Modulation of pharmacokinetics of radioiodinated sugar-conjugated somatostatin analogues by variation of peptide net charge and carbohydration chemistry

Sugar conjugation of biooactive peptides has been shown to be a powerful tool to modulate peptide pharmacokinetics. In the case of radiolabeled somatostatin analogues developed for in vivo scintigraphy of somatostatin receptor (sst) expressing tumors, it generally led to tracers with predominant renal excretion and low uptake in nontarget organs, and in some cases also with enhanced tumor accumulation. Especially with respect to endoradiotherapeutic applicability of these tracers, however, understanding the structural requirements for minimal kidney accumulation and maximal tumor uptake is important. The aim of this study was therefore the evaluation of the potential of specific glycoside structures in combination with reduced peptide net charge to reduce kidney accumulation without affecting tumor accumulation. Three glyco analogues of radioiodinated Tyr(3)-octreotate (TOCA) with z = 0 were evaluated in a comparative study using [(125)I]Mtr-TOCA (z = +1), the maltotriose-Amadori analogue of [(125)I]TOCA, as a reference, [(125)I]Glucuron-TOCA, the Amadori conjugate with glucuronic acid, and [(125)I]Gluc-S- and [(125)I]Gal-S-TOCA, the coupling products with glucosyl- and mannosyl-mercaptopropionate. In cells transfected with sst(1)-sst(5), all three new analogues show sst-subtype binding profiles similar to I-Mtr-TOCA with high, but somewhat reduced, affinity for sst(2). In contrast, internalization into sst(2)-expressing cells (in % of [(125)I]Tyr(3)-octreotide ([(125)I]TOC)) as well as the EC(50,R) of unlabeled TOC for internalization determined in dual-tracer experiments are substantially enhanced for [(123)I]Gal-S-TOCA and [(123)I]Gluc-S-TOCA (internalization, 190% +/- 12% and 265% +/- 20%, respectively, vs 168% +/- 6% of [(125)I]TOC for [(123)I]Mtr-TOCA; EC(50,R), 2.62 +/- 0.07 and 2.96 +/- 0.14, respectively, vs 1.81 +/- 0.07 for [(123)I]Mtr-TOCA). The tumor accumulation of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA in AR42J tumor-bearing nude mice 1 h p.i. is consequently very high (22.6 +/- 2.2 and 26.2 +/- 5.6%ID/g) and comparable to that of [(125)I]Mtr-TOCA (25.1 +/- 4.4%ID/g). [(125)I]Glucuron-TOCA showed lower uptake in sst-expressing tissues than did [(125)I]Mtr-TOCA, but considerably enhanced accumulation in nontarget organs such as liver, intestine, and kidney. Due to increased lipophilicity, hepatic and intestinal uptake 1 and 4 h p.i. of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA was also slightly higher than that of [(125)I]Mtr-TOCA. Kidney accumulation, however, was reduced by approximately 50% for both compounds (2.6 +/- 0.3 and 2.2 +/- 0.4, respectively, vs 4.0 +/- 0.7%ID/g at 1 h p.i.). Because no sugar-specific effect was detected in the latter case, it is concluded that general ligand pharmacokinetics and especially kidney accumulation of the tracers investigated are mainly determined by physicochemical characteristics such as lipophilicity, net charge, and also charge distribution ([(125)I]Glucuron-TOCA vs [(125)I]Gal-S- and [(125)I]Gluc-S-TOCA). With respect to receptor targeting, however, the structure of the carbohydrate moiety plays an important role, leading to dramatically enhanced ligand internalization, especially in the case of [(123)I]Gluc-S-TOCA. Taking into account the combined effects of the Gluc-S-moiety both on kidney and on tumor accumulation, this group seems to be a promising synthon for the synthesis of other radiolabeled peptide analogues with improved pharmacokinetics.     

Radioiodinated azide and isothiocyanate derivatives of cocaine for irreversible labeling of dopamine transporters: synthesis and covalent binding studies

Two novel N-substituted-3beta-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One contains an iodoaryl azide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were prepared in a one-flask procedure by electrophilic radioiodination of the corresponding aniline under no-carrier-added conditions, followed either by diazotization and treatment with sodium azide, or by addition of thiophosgene under basic conditions. Specifically, (-)-N-[4-(3-[(125)I]iodo-4-azidophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ-2-24) and (-)-N-[4-(3-[(125)I]iodo-4-isothiocyanophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ 3-37) were synthesized. Isolation by reversed-phase HPLC and solid-phase extraction gave good average yields of [(125)I]MFZ-2-24 (67%, n = 5) and [(125)I]MFZ-3-37 (45%, n = 3) with high radiochemical purities (96-99%) and specific radioactivities (>2000 mCi/micromol). The utility of the radioligands was demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding to the full-length DAT.     

