Tumours from a cell strain with a high content of metallothionein show enhanced resistance against cis-dichlorodiammineplatinum

Cultured cells with a high content of the extremely cysteine-rich protein metallothionein (MT) have previously been shown to exhibit resistance when exposed to otherwise lethal doses of cis-dichlorodiammineplatinum (cis-DDP), chlorambucil or prednimustine, and to have a significant proportion of intracellular drug associated with MT after such treatment. In order to study this protective mechanism in vivo, cells from a MT-rich variant of a murine fibroblast line resistant to cis-DDP and its parent sensitive line with only trace amounts of MT, were injected subcutaneously into nude mice (24 animals in each group), and tumour growth was compared during cis-DDP treatment. Animals in the treatment groups received 3 intravenous doses of either 4 mg/kg or 8 mg/kg of cis-DDP on day 12, 26 and 33 after inoculation of cells, whereas the control groups received saline. The 8 mg/kg dose produced an almost complete growth inhibition of the tumours derived from the parent cells, as well as from the MT-rich variant. However, following the injections with 4 mg/kg of cis-DDP, tumour volume was reduced by approximately 80% in tumours from the parent cells, whereas the tumours from MT-rich cells were almost completely resistant. This study provides for the first time evidence that metallothionein-conferred protection against cis-DDP toxicity also is mediated in tumours in vivo.     

Stability of cadmium resistance and metallothionein levels in vitro and in vivo

Cultured cells can be adapted to large concentrations of the toxic cadmium ion, apparently by induction of synthesis of the Cd-binding protein metallothionein (MT). One human epithelial line derived from normal skin (HE₁₀₀) and one murine fibroblast line, derived from L cells (C1 1D₁₀₀), were used to study the stability of Cd resistance, the MT levels following omission of Cd, and the inducibility of MT synthesis in cells on reexposure to Cd. In the murine cells there was no significant loss of resistance during a 4-week period either after cultivation in vitro or after growing the cells in nude mice. In the human cells a decrease (50%) in resistance was noted the first week after Cd omission. After removing Cd from the cells, a rapid decrease in MT content was demonstrated. After 3 weeks of cultivation only trace amounts were left in both cell lines. However, approximately 60% (HE₁₀₀) and 80% (C1 1D₁₀₀) of the previous levels were demonstrated after reexposure to maximum tolerable doses for 24 hr. The data indicate that the degree of stability of Cd resistance is dependent on the capacity in cells for an immediate de novo synthesis of large amounts of MT on reexposure to Cd. The animal experiments demonstrate that Cd resistance is maintained even after growing the cells in vivo.     

Mammary tumour inhibition and subacute toxicity in rats of prednimustine and of its molecular components chlorambucil and prednisolone

Prednimustine, a chlorambucil ester of pregnisolone, retarded growth of DMBA-induced mammary tumours in rats and reduced the number of tumours. A combination of chlorambucil and prednisolone (C + P) in the same proportion as in prednimustine, had similar effects, 8 and 26 mg per kg of the C + P combination being equipotnet to 16 and 64 mg per kg of prednimustine, respectively. The mortality figures suggested that prednimustine was considerably less toxic than equipotent doses of C + P. This toxicity difference was confirmed in a parallel investigation of the subacute toxicity in rats of prednimustine and C + P. This study showed that the mortality, reduction of lymphocytes and platelets, and bone marrow depression was much lower after pregnimustine than after equimolar amounts of the C + P combination. The results suggest that the low toxicity of prednimustine makes this drug a better cytostatic agent than the C + P combination treatment.     

Alterations in adenosine 3', 5'-monophosphate-binding protein in Walker carcinoma cells sensitive or resistant to alkylating agents.

