Herbert A. Schmid

Opportunities in somatostatin research: biological, chemical and therapeutic aspects.

Somatostatins — also known as somatotropin-release inhibiting factors (SRIFs) — are a family of cyclopeptides that have broad inhibitory effects on the secretion of hormones such as growth hormone, insulin and glucagon. These effects have formed the basis for the clinical use of SRIF analogues in the treatment of acromegaly and endocrine tumours. The discovery of the five SRIF receptor subtypes in the 1990s further enhanced our understanding of the biological roles of SRIFs, and paved the way for new therapeutic opportunities. Here we review recent advances in SRIF biology, the chemistry of SRIF agonists and antagonists, and the therapeutic potential of such compounds in a wide range of established and novel indications.

Pasireotide (SOM230): Development, mechanism of action and potential applications.

Pasireotide (SOM230) is a multi-receptor ligand somatostatin analogue with high binding affinity for somatostatin receptor subtypes sst(1,2,3) and sst(5). Pasireotide potently suppresses GH, IGF-I and ACTH secretion, indicating potential efficacy in acromegaly and Cushings disease. The prolonged inhibition of hormone secretion by pasireotide in animal models and expression of multiple sst receptors in carcinoid tumors suggests that pasireotide may have clinical advantages over octreotide in patients with carcinoid tumors. Direct and indirect antitumor activity has been observed in vitro with pasireotide, including sst receptor-mediated apoptosis and antiangiogenesis, suggesting a possible role for pasireotide in antineoplastic therapy. In summary, preclinical evidence, as well as preliminary results from clinical studies suggests that pasireotide is a promising new treatment for patients with symptoms of metastatic carcinoid tumors refractory or resistant to octreotide, de novo or persistent acromegaly, and that pasireotide has the potential to be the first directed medical therapy for Cushings disease.

Improving Oral Bioavailability of Peptides by Multiple N-Methylation: Somatostatin Analogues†

Low bioavailability of peptides following oral administration is attributed to their inactivation in the gastro–intestinal tract through enhanced enzymatic degradation in the gut wall by a variety of peptidases expressed at the enterocytes brush border, and to poor intestinal permeation. In addition, the instability of peptides toward peptidases in the systemic blood circulation causes rapid elimination (i.e., short half-life). These factors limit the use of peptides as therapeutic agents in the clinical setting. Several strategies have been used to reduce enzymatic cleavage and uptake into the systemic blood circulation, including prodrug approaches, peptidomimetics, and structural modifications, such as covalent attachment of polyethylene glycol (PEG), lipidation, and chemical modifications, for example, cyclization, d-amino acid substitution, and N-methylation. Cyclic peptides show improved chemical stability and thereby display longer biological half-life compared to their linear counterparts. Yet, additional modifications are required to generate peptides with enhanced enzymatic stability and improved oral bioavailability. One of the techniques suggested to improve the enzymatic stability of peptides is N-methylation. We recently developed a simplified method which allows fast and efficient multiple N-methylation of peptides on solid support. This simplified synthetic capability led us to study the influence of multiple N-methylation of the peptide backbone on its conformation and bioactivity. Inspired by the bioavailability of the highly N-methylated transplantation drug cyclosporin A, which can be administered orally although it violates all Lipinski9s rules on oral bioavailability; we assumed this bioavailability was a result of its multiple N-methylation together with cyclization. Thus, it is possible to overcome the above mentioned bioavailability drawbacks of peptides providing both the biological activity and the receptor selectivity by multiple N-methylation of cyclic peptides. Hence, we planned to screen a complete library of all the possible N-methylated analogues of the Veber–Hirschmann cyclic hexapeptide cyclo(-PFwKTF-) (1; Figure 1) which was reported to be selective towards sst2 and

Functional Activity of the Multiligand Analog SOM230 at Human Recombinant Somatostatin Receptor Subtypes Supports Its Usefulness in Neuroendocrine Tumors

Functional gastroenteropancreatic tumors express all 5 somatostatin receptor subtypes (sst) in different quantities. Octreotide and lanreotide treat patients with these tumors by binding preferentially to sst2 and, to a lesser extent, to sst3 and sst5 receptors, thereby controlling prominent symptoms caused by hormone hypersecretion (diarrhea and flushing). Although symptoms initially improve in most patients, a loss of response occurs in about 50% during continuous treatment. The functional activity at sst receptors of SOM230, a new multiligand somatostatin analog, has been described and compared with that of somatostatin (SRIF-14) and octreotide. These data show that SOM230 is a full agonist with nanomolar potency at sst1,2,3 and sst5 receptors. The multiligand activity profile of SOM230, together with its nondesensitizing inhibitory effect on growth hormone and insulin-like growth factor-I secretion in rats, may underlie its successful use in clinical trials and its potential for use in refractory patients with carcinoid tumors.