High affinity interaction of endothelin-3 with recombinant ETA receptors

Pharmacological evidence has suggested that endothelin-3 (ET-3) may act via a novel form of ET receptor that is shared by ETA receptor antagonists but not by ETB receptor selective agonists. This study analyses the properties of interaction of ET-3 with recombinant bovine ETA receptor. Apparent Kd(ET-3) values as low as 50 nM were defined from [125I]ET-1 binding experiments performed at low (5 microg/ml) protein concentrations in the assays. Larger (up to 1 microM) values were artefactually obtained in experiments performed at larger protein concentrations. The three monoiodo ET-3 derivatives were synthetized. ([125I]Y14)ET-3 did not recognize ETA receptors. ([125I]Y6)ET-3 labelled 18% of [125I]ET-1 binding sites with a Kd value of 320 pM. ([125I]Y13)ET-3 labelled 44% of [125I]ET-1 binding sites with a Kd value of 130 pM. High affinity ([125I]Y6)ET-3 and ([125I]Y13)ET-3 bindings were prevented by ET-1 (Kd = 5-7 pM), ET-3 (Kd = 70-250 pM), BQ-123 (Kd = 2 nM) and FR139317 (Kd = 2 nM) but not by low concentrations of 4-AlaET-1, sarafotoxin S6c or IRL1620. The three monoiodo ET-3 derivatives bound to recombinant rat ETB receptors with a pM affinity. The results suggest that ET-3, ([125I]Y6)ET-3 and ([125I]Y13)ET-3 should not be considered as ETB receptor specific ligands.     

Synthesis and evaluation of a radioiodinated trisaccharide derivative as a synthetic substrate for a sensitive N-acetylglucosaminyltransferase V radioassay

N-acetylglucosaminyltransferase V (GnT-V) is one of the most relevant glycosyltransferases to tumor invasion and metastasis. Based on previous findings of molecular recognition between GnT-V and synthetic substrates, we designed and synthesized a p-iodophenyl-derivatized trisaccharide, 2-(4-iodophenyl)ethyl 6-O-[2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-d-mannopyranosyl]-β-D-glucopyranoside (IPGMG, 1) and its radiolabeled form, [(125)I]IPGMG ([(125)I]1), for use in assays of GnT-V activity in vitro. The tributyltin derivative, 2-[4-(n-tributylstannyl)phenyl]ethyl 6-O-[2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl]-2,3,4-tri-O-acetyl-β-D-glucopyranoside (21), was synthesized as a precursor for the preparation of [(125)I]1. The iododestannylation of 21 using hydrogen peroxide as an oxidant followed by deacetylation yielded [(125)I]1. When [(125)I]1 was incubated in GnT-V-expressing cells with a UDP-GlcNAc donor, the production of β1-6GlcNAc-bearing IPGMG (IPGGMG, 2) was confirmed by radio-HPLC. In kinetic analysis, 1 was found to be a good substrate with a K(m) of 23.7 μM and a V(max) of 159 pmol/h. μg protein. [(125)I]1 would therefore be a useful synthetic substrate for the quantitative determination of GnT-V activity.     

The crystal structure of isopropyl 1-thio-beta-D-galactopyranoside monohydrate at 123 K

The crystal structure of isopropyl 1-thio-beta-D-galactopyranoside monohydrate is orthorhombic, P2(1)2(1)2(1), Z = 4, with cell dimensions at 123 K [293 K] of a = 7.983(1) [8.037(1)], b = 24.574(5) [24.709(4)], c = 6.329(1) [6.3736(8)] A, V = 1241.84 [1265.71] A3. The calculated and measured density is Dx = 1.371 [1.345] g cm-3, Dm = [1.340] g cm-3. Diffraction data were obtained with CuK alpha radiation and a Nonius CAD-4 diffractometer. The structure was solved by using MULTAN, and refined to R(F2) = 0.051, RW(F2) = 0.078, R(F) = 0.029, S = 1.16 for 1502 reflections. The molecule has the 4C1(D) conformation. The orientation of the primary alcohol group is gauche/trans, and that about the glycosidic C-S bond is (-)synclinal relative to the ring C-O bond. Although this compound does not form thermotropic liquid crystals, it has two crystal-to-crystal phase-transitions, at 70 and 104 degrees, prior to melting at 126 degrees. The crystal structure has a characteristic, amphiphilic, head-to-head bilayer molecular packing, with intercalated alkyl groups. The water molecule is included in the hydrogen-bond structure that links the galactoside moieties.     