The extent of binding of 8-[³H]adenosine 3′,5′-monophosphate (cyclic AMP) to specific sites was measured in Walker carcinoma cells of tissue culture lines, having different degrees of resistance to the cytotoxic effects of alkylating agents. When compared with sensitive cells, the resistant lines showed a loss of binding activity with increasing resistance, when measured at either pH 4.0 or pH 6.5. This applied to cell lines made resistant either to 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) or chlorambucil, although the total loss of binding activity was greater in the former cell lines. Scatchard analysis of binding suggested the presence of two sites in all cell lines with K_DI ～ 1–5 × 10^?9M and K_DII ～ 3 × 10^?8M. The decreased binding activity was not due to an increased cAMP phosphodiesterase activity in the resistant cell lines, since the activity of the high affinity form of this enzyme was either the same as (CB 1954-resistant) or less than (chlorambucil-resistant) that found in sensitive cells. Walker cells with acquired resistance to either CB 1954 or chlorambucil showed a degree of cross resistance to the cytostatic effect of cAMP analogues and of other alkylating agents.     

Studies on the pharmacokinetics of chlorambucil and prednimustine in man.

1 Chlorambucil (10 mg) and prednimustine (20 mg), the prednisolone ester of chlorambucil, were administered orally on separate occasions to six patients. 2 Chlorambucil was rapidly absorbed such that the parent compound was observed in the plasma 30 min after administration. 3 A preliminary comparison of chlorambucil levels following oral and intravenous administration, and after repeat oral dosage indicated that chlorambucil was well (greater than 70%) and consistently absorbed. 4 Following prednimustine no parent drug or alkylating metabolites (chlorambucil or phenyl acetic mustard) could be detected in the plasma. 5 In studies with intravenously administered chlorambucil plasma levels of the parent drug were described by a two-compartment open model with first-order kinetics. Significant levels of the cytotoxic metabolite phenyl acetic mustard were detected. 6 It is concluded that: a. the bioavailability of orally administered prednimustine is much lower than that of chlorambucil. Thus the use of prednimustine in routine combination therapy is not recommended. b. due to the lower therapeutic index of phenyl acetic mustard in experimental systems, the production of this metabolite in man may be disadvantageous. Thus research aimed at producing chlorambucil analogues, which cannot be metabolised, seems justified.     

Relationship between glutathione levels and drug or radiation sensitivities in human gastric cancer cell lines in vitro

Summary Permanent cell lines and clones established from an untreated patient (AGS cells) with gastric carcinoma, and from a similar patient who had been treated with Adriamycin, 5FU and cytoxan (SII cells) were used in a study that compared their drug and radiation survival sensitivities to their glutathidine (GSH) values. The SII parental cell line was more resistant than the AGS cells in vitro to chlorambucil, ACT D, Adria, Bleo, and X-rays. This greater resistance was positively correlated with GSH values that were 1.77 times higher than in the AGS parental cell line. By contrast the SII parental cells were more sensitive than the AGS cells to MeCCNU and Melphalan. The drug and radiation sensitives expressed among the clones of the two cell lines were heterogeneous and did not correlate with their GSH values.Abbreviations GSH Glutathione - LD₉₉ Lethal Dose to 99% of the treated population - ACT D Actinomycin D - Mel Melphalan - MeCCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea - Gal Galactitol - Bleo Bleomycin - Adria Adriamycin - 5FU 5-Fluorouracil - D₀ measure of the slope of the survival curve.     

Evaluation of SCH 39166 as PET ligand for central D1 dopamine receptor binding and occupancy in man

