A. Wellstein

Complex dose-response curves of atropine in man explained by different functions of M1- and M2-cholinoceptors

SummaryIn the present study we set out to explain the complex atropine dose-response curves in man in relation to M-cholinoceptor subtype occupancy. In healthy volunteers the effects of atropine on heart rate and salivary flow were quantified. M-cholinoceptor subtype occupancy by antagonist present in plasma samples was detected in an in vitro radioreceptor assay. Atropine effects were studied without and after propranolol (240 mg oral dose) and without and after pirenzepine (1.1 mg i. v.) to differentiate β-adrenoceptor and M-cholinoceptor subtype mediated effects.1.In receptor binding studies, M-cholinoceptors in bovine cerebral cortex membranes were labelled with 3H-pirenzepine (pKd = 8.05), M-cholinoceptors in rat salivary gland membranes with 3H-N-methylscopolamine (pKd = 9.02). Atropine competed for binding of these ligands with a small (2.1-fold) preferential selectivity via the cerebral in comparison to the glandular receptors (pKi = 9.18 versus 8.86). Pirenzepine showed a marked selectivity (40-fold) in this respect with pKi-values of 8.05 (M1: cerebral cortex) and 6.45 (M2: salivary glands).2.At heart rate and at salivary flow, bivalent dose-response curves of atropine were observed with opposite effect vectors. The typical antagonist effects at M-cholinoceptors (i. e. an increase of heart rate and an inhibition of salivary flow) were observed at doses > 1 μg/kg, whereas “paradoxical” cholinomimetic effects of atropine became apparent at lower doses. From a superposition of two isotherms with opposite effect vectors ED50-values were calculated, which were in the range of half-maximal M-cholinoceptor occupancy in the in vitro radioreceptor assay of plasma samples.3.The time-dependent decline of atropine effects after the highest dose (40 μg/kg) and the respective in vitro M-cholinoceptor occupancy were in good agreement with the dose-response data.4.β-Adrenoceptor blockade did not influence the pattern of the atropine dose-response relation. After pirenzepine (∼ 70% of M1-cholinoceptor occupancy), the cholinomimetic effects of atropine were abolished and only monovalent atropine dose-response curves were observed. Maximal effects of atropine were not different in comparison to the respective controls without pirenzepine.5.The standard deviation of the RR-intervals in the ECG as a measure of postsynaptic M-cholinoceptor stimulation was increased after pirenzepine by 58% and decreased after the maximum atropine dose by 85%. It is concluded, that the cholinomimetic effects observed after atropine result from its antagonism at peripheral M1-cholinoceptors. A negative feedback mechanism of acetylcholine release via M1-autoreceptors is discussed as a possible mechanism involved.

Receptor binding of propranolol is the missing link between plasma concentration kinetics and the effect-time course in man

