A microplate binding assay for the somatostatin type-2 receptor (SSTR2)

The clinical importance of somatostatin type-2 receptors (SSTR2) and the study of novel analogues of somatostatin such as OctreoScan or [Tyr3]-octreotide containing DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as metal chelator led us to develop a methodology to monitor the expression of SSTR2 on tumours of pancreatic origin (e.g. rat AR4-2J cancer cells). Usual binding assay protocols using the commercial [125I][Tyr1]-somatostatin radioligand failed, even in the presence of a cocktail of protease inhibitors with a broad spectrum of activity, possibly due to the high susceptibility of this tracer to proteases expressed in pancreatic cells. We prepared our own radioligand [125I][Tyr2]-octreotide which was shown to be much more resistant to degradation after incubation with AR4-2J plasma membranes. As expected, the increased stability of [125I][Tyr3]-octreotide was associated with good binding to SSTR2. Addition of appropriate protease inhibitors further increased the specific binding of [125I][Tyr3]-octreotide to AR4-2J plasma membranes without affecting the stability of the tracer, suggesting that the protease inhibitors also protect the integrity of SSTR2. Optimal conditions (time, temperature, medium) were developed for a binding assay in 96-well plates using AR4-2J plasma membranes in order to make the assay suitable for high-throughput analysis. This protocol was the basis for studying the in vivo regulation of SSTR2 expression in AR4-2J cells implanted into scid mice after exposure to different compounds.     

In vivo application of [111In-DTPA-D-Phe1]-octreotide for detection of somatostatin receptor-positive tumors in rats.

Radioiodinated somatostatin analogues are useful ligands for the in vitro and in vivo detection of somatostatin receptors. [111In-DTPA-D-Phe1]-octreotide, a somatostatin analogue labeled with a different radionuclide, also binds specifically to somatostatin receptors in vitro. In this study we investigated its in vivo application in the visualization of somatostatin receptor-positive tumors in rats. The distribution of the radiopharmaceutical was investigated after intravenous injection in normal rats and in rats bearing the somatostatin receptor-positive rat pancreatic carcinoma CA 20948. After injection the radiopharmaceutical was rapidly cleared (50% decrease in maximal blood radioactivity in 4 min), predominantly by the kidneys. Excreted radioactivity was mainly in the form of the intact radiopharmaceutical. Ex vivo autoradiographic studies showed that specific accumulation of radioactivity occurred in somatostatin receptor-containing tissue (anterior pituitary gland). However, in contrast to the adrenals and pituitary, the tracer accumulation in the kidneys was not mediated by somatostatin receptors. Increasing radioactivity over the somatostatin receptor-positive tumors was measured rapidly after injection and the tumors were clearly visualized by gamma camera scintigraphy. In rats pretreated with 1 mg octreotide accumulation of [111In-DTPA-D-Phe1]-octreotide in the tumors was prevented. Because of its relatively long effective half-life, [111In-DTPA-D-Phe1]-octreotide is a radionuclide-coupled somatostatin analogue which can be used to visualize somatostatin receptor-bearing tumors efficiently after 24 hr, when interfering background radioactivity is minimized by renal clearance. This is an advantage over the previously used [123I-Tyr3]-octreotide which has a shorter effective half-life and shows high abdominal interference due to its hepato-biliary clearance. Therefore, [111In-DTPA-D-Phe1]-octreotide seems a better alternative for scintigraphic imaging of somatostatin receptor-bearing tumors.     

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.     

