Short hairpin rna targeting insulin-like growth fator binding protein-3 restores the bioavailability of insulin-like growth factor-1 in diabetic rats.

PURPOSE: To investigate whether intracavernosal injection of short hairpin RNA for IGFBP-3 could improve erectile function in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: After 12 weeks of IGFBP-3 short hairpin RNA injection treatment, intracavernous pressure responses to electrical stimulation of cavernous nerves were evaluated. The expression of IGFBP-3 and IGF-1 at mRNA and protein levels were detected by quantitative real-time PCR analysis and Western blot, respectively. The concentration of cavernous cyclic guanosine monophosphate was detected by enzyme-linked immunosorbent assay. RESULTS: At 12 weeks after intracavernous administration of IGFBP-3 shRNA, the cavernosal pressure was significantly increased in response to the cavernous nerves stimulation compared to the diabetic group (P<0.05). Cavernous IGFBP-3 expression at both mRNA and protein levels was significantly inhibited. At the same time, cavernous IGF-1 expression was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01). Cavernous cyclic guanosine monophosphate concentration was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01). CONCLUSIONS: Gene transfer of IGFBP-3 shRNA could improve erectile function via the restoration of cavernous IGF-1 bioavailability and an increase of cavernous cGMP concentration in the pathogenesis of erectile dysfunction in streptozotocin-induced diabetic rats.

Short hairpin RNA targeting insulin-like growth factor binding protein-3 restores the bioavailability of insulin-like growth factor-1 in diabetic rats

ABSTRACT Purpose To investigate whether intracavernosal injection of short hairpin RNA for IGFBP-3 could improve erectile function in streptozotocin-induced diabetic rats. Materials and methods After 12 weeks of IGFBP-3 short hairpin RNA injection treatment, intracavernous pressure responses to electrical stimulation of cavernous nerves were evaluated. The expression of IGFBP-3 and IGF-1 at mRNA and protein levels were detected by quantitative real-time PCR analysis and Western blot, respectively. The concentration of cavernous cyclic guanosine monophosphate was detected by enzyme-linked immunosorbent assay. Results At 12 weeks after intracavernous administration of IGFBP-3 shRNA, the cavernosal pressure was significantly increased in response to the cavernous nerves stimulation compared to the diabetic group (P<0.05). Cavernous IGFBP-3 expression at both mRNA and protein levels was significantly inhibited. At the same time, cavernous IGF-1 expression was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01). Cavernous cyclic guanosine monophosphate concentration was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01). Conclusions Gene transfer of IGFBP-3 shRNA could improve erectile function via the restoration of cavernous IGF-1 bioavailability and an increase of cavernous cGMP concentration in the pathogenesis of erectile dysfunction in streptozotocin-induced diabetic rats.

Longitudinal infusion of a complex of insulin-like growth factor-I and IGF-binding protein-3 in five preterm infants: pharmacokinetics and short-term safety.

BACKGROUND: In preterm infants, low levels of insulin-like growth factor-I (IGF-I) and IGF binding protein 3 (IGFBP-3) are associated with impaired brain growth and retinopathy of prematurity (ROP). Treatment with IGF-I/IGFBP-3 may be beneficial for brain development and may decrease the prevalence of ROP. METHODS: In a phase II pharmacokinetics and safety study, five infants (three girls) with a median (range) gestational age (GA) of 26 wk + 6 d (26 wk + 0 d to 27 wk + 2 d) and birth weight of 990 (900-1,212) g received continuous intravenous infusion of recombinant human (rh)IGF-I/rhIGFBP-3. Treatment was initiated during the first postnatal day and continued for a median (range) duration of 168 (47-168) h in dosages between 21 and 111 µg/kg/24 h. RESULTS: Treatment with rhIGF-I/rhIGFBP-3 was associated with higher serum IGF-I and IGFBP-3 concentrations (P < 0.001) than model-predicted endogenous levels. Of 74 IGF-I samples measured during study drug infusion, 37 (50%) were within the target range, 4 (5%) were above, and 33 (45%) were below. The predicted dose of rhIGF-I/rhIGFBP-3 required to establish circulating levels of IGF-I within the intrauterine range in a 1,000 g infant was 75-100 µg/kg/24 h. No hypoglycemia or other adverse effects were recorded. CONCLUSION: In this study, continuous intravenous infusion of rhIGF-I/rhIGFBP-3 was effective in increasing serum concentrations of IGF-I and IGFBP-3, and was found to be safe.