SummaryIn a double-blind, placebo-controlled study in 6 healthy volunteers, the correlation between beta-adrenoceptor binding, the time course of the effect and plasma concentration kinetics was investigated from 0 to 48 h after a single oral dose of propranolol 240 mg. First, the in vitro beta-adrenoceptor interaction of propranolol was investigated. Propranolol inhibited beta-adrenoceptor binding to rat parotid (beta1) and reticulocyte (beta2) membranes in the presence of pooled human plasma with a Ki of about 8 ng/ml plasma. After oral administration of 240 mg propranolol, concentration kinetics in plasma could be described by a Bateman function with a fictive concentration at time 0 of 275 ng/ml plasma, and a mean elimination half-life of 3.5 h. Using the concentration kinetics of propranolol in plasma together with its in vitro beta-adrenoceptor binding characteristics in the presence of placebo plasma from each individual, the time course of antagonism against beta-adrenoceptor mediated effects was predicted. The latter was in agreement with the time course of propranolol-induced inhibition of tachycardia due to orthostasis. After bicycle ergometry, however, the time course of inhibition of tachycardia was shorter than was predicted. Plasma sampled at various times after propranolol administration inhibited beta-adrenoceptor binding of the radioligand 3H-CGP 12177 to rat reticulocyte membranes in a fashion reflecting the time course of inhibition of exercise tachycardia observed in the volunteers. A direct, linear relation was shown between the in vitro inhibition of beta-adrenoceptor binding by the plasma samples withdrawn after propranolol administration and the inhibition of exercise tachycardia observed in parallel. The results show that the concentrations of antagonist present in plasma are representative of the concentrations in the effect compartment. Deep compartments of drug distribution appear irrelevant to the effects of the drugs. The relation between the plasma concentration of propranolol and the reduction in heart rate at various levels of physical effort shows no significant inhibition at rest and increasing IC50-values from orthostasis to 2 min and to 4 min of ergometry. IC50-values after orthostasis are in the range of the Ki-values from in vitro receptor binding studies, whereas the IC50-values after exercise are shifted 2-to 3-fold to the right relative to the Ki-values. This finding is in agreement with increased beta-adrenoceptor stimulation with increasing effort (release of endogenous noradrenaline), which shifts the antagonist concentration-effect curve to the right. Furthermore, the rightward shift can explain why with increasing effort the time course of the inhibitory effect of propranolol becomes shorter. Release of propranolol from presynaptic stores during exercise is irrelevant, since this would result in opposed effects on the concentration-effect relationship (leftward shift) and the time course of antagonism (longer effect) with increasing work load. It is concluded that the receptor interaction of propranolol together with its plasma concentration kinetics can fully explain the time course of effects after a single oral dose, and so receptor interaction will be the missing link in the correlation between concentration kinetics and effect kinetics of propranolol in man. In general, this mode of correlation should be expandable to any drug exerting its effects according to the law of mass action via receptors in the extracellular space. This approach provides a rational basis for the comparison of different drugs from one group irrespective of their receptor affinity and concentration kinetics.

Properties of agonist binding at theβ-adrenoceptor of the rat reticulocyte

SummaryTo study the fundamental differences between agonist and antagonist interaction with the β-adrenoceptor of the rat reticulocyte the radiolabeled agonist3H hydroxybenzylisoprenaline (3H HBI) and the radiolabeled antagonist3H dihydroalprenolol (3H DHA) were used.Equilibrium binding experiments with3H HBI revealed all characteristics expected to a β-adrenoceptor site, i. e. high affinity binding (KDhigh=7.4±0.9×10−9 M), saturability (Bmaxhigh=230±24 fmoles/mg protein), and stereoselectivity. The rank order of potency for competing agonists was isoprenaline > adrenaline > noradrenaline > dopamine.3H HBI high affinity binding sites amounted to about 25% of β-adrenoceptor sites detectable with3H DHA.In competition experiments with3H HBI and (-)isoprenaline[(-)Ipn]aKDhigh-value for (-)Ipn of 3.1±0.6×10−8M was obtained corresponding to theKDhigh-value of (-)Ipn obtained from competition experiments using3H DHA. For (-)propranololKD-values of 0.9±0.5×10−8 M and 1.0 ±1.0×10−8 M were measured using3H HBI and3H DHA respectively.Agonist affinity derived from competition experiments with (-)Ipn versus3H DHA was not affected by temperature changes.Guanylyl-imidodiphosphate [Gpp(NH)p] decreased concentration dependently the number of high affinity binding sites of3H HBI not affecting the respectiveKD-value. Similar effects were observed after omission of Mg2+ from the binding assay or inclusion of Na+ in the Mg2+-free incubation mixture.The association reaction of3H HBI at the β-adrenoceptor revealed two different velocities. The slower phase of the association reaction which represents high affinity binding (80% of equilibrium binding) is not observed in the presence of Gpp(NH)p.A biphasic dissociation of3H HBI binding was induced by 10−4 M (±)propranolol: 25% dissociated with at1/2 of 1.3 min whereas the high affinity binding was reversed with at1/2 of 150 min. This slowly reversible binding of3H HBI however was rapidly reversed by Gpp(NH)p (t1/2<1 min).It is concluded that the agonist ligand3H HBI permits a direct qualitative and quantitative characterization of the agonist induced high affinity state of the β-adrenoceptor. In particular, the kinetic studies strongly support a two step binding model for the agonist-β-adrenoceptor interaction.