Influence of different spacers on the biological profile of a DOTA-somatostatin analogue

Radiolabeled somatostatin analogues have been successfully used for targeted radiotherapy and for imaging of somatostatin receptor (sst1-5)-positive tumors. Nevertheless, these analogues are subject to improving their tumor-to-nontarget ratio to enhance their diagnostic or therapeutic properties, preventing nephrotoxicity. In order to understand the influence of lipophilicity and charge on the pharmacokinetic profile of [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)]-somatostatin-based radioligands such as [DOTA,1-Nal3]-octreotide (DOTA-NOC), different spacers (X) based on 8-amino-3,6-dioxaoctanoic acid (PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG4), N-acetyl glucosamine (GlcNAc), triglycine, beta-alanine, aspartic acid, and lysine were introduced between the chelator DOTA and the peptide NOC. All DOTA-X-NOC conjugates were synthesized by Fmoc solid-phase synthesis. The partition coefficient (log D) at pH = 7.4 indicated that higher hydrophilicity than [111In-DOTA]-NOC was achieved with the introduction of the mentioned spacers, except with triglycine and beta-alanine. The high affinity of [InIII-DOTA]-NOC for human sst2 (hsst2) was preserved with the structural modifications, while an overall drop for hsst3 affinity was observed, except in the case of [InIII-DOTA]-beta-Ala-NOC. The new conjugates preserved the good affinity for hsst5, except for [InIII-DOTA]-Asn(GlcNAc)-NOC, which showed decreased affinity. A significant 1.2-fold improvement in the specific internalization rate in AR4-2J rat pancreatic tumor cells (sst2 receptor expression) at 4 h was achieved with the introduction of Asp as a spacer in the parent compound. In sst3-expressing HEK cells, the specific internalization rate at 4 h for [111In-DOTA]-NOC (13.1% +/- 0.3%) was maintained with [111In-DOTA]-beta-Ala-NOC (14.0% +/- 1.8%), but the remaining derivatives showed <2% specific internalization. Biodistribution studies were performed with Lewis rats bearing the AR4-2J rat pancreatic tumor. In comparison to [111In-DOTA]-NOC (2.96% +/- 0.48% IA/g), the specific uptake in the tumor at 4 h p.i. was significantly improved for the 111In-labeled sugar analogue (4.17% +/- 0.46% IA/g), which among all the new derivatives presented the best tumor-to-kidney ratio (1.9).     

Peptide-chelating agent conjugate for selective targeting of somatostatin receptor type 1: synthesis and characterization

Previously reported results suggest that the analogue of the somatostatin des-AA1,2,5[D-Trp8,IAmp9]-somatostatin (CH-275) peptide bearing chelating agents able to coordinate radioactive metals could be used for scintigraphic imaging of tumor lesions overexpressing sstr1. An efficient synthetic procedure for the preparation of the somatostatin analogue CH-275 and its conjugate DTPAGlu-Gly-CH-275, bearing the chelating agent DTPAGlu (DTPAGlu=N,N-bis[2-[bis(carboxy-ethyl)amino]ethyl]-L-glutamic acid) on the N-terminus, by solid-phase peptide synthesis and 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, is here reported. Rapid and efficient labeling of DTPAGlu-Gly-CH-275 was achieved by addition of 111In(III) to the compound. Typical yields were greater than 97% as determined by reversed phase high performance liquid chromatography (HPLC) at specific activities in the range 4-9 GBq/micromol (100-250 Ci/mmol). A preliminary biological assay of the binding ability of 111In-DTPAGlu-Gly-CH-275 indicates, however, that the labeled compound does not display any specific interaction with somatostatin sstr1 receptors in the tested cell lines. To confirm this unexpected negative result, competition binding experiments were carried out, in which fixed tracer amounts of the 125I-labeled somatostatin-14 were incubated with the receptor-expressing cells in the presence of DTPAGlu-Gly-CH-275 or CH-275 at concentrations ranging from 10(-10) to 10(-3) M. While CH-275 shows a IC50 of 80 nM similar to that already found in displacement experiments on CHO-K1 sstr1-transfected cells, DTPAGlu-Gly-CH-275 displays instead very low or negligible affinity towards this receptor. The NMR solution characterization indicates that the presence of DTPAGlu does not influence the conformational and chemical features of the peptide moiety, thus suggesting that the loss in binding activity should be due to steric hindrance of either the chelating agent DTPAGlu or its indium complex.     