Analysis of the cellular uptake and nuclear delivery of insulin-like growth factor binding protein-3 in human osteosarcoma cells.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) regulates cell proliferation and survival by extracellular interaction and inactivation of the growth factor IGF-I. Beyond that, IGF-independent actions mediated by intracellular IGFBP-3 including nuclear-IGFBP-3, have also been described. We here show, using both confocal and electron microscopy and cell fractionation, that the extracellular addition of IGFBP-3 to living cells results in rapid uptake and nuclear delivery of IGFBP-3, by yet partly unknown mechanisms. IGFBP-3 is internalized through a dynamin-dependent pathway, traffics through endocytic compartments and is finally delivered into the nucleus. We observed docking of IGFBP-3 containing structures to the nuclear envelope and found IGFBP-3 containing dot-like structures to permeate the nuclear envelope. In summary, our findings establish the pathway by which this tumor suppressor protein is delivered from extracellular space to the nucleus.

Insulin-like growth factor-I deficiency in children with growth hormone insensitivity: current and future treatment options.

Growth hormone (GH) acts directly at the growth plate and through the production of insulin-like growth factor (IGF)-I. At least 50% of the hormone circulates bound to GH binding protein, and its secretion is controlled by growth hormone-releasing hormone and somatostatin. Once GH binds to two GH receptors, the janus activated kinase/signal transducers and activators of transcription (JAK/STAT) protein pathway is activated, resulting in the production of IGF-I. Serum IGF-I is produced predominantly in the liver and circulates in a 140 kDa complex, along with its binding protein, IGF binding protein 3, and acid-labile protein. Recombinant human (rh) IGF-I (mecasermin) is approved by the US FDA and the European Medicines Agency for the treatment of patients with severe primary IGF deficiency or for patients with GH1 gene deletion who have developed neutralizing antibodies to GH. It has been shown to increase growth velocity in children with either condition. In the past, there have been adverse events, particularly hypoglycemia, reported with the administration of mecasermin. However, a recent report of long-term therapy with mescasermin in children with severe IGF-I deficiency has concluded that although adverse events are common, they are rarely severe enough to interrupt or modify treatment. The serum half-life of mecasermin is shorter in patients with GH insensitivity syndrome and low serum levels of its binding protein, the insulin-like growth factor binding protein (IGFBP)-3 and acid-labile subunit, compared with the serum half-life in normal volunteers or in patients with an IGF1 gene deletion who have normal levels of IGFBP-3. Mecasermin rinfabate, a complex of rhIGF-I and rhIGFBP-3, appears to prolong the serum half-life and might counteract acute adverse events, particularly hypoglycemia, associated with the administration of mescasermin. Mecasermin rinfabate, however, is no longer available in the USA or Europe for treating conditions involving short stature, because of a legal requirement. Mecasermin has been shown to be effective in increasing height velocity and adult height in patients with severe GH resistance and in IGF1 gene deletion. There has been some interest in using mecasermin to treat patients with partial GH resistance or idiopathic short stature. At the present time, the data are insufficient to make this recommendation.

Efficacy and safety of mecasermin rinfabate.