Simple and reliable radioreceptor assay for beta-adrenoceptor antagonists and active metabolites in native human plasma

SummaryA radioreceptor assay (RRA) for the assay of beta-adrenoceptor antagonists in native human plasma is described. The hydrophilic antagonist3H-CGP 12177 was used as the radioligand. In contrast to the hydrophobic radioligand3H-dihydroalprenolol, which was investigated in parallel, the beta-adrenoceptor binding of3H-CGP 12177 by rat reticulocyte membranes was found not to be affected by inclusion of increasing proportions (0–66% of incubation volume) of human plasma in the assay. Thus, solvent extraction of drug and/or active metabolites was not necessary to avoid binding of the radioligand tracer to plasma added in the RRA. The assay of unprocessed samples was possible. Drug concentrations in plasma after oral administration of propranolol (240 mg) or carteolol (30 mg) to 6 healthy volunteers were measured by the RRA and in parallel by a chemical method. The results from both methods agreed when the plasma concentration kinetics of propranolol were investigated (elimination half-life: 3.9 h). In contrast, plasma concentrations of carteolol were consistently higher according to the RRA after oral administration of the drug. Identical concentrations, however, were found by the RRA and chemical method using plasma samples spiked with carteolol. Plasma concentrations of carteolol detected by the chemical method decline monoexponentially (elimination half-life: 5.4 h). A similar half-life of elimination for parent drug was found by the RRA (5.9 h), but an additional term describing the appearance of an active metabolite was necessary to account for the biphasic drug elimination (elimination half-life of metabolite: 17.3 h). The latter result is in agreement with the appearance of 8-hydroxy-carteolol as an active metabolite, which shows similar affinity for beta-adrenoceptors as the parent drug. The active metabolite, with a 3-fold longer elimination half-life than the parent drug, will prolong the duration of the clinical effects of orally administered carteolol. In conclusion, the RRA permits the determination of beta-adrenoceptor antagonistic activity in native human plasma at concentrations as low as 0.1-fold the IC50-value of the drug or an active metabolite.

Dose-response curves of pirenzepine in man in relation to M1- and M2-cholinoceptor occupancy

SummaryThe aim of the present study was to investigate the M-cholinoceptor subtype selectivity of pirenzepine in man. In parallel with effects on the heart rate and salivary flow, M-cholinoceptor subtype occupancy by antagonist present in plasma samples was detected in radioreceptor assays. Bovine cerebral cortex membranes labelled with 3H-pirenzepine (M1) and rat salivary gland membranes labelled with 3H-N-methylscopolamine (M2) were used in these in vitro assays. A half-maximal occupancy of M1-cholinoceptors in the in vitro assay of plasma samples was detected after 0.25 mg of pirenzepine i.v. The respective half-maximal M2-cholinoceptor occupancy was observed after 10 mg. Doses < 3 mg decreased the heart rate by maximally 10.7 beats/min with an ED50 of about 0.1 mg. An increase in heart rate (relative to control values) was observed at doses > 10 mg. This bivalent dose-response relationship was also observed after β-blockade. Salivary flow tended to increase at doses < 1 mg and was half-maximally inhibited after 10 mg. Combining the in vitro and in vivo results, the typical antimuscarinic effects (tachycardia and inhibition of salivary flow) can be attributed to the blockade of M2-cholinoceptors, whereas the reduction of heart rate coincides with the blockade of the M1-subtype. With respect to the typical antimuscarinic effects, pirenzepine was 70-fold less potent than atropine; in contrast, with respect to the reduction of heart rate, pirenzepine was equipotent with atropine. It is concluded that pirenzepine does not discriminate between cardiac and salivary gland M2-cholinoceptors but shows pronounced selectivity for the M1-cholinoceptors through which it mediates the decrease in heart rate. The latter effect may be explained by inhibitory M1-auto-receptors.