Preclinical evaluation of a dual sstr2 and integrin αvβ3-targeted heterodimer [68Ga]-NOTA-3PEG4-TATE-RGD

PurposeMultiple receptors are co-expressed in many types of cancers. Octreotate (TATE) and Arg-Gly-Asp (RGD) peptides target somatostatin receptor 2 (sstr2) and integrin α_vβ₃, respectively. We developed and synthesized a heterodimer NOTA-3PEG₄-TATE-RGD (3PTATE-RGD) and aimed to investigate its characteristics for dual-targeting sstr2 and integrin α_vβ₃.MethodsTATE and RGD peptides and 1,4,7-triazacylononane-N’,N’’,N’’’-triacetic acid (NOTA) were linked through a glutamate and polyethylene glycol (PEG) linker, then 3PTATE-RGD was labeled with ⁶⁸Ga ion. Receptor-binding characteristics and tumor-targeting efficacy were tested in vitro and in vivo using H69 and A549 lung cancer cell lines and tumor-bearing mice models.Results[⁶⁸Ga]-3PTATE-RGD had comparable sstr2 and integrin α_vβ₃-binding affinity with monomeric TATE and RGD in cell uptake and PET imaging study, respectively. In the competition study, H69 and A549 tumor uptake of [⁶⁸Ga]-3PTATE-RGD was completed inhibited in the presence of an excess amount of unlabeled TATE or RGD, respectively. The blocked level didn’t grow when both of TATE and RGD mixture was co-injected with [⁶⁸Ga]-3PTATE-RGD. The pharmacokinetics of [⁶⁸Ga]-3PTATE-RGD is comparable with [⁶⁸Ga]-TATE and [⁶⁸Ga]-RGD, resulting in a larger application.Conclusion[⁶⁸Ga]-3PTATE-RGD showed improved and wider tumor-targeting efficacy compared with monomeric TATE and RGD peptides, which warrants its further investigation in detection both of sstr2 and integrin α_vβ₃-related carcinomas.     

Synthesis and characterization of α(v)β(3)-targeting peptidomimetic chelate conjugates for PET and SPECT imaging

There is growing interest in small peptidomimetic α(v)β(3) integrin antagonists that are readily synthesized and characterized and can be easily handled using physiological conditions. Peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-β-alanine (IAC) was successfully conjugated to 1-(1-carboxy-3-carbo-t-butoxypropyl)-4,7-(carbo-tert-butoxymethyl)-1,4,7-triazacyclononane (NODA-GA(tBu)(3)) and 1-(1-carboxy-3-carbotertbutoxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA-GA(tBu)(4)) and radiolabeled with (111)In, (67)Ga and (203)Pb. Results of a radioimmunoassay demonstrated binding to purified α(v)β(3) integrin when 1-4equiv of integrin were added to the reaction. Based on this promising result, investigations are moving forward to evaluate the NODA-GA-IAC and DOTA-GA-IAC conjugates for targeting tumor associated angiogenesis and α(v)β(3) integrin positive tumors to define their PET and SPECT imaging qualities as well as their potential for delivery of therapeutic radionuclides.     

2-Phenylimidazo[2,1-i]purin-5-ones: structure-activity relationships and characterization of potent and selective inverse agonists at Human A3 adenosine receptors

