Effect of somatostatin analogs on gastric acid secretion in dogs and rats

The effects of several superactive analogs of somatostatin on gastric acid response to various exogenous and endogenous stimulants were investigated in conscious dogs and rats with gastric fistulae (GF). The inhibition was compared to that induced by somatostatin-14 (S-S-14) at two dose levels. Several octapeptide analogs of somatostatin including D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) and D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (RC-121), which were superactive in tests on suppression of GH levels, were 4-5 times more potent than S-S-14 in inhibiting desglugastrin-stimulated gastric acid secretion in GF dogs. The analog RC-160 also reduced the rise in serum gastrin levels and gastric acid secretion induced by sham feeding (SF) in dogs with gastric and esophageal fistulae (EF), but did not decrease food consumption. Gastric acid secretion induced by histamine (80 micrograms/kg/h) in dogs was not affected by 1-5 micrograms/kg/h of analog RC-121 or by 5 micrograms/kg/h of S-S-14. Analogs RC-160, RC-121, and RC-98-I (D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2) and others also powerfully inhibited desglugastrin-induced potent as S-S-14 in dogs but its activity was higher in rats. The results indicate that octapeptide analogs which are superactive in GH-inhibition tests are also more potent than S-S-14 in suppressing gastric acid secretion. These findings may be of clinical value.     

Octapeptide analogues of somatostatin inhibit the clonal growth and vasoactive intestinal peptide-stimulated cyclic AMP formation in human small cell lung cancer cells.

Two endocrinologically active octapeptide analogues (BIM-23014 C and BIM-23034) of somatostatin (SRIF) containing either an N- or C-terminal 3-(2-naphthyl)-D-Ala residue were examined for their ability to inhibit the in vitro receptor binding, clonal growth, and vasoactive intestinal peptide (VIP)-stimulated cyclic AMP formation in human small cell lung cancer cell (SCLC) line NCI-H345. Both SRIF peptides inhibited [125I]SRIF(Tyr11)-14 binding with IC50 values in the low nM range. Colony formation in the in vitro SCLC growth assay was also inhibited in the same concentration range, as was VIP-stimulated cyclic AMP formation. Therefore, octapeptide analogues of SRIF function as SCLC SRIF receptor agonists.     

Effect of somatostatin and octreotide on proliferation and vascular endothelial growth factor secretion from murine endothelial cell line (HECa10) culture

Angiogenesis, development of new blood vessels, is required for normal tissue repair and also for tumor cell proliferation, extracellular matrix invasion, and hematogenous metastases. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that has been shown to play a key role in neovascularization. Inhibition of angiogenesis in vitro and in vivo was documented by administration of native neuropeptide somatostatin and its analog octreotide. We have studied the effect of somatostatin-14 (SRIF) and ocreotide (sandostatin) on proliferation activity and VEGF release from cultured murine endothelial cells HECa10 in vitro. SRIF in concentrations from 10(-9) to 10(-5) M and ocreotide in concentrations from 10(-9) to 10(-5) M diminished the proliferative activity of cultured cells vs controls. SRIF and ocreotide in concentrations from 10(-14) to 10(-6) M did not change the release of VEGF into supernatants of 24 or 72 h endothelial cell cultures. Although we showed the antiproliferative effect of SRIF and ocreotide on mouse endothelial cells, we were unable to demonstrate the inhibitory effect of tested peptides on VEGF secretion in vitro.     

Bioengineering of human thyrotropin superactive analogs by site-directed "lysine-scanning" mutagenesis. Cooperative effects between peripheral loops

We have previously engineered the first superactive analogs of human thyrotropin (hTSH) by using a novel design strategy. In this study, we have applied homology comparisons focusing on the alphaL3 loop of the common alpha-subunit of human glycoprotein hormones. Seven highly variable amino acid residues were identified, and charge-scanning mutagenesis revealed three previously unrecognized modification permissive domains and four gain-of-function lysine substitutions. Such gain-of-function mutations were hormone- and receptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mutants. In combinations of the most potent alphaL3 loop analog with two previously characterized loop analogs, a higher degree of cooperativity for the alphaL3 loop analog compared with both the alphaL1 loop analog and the hTSH-betaL3 loop analog was observed. We demonstrated that spatially distinct regions of the common alpha-subunit contribute differentially to the interaction of hTSH with its receptor and that combinations of two modified loops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three superactive loops showed cooperativity in receptor binding and activation resulting in the most potent hTSH superactive analog described to date.     