Affinity and selectivity of beta-adrenoceptor antagonists in vitro

&NA; The potency order of the catecholamines (‐)isoprenaline (Iso), ( ‐ )‐noradrenaline (NA), and ( ‐ )‐adrenaline (Adr) in competition for radiolabelled sites is used for their pharmacological classification. It is shown that the radioligand 3H‐CGP 12177 exclusively labels &bgr;1‐adrenoceptors in rat salivary gland membranes (Iso > NA > Adr), and &bgr;2‐adrenoceptors in rat reticulocytes (Iso > Adr ≥ NA). These models are then used to derive the subtype‐selectivity of the classical &bgr;‐adrenoceptor antagonists (±)‐propranolol (prop; twofold &bgr;2‐selective) and (±)‐atenolol (aten; 35‐fold &bgr;1‐selective), as well as of the newer antagonists (±)betaxolol and (±)‐bisoprolol (betax and biso; 35‐fold and 75‐fold &bgr;1‐selective, respectively). The ligand with the highest selectivity is ICI 118,551 (ICI), with a 300‐fold &bgr;2‐subtype selectivity. For comparison with antagonistic effects in humans at given plasma concentrations, the equilibrium dissociation constants of the ligands are measured in the presence of native human plasma and yield values for the relative selectively labelled subtype in the mean (Ki‐values in nmol/l): prop: 20, aten: 250, biso: 24, betax: 23, and ICI: 2.5.

Agonist binding at alpha2-adrenoceptors of human platelets using 3H-UK-14,304: regulation by Gpp(NH)p and cations

SummaryThe agonist/α2-adrenoceptor interactions at human platelet membranes have been examined in radioligand binding studies with the full agonist ligand 3H-UK-14,304 [5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline] and the antagonist ligand 3H-yohimbine. From association kinetics of different concentrations of 3H-UK-14,304 (0.75−8.1 nmol/l) a KD-value of 2.37 nmol/l in agreement with the high-affinity KD-value (KDH = 1.60 ± 0.15 nmol/1) obtained from equilibrium binding studies was derived. In the presence of Gpp(NH)p about 6% of specific radioligand binding was observed in the association reaction. Addition of Gpp(NH)p at equilibrium resulted in a rapid loss (t1/2 < 1 min) of ≈80% of bound radioligand. Dissociation after addition of an excess of phentolamine (10 μmol/l) showed a biphasic time course independent of the radioligand concentration with the proportions of /15 of rapidly (t/12 < 2 min) and /45 of slowly dissociating ligand (k−1 = 0.033±0.004 min−1). Application of a sequential binding model resulted in KD-values from this approach also in agreement with KDH from equilibrium binding studies. The rank order of potency for different agonists and antagonists to compete for binding with 3H-UK-14,304 indicated an α2-adrenoceptor interaction: (−)adrenaline > clonidine > (−)noradrenaline > (−)isoprenaline and yohimbine = rauwolscine > phentolamine > prazosin >- corynanthine > timolol respectively. The analysis of competition isotherms of UK-14,304 versus 3H-yohimbine (Hill-coefficient = 0.59 ± 0.03) showed that the agonist binds to two affinity states of the α2-adrenoceptor, with high (KDH = 1.77 ± 0.50 nmol/l) and low affinity (KDL = 71.2 ± 11.6 nmol/l) respectively. From these experiments a fraction of 56.9%±2.1% of the total number of α2-adrenoceptors (Bmax = 198.4 ± 8.0 fmol/mg of protein) in the high-affinity state was calculated. Similar results were obtained from 3H-UK-14,304 saturation isotherms according to a two-state binding model (KDH = 1.60±0.15 nmol/l; KDL = 66.2±10.7 nmol/l; BmaxH = 57.6% ± 2.3%). Adrenoceptor agonists competed for specific binding of 3H-UK 14,304 and 3H-yohimbine in a manner that suggests that the 3H-UK-14,304 (∼3.5 nmol/l) labeled sites represent predominantly the agonist induced or stabilized high-affinity state of the α2-adrenoceptor. Adrenoceptor antagonists had equal affinities irrespective of the receptor states labeled by the agonist or antagonist radioligand. A loss of the high-affinity binding capacity (BmaxH) of the agonist due to the presence of Gpp(NH)p was delineated from 3H-UK-14,304 saturation isotherms. An IC50-value of 0.181 ± 0.007 μmol/l for this Gpp(NH)p-effect was calculated. Divalent cations such as magnesium and manganese (10 mmol/l) increased specific binding of 3H-UK-14,304 by a factor of 3, without any influence on binding of the antagonist 3H-yohimbine. In contrast, sodium chloride strikingly decreased high-affinity binding of the agonist radioligand (IC50 = 41.9 ± 3.7 mmol/l). Unlike Gpp(NH)p, sodium chloride (> 30 mmol/l) additionally promoted a marked decrease of the affinity of UK-14,304 at the low-affinity binding component. In contrast to the effects on agonist binding, sodium chloride concentrations of 30 to 300 mmol/l increased the binding affinity of the antagonist 3H-yohimbine about 2-fold. The sodium substitute N-methyl-D-glucamine was without effect on binding of 3H-UK-14,304 indicating that the influence of sodium chloride on binding properties was not due to changes in osmolarity. In conclusion these results suggest that 3H-UK-14,304 labels preferentially the agonist induced or stabilized high-affinity state (α2H) of the platelet α2-adrenoceptor.

