Antigenic specificity of a new and potent somatostatin antiserum

The specificity of a new somatostatin antiserum (Ab 6) has been investigated using eight different peptide analogues. For this purpose, somatostatin and all the analogues were tested, in an equimolar concentration range, for their ability to displace 125I-tyr1-somatostatin from its binding to antibodies. Analysis of the displacement curves shows that antiserum Ab 6 predominantly recognizes changes at the central aminoacid sequence of the somatostatin molecule. Its ligand specificity seems to be similar to that of other somatostatin antisera previously described (R-141 and A-101).     

Structure-activity studies with somatostatin: the role of tryptophan in position 8

The gastric acid and pepsin inhibitory activities of 21 analogues of somatostatin, the majority modified at position 8, were determined in conscious cats in order to examine the importance of Trp8 for the activity of somatostatin. Pepsin secretion stimulated by pentagastrin was 5 times more sensitive, compared with the acid secretion, to inhibition by somatostatin. All the analogues showed similar differential sensitivity, indicating a similar specificity of somatostatin receptors involved in the inhibition of these two secretions. Halogenated-Trp8 analogues of somatostatin were only equipotent or slightly more active than somatostatin against gastric secretion in the cat, whilst these analogues are up to 30 times more potent against growth hormone release in the rat, indicating a different specificity of the two groups of receptors. Studies with the position 8 modified analogues suggest that the electron density of the aromatic nucleus of Trp8 may be relatively unimportant in determining the gastric inhibitory activity, whilst it can be concluded that the role of Trp8 in somatostatin depends to a large extent on the indole NH group. The precise role of Trp8 in somatostatin could be an involvement in the binding of somatostatin to its receptors, or involvement in forming the biologically active conformation of somatostatin.     

Pituitary somatostatin receptors. Characterization by binding with a nondegradable peptide analogue

Somatostatin receptors in the rat pituitary gland were characterized by binding analysis with a radioiodinated high affinity somatostatin analogue, 125I-Tyr1[D-Trp8]somatostatin. Receptor binding of this derivative reached equilibrium at 30 min and was maintained at a plateau for at least 60 min. Two L-Trp8- labeled somatostatin analogues. 125I-Tyr1- and [125I-Tyr11]somatostatin, displayed less stable and lower specific uptake and higher nonspecific binding. In contrast to the rapid degradation of the L-Trp8 ligands during binding assay, 125I-Tyr1]D-Trp8]somatostatin retained more than 80% of its binding activity after 90 min of incubation with pituitary particles. Pituitary particles bound 125I-Tyr1]D-Tyr8]somatostatin with high affinity (Ka = 8.6 +/- 1.2 X 10(9) M-1) and capacity of 54.4 +/- 2.6 fmol/mg. These binding sites showed specificity for the native peptide and its active analogues, and other peptide hormones, including angiotensin II, thyrotropin-releasing hormone, vasopressin, oxytocin, substance P, and gonadotropin-releasing hormone, did not inhibit tracer binding. A good correlation was observed between the binding affinities of several somatostatin analogues and their potencies as inhibitors of growth hormone release in rat pituitary cells. These findings emphasize the physiological importance of the pituitary somatostatin receptor in mediating the inhibitory action of the peptide on growth hormone release. The use of Tyr1[d-Trp8]somatostatin as a labeled ligand permits accurate determinations of the binding affinity and concentration of receptors for somatostatin in the normal pituitary gland and provides a basis for further studies of somatostatin receptor regulation and receptor-mediated cellular effects of the tetradecapeptide.     

Conformational studies of somatostatin and selected analogues by Raman spectroscopy

Laser Raman spectroscopy has been used to study the conformations of somatostatin and some selected analogues in aqueous solution. The results indicate that the CS-SC dihedral angles of somatostatin and of these analogues (except [Ala3,14]-SS, which has no disulfide bond) are within 20 degrees of +/- 85 degrees, and the SS-CC dihedral angles are predominantly in the range of 50 degrees-180 degrees. Furthermore, from the behavior of the amide I' and amide III bands, it appears that somatostatin adopts a beta-pleated sheet structure, whereas its analogues are less ordered (to varying degrees).     