There has been interest in using recombinant human (rh) insulin-like growth factor (IGF)-I (rhIGF-I) to treat short stature, either alone or in combination with its binding protein (insulin-like growth factor binding protein [IGFBP]-3). IGF-I has been shown to increase growth velocity in children with IGF deficiency, either as a result of growth hormone insensitivity syndrome (GHIS) or IGF gene deletion. However, there have been adverse events, particularly hypoglycaemia, reported with administration of unbound rhIGF-I. In addition, the serum half-life of unbound rhIGF-I is shorter when administered to patients with GHIS, who have low serum concentrations of its binding proteins IGFBP-3 and acid-labile subunit (ALS), than when administered to normal volunteers or to the patient with an IGF-I gene deletion (who had normal levels of IGFBP-3). iPlex (mecasermin rinfabate), an equimolar mixture of IGF-I and its binding protein IGFBP-3, was developed to prolong the half-life and to counteract acute adverse events (particularly hypoglycaemia) associated with administration of IGF-I. Although there are no published data on the efficacy of mecasermin rinfabate in treating growth disorders, it does appear that mecasermin rinfabate has a longer half-life in patients with GHIS than unbound IGF-I, and fewer reports of adverse events (including hypoglycaemia) when administered to patients with diabetes.

Pharmacokinetic studies of recombinant human insulin-like growth factor I (rhIGF-I)/rhIGF-binding protein-3 complex administered to patients with growth hormone insensitivity syndrome.

CONTEXT: GH insensitivity syndrome (GHIS), Laron syndrome, is characterized by severe short stature, high serum GH levels, and very low serum IGF-I and IGF-binding protein-3 (IGFBP-3) levels associated with a genetic defect of the GH receptor. Recombinant human (rh) IGF-I treatment at doses of 80-120 microg/kg given sc twice daily is effective in promoting growth in these patients. We have investigated a newly developed drug, rhIGF-I/rhIGFBP-3, a 1:1 molar complex of rhIGF-I and rhIGFBP-3. OBJECTIVES: The objectives of the study were to determine IGF-I pharmacokinetics after the administration of rhIGF-I/rhIGFBP-3 in adolescents with GHIS and to evaluate its safety and tolerability. DESIGN: This was an open-label clinical study. SETTING: The study was conducted in a general pediatric ward of a university teaching hospital. PARTICIPANTS: Four patients (one female and three males; mean age, 14.9 yr; mean height sd score, -4.9) with confirmed molecular diagnosis of GHIS agreed to participate in the study. INTERVENTION: rhIGF-I/rhIGFBP-3 was administered in a single sc injection at 0.5 and 1.0 mg/kg.dose (equivalent to 100 and 200 microg/kg rhIGF-I) after breakfast with a 2-d interval between doses. RESULTS: IGF-I levels reached a maximum between 19 +/- 8.3 and 15 +/- 6.2 h for the low and high doses, respectively. The circulating IGF-I levels obtained with the low and high doses were similar, although a discrete dose-dependent increase in circulating IGF-I levels was observed. The IGF-I half-life in four subjects after a dose of 0.5 mg/kg rhIGF-I/rhIGFBP-3 was estimated to be 21+/- 4 h. There were no acute adverse events reported, and all blood glucose measurements were normal. CONCLUSION: These data demonstrated that the rhIGF-I/rhIGFBP-3 complex was effective in increasing levels of circulating total and free IGF-I into the normal range for a 24-h period after a single sc administration in patients with GHIS, and that administration of rhIGF-I/rhIGFBP-3 was safe and well tolerated.

Mecasermin rinfabate: insulin-like growth factor-I/insulin-like growth factor binding protein-3, mecaserimin rinfibate, rhIGF-I/rhIGFBP-3.

