Somatostatin Derivatives Research Articles

Pharmacological properties of hydrophilic and lipophilic derivatives of octreotate

Derivatives of somatostatin (SST) represent the most important peptides for receptor targeting in oncological applications. Whereas the pharmacophor in somatostatin receptor-affine substances has been thoroughly investigated, the influence of modifications at the N-terminal has not yet been systematically studied. In order to investigate the influence of hydrophilic versus lipophilic modifications at the N-terminal end, a series of homologous derivatives of Tyr 3-octreotate modified with oligomers of ethylene glycol or fatty acids were synthesized. For this purpose, Tyr 3-octreotate was assembled using solid phase peptide synthesis and the fatty acids or oligomers of ethylene glycol were conjugated to the N-terminal end. The oligomers of ethylene glycol were activated by 4-nitrophenylchloroformate to obtain carbamate-linked hydrophilic compounds. The receptor affinities of these compounds were determined by competition experiments with [ 125I]Tyr 3-octreotide on rat cortex membranes. The hydrophilic derivatives and the short chain lipophilic derivatives revealed IC 50 values between 0.66 ± 0.02 nM and 2.16 ± 0.31 nM respectively. After labeling with 125I the organ distribution of selected derivatives was investigated in Lewis rats bearing the rat pancreatic tumor CA20948. All of the compounds showed high tumor uptake. The peptides conjugated to oligomers of ethylene glycol showed low uptake into the liver and kidneys. Increasing the length of the fatty acids resulted in a remarkable decrease in kidney uptake. In conclusion, the systematic modifications at the N-terminal result in a low effect on the receptor affinity but allow the modulation of the pharmacokinetic properties of octreotide derivatives.

Effect of intrathecal octreotide on thermal hyperalgesia and evoked spinal c-Fos expression in rats with sciatic constriction injury.

This study was designed to determine whether intrathecal octreotide (sandostatin), a synthetic octapeptide derivative of somatostatin, relieved thermal hyperalgesia and reduced the evoked spinal c-Fos expression in rats with chronic constriction injury (CCI) of the sciatic nerve. Intrathecal catheters were implanted in rats 7 days before CCI of the sciatic nerve over the left hind limb. After confirmation of the development of thermal hyperalgesia by decreased paw withdrawal latencies (PWL) to heat stimulation 7 days after CCI, intrathecal sandostatin at 20, 40, and 80 microg was administered, respectively. Rats in the control group received saline injections intrathecally. PWLs were evaluated at 30, 60, 120, 180, and 240 min after drug administration. Detection of Fos-like immunoreactivity (Fos-LI) neurons in the dorsal horn of the spinal cord following drug administration was performed after mechanical stimulation (stroking of the hind paws) on the 14th day after CCI. The reduction of PWL was attenuated significantly in the groups that received intrathecal sandostatin at 20, 40, and 80 g when compared with the saline group. However, PWL did not return to pre-CCI values in all groups. In the 40 microg group, PWL returned up to 76% of pre-CCI values 120 min after drug administration. Stroking of the hind paw in CCI-treated (ipsilateral) limbs induced a significantly greater expression of spinal Fos-LI neurons than that of non-CCI treated (contralateral) limbs in each group. The number of Fos-LI neurons in animals receiving intrathecal sandostatin was dose-dependently reduced. Expression of Fos-LI neurons in the 80 microg group was nearly completely inhibited. These data suggest that intrathecal sandostatin significantly relieved thermal hyperalgesia behaviorally but with limited effects and dose-dependently reduced spinal Fos-LI neurons expression evoked by stroking stimulation, which may reflect mechanical allodynia in rats with sciatic constriction injury. This implies that intrathecal sandostatin was effective in the treatment of neuropathic pain.

Synthesis, in vitro receptor binding, and in vivo evaluation of fluorescein and carbocyanine peptide-based optical contrast agents.

Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effects of drugs and enhances the contrast between normal and pathologic tissues. One approach to achieve selectivity is to target overexpressed receptors on the membranes of tumor cells and to visualize the tumors by a noninvasive optical imaging method. Accordingly, we conjugated fluorescein and carbocyanine dyes to somatostatin and bombesin receptor-avid peptides and examined their receptor binding affinities. We also prepared potential dual imaging probes consisting of a bioactive peptide for tumor targeting, a biocompatible dye for optical imaging, and a radioactive or paramagnetic metal chelator for scintigraphic or magnetic resonance imaging of tumors. Using these approaches, the resulting carbocyanine derivatives of somatostatin and bombesin analogues retained high binding for their respective receptors. Further evaluation of representative molecules in rats bearing somatostatin- and bombesin-positive tumors showed selective uptake of the agents by the tumor cells. Unlike carbocyanine derivatives, the receptor binding of fluorescein-somatostatin peptide conjugates was highly sensitive to the type of linker and the site of fluorescein attachment on the nonreceptor binding region of the peptide. In general, the presence of flexible linkers disrupted binding affinity, possibly due to the interaction of the linker's thiourea group with the peptide's cyclic disulfide bond. While the receptor binding affinity of the dual probes was not dependent on the type of chelating group examined, it was affected by the relative positions of fluorescein and chelator on the lysine linker. For somatostatin compounds, best results were obtained when the chelator was on the alpha-amino lysine linker and fluorescein was on the epsilon-amino group. In contrast, conjugation of the chelator to epsilon- and fluorescein to the alpha-amino lysine linker of bombesin peptides resulted in high receptor binding. These findings indicate that despite their small size, conjugation of dyes to truncated somatostatin and bombesin peptide analogues results in promising diagnostic agents that retain high receptor binding activity in vitro. The results further show that these contrast agents can selectively and specifically localize in receptor-positive tumors in rat models.

Diagnosis and treatment for carcinoid tumors in the digestive tract

The authors analyzed the characteristics of carcinoid tumors according to their site in the digestive tract. Despite their low speed of growing and metastasis process, such neoplasias are malignant and must be submitted to surgical resection. Advanced cases with metastases are treated with somatostatin derivatives, lessening symptoms, causing occasional regression of lesions and presenting an extended survival. Appendicular and rectal lesions have a better prognosis maybe due to the early diagnosis performed.

Octreoscan in thyroid-associated ophthalmopathy.

Until recently, there was no imaging technique available that could be considered as a reliable measure of inflammation in thyroid-associated opthalmopathy (TAO). Pentetreotide (a synthetic derivative of somatostatin) labeled with 111In has been used to visualize somatostatin receptors in endocrine-related tumours in vivo. It has also been used to measure the orbital uptake in patients with TAO. An increased uptake in the orbit was found in patients with active disease. It was suggested that it is caused by the expression of somatostatin receptors on activated T-lymphocytes. Thus, a positive orbital octreoscan indicates clinically active eye disease in which immunosuppressive treatment might be of therapeutic benefit, in contrast to the fibrotic end stage. Indeed, successful immunosuppression with prednisone, orbital irradiation, or very recently with somatostatin analogues, has been demonstrated in patients with TAO and positive octreoscan. It is inferred that an orbital octreoscan is mainly indicated to select patients with TAO who will benefit from immunosuppression. However, limitations such as cost, nonnegligible radiation burden, nonspecific examination for TAO, and finally, lack of evaluation of eye muscle swelling restrict the widespread use of this technique. It remains to be seen if orbital octreoscan will become a widely available tool in the management of patients with TAO.

Somatostatin receptor sst1-sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands.