Ghrelin acts on leptin-responsive neurones in the rat arcuate nucleus.

Leptin decreases food intake and increases energy expenditure in rodents by inhibiting neurones in the hypothalamic arcuate nucleus. The growth hormone secretagogue (GHS) ghrelin is known to stimulate food intake and to be the endogenous ligand for the GHS‐receptor, which is strongly expressed in the arcuate nucleus, like the leptin receptor (Ob‐R). In this study, we analysed the effect of systemic ghrelin administration on Fos expression in the arcuate nucleus on neurones expressing Ob‐R. Injection of ghrelin (0.2 mg/kg, i.p) significantly increased the number of neurones expressing Fos protein in the ventromedial arcuate nucleus. Fifty‐seven percent of all Fos‐positive cells in the ventromedial arcuate nucleus were also positive for Ob‐R staining. Furthermore, we investigated electrophysiologically the effect of ghrelin and leptin on the activity of arcuate neurones in an in‐vitro slice preparation. Ghrelin stimulated the electrical activity dose‐dependently in 80% of all cells tested (n=49) with a threshold concentration of 10−11 M; only 8% were inhibited and 12% did not respond. The effect of ghrelin (10−7 M) was weakly antagonized by the peptidic GHS‐receptor antagonist (D‐Lys3)‐GHRP‐6 (10−4 M), which also showed a much weaker affinity (IC50, 0.9 × 10−6 M) to the GHS‐receptor than ghrelin (IC50, 0.3 × 10−9 M). Ghrelin increased the electrical activity in 76% of all cells which were inhibited by leptin (n=17). These data show that ghrelin interacts with the leptin hypothalamic network in the arcuate nucleus. The opposite effect of leptin and ghrelin on neurones in the arcuate nucleus may serve as a neurophysiological correlate of the orexigenic and anorectic effects of ghrelin and leptin.

Site-specific effects of ghrelin on the neuronal activity in the hypothalamic arcuate nucleus

The recently discovered hormone ghrelin, which is secreted from the stomach during fasting and hypoglycemia opposes the homeostatic functions of leptin by increasing food intake and decreasing energy expenditure. The hypothalamic arcuate nucleus (Arc) mediates the effects of leptin and contains a high density of ghrelin receptors. The leptin- and ghrelin-responsive network involves the hypothalamic neuropeptide Y/alpha-melanocyte stimulating hormone (NPY/alpha-MSH) system. In the rat, neurons expressing the orexigenic peptide NPY are mainly located in the ventromedial Arc (ArcM), while pro-opiomelanocortin (POMC) neurons, synthesizing the anorectic peptide alpha-MSH, predominate in the ventrolateral Arc (ArcL). In extracellular single unit recordings from in vitro slice preparations of the Arc, superfusion of ghrelin (10(-8) M) exerted predominantly excitatory effects on ArcM neurons (73%, n=93), while a high number ArcL neurons were inhibited in response to ghrelin (42%, n=43). The excitatory effect of ghrelin on neuronal activity was postsynaptic since it was unaffected by synaptic blockade (low Ca(2+)/high Mg(2+) solution). In contrast, the inhibitory response in the ArcL was abolished by the blockade of synaptic interactions indicating a presynaptic mechanism. These results indicate that circulating ghrelin may oppose the actions of leptin by directly activating NPY-neurons of the ArcM and by indirectly inhibiting POMC neurons of the ArcL.

The lamina terminalis and its role in fluid and electrolyte homeostasis.