Evaluation of cleavable (Tyr3)-octreotate derivatives for longer intracellular probe residence

Radioligand targeting of somatostatin receptor subtype 2 (sstr2)-positive tumors with synthetic somatostatin analogues such as octreotide is subject to improvement in tumor to nontumor biodistribution, in part because internalization of such somatostatin analogues is limited by sstr2 recycling to the cell surface. We reasoned that it might be possible to prepare probe-carrying somatostatin analogues that would escape recycling, efficiently depositing probe molecules inside cells and ultimately increasing their intracellular concentration. We have incorporated cathepsin-B-cleavable linkers into (Tyr3)-octreotate chelate conjugates and examined these constructs as to cellular uptake, externalization, subcellular localization, and cleavage in the rat pancreatic tumor cell line AR42J in culture. Comparison of the cleavable radioligands with a noncleavable control indicates that scission of the constituent cathepsin B substrate occurs at a rate faster than ligand externalization, depositing virtually all internalized cleaved radiochelates within lysosomal compartments.     

The therapeutic problem of proliferative diabetic retinopathy: targeting somatostatin receptors.

Clinical management of proliferative diabetic retinopathy has changed very little in the last 5 decades, relying primarily on laser ablation of the retinal vasculature. Several lines of clinical and experimental evidence suggest that somatostatin analogues may be efficacious in inhibiting neovascularization associated with proliferative retinopathy but the mechanism of action for these compounds is unclear. Inhibition of growth hormone secretion and the subsequent suppression of insulin-like growth factor 1 (IGF-1) production by somatostatin has been suggested as the mechanism of action, however, in vitro studies suggest that somatostatin analogues suppress endothelial cell growth through a direct, somatostatin receptor-mediated inhibition of pro-survival signaling pathways. The advent of a new generation of modified peptide and peptidomimetic somatostatin analogues has allowed investigators to more carefully define the receptor subtypes responsible for somatostatin-induced endothelial cell death and may eventually lead to the clinical development of somatostatin analogues that can reduce endothelial cell proliferation, independent of suppression of circulating hormone levels.     

生长抑素受体介导的放射性核素治疗

生长抑素受体介导的放射性核素治疗(peptide receptor radionuclide therapy,PRRT)是用于不能手术治疗或有远处转移的胃、肠和胰腺等神经内分泌肿瘤病人的一种新型治疗方法,本文旨在综述近年来不同核素标记生长抑素类似物的临床评价及相关研究。     

Structure-activity studies with somatostatin: role of lysine in positions 4 and 9 for gastric activity

The gastric inhibitory activity of somatostatin analogues modified in position 4 or 9, was investigated in conscious cats prepared with gastric fistulae. Gastric secretion was stimulated with pentagastrin. Deletion of Lys4, or substitution with an alcoholic (Thr) or acidic (Glu) residue yielded analogues with reduced (10% or less) potency compared with somatostatin. In comparison, [Phe4]somatostatin and modified Phe4 analogues [( p-NH2-Phe4]-, [F5Phe4]- and [Phe4,D-Trp8]somatostatin) were approximately equipotent with somatostatin. The high potency of the Phe4 analogues illustrates that a basic side-chain in position 4 is not essential for gastric activity. In contrast, [Thr9]-, [Glu9]-, [Phe9]- and [p-NH2-Phe9]somatostatin were all inactive (less than 5% potency of somatostatin) in the stomach. Thus the lysyl residue in position 9 is more critical than that in position 4 for somatostatin's gastric activity.     

