Dimitri Iakovidis

Role of β-adrenergic receptor subtypes in Lipolysis

In vitro lipolysis stimulated by low (-)-isopre-naline concentrations (≤30 nM) in epididymal white adipo-cytes from Sprague-Dawley rats was inhibited at least 60–80% by the specific β1-antagonists LK 204-545 and CGP 20712A (1 μM), suggesting that at these low (10 nM) concentrations of (-)-isoprenaline lipolysis was primarily (80%) but not solely mediated via β1-adrenergic receptors. Low concentrations (100 nM) of (-)-noradrenaline and formoterol also confirmed a role for β1-adrenergic receptors in mediating lipolysis at low concentrations of these agonists. At higher agonist concentrations, β3-adrenergic receptors were fully activated and were the dominant β-adrenergic receptor subtype mediating the maximum lipolytic response, and the maximum response was not affected by the β1-antagonists, demonstrating that the β3-receptor is capable of inducing maximum lipolysis on its own. Studies of lipolysis induced by the relatively β2-selective agonist formoterol in the presence of β1-blockade (1 μM CGP 20712A) demonstrated the inability of the β2-selective antagonist ICI 118-551 to inhibit the residual lipolysis at concentrations of ICI 118-551 ≤ 1 μM. Higher concentrations of ICI 118-551 inhibited the residual formoterol-induced lipolysis competetively, but with low affinity (∼500-fold lower than its β2-adrenergic receptor pA2, 7.80 ± 0.21), suggesting that formoterol was not acting via β2-adrenergic receptors. These data are consistent with β1-adrenergic receptors playing an important role in lipolysis at physiological but not pharmacological concentrations of catecholamines and that β2-adrenergic receptors play no obvious direct role in mediating β-adrenergic receptor agonist-induced lipolysis in vitro. Finally, racemic-SR 59230A, unlike the pure (S, S)-isomer (a β3-selective antagonist), was found to be a non-selective antagonist at the three β-adrenergic receptor subtypes, showing that the other enantiomers have different selectivity.

LK 204–545, a highly selective β1-adrenoceptor antagonist at human β-adrenoceptors

LK 204-545 ((+/-)-1-(2-(3-(2-cyano-4-(2-cyclopropyl-methoxy-ethoxy)phenoxy)-2-hydro xy-propyl-amino)-ethyl)-3-(4-hydrxy-phenyl) urea), an antagonist that possesses high beta1-/beta2-selectivity in the rat, and a range of cardio-selective and non-selective beta-adrenoceptor antagonists were examined to compare their radioligand binding affinities for human beta1-, beta2- and beta3-adrenoceptors transfected into CHO cells. LK 204-545 and CGP 20712A displayed the highest beta1-/beta2- (approximately 1800 and approximately 650, respectively) and beta1-/beta3-selectivity (approximately 17000 and approximately 2200, respectively) at human beta-adrenoceptors with LK 204-545 being approximately 2.75-fold more beta1-/beta2-selective and approximately 8-fold beta1-/beta3-selective than CGP 20712A. The high potency of LK 204-545 at transfected human beta1-adrenoceptors and in functional models of rat beta1-adrenoceptors together with its high selectivity, identify it as a useful ligand for studying beta1-adrenoceptors and suggest that it may be the preferred ligand for human beta-adrenoceptor studies.

Partial agonist activity of celiprolol.

Celiprolol given intravenously (i.v.) to pithed rats in the dose range of 0.1–10,000 μg/g produced a dose-dependent increase in heart rate (HR) which was greatest (123 beats/min) at 1,000–3,000 μg/kg. This partial agonist effect was blocked by the selective β1-adrenoceptor antagonist CGP 20712A. Celiprolol also produced a vasodepressor effect in this dose range which was abolished by the relatively selective β1-adrenoceptor antagonist ICI 118551 but not CGP 20712A. The magnitude of this intrinsic sympathomimetic activity (ISA) response was not significantly altered by reserpine pretreatment. Celiprolol also antagonised the effects of isoprenaline 0.05 μg/kg on HR and blood pressure (BP). The β1 selectivity of celiprolol as an antagonist in pithed rats (β1/β2 = 340:1) was similar to that observed in studies with isolated guinea pig atria and trachea (β1/β2 = 63:1), both being considerably greater than that observed with atenolol. Celiprolol, however, like atenolol, potentiated the bronchoconstrictor responses to histamine (3 μg/kg). Metabolic studies of rats and human urine failed to show significant amounts ++of potentially vasoactive metabolites. These data are consistent with celiprolol acting as both a β1- and β2-adrenoceptor partial agonist.