Pharmacological evaluation of (+)-2- [123I]A-69024: a radioligand for in vivo studies of dopamine D1 receptors

(+)-2-[123I] A-69024, [(+)-1-(2-[123I] iodo-4,5-dimethoxy-benzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline], is a specific and enantioselective dopamine D1 receptor radioligand. The in vivo biodistribution of this radioligand in rats showed high brain uptake and a distribution consistent with the density of dopamine D1 receptors. Highest uptake was observed in the striatum (0.65 %ID/g) at 5 min followed by clearance. As a measure of specificity the striatum/cerebellar ratio reached a maximum of 3.9 at 30 min post-injection. Radioactivity in the striatum was reduced by 68% by pre-administration of the D1 antagonist SCH 23390. Pre-administration of other dopamine binding drugs, spiperone (D2), 7-OH-DPAT (D3), and clozapine (D4) displayed no inhibitory effect on (+)-2-[123I]A-69024 accumulation in any brain region. Ketanserin (5-HT2/5-HT2C) and haloperidol (D2 receptor antagonist/sigma receptor ligand) also displayed no inhibitory effect in any brain region studied. With the pharmacologically inactive enantiomer, (-)-2-[123I]A-69024, the brain uptake was determined to be non-specific since a striatum/cerebellar ratio of near 1 was observed throughout the time course of the experiment. (+)-2-[123I]A-69024 displays enantioselectivity for dopamine D1 receptors and may deserve further investigation as a possible SPECT radioligand.     

Synthesis of the 7alpha-cyano-(17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones: potential radioligands for androgen and progesterone receptors

We report the preparation of the 7alpha-cyano derivative of the isomeric (17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones (IVNT) together with their binding affinity for the androgen receptor (AR) and their biodistribution in two different animal models. The cyano group was introduced at the 7alpha-position by hydrocyanation of 4,6-estradien-17beta-ol-3-one with diethylaluminum cyanide. Selective protection of the A-ring enone system as the dienol ether followed by ethynylation and deprotection under base and acid hydrolysis condition gave 7alpha-cyano-17alpha-ethynyl-19-nortestosterone. The stannyl derivatives were prepared by addition of tri-n-butylstannyl hydride and converted stereospecifically to the corresponding [125I]iodovinyl analog using [125I]NaI and H2O2. The [125I]iodovinylsteroids were intravenously administered to male rats and estrogen-primed immature female rats and tissue uptake was measured up to 6h post-injection. Co-administration of NLP-004 or ORG-2058, highly selective ligands for the progesterone receptor, to the female rats did not affect uterus uptake of the 125I-ligands. However co-injection of testosterone to DES-primed male rats induced a marked increase in prostate uptake of the 20Z-isomer of 7alpha-cyano-[125I]-IVNT. The relative binding affinity (RBA) of either 7alpha-cyano-(17alpha,20E/Z)-IVNT isomer for the AR is low (RBA=4 and 3, respectively, versus 100 for 5alpha-dihydrotestosterone (DHT)), suggesting the absence of a possible role of the AR in the localization process. These findings contrast previously reported data for the analogous 7alpha-methyl-[125I]-IVNT where co-administration of testosterone was shown to result in a 50% drop in prostate uptake. These data indicate that the addition of an electron withdrawing 7alpha-cyano group to 123I-labeled nortestosterone derivatives does not improve their potential to serve as SPECT agents for the imaging of AR densities in the prostate.     