Somatostatin receptor biology in neuroendocrine and pituitary tumours: part 1 – molecular pathways

Neuroendocrine tumours (NETs) may occur at many sites in the body although the majority occur within the gastroenteropancreatic axis. Non-gastroenteropancreatic NETs encompass phaeochromocytomas and paragangliomas, medullary thyroid carcinoma, anterior pituitary tumour, broncho-pulmonary NETs and parathyroid tumours. Like most endocrine tumours, NETs also express somatostatin (SST) receptors (subtypes 1–5) whose ligand SST is known to inhibit endocrine and exocrine secretions and have anti-tumour effects. In the light of this knowledge, the idea of using SST analogues in the treatment of NETs has become increasingly popular and new studies have centred upon the development of new SST analogues. We attempt to review SST receptor (SSTR) biology primarily in neuroendocrine tissues, focusing on pituitary tumours. A full data search was performed through PubMed over the years 2000–2009 with keywords ‘somatostatin, molecular biology, somatostatin receptors, somatostatin signalling, NET, pituitary’ and all relevant publications have been included, together with selected publications prior to that date. SSTR signalling in non-neuroendocrine solid tumours is beyond the scope of this review. SST is a potent anti-proliferative and anti-secretory agent for some NETs. The successful therapeutic use of SST analogues in the treatment of these tumours depends on a thorough understanding of the diverse effects of SSTR subtypes in different tissues and cell types. Further studies will focus on critical points of SSTR biology such as homo- and heterodimerization of SSTRs and the differences between post-receptor signalling pathways of SSTR subtypes.     

111In-DTPA-D-Phe1]-octreotide, a potential radiopharmaceutical for imaging of somatostatin receptor-positive tumors: synthesis, radiolabeling and in vitro validation.

Somatostatin receptor-positive human tumors can be detected using radioiodinated analogues of somatostatin, both in vitro and in vivo. [123I-Tyr3]-octreotide has been successfully used in the visualization of somatostatin receptor-positive tumors by gamma camera scintigraphy, but this radiopharmaceutical has some major drawbacks, which can be overcome with other radionuclides such as 111In. As starting material for a potentially convenient radiopharmaceutical, a diethylenetriaminopentaacetic acid (DTPA) conjugated derivative of octreotide (SMS 201-995) was prepared. This peptide, [DTPA-D-Phe1]-octreotide (SDZ 215-811) binds more than 95% of added 111In in an easy, single-step labeling procedure without necessity of further purification. The specific somatostatin-like biologic effect of these analogues was proven by the inhibition of growth hormone secretion by cultured rat pituitary cells in a dose-dependent fashion by octreotide, [DTPA-D-Phe1]-octreotide and non-radioactive [115In-DTPA-D-Phe1]-octreotide. The binding of [111In-DTPA-D-Phe1]-octreotide to rat brain cortex membranes proved to be displaced similarly by natural somatostatin as well as by octreotide, suggesting specific binding of [111In-DTPA-D-Phe1]-octreotide to somatostatin receptors. The binding of the indium-labeled compound showed a somewhat lower affinity when compared with the iodinated [Tyr3]-octreotide, but indium-labeled [DTPA-D-Phe1]-octreotide still binds with nanomolar affinity. In conjunction with in vivo studies, these results suggest that [111In-DTPA-D-Phe1]-octreotide is a promising radiopharmaceutical for scintigraphic imaging of somatostatin receptor-positive tumors.     

New medical approaches in pituitary adenomas

Recently, the medical approach to patients with secreting and clinically non-functioning pituitary adenomas has received great impulse thanks to the availability of new, selective and long-lasting compounds with dopaminergic activity, such as cabergoline, and of somatostatin analogues provided in slow-release formulations, such as lanreotide and octreotide long acting release (LAR). In particular, the use of cabergoline has induced control of hyperprolactinaemia and tumour shrinkage in the great majority of patients with micro- and macroprolactinomas. Cabergoline treatment restores fertility both in women and men, and partially improves osteoporosis, one of the major complications of hyperprolactinaemia. In acromegaly, disease control (growth hormone [GH] <2.5-1.0 microg/l as a fasting or glucose-suppressed value, respectively, together with age-normalised insulin-like growth factor [IGF]-I) is achievable in more than half of patients receiving treatment with lanreotide or octreotide-LAR. Improvement in cardiomyopathy, sleep apnoea and arthropathy has been reported during GH/IGF-I suppression after pharmacotherapy. A synthetic GH analogue, B2036-PEG, that antagonises endogenous GH binding to its receptor-binding sites and a GH-releasing hormone antagonist that blocks the effect of this releasing factor on the hypothalamus and pituitary are presently under investigation in acromegaly. Preliminary studies have clearly demonstrated the effectiveness of the GH receptor antagonist in suppressing IGF-I levels in acromegalic patients previously unresponsive to somatostatin analogues. Beneficial effects of subcutaneous octreotide and lanreotide have also been reported in adenomas secreting thyroid-stimulating hormone, while the results of treatment with dopamine agonists or somatostatin analogues remain disappointing in patients with clinically non-functioning adenomas. In these patients the possibility of visualising in vivo the expression of D(2) receptors using specific radiotracers such as (123)I-methoxybenzamide has allowed selection of patients likely to respond to cabergoline. Scant effects of pharmacotherapy have also been reported in patients with adenomas secreting adrenocorticotropic hormone. However, some preliminary data suggest a potential use of cabergoline in combination with ketoconazole, or alone, in selected cases of Cushing's disease or Nelson's syndrome.     