Insmed is developing mecasermin rinfabate, a recombinant complex of insulin-like growth factor-I (rhIGF-I) and binding protein-3 (rhIGFBP-3) [insulin-like growth factor-I/insulin-like growth factor binding protein-3, rhIGF-I/rhIGFBP-3, SomatoKine], for a number of metabolic and endocrine indications. In the human body, IGF-I circulates in the blood bound to a binding protein-3 (IGFBP-3), which regulates the delivery of IGF-I to target tissues, and particular proteases clip them apart in response to stresses and release IGF-I as needed. IGF-I, a naturally occurring hormone, is necessary for normal growth and metabolism. For the treatment of IGF-I deficiency, it is desirable to administer IGF-I bound to IGFBP-3 to maintain the normal equilibrium of these proteins in the blood. Mecasermin rinfabate (rhIGF-I/rhIGFBP-3) mimics the effects of the natural protein complex in the bloodstream and would augment the natural supply of these linked compounds. The most advanced indication in development of mecasermin rinfabate is the treatment of severe growth disorders due to growth hormone insensitivity syndrome (GHIS), also called Laron syndrome. GHIS is a genetic condition in which patients do not produce adequate quantities of IGF because of a failure to respond to the growth hormone signal. This results in a slower growth rate and short stature. Mecasermin rinfabate also has potential as replacement therapy for IGF-I, which may become depleted in indications such as major surgery, organ damage/failure, traumatic injury, cachexia and severe burn trauma. It also has potential for the treatment of osteoporosis. Mecasermin rinfabate was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on 1 June 2000. Insmed and Avecia of the UK have signed an agreement for manufacturing mecasermin rinfabate and its components, rhIGF-1 and rhIGFBP-3. CGMP clinical production of mecasermin rinfabate and its components will be carried out in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines at the capacity of up to 1000L. In April 2004, Insmed announced that it acquired a lease to operate the manufacturing facility formerly operated by Baxter for the commercial production of SomatoKine in Boulder, CO, USA. With the two manufacturing facilities for SomatoKine, Insmed plans to meet the development and commercial demands for the product over the next several years. In its 2003 Form-10K, Insmed announced plans to conduct comparative studies with the previously used drug substance and the new substance produced by Avecia. The comparative data will be included in the regulatory filing for mecasermin rinfabate. Mecasermin rinfabate was originally licensed to Welfide for Japan. On 1 October 2001, Welfide Corporation merged with Mitsubishi-Tokyo Pharmaceuticals to form Mitsubishi Pharma Corporation. The new company is a subsidiary of Mitsubishi Chemical. In October 2004, Insmed announced that Tzamal Pharma has been granted exclusive distribution and marketing rights for mecasermin rinfabate in certain Middle Eastern territories including Israel. Tzamal Pharma also acquired exclusive rights to Insmed's named patient programme for the agent in these territories. Tzamal Pharma intends to begin the appropriate registration activities for mecasermin rinfabate in the treatment of children with growth hormone-insensitivity syndrome. This pivotal, 12-month, multicentre, open-label trial in 30 children with GHIS was initiated in June 2003 and was designed to evaluate the safety and efficacy of the agent in prepubescent children with GHIS. The 6-month endpoint data analysis showed that mecasermin rinfabate given as a once-daily injection was safe and well tolerated. The agent demonstrated a significant increase in height velocity in children with GHIS similar to that observed by Pfizer in their pivotal study with twice-daily injections of rhIGF-I. The full results from the pivotal trial are expected in 2005. In April 2003 Insmed initiated a named patient programme in Europe that will make available mecasermin rinfabate for the treatment of GHIS-Laron syndrome. The treatment of patients was initiated in Scandinavia, with authorisation pending in several other European countries. Mecasermin