Meta-iodobenzylguanidine derivatives containing a second guanidine moiety

Radioiodinated meta-iodobenzylguanidine (MIBG) is used in the diagnosis and therapy of various neuroendocrine tumors. To investigate whether an additional guanidine function in the structure of MIBG will yield analogues that may potentially enhance tumor-to-target ratios, two derivatives-one with a guanidine moiety and another with a guanidinomethyl group at the 4-position of MIBG-were prepared. In the absence of any uptake-1 inhibiting conditions, the uptake of 4-guanidinomethyl-3-[(131)I]iodobenzylguanidine ([(131)I]GMIBG) by SK-N-SH cells in vitro was 1.7+/-0.1% of input counts, compared to a value of 40.3+/-1.4% for [(125)I[MIBG suggesting that guanidinomethyl group at the 4-position negated the biological properties of MIBG. On the other hand, 4-guanidino-3-[(131)I]iodobenzylguanidine ([(131)I]GIBG) had an uptake (5.6+/-0.3%) that was 12-13% that of [(125)I]MIBG (46.1+/-2.7%), and the ratio of uptake by control over DMI-treated (nonspecific) cultures was higher for [(131)I]GIBG (20.9+/-0.3) than [(125)I]MIBG itself (15.0+/-2.7). The exocytosis of [(131)I]GIBG and [(125)I]MIBG from SK-N-SH cells was similar. The uptake of [(131)I]GIBG in the mouse target tissues, heart and adrenals, as well as in a number of other tissues was about half that of [(125)I]MIBG. These results suggest that substitution of guanidine functions, especially a guanidinomethyl group, in MIBG structure may not be advantageous.     

Inhibitory effects of calcium channel blockers on thyroid hormone uptake in neonatal rat cardiomyocytes

The effects of the Ca2+ channel blockers verapamil, nifedipine, and diltiazem on triiodothyronine (T3) and thyroxine (T4) uptake were tested in cultured cardiomyocytes from 2-day-old rats. Experiments were performed at 37 degrees C in medium with 0.5% BSA for [125I]T3 (100 pM) or 0.1% BSA for [125I]T4 (350 pM). The 15-min uptake of [125I]T3 was 0.124 +/- 0.013 fmol/pM free T3 (n = 6); [125I]T4 uptake was 0.032 +/- 0.003 fmol/pM free T4 (n = 12). Neither T3 nor T4 uptake was affected by 1% DMSO (diluent for nifedipine and verapamil). Uptake of [125I]T3 but not of [125I]T4 was dose dependently reduced by incubation with 1-100 microM verapamil (49-87%, P < 0.05) or nifedipine (53-81%, P < 0.05). The relative decline in [125I]T3 uptake after 4 h of incubation with 10 microM verapamil or nifedipine was less than after 15 min or 1 h, indicating that the major inhibitory effect of the Ca2+ channel blockers occurred at the level of the plasma membrane. The reduction of nuclear [125I]T3 binding by 10 microM verapamil or nifedipine was proportional to the reduction of cellular [125I]T3 uptake. Diltiazem (1-100 microM) had no dose-dependent effect on [125I]T3 uptake but reduced [125I]T4 uptake by 45% (P < 0.05) at each concentration tested. Neither the presence of 20 mM K+ nor the presence of low Ca2+ in the medium affected [125I]T3 uptake. In conclusion, the inhibitory effects of Ca2+ channel blockers on T3 uptake in cardiomyocytes are not secondary to their effects on Ca2+ influx but, rather, reflect interference with the putative T3 carrier in the plasma membrane.     

In vitro binding properties and autoradiographic imaging of 3-iodobenzamide ([125I]-IBZM): a potential imaging ligand for D-2 dopamine receptors in SPECT

The in vitro binding properties of the [125I] labeled benzamide (S(-)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-hydroxy-3-iodo-6-methoxy- benzamide, IBZM) were determined in bovine and mouse caudate membrane homogenates and by autoradiography of mouse brain slices. [125I]-IBZM binding is saturable and reversible with a Bmax of 373 +/- 51 fmol/mg protein and a Kd of 3.1 +/- 0.62 nM (mean +/- SD, Scatchard analyses) and 0.56 nM as calculated by association and dissociation time constants. In competition experiments, Ki values for the D-2 antagonists YM-09151-2 and spiperone are 4 orders of magnitude lower than the Ki value for the D-1 antagonist SCH-23390 and S(-)-IBZM is ten-fold more potent than R(+)-IBZM. [125I]-IBZM has a low affinity for serotonin S-2 and for alpha receptors. Therefore, it is a highly selective ligand for dopamine D-2 receptors. Autoradiographic images of brain sections incubated with [125I]-IBZM show the dopamine D-2 receptors of the striatum, nucleus accumbens and olfactory tubercle with a high ratio of specific to nonspecific binding. Thus, S(-)-IBZM, when labeled with [123I], may be useful for in vivo imaging of dopamine D-2 receptors by single photon emission computerized tomography (SPECT).     