New approaches to the therapy of various tumors based on peptide analogues.

The discovery of hypothalamic hormones was briefly reviewed. The development of new hormonal methods for the therapy of various cancers based on analogues of hypothalmic hormones is then presented. My group isolated luteininzing hormone-releasing hormone (LH-RH), also known as Gn-RH, from pig hypothalmi, elucidated its amino acid sequence, and synthesized it in 1971. The interest in medical applications of LH-RH led to the synthesis of LH-RH analogues by various groups. LH-RH agonists substituted in positions 6 or 10 including Decapeptyl, Leuprolide and Zoladex are much more active than LH-RH and on continuous administration produce inhibition of pituitary and gonads. Chronic administration of LH-RH agonists is being utilized for the treatment of prostate and breast cancer. Octapeptide analogues of somatostatin have various applications in Oncology. In 1980 we developed a new endocrine therapy for advanced prostate cancer based on agonists of LH-RH, which is now preferred by 70-90% of prostate cancer patients for primary treatment. LH-RH antagonists such as Cetrorelix can be used for therapy of BPH. On the basis of the presence of specific receptors for hypothalamic peptides on human cancers, we developed targeted cytotoxic analogues of LH-RH, somatostatin, and bombesin/GRP linked to doxorubicin or 2-pyrrolinodoxorubicin. These analogues inhibit the growth of experimental human prostate, breast, ovarian and endometrial cancer, renal cell carcinoma, pancreatic, colorectal and gastric cancers, small cell lung carcinoma (SCLC) and non-SCLC, brain tumors, melanomas, and lymphomas. Cytotoxic LH-RH analogues are now in clinical trials. Recently we demonstrated that growth hormone-releasing hormone (GH-RH) also serves as an autocrine growth factor in many cancers. Antagonistic analogues of GH-RH synthesized in our laboratory inhibit the growth of diverse tumors. The discovery of LH-RH and somatostatin has led to clinical use of their analogues in the field of cancer treatment and GH-RH antagonists also show a great promise.     

Le DOTATOC-(68Ga) pour l’imagerie TEP des tumeurs endocrines digestives : présentation d’un cas et revue de la littérature

⁶⁸Ga is a positron emitter, obtained from a ⁶⁸Ge/⁶⁸Ga generator, which can be used to label peptides of clinical interest. DOTATOC is a tracer of high affinity for the type 2 somatostatin receptors and is used for imaging of tumours which are expressing them, including endocrine tumours. We report on the case of a patient with a history of small bowel carcinoid tumour with hepatic metastases, treated by somatostatin analogues, in whom somatostatin receptor scintigraphy showed three liver foci and DOTATOC-(⁶⁸Ga) PET/CT highlighted much more liver lesions. Two recent studies emphasised on the superiority of DOTATOC-(⁶⁸Ga) PET over somatostatin receptor scintigraphy, and its complementarity with CT, especially for the diagnosis of bone metastases. DOTATOC-(⁶⁸Ga) PET/CT, which associates the specificity of somatostatin receptor scintigraphic detection with the spatial resolution of PET and the anatomical precision of CT, seems to be promised to a brilliant future, the more so as it offers many advantages to the patient: a shorter waiting time, one single image acquisition and a satisfying dosimetry.     