rinfabate will be made available to those GHIS patients who, in the opinion of their doctor, may benefit from IGF-I therapy. At precommercial scale quantities, the drug will be available on a limited basis.A phase II dose-ranging study in children with GHIS was completed at Saint Bartholomew's and the Royal London School of Medicine, London, UK. A single dose of mecasermin rinfabate delivered the same amount of IGF-1 as two daily injections of unbound IGF-1. No adverse events were reported. Insmed has acquired an exclusive licence to Pharmacia's regulatory filings concerning yeast-derived insulin-like growth factor 1 (IGF-1). These filings were used by Pharmacia to receive marketing approvals in several European countries and also in the IND application with the US FDA. Insmed believes that this licence will facilitate the development of mecasermin rinfabate for the treatment of children with GHIS. In January 2003, Insmed announced positive results from a double-blind, placebo-controlled, dose-ranging study of mecasermin rinfabate in adolescent patients with type 1 diabetes receiving insulin therapy. The study was conducted at the University of Cambridge, Cambridge, UK, under supervision of Prof. D. Dunger. The researchers from The Robarts Research Institute and the University of Western Ontario, Canada (leading investigator T.L. Delovitch, the Sheldon H. Weinstein scientist in Diabetes at the University of Western Ontario) have found that mecasermin rinfabate complex was significantly more effective than IGF-1 in reducing the severity of insulitis, beta cell destruction and delaying the onset of type 1 diabetes. The study was supported by grants from Canadian Institutes of Health and the Juvenile Diabetes Research Foundation. Insmed plans to initiate large-scale phase II clinical studies in this indication. At the BIO 2004 Annual International Convention (BIO-2004) in June 2004, Insmed announced that it has received a grant from the US National Institutes of Health (NIH)/Muscular Dystrophy Association (MDA) worth USD $6.5 million to investigate the efficacy of mecasermin rinfabate for the treatment of myotonic dystrophy. It has also been granted orphan drug status for the treatment of GHIS-Laron syndrome in the US and Europe. In December 2003, Insmed announced that mecasermin rinfabate was designated orphan drug status by the FDA for the treatment of extreme insulin resistance. This provides Insmed with 7 years of market exclusivity following approval of mecasermin rinfabate for this indication. Insmed has received orphan drug designation for mecasermin rinfabate in the treatment of extreme insulin resistance in Europe (October 2004). In November 2004, Insmed was granted the European patent EP1183042 entitled "Methods for Treating Diabetes". This patent corresponds with the US patent US 6,040,292 also entitled "Methods for Treating Diabetes". Both patents cover type 1 and type 2 diabetes mellitus and insulin resistant diabetes including type A insulin resistance (the least severe form of extreme insulin resistance syndromes). In January 2004, Insmed obtained a non-exclusive licence to the patents for use of IGF-I for the treatment of extreme or severe insulin-resistant diabetes from Fujisawa Pharmaceutical. Insmed will have worldwide rights in territories (excluding Japan) with existing valid patent claims including the US and Europe. Insmed holds 28 US issued or allowed patents for the composition, production, antibodies and methods of use of mecasermin rinfabate. These US patents expire at various times between the years 2010 and 2019. Insmed through their lawyers filed its defense and counterclaim to the alleged patent infringement brought by Tercica against Insmed in the London High Court of Justice. Insmed asserted that it did not infringe any valid patent claims as none of the claims of the patent were patentable because the subject matter was not new. Insmed also stated that the patent did not involve an inventive step, did not have capability of industrial application and had no clear description of the invention so that invention can be performed by the person skilled in the art. Insmed is seeking revocation of the patent on these grounds.