Diazontized (125I) diiodosulafanilic acid as a label for cell surface membranes. Studies on erythrocytes.

1. Diazotized 2,6-diiodosulfanilic acid (DDISA) appears to have properties suitable to serve as an artificial, non-penetrating label of cell surface membranes. Therefore, the conditions for selective labeling of cell surface membranes as compared to intracellular proteins as well as a method for its chemical determination were explored in the present study. 2. DDISA reacts with alpha-naphthol at neutral pH to produce a compound (1-hydroxy-4-(2,6-diiodo-4-sulfo-1-phenylazo-(naphthylene)), DSPN) with a characteristic spectrum in the visible range (Amax 430 nm). The absorbance of the reaction product, DSPN, is linearly proportional to the concentration of DDISA and can be used as a method for the colorimetric determination of DDISA. Reaction of DDISA with a molar excess of alpha-naphthol was also used as a method for inactivating unreacted DDISA to terminate labeling prior to cell fractionation. 3. [125I]DDISA reacts avidly with a variety of basic, neutral and acidic proteins as well as with cell membranes to form an acid-stable covalent azo linkage. 4. Effectiveness of labeling of the surface membrane of intact erythrocytes after incubation with [125I]DDISA was assessed by th ratio of 125I incorporated into membrane proteins compared to intracellular proteins. When intact erythrocytes were exposed to [125I]DDISA, the optimal labeling of membranes occurred at 37 degrees C after 20 min of incubation time and at a concentration of 10(-4) M [125I]DDISA in the incubation media. Under these conditions the ratio of the specific activity (cpm 125I/mg protein) of the membrane fraction to the specific activity of the soluble protein fraction (membrane/supernatant ratio) was greater than 500. When incubations were conducted at 4 degrees C this ratio was less than 50. However, when osmotically lysed erythrocytes were incubated with [125I]DDISA the majority of the label reacted with the soluble protein fraction resulting in a membrane/supernatant ratio of 0.14. 5. The results thus suggest that [125I]DDISA used under the appropriate incubation conditions, including the inactivation and removal of [125I]DDISA by washing with alpha-naphthol, can serve as a highly selective membrane label with minimal incorporation into intracellular soluble proteins. The general applicability of this method for other cell types remains to be explored.     

Evaluation of chemical, physical, and biologic properties of tumor-targeting radioiodinated quinazolinone derivative

Our group is developing a novel technology, enzyme-mediated cancer imaging and therapy (EMCIT), that aims to entrap radioiodinated compounds within solid tumors for noninvasive tumor detection and therapy. In this approach, a water-soluble, radioiodinated prodrug is hydrolyzed in vivo to a highly water-insoluble compound by an enzyme overexpressed extracellularly by tumor cells. We have synthesized and characterized the water-soluble prodrug, 2-(2'-phosphoryloxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]5, which is readily hydrolyzed by alkaline phosphatase, an enzyme expressed by many tumor cell lines, to a water-insoluble drug, 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]1. In the course of our study, we discovered that ammonium 2-(2'-phosphoryloxyphenyl)-6-tributylstannyl-4-(3H)-quinazolinone, an intermediate in the radioiodination of the prodrug, exists as two isomers (3 and 4) whose radioiodination leads, respectively, to [(125)I]6 and [(125)I]5. These prodrugs have different in vitro and in vivo biologic activities. Compound 6 is not hydrolyzed by alkaline phosphatase (ALP), whereas 5 is highly soluble (mg/mL) in aqueous solution and is rapidly dephosphorylated in the presence of ALP to 1, a water-insoluble molecule (ng/mL). Mouse biodistribution studies indicate that [(125)I]6 has high uptake in kidney and liver and [(125)I]5 has very low uptake in all normal organs. Compounds 3 and 6 are converted, respectively, to 4 and 5 after incubation in DMSO. The stability of 5 in human serum is high. The minimum ALP concentration needed to hydrolyze 5 is much greater than the ALP level in the blood of patients with cancer, and the latter should not affect the pharmacokinetics of the compound. Incubation of 5 with viable human and mouse tumor-cell lines--but not with normal human cells and mouse tissues--leads to its hydrolysis and the formation of large crystals of 1. We expect that 5 will also be hydrolyzed in vivo by tumor cells that express phosphatase activity extracellularly and anticipate the specific precipitation of radioiodinated 1 within tumor cell clusters. This should lead to high tumor-to-normal-tissue ratios and enable imaging (SPECT/PET) and radionuclide therapy of solid tumors.     