SCH 39166 is the first selective D₁-dopamine receptor antagonist developed for clinical trials in schizophrenia. SCH 39166 was evaluated as a radioligand for PET, labeled with¹¹C, and as a D₁-dopamine receptor antagonist after single oral doses in healthy men. After intravenous injection of [¹¹C]SCH 39166 distribution of radioactivity in brain grossly reflected D₁-dopamine receptor density. The putamen to cerebellum ratio at equilibrium was low (1.54±0.18 SD), which makes [¹¹C]SCH 39166 less suitable as a radioligand for applied PET studies. Saturability of specific binding was demonstrated after IV injection of [¹¹C]SCH 39166 with low specific radioactivity. Stereospecificity of binding was examined using the stereoisomer [¹¹C]SCH 39165. D₁-Receptor occupancy was demonstrated with [¹¹C]SCH 39166 2 h after administration of single oral doses of unlabeled SCH 39166 to each of three healthy subjects (25, 100 and 400 mg). There was a substantial reduction of specific [¹¹C]SCH 39166 uptake in the putamen after all doses. Single oral doses of 100 mg induced approximately 70% D₁-dopamine receptor occupancy in the basal ganglia, which should be sufficient to investigate the antipsychotic potential of D₁-dopamine receptor antagonism in clinical studies.     

Uptake and decomposition of chlorambucil by L5178Y lymphoblasts in vitro

The uptake of [¹⁴C]chlorambucil by L5178Y lymphoblasts was studied using thin-layer chromatography to identify the various radioactive components that enter or leave cells. Theoretical calculations predicted that entry of chlorambucil into cells by simple diffusion would be rapid and essentially complete in 45 sec or less. Uptake of intact chlorambucil was rapid, reaching a cell/medium ratio of approximately 1.5 in less than 15 sec at both 37° and 4°, consistent with a simple diffusion mechanism. In cells treated with [¹⁴C]chlorambucil for 60 min, the intracellular level of intact drug decreased with time, and this decay was attributed to hydrolysis and alkylation. The level of intact drug in the medium decreased at a similar rate resulting in a nearly constant cell/medium distribution ratio. Intact chlorambucil in the cells was found to be entirely ethanol- and trichloroacetic acid-soluble. Efflux of intact chlorambucil was very rapid and temperature-insensitive. These findings suggest that chlorambucil efflux, as well as influx, is by a simple diffusion mechanism. A derivative of chlorambucil was found in ethanol solutions of the drug. This derivative, which may be the ethyl ester of the drug, is highly concentrated in cells and may interfere with pharmacological investigations of chlorambucil.     

Optimization and synthesis of an 18F‐labeled dopamine D3 receptor ligand using [18F]fluorophenylazocarboxylic tert‐butylester

There is still no efficient fluorine‐18‐labeled dopamine D₃ subtype selective receptor ligand for studies with positron emission tomography. We aim at improving the D₃ selectivity and hydrophilicity of a candidate ligand by changing the substitution pattern to a 2,3‐dichlorophenylpiperazine and hydroxylation of the butyl chain. The compound [¹⁸F]3 exhibited D₃ affinity of K_i = 3.6 nM, increased subtype selectivity (K_i(D₂/D₃) = 60), and low affinity to 5‐HT_1A and α₁ receptors (K_i (5‐HT_1A/D₃) = 34; K_i (α₁/D₃) = 100). The two‐step radiosynthesis was optimized for analog [¹⁸F]4 by reducing the necessary concentration of the precursor amine (57 mM), which reacted with [¹⁸F]fluorophenylazocarboxylic tert‐butylester under basic conditions. The optimization of the base (Cs ₂CO₃, 23 mM) and the adjustment of reaction temperature led to the radiochemical yield of 63% after 5 min at 35°C. The optimized reaction conditions were transferred on to the synthesis of [¹⁸F]3 with an overall non‐decay corrected yield of 8‐12% in a specific activity of 32‐102 GBq/µmol after a total synthesis time of 30‐35 min. This provides a D ₃ radioligand candidate with improved attributes concerning selectivity and radiosynthesis for further preclinical studies.     