Somatostatin receptors are known to be expressed in a large number of human tumours and represent the basis for in vivo tumour targeting. Stable somatostatin derivatives such as octreotide or lanreotide are the most frequently used radiopharmaceuticals acting through specific binding to somatostatin receptors; however, they do not bind with high affinity to all five receptor subtypes. Whereas the mRNAs for most receptor subtypes have been detected in tumours, it is in most cases unclear which of the receptor subtype proteins are expressed. Since in vitro receptor binding methods are close correlates and predictors of in vivo peptide receptor targeting, we took advantage of the recently developed subtype-selective analogues and evaluated approximately 200 tumours for their receptor subtype protein expression in specific binding assays using autoradiography with 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28 and displacement by subtype-selective analogues. The majority of the tested neuroblastomas, meningiomas, medulloblastomas, breast carcinomas, lymphomas, renal cell carcinomas, paragangliomas, small cell lung carcinomas and hepatocellular carcinomas predominantly expressed sst2. The prostate carcinomas and sarcomas preferentially expressed sstl, while a majority of inactive pituitary adenomas displayed sst3 and, to a lesser extent, sst2. Growth hormone-secreting pituitary adenomas preferentially expressed sst2 and sst5; gastroenteropancreatic tumours and phaeochromocytomas frequently displayed sst2 and/or sstl. Non-neoplastic human tissues such as vessels, nerve plexus, pancreatic islets, prostatic stroma, adrenal medulla, spleen and germinal centres of the lymphoid tissues preferentially expressed sst2. However, the human gastric mucosa predominantly expressed sst1 while colonic mucosa displayed sst2. Interestingly, a minority of tumours showed a strong 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28 binding, of which less than 50% could be displaced by the sum of the five subtype-selective analogues. This observation suggests the existence of an as yet unknown subtype in selected tumours. This study is the first report to analyse the somatostatin receptor subtype expression in tumours with binding methods. We conclude that sst2, with high affinity for current radiopharmaceuticals such as Octreoscan, is predominantly expressed in a majority of tumours. Fewer tumour types (sarcomas, prostate cancers, inactive pituitary adenomas) preferentially express another subtype. This information is of importance with regard to the clinical applications and development of somatostatin analogues with distinct receptor subtype selectivities.

Biodistribution, blood half-life, and receptor binding of a somatostatin-dextran conjugate.

Derivatives of somatostatin (sms) are attracting increasing interest as part of the treatment of several cancer diseases expressing sms receptors (srs). Radiolabeled sms analogs can additionally be used for systemic radiotherapy and for diagnostic investigations. Glycosylated sms-14 (sms-dextran70) was characterized regarding in vitro srs binding, biodistribution, and blood half-life in mice. Rat brain cortex membranes (expressing srs 2) were used for the srs binding study. Tyr3-Octreotide was used as positive control. The binding data were analyzed by competition curve analysis. In the biodistribution study, the Bolton-Hunter reagent was used for the radioiodination of sms-dextran70. Organs and blood were collected at different time-points and the percentage of the injected dose per gram of tissue (%ID/g) was calculated. The conjugate was administered subcutaneously (sc). The sms-dextran70 showed high srs binding affinity (i.e., in the same nanomolar range as the reference ligand Tyr3-octreotide (IC50 approximately 2.5 nM). The blood half-life was approx 27 h after reaching maximum blood concentration 24 h postinjection. Because of the molecular weight of the conjugate (i.e., approx 75,000) being above the kidney threshold for dextran (i.e., 50,000), the digestion and excretion is assumed to be mainly through the hepatobiliary system. Increased uptake was seen in the adrenals, which are receptor-positive organs. Some accumulation was seen in the stomach, indicating certain deiodination of the conjugate label. The sms-dextran70 showed promising properties and its clinical relevance is currently being evaluated in clinical phase I-II studies.

The role of octreoscan in thyroid eye disease.

Until recently there was no imaging technique available which could demonstrate pathological changes in orbital tissues and could be regarded as a reliable measure of inflammation in thyroid eye disease (TED). Pentetreotide (a synthetic derivative of somatostatin) labelled with 111In has been used to localize tumours which possess surface or membrane receptors for somatostatin in vivo using a gamma camera (1). This technique visualizes somatostatin receptors in endocrine-related tumours in vivo and predicts the inhibitory effect of the somatostatin analogue octreotide on hormone secretion by the tumours (1). By applying 111In-DTPA-d-Phe octreotide scintigraphy (octreoscan), accumulation of the radionuclide was also detected in both the thyroid and orbit of patients with Graves' disease (2-4). If peak activity in the orbit 5h after injection of radiolabelled octreotide is set at 100%, a decrease to 40+/-4% is found at 24h, significantly different from the decrease in blood pool radioactivity, which is 15+/-4% at 24h. Accumulation of the radionuclide is most probably due to the presence in the orbital tissue of activated lymphocytes bearing somatostatin receptors (5). Alternative explanations are binding to receptors on other cell types (e.g. myoblasts, fibroblasts or endothelial cells) or local blood pooling due to venous stasis by the autoimmune orbital inflammation.