Structure-activity relationships of 2-phenyl-imidazo[2,1-i]purin-5-ones as ligands for human A(3) adenosine receptors (ARs) were investigated. An ethyl group in the 8-position of the imidazoline ring of 4-methyl-2-phenyl-imidazopurinone leading to chiral compounds was found to increase affinity for human A(3) ARs by several thousand-fold. Propyl substitution instead of methyl at N4 decreased A(3) affinity but increased A(1) affinity leading to potent A(1)-selective AR antagonists. The most potent A(1) antagonist of the present series was (S)-8-ethyl-2-phenyl-4-propyl-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (S-3) exhibiting a K(i) value of 7.4 nM at rat A(1) ARs and greater than 100-fold selectivity versus rat A(2A) and human A(3) ARs. At human A(1) ARs 2-phenylimidazo[2,1-i]purin-5-ones were generally less potent and therefore less A(1)-selective (S-3: K(i)=98 nM). 2-, 3-, or 4-Mono-chlorination of the 2-phenyl ring reduced A(3) affinity but led to an increase in affinity for A(1) ARs, whereas di- (3,4-dichloro) or polychlorination (2,3,5-trichloro) increased A(3) affinity. The most potent and selective A(3) antagonist of the present series was the trichlorophenyl derivative (R)-8-ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (R-8) exhibiting a subnanomolar K(i) value at human A(3) ARs and greater than 800-fold selectivity versus the other AR subtypes. Methylation of 4-alkyl-2-phenyl-substituted imidazo[2,1-i]purin-5-ones led exclusively to the N9-methyl derivatives, which exhibited largely reduced AR affinities as compared to the unmethylated compounds. [35S]GTP gamma S binding studies of the most potent 2-phenyl-imidazo[2,1-i]purin-5-ones at membranes of Chinese hamster ovary cells expressing the human A(3) AR revealed that the compounds were inverse agonists at A(3) receptors under standard test conditions. Due to their high A(3) affinity, selectivity, and relatively high water-solubility, 2-phenyl-imidazo[2,1-i]purin-5-ones may become useful research tools.     

Synthesis, in vitro and in vivo evaluation of [O-methyl-11C] 2-{4-[4-(3-methoxyphenyl)piperazin-1-yl]-butyl}-4-methyl-2H-[1,2,4]-triazine-3,5-dione: a novel agonist 5-HT1A receptor PET ligand

Synthesis and in vivo evaluation of 2-{4-[4-(3-methoxyphenyl)piperazin-1-yl]-butyl}-4-methyl-2H-[1,2,4]triazine-3,5-dione (5 or MMT), a high affinity and selective serotonin 5-HT1AR agonist PET tracer, are described. GTPgammaS assay shows that MMT is an agonist with an EC50 comparable to 5-HT. Radiolabeling of 5 was achieved in 30% yield (EOS) from desmethyl-MMT (4) with >99% chemical and radiochemical purities and a specific activity >1000 Ci/mmol. PET studies in baboon show that [11C]5 penetrates the blood-brain barrier but, because of low specific binding and fast clearance of radioactivity it is not a suitable PET tracer for the in vivo quantification of 5-HT1AR.     

Binding of somatostatin to guinea-pig pancreatic membranes: regulation by ions

Somatostatin receptors were characterized on guinea-pig pancreatic acini membranes using 125I-[Tyr11] somatostatin 14 as a radioligand. In 0.1 mM Ca2+ buffer the binding was saturable and slowly reversible, exhibiting a single class of high affinity binding sites (KD = 0.15 +/- 0.03 nM) with a maximal binding capacity (B max) of 178 +/- 18 fmol/mg protein. In 30 nM) free Ca2+ buffer, the binding was highly reversible. Affinity and B max were decreased by about 2-fold. Ca2+ exhibited an EC50 of 2.4 +/- 0.9 microM to potentiate the binding of somatostatin. Na+, but not K+, inhibited the binding: Bmax was decreased with no change in affinity. Somatostatin analogs inhibited the binding of 125I-[Tyr11] somatostatin 14. The relative potencies were: somatostatin 14 greater than somatostatin 28 = [Nle8]somatostatin 28 greater than [D Tryp8, D Cys14]somatostatin 14.     