β-Adrenoceptor agonists and antagonists: conformational analysis of the ethanolamine and propanolamine side-chain

Abstract The solid state conformations of the ethanolamine or propanolamine side-chain for a variety of β-adrenoceptor agonists and antagonists have been analysed and compared to those conformations determined in solution and theoretical gas states. For the arylethanolamine (AEA)-type compounds the ethanolamine side-chain conformations adopted in the solid state were found to be independent of pharmacological action, crystallization method or substitution on the phenyl ring and arnino groups. Common conformations were observed in the solid, theoretical gas and solution states. This was also the case for the propanolamine side-chain of aryloxypropanolamine (AOPA)-type compounds. The propanolamine side-chain of oxime ether and branched alkyl ether β-adrenoceptor antagonists were also found to adopt conformations in the solid and theoretical gas states which were similar to those of the AOPA class.

Synthesis, β-adrenoceptor pharmacology and toxicology of S-(−)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride, a short acting β1-specific antagonist

The synthesis of S-(-)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride (D140S.HCl 6), a novel short acting beta(1)-specific adrenoceptor antagonist, has been described. The antagonist potency for D140S.HCl 6 has been compared with esmolol, another short acting agent, and other well known beta-adrenoceptor antagonists in isolated rat tissue preparations. The pharmacokinetics of D140S.HCl 6 in 7 day continuous intravenous infusions and 4 weeks intravenous bolus injection studies in conscious rats and dogs have been examined in toxicology studies. The effect on the isoprenaline-induced heart rate increase and the pharmacodynamic half-life of D140S.HCl 6 has been compared with esmolol in a conscious rat model. In addition, the results of a range of toxicological studies are presented. The results indicate that D140S.HCl 6 is a highly specific beta(1)-adrenoceptor antagonist (pA(2) = 8.15+/-0.22, beta(1)/beta(2) selectivity > 4400). The in vitro studies suggest D140S.HCl is ca. ten times more potent and 60 times more beta(1)-specific than racemic esmolol. Pharmacokinetic non-linearity was seen when given as a 7 day intravenous infusion at toxicological doses above 10 mg kg(-1) h(-1) in the rat and 2.5 mg kg(-1) h(-1) in the dog. Both D140S.HCl 6 and esmolol have very short durations of action after intravenous infusion in the rat (pharmacodynamic half-life is < 15 min for D140S.HCl and 10 min for esmolol). The toxicological tests indicate that D140S.HCl 6 shows no unexpected toxicity and none of the tissue irritancy problems reported for esmolol formulations.

β1- and β2-Adrenoceptor antagonist activity of a series of para-substituted N-isopropylphenoxypropanolamines

Abstract To further explore the structure-activity relationships of β-adrenoceptor (β-AR) antagonists, a series of 25 para -substituted N -isopropylphenoxy-propanolamines were synthesised, nine of which are new compounds. All have been examined for their ability to antagonise β 1 -ARs in rat atria and β 2 -ARs in rat trachea. Substitution in the para -position of the phenyl ring is thought to confer β 3 -specificity and the selectivity of these compounds for the β 1 -AR ranges from 1.5–234. None of the compounds tested were selective for the β 2 -AR. Of the 25 compounds studied, 22 had reasonable (pA 2 > 7) potencies for the rat β 1 -AR. Only compound 1 displayed reasonable (pA 2 > 7) potency for the rat β 2 -AR. Twenty two compounds were used as the training set for comparative molecular field analysis (CoMFA) of antagonist potency (pA 2 ) at the rat β 1 - and β 2 -ARs. The inclusion of a number of additional physical characteristics improved the QSAR analysis over models derived solely using the CoMFA electrostatic and steric fields. The final models predicted the β 1 - and β 2 -AR potency of the compounds in the training set with high accuracy (r 2 = 0.93 and 0.86 respectively). The final β 1 -AR model predicted the β 1 -potencies of two out of the three test compounds, not included in the training set, with residual pA 2 values 2 -AR model (residual pA 2 values

[3H]Rilmenidine-labelled imidazoline-receptor binding sites co-localize with [3H]2-(benzofuranyl)-2-imidazoline-labelled imidazoline-receptor binding sites and monoamine oxidase-B in rabbit, but not rat, kidney