Nonpeptide Somatostatin Agonists with sst4 Selectivity: Synthesis and Structure−Activity Relationships of Thioureas

Utilizing NNC 26-9100 (11) as a structural lead, a variety of nonpeptide derivatives of somatostatin were synthesized and evaluated for sst2 and sst4 receptor binding affinity. A novel thiourea scaffold was utilized to attach (1) a heteroaromatic nucleus to mimic the Trp8 residue, (2) a nonheteroaromatic nucleus to mimic Phe7, and (3) a primary amine or other basic group to mimic the Lys9 residue of somatostatin. Displacement studies were carried out using membranes from cell lines expressing ssts [BHK cells (sst4) and HEK 293 cells (sst2)] utilizing [125I]Tyr11-SRIF as the radioligand. Several thioureas (11, 38, 39, 41, and 42) and the urea 66 exhibited Ki values of less than 100 nM. The thioureas 11 (Ki = 6 nM) and 41 (Ki = 16 nM) and the urea 66 (Ki = 14 nM) are believed to be the most potent nonpeptide sst4 agonists known. Since the thiourea 11 and the urea 66 exhibit high sst4 selectivity, these novel nonpeptide derivatives may be useful tools for studying the sst4 receptor. Studies are currently in progress to evaluate the therapeutic potential of NNC 26-9100 (11) in the treatment of glaucoma.

Differential Internalization of Somatostatin in COS-7 Cells Transfected with SST1 and SST2 Receptor Subtypes: A Confocal Microscopic Study Using Novel Fluorescent Somatostatin Derivatives

A growing body of evidence suggests that neuropeptide binding to G protein-linked receptors may result in internalization of receptor-ligand complexes, followed by intracellular mobilization and degradation of the ligand into its target cells. Because of discrepant results in the literature concerning the occurrence of such a mechanism for the tetradecapeptide somatostatin (SRIF), we have reinvestigated this question by comparing the binding and internalization of iodinated and fluorescent derivatives of the metabolically stable analog of SRIF,[ d-Trp8]SRIF, in COS-7 cells transfected with complementary DNA encoding the sst1 or sst2A receptor subtype. A series of fluoresceinyl and Bodipy fluorescent derivatives of[ d-Trp8]SRIF-14 was purified by HPLC, analyzed for purity by mass spectrometry, and tested for biological activity in a membrane binding assay. Of the six compounds tested, fluoresceinyl and Bodipy derivatives labeled in position α (fluo-SRIF) retained high affinity for SRIF receptors. COS-7 cells transfected with complementary DNA encoding either sst1 or sst2A receptors both displayed specific, high affinity binding of iodinated and fluo-SRIF. At 4 C, the labeling was confined to the cell surface in both cell types, as indicated by the fact that it was entirely removable by a hypertonic acid wash and assumed a pericellular distribution in the confocal microscope. At 37 C, the fate of specifically bound ligand varied markedly according to the type of receptor transfected. In cells encoding the sst1 receptor, approximately 20% of specifically bound ligand was recovered in the acid-resistant (i.e. intracellular) fraction. This fraction remained clustered at the periphery of the cell, suggesting that it was being sequestered either within or immediately beneath the plasma membrane. By contrast, in cells transfected with sst2A receptors, up to 75% of specifically bound ligand was recovered inside the cells, where it clustered into small endosome-like particles. These particles increased in size and moved toward the nucleus with time, suggestive of receptor-ligand complexes proceeding down the endocytic pathway. These results demonstrate that neuropeptides may be processed differently depending on the subtype of receptor expressed in their target cells and suggest that these different processing patterns may reflect different modes of sensitization/desensitization and recycling of the receptors, and thereby of transmembrane signaling.

Differential internalization of somatostatin in COS-7 cells transfected with SST1 and SST2 receptor subtypes: a confocal microscopic study using novel fluorescent somatostatin derivatives.