Growth inhibition of human colon adenocarcinoma-derived Caco-2 cells by 1,25-dihydroxyvitamin D3 and two synthetic analogs: relation to in vitro hypercalcemic potential

Summary The effect of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and of two synthetic analogs, 1,25S,26-tri-hydroxy-22-vitamin D₃ (1,25,26(OH)₃-22ene-D₃, Ro 23-4319) and 1,25-dihydroxy-16-23yne-vitamin D₃ (1,25(OH)₂-16ene-23yne-D₃, Ro 23-7553) on cell growth was evaluated by determination of [³H]thymidine incorporation into DNA of human colon adenocarcinomaderived Caco-2 cells. The extent of growth inhibition by the vitamin D compounds varied between 20–40% (at 10^–8 M), depending on particular growth conditions of Caco-2 cells as well as on the molecular structure of the vitamin D sterols. In confluent, i.e., rather quiescent cells, all three vitamin D compounds were equipotent in suppressing growth. In rapidly dividing log phase cells, 1,25(OH)₂-16ene-23yne-D₃ or 1,25,26(OH)₃-22ene-D₃ were ten or five times, respectively, more efficient than 1,25(OH)₂D₃. A substantial effect on induction of the colonocyte differentiation marker alkaline phosphatase was only elicited by 1,25(OH)₂-16ene-23yne-D₃.The ability of the vitamin D compounds to raise intestinal calcium absorption was evaluated by determination of ⁴⁵Ca²⁺ accumulation in embryonic chick duodenal explants. In this assay, both synthetic analogs were less effective than 1,25(OH)₂D₃ by a factor of 20.The intrinsic bone resorbing activities of the vitamin D analogs were compared in organ-cultured neonatal mouse calvariae. The most effective antiproliferative compound, 1,25(OH)₂-16ene-23yene-D₃, stimulated calcium release from cultured bones at concentrations less than 10^–11 M, and was thus ten times more potent than 1,25(OH)₂D₃ and hundred times more than 1,25,26-(OH)₃-22ene-D₃.A preliminary report of this study was presented at the International Conference on Calcium Regulating Hormones, April 24–29, 1992, Florence, ItalyCorrespondence to M. Peterlik at the above address     

Central 5-HT2A and D2 dopamine receptor occupancy after sublingual administration of ORG 5222 in healthy men

It has been suggested that a combined blockade of 5-HT_2A and D₂-dopamine receptors improves efficacy and reduces the risk for extrapyramidal symptoms when treating schizophrenic patients with antipsychotic drugs. ORG 5222 is a new potential anti-psychotic drug which has high affinity for 5-HT_2A, D₂-dopamine and α₁ adrenergic receptors in vitro. The objective of this study was to examine if ORG 5222 occupies 5-HT_2A and D₂-dopamine receptors in human subjects in vivo. Central receptor occupancy was measured by positron emission tomography (PET) in three healthy subjects after sublingual administration of 100 μg ORG 5222. [¹¹C]N-methylspiperone ([¹¹C] NMSP) was the radioligand used to measure 5-HT_2A receptor binding in the neocortex and [¹¹C]raclopride to measure D₂-dopamine receptor binding in the striatum. The 5-HT_2A occupancy was 15–30% and the D₂-dopamine receptor occupancy was 12–23%. The study confirms that ORG 5222 binds to 5-HT_2A and D₂-dopamine receptors in human brain. Since receptor occupancy of ORG 5222 is rather low, doses higher than 100 μg are suggested in future clinical trials to evaluate the antipsychotic drug effect of ORG 5222. Received: 9 September 1996 / Final version: 2 January 1997     