The distribution and relative densities of imidazoline-receptor binding sites (I-RBS) and monoamine oxidase (MAO)-A and -B enzyme(s) in rat and rabbit kidney were compared autoradiographically using fixed nanomolar concentrations of [3H]rilmenidine and [3H]2-(benzofuranyl)-2-imidazoline ([3H]2-BFI) to label I-RBS, and [3H]RO41-1049 and [3H]RO19-6327 to label MAO-A and -B isoenzymes, respectively. In rat kidney, high densities of I-RBS labelled by [3H]rilmenidine were observed in the cortex and outer stripe (120-280 fmol/mg tissue), in contrast to low I-RBS densities labelled by [3H]2-BFI (<4 fmol/mg). A relatively high density of [3H]RO41-1049 binding to MAO-A enzyme was present in all regions of the rat kidney (160-210 fmol/mg) compared with a low density of [3H]RO19-6327 binding to MAO-B (< 25 fmol/mg). Comparison of MAO-A and -B distributions with that of [3H]rilmenidine-labelled I-RBS strongly suggests a lack of association in rat kidney. Similarly, the extremely low densities of [3H]2-BFI-labelled I2-RBS in rat kidney contrasts with the density of MAO-A, but is consistent with the low density of MAO-B. Rabbit kidney cortex and outer stripe contained high relative densities of [3H]rilmenidine-labelled I-RBS (200-215 fmol/mg) and [3H]2-BFI-labelled I2-RBS (45-60 fmol/mg) with lower densities in the inner stripe and inner medulla (< or = 100 and 30 fmol/mg respectively). A high density of MAO-A binding was observed in the inner stripe (515 fmol/mg) with lower levels in the cortex and outer stripe (100-240 fmol/mg), while high densities of MAO-B binding were observed in the cortex and outer stripe (290-450 fmol/mg) with lower levels in the inner stripe (65 fmol/mg). The correlation between the localization of [3H]rilmenidine-labelled I-RBS and [3H]RO19-6327-labelled MAO-B in rabbit kidney (r = 0.87, P = 0.057) suggest that [3H]rilmenidine may label a binding site co-existent with MAO-B, but not MAO-A (n.s.), in this tissue, but rilmenidine did not inhibit [3H]RO41-1049 or [3H]RO19-6327 binding. The distribution of [3H]2-BFI-labelled I2-RBS overlapped the combined distributions of both MAO-A and -B isoenzymes, suggesting that [3H]2-BFI may label sites on both enzymes in the rabbit, but [3H]2-BFI binding only correlated with [3H]RO19-6327 (r = 0.84, P = 0.07), not [3H]RO41-1049 binding (n.s.). Moreover, 2-BFI only inhibited [3H]RO19-6327, not [3H]RO41-1049 binding. These data are consistent with reports that I2-RBS are located on MAO-B and allosterically influence the catalytic site. The relationship of [3H]rilmenidine- and [3H]2-BFI-labelled I-RBS and the identity of non-MAO-associated [3H]rilmenidine-labelled I-RBS requires further investigation.

CoMFA analysis of the human β1-adrenoceptor binding affinity of a series of phenoxypropanolamines

A series of 36 phenoxypropanolamines was examined to determine the structure--activity relationships of beta-adrenoceptor (beta-AR) antagonists for the human beta(1)-AR. The binding affinities of all the compounds were determined for human beta(1)-ARs expressed in Chinese hamster ovary cells and the antagonist potency for rat atrial beta(1)-ARs was determined for 32 of these compounds for comparative purposes. The compounds, based upon a phenoxypropanolamine core structure with various meta-, ortho-, para- and amine-substituents, displayed binding affinities (pK(i)) for the human beta(1)-AR ranging from 5.49 to 9.35. Antagonist potencies (pA(2)) in the rat ranged from 5.52 to 9.56 and correlated with the human binding affinities (r(2)=0.86). Twenty-six compounds were used as the training set for comparative molecular field analysis (CoMFA) of antagonist binding affinity at the human beta(1)-AR and also of antagonist potency for rat atrial beta(1)-ARs. The CoMFA models were derived using both the CoMFA electrostatic and steric field parameters. The initial human beta(1)-AR model (n=26, q(2)=0.59, ONC=6, SE(CV)=0.70, r(2)=0.98, SE(non-CV)=0.16, F(6,19)=148) predicted the binding affinities of seven out of ten test compounds, not included in the training set, with residual pK(i) values less-than-or-equal0.50. The final human beta(1)-AR model (n=36, q(2)=0.66, ONC=5, SE(CV)=0.61, r(2)=0.95, SE(non-CV)=0.24, F(5,30)=107), consisting of the training set plus the test set of compounds, may prove useful in the design of new phenoxypropanolamine type beta(1)-AR antagonists. The initial rat beta(1)-AR model (n=26, q(2)=0.42, ONC=6, SE(CV)=0.76, r(2)=0.94, SE(non-CV)=0.25, F(6,19)=47) predicted the affinities of five out of six test compounds with residual pA(2) values less-than-or-equal0.50. The final rat beta(1)-AR model (i.e. training set plus test set of compounds) (n=32, q(2)=0.38, ONC=5, SE(CV)=0.69, r(2)=0.93, SE(non-CV)=0.24, F(5,26)=67) in particular has a low q(2) value, indicating that, at least for the rat, the biologically active phenoxypropanolamine conformation may be quite different to the low energy extended conformation chosen for this CoMFA study.