Structural and in vivo studies of metal chelates of Ga(III) relevant to biomedical imaging

The solution chemistry and structure of the complex of the triazamacrocyclic ligand NOTP (1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonate)) with Ga3+ in D2O have been investigated by 1H, 71Ga and 31P NMR spectroscopy. These NMR results show the presence of a 1:1 Ga(NOTP)3- complex, with a highly symmetrical, pseudo-octahedral geometry, possibly with a C3 axis. The 1H spectrum shows that the triazamacrocyclic chelate ring is very rigid, with all the ring protons non-equivalent. The complex is stable in aqueous solution in a wide pH range. Its high thermodynamic stability agrees well with previous results from biodistribution and gamma imaging studies in Wistar rats with 67Ga3+ chelates of triaza macrocyclic ligands, which showed that the neutral chelates 67Ga(NOTA) (where NOTA is 1,4,7-triazacyclononane-1,4,7-triacetate) and 67Ga(NOTPME) (where NOTPME is 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonate monoethylester)) have similar in vivo behaviour, with high stability and rapid renal excretion, but the high negatively charged 67Ga(NOTP)3- has a considerably slower kidney uptake and elimination.     

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.     

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.     

Synthesis and evaluation of glycosylated octreotate analogues labeled with radioiodine and 211At via a tin precursor

Carbohydration of N-terminus and substitution of a threonine for the threoninol residue at the C-terminus of Tyr3-octreotide (TOC) has resulted in improved pharmacokinetics and tumor targeting of its radioiodinated derivatives. Yet, these peptides are very susceptible to in vivo deiodination due to the similarity of monoiodotyrosine (MIT) to thyroid hormone. The goal of this work was to develop octreotate analogues containing both a sugar moiety and a nontyrosine prosthetic group on which a radioiodine or 211At can be introduced. Solid-phase synthesis and subsequent modifications delivered an iodo standard of the target peptide N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-iodobenzoyl)-Lys0-octreotate (GIBLO) and the corresponding tin precursor N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-[(3-tri-n-butylstannyl)benzoyl]-Lys0-octreotate (GTBLO). GIBLO displaced [125I]TOC from somatostatin receptor subtype 2 (SSTR2)-positive AR42J rat pancreatic tumor cell membranes with an IC50 of 0.46 +/- 0.05 nM suggesting that GIBLO retained affinity to SSTR2. GTBLO was radiohalogenated to [131I]GIBLO and N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-[211At]astatobenzoyl)-Lys0-octreotate ([211At]GABLO) in 21.2 +/- 4.9% and 46.8 +/- 9.5% radiochemical yields, respectively. From a paired-label internalization assay using D341 Med medulloblastoma cells, the maximum specific internalized radioactivity from [131I]GIBLO was 1.78 +/- 0.8% of input dose compared to 9.67 +/- 0.43% for N(alpha)-(1-deoxy-D-fructosyl)-[125I]iodo-Tyr3-octreotate ([125I]I-Gluc-TOCA). Over a 4 h period, the extent of internalization of [131I]GIBLO and [211At]GABLO was similar in this cell line. In D341 Med murine subcutaneous xenografts, the uptake of [125I]I-Gluc-TOCA at 0.5, 1 and 4 h was 21.5 +/- 4.0% ID/g, 18.8 +/- 7.7% ID/g, and 0.9 +/- 0.4% ID/g, respectively. In comparison, these values for [131I]GIBLO were 6.9 +/- 1.2% ID/g, 4.7 +/- 1.4% ID/g, and 0.8 +/- 0.5% ID/g. Both in vitro and in vivo catabolism studies did not suggest the severance of the lys0 along with its appendages from the peptide. Taken together, although GIBLO maintained affinity to SSTR2, its tumor uptake both in vitro and in vivo was substantially lower than that of I-Gluc-TOCA suggesting other factors such as net charge and overall geometry of the peptide may be important.     