The lamina terminalis, which forms most of the anterior wall of the third ventricle, consists of the median preoptic nucleus and two circumventricular organs (CVOs), the subfornical organ and organum vasculosum of the lamina terminalis. These latter two regions lack a blood-brain barrier and, unlike other regions of the brain, are influenced by the hormonal and ionic composition of the blood. The CVOs of the lamina terminalis are rich in receptors for a number of circulating peptides and the subfornical organ and the OVLT are clearly established as the prime cerebral targets for circulating angiotensin II, atrial natriuretic peptide (AVP) and relaxin to influence central nervous system pathways regulating body fluid homeostasis. Together with the median preoptic nucleus, these two CVOs also detect changes and relay neural signals relating to the tonicity of body fluids and play important roles in osmoregulatory fluid intake and excretion. The neural circuitry of the lamina terminalis involves both afferent and efferent connections to several other regions of the brain, and neurons within the individual components of lamina terminalis are reciprocally connected with each other. This neural circuitry subserves the influence that the lamina terminalis exerts on vasopressin secretion, thirst, the appetite for salt, renal sodium excretion and renin secretion by the kidney. Copyright 1999 Harcourt Publishers Ltd.

Circulating relaxin acts on subfornical organ neurons to stimulate water drinking in the rat

Relaxin, a peptide hormone secreted by the corpus luteum during pregnancy, exerts actions on reproductive tissues such as the pubic symphysis, uterus, and cervix. It may also influence body fluid balance by actions on the brain to stimulate thirst and vasopressin secretion. We mapped the sites in the brain that are activated by i.v. infusion of a dipsogenic dose of relaxin (25 μg/h) by immunohistochemically detecting Fos expression. Relaxin administration resulted in increased Fos expression in the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and magnocellular neurons in the supraoptic and paraventricular nuclei. Ablation of the SFO abolished relaxin-induced water drinking, but did not prevent increased Fos expression in the OVLT, supraoptic or paraventricular nuclei. Although ablation of the OVLT did not inhibit relaxin-induced drinking, it did cause a large reduction in Fos expression in the supraoptic nucleus and posterior magnocellular subdivision of the paraventricular nucleus. In vitro single-unit recording of electrical activity of neurons in isolated slices of the SFO showed that relaxin (10−7 M) added to the perfusion medium caused marked and prolonged increase in neuronal activity. Most of these neurons also responded to 10−7 M angiotensin II. The data indicate that blood-borne relaxin can directly stimulate neurons in the SFO to initiate water drinking. It is likely that circulating relaxin also stimulates neurons in the OVLT that influence vasopressin secretion. These two circumventricular organs that lack a blood–brain barrier may have regulatory influences on fluid balance during pregnancy in rats.

Amylin and glucose co-activate area postrema neurons of the rat

Glucose is an important metabolic factor controlling feeding behavior. There is evidence that physiologically relevant glucose sensors reside in the caudal hindbrain. The area postrema (AP) in particular, which has been characterized as a receptive site for the anorectic hormone amylin, may monitor blood glucose levels. To determine whether glucose and amylin co-activate the same subset of AP neurons, we performed extracellular single unit recordings from a rat AP slice preparation. In 53% of all AP neurons tested (n=32), the activity was positively correlated to the glucose concentration. Interestingly, there was a coincidental sensitivity (94%) of AP neurons to glucose and amylin, which exerted excitatory effects on these cells. We conclude that the co-sensitivity of AP neurons to glucose and amylin, both increasing in response to food intake, points to the AP as an important hindbrain center for the integration of the metabolic and hormonal control of nutrient intake.

Orexin A activates leptin-responsive neurons in the arcuate nucleus

Orexins, also named hypocretins, are newly described neuropeptides, which are produced almost exclusively in neurons of the lateral hypothalamus and have been shown to increase food intake after intracerebroventricular injection. Leptin, the ob-gene product released from white adipocytes, is suspected to reduce food intake mainly by acting on neurons in the arcuate nucleus of the hypothalamus. Application of orexin A activated 85% (66 out of 78) of all neurons of the rat arcuate nucleus investigated electrophysiologically in an in vitro slice preparation, by a direct excitatory postsynaptic effect. Leptin inhibited electrical activity in 10 out of 22 orexin-sensitive neurons in this brain region and excited only 3 neurons. These data give the first indication as to where and how orexin might interact with the leptin-responsive hypothalamic network.