Opiate-like naloxone-reversible actions of somatostatin given intracerebrally

The latency to tail-flick response in the rat was significantly prolonged by cerebroventricular infusion of 1.0 microgram of somatostatin (SRIF) and more so with 10.0 microgram. The D-tryptophan analog was less effective than native SRIF. Pretreatment with naloxone eliminated analgesia but not seizures induced by SRIF. Recording of the EEG activity enabled determination of the specific state of the sleep-waking cycle in which the repeated tail-flick responses were tested: latency was generally longer in both control and test animals when tail immersion was performed during the state of sleep or drowsiness rather than during the awake state. Although animals receiving SRIF were less likely to fall asleep between subsequent test trails, the average latency was actually longer than after control saline infusion when the animals slept more. SRIF, unlike other releasing factors and peptides tested, showed significant activity in an opiate radioreceptor assay. The blockade of SRIF action by naloxone pretreatment, along with binding of SRIF to opiate receptors in vitro, suggest opiate receptors to be involved in the mediation of analgesia observed in present study.     

Somatostatin analog SMS 201995 inhibits proliferation in human leukemia T-cell line: relevance of the adenylyl cyclase stimulation

Octreotide SMS 201995 is a stable somatostatin (SRIF) analog with potent antiproliferative actions in numerous cell types including normal T lymphocytes. It is currently used in the clinical treatment of different malignancies. However, the possible beneficial actions of octreotide in T-cell leukemia have not been addressed before, although these cells express SRIF receptors. For instance, human leukemia Jurkat T cells have been shown to express a single SRIF receptor isotype: sst3 that can be pharmacologically targeted by octreotide. In this study, we therefore studied SMS 201995 effects on in vitro [(3)H-CH3]thymidine incorporation in Jurkat T cells. Our data show that octreotide inhibits the proliferation of Jurkat cells both in the absence and in the presence of mitogens. By contrast, SRIF28, an endogenous SRIF analog sharing with SMS 201995 an almost identical affinity for somatostatin sst3 receptors, increases [(3)H-CH3]thymidine uptake in both mitogen-activated and nonactivated cells. To assess the mechanisms of the opposite actions of these two analogs on leukemia T-cell proliferation, we next studied their effects on adenylyl cyclase activity in whole Jurkat cells. At least in the presence of mitogens, SMS 201995 significantly enhances the adenylyl cyclase activity whereas SRIF28 inhibits it. Taken together these data are in accordance with the current hypothesis according to which increase and decrease in cAMP production are required to allow the inhibition and stimulation of T-cell proliferation, respectively. They also point to a potential therapeutic benefit of SMS 201995 in the management of human T-cell leukemia.     

In vivo and in vitro effects of cholecystokinin octapeptide on the release of growth hormone in rats

The effect of cholecystokinin octapeptide (CCK-8) on the release of growth hormone (GH) in rats was studied in vivo and in vitro. Intravenous injection of 5 micrograms/100 g BW of CCK-8 resulted in significant increase in the plasma GH level after 10 and 20 min. CCK-8 at concentrations of 10(-11)M to 10(-7)M also caused dose-dependent stimulation of GH release from dispersed cells of rat anterior pituitary. On the other hand, somatostatin (SRIF) inhibited GH release from dispersed cells of rat anterior pituitary in a dose-related manner at concentrations of 10(-7)M to 10(-9)M. Release of GH from the cells was increased by addition of K+ at high concentration (50 mM) in a Ca++-dependent manner. Addition of 10(-3)M verapamil to the incubation medium inhibited CCK-8-induced GH release from the cells. Addition of SRIF (10(-7)M) to the incubation medium inhibited GH release from the cells induced by CCK-8 or high K+ (50 mM). These results indicate that CCK-8 acts directly on the anterior pituitary cells to stimulate GH release and that calcium ion is involved in the mechanism of this effect.     