Solubilization and characterization of guinea-pig pancreatic somatostatin receptors

The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I-[Tyr11]somatostatin or its analogue 125I-[Leu8,DTrp22,Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) and octyl beta-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr = 93,000 which interacted with the two radioligands. In addition, a similar component of Mr = 88,000 was characterized after analysis by SDS-PAGE of membrane receptors covalently cross-linked with 125I-[Leu8,DTrp22,Tyr25]somatostatin 28 by different heterobifunctional reagents: N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl 4-azidobenzoate, N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate. Optimal cross-linking results were obtained with N-5-azido-2-nitrobenzoyloxysuccinimide. The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars. We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent Mr approximately 85,000-90,000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.     

Binding of [H][D-Ala, MePhe, Gly-ol] Enkephalin, [H][D-Pen, D-Pen]Enkephalin, and [H]U-69,593 to Airway and Pulmonary Tissues of Normal and Sensitized Rats

Bhargava, H. N., V. M. Villar, J. Cortijo and E. J. Morcillo. Binding of [³H][D-Ala², MePhe⁴, Gly-ol⁵]enkephalin, [³H][D-Pen², D-Pen⁵]enkephalin, and [³H]U-69,593 to airway and pulmonary tissues of normal and sensitized rats. Peptides 18(10) 1603–1608, 1997.—The role of endogenous opioid peptides in the regulation of bronchomotor tone, as well as in the pathophysiology of asthma is uncertain. We have studied the binding of highly selective [³H]labeled ligands of μ-([D-Ala², MePhe⁴, Gly-ol⁵]enkephalin; DAMGO), δ ([D-Pen², D-Pen⁵]enkephalin; DPDPE), and κ-(U-69,593) opioid receptors to membranes of trachea, main bronchus, lung parenchyma and pulmonary artery obtained from normal (unsensitized) and actively IgE-sensitized rats acutely challenged with the specific antigen. [³H]DAMGO, [³H]DPDPE and [³H]U-69,593 bound to membranes of normal and sensitized tissues at a saturable, single high-affinity site. The rank order of receptor densities in normal tissues was δ- ≥ κ- ≥ μ-, with lung parenchyma exhibiting the greatest binding capacity for δ- and μ- receptors compared to the other regions examined. The K_d values showed small differences between ligands and regions tested. The μ- and δ-opioid receptor densities were decreased in sensitized main bronchus and lung parenchyma, respectively, compared to normal tissues. By contrast, κ-opioid receptor density was augmented in sensitized lung parenchyma but an increase in K_d values was also observed. These differential changes in the density and affinity of opioid receptor types may be related to alterations in endogenous opioid peptides during the process of sensitization.     

Effects of trypsin on binding of [3H]epinephrine and [3H]-dihydroergocryptine to rat liver plasma membranes. Evidence for interconversion of binding sites

Treatment of liver plasma membranes with trypsin at low concentrations (1 to 2 microgram/mg of protein) caused at 3- to 4-fold increase in alpha-specific [3H]epinephrine binding. The change was due to an increase in the number of high affinity binding sites, with no change in the dissociation constant. With increasing trypsin concentrations, the dissociation constant was decreased and there was a progressive loss of binding. Elastase, papain, and thermolysin caused similar effects, whereas the thrombin, leucine aminopeptidase, phospholipase A2, phospholipase C, phospholipase D, and detergents did not cause an increase in [EH]epinephrine binding. The increase in epinephrine high affinity binding sites was correlated with a loss of high affinity [3H]-dihydroergocryptine binding sites which also bind [3H]epinephrine with low affinity (El-Refai, M. F., Blackmore, P. F., and Exton, J. H. (1979) J. Biol. Chem. 254, 4375-4386). Incubation of membranes with the alpha blockers dihydroergocryptine (50 nM) and phenoxybenzamine (20 nM) prior to protease treatment diminished the increase in [3H]epinephrine binding induced by trypsin (1.5 microgram/mg). The concentration dependence and time course of trypsin actions on 70 nM [3H]epinephrine binding and 10 nM [3H]dihydroergocryptine binding are consistent with a trypsin-mediated conversion of low affinity epinephrine binding sites to high affinity epinephrine binding sites.     

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.     