Some kinetic aspects in the immunoradiometric assay of insulin-like growth factor binding protein-3.

In this article the kinetics of the insulin-like growth factor binding protein-3 (IGFBP-3) reaction with its specific antibody immobilised on the inner wall of the reaction tube, and the subsequent binding of the immunocomplex formed with a second (125)I-labelled antibody are described. These reactions are used in the immunoradiometric determination of IGFBP-3. Independent variables were analyte and labelled antibody, temperature, viscosity, and the ionic strength of the medium. For the global process mono-exponential kinetics were found to be dependent on the concentrations, such dependence fitting with the models discussed in this paper. Viscosity results clearly indicate its negative influence on the direct reaction rate. Ionic strength shows noticeable but not too relevant effects, which suggests that the variation caused by the glycerol addition is not due to the influence of the dielectric constant of the solutions used. The effect of temperature shows activation parameters similar to the viscous flow energy of water, which suggests diffusion control for the global process.

Greater potency of IGF-I than IGF-I/BP-3 complex in catabolic parenterally fed rats.

We compared the anabolic effects of recombinant human insulin-like growth factor I (rhIGF-I, 2.5 mg/kg) and equimolar amounts of rhIGF-I prebound to rhIGF binding protein-3 (rhIGF-I/BP-3) coinfused continuously with total parenteral nutrition (TPN) solution in dexamethasone (Dex, 70 microg/day ip)-treated male rats for 6 days. The four TPN groups included control, Dex, Dex + IGF-I, and Dex+IGF-I/BP-3. Pharmacokinetic analysis indicated reduced clearance of IGF-I when infused as IGF-I/BP-3 compared with free IGF-I (0.91 +/- 0.09 vs. 2.01 +/- 0.19 ml serum/min, P < 0.001) and this was associated with significantly greater serum IGF-I concentrations in the Dex+IGF-I/BP-3 group. Despite greater total serum IGF-I levels, infusion of free IGF-I produced greater anabolic responses than IGF-I/BP-3 based on body weight, nitrogen balance, and jejunal cellularity. Treatment with free IGF-I, but not IGF-I/BP-3, significantly reduced serum insulin and glucose levels that were elevated due to Dex. There were no significant differences in liver IGF-I mRNA levels between groups. Serum IGFBP-3 levels were elevated with infusion of IGF-I/BP-3 compared with IGF-I. These results indicate greater anabolic potency of IGF-I compared with IGF-I/BP-3 when administered by continuous parenteral infusion with TPN solution in catabolic rats.

Dietary fat and carbohydrates are independently associated with circulating insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 concentrations in healthy adults.

PURPOSE: To evaluate and quantify the association between consumption of specific food groups/macronutrients and concentrations of serum insulin-like growth factor 1 (IGF-1) and insulin-like growth factor-binding protein 3 (IGFBP-3). SUBJECTS AND METHODS: Data from a comprehensive food-frequency questionnaire administered to 115 healthy subjects were used to study cross-sectionally the relationship between nutritional factors and circulating IGF-1 and IGFBP-3 concentrations. Adjustment for the effect of total energy intake and a series of epidemiologic parameters (age, sex, height, body mass index, smoking, alcohol consumption, and coffee drinking) was implemented through multivariate linear regression. RESULTS: We observed that serum levels of IGF-1 are positively associated with consumption of red meats, fats, and oils. In addition, serum levels of IGF-1 are independently and positively associated with energy intake from lipids and negatively associated with energy intake from carbohydrates. Finally, serum levels of IGFBP-3 are independently and negatively associated with energy intake from saturated fat. CONCLUSION: Serum IGF-1 and/or IGFBP-3 concentrations are associated with red meat, carbohydrate intake, and fat intake and, thus, may mediate the effect of these dietary factors on the pathogenesis of several disease states. Additional studies are needed to further quantify these associations and elucidate the underlying mechanisms.

Age- and growth hormone-induced alterations in renal sulfate transport.

The effects of growth hormone (GH) treatment on renal sodium sulfate cotransport (NaSi-1) were studied in adult (9-10 months) and old (22-23 months) male Fischer 344 rats. All animals received twice-daily s.c. injections of recombinant human GH (hGH; 4 mg/kg) for up to 6 days. Animals were sacrificed by exsanguination on days 0, 1, 2, 3, 4, 5, and 6. Kidneys were removed, and kidney cortex was trimmed immediately and used for RNA and membrane preparations. Plasma hGH concentrations were significantly lower in old rats during the hGH treatment (P <.05). Insulin-like growth factor-I (IGF-I) levels were significantly increased and remained stable after day 2 of hGH treatment in both age groups (P <.05). There was no significant difference in plasma IGF-I levels between age groups. Plasma IGF-I binding protein 3 (IGFBP-3) concentrations were significantly higher in 9- to 10-month-old rats compared with that in 22- to 23-month-old animals (P <.001). There were no significant differences in plasma IGFBP-3 concentrations between days of hGH treatment. The NaSi-1 mRNA levels were significantly lower in 22- to 23-month-old rats compared with that in 9- to 10-month-old animals (P <.001). The NaSi-1 mRNA levels were significantly increased on days 2 and 3 of hGH treatment (P <.05) and then gradually decreased to the control value. The NaSi-1 protein levels in old animals (22-23 months) were also significantly lower than that of 9- to 10-month-old animals and were significantly increased from day 2 of hGH treatment, reaching a maximum level on day 3 or 4 and then returning to the baseline level in both age groups. From these results, it was concluded that 1) NaSi-1 mRNA and protein levels are lower in old animals and increase in both adult and aged rats after hGH treatment, 2) plasma IGF-I levels are similar in adult and aged rats and increase after hGH treatment, and 3) plasma IGFBP-3 levels are lower in old rats and remain unchanged after hGH treatment.