Buccal films of prednisolone with enhanced bioavailability

Abstract

The conventional formulation of prednisolone is considered to be low in efficacy, primarily on account of their failure in providing and maintaining effective therapeutic drug levels. This study aims to focus on development of a mucoadhesive buccal delivery system with a twofold objective of offering a rapid as well as a prolonged delivery of prednisolone coupled with enhanced therapeutic efficacy. Buccoadhesive films of prednisolone were prepared by solvent-casting method using hydroxyl propyl methyl cellulose (K100), Carbopol 940 and/or Eudragit® NE 40?D. Placebo films possessing the most desirable physicomechanical properties were selected for drug loading. The effect of polymer and its content on film properties, i.e. mucoadhesive strength, swelling and hydration, in vitro drug release was studied. Based on these studies, film F7D was selected for ex vivo permeation across porcine cheek mucosa. The steady state flux of prednisolone across the buccal mucosa was found to be 105.33?±?32.07?µg/cm²/h. A comparative pharmacokinetic study of prepared film (F7D) and oral suspension of prednisolone was conducted. In vivo data of buccal film show greater bioavailability (AUC_0–α: 24.26?±?4.06?µg.h/ml versus 10.65?±?2.15?µg.h/ml) and higher C_max (2.70?±?0.38?µg/ml versus 2.29?±?0.32?µg/ml) value when compared to oral suspension. The data observed from this study highlight the feasibility of the buccal route as a viable option for delivery of prednisolone.     

Effects of NZ-107 on Airway Inflammation and Cell Activation in Guinea-pigs

Abstract— The effects of NZ-107 on some airway inflammation models and the generation of superoxide anion (O₂^?) were studied in guinea-pigs. Airway inflammation was caused by intra-tracheal injection of murine recombinant interleukin-5 (mrIL-5, 15 μg/animal), inhalation of platelet-activating factor (PAF, 0·003%) and intra-tracheal injection of leukotriene B₄ (LTB₄, 10 μg/animal). NZ-107 (4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone) at a dose of 50 mg kg^?1, intraperitoneally reduced mrIL-5- and PAF-induced eosinophilia. This compound at a dose of 25 and 50 mg kg^?1 also suppressed LTB₄-induced eosinophilia and neutrophilia in bronchoalveolar lavage fluid (BALF). On the other hand, prednisolone at a dose of 20 mg kg^?1, i.p., prevented the increased number of macrophages, eosinophils and neutrophils induced by mrIL-5, the increased number of eosinophils induced by PAF and the increased number of eosinophils and neutrophils induced by LTB₄ in BALF. Furthermore, both drugs reduced mrIL-5- or PAF-induced increase in the number of airway epithelial cells in BALF. The generation of O₂^? was measured by the method of cytochrome C reduction. NZ-107 (10–100 μg mL^?1) attenuated PAF- and FMLP-induced O₂^? production from macrophages and reduced PAF-induced O₂^? generation by eosinophils but had no effect on that from neutrophils. These results indicate that NZ-107 prevents the increased number of pulmonary eosinophils and airway epithelial cells and the activation of macrophages and eosinophils, suggesting that NZ-107 may be useful as a remedy for airway inflammatory diseases such as bronchial asthma.     

Effect of dopamine D<Subscript>3</Subscript> receptor blockade on renal function and glomerular size in diabetic rats

Dopamine D₂-like receptors, including D₂, D₃, and D₄ receptors, are involved in the regulation of glomerular hyperfiltration due to diabetes mellitus. These hemodynamic alterations represent a risk factor for the later development of diabetic nephropathy. The aim of the present study was to determine whether the D₃ receptor subtype modulates the diabetes-induced increase in glomerular filtration rate (GFR) in rats. Renal function was studied in Sprague–Dawley rats 14 days after induction of a moderate diabetes mellitus (DM) by streptozotocin and in non-diabetic controls (CON). Rats were orally treated either with the peripherally acting, selective dopamine D₃ receptor antagonist BSF 135170 (BSF, 10 mg/kg per day for 2 weeks) or with vehicle (VHC). Perfusion-fixed kidneys were used for estimation of glomerular volume. In conscious rats, which were treated with BSF, the DM-induced increase in fluid intake, urinary output, and renal sodium excretion was significantly less pronounced than in the vehicle group (DM-VHC). In the clearance experiments, GFR in CON was about 0.84±0.04 ml/min per 100 g body weight. The DM-VHC group presented a significant glomerular hyperfiltration (1.09±0.04 ml/min per 100 g body weight). Treatment with BSF significantly lowered GFR towards levels of CON. The estimated glomerular volume was 0.73±0.03×10⁶ m³ in the CON-VHC group and 0.86±0.04×10⁶ m³ in the DM-VHC animals. Interestingly, treatment with BSF decreased the glomerular volume in both groups. Irrespective of BSF treatment, kidney wet weight related to body weight was about 36% higher in DM animals compared with CON animals. We conclude that dopamine D₃ receptors represent a target for the modulation of diabetes-induced glomerular hyperfiltration. Therefore, the results encourage the testing of the possible beneficial effects of long-term D₃ receptor blockade on the development of diabetic nephropathy.     