Potent GnRH agonists containing L-amino acid derivatives in the six position

New agonists related to gonadotropin-releasing hormone (GnRH) have been synthesized that are comparable in potency to the GnRH and its superagonists for release of LH and estrus suppression without substitutions with D- or unnatural amino acids in position 6. We now report a series of L-beta-aspartyl-6 GnRH analogs containing only naturally occurring L-amino acids in the whole sequence, exhibiting considerable in vivo biological activity. Dose and time dependent LH release capability of the different analogs in adult male mice, estrus suppression comparisons and blockade of ovulation in female rats are given. The incorporation of L-Asp-OMe and L-Asp-OBzl in position 6 of GnRH resulted in the most potent GnRH agonists (to 12-20xGnRH potency) in this series inducing a biphasic biological response similar to the D-amino acid-6 substituted superactive GnRH analogs. A correlation between the LH releasing potencies of the analogs and their HPLC retention times was also investigated. Peptide synthesis were achieved using either solid phase or solution phase methodology.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Summary Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.     

In vitro and in vivo inhibition of human small cell lung carcinoma (NCI-H69) growth by a somatostatin analogue

An endocrinologically-potent octapeptide analogue of somatostatin (SRIF), 3-(2-naphthyl)-D-Ala-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (BIM-23014 C), was examined for its ability to inhibit the in vitro and in vivo growth of the human small cell lung carcinoma (SCLC) line, NCI-H69. When cultured cells were implanted into athymic nude mice, treatment (500 micrograms/injection, twice daily) resulted in a prolongation of lag time for the appearance of measurable tumors, and there was a marked inhibition of the growth rate. Indeed, peptide injection in the region of the tumor resulted in a complete regression of the NCI-H69 tumors. Withdrawal of BIM-23014 C treatment resulted in an acceleration of tumor growth indicating an antiproliferative rather the oncolytic action. A similar inhibition of tumor growth was also observed when solid tumors obtained from the first implantation were used as the donor tissues. In cell culture, the proliferation in the presence of a low concentration (10nM) of BIM-23104 C was also significantly retarded suggesting a direct mechanism of action.     

Comparison of somatostatin and its highly potent hexa- and octapeptide analogs on exocrine and endocrine pancreatic secretion

The effects on pancreatic responses of highly potent cyclic hexapeptide (cyclo (N-Me-Ala-Phe-D-Trp-Lys-Thr-Phe)) (Veber analog) and octapeptide analogs of somatostatin such as D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (SMS 201-995), D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (RC-121), and D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) have been compared with somatostatin tetradecapeptide (SS-14) and atropine. The parameters evaluated were pancreatic responses to secretin and meat feeding in conscious dogs with chronic pancreatic fistula and amylase release from the dispersed pancreatic acini. The analogs were administered intravenously or intraduodenally. The cyclic hexapeptide and octapeptide analogs, given iv in graded doses against a constant background stimulation with secretin, produced similar and dose-dependent inhibition of pancreatic HCO3- and protein secretion. Analogs RC-121, RC-160, and the Veber analog were about two to four times more active than SS-14 in suppressing HCO3- secretion and equipotent in reducing protein secretion, but SMS 201-995 was only about half as potent as somatostatin in inhibiting HCO3-. RC-160 was effective in inhibiting secretin-induced protein secretion at lower doses than other analogs. In tests with feeding, SMS 201-995, the Veber analog, RC-121, and RC-160 were more potent inhibitors of exocrine pancreatic secretion of HCO3- and protein and exhibited more prolonged inhibitory effects than SS-14. The Veber analog, RC-121, and RC-160 were also more effective after intraduodenal administration. Atropine also caused significant inhibition of both HCO3- and protein responses to secretin and meal feeding. All four analogs decreased the postprandial insulin and pancreatic polypeptide release to a similar degree as SS-14. Neither SS-14 nor the analogs tested significantly affected basal or caerulein-, gastrin-, secretin-, or bethanechol-stimulated amylase release from the dispersed canine pancreatic acini. Atropine reduced amylase release induced by bethanechol, but not that stimulated by caerulein, gastrin, or secretin. This indicated that the analogs, as somatostatin, are ineffective as secretory inhibitors in vitro. We conclude that cyclic hexapeptide and octapeptide analogs are more potent and longer acting inhibitors of pancreatic secretion than somatostatin-14 in vivo.     