Inhibition of angiotensin‐I response by cilazapril and its time course in normal volunteers*

Cilazapril is a new angiotensin‐converting enzyme (ACE) inhibitor. In a double‐blind crossover study six normal male volunteers received single oral doses of cilazapril, 4 mg, captopril, 25 mg, enalapril, 10 mg, and placebo. The response of diastolic blood pressure to an intravenous infusion with increasing doses of angiotensin I (AT‐I) (0.1 to 18 μg/min) was determined at control and up to 36 hours after oral drug intake. Additionally the response to AT‐I was established before, during, and after cessation of a 15‐day 2.5 mg/day cilazapril administration. The ACE inhibitors antagonized the AT‐I effects and shifted the AT‐I dose‐effect curves rightward, whereas placebo was not effective. After single doses the effects of cilazapril and enalapril declined with a similar elimination half‐life of ~4 hours; with captopril ~2 hours was observed. After multiple administration of cilazapril there was no evidence of cumulative effects. Cilazapril is an orally active ACE inhibitor that does not show pharmacodynamically relevant accumulation.

Transdermal delivery of bupranolol: Pharmacodynamics and beta-adrenoceptor occupancy

SummaryBupranolol is a non-selective beta-adrenoceptor antagonist with a Ki-value of 6–15 nmol/l (equivalent to 1.5–4 ng/ml in plasma) at beta1- (rat salivary gland) and beta2-adrenoceptors (rat reticulocytes) in receptor binding studies with3H-CGP 12177 in the presence of human plasma. After oral administration of 200 mg bupranolol to healthy volunteers, the maximal plasma concentration was observed within 1.2 h but it only reached a level close to the Ki-value. Elimination from plasma was rapid (t1/2=2.0 h).Administration of 30 mg bupranolol in a transdermal delivery system (TTS) every 24 h to 6 healthy volunteers for 72 h yielded steady state plasma concentrations 4- to 5-times above the Ki-value as shown by in vitro inhibition of beta-adrenoceptor binding by plasma samples. The pharmacodynamic effect, measured as the reduction in exercise tachycardia, showed a stable inhibitory effect; antagonism of a bolus injection of isoprenaline indicated a 10- to 15-fold right shift of the dose-response curve during the observation period of 72 h.It is concluded that steady-state plasma concentrations and effect of the elsewise rapidly eliminated beta-blocker bupranolol can be achieved by a transdermal delivery system applied each day.

Hemodynamic responses to angiotensin I in normal volunteers and the antagonism by the angiotensin-converting enzyme inhibitor cilazapril

According to classic pharmacologic theory, agonist/antagonist competition can be used to quantify an antagonists potency by measurement of agonist dose- response curves in the presence of varying doses of the antagonist. We used this principle to characterize the interaction between angiotensin I (AI) and the angiotensinconverting enzyme (ACE) inhibitor cilazapril in humans. In addition, by comparing the effects of AI and angiotensin II before and after administration of a 30-mg dose of cilazapril, we could show the specific AI antagonism of the ACE inhibitor in humans. To obtain the antagonists dose-response curves, six healthy male volunteers received five single oral doses of cilazapril, 0.5–8.0 mg. Enalapril, 10 mg, and captopril, 12.5 mg, served as positive controls and placebo as the negative control. Dose-response curves following intravenous infusions of AI were established 4 h after oral ingestion of the ACE inhibitors. Noninvasively measured systolic and diastolic blood pressures and total peripheral resistance assessed AI effects. Cilazapril dose dependently shifted the AI dose-response curve rightward, with 1.0 mg inducing a twofold shift. Enalapril and captopril appear less potent, on a milligram basis, in antagonizing AI effects 4 h after drug intake. The methodology could be a useful tool for a rational testing and comparison of ACE inhibitors in clinical pharmacology.