3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes

Summary The binding properties of 3- and 4-O-sulfoconjugated dopamine (DA-3-0-S, DA-4-0-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D₂ receptors were investigated. ³H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D₂ and serotoninergic 5HT₂ receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT₂ and D₂ receptors, respectively, in order to discriminate between ³H-spiperone binding to D₂ and to 5HT₂ receptors. Under our particular membrane preparation and assay conditions, ³H-spiperone binds to D₂ and 5HT₂ receptors with a maximal binding capacity (B _max) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants K _D (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (K _D = 24 nmol/l) and 80% low (K _D = 420 nmol/l) affinity binding sites corresponding to 5HT₂ and D₂ receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mol/l ketanserin to mask the 5HT₂ receptors. DA competition curves were best fitted assuming two binding sites, with high (K _H = 0.12 mol/l) and low (K_L = 18 mol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (K _D = 2.8 mol/l). Competition experiments with various compounds confirmed the binding of ³H-spiperone to D₂ receptors. DA-3-O-S, DA-4-O-S, and MT were more than 5,000-, more than 10,000-, and 530-fold less potent in competing for ³H-spiperone binding when compared with DA at the high affinity binding site which mediates biological effects. Therefore, it is concluded that these DA metabolites are biologically ineffective at central D₂ receptors. Send offprint requests to E. Werle at the above address     

Sequence specificity for DNA interstrand cross-linking induced by anticancer drug chlorambucil

Chlorambucil is known to alkylate primarily N7 of guanine and N3 of adenine to induce DNA monofunctional adducts and interstrand cross-links (ISC). We have investigated the sequence specificity for DNA ISC induced by chlorambucil using duplex oligomers containing a difined cross-linkable sequences 5′-A^*TT, 5′-G^*TT, or 5′-G^*CC in which asterisk indicates the potential cross-linking site and underlined base indicates the potential cross-linking site on the opposite strand. An analysis of 20% denaturing polyacrylamide gel electrophoresis showed that chlorambucil was able to induce DNA ISC in the duplex oligomers containing a sequence 5′-GCC. The formation of DNA ISC was not observed in the duplex oligomers containing sequences 5′-ATT or 5′-GTT. These results indicate that chlorambucil induces guanineguanine DNA ISC but not guanine-adenine or adenine-adenine DNA ISC. In addition, we have tested the ability of chlorambucil to induce DNA ISC within 5′-GNNC or 5′-GC sequences using duplex oligomers containing the sequence 5′-G⁴G³G²C. The result of DNA strand cleavage assay showed that DNA ISC was formed at the 5′-GGC sequence (an 1,3 cross-link, G₁-G₃) but not at 5′-GGGC (an 1,4 cross-link, G₁-G₄) or 5′-GC sequence (an 1,2 cross-link, G₁-G₂).     