Biological activities of des-His[Glu]glucagon amide, a glucagon antagonist

Hyperglycemia in diabetes mellitus is generally associated with elevated levels of glucagon in the blood. A glucagon analog, des-His¹[Glu⁹] glucagon amide, has been designed and synthesized and found to be an antagonist of glucagon in several systems. It has been a useful tool for investigating the mechanisms of glucagon action and for providing evidence that glucagon is a contributing factor in the pathogenesis of diabetes. The in vitro and in vivo activities of the antagonist are reported here. The analog bound 40% as well as glucagon to liver membranes, but did not stimulate the release of cyclic AMP even at 10⁶ higher concentration. However, it did activate a second pathway, with the release of inositol phosphates. In addition, the analog enhanced the glucose-stimulated release of insulin from pancreatic islet cells. Of particular importance were the findings that the antagonist also showed only very low activity (<0.2%) in the in vivo glycogenolysis assay, and that at a ratio of 100:1 the analog almost completely blocked the hyperglycemic effects of added glucagon in normal rabbits. In addition, it reduced the hyperglycemia produced by endogenous glucagon in streptozotocin diabetic rats. Thus, we have an analog that possesses properties that are necessary for a glucagon antagonist to be potentially useful in the study and treatment of diabetes.     

In vitro actions on hemopoietic cells of recombinant murine GM-CSF purified after production in Escherichia coli: comparison with purified native GM-CSF

Recombinant murine GM-CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM-CSF. Both recombinant and native GM-CSF stimulated granulocyte and/or macrophage colony formation by adult and fetal mouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM-CSF (25 X 10(8) U/mg) was similar to that of the native form (15 X 10(8) U/mg). At high concentrations (greater than 200 U/ml), both forms of GM-CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (greater than 800 U/ml), megakaryocyte and some erythroid and mixed-erythroid colony formation. Recombinant GM-CSF was as effective in stimulating the proliferation of the GM-CSF-dependent cell line FD as the native molecule. Both recombinant and native GM-CSF were able to induce partial differentiation in colonies of WEHI-3B myeloid leukemic cells. Recombinant GM-CSF competed effectively for the binding of 125I-labeled native GM-CSF to hemopoietic cells, and antiserum to recombinant GM-CSF also neutralized the biological activity of native GM-CSF. The bacterially synthesized GM-CSF was a slightly more effective stimulus for megakaryocyte colony formation than the native molecule. The demonstration that purified bacterially synthesized GM-CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.     

Design, synthesis, and biological evaluation of chicoric acid analogs as inhibitors of HIV-1 integrase

A series of analogs of the potent HIV-1 integrase (HIV IN) inhibitor chicoric acid (CA) was designed with the intention of ameliorating some of the parent natural product's undesirable properties, in particular its toxicity, instability, and poor membrane permeability. More than 70 analogs were synthesized and assayed for three types of activity: (1) the ability to inhibit 3'-end processing and strand transfer reactions using recombinant HIV IN in vitro, (2) toxicity against the CD4+ lymphoblastoid cell line, MT2, and (3) anti-HIV activity against HIV(LAI). CA analogs lacking one of the carboxyl groups of CA and with 3,4,5-trihydroxycinnamoyl sidechains in place of the caffeoyl group of CA exhibited the most potent inhibition of HIV replication and end-processing activity. Galloyl-substituted derivatives also displayed very potent in vitro and in vivo activities, in most cases exceeding the inhibitory effects of CA itself. Conversely, analogous monocarboxy caffeoyl analogs exhibited only modest inhibition, while the corresponding 3,4-dihydroxybenzoyl-substituted compounds were devoid of activity.     