A growing body of evidence suggests that neuropeptide binding to G protein-linked receptors may result in internalization of receptor-ligand complexes, followed by intracellular mobilization and degradation of the ligand into its target cells. Because of discrepant results in the literature concerning the occurrence of such a mechanism for the tetradecapeptide somatostatin (SRIF), we have reinvestigated this question by comparing the binding and internalization of iodinated and fluorescent derivatives of the metabolically stable analog of SRIF, [D-Trp8]SRIF, in COS-7 cells transfected with complementary DNA encoding the sst1 or sst2A receptor subtype. A series of fluoresceinyl and Bodipy fluorescent derivatives of [D-Trp8]SRIF-14 was purified by HPLC, analyzed for purity by mass spectrometry, and tested for biological activity in a membrane binding assay. Of the six compounds tested, fluoresceinyl and Bodipy derivatives labeled in position alpha (fluo-SRIF) retained high affinity for SRIF receptors. COS-7 cells transfected with complementary DNA encoding either sst1 or sst2A receptors both displayed specific, high affinity binding of iodinated and fluo-SRIF. At 4 C, the labeling was confined to the cell surface in both cell types, as indicated by the fact that it was entirely removable by a hypertonic acid wash and assumed a pericellular distribution in the confocal microscope. At 37 C, the fate of specifically bound ligand varied markedly according to the type of receptor transfected. In cells encoding the sst1 receptor, approximately 20% of specifically bound ligand was recovered in the acid-resistant (i.e. intracellular) fraction. This fraction remained clustered at the periphery of the cell, suggesting that it was being sequestered either within or immediately beneath the plasma membrane. By contrast, in cells transfected with sst2A receptors, up to 75% of specifically bound ligand was recovered inside the cells, where it clustered into small endosome-like particles. These particles increased in size and moved toward the nucleus with time, suggestive of receptor-ligand complexes proceeding down the endocytic pathway. These results demonstrate that neuropeptides may be processed differently depending on the subtype of receptor expressed in their target cells and suggest that these different processing patterns may reflect different modes of sensitization/desensitization and recycling of the receptors, and thereby of transmembrane signaling.

Modified method using a somatostatin analogue, octreotide acetate (Sandostatin) to assess in vivo insulin sensitivity.

In order to evaluate the steady state plasma glucose (SSPG) method by using a new somatostatin derivative, octreotide acetate (Sandostatin) instead of somatostatin that we had used for the insulin sensitivity test, we examined whether octreotide was able to suppress C-peptide (CPR), glucagon (IRG), and GH to a similar degree to that achieved with somatostatin. A total of 52 studies were performed in 45 essential hypertensive subjects and 7 healthy subjects. Octreotide was given subcutaneously in a does of 50 micrograms or 100 micrograms 10 min before the test (sc 50, sc 100 groups) or intravenously infused over 2 h (10 micrograms in bolus followed by a constant infusion, 50, 100, or 150 micrograms/2 h: i.v. 50, i.v. 100, i.v. 150 groups). In all of the groups the plasma immunoreactive insulin (IRI) concentration increased gradually after insulin injection and reached the steady state plasma insulin (SSPI) level between 40 and 60 microU/ml at 60 min through 120 min. Plasma CPR at 120 min was the most suppressed (by 67% of the basal level in i.v. 150 group during the study period), but on the other hand in both the sc 100 and i.v. 100 groups the plasma CPR concentration at 120 min was suppressed by nearly 40%, but not significantly suppressed in either the sc 50 or the i.v. 50 group. Plasma IRG and GH were strongly suppressed after 60 min in all groups during the study period. Plasma glucose had increased significantly at 30 min and reached the steady state at 90 min through 120 min in hypertensive and healthy subjects. The results indicated that the modified SSPG method with continuous intravenous infusion of Octreotide at 150 micrograms/2 h was adequate for the measurement of insulin sensitivity.

The somatostatin analogue octreotide protects against ethanol-induced microcirculatory stasis and elevated vascular permeability in rat gastric mucosa