Activation of recombinant h 5-HT1B and h 5-HT1D receptors stably expressed in C6 glioma cells produces increases in Ca2+-dependent K+ current

The putative coupling between stably expressed recombinant h 5-HT_1B or h 5-HT_1D receptors and K⁺ channels which regulate excitability was investigated in C6 glioma cells. Outward K⁺ currents (I _K) were examined in non-transfected C6 glioma cells and in cells expressing cloned h 5-HT_1B or h 5-HT_1D receptors using the patch-clamp technique in the whole-cell configuration. I _K was elicited by a depolarizing step from a holding potential of –60 mV. In C6 glioma cells expressing either recombinant h 5-HT_1B or h 5-HT_1D receptors, sumatriptan similarly increased I _K in a concentration-dependent manner (maximum increase 19.4±7.2%, n=8, P<0.05 and 25.1±3.9%, n=6, P<0.001, respectively) with EC₅₀ values (geometric mean with 95% confidence intervals in parentheses) of 56.3 nM (7.9–140 nM) and 68.7 nM (16–120 nM), respectively. Sumatriptan failed to elicit increases in I _K in non-transfected cells, confirming a specific involvement of the respective membrane h 5-HT_1B and h 5-HT_1D receptors in transfected C6 cells. In the presence of the mixed 5-HT_1B/D receptor antagonist GR 127935 (0.1 μM), sumatriptan (1 μM) failed to significantly increase I _K in C6 cells expressing h 5-HT_1B receptors (–7.5±3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 μM) was required to significantly inhibit sumatriptan-evoked increases in I _K in C6 cells expressing h 5-HT_1D receptors (–1.8±3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-HT_1B and h 5-HT_1D receptors, respectively. In C6 cells expressing either cloned h 5-HT_1B or h 5-HT_1D receptors, sumatriptan-induced increases in I _K were prevented by the calcium chelator EGTA (5 mM) when included in the patch pipette (maximum increase 0.57±0.6%, n=3, P=NS and –2.8±1.6%, n=5, P=NS, respectively). In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _K in the presence of dibutyryl cAMP (10 μM) or when a nominally Ca²⁺-free medium was included in the patch pipette (–19.4±5.1%, n=5 and –5.2±4.3%, n=5, respectively, P=NS in each case). In addition, the Ca²⁺-dependent K⁺ channel blockers iberiotoxin (0.1 μM) and tetraethylammonium (TEA, 1 mM) abolished sumatriptan-induced increases in I _K (–0.5±1.0%, n=4 and –3.9±3.1%, n=4, respectively, P=NS in each case) in C6 cells expressing h 5-HT_1B receptors, confirming the involvement of Ca²⁺-dependent K⁺ channels. In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _k after 30-min incubation with thapsigargin (1 μM) or when heparin (2 mg/ml) was included in the patch pipette (1.1±0.4%, n=5 and 1.2±2.4%, n=5, respectively, P=NS). In conclusion, evidence is provided that both recombinant h 5-HT_1B and h 5-HT_1D receptors stably transfected in C6 glioma cells are positively coupled to Ca²⁺-dependent K⁺ channels, and the outward hyperpolarizing current mediated by these channels is dependent upon IP₃ receptor-mediated intracellular Ca²⁺ release. Received: 15 April 1998 / Accepted: 9 September 1998     

Syntheses and radiofluorination of two derivatives of 5‐cyano‐indole as selective ligands for the dopamine subtype‐4 receptor