Synthesis, biologic activity, and protein binding characteristics of a new vitamin D analog, 22-hydroxyvitamin D3

We synthesized a novel vitamin D analog, 22-hydroxyvitamin D3 9 and tested its biologic activity (and antivitamin properties) in vivo in vitamin D-deficient rats, and in vitro in the chick embryonic duodenum. We examined its ability to bind to the sterol carrier protein, vitamin D binding protein and the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. The new vitamin 9 was synthesized from 3 beta-hydroxy-22,23-dinorcholenic acid 1 in 12 steps. The vitamin 9 displayed no vitamin D agonist activity in the intestine or in bone in vivo and did not block the activity of vitamin D3 or 25-hydroxyvitamin D3. It was a weak vitamin D3 agonist in the chick embryonal duodenum in vitro. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. Vitamin 9 bound to the chick intestinal cytosol receptor with low affinity. 22-Hydroxyvitamin D3 and various vitamin D sterols were bound to vitamin D binding protein in the following order: 25-hydroxyvitamin D3. (24R)-24,25-dihydroxyvitamin D3, and (25S)-25,26-dihydroxyvitamin D3 greater than 22-hydroxyvitamin D3 greater than 11 alpha-hydroxyvitamin D3 greater than 1,25-dihydroxyvitamin D3 greater than vitamin D3. We conclude that the introduction of a hydroxyl group at C-22 in the side chain of the vitamin D3 molecule decreases its biological activity.     

Thymic humoral factor gamma 2: purification and amino acid sequence of an immunoregulatory peptide from calf thymus

Thymic humoral factor gamma 2 (THF-gamma 2), an octapeptide essential for immune regulation, was purified from calf thymus. The purification of THF-gamma 2, monitored in vitro and in vivo in mouse splenocyte proliferation assays, was achieved by gel filtration of low molecular weight thymus extracts followed by ion-exchange chromatography and sequential reversed-phase high-performance liquid chromatography. The process yielded 5 micrograms of THF-gamma 2/1000 kg of thymus tissue. The concentration of THF-gamma 2 required for augmentation of lymphocyte proliferation and interleukin 2 production was 5 ng/mL in vitro and 10 ng/kg per mouse in vivo. THF-gamma 2 has the amino acid sequence Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. The proposed structure has been confirmed because a peptide was synthesized on the basis of this sequence that showed activity identical with that of the biological molecule. It shows no homology to the amino acid sequence of other thymic hormones nor is it part of any peptide or protein of known sequence. THF-gamma 2 retains essentially all of the biological activity of the thymus extract from which it is derived.     

Mutational analysis of autonomously functioning trans-activating peptides encoded by adenovirus E1A: evidence for two subdomains.

We have shown previously that a chemically synthesized adenovirus E1A region 3 peptide of 49 amino acids, protein domain 3 (PD3; residues 140 to 188 of the 289-amino-acid protein), trans activates viral genes in vitro and in vivo. To study structure-function relationships, we synthesized N-terminal deletion and cysteine substitution mutant peptides and tested their activities in a cell microinjection assay. Peptides lacking 1 to 12 N-terminal residues exhibited 5- to 50-fold-reduced molar specific activities, whereas those lacking 16 or 18 residues were inactive. Substitution of each of five PD3 cysteine residues with alanine resulted in substantial losses of activity: mutants in the PD3 N-terminal portion showed 40 to 55% of wild-type activity but required a 20-fold-higher concentration than PD3, whereas those in the C-terminal half were as much less active. These peptide mutant studies suggest the existence of two PD3 functional regions: one, localized in the C-terminal 70 to 75% of the molecule, is essential for trans activation; the other, localized in the N-terminal 25 to 30%, can be overridden to a significant extent at high peptide concentrations.