Synthesis and biological properties of 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl- and 16alpha-[125I]iodo-estradiols

The synthesis, receptor binding affinity, estrogenic potency and tissue distribution of the 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl-(CIVE) and 16alpha-[125I]iodo-estradiols (CIE) are reported. The iodovinyl derivatives were prepared via the (17alpha,20E/Z)-tri-n-butylstannyl intermediates, derived from the addition of tri-n-butyl tin hydride to the 17alpha-ethynyl group of the 7alpha-cyano-17alpha-ethynylestradiol, using triethylborane as a catalyst. The no-carrier-added [125I]-CIVE isomers were prepared via the same stereospecific reaction. [125I]-CIE was prepared from 7alpha-cyano-16beta-bromoestradiol via halogen exchange with Na125I. Addition of the 7alpha-cyano group to 16alpha-iodoestradiol did not affect estrogen receptor binding affinity (RBA of CIE is 115). However the estrogenic potential of CIE, as measured by the capacity to stimulate the expression of the pS2 gene, was reduced to 1% as compared to that of estradiol. Addition of a 7alpha-cyano group to the (17alpha,20E/Z)-IVE isomers reduced the RBA to 21 and 36, respectively, while the estrogenic potential was reduced to 2-3% of that of estradiol. Uterus uptake in immature rats of the 125I-labeled CIVE 20E-isomer and the 16alpha-iodo CIE peaked at 0.5h post injection while the (17alpha,20Z)-CIVE isomer showed a maximum only past 5h post injection. Uptake of all three 125I-labeled 7alpha-cyanoestrogens was suppressed by the co-injection of non-radioactive estradiol confirming the role of estrogen receptors in the localization process. Uterus retention pattern differ substantially from those of the analogues 7alpha-methylestrogens, which were previously shown to give high maximum 125I-uptake values at 2h post injection. Overall our data indicate that addition of a 7alpha-cyano group to 123I-labeled estrogens does not improve their potential to serve as SPECT agents for the imaging of estrogen receptor densities in breast cancer.     

Characterization of the AT(4) receptor in a human neuroblastoma cell line (SK-N-MC)

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II (Ang II), binds to a distinct receptor designated the AT(4) receptor. The peptide elicits a range of vascular and central actions including facilitation of memory retention and retrieval in several learning paradigms. The aim of this study was to characterize the AT(4) receptor in a human cell line of neural origin. Receptor binding studies indicate that the human neuroblastoma cell line SK-N-MC cells express a high-affinity Ang IV binding site with a pharmacological profile similar to the AT(4) receptor: (125)I]-Ang IV and (125)I]-Nle(1)-Ang IV bind specifically to the SK-N-MC cell membranes (K(d) = 0.6 and 0.1 nM) in a saturable manner (B(max) = 1.2 pmol/mg of protein). AT(4) receptor ligands, Nle(1)-Ang IV, Ang IV and LVV-haemorphin 7 (LVV-H7), compete for the binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to the SK-N-MC cell membranes with rank order potencies of Nle(1)-Ang IV > Ang IV > LVV-H7 with IC(50) values of 1.4, 8.7 and 59 nM ([(125)I]-Ang IV) and 1.8, 20 and 168 nM ([(125)I]-Nle(1)-Ang IV), respectively. The binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to SK-N-MC cell membranes was not affected by the presence of GTP gamma S. Both Ang IV and LVV-H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT(4) receptor in the SK-N-MC cells is a 180-kDa glycoprotein; under non-reducing conditions a 250-kDa band was also observed. In summary, the human neuroblastoma cell line, SK-N-MC, expresses functional AT(4) receptors that are responsive to Ang IV and LVV-H7, as indicated by an increase in DNA synthesis. This is the first human cell line of neural origin shown to express the AT(4) receptor.     