Cyclic somatostatin octapeptide analogues with high affinity and selectivity toward mu opioid receptors

A series of cyclic conformationally restricted penicillamine containing somatostatin octapeptide analogues have been prepared by standard solid phase synthetic techniques and tested for their ability to inhibit specific [125I]CGP 23,996 (des-Ala1-,Gly2-[desamino-Cys3Tyr11]-dicarba3, 14-somatostatin), [3H]naloxone or [3H]DPDPE ([D-Pen2-D-Pen5]enkephalin) binding in rat brain membrane preparations. We now report structure-activity relationship studies with the synthesis of our most potent and selective mu opioid receptor compound D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, which we refer to as Cys2Tyr3Orn5Pen7-amide. While this octapeptide exhibited high affinity (IC50 = 2.80 nM) for an apparently single population of binding sites (nH = 0.89 +/- 0.1) and exceptional selectivity for mu opioid receptors with an IC50(DPDPE)/IC50 (naloxone) ratio of 4,829, it also displayed very low affinity for somatostatin receptors (IC50 = 22,700 nM). Thus, Cys2Tyr3Orn5Pen7-amide may be the ligand of choice for further characterization of mu opioid receptors and for examining the physiological role of this class of receptors.     

Synthesis and evaluation of Re/99mTc(I) complexes bearing a somatostatin receptor-targeting antagonist and labeled via a novel [N,S,O] clickable bifunctional chelating agent

The somatostatin receptor subtype 2 (SSTR2) is often highly expressed on neuroendocrine tumors (NETs), making it a popular in vivo target for diagnostic and therapeutic approaches aimed toward management of NETs. In this work, an antagonist peptide (sst₂-ANT) with high affinity for SSTR2 was modified at the N-terminus with a novel [N,S,O] bifunctional chelator (2) designed for tridentate chelation of rhenium(I) and technetium(I) tricarbonyl cores, [Re(CO)₃]⁺ and [^99mTc][Tc(CO)₃]⁺. The chelator-peptide conjugation was performed via a Cu(I)-assisted click reaction of the alkyne-bearing chelator (2) with an azide-functionalized sst₂-ANT peptide (3), to yield NSO-sst₂-ANT (4). Two synthetic methods were used to prepare Re-4 at the macroscopic scale, which differed based on the relative timing of the click conjugation to the [Re(CO)₃]⁺ complexation by 2. The resulting products demonstrated the expected molecular mass and nanomolar in vitro SSTR2 affinity (IC₅₀ values under 30?nM, AR42J cells, [¹²⁵I]iodo-Tyr¹¹-somatostatin-14 radioligand standard). However, a difference in their HPLC retention times suggested a difference in metal coordination modes, which was attributed to a competing N-triazole donor ligand formed during click conjugation. Surprisingly, the radiotracer scale reaction of [^99mTc][Tc(OH₂)₃(CO)₃]⁺ (^99mTc; t_½?=?6?h, 141?keV γ) with 4 formed a third product, distinct from the Re analogues, making this one of the unusual cases in which Re and Tc chemistries are not well matched. Nevertheless, the [^99mTc]Tc-4 product demonstrated excellent in vitro stability to challenges by cysteine and histidine (≥98% intact through 24?h), along with 75% stability in mouse serum through 4?h. In vivo biodistribution and microSPECT/CT imaging studies performed in AR42J tumor-bearing mice revealed improved clearance of this radiotracer in comparison to a similar [^99mTc][Tc(CO)₃]-labeled sst₂-ANT derivative previously studied. Yet despite having adequate tumor uptake at 1?h (4.9% ID/g), tumor uptake was not blocked by co-administration of a receptor-saturating dose of SS-14. Aimed toward realignment of the Re and Tc product structures, future efforts should include distancing the alkyne group from the intended donor atoms of the chelator, to reduce the coordination options available to the [M(CO)₃]⁺ core (M?=?Re, ^99mTc) by disfavoring involvement of the N-triazole.