Two fluoroethoxy substituted derivatives, namely 2‐[4‐(2‐(2‐fluoroethoxy)phenyl)‐piperazin‐1‐ylmethyl]indole‐5‐carbonitrile ( 5a ) and 2‐[4‐(4‐(2‐fluoroethoxy)‐phenyl)piperazin‐1‐ylmethyl]indole‐5‐carbonitrile ( 5b ) were synthesized as analogs of the selective D₄ receptor ligand 2‐[4‐(4‐fluorophenyl)piperazin‐1‐ylmethyl]indole‐5‐carbonitrile (FAUC 316). In vitro characterization using CHO‐cells expressing different dopamine receptor subtypes gave K_i values of 2.1 ( 5a ) and 9.9 nM ( 5b ) for the dopamine D₄ subtype and displayed a 420‐fold D₄‐selectivity over D₂ receptors for 5b . The para‐fluoroethoxy substituted candidate 5b revealed substantially reduced α₁ and serotoninergic binding affinities in comparison to the ortho‐fluoroethoxy substituted compound. In order to provide potential positron emission tomography (PET) imaging probes for the dopamine D₄ receptor, ¹⁸F‐labelling conditions using [¹⁸F]fluoroethyl tosylate were optimized and led to radiochemical yields of 81 ± 5% ( [¹⁸F]5a ) and 47 ± 4% ( [¹⁸F]5b ) (n = 3, decay‐corrected and referred to labelling agent), respectively. Thus, ¹⁸F‐fluoroethylation favourably at the para position of the phenylpiperazine moiety of the 5‐cyano‐indole framework proved to be tolerated by D₄ receptors and could also be applied to alternative scaffolds in order to develop D₄ radioligand candidates for PET with improved D₄ receptor affinity and selectivity. Copyright © 2005 John Wiley & Sons, Ltd.     

Comparison of human α1-adrenoceptor subtype coupling to protein kinase C activation and related signalling pathways

The coupling of human α₁-adrenoceptor subtypes to protein kinase C (PKC) and PKC-related signalling events were investigated in rat-1 fibroblasts stably expressing α_1A-, α_1B- or α_1D-adrenoceptors at densities of 1328 ± 200, 5030 ± 703 and 150 ± 14 fmol/mg protein respectively. In functional assays the α₁-adrenoceptor agonist phenylephrine significantly stimulated PKC (assessed as increased activity in the membrane fraction) in cells expressing α_1A- or α_1B- but not α_1D-adrenoceptors. In immunoblot assays phorbol ester treatment enhanced membrane-associated immunoreactivity of PKCα, PKCδ and PKCɛ to a similar extent in all three cell lines. Stimulation of α_1A- and α_1B-adrenoceptors also increased immunoreactivity of PKCα, PKCδ and PKCɛ in the membrane fraction, while α_1D-adrenoceptor stimulation yielded only very small and inconsistent alterations. Immunoreactivity of PKCζ was not consistently affected by phorbol ester or phenylephrine in any of the cell lines. Stimulation of all three α₁-adrenoceptors time- and concentration-dependently increased inositol phosphate formation. Maximum activation occurred with the order α_1A≈α_1B > α_1D. Phenylephrine also concentration dependently elevated free intracellular [Ca²⁺] in all three cell lines with the order of efficacy α_1A > α_1B > α_1D. In the presence of ethanol, phenylephrine stimulated phosphatidylethanol formation in α_1A- and α_1B-adrenoceptor-expressing cells time and concentration dependently but only weakly and inconsistently in α_1D-adrenoceptor-expressing cells. The efficacy of phenylephrine (100 μM) relative to that of noradrenaline (100 μM) for stimulation of phosphatidylethanol formation was similar (≥ 75%) for all three subtypes. The alkylating agent phenoxybenzamine concentration dependently reduced α_1A-adrenoceptor density and phenylephrine-stimulated Ca²⁺ elevations to levels seen with α_1D-adrenoceptors but reductions of phenylephrine-stimulated phosphatidylethanol formation were weaker. We conclude that human α_1A- and α_1B-adrenoceptors expressed in rat-1 cells couple to activation of PKCα, PKCδ and PKCɛ but not PKCζ; this may involve stimulation of phospholipases C and D and intracellular Ca²⁺ elevations. Activation of these pathways by α_1D-adrenoceptors appears to be much weaker and was not detected consistently; this was not fully explained by weak partial agonism of phenylephrine at this subtype or by lower receptor densities. Overall the α_1A-adrenoceptor may have the highest efficiency of stimulus-response coupling among human α₁-adrenoceptor subtypes. Received: 25 July 1997 / Accepted: 29 September 1997