Complete clinical remission and disappearance of liver metastases after treatment with somatostatin analogue in a 40-year-old woman with a malignant insulinoma positive for somatostatin receptors type 2

Insulinoma is the most common pancreatic endocrine tumor, accounting for 40% of all pancreatic functional neoplasm, and is characterized by hypersecretion of insulin and hypoglycemia. Elective treatment for insulinomas is surgical enucleation. Medical therapy with diazoxide, followed by somatostatin analogues in some cases, may be necessary to treat the hypoglycemic symptoms. We report a case of a patient affected by metastatic insulinoma with severe hypoglycemia. After surgery, histopathology confirmed the presence of a malignant insulinoma with multiple metastases in the liver. Due to the persistence of hypoglycemia, the patient was started on octreotide LAR treatment, which determined a complete clinical remission with regression of the metastatic lesions in the liver after one year. Repeated CT scans 2 and 3 years after surgery confirmed the remission. To our knowledge, the complete regression of the disease in insulinomas treated with long-standing somatostatin analogue therapy has never been reported. Immunohistochemical analysis in tissue specimens showed a strong membrane immunoreactivity for somatostatin receptors type 2 (SSTR2) in both the primary nodule and the metastases. The capacity of somatostatin analogues to negatively regulate cell proliferation through indirect and direct mechanisms has been experimentally demonstrated. Furthermore, SSTR2 activation may exert pro-apoptotic effects in neoplastic cells. Thus, both mechanisms may have been responsible of the remission of the disease in this patient. This case underlies the potential impact of the treatment of pancreatic insulinomas with somatostatin analogues, and, if confirmed, the usefulness of SSTR determination in these neoplastic specimens.     

生长抑素受体介导的肿瘤靶向治疗

许多肿瘤,如神经内分泌肿瘤、甲状腺癌、乳腺癌等能高表达生长抑素受体,从而为生长抑素及其类似物的治疗提供了靶点。生长抑素受体介导的肿瘤靶向治疗主要包括放射性核素治疗、放射导向手术治疗、细胞毒素治疗和溶瘤病毒治疗。目前,生长抑素受体介导的放射性核素治疗已应用于神经内分泌肿瘤,尤其在胃肠胰神经内分泌肿瘤的诊断和治疗中占据重要地位。另外,生长抑素受体介导的放射导向手术治疗、细胞毒素治疗和溶瘤病毒治疗的潜在价值也越来越引起研究者们的重视。本文通过查阅近五年来关于生长抑素受体介导的肿瘤靶向治疗的国内外文献,综述了生长抑素受体介导的肿瘤靶向治疗的最新研究进展。     

Strategies to improve plasma half life time of peptide and protein drugs

Summary. Due to the obvious advantages of long-acting peptide and protein drugs, strategies to prolong plasma half life time of such compounds are highly on demand. Short plasma half life times are commonly due to fast renal clearance as well as to enzymatic degradation occurring during systemic circulation. Modifications of the peptide/protein can lead to prolonged plasma half life times. By shortening the overall amino acid amount of somatostatin and replacing l-analogue amino acids with d-amino acids, plasma half life time of the derivate octreotide was 1.5 hours in comparison to only few minutes of somatostatin. A PEG_2,40 K conjugate of INF-α-2b exhibited a 330-fold prolonged plasma half life time compared to the native protein. It was the aim of this review to provide an overview of possible strategies to prolong plasma half life time such as modification of N- and C-terminus or PEGylation as well as methods to evaluate the effectiveness of drug modifications. Furthermore, fundamental data about most important proteolytic enzymes of human blood, liver and kidney as well as their cleavage specificity and inhibitors for them are provided in order to predict enzymatic cleavage of peptide and protein drugs during systemic circulation.     

A short acute octreotide test for response prediction of long-term treatment with somatostatin analogues in acromegalic patients.