Infused IGF-I/IGFBP-3 complex causes glomerular localization of IGF-I in the rat kidney.

Insulin-like growth factor I (IGF-I) increases renal blood flow, glomerular filtration rate (GFR), and proximal tubule reabsorption of phosphate in humans and rodents. The biological effects of IGF-I are likely to be influenced by cellular localization of IGF-I within the kidney. We therefore tested whether the renal localization of infused IGF-I could be altered if given with selected IGF-binding proteins (IGFBPs). Rats were treated with intravenous injections of 125I-labeled IGF-I, 125I-IGFBP-3, or 125I-IGFBP-4 alone or with complexes of 125I-IGF-I and IGFBP-3 or IGFBP-4. The cellular localization of IGF and the IGFBP within the kidney was then determined. 125I-IGF-I, 125I-IGFBP-4, and 125I-IGF-I/IGFBP-4 complexes were found almost exclusively in vacuolar structures (endosomes) of proximal renal tubules. In contrast, about one-third of renal 125I-IGFBP-3 and 125I-IGF-I/IGFBP-3 was localized to glomeruli. When 125I-IGF-I was given alone, 3% was found in glomeruli and 89% in proximal tubules. When given as 125I-IGF-I/IGFBP-3, 29% was in glomeruli and 65% in proximal tubules. We conclude that the cellular localization of IGF-I within the kidney can be directed to glomerular elements if the IGF-I is given with IGFBP-3.

Serum insulin-like growth factors (IGFs) and IGF binding protein (IGFBP)-3 in patients with gastric cancer: IGFBP-3 protease activity induced by surgery.

Recent findings have indicated that insulin-like growth factors (IGF-I and IGF-II) may play a role in neoplasia. Alteration of serum IGFs or IGF Binding Proteins (IGFBPs) have been reported in some tumors. In this study, we measured serum IGF-I, IGF-II and IGFBPs profile in gastric cancer by radioimmunoassay and Western ligand blots. The serum IGF-I level in gastric cancer was significantly lower than in control subjects (65.2 +/- 26.5 vs 148.4 +/- 55.2 ng/ml, p < 0.01) and was further decreased to 45.5 +/- 20.9 ng/ml after surgery. The serum IGF-II level was slightly higher than that in control subjects (826.3 +/- 360.2 vs 735.7 +/- 154.6 ng/ml) but it was significantly decreased after surgery (525.7 +/- 220.1 ng/ml, p < 0.05). The serum IGFBP-3 level was not significantly different from those in control subjects. However, we observed a decreased level of serum IGFBP-3 after surgery, and incubation of postoperative serum with control serum resulted in a significant reduction of IGFBP-3 level. The reduction of IGFBP-3 in postoperative serum was mainly due to surgery associated IGFBP-3 protease activity. This protease activity was totally inhibited by aprotinin, EDTA and PMSF but not by pepstatin and leupeptin. This inhibition pattern is consistant with cation dependent serine protease. We speculate that proteolysis of IGFBP-3 may contribute to increase the bioavailability of IGFs.

