D. Palm

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A direct, linear relation was shown between the in vitro inhibition of beta-adrenoceptor binding by the plasma samples withdrawn after propranolol administration and the inhibition of exercise tachycardia observed in parallel.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.

Correlation between increased dopamine-β- hydroxylase activity and catecholamine concentration in plasma: Determination of acute changes in sympathetic activity in man

SummaryIn 11 healthy untrained volunteers the increase in plasma dopamine-β-hydroxylase (DBH) activity during graded physical exercise has been examined as a true measure of increased activity of the sympathetic nervous system. The correlation between DBH activity, catecholamine concentration (CA) in plasma and heart rate was studied. When work on an electrically braked bicycle ergometer was gradually increased from 12.5 to 100, 200 and 300 watts there was a linear increase in DBH activity and heart rate; the increase in CA concentrations followed an exponential function. The peak values for DBH activity and CA concentration in plasma after the 300 watt work load (as percentages of the resting levels) were 130±3% and 820±71%, respectively; the adrenaline concentration in plasma increased only to 150±19% (p>0.05). There were significant correlations between heart rate and work load, DBH and work load and log CA and work load. The data imply direct correlations between heart rate and DBH, heart rate and log CA and DBH and log CA. The exponential increase in noradrenaline concentration in plasma might be due either to a greater net “overflow” from sympathetic nerve endings, and/or to increased secretion by the adrenal medulla. In the latter case, the release of noradrenaline would not be accompanied by secretion either of adrenaline or DBH. After work ceased there were sharp falls in heart rate and CA concentration, which indicate an immediate drop in sympathetic activity. DBH activity in plasma returned to normal very slowly; it reached half maximum values after 20 – 22 min. It is concluded that increased sympathetic activity in man can be estimated in vivo as changes in DBH and/or CA concentration in plasma. In contrast, a rapid decrease in sympathetic activity is directly reflected only by a rapid fall in the plasma concentrations of CA.

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.

Rates of recovery of irreversibly inhibited monoamine oxidases: A measure of enzyme protein turnover

Summary1.After irreversible inhibition of monoamine oxidase (MAO) in rats with hydrazine derivatives (3-amino-2-oxazolidinone, furazolidone, benmoxine) and the non-hydrazine pargyline, recovery of enzyme activity occurred at rates which were characteristic for the organ investigated and independent of the chemical structure of the irreversible inhibitor. The respective half times were the same in homogenates and in mitochondria and amounted to about 10 days in the brain and 3 to 4 days in the liver; in the small intestine, mucosal MAO activity recovered with a half time of 0.5 days, whereas in the residual intestinal layers a half time of about 4 days was found.\3-Tranylcypromine is not an irreversible inhibitor: the half times of MAO recovery were 3.6 days in the brain and 2.4 days in the liver.Thus, long duration of inhibition and organ-specific half times of recovery of MAO inhibition are characteristic features of irreversible inhibitors.2.When mitochondrial protein was labelled by i.v. injection of 14C-leucine, specific radioactivity declined with a half time of 9.4 days in the brain and 4.0 days in the liver; for the intestinal mucosa and for the residual intestinal layers, a half time of 0.5 and 4.4 days, respectively, was found.As these values are nearly identical with those found for the rates of MAO recofery after irreversible inhibition, the assumption is supported that irreversibly inhibited MAO must be replaced by newly synthetized enzyme. Vice versa, the rates of MAO recovery after irreversible inhibition seem to reflect the rates of mitochondrial protein turnover. Measurements of the rate of recovery of irreversibly inhibited MAO activity may therefore be useful to determine the turnover of the enzyme protein.