The usefulness of the acute octreotide test in the selection of acromegalic patients for chronic somatostatin depot analogues treatment is controversial. The aim of the present study was to determine its accuracy for chronic response prediction, and the reliability of a short version of the classic 6-hour test. The data from 26 acromegalics (19 women, 7 men, mean age 52.6+/-13.1 years) studied with an acute octreotide test (6 hours sampling for GH measurement after octreotide 100 microg s. c.) were retrospectively analyzed. Eighteen of them followed chronic somatostatin depot analogues treatment for 12 months. GH nadir was always detected at 2 hours (mean decrease 75.9+/-24%). GH levels at 2 hours positively correlated with the other time-points (r(s) 0.97, 0.98, 0.97, 0.96 at 3, 4, 5 and 6 h, respectively; p<0.0001). During chronic treatment with maximal effective dose for 12 months, 61% of the patients achieved IGF1 <3 SD and 22% reached IGF1 <2 SD. GH nadir correlated with IGF1 decrease at 12 months (r(s) 0.76, p<0001). GH nadir of 9.2 ng/ml predicts IGF1 <3 SD with 82% sensitivity and 58% specificity (75% PPV, 67% NPV); for IGF1<2 SD, 75% sensitivity and 58% specificity are obtained for GH nadir 3.6 ng/ml, with 33% PPV and 89% NPV. Acute octreotide test reliably predicts response to long-term treatment; the short, 2-hour version is fully informative for therapeutic decisions in acromegalic patients.     

Synthesis of a cyclic retro analogue of somatostatin suitable for photoaffinity labelling

Cyclic somatostatin analogues containing the modified retro sequence of the amino acids Phe7 to Phe11 of the natural compound have been found to exhibit high activity for cytoprotection of rat hepatocytes against cell poisons such as phallotoxins and galactosamine. Cyclo(-Phe(p-NH(1-14C)Ac)-Thr-Lys(CO(p-N3)C6H4)-Trp-Phe-D-Pro), a photoreactive and radioactive analogue of one of the most active cyclohexapeptides, was synthesized by a combination of solid phase technique and classical solution peptide synthesis. This peptide labels the same proteins in rat liver cell membrane that are modified by photolysable derivatives of bile acids, phalloidin and antamanide.     

The importance of presurgical somatostatin analogue therapy in acromegaly

Different types of treatment, including surgery, medical therapy and radiotherapy, are possible in achieving control of acromegaly. Of the medical therapies available, somatostatin analogues are effective in the majority of patients and can induce pituitary tumour shrinkage. The rationale and outcome of somatostatin analogue treatment before surgery in patients with acromegaly is briefly presented. In summary, the benefits of somatostatin analogues given preoperatively should be considered carefully as optimisation of cardiovascular, respiratory and metabolic functions is clinically relevant for perioperative morbidity. Somatostatin analogues also induce significant shrinkage of GH-secreting pituitary tumours, although this does not seem to be helpful in terms of improved surgical outcome.     

Partial retro-inverso analogues of somatostatin: pairwise modifications at residues 7 and 8 and at residues 8 and 9

Peptide bonds between residues 7 and 8 residues 8 and 9, postulated internal cleavage sites of the peptide hormone somatostatin, were subjected to "pairwise" retro-inverso modification, where atoms of these peptide bonds were interchanged to give the analogues [gPhe7-m-(RS)-Trp8]somatostatin (I) and [gTrp8-m-(RS)-Lys9]somatostatin (II). Key fragments containing the modifications were synthesized by using [bis(trifluoroacetoxy)iodo]benzene for the generation of gem-diaminoalkyl-containing precursors from peptide amides. The versatility of solution synthetic methods was utilized to allow the incorporation of the modified segments. Protecting groups, removable selectively and under mild conditions, included tert-butyl-based groups for the side chains and the tert-butylmercapto group for the cysteine thiols. The excellent results obtained in the syntheses of analogues I and II, and previously of somatostatin on a larger scale [Moroder, L., Gemeiner, M., Goehring, W., Faeger, E., Thamm, P., & Wunsch, E. (1981) Biopolymers 20, 17-31], suggest the general feasibility of this route for the synthesis of centrally modified analogues. The purification of the products by Sephadex LH-20 chromatography afforded the separation of diastereomers of both analogues. The two isomers of I showed significant but different activities while those of analogue II were marginally active.