Rapid clearance of human insulin-like growth factor binding protein-3 from the rat circulation and cellular localization in liver, kidney and stomach.

The gastrointestinal tract represents a major site for the trophic actions of insulin-like growth factors (IGFs), which may be derived in vivo from a large circulating pool. The high capacity binding protein for IGFs in blood is IGF binding protein-3 (IGFBP-3), which is largely complexed with an acid-labile subunit (ALS). However, we and others have shown that IGFBP-3 not complexed with ALS can rapidly leave the circulation, and may carry IGFs to peripheral tissues. In this study we investigated the transfer of recombinant, glycosylated human (h)IGFBP-3 from the rat circulation to the stomach and intestine, compared with liver and kidney. [125I]-labeled IGFBP-3 was administered into the tail vein of conscious male rats, which were killed between 5 min and 2 h later. Blood was taken for the preparation of plasma, and the liver, kidneys, stomach and intestine were removed either for estimation of the associated radioactivity, or fixed for autoradiographic analysis of histological sections. Following injection, [125I]-labeled IGFBP-3 was associated, in part, with a 150 kDa complex in plasma within 10 min when analyzed by gel filtration chromatography. However, 84% of the administered IGFBP-3 had already left the circulation, and 40% of the initial injected dose was accumulated in liver by 5 min, with a further 4% localized in the kidneys. Autoradiographic analysis showed that IGFBP-3 was selectively accumulated within Kupffer cells of the liver, and by the glomeruli and proximal tubules of the kidney. Little radiolabeled IGFBP-3 was recovered from the small intestine, but 14% of the initial injected dose was found within the stomach after 2 h, and a further 12% within the stomach contents. Autoradiographic localization within the stomach showed that the [125I]-labeled IGFBP-3 was primarily associated with the mucosal lining and gastric glands. Separation on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that the majority of the radioactivity associated with the stomach contents represented small, degraded peptides. These results suggest that while a rapid clearance of IGFBP-3 is achieved by the liver and kidney, a longer term accumulation occurs in the stomach with a luminal secretion. This may represent a delivery system by which circulating IGFs may reach gastric tissue.

Pharmacokinetics and bioavailability of rhIGF-I/IGFBP-3 in the rat and monkey.

Circulating IGF-I exists primarily as part of a ternary 150 kDa complex comprising equimolar amounts of IGF-I, IGFBP-3 and acid labile subunit (ALS). It is also known that in contrast to IGF-I and IGFBP-3 there exists a substantial quantity of unbound ALS in the circulation. As part of our preclinical development program, we have investigated the pharmacokinetic properties of a complex of human recombinant IGF-I and IGFBP-3. Systematic administration of rhIGF-I/IGFBP-3 results in binding of this binary complex to endogenous free ALS and thus leads to increased circulating levels of 150 kD ternary complex (IGF-I/IGFBP-3/ALS). In contrast to the administration of free IGF-I, IGF-I/IGFBP-3 dosing leads to increased systemic IGF-I exposure (i.e. increased area under the time vs. serum concentration curve or AUC) and decreased clearance (CL). Upon administration of equimolar doses of IGF-I/IGFBP-3 to the rat and monkey, it was found that in the monkey AUC was increased and CL decreased when compared to the rat. In addition, the pharmacokinetic profile suggests that saturation of excess ALS occurs at considerably lower doses of rhIGF-I/IGFBP-3 in the monkey than in the rat. Finally, the bioavailability of rhIGF-I/IGFBP-3 was assessed in the rat and found to be approximately 85% and 50% after intramuscular and sub-cutaneous administration, respectively. It was concluded that the formation of the 150 kD ternary complex had a significant impact on increasing the systemic exposure to rhIGF-I when administered as the binary complex (rhIGF-I/IGFBP-3). In addition, IGFBP-3 increases the therapeutic index of rhIGF-I even at doses that significantly exceed the saturation of ALS.