Synthesis of radioiodine labeled dibenzyl disulfide for evaluation of tumor cell uptake

Benzyl 4-halobenzyl and ally benzyl disulfide were synthesized as diallyl disulfide analogues and their tumor growth inhibitory effects on the cancer cells (SNU C5 and MCF-7) were comparable to that of diallyl disulfide, indicating that the disulfide functional group was responsible for the tumor growth inhibitory effects. Cu(I)-assisted radioiodination of benzyl 4-bromobenzyl disulfide gave benzyl 4-[123I/125I]iodobenzyl disulfide in 30-40% radiochemical yield. The radiolabeled disulfide was taken up by the cancer cells in a time-dependent manner, and the uptake was inhibited by the pretreatment of S-methyl methanethiosulfonate (MMTS), phorone and diallyl disulfide. This study suggested that the radiolabeled dibenzyl disulfide was taken up by the cancer cells via thiol-disulfide exchange and retained inside the cells.     

Molecular characteristics and evidence for internalization of vasoactive-intestinal-peptide (VIP) receptors in the tumoral rat-pancreatic acinar cell line AR 4-2 J

1. Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas [125I]Iodo-VIP binding to cell membranes showed the following IC50 values for unlabeled peptides: VIP, 0.3 nM; peptide His-IleNH2, 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [125I]iodo-VIP binding to intact cells reached steady state after 5 min at 37 degrees C, while the sequestration-internalization of the [125I]iodo-VIP-receptor complex (tested by cold acid washing) increased progressively, reaching 75% of total binding after 1 h. This phenomenon was blocked at 4 degrees C. Further data with dexamethasone, tunicamycin, cycloheximide, low temperature, and/or phenylarsine oxide, suggested a half-life of 2 days for VIP receptors and the necessity of N-glycosylation for proper translocation. 2. For chemical [125I]iodo-VIP cross-linking bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone gave the best yield when compared with five other bifunctional reagents. In membranes, the main specifically cross-linked peptide had Mr 66,000 under nonreducing conditions, and migrated with lower velocity (-5%) under reducing conditions. Cross-linking was suppressed by VIP, peptide His-IleNH2 and helodermin (competitively) and also by GTP. In intact cells, the Mr of [125I]iodo-VIP-cross-linked peptides depended on the mode of cell solubilization. After direct solubilization, the major cross-linked radioactivity migrated as a smear of Mr 130,000-180,000 but an Mr-66,000 peptide was also detectable. In contrast, the solubilization of cross-linked cells detached by mild trypsinisation gave mainly the Mr-66,000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high-Mr complex and that mild cell treatment was sufficient to disrupt this complex.     

Photoaffinity labeling of dopamine D1 receptors

A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.     

Different distribution of endothelin receptor subtypes in pulmonary tissues revealed by the novel selective ligands BQ-123 and [Ala1,3,11,15]ET-1.

We have demonstrated the different distribution of two distinct endothelin (ET) receptor subtypes in porcine pulmonary tissues using a radioligand binding assay. The clear differentiation of the subtypes was made possible by the discovery of two compounds, BQ-123 and [Ala1,3,11,15]ET-1 (4AlaET-1), that are highly selective for ETA and ETB receptors, respectively. In the bronchus and lung parenchyma, BQ-123 inhibited 65% and 30% of [125I]ET-1 binding on the sensitive sites, while 4AlaET-1 displaced 25% and 60%, respectively. The combination of the two compounds completely inhibited ET-1 binding in both tissues. An autoradiographic study of [125I]ET-1 binding using BQ-123 and 4AlaET-1 also supported the different localization of two ET receptor subtypes in pulmonary tissues. In particular, the blood vessels and bronchi are rich in ETA, but the lung parenchyma is rich in ETB.     

A novel ETA antagonist (BQ-123) inhibits endothelin-1-induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells.

The effects of a novel cyclic pentapeptide (BQ-123), an endothelin (ET) antagonist selective for the ETA receptor subtype, on phosphoinositide breakdown and DNA synthesis stimulated by ET-1 were studied in cultured rat vascular smooth muscle cells (VSMC). BQ-123 competitively inhibited the binding of [125I]ET-1 to VSMC with the apparent Ki of 4 x 10(-9) M. BQ-123 dose-dependently inhibited formation of inositol-1,4,5-trisphosphate and [3H]thymidine uptake stimulated by ET-1. These data suggest that the ET-1-induced DNA synthesis in VSMC is mainly mediated by ETA receptor subtype.     

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.