NLDC-145 (red pulp). IGF-I and both IGF receptors were found in these organs in nearly all cell types, with a similar pattern in transgenic mice and in control animals. Histomorphometric analysis revealed a marked increase of thymus cortex size and an increased trabecular size in the spleen. This suggests that IGF-II overproduction induces local effects (auto/paracrine) in the thymic cortex, but not in the thymic medulla. Trabecular growth in the spleen most likely is a distant effect (paracrine or endocrine) of IGF-II overproduction.

Overexpression of human insulin-like growth factor-II in transgenic mice causes increased growth of the thymus.

In order to determine the effects of IGF-II overexpression on growth of mice, transgenic mice were produced carrying one of three different H-2Kb human IGF-II minigenes in which different non-coding exons (exon 5, truncated exon 5 or exon 6) preceded the coding exons 7, 8 and 9. These were spaced by truncated introns and for proper polyadenylation an SV40 polyadenylation signal was incorporated. The highest levels of IGF-II minigene mRNA expression were found in lines containing the truncated exon 5 construct (II5'). Those containing exon 6 (II6) had less expression and 5 constructs (II5) gave only moderate levels of mRNA expression. In general mRNA expression was highest in thymus and spleen, low in liver and kidney and absent in the brain. In addition, one II5' line showed expression in the brain. Serum IGF-II levels at 8 weeks of age were increased 7- to 8-fold in homozygous transgenic lines with construct II5' without brain expression and 2- to 3-fold in the one that showed expression in the brain; serum IGF-I levels were unchanged. Serum IGFs in the lines containing the constructs II5 and II6 were not different from those of the controls. In all cases body length and weight as well as the weight of several organs such as brain, liver, kidneys, heart and spleen when expressed as a function of age did not differ from controls. Only the thymus showed a significant increase in weight in the transgenics II5'. Inbreeding of 2 lines containing construct II5' with pituitary deficient Snell dwarf mice did not influence body length or weight despite increased serum IGF-II levels. Again the thymus showed a marked increase in growth. The biological activity of the IGF-II peptide was further demonstrated by increased serum IGF-binding protein-3 in the transgenic dwarf mice, as shown by Western ligand blotting. In summary, overexpression of IGF-II in transgenic normal and dwarf mice does not affect overall body growth, but causes increased growth of the thymus. This suggests a role for IGF-II in thymic development by paracrine/autocrine action.

Insulin-like growth factors-I and -II and their binding proteins during postnatal development of dwarf Snell mice before and during growth hormone and thyroxine therapy.

The ontogeny of serum insulin-like growth factors (IGFs)-I and -II and their binding proteins (IGFBPs) was studied in normal and dwarf Snell mice. IGF-I concentrations in serum of normal mice increased between 4 and 8 weeks of age; dwarf mice had very low serum IGF-I levels. In both normals and dwarfs, serum IGF-II levels were highest soon after birth and dropped steadily thereafter. Western ligand blots of serum IGFBPs with 125I-IGF-II as tracer revealed the expected bands of 41.5, 38.5, 30-32 and 24 kDa. In normal mice the IGFBP-3 doublet was already detectable at 2 weeks of age, and its intensity increased with age. In dwarf mice the IGFBP-3 doublet was hardly detectable. The changes of IGFs and their IGFBPs were studied in sera of dwarf mice after treatment with growth hormone (GH) and/or thyroxine (T4) for 4 weeks. In spite of a comparable growth response obtained using these hormones, serum IGF-I was increased only by GH treatment; a small but significant decrease of serum IGF-II was obtained following GH or T4 treatment. An increase of the IGFBP-3 doublet was only obtained with GH; T4 and GH + T4 had no effect. The rise of IGFBP-3 after GH treatment was accompanied by the formation of the IGFBP 150 kDa complex, as measured by neutral gel chromatography. The size distribution of 125I-IGF-II was restored to normal, while with 125I-IGF-I only a small peak at 150 kDa was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant insulin-like growth factor-II inhibits the growth-stimulating effect of growth hormone on the liver of Snell dwarf mice.

The actions and interactions of recombinant insulin-like growth factor-I and -II (IGF-I and IGF-II), alone or in combination with human GH on body growth and the growth of several organs were studied in the Snell dwarf mouse. IGF-I and -II stimulate to a similar extent sulfate incorporation into cartilage, and both IGFs increase body length and weight. IGF-II as well as IGF-I have clear effects on the size of the submandibular salivary glands, kidneys, and spleen. IGF-II, however, did not influence the weight of the lung, in contrast with IGF-I. GH treatment alone resulted in growth of the liver, whereas both IGFs were inactive. Surprisingly, IGF-II and, to a lesser extent, IGF-I inhibited GH-induced growth of the liver. Glycogen storage in the liver was decreased by treatment with IGF-II alone or in combination with GH, as shown by histological examination. It was not affected by GH, IGF-I, or GH plus IGF-I. Also, the size of the centrilobular hepatocytes was decreased by treatment with IGF-II and IGF-II plus GH; GH alone had a hypertrophic effect, whereas IGF-I or GH plus IGF-I had none. In contrast to GH, IGFs did not increase polyploidy. Treatment with IGF-II increased the level of IGFBP-3, as did IGF-I or GH treatment, as shown by Western ligand blotting. The IGFs appeared to have a greater effect on the induction of 38.5-kilodalton IGFBP-3 than GH, suggesting a different role in the regulation of glycosylation. In conclusion, IGF-I and IGF-II as well as GH have a stimulatory effect on general body growth and are effective in the stimulation of serum IGFBP-3, sulfate incorporation into cartilage, as well as the growth of specific organs in Snell dwarf mice. Both IGFs, alone or in combination with GH, show distinct effects on the growth of the liver with respect to several histological parameters, which require further exploration.