Occurrence of adenyl cyclase activity in human erythrocytes

SummaryRed cell ghosts from healthy volunteers and from patients suffering from haemopoietic or haemolytic disorders were differentiated with respect to their basal, sodium fluoride- and isoproterenol-stimulated adenyl cyclase activities; it was investigated whether adenyl cyclase activities are correlated with the reticulocyte counts in the respective blood samples. In the experiments presented, it has been established for the first time that significant adenyl cyclase activity is present in ghosts from human red blood cells.In patients with pernicious anaemia, both reticulocyte counts and sodium fluoridestimulated adenyl cyclase activities were found to be enhanced during treatment with cyanocobalamine. In patients with haemolytic diseases (reticulocyte counts about 10%) adenyl cyclase activities did not differ from those found in healthy volunteers (about 1% reticulocytes). In ghost preparations from patients with chronic renal insufficiency, sodium fluoride- and isoproterenol-stimulated adenyl cyclase activities were slightly depressed. The possible significance of these findings is discussed.ZusammenfassungAn Erythrozytenschatten-Präparationen von Gesunden und von Patienten mit Erkrankungen des hämopoetischen Systems oder mit hämolytischen Erkrankungen wurden die Aktivitäten der Adenylzyklase (basale, Natriumfluorid- und Isoproterenol-stimulierte Aktivität) bestimmt; es wurde untersucht, inwieweit eine Korrelation zwischen der Aktivität der Adenylzyklase und den Retikulozytenzahlen im peripheren Blut besteht.In den vorliegenden Untersuchungen konnte erstmals nachgewiesen werden, daß in menschlichen Erythrozytenschatten signifikante Adenylzyklase-Aktivität vorhanden ist. Bei Patienten mit perniziöser Anämie waren unter der Behandlung mit Cyanocobalamin sowohl die Retikulozytenzahlen als auch die Natriumfluorid-stimulierten Adenylzyklase-Aktivitäten gegenüber den gesunden Kontrollpersonen erhöht. Patienten mit hämolytischer Anämie (10% Retikulozyten) wiesen in ihren Schattenpräparationen die gleichen Adenylzyklase-Aktivitäten auf wie Normalpersonen (1% Retikulozyten). In Erythrozytenschatten von Patienten mit chronischer Niereninsuffizienz fanden sich erniedrigte Natriumfluorid- und Isoproterenol-stimulierte Adenylzyklase-Aktivitäten. Die mögliche Bedeutung dieser Befunde wird diskutiert.

Dose‐effect and pharmacokinetic‐pharmacodynamic relationships of the β1adrenergic receptor blocking properties of various doses of carvedilol in healthy humans

To evaluate the pharmacodynamic properties of carvedilol across a broad range of doses in relation to its enantiospecific kinetics and adrenergic receptor occupancies, relative to placebo and propranolol.

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.

Esmolol, an ultrashort-acting, selective β1-adrenoceptor antagonist: pharmacodynamic and pharmacokinetic properties

The effects of esmolol at different rates of infusion (100, 250 and 500 μg·kg−1 BW·min−1) were compared with β-adrenoceptor occupancy (β1 and β2, estimated by a subtype selective radioreceptor assay) and plasma concentrations of esmolol and its acid metabolite were measured by HPLC. Up to a rate of infusion of esmolol of 500 μg·kg−1 BW·min−1 there was a maximal β1-receptor occupancy of 84.7% while β2-receptor occupancy was below the detection limit; confirming the β1 selectivity of esmolol. Exercise-induced increases in heart rate and systolic blood pressure were reduced by esmolol in a dose-dependent manner. The estimated EC50 values of rate of infusion for the reduction in heart rate and systolic blood pressure during exercise were 113 and 134 μg·kg−1 BW · min−1, respectively. Additionally, heart rate and systolic blood pressure were reduced moderately at rest. Because of the short elimination half-life of esmolol caused by the rapid hydrolysis to its acid metabolite, 45 min after end of infusion high plasma concentrations of the metabolite (maximally 80 μg·ml−1) but no esmolol were detectable. Since no in vivo effects have been observed, despite the presence of high plasma concentrations of the metabolite, the metabolite did not participate in the observed effects up to an infusion rate of esmolol of 500 μg·kg−1 BW·min−1. The plasma concentrations of antagonist detected by radioreceptor assay and plasma concentrations of esmolol detected by HPLC showed a good correlation (r=0.97). Since the cardiovascular effects, determined before and 45 min after termination of infusion of esmolol were similar, it can be concluded that the observed effects on heart rate and systolic blood pressure are exclusively mediated by esmolol.

Adenyl cyclase activities in rat erythrocytes during stress erythropoiesis: Localization of the enzyme in the reticulocytes

Abstract NaF- and isoproterenol-stimulated adenyl cyclase activity found in preparations from rat erythrocytes is obviously localized mainly in the reticulocytes. After treatment with acetylphenyl hydrazine, both reticulocyte counts and adenyl cyclase activity increase dose-dependently. Evidence is given that adenyl cyclase activity in the red blood cell is lost during the maturation process.

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.