Somatostatin 14 and various derivatives protect rat gastric mucosa against ethanol-induced lesions. Their mechanism of action is unknown. We investigated the effect of two somatostatin derivatives, octreotide and 5-( l)-citrullin-octreotide, on ethanol-induced hemorrhagic lesions, microcirculatory stasis and elevated vascular permeability in the rat stomach, with the goal to elucitate the pharmacological and microcirculatory mechanisms behind the gastroprotective effect. Radioligand studies revealed a high affinity of octreotide for the somatostatin receptor (IC 50 = 5 × 10 −10 mol/l), in contrast to 5-( l)-citrullin-octreotide (IC 50 = 3 × 10 −6 mol/l). This was in good agreement with the inhibition of growth hormone release from rat anterior pituitary cells (octreotide: IC 50 = 1.2 × 10 −10 mol/l; 5-( l)-citrullin-octreotide: IC 50 = 3 × 10 −6 mol/l. Intragastric administration of ethanol to rats resulted in lesions of the gastric mucosa affecting 18.9 ± 3.1% of the area of the glandular stomach. Octreotide reduced the area to 6.4 ± 1.7%( P < 0.05). The dose-response curve was bell-shaped. 5-( l)-citrullin-octreotide was totally devoid of any protective activity (dose range: 0.1 ng/kg to 0.1 mg/kg). We further investigated the effect of the two peptides on ethanol-induced mic increase in microvascular permeability, measured by the extravasation of the tracer fluorescein-isothiocyanate-dextran (molecular weight 150 000). Pretreatment with octreotide (0.1 ng/kg s.c.) prevented stasis and reduced capillary permeability significantly. 5-( l)-citrullin-octreotide had no effect on ethanol-induced microcirculatory stasis and elevated vascular permeability in rat gastric mucosa. In summary, very low doses of octreotide have a benefical effect on ethanol-induced hemorrhagic lessions, microcirculatory stasis and increased capillary permeability. This effect is most likely mediated by somatostatin receptors.

SDZ CO 611: a highly potent glycated analog of somatostatin with improved oral activity

To obtain orally active octreotide (Sandostatin ®, SMS 201-995) analogs a new class of glycated somatostatin derivatives were synthesized by the Amadori reaction (Maillard reaction). The synthesis, chemical and biological characterization of a series of new compounds is described. These oligopeptides bind with high affinity to somatostatin receptors and retain full biological activity. Whereas generally polypeptide hormones are almost completely inactive after oral administration, we report here for the first time that these analogs show remarkably high activity by the oral route. Thus for example SDZ CO 611, the3D(+)-maltose Amadori derivative of octreotide, has about 10 times higher oral effect bioavailability than octreotide while maintaining the selectivity, metabolic stability and long duration of action of the parent compound.

Octreotide scintigraphy localizes somatostatin receptor-positive islet cell carcinomas

Tyr-3-Octreotide is a synthetic derivative of somatostatin and a somatostatin-receptor analogue. The iodine-123-labelled compound localizes somatostatin-receptor-positive tumours. In this paper two patients are reported in whom somatostatin receptors were demonstrated in vitro. In a 60-year-old female with an islet cell carcinoma of the pancreas, multiple liver metastases and previously unrecognized bone metastases in the right acetabulum could be diagnosed as the reason for a persistent hypoglycaemia. In a 60-year-old male an islet cell carcinoma of the pancreas was localized with 123I-Tyr-3-octreotide. The somatostatin receptors were demonstrated in vitro and the tumour was successfully treated with somatostatin. These studies demonstrate that 123I-Tyr-3-octreotide offers the possibility of localizing somatostatin-receptor-positive tumours and their metastases. Moreover the method makes it possible to determine the receptor status of a tumour in vivo.

Growth Progression and 24-Hour Hormone Profile in an Infant Treated Chronically with a Long-Acting Somatostatin Derivative

The diagnosis of nesidioblastosis was established in a 9-month-old male child with a history of recurrent convulsive seizures and hypoglycemia. After unsuccessful subtotal pancreatectomy, treatment was started with the long-acting somatostatin derivative Sandostatin (Octreotide, Sandoz) at a dosage of 25 micrograms t.i.d. spaced between carbohydrate-enriched meals. With this regime, blood glucose was maintained at the low normal range and seizures ceased. During a 30-month observation period, growth velocity and weight progression were well within the predicted limits. A 24-hour hormone profile recorded at the end of the observation period revealed the following: (1) failure to improve blood glucose with carbohydrate-enriched food due to reactive hyperinsulinemia; (2) hyperglycemic reaction after administration of Sandostatin caused by a reduction of plasma insulin; this effect was particularly marked during sleep; (3) low mean GH, decreased spiking frequency and reduced area covered by the nocturnal peaks by recognized standards, and (4) normal somatomedin C levels for age. Interpretation of growth hormone (GH) data is hindered by the lack of pertinent information from the patient's age group. Recording of normal growth progression in the case illustrated here can only be explained by the capability of a reduced GH secretory rate to maintain full biological activity as shown by the normal plasma level of somatomedin C. Indeed, recent evidence has been provided elsewhere for normal growth progression in the presence of low GH secretion, although other factors unrelated to this hormone may also be operative at this early age. Further reports concerning the treatment of non-GH-dependent conditions with somatostatin derivatives will certainly contribute to the better understanding of the mechanisms governing growth in the postnatal period.

Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog.

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.

GASTROINTESTINAL HORMONES: FROM BASIC SCIENCE TO A CLINICAL PERSPECTIVE

The gastrointestinal tract is the largest endocrine organ in the body. However, gastrointestinal hormones are not confined to the gut and many of them are delivered to their target tissue by neural and paracrine routes as well as the circulation. Regulatory peptide is therefore a more appropriate term than gastrointestinal hormone. The functions of these regulatory peptides include effects on intake, digestion and absorption of food, and changes in gut secretions, motility and growth. Since these peptides do not act alone but in concert it has been difficult to ascribe particular functions to individual peptides. However, the recent and on-going development of specific regulatory peptide agonists and antagonists has resulted in major advances in our understanding of the physiology of these peptides. In turn these findings are creating new therapeutic avenues providing some return from all the research on these gastrointestinal regulatory peptides. The somatostatin derivative (octreotide or sandostatin) is the most obvious example. Although only approved in Australia for treatment of carcinoids and VIPomas, the prospects include treatment of other gastroenteropancreatic tumours, acromegaly, idiopathic diarrhoea, fistula closure, dumping, and ERCP or post-operative pancreatitis. A new gastrokinetic agent, that acts via the motilin receptor, is undergoing trials for the treatment of impaired gastric emptying. The trophic effect of gastrointestinal peptides has clinical significance. For instance, gastrin antagonists inhibit cell proliferation of colon carcinoma cell lines. Furthermore the trophic effect of gastrin must be considered when potent gastric acid inhibitors, which cause a reflex increase in gastrin, are used. The outlook is for more mammalian regulatory peptides to be discovered adding further to the complexity.(ABSTRACT TRUNCATED AT 250 WORDS)

LOCALISATION OF ENDOCRINE-RELATED TUMOURS WITH RADIOIODINATED ANALOGUE OF SOMATOSTATIN

Various endocrine-related tumours contain large numbers of high-affinity somatostatin receptors. 123I-labelled tyr-3-octreotide (tyr-3-SMS 201-995, a synthetic derivative of somatostatin) was used to localise such tumours in vivo with a gamma-camera. Positive scans were obtained for two meningiomas, two gastrinomas, and one carcinoid; negative scans were obtained for one insulinoma (in which unlabelled octreotide had no effect on insulin levels), one phaeochromocytoma, one adrenal carcinoma (octreotide had no effect on cortisol levels), and three medullary thyroid carcinomas (octreotide had no effect on calcitonin levels). Thus radioiodinated tyr-3-octreotide can label somatostatin receptors in endocrine-related tumours in vivo and can therefore be used for tumour localisation.

Proton n.m.r. investigation of conformational influence of penicillamine residues on the disulfide ring system of opioid receptor selective somatostatin derivatives.

Three cyclic disulfide analogs related to somatostatin, D-Phe(1)-cyclo(Cys(2)-Tyr(3)-D-Trp(4)-Lys(5)-Thr(6)-Xxx(7))-Thr(8)- NH2 (where Xxx = L-Pen 1; L-Cys 3; or D-Pen 4) were examined in DMSO-d6 by one- and two-dimensional proton n.m.r. spectroscopy in order to analyze the conformational influence of the position-7 residue on the 20-membered disulfide ring. From these studies it was concluded that all three analogs maintain a beta II' turn solution conformation for the core tetrapeptide -Tyr(3)-D-Trp(4)-Lys(5)-Thr(6)-. However, the disulfide conformation differs in the analogs, with 1 and 3 having a left-handed and 4 a right-handed disulfide chirality.